7582_FM_i-xxiv 19/08/20 1:39 PM Page i

Urinalysis
and Body Fluids
SEVENTH EDITION
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7582_FM_i-xxiv 19/08/20 1:39 PM Page iii

Urinalysis
and Body Fluids
SEVENTH EDITION

Susan King Strasinger, DA, MT(ASCP)

Retired Faculty Associate
Clinical Laboratory Sciences Program
The University of West Florida
Pensacola, Florida

Marjorie Schaub Di Lorenzo, BS, MT(ASCP)SH

Phlebotomy Technician Program Coordinator
Nebraska Methodist College
Omaha, Nebraska
7582_FM_i-xxiv 19/08/20 1:39 PM Page iv

F. A. Davis Company
1915 Arch Street
Philadelphia, PA 19103
www.fadavis.com

Copyright © 2021 by F. A. Davis Company

Copyright © 2021 by F. A. Davis Company. All rights reserved. This product is protected by copyright. No part of it may be reproduced,
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Printed in the United States of America

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Manager of Content Development: George W. Lang
Developmental Editor: Stephanie Kelly
Content Project Manager: Megan Suermann
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As new scientific information becomes available through basic and clinical research, recommended treatments and drug therapies undergo
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Library of Congress Cataloging-in-Publication Data

Names: Strasinger, Susan King, author. | Di Lorenzo, Marjorie Schaub, 1953-

author.
Title: Urinalysis and body fluids / Susan King Strasinger, Marjorie Schaub
Di Lorenzo.
Description: Seventh edition. | Philadelphia: F. A. Davis Company, [2021] |
Includes bibliographical references and index.
Identifiers: LCCN 2020022498 (print) | LCCN 2020022499 (ebook) | ISBN
9780803675827 (paperback) | ISBN 9780803675834 (ebook)
Subjects: MESH: Urinalysis—methods | Body Fluids—chemistry
Classification: LCC RB53 (print) | LCC RB53 (ebook) | NLM QY 185 | DDC
616.07/566—dc23
LC record available at https://lccn.loc.gov/2020022498
LC ebook record available at https://lccn.loc.gov/2020022499

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7582-7/20 0 + $.25.
7582_FM_i-xxiv 19/08/20 1:39 PM Page v

To Harry—you will always be my editor-in-chief

—SKS

To my husband, Scott, for his support

and encouragement
—MSD
7582_FM_i-xxiv 19/08/20 1:39 PM Page vi
7582_FM_i-xxiv 19/08/20 1:39 PM Page vii

Preface

As will be apparent to readers, the seventh edition of Urinalysis bronchoalveolar lavage, and amniotic fluids, Chapters 10
and Body Fluids has been revised and enhanced substantially. through 17 have been enhanced. Sections covering anatomy
However, the objective of the text—to provide concise, com- and physiology; specimen collection, handling, and processing;
prehensive, and carefully structured instruction in the analysis current testing methodology; and associated clinical significance
of nonblood body fluids—remains the same. have been added for each of these fluids. A new chapter
This seventh edition has been redesigned to meet the (Chapter 14) discusses the collection, analysis, and significance
changes occurring in both laboratory medicine and instruc- of specimens for bronchoalveolar lavage.
tional methodology. The book is organized into three parts: Each chapter opens with learning outcomes and key terms
Basic Principles, Urinalysis, and Other Body Fluids. and concludes with multiple choice questions, case studies,
To meet the expanding technical information required by and clinical situations for student review. In response to read-
students in laboratory medicine, all of the chapters have been ers’ suggestions, the number of color images and figures have
updated. Part One focuses on basic principles associated with been increased significantly. The text has been supplemented
urinalysis and body fluids. Chapter 1 covers overall laboratory extensively with tables, summaries, and procedure boxes. Case
safety, precautions relating to analysis of urine and body fluids, studies in the traditional format, as well as clinical situations
and the importance of quality assessment and management in relating to technical considerations included at the end of
the urinalysis laboratory. Preexamination (preanalytical), ex- the chapters, offer students an opportunity to think critically
amination (analytical), and postexamination (postanalytical) about the material. A feature titled Historical Notes provides
variables; procedure manuals; and current regulatory issues are a reference for topics or tests that are no longer performed rou-
stressed. Chapter 2 is new and introduces the student to the tinely. Another feature, Technical Tips, emphasizes informa-
most up-to-date automation tools for analysis of urine and tion important to performing procedures. Procedures have
body fluids. This chapter covers the ever-increasing variety of been reformatted into step-by-step instructions. The F. A. Davis
automated instrumentation available by different manufactur- logo icon that appears in various chapters will direct the students
ers for the analysis of urine and body fluids. The basic princi- to the procedural videos found at www.fadavis.com under the
ples used in urine chemical automation, such as reflectance Instructor Resources. Reagent Strip Color Charts enhance
photometry, light scatter, fluorescence light, and refractometry, Chapter 6, Chemical Examination of Urine, highlighting the
are explained. Included in this chapter are the significant timing, specificity, and sensitivity of each chemical reaction.
advancements in the automated microscopic analysis of urine New to this edition is a two-part, full-color Atlas of Images
that cover the principles of flow cytometry and digital image with images for urine and body fluid structures. Over 130 new
analysis. Chapter 3 discusses the importance of urine specimen images are included in the atlas for use as a reference in
collections, handling, and preservation. Chapter 4 covers renal the identification of cellular components in both urine and
physiology and the various renal function tests. These chapters other body fluids. Images showing real urine sediments with
provide the background for discussing the three components mixed field components can be used for identifying cells that
of a complete urinalysis and associated diseases that are are often misidentified and serve as an excellent teaching re-
discussed in the next part. source. The atlas provides a description; clinical significance;
In Part Two, Chapter 5 covers the physical examination of physical, chemical, and microscopic correlations; and tips for
urinalysis, whereas Chapter 6 describes the chemical exami- identification for student reference. Boxes and Tables sum-
nation and includes the chemical reaction principle, clinical marize important information and correlations. An Answer
significance, and reaction interference for each chemical test. Key for the study questions, case studies, and clinical situations
Chapter 7 includes numerous additional images showing the is included at the end of the book. Key terms appear in bold-
various urine microscopic components. In Chapters 8 and 9, face blue color within the chapters. General medical terms ap-
the diseases of glomerular, tubular, interstitial, vascular, and pear in boldface black in the text and also are included in the
hereditary origin that are encountered most frequently are Glossary. The abbreviations noted in boldface red color
related to their associated laboratory tests. throughout the book have been collected in a convenient
Part Three is dedicated to a discussion of other body flu- Abbreviations list at the back of the book. The online Instructor
ids. To accommodate advances in laboratory testing of those Resources at www.fadavis.com include example lecture and lab-
fluids, including cerebrospinal, seminal, synovial, serous, oratory schedules, procedures for student laboratory simulated

vii
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viii Preface

specimens, class activities, chapter outlines, case studies and have access to online review questions, animations, videos, in-
clinical situations with answers, study questions and answers, teractive exercises, and robust case studies at www.fadavis.com.
a test question bank, image bank, chapter-by-chapter Power- As always, we thank our readers for their valuable sugges-
Points, an electronic version of the textbook, the Instructor’s tions, which have guided us in creating this exciting new edi-
Guide, animations, and videos. For self-study, students will tion as well as the student and instructor resources.
Susan King Strasinger
Marjorie Schaub Di Lorenzo
7582_FM_i-xxiv 19/08/20 1:39 PM Page ix

Contributor and Special Content Reviewer

Kathleen Finnegan, MS, MT(ASCP)SH

Clinical Associate Professor
Clinical Laboratory Sciences
Stony Brook University
Stony Brook, New York

ix
7582_FM_i-xxiv 19/08/20 1:39 PM Page x
7582_FM_i-xxiv 19/08/20 1:39 PM Page xi

Reviewers

Wayne Aguiar, MS, MLS(ASCP) Linda Bau, MPA, MT(ASCP)

Director of Health Sciences and Medical Laboratory MLT Program Director
Science Allied Health
College of Arts and Sciences Moraine Park Technical College
University of Bridgeport Fond du Lac, Wisconsin
Bridgeport, Connecticut
Annette E. Bednar, MSE, MT(ASCP)
Laura Ahonen, MS, MT(ASCP) Assistant Professor of Clinical Laboratory Sciences
MLT Program Director College of Nursing and Health Professions
Phlebotomy Program Director Arkansas State University
Northcentral Technical College Jonesboro, Arkansas
Wausau, Wisconsin
Angela R. Bell, MS, MLS(ASCP)SM, DLM
Alison Albertson, MS, MT(ASCP) Medical Laboratory Technology and Phlebotomy
MLT Program Director Program Director
Medical Laboratory Health Professions
Lake Area Technical Institute Tidewater Community College
Watertown, South Dakota Norfolk, Virginia

Lois C. Anderson, MA, MT(ASCP) Jan Bradley, MT(ASCP)

Associate Professor Program Director and Instructor
Minnesota State University, Mankato Medical Laboratory Technology
Mankato, Minnesota Wayne Community College
Goldsboro, North Carolina
Beverly J. Barham, PhD, MPH, MT(ASCP)
Professor Nicholas Brehl, MLS(ASCP)CM
Medical Laboratory Science Program Assistant Clinical Professor
Department of Health Sciences Pathology and Laboratory Medicine
Illinois State University Indiana University School of Medicine
Normal, Illinois Indianapolis, Indiana

Brenda C. Barnes, PhD, MT(ASCP)SBB Carol Brennan, BS, MT(ASCP)

Director, Medical Laboratory Science Program Clinical Instructor, Microbiology and Serology
Director, EdD Health Professions Education Program Medical Laboratory Science Program
Professor Methodist Hospital Pathology
Allen College – UnityPoint Health Omaha, Nebraska
Waterloo, Iowa
Michelle Briski, MEd, MT(ASCP)
Crystal E. Barrett, MAED/T, MLS(ASCP)CM MLT Program Director
Adjunct Faculty Saint Paul College
Nursing and Allied Health Saint Paul, Minnesota
J. Sargeant Reynolds Community College
Richmond, Virginia

xi
7582_FM_i-xxiv 19/08/20 1:39 PM Page xii

xii Reviewers

Keri Brophy-Martinez, MT(ASCP) Christy A. Cole, MT(ASCP)

Department Chair/Medical Laboratory Technology Instructor/Phlebotomy Advisor
Professor Clinical Laboratory Technology
Medical Laboratory Technology West Georgia Technical College
Austin Community College Waco, Georgia
Austin, Texas
Janet E. Cooper, MSNS, BS, MT(ASCP)H
Karen Brown, MS, MLS(ASCP)CM Program Director
Professor Medical Laboratory Technology
Pathology Mississippi Delta Community College
University of Utah Moorhead, Mississippi
Salt Lake City, Utah
Rhonda Craver, MLS(ASCP)
Renelle Brun, MLT Director
Clinical Biochemistry Instructor Montana Medical Laboratory Science Program
Medical Laboratory Sciences Montana State University
CCNB Dieppe Campus Bozeman, Montana
Dieppe, New Brunswick
Canada Diane Davis, PhD, MLS (ASCP)CM, SCCM, SLSCM
Professor and Chair
Jennifer Bushnell, MEd, MLS(ASCP) Medical Laboratory Science Program
MLS Program Director Salisbury University
RMLS Salisbury, Maryland
McNeese State University
Lake Charles, Louisiana Linda J. Dickson, MSPH, MT(ASCP)
MLT Program Director
Tracy Camara, MBA, MT(ASCP)SM Medical Clinical Laboratory Technician
Program Director, School of Medical Laboratory Science Erwin Technical College
System Director, Laboratory Education and Performance Tampa, Florida
Improvement
Baptist Health – South Patricia Ellinger, MSEd, MLS(ASCP)CM, SBBCM
Montgomery, Alabama
MLS Faculty
Medical Laboratory Science Program
Leslie Cameron, MLT St. Cloud State University
Program Instructor St. Cloud, Minnesota
Medical Laboratory Technology
Chemistry Mary Emes, MMedSc (Pathology)
Nova Scotia Community College Certificate in Adult Education, MLT, ART (CC, MI, TS)
Halifax, Nova Scotia Professor
Canada Medical Laboratory Science and Continuing Education
The Michener Institute of Education at University Health
Katie Cavnar, MS, MT(ASCP) Network
Chair Toronto, Ontario
Medical Laboratory Sciences Canada
University of West Florida
Pensacola, Florida Julie Foley, CLS, MT(ASCP)
Education Coordinator
Carol E. Church, MAT, MT(ASCP)SM CLS/CGMBS Training Programs
Assistant Professor California State University, Los Angeles
Clinical Laboratory Technologies Los Angeles, California
SUNY Broome Community College
Binghamton, New York
7582_FM_i-xxiv 19/08/20 1:39 PM Page xiii

Reviewers xiii

Kristin A. Forsberg, MEd, MLS(ASCP)CM Virginia Haynes, MS, MLS(ASCP)CM

MLT Program Director Program Director
MLT Program MLT
Laurel Technical Institute Lake Superior College
Hermitage, Pennsylvania Duluth, Minnesota

Karen Gatewood, MEd, MLS(ASCP)CM Tamara Henderson, CLS(ASCP)

MLT Assistant Program Director Medical Laboratory Technology
MLT Program Pearl River Community College
Harcum College Poplarville, Mississippi
Bryn Mawr, Pennsylvania
Candy Hill, MAEd, MT(ASCP)
Lynette K. Gayle, MLS(ASCP), JD, MA Ed MLT Program Coordinator
CLS Program Director/Assistant Professor Center for Professional, Career and Technical Education
Allied Health Jefferson State Community College
Tuskegee University Birmingham, Alabama
Tuskegee, Alabama
Elizabeth Hoffman, MA Ed, CMA(AAMA),
Jeffrey S. Gillette, PhD, MT(ASCP) CPT(ASPT)
Program Director Medical Assistant Program Director
Professor, Medical Laboratory Technology Henry Ford College
Virginia Western Community College Dearborn, Michigan
Roanoke, Virginia
Ruth Jacobs, MT(ASCP)
Karen Gordon, MS, MLS(ASCP)CM, SLS Assistant Professor/Department Chair
Professor Emeritus Allied Health
Retired MLT Program Head/Assistant Dean BridgeValley Community and Technical College
Northern Virginia Community College – Medical South Charleston, West Virginia
Education Campus
Springfield, Virginia Haywood B. Joiner Jr., EdD, MLS(ASCP)
Chair
Cheri Goretti, MA, MT(ASCP), CMA(AAMA) Department of Allied Health
Professor Emeritus Louisiana State University of Alexandria
Medical Assisting and Allied Health Alexandria, Louisiana
Quinebaug Valley Community College
Danielson, Connecticut Minh Do Kosfeld, PhD, MLT(ASCP)CM
Investigative and Medical Sciences Program Director
Nancy Grandbois, MS, MLS(ASCP)CM Assistant Professor
Instructor Department of Clinical Health Sciences
Biomedical and Nutritional Sciences Saint Louis University
University of Massachusetts Lowell St. Louis, Missouri
Lowell, Massachusetts
Heather Lai, MLS(ASCP)SH
Tammy Hardie, BA, MLT, RT Clinical Instructor of Hematology, Coagulation,
Instructor Urinalysis, and Body Fluids
Medical Laboratory Sciences Unity Point Health – St. Luke’s Medical Laboratory
British Columbia Institute of Technology Science Program
Burnaby, British Columbia Cedar Rapids, Iowa
Canada
7582_FM_i-xxiv 19/08/20 1:39 PM Page xiv

xiv Reviewers

Karlyn Lange, MT(ASCP) Lori Moore, MEd, MT(ASCP)

Program Director Program Director, Instructor
Mercy Integrated CLS Program School of Medical Technology
Toledo, Ohio Berkshire Medical Center
Pittsfield, Massachusetts
Laura R. Link, MS, MLS(ASCP)CM
Program Director and Assistant Professor Deirdre Parsons, MS, MT(ASCP)SBB
Medical Laboratory Science Program Director
Radford University University of Maryland
Radford, Virginia Baltimore, Maryland

Janis P. Livingston, MT(ASCP) Srinivas Pentyala, PhD

MLT Clinical Education Coordinator Professor and Director of Translational Research of
Medical Laboratory Technology Anesthesiology
Midlands Technical College Faculty Member in Urology, Health Sciences, Physiology
Columbia, South Carolina and Biophysics
Stony Brook Medical Center
Patricia Longpre, MLT Stony Brook, New York
Professor
Health Sciences Lori Rager, CMA (AAMA), COC, CPC (AAPC)
Cambrian College of Applied Arts and Technology Lead MA Instructor
Sudbury, Ontario Medical Assistant
Canada Utah State University Eastern
Price, Utah
Nina Maniotis, MT(ASCP)
Phlebotomy Program Director/Instructor Mary Sadlowski, BS, MT
Health and Human Sciences Adjunct Teacher
Weatherford College Health Department
Weatherford, Texas Community College of Baltimore County
Baltimore, Maryland
Teresa Maxwell, BS, MT(ASCP)
Technical Coordinator Salle Sappington, MLS(ASCP)
Methodist Hospital Pathology Teacher
Omaha, Nebraska Monroe Technology Center
Loudoun County Schools
Ann C. McConnell, MLS(ASCP)CM Leesburg, Virginia
Chair and Program Director
Clinical Associate Professor Rebbecca Silva, MS, MT(ASCP)
East Carolina University Assistant Professor and Chair
Greenville, North Carolina Medical Laboratory Technology
New England Institute of Technology
Carol McCoy, PhD, MLS(ASCP)CM East Greenwich, Rhode Island
Adjunct Professor
Oklahoma Christian University Rosalyn H. Singleton, MHS, MT(ASCP), BS
Oklahoma City, Oklahoma Clinical Coordinator
Medical Laboratory Technology
Dana McGuire, MPH, BS, MLS(ASCP)CM Northeast Mississippi Community College
Booneville, Mississippi
Technical Coordinator
Methodist Women’s Hospital Pathology
Omaha, Nebraska
7582_FM_i-xxiv 19/08/20 1:39 PM Page xv

Reviewers xv

C. Thomas Somma, EdD Karlien Smith Tebbetts, MLS(ASCP)

Associate Professor Faculty
Clinical Laboratory Sciences Medical Technology
Old Dominion University Houston Community College
Norfolk, Virginia Houston, Texas

Rose Marie Spangler, MPH, MLS(ASCP) M. Lorraine Torres, EdD, MT(ASCP)

Program Director/Assistant Professor Program Director
Health-Related Professions Clinical Laboratory Sciences Program Director
Northeast State Community College The University of Texas at El Paso
Blountville, Tennessee El Paso, Texas

Donna Spannaus-Martin, PhD, MLS(ASCP)CM Maria Torres-Pillot, MA, MLS (ASCP)CM

Professor Program Director of Medical Laboratory Technology
Medical Laboratory Sciences Health Sciences
University of Minnesota Argosy University – Dallas
Minneapolis, Minnesota Farmers Branch, Texas

Brian Spezialetti, MS, MT(ASCP) Misty D. Turner, MT(ASCP)

Program Director Education Coordinator
School of Medical Laboratory Sciences School of Clinical Laboratory Science
Guthrie Robert Packer Hospital Augusta Health
Sayre, Pennsylvania Fishersville, Virginia

John Strous, MS, MLS(ASCP)CM Meridee Van Draska, MSMHA, MLS (ASCP),
Emeritus AHI (AMT)
Program Director Associate Professor
Medical Technology Program Medical Laboratory Science Program Director
University of Wisconsin Oshkosh Illinois State University
Oshkosh, Wisconsin Normal, Illinois

Harvey Suski, MLT WenFang Wang, PhD, C(ASCP)CM

Instructor Clinical Assistant Professor
Medical Laboratory Sciences Department of Clinical Laboratory Sciences
Red River College Kansas University Medical Center
Winnipeg, Manitoba Kansas City, Kansas
Canada
Lanette Hall Washington, MSM, MLS(ASCP)CM,
MaryEllen Tancred, PhD, MLS(ASCP)CMSHCM MT (AMT)
NAACLS Program Director/Coordinator MLT Program Director
Associate Professor Education
Allied Health – Medical Laboratory Technology Fortis College – Baton Rouge
Columbus State Community College Baton Rouge, Louisiana
Columbus, Ohio
Tiffany H. Whittle, MSHS, MLS(ASCP)CM
Angela Tarrant, MEd, MT(ASCP)SMCM MLT Associate Professor
Program Director Medical Laboratory Technology
Medical Laboratory Technician Greenville Technical College
Otero Junior College Greenville, South Carolina
La Junta, Colorado
7582_FM_i-xxiv 19/08/20 1:39 PM Page xvi

xvi Reviewers

Marie L. Wilson, MT(ASCP) Kimberly G. Yarborough, MLS(ASCP)CMSHCM

Laboratory Supervisor Medical Laboratory Science
Daviess Community Hospital Carolinas College of Health Sciences
Washington, Indiana Charlotte, North Carolina

Kathi Winker, MS, MT(ASCP)

MLT Program Director
Health and Public Safety
Blackhawk Technical College
Janesville, Wisconsin
7582_FM_i-xxiv 19/08/20 1:39 PM Page xvii

Acknowledgments

Many people deserve credit for the help and encouragement illustrations, and the students from the University of West Florida
they have provided us in the preparation of this seventh edi- who worked under the guidance of Sherman Bonomelli to
tion. Our continued appreciation is also extended to all of the produce many of those images. Images for Chapter 16 had been
people who were instrumental in the preparation of previous provided previously by Carol Brennan, MT(ASCP); Diane Siedlik,
editions. We want to especially thank Kathleen Finnegan of MT(ASCP); Christian Herdt, MT(ASCP); and Teresa Karre, MD,
Stony Brook, New York, for her countless hours capturing from Methodist Hospital. Images for the atlas were provided
urine and body fluid images for the Atlas of Images that is new by Dawn Plank, MLS(ASCP)CM, Methodist Hospital, Omaha,
to the seventh edition. We are forever indebted to her for her Nebraska, and Kathleen Finnegan, MS, MT(ASCP)SH, Clinical
expertise, enthusiasm, and commitment in the development Associate Professor, Stony Brook University, New York, as well
of this project. as the Stony Brook University Clinical Laboratory Science 2020
The valuable suggestions from previous readers and the senior students, who helped find the urinalysis elements that
support from our colleagues at Methodist Hospital, Omaha, were photographed for the atlas.
Nebraska, have been a great asset to us in the production of We would like to express our gratitude for the help, patience,
this new edition. We thank each and every one of you. guidance, and understanding of our editors at F. A. Davis: Christa
We extend special thanks to the people who have provided Fratantoro, Publisher; Megan Suermann, Content Project
us with so many beautiful photographs for the text over the years: Manager; and Stephanie Kelly, Developmental Editor. We thank
Donna L. Canterbury, BA, MT(ASCP)SH; Joanne M. Davis, BS, all the members of the F. A. Davis team who were instrumental
MT(ASCP)SH; M. Paula Neumann, MD; Gregory J. Swedo, MD; in bringing this edition to fruition: George Lang, Manager
and Scott Di Lorenzo, DDS. We also remember and appreciate of Content Development; Carolyn O’Brien, Art and Design
Sherman Bonomelli, MS, who had contributed original visual Manager; Sharon Lee, Production Manager; Kate Margeson,
concepts that became the foundation for many of the line Illustrator Coordinator; and Julie Mangoff of Graphic World, Inc.

xvii
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7582_FM_i-xxiv 19/08/20 1:39 PM Page xix

Contents

Atlas of Images 1 Automated Microscopy Analyzers 79

Automated Urinalysis Systems 86
PART ONE: Basic Principles 48 Body Fluid Analysis Automation 87
CHAPTER 1 CHAPTER 3

Safety and Quality Management 49 Introduction to Urinalysis 91

Safety 50 History and Importance 92

Biological Hazards 50 Urine Formation 93
Infectious Agent (Pathogen) 50 Urine Composition 93
Reservoir 50 Urine Volume 93
Portal of Exit 51 Specimen Collection 94
Means of Transmission 51 Containers 94
Portal of Entry 51 Labels 95
Susceptible Host 51 Requisition Form 95
Standard Precautions 51 Specimen Rejection 95
Sharp Hazards 56 Specimen Handling 95
Chemical Hazards 56 Specimen Integrity 95
Chemical Spills and Exposure 56 Specimen Preservation 96
Chemical Handling 57 Types of Specimens 96
Chemical Hygiene Plan 57 Random Specimen 96
Chemical Labeling 57 First Morning Specimen 96
Safety Data Sheets 57 24-Hour (or Timed) Specimen 97
The Globally Harmonized System Catheterized Specimen 98
of Classification and Labeling of Chemicals 58 Midstream Clean-Catch Specimen 98
Chemical Waste Disposal 58 Suprapubic Aspiration 98
Radioactive Hazards 58 Prostatitis Specimen 98
Electrical Hazards 59 Pediatric Specimens 100
Fire/Explosive Hazards 60 Drug Specimen Collection 100
Physical Hazards 61 CHAPTER 4
Quality Management 61 Renal Function 105
Urinalysis Procedure Manual 62 Introduction 106
Preexamination (Preanalytical) Variables 62 Renal Physiology 106
Examination (Analytical) Variables 64 Renal Blood Flow 106
Postexamination Variables 68 Glomerular Filtration 106
CHAPTER 2 Tubular Reabsorption 110
Urine and Body Fluid Analysis Automation 75 Tubular Secretion 111
Urinalysis Automation 76 Renal Function Tests 113
Reflectance Photometry 76 Glomerular Filtration Tests 113
Analyzers 76 Tubular Reabsorption Tests 116
Fully Automated Urine Chemistry Analyzers 78 Tubular Secretion and Renal Blood Flow Tests 119

xix
7582_FM_i-xxiv 19/08/20 1:39 PM Page xx

xx Contents

PART TWO: Urinalysis 124 Bilirubin 154

Bilirubin Production 154
CHAPTER 5
Clinical Significance 155
Physical Examination of Urine 125 Reagent Strip (Diazo) Reactions 155
Introduction 126 Reaction Interference 156
Color 126 Urobilinogen 156
Normal Urine Color 126 Clinical Significance 157
Abnormal Urine Color 127 Reagent Strip Reactions and Interference 157
Clarity 129 Reaction Interference 157
Normal Clarity 129 Nitrite 157
Nonpathological Turbidity 129 Clinical Significance 157
Pathological Turbidity 130 Reagent Strip Reactions 158
Specific Gravity 131 Reaction Interference 158
Refractometer 131 Leukocyte Esterase 159
Osmolality 132 Clinical Significance 159
Reagent Strip Specific Gravity 133 Reagent Strip Reaction 160
Odor 134 Reaction Interference 160
CHAPTER 6 Specific Gravity 160
Chemical Examination of Urine 139 Reagent Strip Reaction 160
Introduction 140 Reaction Interference 160
Reagent Strips 140 CHAPTER 7
Reagent Strip Technique 140 Microscopic Examination of Urine 167
Handling and Storing Reagent Strips 141 Introduction 168
Quality Control of Reagent Strips 142 Macroscopic Screening 168
Confirmatory Testing 142 Specimen Preparation 168
pH 142 Specimen Volume 168
Clinical Significance 143 Centrifugation 168
Reagent Strip Reactions 143 Sediment Preparation 169
Protein 143 Volume of Sediment Examined 169
Clinical Significance 144 Commercial Systems 169
Prerenal Proteinuria 144 Examining the Sediment 169
Renal Proteinuria 144 Reporting the Microscopic Examination 170
Postrenal Proteinuria 145 Correlating Results 170
Reagent Strip Reactions 146 Sediment Examination Techniques 170
Reaction Interference 146 Sediment Stains 171
Glucose 148 Cytodiagnostic Urine Testing 173
Clinical Significance 148 Microscopy 173
Reagent Strip (Glucose Oxidase) Reaction 149 Types of Microscopy 176
Reaction Interference 150 Urine Sediment Constituents 178
Copper Reduction Test (Clinitest) 150 Red Blood Cells 179
Clinical Significance of Clinitest 151 White Blood Cells 181
Ketones 151 Epithelial Cells 182
Clinical Significance 151 Bacteria 187
Reagent Strip Reactions 152 Yeast 189
Reaction Interference 152 Parasites 189
Blood 152 Spermatozoa 189
Clinical Significance 153 Mucus 190
Reagent Strip Reactions 153 Casts 191
Reaction Interference 154
7582_FM_i-xxiv 19/08/20 1:39 PM Page xxi

Contents xxi

Urinary Crystals 199 WBC Count 258

Urinary Sediment Artifacts 209 Quality Control of CSF and Other Body Fluid Cell
CHAPTER 8 Counts 258
Renal Disease 219 Differential Count on a CSF Specimen 258
Cytocentrifugation 259
Introduction 220
CSF Cellular Constituents 259
Glomerular Disorders 220
Chemistry Tests 266
Glomerulonephritis 220
Cerebrospinal Protein 266
Nephrotic Syndrome 222
CSF Glucose 268
Tubular Disorders 222
CSF Lactate 268
Acute Tubular Necrosis 222
CSF Glutamine 268
Hereditary and Metabolic Tubular Disorders 225
Microbiology Tests 268
Interstitial Disorders 226
Gram Stain 269
Urinary Tract Infection 226
Immunologic Assays 270
Acute Pyelonephritis 226
PCR Molecular Diagnostic Testing 270
Chronic Pyelonephritis 227
Serological Testing 271
Acute Interstitial Nephritis 228
CHAPTER 11
Renal Failure 228
Renal Lithiasis 229 Semen 277
Introduction 278
CHAPTER 9
Physiology 278
Urine Screening for Metabolic Disorders 235
Specimen Collection 278
Introduction 236
Specimen Handling 280
Overflow Versus Renal Disorders 236
Semen Analysis 280
Newborn Screening Tests 236
Appearance 280
Amino Acid Disorders 236
Liquefaction 280
Phenylalanine-Tyrosine Disorders 236
Volume 280
Branched-Chain Amino Acid Disorders 239
Viscosity 281
Tryptophan Disorders 241
pH 281
Cystine Disorders 242
Sperm Concentration and Sperm Count 281
Porphyrin Disorders 243
Sperm Motility 283
Mucopolysaccharide Disorders 246
Automated Semen Analysis Systems 283
Purine Disorders 247 Sperm Morphology 283
Carbohydrate Disorders 247 Additional Testing 285
Sperm Vitality 286
PART THREE: Other Body Fluids 252
Seminal Fluid Fructose 286
CHAPTER 10 Antisperm Antibodies 286
Cerebrospinal Fluid 253 Microbial and Chemical Testing 287
Introduction 254 Postvasectomy Semen Analysis 287
Formation and Physiology 254 Sperm Function Tests 288
Specimen Collection and Handling 254 Semen Analysis Quality Control 288
Appearance 255 CHAPTER 12

Traumatic Collection (Tap) 256 Synovial Fluid 293

Uneven Blood Distribution 256 Physiology 294
Clot Formation 257 Specimen Collection and Handling 294
Xanthochromic Supernatant 257 Color and Clarity 295
Cell Count 257 Viscosity 296
Methodology 257 Cell Counts 296
Total Cell Count 258 Differential Count 296
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xxii Contents

Crystal Identification 297 CHAPTER 15

Types of Crystals 297 Amniotic Fluid 327
Slide Preparation 297 Introduction 328
Crystal Polarization 298 Physiology 328
Chemistry Tests 300 Function 328
Glucose 300 Volume 328
Total Protein 300 Chemical Composition 329
Uric Acid 301 Differentiating Maternal
Lactate 301 Urine From Amniotic Fluid 329
Enzymes 301 Specimen Collection 329
Microbiological Tests 301 Indications for Amniocentesis 329
Serological Tests 301 Collection 330
CHAPTER 13 Specimen Handling and Processing 330
Serous Fluid 305 Color and Appearance 331
Introduction 306 Tests for Fetal Distress 331
Formation 306 Hemolytic Disease of the Fetus
and Newborn 331
Specimen Collection and Handling 306
Neural Tube Defects 332
Transudates and Exudates 307
Tests for Fetal Maturity 333
General Laboratory Procedures 307
Fetal Lung Maturity 333
Pleural Fluid 308 Lecithin-Sphingomyelin Ratio 333
Appearance 308 Phosphatidyl Glycerol 334
Hematology Tests 309 Foam Stability Index 334
Chemistry Tests 310 Lamellar Bodies 334
Microbiological and Serological Tests 312
CHAPTER 16
Pericardial Fluid 312
Fecal Analysis 339
Appearance 312
Introduction 340
Laboratory Tests 312
Physiology 340
Peritoneal Fluid 314
Diarrhea and Steatorrhea 341
Transudates Versus Exudates 314
Diarrhea 341
Appearance 314
Steatorrhea 342
Laboratory Tests 314
Specimen Collection 342
CHAPTER 14
Macroscopic Screening 342
Bronchoalveolar Lavage Fluid 321
Color 343
Clinical Significance 322
Appearance 343
Specimen Collection 322
Microscopic Examination of Feces 343
Specimen Handling and Transport 322
Fecal Leukocytes 343
Color and Clarity 322 Muscle Fibers 344
Cell Counts 322 Qualitative Fecal Fats 344
Leukocytes 323 Chemical Testing of Feces 345
Macrophages 323 Occult Blood 345
Lymphocytes 323 Quantitative Fecal Fat Testing 347
Neutrophils 323 APT Test (Fetal Hemoglobin) 348
Eosinophils 323 Fecal Enzymes 349
Mast Cells 324 Carbohydrates 349
Erythrocytes 324
Epithelial Cells 324
Microbiological Tests 324
Cytology 324
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Contents xxiii

CHAPTER 17 Desquamative Inflammatory Vaginitis 365

Vaginal Secretions 355 Atrophic Vaginitis 365
Introduction 356 Additional Procedures for Vaginal Secretion
Specimen Collection and Handling 357 Associated With Pregnancy 365
Fetal Fibronectin Test 365
Color and Appearance 357
AmniSure Test 366
Diagnostic Tests 357
Actim PROM 366
pH 357
ROM Plus 366
Microscopic Procedures 358
Answers to Study Questions, Case Studies,
Other Diagnostic Tests 363
and Clinical Situations 371
Vaginal Disorders 364
Abbreviations 381
Bacterial Vaginosis 364
Glossary 385
Trichomoniasis 364
Index 393
Candidiasis 364
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7582_Atlas_001-047 29/07/20 4:17 PM Page 1

Atlas of Images
Kathleen Finnegan

URINALYSIS 2 Crystals/Acid 26
Cells 2 Uric Acid Crystals 26
Red Blood Cells 2 Amorphous Urate Crystals 27
White Blood Cells 3 Calcium Oxalate Crystals 28
Epithelial Cells 5 Sodium Urate Crystals 29
Transitional Epithelial (Urothelial) Cell 7 Crystals/Alkaline 30
Renal Tubular Epithelial Cells 8 Amorphous Phosphate Crystals 30
Oval Fat Bodies 10 Triple Phosphate Crystals 30
Microorganisms 11 Calcium Phosphate Crystals 32
Bacteria 11 Calcium Carbonate Crystals 32
Yeast 12 Ammonium Biurate Crystals 33
Parasites 14 Abnormal Crystals 34
Trichomonads 14 Cystine Crystals 34
Pinworm 14 Leucine Crystals 34
Schistosoma Haematobium 15 Tyrosine Crystals 35
Miscellaneous Structures 16 Cholesterol Crystals 35
Sperm 16 Bilirubin Crystals 36
Mucus 16 Sulfonamide Crystals 37
Casts 17 Ampicillin Crystals 37
Hyaline Casts 17 Artifacts 38
Red Blood Cell Casts 18 Radiographic Dye Crystals 38
White Blood Cell Casts 19 Starch 38
Bacterial Casts 20 Fibers 39
Epithelial Cell Casts 20 Oil Droplets or Air Bubbles 40
Granular Casts 21 BODY FLUIDS 41
Mixed Cell Casts 23 Cerebrospinal Fluid 41
Fatty Casts 23 Peritoneal Fluid 43
Waxy Casts 24 Pleural Fluid 44
Broad Casts 25 Synovial Fluid 46

1
7582_Atlas_001-047 29/07/20 4:17 PM Page 2

URINALYSIS
Cells

Red Blood Cells Clinical Significance

Description • Increased RBCs are seen with urinary tract infections (UTIs),
• Normal: Pale nonnucleated biconcave discs, membrane regular, trauma to the kidneys, or menstrual contamination
6 to 8 µm in size • Dysmorphic RBCs are associated with glomerular membrane
• Hypotonic urine: Red blood cells (RBCs) appear colorless with damage
membrane intact and regular; often lose their hemoglobin in a
UA Correlations
dilute urine with low specific gravity
• Color: Pale pink to bright red
• Hypertonic urine: RBCs have lost their biconcave shape, the
• Specific gravity
membrane becomes irregular, and the cells appear crenated;
• Blood
they appear like small spheres with spicules in a urine with
high specific gravity Tips
• Dysmorphic: RBCs vary in size and shape, are often • RBCs can be confused with yeast cells (round to oval and
fragmented, or have cellular protrusions (burr cells or smaller in shape and tend to bud) or oil droplets and air
acanthrocytes) bubbles (tend to be refractile)

A-01. Normal RBCs. Note uric acid crystals. A-03. Ghost RBCs.

A-02. Normal and crenated RBCs. A-04. Crenated RBCs.

2
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Atlas of Images | Urinalysis 3

A-05. Dysmorphic RBCs.

White Blood Cells • Lymphocytes are associated with renal transplant

Description • Monocytes are associated with chronic inflammation
• Colorless, 12 µm in size
UA Correlations
• Neutrophils are granular and multinucleated; the majority of
• Clarity
white blood cells (WBCs) in the peripheral blood are neutrophils
• Leukocyte esterase
• Lymphocytes and monocytes are agranular
• Nitrite with bacterial infection
• Hypotonic urine: Sparkling cytoplasm in the neutrophils
associated with Brownian movement are called glitter cells; Tips
they absorb water and swell • Neutrophils lose their nuclear detail in dilute urine and can be
confused with renal tubular epithelial (RTE) cells, which tend to
Clinical Significance
be larger with an eccentric nucleus
• Neutrophils are associated with bacterial infection, cystitis, and
• During active inflammation of the urinary tract, the WBCs may
inflammation
have extended pseudopodia
• Eosinophils are associated with drug-induced interstitial
nephritis

A-06. One-segmented WBC. Note the multilobed nucleus. A-07. Clump of WBCs.
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4 Atlas of Images | Urinalysis

A-08. WBCs and a single transitional epithelial cell. A-11. WBCs with three RBCs.

A-09. WBCs with a fine granular cast. A-12. Numerous WBCs, bacteria, and two RBCs.

A-10. Segmented WBCs. A-13. WBCs, RBCs, squamous epithelial cells, and two calcium
oxalate crystals.
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Atlas of Images | Urinalysis 5

A-14. Glitter cell. Observe the granules. RBCs are also present.

Epithelial Cells • Clue cells are pathological and indicative of vaginal infection
Description caused by Gardnerella vaginalis
• Large cells, 60 to 100 µm in size, with abundant irregular
UA Correlations
cytoplasm and wrinkled borders
• Clarity
• Nucleus is in the center and is the size of an RBC; can be
binucleated, tend to have multiridge borders that tend to fold Tips
or curl • Squamous epithelial cells tend to resemble a fried egg
• Clue cell: Variation of squamous epithelial cells; covered with • When rolled or curled, they tend to look like casts
Gardnerella coccobacillus, which gives the cell a granular • Casts do not have a nucleus
appearance • When seen in high numbers, they can indicate contamination
from vaginal secretions or poor collection
Clinical Significance
• Originate from the mucous membrane around the urethra
• Squamous epithelial cells can be associated with wear and tear
from urethra or vaginal mucosa; seen more frequently in females

A-15. Clump of squamous epithelial cells. Note normal RBCs and A-16. Rolled squamous epithelial cell. Note bacteria.
one crenated RBC.
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6 Atlas of Images | Urinalysis

A-17. Clue cell. Note bacteria covering the epithelial cell. A-20. Three squamous epithelial cells and one WBC.

A-18. Squamous epithelial cells with WBCs and one calcium oxalate A-21. Squamous epithelial cell. Note the abundant cytoplasm and
crystal. Note refractile fiber. prominent centrally located nucleus.

A-19. Two squamous epithelial cells, one calcium oxalate crystal, A-22. Squamous epithelial cell and transitional epithelial cell. The
and one RBC. amount of cytoplasm is much larger in the squamous epithelial cell.
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Atlas of Images | Urinalysis 7

Transitional Epithelial (Urothelial) Cell UA Correlations

• Clarity
Description
• Blood, if malignancy associated
• Smaller than squamous epithelial cells
• Can be spherical, pear-shaped, polyhedral, or caudate shaped Tips
with a centrally located nucleus • Spherical forms resemble RTE cells
• In elongated cells, the nucleus tends to be off-center • When seen in clusters or sheets, they are associated with
• Cells contain a moderate amount of cytoplasm catheterized specimens
• Transitional epithelial cells tend to take on water and can look
Clinical Significance
like large round balloons
• Originate from mucosa of renal pelvis, ureter, and bladder
• Appear in urine with infection, inflammation, renal stones,
bladder cancer, and catheterization

A-23. Pear-shaped transitional epithelial cell. Note bacteria. A-25. Two spherical transitional epithelial cells. WBCs, RBCs, and
yeast (refractile) are also present.

A-24. Clump of transitional epithelial cells. Note RBC. A-26. Squamous and transitional epithelial cells. Notice the central
location of the nucleus of the epithelial cells. The squamous epithe-
lial cell has a greater amount of cytoplasm. Many WBCs and bacteria
are also present.
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8 Atlas of Images | Urinalysis

A-27. A clump of transitional epithelial cells. A-28. Spherical transitional epithelial cell with a hyaline cast in the
left lower corner.

Renal Tubular Epithelial Cells (RTE) UA Correlations

Description • Clarity
• Originate from proximal renal tubules, loop of Henle, distal • Color
convoluted renal tubules, and collecting ducts; vary in size and • Protein
shape due to different origins • Blood
• Proximal tubular cells are large in size and oblong in shape with • Bilirubin (hepatitis)
grainy cytoplasm; nucleus is eccentric • Leukocyte esterase (pyelonephritis)
• Distal tubular cells are smaller and more round to oval, with • Nitrite (pyelonephritis)
small, round eccentric nucleus
Tips
• Collecting duct cells are polygonal or cuboidal with a large
• RTEs from the collecting ducts have a flat edge to one side of the
nucleus
membrane; cells from the proximal convoluted tubules are more
Clinical Significance rectangular and can be mistaken for casts
• Increased amounts are indicative of necrosis of the renal • Cells from the distal convoluted tubule are small and can be
tubules, renal tubular damage, exposure to heavy metals, drug- mistaken for WBCs
induced toxicity, hemoglobin and myoglobin toxicity, ischemic
events, and viral infections

A-29. Single and double RTE cells. Note fiber. A-30. One RTE cell with two squamous epithelial cells.
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Atlas of Images | Urinalysis 9

A-31. One RTE cell. Note bacteria. A-34. Three RTE cells. Notice the eccentrically placed nuclei.

A-32. Clump of RTE cells. A-35. RTE cells. Fiber artifact present.

A-33. RTE cell. A-36. RTE cell and WBCs.

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10 Atlas of Images | Urinalysis

Oval Fat Bodies • Blood

Description • Free fat droplets
• Oval fat bodies are renal tubular cells laden with fat • Fatty casts
• They are highly refractile and globular in shape, with a nucleus
Tips
that is difficult to see
• Oval fat bodies can be seen in conjunction with free floating fat
• Polarizing microscopy demonstrates a Maltese cross
droplets
formation
• Lubricants used in specimen collection can be confused with
Clinical Significance fat droplets
• Oval fat bodies can be associated with nephrotic syndrome,
glomerular dysfunction, and renal tubular cell death
UA Correlations
• Clarity
• Protein

A-37. Oval fat body.

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Microorganisms

Bacteria • Nitrite
Description • WBCs
• Small, spherical cocci and rod-shaped (bacilli) structures • WBCs in clumps
• Vary in size from short to long
Tips
• Can appear single or in pairs or chains
• Motility will help distinguish bacteria from amorphous urates
Clinical Significance (pink-colored sediment) or amorphous phosphates (white-
• Increased bacteria indicate a UTI colored sediment) or calcium carbonate crystals, which can look
• Bacteria should be seen with increased WBCs, positive like bacteria
leukocyte esterase, an alkaline pH, and a +/– nitrite • Bacteria multiply in urine if left at room temperature for several
hours
UA Correlations
• Alkaline pH
• Leukocyte esterase

A-38. Bacteria in chains with WBCs and RBCs. A-40. Many bacteria and WBCs.

A-39. Rod-shaped bacteria with WBCs.

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12 Atlas of Images | Urinalysis

Yeast Tips
Description • Yeast can be confused with RBCs
• Appear as colorless, small, refractile, round to oval structures • Budding yeast can help differentiate
that may be single, budding, or with pseudohyphae and • Yeast does not dissolve when acetic acid is added
branching • As with bacteria, yeast will multiply in urine if left at room
temperature for several hours
Clinical Significance
• Candida albicans is yeast found commonly in urine of women, it
most often indicates vaginal secretion contamination or vaginal
infection
• It can also be seen in patients who are diabetic or
immunocompromised
UA Correlations
• Glucose
• Increased WBCs
• Leukocyte esterase

A-41. Packed field of budding yeast and pseudohyphae. A-42. Budding yeast with WBCs and one RBC.
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A-43. Budding yeast with RBCs, WBCs, and one squamous epithelial A-45. Budding yeast with pseudohyphae and RBCs.
cell. Note the halo around RBCs.

A-44. Clump of budding yeast and WBCs.

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Parasites

Trichomonads • WBC clumps may be present

Description • Leukocyte esterase
• A trophozoite that is a pear-shaped flagellate with an
Tips
underlying membrane that causes a rapid flitting motion;
• T. vaginalis must be identified in fresh urine
resembles WBC with tail-like flagella
• They begin to die, and when not moving, they resemble WBCs
Clinical Significance • Organisms must be moving or have motility not to be confused
• Trichomonas vaginalis is a sexually transmitted pathogen with WBCs and RTE cells
associated with vaginal or urethral inflammation
• In males, it is asymptomatic
UA Correlations
• Increased WBCs
• Increased epithelial cells

A-46. A trichomonad. Notice the flagella and undulating membrane.

Pinworm UA Correlations
Description • N/A
• Eggs or ova are football shaped with one flattened side and one
Tips
round side
• Eggs or ova tend to be quite large compared with WBCs and
• Eggs are transparent, and developing larva can be seen
epithelial cells
sometimes
Clinical Significance
• Can be found as a result of fecal contamination
• Usually seen with school-age children
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Atlas of Images | Urinalysis 15

A-47. Enterobius vermicularis worm (pinworm). A-48. Enterobius vermicularis ova.

Schistosoma Haematobium UA Correlation

Description • Increased RBCs
• Very large in size and football shaped with a terminal spine • Increased WBCs
• Cell wall looks thick Tips
Clinical Significance • Sometimes the terminal spine can be small and hard to see
• Eggs can be found in urine and are endemic to Africa and the • Examine several ova for confirmation
Middle East

A-49. Schistosoma haematobium ova.

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16 Atlas of Images | Urinalysis

Miscellaneous Structures

Sperm UA Correlations
Description • Protein
• Spermatozoa have tapered oval heads with long, delicate,
Tips
flagella-like tails
• Urine is very toxic to sperm, so it rarely shows motility in urine
Clinical Significance
• Sperm can be found in the urine of both males and females
after sexual intercourse, masturbation, and nocturnal emission;
rarely have any clinical significance

A-50. Sperm and bacteria in urine sediment. A-51. Two sperm and a hyaline cast.

Mucus UA Correlations
Description • Clarity
• Single or clumped, ribbon-like or thread-like structures that
Tips
have irregular or serrated ends with low refractive index
• Mucus is hard to see with increased light due to its low
• Subdued light is required when using bright-field microscopy
refractive index
Clinical Significance • Heavy mucus threads have WBCs among them
• No clinical significance • These structures can be misidentified as hyaline casts
• Mucus can be abundant in the presence of inflammation or • Casts have rounded edges and are more cylindrical
irritation to the urinary tract

A-52. Mucus with sperm present.

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Casts

Hyaline Casts • Can be seen in patients with chronic renal disease and
Description congestive heart failure
• Composed of uromodulin protein and appear colorless and
UA Correlations
transparent under low power (100×)
• Protein
• Low refractive index similar to urine, making them difficult to
• Blood
see using bright-field microscopy
• Consist of parallel sides with rounded ends, with a variety of Tips
sizes and shapes • Hyaline casts have a low refractive index and should be viewed
• May contain incisions or have adhering cells or granules under under low light
them • Under bright-light microscopy, they can be missed
• Hyaline casts can be confused with mucus and fibers
Clinical Significance
• Mucus is thin and ribbon-like, whereas fibers do not have
• Can be found in normal individuals; increased numbers with
parallel sides or rounded edges and tend to have dark edges
strenuous exercise, stress, fever, and dehydration

A-53. Hyaline cast. A-55. Hyaline cast. Note refractile fiber in the top left corner.

A-54. Hyaline cast. Several RBCs present. A-56. Hyaline cast and numerous squamous epithelial cells.
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18 Atlas of Images | Urinalysis

A-57. Cylindroid.

Red Blood Cell Casts Tips

Description • RBC clumps can be confused with RBC casts
• There are few to many RBCs dispersed in the protein matrix • Casts have parallel sides with rounded edges
• They appear red to brown but vary in size and shape • The RBCs should be distinct and inside the protein matrix to be
called a cast
Clinical Significance
• If the RBCs have degenerated to a yellow-brown homogenous
• RBC casts are associated with damage to the glomerulus or
structure, it is called a hemoglobin cast
glomerulonephritis, bleeding into the nephron, and lupus
UA Correlations
• Increased RBCs
• Protein
• Blood

A-58. Hemoglobin cast.

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Atlas of Images | Urinalysis 19

White Blood Cell Casts UA Correlations

Description • Protein
• WBCs with prominent nuclei are found in a cylindrical matrix • Leukocyte esterase
with parallel sides and rounded edges that vary in size and • Increased WBCs
length Tips
Clinical Significance • WBC clumps can be confused with WBC casts
• The presence of WBC casts can indicate renal inflammation, • To identify the cast, the WBCs are intact with visible nuclei
infection, acute pyelonephritis, or interstitial nephritis within the protein matrix
• The presence of WBC casts can distinguish pyelonephritis or
upper UTI from cystitis or lower UTI

A-59. WBC cast. Notice the WBCs within the matrix of the cast and A-61. Broad WBC cast with many free-floating WBCs, two RBCs, and
free-floating in the urine sediment. budding yeast present.

A-60. Degenerating WBC cast. WBCs, RBCs, a hyaline cast, a RTE, and
an artifact fiber are also present.
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20 Atlas of Images | Urinalysis

Bacterial Casts • Protein

Description • Leukocyte esterase
• Casts contain bacilli within the protein matrix • Nitrite
Clinical Significance Tips
• Diagnostic for pyelonephritis or upper UTI • These casts can also contain WBCs and are called mixed casts
• These casts can be confused with granular casts; confirmation is
UA Correlations
through a Gram stain
• WBCs
• Bacteria

A-62. Mixed granular cast with bacteria embedded in the matrix.

Note bacteria surrounding in cast. Bacterial casts are not common.

Epithelial Cell Casts UA Correlations

Description • Protein
• RTE cells with large nuclei are seen within protein matrix; they • RTE cells
have a high refractive index
Tips
• As the RTE cells undergo degenerating changes, they become
• Epithelial cell casts can be confused with WBC casts
smaller and round resembling WBCs
• A negative leukocyte esterase can help distinguish the
Clinical Significance epithelial cell cast from a WBC cast
• Most often associated with acute tubular necrosis, advanced
tubular destruction, and heavy metal or drug toxicity

A-64. Cast with oval fat bodies.

A-63. Renal epithelial cell cast.

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Atlas of Images | Urinalysis 21

Granular Casts UA Correlations

Description • Protein
• Casts are a degeneration of cellular casts; granules begin large • RBCs
and are coarse and granular • WBCs
• As stasis continues, granules break down further and are called • Cellular casts
fine granular casts Tips
• Coarse granules are dark in color, and fine granules tend to • Examine under low power, but casts are identified under high
be pale power
• Casts can be of various sizes and shapes • These casts can be reported as coarse, fine, or just granular
Clinical Significance • They can be confused with fibers or clumps of sediment
• These casts can indicate urinary stasis, tubular disease, • For it to be a cast, granules must be in a surrounding protein
glomerulonephritis, pyelonephritis, or may be seen in stress or matrix
after exercise

A-65. Granular cast. A-67. Granular cast with a squamous epithelial cell and RBC.

A-66. Granular cast. Note RBCs, WBCs, and bacteria. A-68. One granular cast. Numerous sperm present.
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22 Atlas of Images | Urinalysis

A-69. Granular cast. A-72. Granular cast.

A-70. Granular cast. RBCs are also present. A-73. One granular cast and two hyaline casts.

A-71. One granular cast with four squamous epithelial cells. Note A-74. Granular cast. Sperm present at the top of image. Amorphous
the highly refractile artifact. is also present.
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Mixed Cell Casts • RBCs

Description • WBCs
• Contain multiple cellular constituents, which can include RBCs, • RTE cells
WBCs, RTE cells, and bacteria
Tips
Clinical Significance • Mixed casts should have separate free-floating cellular elements
• Can indicate renal disease and are usually seen with other present
cellular casts
UA Correlations
• Protein
• Leukocyte esterase

A-75. Mixed-cell cast. Note free-floating RBCs and WBCs.

Fatty Casts UA Correlations

Description • Protein
• Highly refractile • Free fat droplets
• Casts may contain fat droplets and intact oval fat bodies of • Oval fat bodies
varying sizes within the protein matrix
Tips
Clinical Significance • Maltese cross formations can be seen with cholesterol under
• Associated with nephrotic syndrome, acute tubular necrosis, polarization
diabetes mellitus, toxic tubular necrosis, and crushing injuries

A-76. Fatty cast. Note intact oval fat bodies within the matrix.
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Waxy Casts • Cellular casts

Description • Granular casts
• Highly refractile with broken, jagged, or blunt ends with cracks
Tips
on the margin of the matrix
• These casts tend to look like paraffin shavings sitting on top of
• May be colorless to gray
the urine
Clinical Significance • They are often broad in size due to renal stasis
• Casts indicate urinary stasis, chronic renal failure, or end-stage
renal disease
UA Correlations
• Protein
• Increased RBCs
• Increased WBCs

A-77. Waxy cast with a fragmented end and notches on its side. A-79. Waxy cast.
WBCs are also present.

A-78. A waxy cast is located in the center with four granular casts to
the side. Also present are RTE cells, RBCs, and squamous epithelial cells.
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Broad Casts • Granular casts

Description • Waxy casts
• Named for their wide size
Tips
• Formed due to distention of the collecting tubules where they
• Granular or waxy casts may be classified as broad
are formed
Clinical Significance
• Represent extreme urinary stasis and renal failure
UA Correlations
• Protein
• Increased RBCs
• Increased WBCs

A-80. Broad granular cast.

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Crystals/Acid

Uric Acid Crystals UA Correlations

Description • Acid pH
• Yellow to brown in color; can occur in many shapes, including
Tips
rhombic, four-sided flat plates, rosettes, barrels, and wedges
• These crystals can be confused with cystine crystals
Clinical Significance • Uric acid crystals are birefringent under polarized light
• Can be found in normal individuals • Crystals have four sides and look more like a football or lemon
• Can be associated with pathological conditions, such as urinary wedge
stones, gout, high purine metabolism, acute febrile conditions, • Cystine have six sides or look like a stop sign
newborns, and patients with leukemia receiving chemotherapy

A-81. Uric acid crystals, with barrel shape. A-83. Uric acid crystals. Note the rosette and wedge shapes.

A-82. Uric acid crystal clump. A-84. Two uric acid crystals observed using high-power magnifica-
tion. One hyaline cast, WBCs, and bacteria are also present.
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A-85. Uric acid crystals observed using low-power magnification.

Note the numerous hyaline casts.

Amorphous Urate Crystals Tips

Description • When amorphous urates clump, they tend to look like a
• Appear as yellow to reddish granules granular cast
• Always look for the protein matrix to define a cast
Clinical Significance
• They also can hide bacteria and RBCs
• No clinical significance
• Amorphous urates dissolve when heated to 60°C
• These are salts of sodium, potassium, magnesium, and calcium;
usually appear in older urine
UA Correlations
• Acid pH
• Sediment has a pink to brick dust–like color after centrifugation

A-86. Amorphous urates distinguished by acid pH.

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Calcium Oxalate Crystals UA Correlations

Description • Acid or neutral pH
• Often appear as colorless, envelope-shaped crystals with an X
Tips
or cross in the middle
• The more common calcium oxalate crystals have the shape of
• Can appear as dumbbell- or small oval–shaped
an emerald-cut diamond
Clinical Significance • The oval shapes tend to look like RBCs, but they are more
• Frequently can be found in normal urine after ingesting elliptical and tend to have a dark outline along their edge
oxalate-rich food, such as tomatoes, spinach, oranges, fruit juice,
vitamin C, and chocolate
• Pathological conditions include ethylene glycol poisoning, liver
disease, and renal calculi

A-87. Calcium oxalate crystals. Note RBCs and budding yeast. A-89. Monohydrate calcium oxalate crystals.

A-88. Clump of calcium oxalate crystals. A-90. One monohydrate calcium crystal and two classic dihydrate
calcium oxalate crystals.
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Atlas of Images | Urinalysis 29

A-91. Calcium oxalate crystal. A-92. Monohydrate calcium oxalate crystals.

Sodium Urate Crystals Tips

Description • These are usually seen when urine has been standing
• Colorless or yellowish, slender needles with blunt ends that can • When found in clusters, they look like “pick-up sticks”
be found singly or in clusters
Clinical Significance
• No clinical significance
UA Correlations
• Acid pH

A-93. Sodium urate crystals.

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30 Atlas of Images | Urinalysis

Crystals/Alkaline

Amorphous Phosphate Crystals Tips

Description • Amorphous phosphates can interfere with the microscopic
• Fine colorless granules that can be hard to distinguish from examination
amorphous urates; differentiated by sediment color and • RBCs and bacteria may not be visualized
urine pH • Amorphous phosphates do not dissolve when heated at 60°C
• Refrigeration increases their presence
Clinical Significance
• They are distinguished from urates by urine pH
• No clinical significance
UA Correlations
• Urine is cloudy when many crystals are present
• Alkaline or neutral pH
• White sediment after centrifugation

A-94. Amorphous phosphate crystals.

Triple Phosphate Crystals Tips

Description • Triple phosphate crystals vary in size and sometimes have the
• Most common form is colorless prisms with three to six sides shape and size of a calcium oxalate
referred to as coffin covers • The cross on top of the crystal overlaps or meets in the middle
• Vary in size of the triple phosphate crystal
• When they denature, they become feathery or leaf-like • For calcium oxalate crystals, the X crosses in the middle
Clinical Significance
• Can be seen in healthy individuals with no clinical significance
• Can be associated with urinary stones and UTI
UA Correlations
• Alkaline or neutral pH
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Atlas of Images | Urinalysis 31

A-95. Triple phosphate crystals. A-98. Square triple phosphate crystals with bacteria and yeast
present in urine sediment.

A-96. Clump of triple phosphate crystals. A-99. Triple phosphate crystals.

A-97. Triple phosphate crystal with RBCs.

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32 Atlas of Images | Urinalysis

Calcium Phosphate Crystals UA Correlations

Description • Alkaline or neutral pH
• Colorless to grayish-white thin needles
Tips
• Arranged in bundles or thin, long prisms and can be arranged
• Sometimes the sheets resemble large degenerating
in rosettes or can be flat plates
squamous cells
• Tend to float on the top of the urine
Clinical Significance
• Can be found in normal urine and are associated with renal
stones

A-100. Calcium phosphate crystals.

Calcium Carbonate Crystals UA Correlations

Description • Alkaline pH
• Small, colorless, dumbbell-shaped; appear in pairs or may have Tips
a spherical shape • Calcium carbonate can appear in clumps and resemble
Clinical Significance amorphous phosphates or be mistaken for bacteria
• No clinical significance • They tend to be a little larger than amorphous phosphates

A-101. Calcium carbonate crystals.

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Atlas of Images | Urinalysis 33

Ammonium Biurate Crystals UA Correlations

Description • Alkaline pH
• Appear as yellow to brown spheres with long spicules
Tips
surrounding the crystal; resemble a “thorny apple”
• Usually encountered in old specimens
Clinical Significance
• Usually seen in urines that have not been stored properly
• May be associated with the presence of ammonia produced by
urea-splitting bacteria

A-102. Two ammonium biurate crystals. A-104. Ammonium biurate crystals.

A-103. Ammonium biurate crystal. Note bacteria.

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34 Atlas of Images | Urinalysis

Abnormal Crystals

Cystine Crystals Tips

Description • Cystine crystals can be distinguished from uric acid crystals
• Appear as colorless hexagonal plates with sides that are not using polarization
always even, resembling a stop sign • Uric acid crystals are birefringent under polarization, whereas
• Can be found single or overlapping each other to form rosettes cystine crystals are not
• Cystine crystals appear as a six-sided stop sign, whereas uric
Clinical Significance
acid crystals appear as a four-sided stop sign
• Clinically significant and indicate a metabolic hereditary
disorder called cystinosis or cystinuria
UA Correlations
• Acid pH

A-105. Cystine crystals.

Leucine Crystals Tips

Description • These crystals can sometimes resemble fat globules
• Amino acid crystals • Under polarization, they produce a pseudo–Maltese cross
• Appear as oily or highly refractile yellow to brown spheres with pattern
radial striations • They also can resemble wagon wheels or concentric circles
resembling a tree’s growth pattern or striations of a tree
Clinical Significance • These crystals are rarely seen in urinalysis
• Can appear in patients with severe liver disease, terminal
cirrhosis, viral hepatitis, and maple syrup disease
• Can also be seen in patients with tyrosine crystals
UA Correlations
• Acid or neutral pH
• Bilirubin
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Atlas of Images | Urinalysis 35

A-106. Leucine crystals (×400). Note the concentric circles.

Tyrosine Crystals UA Correlations

Description • Acid or neutral pH
• Amino acid crystals • Bilirubin
• Appear as fine needles occurring in shelves or clusters that are Tips
colorless or yellow • This is a rare crystal not seen very often in urine sediment
Clinical Significance • They are often observed with leucine crystals
• Associated with liver disease and with inherited disorders of
amino acid metabolism

A-107. Tyrosine crystal.

Cholesterol Crystals UA Correlations

Description • Acid pH
• Appear as irregular, rectangular, large transparent plates with a • Protein
notch in one or more corners • Oval fat bodies
• Often appear in stacks • Fatty casts
• Fat globules
Clinical Significance
• Can be seen in patients with nephrotic syndrome, chyluria, and Tips
disorders producing lipiduria • These crystals are seen after refrigeration
• They are highly birefringent with polarized light
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36 Atlas of Images | Urinalysis

A-108. Cholesterol crystals.

Bilirubin Crystals UA Correlations

Description • Acid pH
• Appear as small clusters of fine clumped needles or spheres • Bilirubin
• Dark yellow in color
Tips
Clinical Significance • A positive result for bilirubin must be present for these crystals
• Associated with hepatic disease, producing large amounts of to be identified
bilirubin

A-109. Bilirubin crystal. Note the classic bright yellow color.

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Atlas of Images | Urinalysis 37

Sulfonamide Crystals UA Correlations

Description • Acid pH
• Can appear in many different forms; most shapes are bundles of
Tips
needles that resemble stacks of wheat or have a fan shape
• These crystals can be confused with ammonium biurate
• Can be brown in color
• Ammonium biurate crystals are found with an alkaline pH
Clinical Significance
• With the development of more soluble sulfur drugs, these
crystals are not found often
• Patients with UTIs are treated with sulfonamides
• Also can be associated with renal calculi or tubular damage

A-110. Sulfa crystal.

Ampicillin Crystals UA Correlations

Description • Acid or neutral pH
• Appear as colorless long needles that tend to form bundles
Tips
after refrigeration
• Knowledge of patient history is helpful to identify these crystals
Clinical Significance
• Indicative of large doses of ampicillin without adequate
hydration

A-111. Ampicillin crystals.

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38 Atlas of Images | Urinalysis

Artifacts

Radiographic Dye Crystals UA Correlations

Description • High specific gravity when using a refractometer
• When ionic radiographic crystals precipitate out in urine,
Tips
they appear as flat, elongated rectangles that are strongly
• These have a similar appearance to cholesterol crystals
birefringent
• Cholesterol crystals are accompanied by other lipid elements
Clinical Significance • May persist for 3 days after the procedure
• X-ray procedures, such as a pyelogram, use dye to visualize the
urinary tract

A-112. Radiographic dye crystals.

Starch UA Correlations
Description • N/A
• Vary in size
Tips
• Have a Y indentation in the center
• Starch or pollen granules can be confused with fat droplets
• Not perfectly round, but have more scalloped edges
• Under polarized light, starch can appear as the Maltese cross
Clinical Significance
• Caused by contamination from baby powder or powder from
health care worker gloves

A-113. Starch granule. A-114. Starch granules. Notice the dimpled centers.
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Atlas of Images | Urinalysis 39

Fibers Tips
Description • Fibers can be plant, vegetable, fabric threads, toilet paper,
• Large with distinct, flat, dark edges cotton, or diaper
• Refractile • They can resemble hyaline casts
• Fibers are thicker at their margins and polarize
Clinical Significance
• Considered contaminants
UA Correlations
• N/A

A-115. Fiber. Note RBCs. A-117. Fiber. Note refractibility.

A-116. Fiber. A-118. Fiber.

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40 Atlas of Images | Urinalysis

A-119. Refractile fiber, dysmorphic RBCs, and an RTE cell.

Oil Droplets or Air Bubbles UA Correlations

Description • N/A
• Highly refractile; may resemble RBCs
Tips
Clinical Significance • Oil droplets and air bubbles can look like RBCs; however, their
• Contamination; oil droplets from immersion oil and air bubbles high refractibility distinguishes them
formed when the specimen is placed under a cover slip are
highly refractile using low power

A-121. Note the oil droplet indicated by the arrow.

A-120. Air bubble. Note high refractibility.

7582_Atlas_001-047 29/07/20 4:18 PM Page 41

BODY FLUIDS
Cerebrospinal Fluid

A-122. Bloody CSF with neutrophils, lymphocytes, and one A-124. Bloody CSF with neutrophils, with cytoplasmic vacuoles
monocyte. resulting from cytoplasmic centrifugation.

A-123. Bloody CSF with neutrophils. A-125. Ependymal cells in CSF. Notice the nucleoli and less distinct
borders.

41
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42 Atlas of Images | Body Fluids

A-126. Neutrophils with intracellular and extracellular bacteria

in CSF.
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Atlas of Images | Body Fluids 43

Peritoneal Fluid

A-127. Two large mesothelial cells and three monocytes in A-128. Two monocytes and one lymphocyte in peritoneal fluid.
peritoneal fluid.
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44 Atlas of Images | Body Fluids

Pleural Fluid

A-129. Two monocytes, one eosinophil, and one neutrophil in A-131. Lymphocytes, neutrophils, and monocytes in pleural fluid.
pleural fluid. Note the large reactive lymphocyte.

A-130. Reactive lymphocytes in pleural fluid. A-132. Reactive lymphocytes in pleural fluid.
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Atlas of Images | Body Fluids 45

A-133. Mesothelial cells in pleural fluid. A-135. RBCs, lymphocyte, and neutrophil in pleural fluid.

A-134. Eosinophil and RBCs in pleural fluid.

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46 Atlas of Images | Body Fluids

Synovial Fluid

A-136. Wright-stained neutrophils containing CPPD crystals in A-138. Wright-stained eosinophil in synovial fluid.
synovial fluid.

A-139. Polarized urate crystals in synovial fluid.

A-137. Numerous Wright-stained neutrophils with vacuoles in

synovial fluid, indicative of inflammation.
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7582_Ch01_048-074 24/06/20 5:42 PM Page 48

PART ONE

Basic Principles
Chapter 1: Safety and Quality Management
Chapter 2: Urine and Body Fluid Analysis Automation
Chapter 3: Introduction to Urinalysis
Chapter 4: Renal Function
7582_Ch01_048-074 24/06/20 5:42 PM Page 49

CHAPTER 1
Safety and Quality
Management
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
1-1 Define and give an example of the types of safety haz- 1-9 Describe the use of the Globally Harmonized
ards encountered in the laboratory. System (GHS).
1-2 List the six components of the chain of infection and 1-10 Recognize standard hazard warning symbols.
the laboratory safety precautions that break the chain.
1-11 State and interpret the components of the National
1-3 State the purpose of the Standard Precautions policy, Fire Protection Association (NFPA) hazardous material
and describe its guidelines. labeling system.
1-4 State the requirements mandated by the Occupational 1-12 Describe precautions that laboratory personnel should
Exposure to Bloodborne Pathogens Compliance take with regard to radioactive, electrical, fire, and
Directive. physical hazards.
1-5 Describe the types of personal protective equipment 1-13 Explain the RACE and PASS actions to be taken when
(PPE) that laboratory personnel wear, including when, a fire is discovered.
how, and why each article is used.
1-14 Explain the role of quality management (QM) in the
1-6 Correctly perform hand-hygiene procedures following urinalysis laboratory.
guidelines provided by the Centers for Disease Control
1-15 Define the preexamination (preanalytical), examina-
and Prevention (CDC).
tion (analytical), and postexamination (postanalytical)
1-7 Describe the acceptable methods for handling and dis- components of QM.
posing of biological waste and sharp objects in the uri-
1-16 Distinguish among internal, external, and electronic
nalysis laboratory.
quality control, as well as external quality assessment
1-8 Discuss the components and purpose of a chemical (proficiency testing) in a QM program.
hygiene plan and a Safety Data Sheet (SDS).

KEY TERMS
Accreditation Clinical and Laboratory Standards Globally Harmonized System (GHS)
Accuracy Institute (CLSI) Infection control
Autoverification Delta check Internal quality control
Biohazardous Electronic quality control Occupational Safety and Health Ad-
Chain of infection Examination variable ministration (OSHA)
Chemical hygiene plan (CHP) External quality assessment (EQA) Personal protective equipment
External quality control (PPE)
Clinical Laboratory Improvement
Amendments (CLIA) Fomite Postexamination variable
Continued
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50 Part One | Basic Principles

K E Y T E R M S —cont’d
Postexposure prophylaxis (PEP) Quality management (QM) Specificity
Precision Quality management system (QMS) Standard Precautions (SP)
Preexamination variable Radioisotope Test utilization
Preventive maintenance (PM) Reliability Turnaround time (TAT)
Proficiency testing (PT) Root cause analysis (RCA) Universal Precautions (UP)
Quality assessment (QA) Safety Data Sheet (SDS)
Quality control (QC) Sensitivity

Safety throughout life. Safety procedure manuals that describe the

safety policies mandated by the Centers for Disease Control and
The clinical laboratory contains a variety of safety hazards, Prevention (CDC) and the Occupational Safety and Health
many capable of producing serious injury or life-threatening Administration (OSHA) must be readily available in the labo-
disease. To work safely in this environment, laboratory person- ratory, and it is essential for laboratory personnel to adhere
nel must learn what hazards exist, the basic safety precautions closely to these guidelines. The laboratory director must update
associated with them, and how to apply the basic rules of com- and review the manual annually. The Clinical and Laboratory
mon sense required for everyday safety for patients, coworkers, Standards Institute (CLSI) provides the guidelines for writing
and themselves. these procedures and policies.1-3
As seen in Table 1-1, some hazards are unique to the health-
care environment, whereas others are encountered routinely
Biological Hazards
The health-care setting provides abundant sources of
Table 1–1 Types of Safety Hazards potentially harmful microorganisms. These microor-
ganisms are frequently present in the specimens re-
Type Source Possible Injury
ceived in the clinical laboratory. Understanding how
Biological Infectious Bacterial, fungal, microorganisms are transmitted (chain of infection) is essential
agents viral, or parasitic to preventing infection. All health-care facilities have developed
infections procedures to control and monitor infections occurring within
Sharps Needles, lancets, Cuts, punctures, their facilities, which is referred to as infection control. The
broken glass or exposure to chain of infection requires a continuous link among six elements:
bloodborne an infectious agent, a reservoir, a portal of exit, a means of trans-
pathogens mission, a portal of entry, and a susceptible host.4
Chemical Preservatives Exposure to toxic, Infectious Agent (Pathogen)
and reagents carcinogenic, or
caustic agents Infectious agents can include bacteria, fungi, parasites, and
viruses. The chain of infection is broken by early detection of
Radioactive Equipment and Exposure to
one of these agents. The treatment of infectious agents can
radioisotopes radiation
reduce their opportunity for growth.
Electrical Ungrounded or Burns or shock
wet equip- Reservoir
ment; frayed
cords The reservoir is the location of potentially harmful microor-
Fire/ Open flames, Burns or ganisms, such as a contaminated clinical specimen or an in-
explosive organic dismemberment fected patient. It is the place where the infectious agent can
chemicals live and possibly multiply. Humans and animals make excellent
reservoirs. Equipment and other soiled inanimate objects,
Physical Wet floors, Falls, sprains, or called fomites, will serve as reservoirs, particularly if they con-
heavy boxes, strains tain blood, urine, or other body fluids. Some microorganisms
patients form spores or become inactive when conditions are not ideal
From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, and wait until a suitable reservoir is available. Disinfecting the
Fourth Edition, FA Davis, Philadelphia, 2019, p. 44, with permission. work area kills the infectious agent and eliminates the reservoir,
thereby breaking the chain of infection.
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Chapter 1 | Safety and Quality Management 51

Portal of Exit Preventing completion of the chain of infection is a primary

objective of biological safety. Figure 1-1 illustrates the universal
The infectious agent must have a way to exit the reservoir to symbol for biohazardous material and demonstrates how
continue the chain of infection. This can be through the following prescribed safety practices can break the chain of
mucous membranes of the reservoir’s nose, mouth, and eyes, infection. Figure 1-1 places particular emphasis on laboratory
as well as in blood or other body fluids. The chain of infection practices.
is broken when contaminated materials are placed in biohazard
containers and/or when tubes and specimen containers are
sealed. When contaminated materials are in the appropriate
Standard Precautions
containers that remain sealed, the infectious agent still has a Proper hand hygiene, correct disposal of contaminated materials,
reservoir but no means of exit. and wearing personal protective equipment (PPE) are of major
importance in the laboratory. Concern over exposure to blood-
Means of Transmission borne pathogens, such as hepatitis B virus (HBV), hepatitis C
Once the infectious agent has left the reservoir, it must have a virus (HCV), and human immunodeficiency virus (HIV), caused
way to reach a susceptible host. Means of transmission include: the CDC and OSHA to draft guidelines and regulations to
prevent exposure. In 1987, the CDC instituted Universal
1. Direct contact: the unprotected host touches the patient, Precautions (UP), in which all patients are considered to be
specimen, or a contaminated object (reservoir) possible carriers of bloodborne pathogens. The guideline rec-
2. Airborne: the host inhales dried aerosol particles circu- ommends wearing gloves when collecting or handling blood
lating on air currents or attached to dust particles and body fluids contaminated with blood and wearing face
3. Droplet: the host inhales infected aerosol droplets from shields when there is danger of blood splashing on mucous
the reservoir (e.g., aerosol droplets from a patient or an membranes and when disposing of all needles and sharp ob-
uncapped centrifuge tube, or when specimens are jects in puncture-resistant containers. The CDC excluded urine
aliquoted or spilled) and body fluids not visibly contaminated by blood from UP,
4. Vehicle: the host ingests a contaminated substance although many specimens can contain a considerable amount
(e.g., food, water, specimen) of blood before it becomes visible. UP was modified for body
substance isolation (BSI) to help alleviate this concern. BSI
5. Vector: from an animal or insect bite guidelines are not limited to bloodborne pathogens; they
Hand sanitizing and adhering to Standard Precautions consider all body fluids and moist body substances to be
(SP) are methods to break the chain of infection. potentially infectious. According to BSI guidelines, personnel
must wear gloves at all times when encountering moist body
Portal of Entry substances. A major disadvantage of BSI guidelines is that they
After the infectious agent has been transmitted to a new reser- do not recommend hand sanitizing after removing gloves
voir, it must have a means to enter the reservoir. The portal of unless visual contamination is present.
entry can be the same as the portal of exit, which includes the In 1996, the CDC and the Healthcare Infection Control
mucous membranes of the reservoir’s nose, mouth, and eyes, Practices Advisory Committee (HICPAC) combined the major
as well as breaks in the skin and open wounds. Disinfection features of UP and BSI guidelines and called the new guidelines
and sterilization and strict adherence to SPs block the portal Standard Precautions. Although SP, as described next, stress
of entry, thereby breaking the chain of infection. patient contact, the principles also can be applied to handling
patient specimens in the laboratory.5
Susceptible Host SP are as follows:

The susceptible host can be another patient during invasive pro- 1. Hand hygiene: Hand hygiene includes both hand washing
cedures, visitors, and health-care personnel when exposed to and the use of alcohol-based antiseptic cleansers. Sanitize
infectious specimens or needlestick injuries. Immunocompro- hands after touching blood, body fluids, secretions, excre-
mised patients, newborns and infants, and the elderly are often tions, and contaminated items, whether or not gloves are
more susceptible hosts. Stress, fatigue, and lack of proper nu- worn. Sanitize hands immediately after removing gloves,
trition depress the immune system and contribute to the sus- between patient contacts, and when otherwise indicated to
ceptibility of patients and health-care providers. Once the chain avoid transferring microorganisms to other patients or en-
of infection is complete, the infected host becomes another vironments. Sanitizing hands may be necessary between
source able to transmit the microorganisms to others.1 To break tasks and procedures on the same patient to prevent cross-
the chain of infection, health-care personnel must sanitize contamination of different body sites.
hands often, wear gloves, stay current with the required immu- 2. Gloves: Wear gloves (clean, nonsterile, latex-free gloves
nizations and tests, and maintain a healthy lifestyle. are adequate) when touching blood, body fluids, secre-
In the clinical laboratory, the most direct contact with a tions, excretions, and contaminated items. Put on gloves
source of infection is through contact with patient specimens, just before touching mucous membranes and nonintact
although contact with patients and infected objects also occurs. skin. Change gloves between tasks and procedures on
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52 Part One | Basic Principles

Break the link Break the link

• Immunizations • Disinfection
• Patient isolation Infectious agent • Hand hygiene
• Nursery • Bacteria
precautions • Fungi
• Healthy lifestyle • Parasites
Susceptible • Viruses
host Reservoir
• Patients • Humans
• Elderly • Animals
• Newborns • Insects
• Immuno- • Fomites
compromised • Blood/body
• Health-care fluids
workers

Portal of exit
Portal of
entry • Nose
• Mouth
• Nose
• Mucous
• Mouth
membranes
• Mucous
• Specimen
membranes
collection
• Skin
• Unsterile
equipment

Means of transmission
• Droplet
• Airborne
Break the link • Contact Break the link
• Hand hygiene • Vector • Sealed biohazardous
• Standard precautions • Vehicle waste containers
• PPE • Sealed specimen
• Sterile equipment containers
• Hand hygiene
• Standard precautions
Break the link
• Hand hygiene
• Standard precautions
• PPE
• Patient isolation

Figure 1–1 Chain of infection and safety practices related to the biohazard symbol. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy
Textbook, Fourth Edition, FA Davis, Philadelphia, 2011, with permission.)

the same patient after contact with material that may used during patient care activities related to suspected
contain a high concentration of microorganisms. Mycobacterium exposure.
Remove gloves promptly after use, before touching 4. Gown: Wear a gown (a clean, nonsterile gown is ade-
noncontaminated items and environmental surfaces, quate) to protect skin and to prevent soiling of cloth-
and between patients. Always sanitize your hands imme- ing during procedures and patient care activities that
diately after glove removal to avoid transferring microor- are likely to generate splashes or sprays of blood, body
ganisms to other patients or environments. fluids, secretions, or excretions. Select a gown that is
3. Mouth, nose, and eye protection: Wear a mask and eye appropriate for the activity and the amount of fluid
protection or a face shield to protect mucous mem- likely to be encountered (e.g., fluid-resistant labora-
branes of the eyes, nose, and mouth during procedures tory coat in the laboratory). Remove a soiled gown as
and patient care activities that are likely to generate promptly as possible, and sanitize hands to avoid
splashes or sprays of blood, body fluids, secretions, or transferring microorganisms to other patients or
excretions. A specially fitted respirator (N95) must be environments.
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Chapter 1 | Safety and Quality Management 53

5. Patient care equipment: Handle used patient care The Occupational Exposure to Bloodborne Pathogens
equipment soiled with blood, body fluids, secretions, Standard is a law monitored and enforced by OSHA.6,7 OSHA
and excretions in a manner that prevents skin and requires these controls to be provided by or mandated by the
mucous membrane exposure, clothing contamination, employer for all employees. Specific requirements of this
and transfer of microorganisms to other patients or OSHA standard include the following:
environments. Ensure that reusable equipment is not
used for the care of another patient until it has been Engineering Controls
cleaned and reprocessed appropriately. Ensure that 1. Providing sharps disposal containers and needles with
single-use items are discarded properly. safety devices
6 Environmental control: Ensure that the hospital has 2. Requiring discarding of needles with the safety device
adequate procedures for the routine care, cleaning, and activated and the holder attached
disinfection of environmental surfaces, beds, bed rails,
3. Labeling all biohazardous materials and containers
bedside equipment, and other surfaces that are touched
frequently. Ensure that these procedures are being
Work Practice Controls
followed.
7. Linen: Handle, transport, and process linen soiled 4. Requiring all employees to practice SP and documenting
with blood, body fluids, secretions, and excretions in training on an annual basis
a manner that prevents skin and mucous membrane 5. Prohibiting eating, drinking, smoking, and applying
exposures and clothing contamination and that avoids cosmetics in the work area
the transfer of microorganisms to other patients and 6. Establishing a daily work surface disinfection protocol
environments.
8. Occupational health and bloodborne pathogens: Take Personal Protective Equipment
care to prevent injuries when using needles, scalpels, 7. Providing laboratory coats, gowns, face shields, and
and other sharp instruments or devices; when handling gloves to employees and laundry facilities for nondispos-
sharp instruments after procedures; when cleaning used able protective clothing
instruments; and when disposing of used needles. Never
recap used needles or otherwise manipulate them using Medical
both hands or use any other technique that involves
directing the point of a needle toward any part of the 8. Providing immunization for HBV free of charge
body; rather, use self-sheathing needles or a mechanical 9. Providing medical follow-up to employees who have
device to conceal the needle. Do not remove used un- been exposed accidentally to bloodborne pathogens
sheathed needles from disposable syringes by hand,
and do not bend, break, or otherwise manipulate used Documentation
needles by hand. Place used disposable syringes and
10. Documenting annual training of employees in safety
needles, scalpel blades, and other sharp items in appro-
standards
priate puncture-resistant containers, which are located
as close as practical to the area in which the items were 11. Documenting evaluations and implementation of safer
used, and place reusable syringes and needles in a needle devices
puncture-resistant container for transport to the repro- 12. Involving employees in the selection and evaluation of
cessing area. Use mouthpieces, resuscitation bags, or new devices and maintaining a list of those employees
other ventilation devices as an alternative to mouth-to- and the evaluations
mouth resuscitation methods in areas where the need 13. Maintaining a sharps injury log, including the type and
for resuscitation is predictable. brand of safety device, location and description of the
9. Patient placement: Place a patient in a private room if incident, and confidential employee follow-up
he or she contaminates the environment or does not (or Any accidental exposure to a possible bloodborne pathogen
cannot be expected to) assist in maintaining appropriate through needlestick, mucous membranes, or nonintact skin
hygiene or environment control. If a private room is not must be immediately reported to a supervisor. A confidential
available, consult with infection control professionals evaluation of the incident must begin right away to ensure
regarding patient placement or other alternatives. appropriate postexposure prophylaxis (PEP) is initiated
10. Respiratory hygiene/cough etiquette: Educate within 24 hours. If it is determined that a significant expo-
health-care personnel, patients, and visitors to contain sure occurred, an incident report will be completed; blood
respiratory secretions to prevent droplet and fomite testing for HIV, HBV, and HCV on both the employee and
transmission of respiratory pathogens. Offer masks to the source patient is performed; and employee treatment
coughing patients, distance symptomatic patients from and counseling are initiated. The CDC provides periodically
others, and practice good hand hygiene to prevent the updated guidelines for the management of exposures and
transmission of respiratory pathogens. recommended PEP.8,9
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54 Part One | Basic Principles

Personal Protective Equipment Hand Hygiene

PPE used in the laboratory includes gloves, fluid-resistant Hand hygiene is emphasized in Figure 1-1 and in the SP guide-
gowns, eye and face shields, and Plexiglas countertop shields. lines. Hand contact is the primary method of infection trans-
Shoes must be closed-toed and cover the entire foot. mission. Laboratory personnel must always sanitize hands in
Gloves should be worn when in contact with patients, spec- the following situations:
imens (urine, blood, body fluids, secretions, excretions), con- • Before patient contact
taminated items, and laboratory equipment or fixtures. When
• After gloves are removed
specimens are collected, gloves must be changed between every
patient. In the laboratory, they are changed whenever they • Before leaving the work area
become noticeably contaminated or damaged and are always • At any time when hands have been knowingly
removed when leaving the work area. Wearing gloves is not a contaminated
substitute for hand hygiene, and hands must be sanitized after • Before going to designated break areas
gloves are removed.
• Before and after using bathroom facilities
A variety of gloves is available, including sterile and
nonsterile, powdered and unpowdered, and latex and non- Hand hygiene includes both hand washing and using
latex. Allergy to latex is increasing among health-care work- alcohol-based antiseptic cleansers. Alcohol-based cleansers can
ers, and laboratory personnel should be alert for symptoms be used when hands are not visibly contaminated, but those
of reactions associated with latex. Reactions to latex include cleansers are not recommended after contact with spore-
irritant contact dermatitis, which produces patches of dry, forming bacteria, including Clostridium difficile and Bacillus sp.
itchy irritation on the hands; delayed hypersensitivity reac- because the spores formed by these bacteria are not killed by
tions resembling poison ivy that appear 24 to 48 hours after alcohol-based cleanser. Hand washing with soap and water and
exposure; and true, immediate hypersensitivity reactions scrubbing to create a friction can remove these spores.
often characterized by facial flushing and breathing difficul- When using alcohol-based cleansers, apply the cleanser to
ties. Hand sanitizing immediately after removing gloves and the palm of one hand. Rub your hands together and over the
avoiding powdered gloves may aid in preventing the devel- entire cleansing area, including the area between the fingers
opment of latex allergies. Replacing latex gloves with nitrile and thumbs. Continue rubbing until the alcohol dries.
or vinyl gloves provides an alternative. Any symptoms of latex The CDC has developed guidelines to be followed for cor-
allergy should be reported to a supervisor because true latex rect hand washing.1,11 Procedure 1-1 demonstrates CDC rou-
allergy can be life-threatening.10 tine hand washing guidelines.4 More stringent procedures are
used in surgery and in areas with patients who are highly sus-
Laboratory Coats ceptible, such as patients who are immunocompromised
Full-length, fluid-resistant laboratory coats with wrist cuffs and/or burn patients.
are worn to protect clothing and skin from exposure to
patients’ body substances. These coats should always be Biological Waste Disposal
completely buttoned, and gloves should be pulled over the
All biological waste, except urine, must be placed in appropri-
cuffs. They are worn at all times when working with patient
ate leakproof containers labeled with the biohazard symbol
specimens and are removed before leaving the work area.
(Fig. 1-2) or with red or yellow color coding. This includes
They are changed when they become visibly soiled. Dispos-
both specimens and the materials with which the specimens
able coats are placed in containers for biohazardous waste,
come in contact. Then waste is decontaminated following
and nondisposable coats are placed in designated laundry
facility policy: incineration, autoclaving, or pickup by a certified
receptacles.
hazardous waste company.
Urine may be discarded by pouring it into a laboratory
Masks, Goggles, and Face Shields
sink under a Plexiglas countertop shield. Care must be taken
The mucous membranes of the eyes, nose, and mouth must
to avoid splashing, and the sink should be flushed with water
be protected from specimen splashes and aerosols. A variety
after specimens are discarded. Disinfection of the sink using a
of protective equipment is available, including masks and gog-
1:5 or 1:10 dilution of sodium hypochlorite (bleach) should
gles, full-face plastic shields that cover the front and sides of
be performed daily. Sodium hypochlorite dilutions stored in
the face, masks with attached shields, and Plexiglas counter-
plastic bottles are effective for 1 month if protected from light
top shields. Particular care should be taken to avoid splashes
after preparation.12 Empty urine containers can be discarded
and aerosols when removing container tops, pouring speci-
as nonbiologically hazardous waste (Fig. 1-3).
mens, and centrifuging specimens. Transfer pipettes should
be used when aliquoting samples from a specimen container.
Decontamination
Specimens must never be centrifuged in uncapped tubes or
in uncovered centrifuges. When specimens are received in Regular disinfection is needed for contaminated nondisposable
containers with contaminated exteriors, the exterior of the equipment, blood spills, and laboratory areas that process
container must be disinfected or, if necessary, a new specimen blood and body fluids. Laboratory countertops should be dis-
may be requested. infected after every work shift and after any spill occurs. The
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Chapter 1 | Safety and Quality Management 55

CDC recommends the use of a 1:10 dilution of sodium must be confirmed that the product is effective enough to elim-
hypochlorite for routinely disinfecting countertops and acci- inate most bacteria, including Mycobacterium tuberculosis, fungi,
dental spills. The solution should be allowed to air-dry on the and viruses. It is also important to know the contact time
contaminated area. Absorbent materials used for cleaning needed for disinfectant chemical products to work effectively
countertops and removing spills must be discarded in biohaz- on laboratory surfaces as prescribed by the manufacturer.
ard containers. Other products are commercially available both When spills occur, do not mop or wipe the fluid; instead,
in prefilled spray bottles and single-use wipes. However, it use an absorbent powder (e.g., Zorbitrol) or paper towels to

PROCEDURE 1-1
Hand Washing Procedure fingernails for at least 20 seconds; include your thumbs
Visit www.fadavis.com for Phlebotomy Video 3-1 and and wrists in the cleaning.
Video 3-2 (Hand Washing).

Equipment
Antimicrobial soap
Paper towels
Running water
Waste container
Procedure
1. Wet your hands with warm water. Do not allow parts of
your body to touch the sink.

4. Rinse your hands in a downward position to prevent

recontamination of your hands and wrists.

2. Apply soap, preferably antimicrobial.

5. Obtain a paper towel from the dispenser.

3. Rub to form a lather, create friction, and loosen debris.

Thoroughly clean between your fingers and under your

Continued
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56 Part One | Basic Principles

PROCEDURE 1-1—cont’d
6. Dry your hands with a paper towel. 7. Turn off the faucets with a clean paper towel to prevent
recontamination.

remove as much as possible before disinfecting. When using an

absorbent powder, the liquid will solidify and then can be
scooped up. If paper towels are used to absorb the liquid, pour
bleach over the towels. Dispose of absorbent material and paper
towels used for disinfection in the appropriate biohazard con-
tainer. Disinfect the spill area with bleach or a phenol solution.

Sharp Hazards
Sharp objects in the laboratory, including needles,
lancets, and broken glassware, present a serious
biological hazard, particularly for the transmission of
bloodborne pathogens. All sharp objects must be dis-
posed in puncture-resistant, leakproof container with the biohaz-
ard symbol. Puncture-resistant containers should be conveniently
located within the work area. The biohazard sharp containers
should not exceed three-fourths full and must always be replaced
when the level of waste inside reaches the safe capacity mark.

Chemical Hazards
The same general rules for handling biohazardous
materials apply to chemically hazardous materials;
that is, to avoid getting these materials in or on bod-
ies, clothes, or the work area. Every chemical in the
workplace should be presumed to be hazardous.

Chemical Spills and Exposure

When skin or eye contact occurs, the best first aid is to flush
Figure 1–2 Biohazard symbol. (From Strasinger, SK, and DiLorenzo, the area with large amounts of water for at least 15 minutes
MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, and then seek medical attention. For this reason, all labora-
2019, p. 60, with permission.)
tory personnel should know the location and proper use of
emergency showers and eye wash stations. Contaminated
clothing should be removed as soon as possible. No attempt
should be made to neutralize chemicals that come in contact
with the skin.
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Chapter 1 | Safety and Quality Management 57

Chemical Handling
Chemicals should never be mixed together unless specific in-
structions are followed, and they must be added in the order
specified. This is particularly important when combining acid
and water. Acid should always be added to water to avoid the
possibility of sudden splashing caused by the rapid generation
of heat in some chemical reactions. Wearing goggles and
preparing reagents under a fume hood are recommended safety
precautions. Chemicals should be used from containers that
are of an easily manageable size. Pipetting by mouth is unac-
ceptable in the laboratory.

Chemical Hygiene Plan

A OSHA also requires all facilities that use hazardous chemicals to
have a written chemical hygiene plan (CHP) available to em-
ployees.13 The purpose of the plan is to detail the following:
1. Appropriate work practices
2. Standard operating procedures
3. PPE
4. Engineering controls, such as fume hoods and flamma-
bles safety cabinets
5. Employee training requirements
6. Medical consultation guidelines
Each facility must appoint a chemical hygiene officer, who
is responsible for implementing and documenting compliance
with the plan. Examples of required safety equipment and in-
formation are shown in Figure 1-4.

Chemical Labeling
Hazardous chemicals should be labeled with a description of
their particular hazard, such as poisonous, corrosive, flamma-
ble, explosive, teratogenic, or carcinogenic (Fig. 1-5). The
National Fire Protection Association (NFPA) has developed the
Standard System for the Identification of the Fire Hazards of
B Materials, NFPA 704.14 This symbol system is used to inform
firefighters of the hazards that they may encounter with fires
Figure 1–3 Technologist disposing of urine (A) sample and in a particular area. The diamond-shaped, color-coded symbol
(B) container. contains information relating to health (blue), flammability
(red), reactivity (yellow), and specific hazards (white). Each
category is graded on a scale of 0 to 4, based on the extent of
Chemical spill kits containing protective apparel, nonre-
concern. These symbols are placed on doors, cabinets, and
active absorbent material, and disposable bags and waste tags
containers. The federal Department of Transportation (DOT)
for disposing of contaminated materials should be available for
regulations state that all chemicals are required to have the
cleaning up spills. Liquids are absorbed using a neutralizer,
NFPA 704-M Hazard Identification System warnings on their
such as sodium bicarbonate, and a sand mixture or ground
shipping containers. An example of this system is shown in
clay. After the liquid is absorbed, it is scooped up and placed
Figure 1-6.
into a waste bag and labeled appropriately for disposal. Then
the area is cleaned thoroughly.
A chemical spill or exposure must be reported to the em-
Safety Data Sheets
ployee health department and an incident (variance) report The OSHA Federal Hazard Communication Standard requires
submitted that documents the exposure according to the facil- that all employees have a right to know about all chemical haz-
ity policy. Examples of chemical exposure include any time a ards present in their workplace. All chemicals and reagents
chemical was breathed in or touched the skin, two or more containing hazardous ingredients in a concentration greater
chemicals that came in contact with each other and created an than 1% are required by OSHA to have a Safety Data Sheet
odor or fume, or an identified substance spill. (SDS) on file in the workplace. By law, vendors are required
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58 Part One | Basic Principles

to provide these sheets to purchasers; however, the facility

itself is responsible for obtaining and making SDSs available
to employees. Information contained in an SDS includes the
following:
1. Physical and chemical characteristics
2. Fire and explosion potential
3. Reactivity potential
4. Health hazards and emergency first aid procedures
5. Methods for safe handling and disposal
6. Primary routes of entry
7. Exposure limits and carcinogenic potential

The Globally Harmonized System

of Classification and Labeling of Chemicals
The Globally Harmonized System (GHS) of Classification
and Labeling of Chemicals is an international effort to stan-
dardize both the classification of hazardous chemicals and
the symbols used to communicate these hazards on labels
and in SDS documentation (Fig. 1-7). It includes criteria for
the classification of health, physical, and environmental
hazards. The standard label elements include GHS hazard
pictograms, signal words, and a GHS hazard statement. The
A SDS under GHS provides a clear description of the data used
to identify hazards and has 16 sections in a specified order
(Box 1-1).
OSHA aligned its Hazard Communication Standard with
the GHS and requires that all employees be trained on the new
label elements and SDS format. The adoption of the GHS in
the United States has increased awareness and understanding
of hazards in the workplace.

Chemical Waste Disposal

State and federal regulations are in place for the disposal of
chemicals and should be consulted. Any hazardous chemical
waste should be disposed of per current Environmental Pro-
tection Agency (EPA) regulations. Most reagents used in the
laboratory come with an SDS, which provides specific infor-
mation for the disposal of particular chemicals. All chemicals
should be disposed of by following the SDS directions. Sinks
and drains should be flushed with large amounts of water after
disposal of chemical waste.

Radioactive Hazards
Radioactivity may be encountered in the clinical lab-
oratory when procedures using radioisotopes are
performed. The amount of radioactivity present in
B the clinical laboratory is very small and represents
little danger; however, the effects of radiation are cumulative
Figure 1–4 Chemical safety aids. A. Emergency shower. B. Eye related to the amount of exposure. The amount of radiation
wash station. (From Strasinger, SK, and DiLorenzo, MA: The Phle- exposure is related to a combination of time, distance, and
botomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, p. 64, shielding. People working in a radioactive environment are re-
with permission.) quired to wear measuring devices to determine the amount of
radiation that they are accumulating.
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Chapter 1 | Safety and Quality Management 59

A B

Figure 1–5 Chemical hazard symbols. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis,
Philadelphia, 2019, p. 65, with permission.)

Laboratory personnel should be familiar with the radioac- setting. Equipment should not be operated with wet hands.
tive hazard symbol shown here. This symbol must be displayed Designated hospital personnel monitor electrical equipment
on the doors of all areas where radioactive material is present. closely; however, laboratory personnel should continually
Exposure to radiation during pregnancy presents a danger to observe for any dangerous conditions, such as frayed cords and
the fetus; personnel who are pregnant or think they may be overloaded circuits, and report them to the supervisor. Equip-
should avoid areas with this symbol. ment that has become wet should be unplugged and allowed
to dry completely before reusing. Equipment also should be
unplugged before cleaning. All electrical equipment must be
Electrical Hazards grounded with three-pronged plugs.
When an accident involving electrical shock occurs, the
The laboratory setting contains electrical equipment electrical source must be removed immediately. This must be
with which workers have frequent contact. The same done without touching the person or the equipment involved
general rules of electrical safety observed outside to avoid transferring the current. Turning off the circuit
the workplace apply. The danger of water or fluid breaker, unplugging the equipment, or moving the equipment
coming in contact with equipment is greater in the laboratory using a nonconductive glass or wood object are safe procedures
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60 Part One | Basic Principles

HAZARDOUS MATERIALS to follow. The victim should receive immediate medical assis-
CLASSIFICATION tance after discontinuation of the electricity. Cardiopulmonary
HEALTH HAZARD FIRE HAZARD resuscitation (CPR) may be necessary.
Flash Point
4 Deadly 4 Below 73°F
3 Extreme danger 3 Below 100°F
2 Hazardous
1 Slightly hazardous
2
1
Below 200°F
Above 200°F Fire/Explosive Hazards
0 Normal material 0 Will not burn

2 The Joint Commission (TJC) requires that all health-

care facilities post evacuation routes and detailed
plans to follow in the event of a fire. Laboratory per-

3 1 sonnel should be familiar with these procedures.

When a fire is discovered, all employees are expected to take
the actions to protect themselves and others, following the

SPECIFIC
HAZARD
W REACTIVITY
acronym RACE:
Rescue—rescue anyone in immediate danger
4 May deteriorate Alarm—activate the institutional fire alarm system
3 Shock and heat
Oxidizer OXY may deteriorate Contain—close all doors to potentially affected areas
Acid ACID 2 Violent chemical
Alkali ALK change Extinguish/Evacuate—attempt to extinguish the fire, if possible,
Corrosive COR 1 Unstable if
Use No Water W heated
or evacuate, closing the door
Radiation 0 Stable As discussed previously, laboratory workers often use po-
tentially volatile or explosive chemicals that require special pro-
cedures for handling and storage. Flammable chemicals should
Figure 1–6 NFPA hazardous material symbols. be stored in safety cabinets and explosion-proof refrigerators,

Health hazard Flame Exclamation mark

• Carcinogen • Flammables • Irritant (skin and eye)

• Mutagenicity • Pyrophorics • Skin sensitizer
• Reproductive toxicity • Self-heating • Acute toxicity (harmful)
• Respiratory sensitizer • Emits flammable gas • Narcotic effects
• Target organ toxicity • Self-reactives • Respiratory tract irritant
• Aspiration toxicity • Organic peroxides • Hazardous to ozone
layer (nonmandatory)

Gas cylinder Corrosion Exploding bomb

• Gases under pressure • Skin corrosion/burns • Explosives

• Eye damage • Self-reactives
• Corrosive metals • Organic peroxides

Flame over circle Environment Skull and crossbones

(nonmandatory)

• Oxidizers • Aquatic toxicity • Acute toxicity

(fatal or toxic)

Figure 1–7 GHS pictograms and hazards chart. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis,
Philadelphia, 2019, Figure 3-16, p. 66, with permission.)
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Chapter 1 | Safety and Quality Management 61

Box 1–1 Globally Harmonized System (GHS) and Safety Data Sheet (SDS) Format
Sixteen-section SDS that includes the following headings in the order 9. Physical and chemical properties
specified: 10. Chemical stability and reactivity
1. Identification 11. Toxicological information
2. Hazard(s) identification 12. Ecological information
3. Composition/information on ingredients 13. Disposal considerations
4. First-aid measures 14. Transport information
5. Firefighting measures 15. Regulatory information
6. Accidental release measures 16. Other information
7. Handling and storage
From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook,
8. Exposure control/personal protection
Fourth Edition, FA Davis, Philadelphia, 2019, p. 67, with permission.

and cylinders of compressed gas should be located away from floors; bend the knees when lifting heavy objects; keep long
heat and securely fastened to a stationary device to prevent ac- hair pulled back; avoid dangling jewelry; and maintain a clean,
cidental capsizing. Fire blankets may be present in the labora- organized work area. Closed-toed shoes that provide maxi-
tory. Persons with burning clothes should be wrapped in the mum support are essential for safety and comfort.
blanket to smother the flames.
The NFPA classifies fires with regard to the type of burning
material. It also classifies the type of fire extinguisher that is used Quality Management
to control them. This information is summarized in Table 1-2.
The term quality management (QM) refers to the overall
The multipurpose ABC fire extinguishers are the most common,
process of guaranteeing quality patient care, and it is regulated
but the label should always be checked before using. It is im-
throughout the total testing system. A quality management
portant to be able to operate the fire extinguishers. The acronym
system (QMS) refers to all of the laboratory’s policies,
PASS is about handling a fire extinguisher and can be used to
processes, procedures, and resources needed to achieve quality
remember the steps in the operation:
testing.15 In a clinical laboratory, a QMS includes not only test-
1. Pull pin ing controls, referred to as quality control (QC), but also
2. Aim at the base of the fire encompasses preexamination (preanalytical) variables (e.g.,
3. Squeeze handles specimen collection, handling, and storage), examination (an-
alytical) variables (e.g., reagent and test performance, instru-
4. Sweep nozzle side to side
ment calibration and maintenance, personnel requirements,
and technical competence), postexamination (postanalytical)
Physical Hazards variables (e.g., reporting of results and interpretation), and
documentation that the program is being followed meticu-
Physical hazards are not unique to the laboratory, lously. The original terms preanalytical, analytical, and post-
and routine precautions observed outside the work- analytical have been replaced with the standard terms of
place apply. General precautions to consider are to preexamination, examination, and postexamination from the
avoid running in rooms and hallways; watch for wet International Organization for Standardization (ISO).

Table 1–2 Types of Fires and Fire Extinguishers

Fire Type Extinguishing Material Type/Composition of Fire Extinguisher
Class A Wood, paper, clothing Class A Water
Class B Flammable organic chemicals Class B Dry chemicals, carbon dioxide, foam, or halon
Class C Electrical Class C Dry chemicals, carbon dioxide, or halon
Class D Combustible metals None Sand or dry powder
Class ABC Dry chemicals
Class K Grease, oils, fats Class K Liquid designed to prevent splashing and cool
the fire

From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook, Fourth Edition, FA Davis, Philadelphia, 2019, with permission.
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62 Part One | Basic Principles

All of the following are included in a QMS: • Patient preparation

• Procedure manuals • Specimen type and method of collection
• Internal quality control • Specimen acceptability and criteria for rejection
• External quality control • Reagents
• Electronic quality control • Standards and controls
• Calibration or calibration verification • Instrument calibration and maintenance protocols and
• Standardization schedules
• Proficiency testing (PT), more formally known as • Step-by-step procedure
external quality assessment (EQA)16 • Calculations
• Record-keeping • Frequency and tolerance limits for controls and correc-
• Equipment maintenance tive actions
• Safety programs • Reference values and critical values
• Training • Interpretation of results
• Education and competency assessment of personnel • Specific procedure notes
• A review process that is scheduled and documented • Limitations of the method
Essentially, QM is the continual monitoring of the entire • Method validation
test process, from test ordering and specimen collection • Confirmatory testing
through reporting and interpreting results. Written policies and • Recording of results
documented actions as they relate to the patient, the laboratory,
• References
ancillary personnel, and the health-care provider are required.
Having written remedial actions mandating the steps to take • Effective date
when any part of the system fails is essential to a QM program. • Author
QM in the urinalysis laboratory—or any other laboratory • Review schedule
department—is an integration of many factors used to monitor,
Current package inserts should be reviewed and available
evaluate, and improve the quality of laboratory services
at the workplace. Electronic manuals are acceptable and must
through all three phases of patient testing. This section of the
be readily available to all personnel. As with written procedural
text will provide a collection of the procedures essential for
manuals, electronic versions must be subjected to proper doc-
providing quality urinalysis. In the following chapters, the
ument control (i.e., only authorized persons may make
methods of ensuring accurate results will be covered on an
changes, changes are dated/signed [manually or electronically],
individual basis for each of the tests.
and there is documentation of periodic review).17,18
Documentation of QM procedures is required by all labo-
Evaluating procedures and adopting new methodologies
ratory accreditation agencies, including TJC, College of Amer-
is an ongoing process in the clinical laboratory. Whenever
ican Pathologists (CAP), American Association of Blood Banks
changes are made (Fig. 1-8), a person with designated author-
(AABB), American Osteopathic Association (AOA), American
ity, such as the laboratory director or section supervisor, should
Society of Histocompatibility and Immunogenetics (ASHI),
review and reference the procedure and then sign off on the
American Association for Laboratory Accreditation (A2LA),
change, and personnel should be notified of the changes. There
and the Commission on Laboratory Assessment (COLA); it is
must be documentation of an annual review of all procedures
also required for Medicare and Medicaid reimbursement.
by the designated authority.
Guidelines published by CAP and the CLSI provide very com-
plete instructions for documentation and are used as a refer- Preexamination (Preanalytical) Variables
ence for the ensuing discussion of the specific areas of
urinalysis QC and QM.16-18 Preexamination (preanalytical) variables occur before the actual
Documentation in the form of a procedure manual is re- testing of the specimen and include test requests, patient
quired in all laboratories, and this format is used as the basis preparation, timing, specimen collection, handling, and stor-
for the following discussion. age. Health-care personnel outside the clinical laboratory con-
trol many of these factors, such as ordering tests and specimen
collection. Communication between departments and ade-
Urinalysis Procedure Manual quate training on the correct procedures for ordering a test
(test utilization), collecting, and transporting the specimen
A procedure manual containing all the procedures performed
improves the turnaround time (TAT) of results, avoids dupli-
in the urinalysis section must be available for reference in the
cation of test orders, and ensures a high-quality specimen. TAT
working area and must comply with the CLSI guidelines. The
is defined as the amount of time required from the point at
following information is included for each procedure:
which a test is ordered by the health-care provider until the
• Principle or purpose of the test results are reported to the health-care provider. Laboratories
• Clinical significance determine the TAT for tests, including both stat and routine
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Chapter 1 | Safety and Quality Management 63

tests, as appropriate. Then the laboratory can monitor the TATs

to determine areas in the process that need improvement. This
URINALYSIS SECTION can be determined by creating a cause-and-effect diagram, as
shown in Figure 1-9.
SPECIMEN ACCEPTABILITY/LABELING
Specimen Collection and Handling
Prepared by:
Specific information on specimen collection and handling
Initial approval:
should be stated at the beginning of each procedure listed in
Procedure placed in use: the manual. Requisition forms and computerized entry forms
should designate the type of urine specimen to be collected
Revised:
and the date and time of collection. The form should include
Reason for revision: space for recording
Effective Date Supervisor Approval Medical Director Approval
1. The actual date and time of specimen collection
Reviewed
2. Whether the specimen was refrigerated before transporting
Reviewed
3. The time the specimen was received in the laboratory
and the time the test was performed
Reviewed
4. Tests requested
Reviewed
5. An area for specific instructions that might affect the re-
Reviewed sults of the analysis
6. Patient identification information18
The patient’s sex, age or date of birth, and, when appro-
Figure 1–8 Example of procedure review documentation. priate, the source of the specimen and the time it was collected
(Adapted from the Department of Pathology, St. Joseph Hospital, must be documented.15
Omaha, NE.) Along with the specific procedure, the requisition form
must detail patient preparation (e.g., fasting or elimination
of interfering medications), type and volume of specimen
required, and the need for sterile or opaque containers. All

CAUSE-AND-EFFECT DIAGRAM URINALYSIS TURN-AROUND-TIME (TAT)

Collection Transport
Missed collection Delayed delivery
Incorrect specimen type
Contaminated specimen container
Missed pick-up Distance
Insufficient urine volume Insufficient container Specimen misplaced
Patient misidentification Transport staff
unavailable
Special conditions not followed Lost label No requisition Leaking closure lid Incorrect storage

Incorrect container Patient preparation No chemical preservative

Not refrigerated
Time of collection not indicated Not fasting Medications
Incomplete requisition
UA results
TAT
Interfering substances
Instrument malfunction Report not charted
Computer down
Procedure failure
Wrong specimen tested Printer error
Failure to send report

Inadequate volume

Test analysis Reporting

Figure 1–9 Cause-and-effect diagram for analyzing urinalysis TAT.

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64 Part One | Basic Principles

urine specimens should be examined within 2 hours. If this determine the root cause analysis (RCA) and develop a pre-
is not possible, written instructions for preserving the speci- ventive or corrective action plan. Laboratory information sys-
men must be available.18 tems have the capability to electronically generate these forms
The manual should also include instructions of a general for review. An acceptable specimen requires verification of the
nature, such as procedures for collecting clean-catch midstream patient’s identification information on the requisition form and
and timed specimens, as well as information on specimen pro- the container label, timely transport to the laboratory, the pres-
cessing and printed instructions that are given to patients. ence of refrigeration or recommended preservative if transport
Criteria for specimen rejection for both physical character- was delayed, and collection of an adequate amount of the cor-
istics and labeling errors must be present. Box 1-2 gives an ex- rect urine specimen type in a noncontaminated, tightly closed
ample of a policy for handling mislabeled specimens. Written container.18
criteria for rejecting specimens must be documented and avail-
able to the health-care provider and nursing staff.18 Box 1-3 lists Examination (Analytical) Variables
the criteria for urine specimen rejection.
The examination (analytical) variables are the processes that
Laboratory personnel must determine the suitability of a
directly affect the testing of specimens. They include reagents,
specimen and document any problems and corrective actions
instrumentation and equipment, testing procedure, QC,
taken. An example of an internal laboratory quality improve-
preventive maintenance (PM), access to procedure manuals,
ment form is shown in Figure 1-10. It is used as a tool to doc-
and competency of personnel performing the tests.
ument a problem at the point of discovery, describing what
happened and the immediate corrective action taken. This
Reagents
enables the laboratory director to capture the information to
The manual should state the name and chemical formula of
each reagent used; instructions for preparation, when necessary,
or company source of prepared materials; storage requirements;
Box 1–2 Policy for Handling Mislabeled and procedures for reagent QC. The type of water used for
Specimens preparing reagents and controls must be specified. Distilled or
deionized water or clinical laboratory reagent water (CLRW)
Do NOT assume any information about the specimen or patient.
must be available. CLRW quality must be monitored regularly
Do NOT relabel an incorrectly labeled specimen. for microbial contamination as well as ionic and organic impu-
Do NOT discard the specimen until investigation is complete. rities.19 A written policy must be available with the procedures
Leave specimen EXACTLY as you receive it; put in the refrigerator to follow when the CLRW quality is not within the tolerance
for preservation until errors can be resolved. limits. A bold-type statement of any safety or health precautions
Notify nursing station, doctor’s office, etc. of problem and why it
associated with reagents should be present. An example of this
must be corrected for analysis to continue. would be the heat produced in the Clinitest reaction.
All reagents and reagent strips must be properly labeled
Identify problem on specimen requisition form with date, time, and
with the date of preparation or opening, purchase and received
your initials.
date, expiration date, and appropriate safety information.
Make person responsible for specimen collection participate in Reagent strips should be checked against known negative and
solution of problem(s). Any action taken should be documented positive control solutions on each shift or, at a minimum, once
on the requisition slip.
a day and whenever a new bottle is opened. Reagents are
Report all mislabeled specimens to the appropriate supervisor. checked daily or when tests requiring their use are requested.
After checking is performed, laboratory personnel should
From Schweitzer, SC, Schumann, JL, and Schumann, GB: Quality
assurance guidelines for the urinalysis laboratory. Journal of
properly record the results of all reagent checks. Reagent strips
Medical Technology 3(11): 568, 1986, with permission. must never be refrigerated, must be protected from light, and
must be recapped immediately after removing each strip.

Instrumentation and Equipment

The procedure manual should clearly state the instructions re-
Box 1–3 Criteria for Urine Specimen Rejection
garding the operation, performance, and frequency of calibra-
Unlabeled containers tion; limitations; and procedures to follow when limitations or
Nonmatching labels and requisition forms linearity are exceeded, such as dilution procedures. Instruc-
Specimens contaminated with feces or toilet paper tions detailing the appropriate recording procedures must be
included.
Containers with contaminated exteriors
The instruments encountered in the urinalysis labora-
Insufficient volume of urine tory most frequently are refractometers, osmometers, auto-
Specimens that were transported or preserved improperly mated reagent strip readers, and automated microscopy
Delay between time of collection and receipt in the laboratory instruments. Refractometers are calibrated daily or when
used against deionized water (1.000) and a known control,
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Chapter 1 | Safety and Quality Management 65

Quality Improvement Follow-up Report

CONFIDENTIAL

Instructions: Section I should be completed by the individual identifying the event.

Date of report: Reported by:

Date of incident: Date/time of discovery:
Patient MR# Patient accession#

Section I.
Summary of incident — describe what happened

What immediate corrective action was taken?

Provide the ORIGINAL to team leader/technical specialist within 24 hours of incident discovery
Date:
To:
Forwarded for follow-up:
Date:
To:

Section II. Management investigation: Tracking #

Instructions: Section II should be completed by laboratory management within 72 hours

Check appropriate problem category

Unacceptable patient samples Wrong tube type

(Due to hemolysis, QNS, or contaminated)

Equipment-related event Misidentified sample

Standard operating procedure deviation Wrong location

Communication problem/complaint Other (explain)

Accident

Explain answers:

Preventive/corrective action recommendations:

Technical specialist/team leader: Date:

Medical director review: Date:

Quality assurance review: Date:

FDA reportable: Yes or no Date reported:

Figure 1–10 Sample of quality improvement follow-up report form. (From Danville Regional Medical Center Laboratory, Danville, VA, with
permission.)
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66 Part One | Basic Principles

such as 5% saline (1.022 ± 0.001) or 9% sucrose (1.034 ± including reasons for special precautions; sources of error and
0.001). Two levels of commercial controls are available for interfering substances; criteria for the correlation of the phys-
the osmometer, urine reagent strip tests, and human chori- ical, chemical, and microscopic urinalysis results; helpful hints;
onic gonadotropin (hCG) kit tests. All control values must clinical situations that influence the test; alternative proce-
be recorded. Automated urinalysis systems and reagent strip dures; and acceptable TATs for stat tests are listed under the
readers are calibrated using manufacturer-supplied calibration title of Procedure Notes following the step-by-step procedure.
materials, following the protocol specified by the manufac- Reference sources should be listed. Manufacturer’s package
turer. Both positive and negative control values must be run inserts may be included but cannot replace the written procedure.
and recorded (Fig. 1-11). There must be documentation The laboratory director must sign and date new procedures and
showing evidence of corrective action for any failed QC tests. all modifications of procedures before they are used.15
No patient’s testing may be performed until QC is acceptable.
Other common equipment found in the urinalysis labo- Quality Control
ratory includes refrigerators, freezers, pipettes, centrifuges, mi- QC refers to the materials, procedures, and techniques that
croscopes, and water baths. Temperatures of refrigerators and monitor the accuracy, precision, and reliability of a labora-
water baths should be taken daily and recorded. Calibration tory test. QC procedures are performed to ensure that accept-
of centrifuges and accuracy of timers are customarily per- able standards are met during the process of patient testing.
formed every 3 months, and there should be a record of the The step-by-step instructions for each test should include
appropriate relative centrifugal force for each setting. Disinfec- specific QC information regarding the type of control specimen
tion of centrifuges should be done routinely on a weekly basis preparation and handling, frequency of use, tolerance levels,
or after spills. Microscopes should be kept clean at all times and methods of recording. QC is performed at scheduled
and have an annual professional cleaning. Automatic pipettes times, such as at the beginning of each shift or before testing
are initially and periodically checked for accuracy and repro- patient specimens, and it must always be performed if reagents
ducibility. As mandated by the TJC or CAP guidelines, a rou- are changed, an instrument malfunction has occurred, or test
tine PM schedule for instruments and equipment should be results are questioned by the health-care provider. Control
prepared and records kept of all routine and nonroutine main- results must be recorded in a log, either paper or electronic.
tenance performed. Patient test results may not be reported until the QC is verified.
Deionized water used for reagent preparation is quality Both external quality control monitoring and internal and elec-
controlled by checking pH and purity meter resistance on a tronic quality control processes are practiced in the urinalysis
weekly basis and the bacterial count on a monthly schedule. laboratory.
All results must be recorded on the appropriate forms.
External Quality Control
Testing Procedure
External quality controls are used to verify the accuracy (ability
Detailed, concise testing instructions are written in a step-by- to obtain the expected result) and precision (ability to obtain
step manner. Instructions should begin with specimen prepa- the same result on the same specimen) of a test, and they are
ration, such as time and speed of centrifugation, and include exposed to the same conditions as the patient specimens. Re-
types of glassware needed, time limitations and stability of liability is the ability to maintain both precision and accuracy.
specimens and reagents, calculation formulas and a sample cal- Commercial controls are available for the urine chemistry tests,
culation, health and safety precautions, QC checks, reference specific gravity, and certain microscopic constituents. Analysis
ranges, and procedures. Additional procedure information, of two levels of control material is required. The concentration

QUALITY CONTROL Month:__________20_____

Positive control Negative control
GLU BIL KET SP BLD PH PROT NIT LEU GLU BIL KET SP BLD PH PROT NIT LEU
GR EST GR EST
mg/dL mg/dL 1.0 mg/dL mg/dL mg/dL 1.0 mg/dL
Control values

Reagent TECH DATE

Lot #
EXP

Figure 1–11 Sample instrument QC recording sheet. (Adapted from the Department of Pathology, Methodist Hospital, Omaha, NE, with
permission.)
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Chapter 1 | Safety and Quality Management 67

of controls should be at medically significant levels and should

Day
be as much like the human specimen as possible. Documenta-
2 4 6 8 10 12 14 16 18 20
tion of QC includes dating and initialing the material when it
is first opened and recording the manufacturer’s lot number + 2 SD
and the expiration date each time a control is tested and the + 1 SD
test result is obtained. Food and Drug Administration (FDA) X
– 1 SD
standards require that control material test negative for HIV
– 2 SD
and HBV. The same person who is performing the patient test-
In Control
ing should perform the testing and interpretation of external
controls in the laboratory.
Control data are evaluated before releasing patient results. + 2 SD
Data obtained from repeated measurements have a Gaussian + 1 SD
X
distribution or spread in the values that indicate the ability to
– 1 SD
repeat the analysis and obtain the same value. The laboratory, – 2 SD
after repeated testing, establishes the value for each analyte, and Shift
then it should calculate the mean and standard deviation (SD).
The control mean is the average of all data points, and the SD
is a measurement statistic that describes the average distance + 2 SD
+ 1 SD
each data point in a normal distribution is from the mean. The X
coefficient of variation (CV) is the SD expressed as a percent- – 1 SD
age of the mean. The CV indicates whether the distribution of – 2 SD
values about the mean is in a narrow versus broad range and Trend
should be less than 5%. Confidence intervals are the limits be-
tween which the specified proportion or percentage of results Figure 1–12 Levy-Jennings charts showing in-control, shift, and
will lie. Control ranges are determined by setting confidence trend results.
limits that are within ±2 SD or ±3 SD of the mean, which in-
dicates that 95.5% to 99.7% of the values are expected to be
within that range.
Values are plotted on Levy-Jennings control charts to
visually monitor control values. Immediate decisions about Electronic Controls
patient results are based on the ability of control values to Electronic controls use a mechanical or electrical device in
remain within a preestablished limit. Changes in the accuracy place of a liquid QC specimen. This type of QC can be an in-
of results are indicated by either a trend, which is a gradual ternal or an external component inserted into a point-of-care
changing in the mean in one direction, or a shift, which is an (POC) instrument. Electronic controls verify the functional
abrupt change in the mean (Fig. 1-12). Changes in precision ability of a testing device, but they do not verify the integrity
are shown by a large amount of scatter about the mean and an of the testing supplies. Many test systems use a combination
uneven distribution above and below the mean that are most of external and internal controls to verify that the entire test
often caused by errors in technique. system is working properly.
Corrective action, including the use of new reagents, Proficiency Testing (External Quality Assessment)
reagent strips, or controls and the verification of lot numbers PT, or EQA, is the testing of unknown specimens received from
and expiration dates, must be taken when control values are an outside agency and provides unbiased validation of the
outside the tolerance limits. All corrective actions taken are quality of patient test results. Several commercial vendors, such
documented. A protocol for corrective action is shown in as CAP, provide PT. Laboratories subscribing to these programs
Figure 1-13. A designated supervisor reviews all QC results. receive lyophilized or ready-to-use specimens for routine uri-
Laboratories may participate in a commercial QC pro- nalysis and digital images or photomicrographs for sediment
gram. Participating laboratories return results from the same constituent identification. Personnel in subscribing laboratories
lot of QC material sent by the manufacturer back to the man- test these proficiency survey specimens in the same manner as
ufacturer for statistical analysis and comparison with other patient specimens. No communication with other laboratories
laboratories using the same methodology. is permitted. The subscribing laboratory returns the results to
the PT vendors, where they are statistically analyzed with those
Internal Quality Control from all participating laboratories. Then the director of the sub-
Internal quality control consists of internal monitoring systems scribing laboratory receives a report that evaluates laboratory
built in to the test system and are called internal or procedural accuracy and compares it with other laboratories using the
controls.16,20 Internal controls monitor the sufficient addition same method of analysis. Corrective action must be taken for
of a patient sample or reagent, the instruments/reagents inter- unacceptable results.16 The Clinical Laboratory Improve-
action, and, for lateral flow test methods, whether the sample ment Amendments (CLIA) mandates comparison testing for
migrated through the test strip properly.16 laboratory accreditation.20
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68 Part One | Basic Principles

A. Record all actions taken and the resolution of any problems An IQCP requires risk assessment (RA), a quality control
plan (QCP), and quality assessment (QA).21 RA identifies and
B. Use the flow diagram below:
evaluates potential problems that may occur in the entire test-
1. Run control ing process from preexamination through the examination and
postexamination processes. Components that are assessed for
In control Out of control
potential errors are the specimen, test system, reagents, envi-
ronment, and testing personnel. QCP establishes control pro-
Proceed with Go to Step 2 cedures to reduce the possibility of reporting an inaccurate
testing
patient test result. It must include the number, type, and fre-
quency of testing, as well as criteria for acceptable results of
2. Inspect control for: Outdate (age), proper storage, the QCs. IQCP QA involves selecting quality monitors that
correct lot number, signs of contamination include:
Yes, there is a problem No obvious explanation • QC records
• PT records
Make new control Retest • Review of patient results
and retest
In control Out of control • Specimen rejection logs
• TAT reports
Proceed with Go to Step 3 • Records of preventive measures, corrective actions, and
testing
follow-up
• Personnel competency records
3. Make up new bottle of control

In control Out of control Personnel and Facilities

QC is only as good as the personnel performing and monitor-
Discard old control Go to Step 4
and proceed with testing
ing it. Personnel assessment includes education and training,
continuing education, competency assessment, and perform-
ance appraisals. Each new employee must have documentation
4. Open new can of reagent strips and test with new control of training during orientation to the laboratory. This is a check-
In control Out of control
list of procedures and must include the date and initials of the
person doing the training and of the employee being trained.
Up-to-date reference materials and atlases should be readily
Discard bad reagent strips Switch lot numbers
and proceed with testing and retest available, and documentation of continuing education must be
maintained.16
In control Out of control An adequate, uncluttered, safe working area is also
essential for both quality work and personnel morale.
Discard entire lot, Notify supervisor or
notify manufacturer, resource person: do not
Standard precautions for handling body fluids must be
and proceed with testing proceed with testing followed at all times.

Figure 1–13 “Out-of-control” procedures. (From Schweitzer, SC, Postexamination Variables

Schumann, JL, and Schumann, GB: Quality assurance guidelines
for the urinalysis laboratory. Journal of Medical Technology 3(11): Postexamination variables are processes that affect the
567–572, 1986, with permission.) reporting of results and correct interpretation of data. Re-
porting of test results to the appropriate health-care provider
in an efficient and accurate manner is essential to quality
Individualized Quality Control Plan patient care. Reports may be handwritten or instrument
An individualized quality control plan (IQCP) is an alterna- printouts. Reports may be delivered by telephone or fax
tive CLIA QC option that provides equivalent quality testing or transmitted electronically to the requesting health-care
to meet the CLIA regulations for moderate- and high- provider.
complexity tests and provider-performed microscopy proce- Laboratory reports must be present in the patient’s record.
dures (PPMP) tests. IQCP considers the entire testing process— Required information includes:
preexamination, examination, and postexamination—to en- • Patient’s first and last name
sure quality testing, not just the frequency of QC and number • Patient’s unique identification number
of QC materials. With this option, the laboratory can deter-
• Specimen collection date and time
mine the frequency of QC testing based on information about
the test, the risk assessment, and the accreditation agencies’ • Specimen source (if pertinent to the test)
requirements. • Condition of unsatisfactory specimen
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Chapter 1 | Safety and Quality Management 69

• Tests performed, the results, and the reference ranges of instrument to the designated health-care provider. It is essential
the tests that the operator carefully review results before transmittal.
• Date and time of the final results generated The operator also may enter the results manually into the
laboratory computer system and then transmit them to the
• Facility where the test was performed
health-care provider.
Written Reports Telephone (Verbal) Results
Each procedure covered in the procedure manual should The telephone is frequently used to transmit results of stat tests
include standardized reporting formats and, when applicable, and critical values. Calls requesting additional results may be
reference ranges. There must be a written procedure for report- received from personnel on hospital units and from health-care
ing, reviewing, and correcting errors. providers. When telephoning results, confirm that the results
Forms for reporting results should provide adequate are being reported to the appropriate person. The time of the
space for writing and should present the information in a call and the name of the person receiving the results must be
logical sequence. Standardized reporting methods minimize documented according to the facility’s policy.
health-care provider confusion when interpreting results
(Fig. 1-14). Result Errors
Electronic Results Errors may be discovered in the laboratory through a QM pro-
cedure known as the delta check that compares a patient’s
Electronic transmission is now the most common method for test results with the previous results. Variation outside the
reporting results. Many urinalysis instruments have the capa- established parameters alerts laboratory personnel to the pos-
bility for the operator to transmit results directly from the sibility of an error that occurred during the testing procedure
or during patient identification. Many laboratory analyzers
now have autoverification, an automated comparison of
patient results with predetermined preapproved criteria,
MICROSCOPIC QUANTITATIONS programmed into them.4
Erroneous results must be corrected in a timely manner to
Quantitate an average of 10 representative fields. Do not quantitate
budding yeast, mycelial elements, Trichomonas, or sperm, but do assure that the patient does not receive treatment based on in-
note their presence with the appropriate LIS code. correct results. Errors can occur in patient identification, speci-
men labeling, or result transcription. The patient’s record should
Epithelial cells/LPF
None: 0 be corrected as soon as the error is detected; however, the original
Rare: 0–5 result must not be erased in the event that the health-care
Few: 5–20 provider treated the patient based on the erroneous results. An
Moderate: 20–100
Many: >100 incident (variance) report documenting the problem and correc-
Casts/LPF tive action may need to be submitted. Appropriate documenta-
None: 0 tion of erroneous results should follow facility protocol.
Numerical ranges: 0–2, 2–5, 5–10, >10
RBCs/HPF
None: 0 Critical Results
Numerical ranges: 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100
WBCs/HPF There should be written procedures available for the reporting
None: 0 of critical values. Critical values are test results that are signif-
Numerical ranges: 0–2, 2–5, 5–10, 10–25, 25–50, 50–100, >100
Crystals/HPF icantly lower or higher than the normal reference range. They
None: 0 can be life-threatening and must be relayed to the health-care
Rare: 0–2 provider immediately, following facility policy. Figure 1-15 is
Few: 2–5
Moderate: 5–20 an example of the procedure instructions for reporting critical
Many: >20 values in the urinalysis department analyzing pediatric speci-
Bacteria/HPF mens; this should include the presence of ketones or reducing
None: 0
Rare: 0–10 substances in newborns.
Few: 10–50
Moderate: 50–200 Interpreting Results
Many: >200
Mucous threads The specificity and the sensitivity for each test should be in-
Rare: 0–1
Few: 1–3 cluded in the procedure manual for correct interpretation of
Moderate: 3–10 results. Sensitivity (the lowest level of an analyte that a test can
Many: >10 detect) and specificity (the likelihood of measuring the analyte
desired) vary among manufacturers. The manual should list all
Figure 1–14 Sample standardized urine microscopic reporting for- known interfering substances for evaluation of patient test data.
mat. (From University of Nebraska Medical Center, Omaha, NE, with A well-documented QM program ensures quality test results
permission.) and patient care data.
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70 Part One | Basic Principles

SUMMARY 1-1 Quality Management Errors

Preexamination Instrument malfunction
Patient misidentification Interfering substances present
Wrong test ordered Misinterpretation of QC data
Incorrect urine specimen type collected
Postexamination
Insufficient urine volume Patient misidentification
Delayed transport of urine to the laboratory Poor handwriting
Incorrect storage or preservation of urine Transcription error
Examination Poor quality of instrument printer
Sample misidentification Failure to send report
Erroneous instrument calibration Failure to call critical values
Reagent deterioration Inability to identify interfering substances
Poor testing technique

References
Reporting Critical Results In Urinalysis
1. Clinical and Laboratory Standards Institute: Protection of
POSITIVE KETONES Laboratory Workers from Occupationally Acquired Infections:
For children age 2 years and younger: Approved Guideline, ed 4, CLSI Document M29-A4, Clinical
and Laboratory Standards Institute, Wayne, PA, 2014, CLSI.
All results positive for ketones should be telephoned to the 2. Clinical and Laboratory Standards Institute: Clinical Laboratory
appropriate nursing unit. Safety: Approved Guideline, ed 3, CLSI Document GP17-A3,
Document the following information in the computer as a Clinical and Laboratory Standards Institute, Wayne, PA,
chartable footnote appended to the result: 2012, CLSI.
Time of telephone call
3. Clinical and Laboratory Standards Institute: Clinical Laboratory
Waste Management: Approved Guideline, ed 3, CLSI Document
Initials of the person making the call GP05-A3, Clinical and Laboratory Standards Institute, Wayne,
Name of the person receiving the telephone call PA, 20011, CLSI.
4. Strasinger, SK, Di Lorenzo, MS: The Phlebotomy Textbook,
POSITIVE CLINITEST ed 4. F.A. Davis, Philadelphia, 2019.
For children age 2 years and younger: 5. Centers for Disease Control and Prevention. Guidelines for Isola-
tion Precautions: Preventing Transmission of Infectious Agents
All Clinitest results should be telephoned to the appropriate in Healthcare Settings, 2007. Web site: https://www.cdc.gov/
nursing unit. infectioncontrol/guidelines/isolation/index.html. Accessed
Document the following information in the computer as a April 18, 2019.
chartable footnote appended to the result: 6. Occupational Exposure to Blood-Borne Pathogens, Final Rule.
Time of telephone call
Federal Register 29 (Dec 6), 1991.
7. Occupation Safety and Health Administration. Revision to
Initials of the person making the call OSHA’s Bloodborne Pathogens Standard. Department of Labor.
Name of the person receiving the telephone call Web site: https://www.osha.gov/SLTC/bloodbornepathogens/
standards.html. Accessed April 18, 2019.
8. Centers for Disease Control and Prevention. Updated U.S. Public
Figure 1–15 An example of procedure instructions for reporting Health Service Guidelines for the Management of Occupational
critical values in the urinalysis section. A procedure review docu- Exposures to HBV, HCV and HIV and Recommendations for
ment similar to that shown in Figure 1–8 would accompany this Post-exposure Prophylaxis. MMWR 50(RR11):1–42, 2001.
instruction sheet. Web site: https://www.cdc.gov/mmwr/preview/mmwrhtml/
rr5011a1.htm. Accessed April 18, 2019.
9. Centers for Disease Control and Prevention. Updated U.S.
Public Health Service Guidelines for the Management of
Occupational Exposure to HIV and Recommendations for
For additional resources please visit Post-exposure Prophylaxis. MMWR 54(RR09):1–17, 2005.
www.fadavis.com Web site: https://www.cdc.gov/mmwr/preview/mmwrhtml/
rr5409a1.htm. Accessed April 18, 2019.
7582_Ch01_048-074 24/06/20 5:43 PM Page 71

Chapter 1 | Safety and Quality Management 71

10. NIOSH Alert. Preventing Allergic Reactions to Natural Rubber 16. Clinical and Laboratory Standards Institute (CLSI): Quality
Latex in the Workplace. DHHS (NIOSH) Publication 97-135. Practices in Noninstrumented Point of Care Testing: An In-
National Institute for Occupational Safety and Health, structional Manual and Resources for Health Care Workers.
Cincinnati, OH, 1997. Approved Guideline. CLSI Document POCT08-A, Wayne, PA,
11. Centers for Disease Control and Prevention. Guideline for 2010.
Hand Hygiene in Health-Care Settings: Recommendations of 17. College of American Pathologists: Commission on Laboratory
the Healthcare Infection Control Practices Advisory Committee Accreditation, Urinalysis Checklist. College of American
and the HICPAC/SHEA/APIC/IDSA Hand Hygiene Task Force, Pathologists, Skokie, IL, 2007. Web site: http://webapps.cap.
MMWR 51(pages 21-26), 2002. Web site: https://www.cdc.gov/ org/apps/docs/laboratory_accreditation/checklists/urinalysis_
mmwr/preview/mmwrhtml/rr5116a1.htm. Accessed April 18, clinical_microscopy_sep07.pdf. Accessed April 18, 2019.
2019. 18. Clinical and Laboratory Standards Institute (CLSI), Urinalysis:
12. Centers for Disease Control and Prevention. Guideline for Approved Guideline – Third Edition, CLSI Document GP16-A3,
Disinfection and Sterilization in Healthcare Facilities, 2008. Wayne, PA, 2009.
Web site: https://www.cdc.gov/infectioncontrol/pdf/guidelines/ 19. Clinical and Laboratory Standards Institute (CLSI): Preparation
disinfection-guidelines.pdf. Accessed April 18, 2019. and Testing of Reagent Water in the Clinical Laboratory: Ap-
13. Occupational Exposure to Hazardous Chemicals in Laboratories proved Guideline, Fourth Edition, CLSI Document GP40-A4-
(Non-Mandatory Appendix); Technical Amendment. January 22, AMD, Wayne, PA 2006.
2013. Office of the Federal Register. Web site: https://www. 20. Centers for Medicare & Medicaid Services, Clinical Laboratory
federalregister.gov/documents/2013/01/22/2013-00788/ Improvement Amendments (CLIA). Proficiency Testing and PT
occupational-exposure-to-hazardous-chemicals-in-laboratories- Referral. Dos and Don’ts. Web site: https://www.cms.gov/
non-mandatory-appendix-technical. Accessed April 18, 2019. Regulations-and-Guidance/Legislation/CLIA/Downloads/
14. National Fire Protection Association: Hazardous Chemical CLIAbrochure8.pdf. Accessed April 18, 2019.
Data, No. 49. Boston, NFPA, 1991. 21. Centers for Medicare & Medicaid Services (CMS). Clinical
15. Centers for Medicare & Medicaid Services, Department of Laboratory Improvement Amendments (CLIA). Individualized
Health and Human Services: Clinical Laboratory Improvement Quality Control Plan (IQCP). Web site: https://www.cms.gov/
Amendments. Web site: www.cms.hhs.gov/CLIA/05_CLIA_ Regulations-and-Guidance/Legislation/CLIA/Individualized_
Brochures.asp Accessed April 18, 2019. Quality_Control_Plan_IQCP.html. Accessed April 15, 2019.

Study Questions
1. Which of the following organizations publishes 4. The best way to break the chain of infection is:
guidelines for writing procedures and policies in A. Hand sanitizing
the urinalysis?
B. Personal protective equipment
A. CDC
C. Aerosol prevention
B. OSHA
D. Decontamination
C. CLSI
5. The current routine infection control policy developed by
D. CLIA
CDC and followed in all health-care settings is:
2. Exposure to toxic, carcinogenic, or caustic agents is what A. Universal Precautions
type of laboratory safety hazard?
B. Isolation Precautions
A. Biological
C. Blood and Body Fluid Precautions
B. Sharps
D. Standard Precautions
C. Chemical
6. An employee who is accidentally exposed to a possible
D. Fire/explosive
bloodborne pathogen should immediately:
3. In the urinalysis laboratory, the primary source in the A. Report to a supervisor
chain of infection would be:
B. Flush the area with water
A. Patients
C. Clean the area with disinfectant
B. Needlesticks
D. Receive HIV prophylaxis
C. Specimens
D. Biohazardous waste
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72 Part One | Basic Principles

7. Personnel in the urinalysis laboratory should wear 14. When combining acid and water, ensure that:
laboratory coats that: A. Acid is added to water
A. Do not have buttons B. Water is added to acid
B. Are fluid-resistant C. They are added simultaneously
C. Have short sleeves D. Water is slowly added to acid
D. Have full-length zippers
15. An employee can learn the carcinogenic potential of
8. All of the following should be discarded in biohazardous potassium chloride by consulting the:
waste containers except: A. Chemical hygiene plan
A. Urine specimen containers B. Safety Data Sheet
B. Towels used for decontamination C. OSHA standards
C. Disposable laboratory coats D. Urinalysis procedure manual
D. Blood collection tubes
16. Employees should not work with radioisotopes
9. An employer who fails to provide sufficient gloves for if they are:
the employees may be fined by the: A. Wearing contact lenses
A. CDC B. Allergic to iodine
B. NFPA C. Sensitive to latex
C. OSHA D. Pregnant
D. FDA
17. All of the following are safe to do when removing the
10. An acceptable disinfectant for decontamination of blood source of an electric shock except:
and body fluids is: A. Pulling the person away from the instrument
A. Sodium hydroxide B. Turning off the circuit breaker
B. Antimicrobial soap C. Using a glass container to move the instrument
C. Hydrogen peroxide D. Unplugging the instrument
D. Sodium hypochlorite
18. The acronym PASS refers to:
11. Correct hand washing includes all of the following A. Presence of vital chemicals
except:
B. Operation of a fire extinguisher
A. Using warm water
C. Labeling of hazardous material
B. Rubbing to create a lather
D. Presence of radioactive substances
C. Rinsing hands in a downward position
19. The system used by firefighters to assess the risk
D. Turning on the water with a paper towel
potential when a fire occurs in the laboratory is:
12. Centrifuging an uncapped specimen may produce a A. SDS
biological hazard in the form of:
B. RACE
A. Vectors
C. NFPA
B. Sharps contamination
D. PASS
C. Aerosols
20. A class ABC fire extinguisher contains:
D. Specimen contamination
A. Sand
13. An employee who accidentally spills acid on his arm
B. Water
should immediately:
C. Dry chemicals
A. Neutralize the acid with a base
D. Acid
B. Hold the arm under running water for 15 minutes
C. Consult the SDS 21. The first thing to do when a fire is discovered is to:
D. Wrap the arm in gauze and go to the emergency A. Rescue people in danger
department B. Activate the alarm system
C. Close doors to other areas
D. Extinguish the fire if possible
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Chapter 1 | Safety and Quality Management 73

22. If a red rash is observed after removing gloves, the 30. During laboratory accreditation inspections, procedure
employee: manuals are examined for the presence of:
A. May be washing her hands too often A. Critical values
B. May have developed a latex allergy B. Procedure references
C. Should apply cortisone cream C. Procedures for specimen preservation
D. Should not rub her hands so vigorously D. All of the above
23. Pipetting by mouth is: 31. As the supervisor of the urinalysis laboratory, you have
A. Acceptable for urine but not serum just adopted a new procedure. You should:
B. Not acceptable without proper training A. Put the package insert in the procedure manual
C. Acceptable for reagents but not specimens B. Put a complete, referenced procedure in the manual
D. Not acceptable in the laboratory C. Notify the microbiology department
D. Put a cost analysis study in the procedure manual
24. The NPFA classification symbol contains information on
all of the following except: 32. Indicate whether each of the following would be
A. Fire hazards considered a 1) preexamination, 2) examination, or
3) postexamination factor by placing the appropriate
B. Biohazards
number in the blank:
C. Reactivity
Reagent expiration date
D. Health hazards
Rejecting a contaminated specimen
25. The GHS requires the following on a chemical label: Constructing a Levy-Jennings chart
A. Biohazard symbol, warning sign, environmental Telephoning a positive Clinitest result on a
impact newborn
B. Hazard pictogram, signal words, hazard statement Calibrating the centrifuge
C. Biological symbol, hazard pictogram, long-term Collecting a timed urine specimen
effects
33. The testing of a specimen from an outside agency and
D. Signal words, hazard statement, biological symbol
the comparison of results with participating laboratories
26. The classification of a fire that can be extinguished with is called:
water is: A. External QC
A. Class A B. Electronic QC
B. Class B C. Internal QC
C. Class C D. Proficiency testing
D. Class D
34. A color change indicating that a sufficient amount of a
27. Employers are required to provide free immunization for: patient’s specimen or reagent is added correctly to the
A. HIV test system would be an example of:
B. HTLV-1 A. External QC
C. HBV B. Equivalent QC
D. HCV C. Internal QC
D. Proficiency testing
28. A possible physical hazard in the hospital is:
A. Wearing closed-toed shoes 35. What steps are taken when the results of reagent strip
QC are outside the stated confidence limits?
B. Not wearing jewelry
A. Check the expiration date of the reagent strip
C. Having short hair
B. Run a new control
D. Running to answer the telephone
C. Open a new reagent strips container
29. Quality management refers to:
D. All of the above
A. Analysis of testing controls
B. Increased productivity
C. Precise control results
D. Quality of specimens and patient care
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74 Part One | Basic Principles

36. When a new bottle of QC material is opened, what 37. When a control is run, what information is
information is placed on the label? documented?
A. The supervisor’s initials A. The lot number
B. The lot number B. Expiration date of the control
C. The date and the laboratory worker’s initials C. The test results
D. The time the bottle was opened D. All of the above

Case Studies and Clinical Situations

1. State a possible reason for an accreditation team to report scientist was waiting too long to read the glucose results
a deficiency in the following situations: and therefore reporting erroneous results.
a. The urine microscopic reporting procedure has been a. What is wrong with this scenario?
recently revised. b. Who should run the QC for each test? Why?
b. An unusually high number of urine specimens are c. When should controls be run?
being rejected because of improper collection.
d. What do you do when the QC is out of range?
c. A key statement is missing from the Clinitest
e. When can you report patient results?
procedure.
d. Open control bottles in the refrigerator are examined. 4. An outpatient urine specimen was delivered to the labora-
tory at 0800 and placed on the counter in the urinalysis
2. As the new supervisor of the urinalysis section, you en- department. The medical laboratory scientist performed
counter the following situations. Explain whether you urinalysis on the specimen at 1130. The following results
would accept them or take corrective action. were abnormal:
a. You are told that the supervisor always performs the Clarity: Cloudy
CAP proficiency survey.
pH: 9.0
b. QC is not performed daily on the reagent strips.
Nitrite: Positive
c. The urinalysis section is primarily staffed by personnel
assigned to other departments for whom you have no The patient was a known diabetic; however, the glucose
personnel data. result was negative.
a. What could be a possible cause for the abnormal
3. The medical laboratory scientist was assigned to test 10
results?
urine specimens chemically. She removed 10 strips from
the container and proceeded with testing. Several pa- b. Where would the information concerning what
tients’ urine indicated a trace positive glucose in the should have been done with this specimen be found,
urine. She then opened a new bottle of reagent dipsticks as well as the criteria for rejection?
and proceeded to perform the QC. The negative control c. What QM procedure may have detected this error?
also tested as a trace positive for glucose. The medical d. What form will need to be completed for this
laboratory scientist consulted the supervisor. The supervi- scenario?
sor ran the QC, and the results were in the correct range.
e. How might this affect this patient’s care?
After observing the medical laboratory scientist’s tech-
nique, the supervisor realized that the medical laboratory f. How will the corrected results be documented?
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CHAPTER 2
Urine and Body Fluid
Analysis Automation
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
2-1 Explain the principle of reflectance photometry. 2-4 Describe laser-based flow cytometry, digital imaging,
and auto particle recognition technologies used in auto-
2-2 Differentiate between semiautomated urine chemistry
mated urine microscopy analyzers.
analyzers and fully automated urine chemistry analyzers.
2-5 Discuss the advantages of automated body fluid analyz-
2-3 State the advantages of automated microscopy methods
ers over the Neubauer hemocytometer for body fluid
over manual microscopy methods for analyzing urine
cell counts.
sediment.

KEY TERMS
Auto-checks Digital imaging Light-emitting diode (LED)
Auto particle recognition (APR) Flow cytometry Reflectance photometry
Autovalidated Histograms Scattergrams
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76 Part One | Basic Principles

Urinalysis Automation printed report; flagging abnormal results; storing patient and con-
trol results; and minimal calibration, cleaning, and maintenance.
Studies have shown that the major variable in urinalysis testing Automated instruments in urinalysis include semiauto-
is the conscientiousness of the laboratory personnel in their mated and fully automated chemistry analyzers, automated
timing and interpretations of the color reactions. Correct color urine cell microscopy analyzers, and systems that are com-
readings depend on the accuracy of the timing. The ultimate pletely automated. Semiautomated instruments still depend on
goal of urinalysis automation is to improve reproducibility and an operator for specimen mixing, test strip dipping, and mi-
color discrimination while increasing productivity and stan- croscopic results input. In a fully automated chemistry ana-
dardization for reporting urinalysis results. lyzer, the tubes of urine are placed on a rack or a carousel and
moved automatically through the instrument. Automated urine
Reflectance Photometry cell microscopy analyzers mix, aspirate, dilute, and stain urine
Subjectivity associated with visual discrimination among colors to classify urine sediment particles. Automated urine systems
has been alleviated by the development of automated reagent perform a complete urinalysis that includes the physical, chem-
strip readers that use a spectrophotometric measurement of ical, and microscopic parts of a routine urinalysis by integrating
light reflection, termed “reflectance photometry.” Reflectance a fully automated chemistry analyzer with an automated urine
photometry uses the principle that light reflection from the test cell microscopy analyzer. The automated urinalysis instru-
pads decreases in proportion to the intensity of the color ments currently available are listed in Table 2-2; however, new
produced by the concentration of the test substance. In re- instruments are being developed continually.1,2
flectance photometry, a monochromatic light source is directed
toward the reagent pads by placing a filter between the light Analyzers
source and the reflective surface of the pad or by using a light-
Semiautomated Urine Chemistry Analyzers
emitting diode (LED) to provide the specific wavelength
needed for each test pad color reaction. The light is reflected Semiautomated urine analyzers test for the chemical compo-
to a photodetector, as well as a converter that is either analog nents of urine. The instruments read and interpret the reagent
or digital. The instruments compare the amount of light reflec- strip results consistently, thereby standardizing the interpreta-
tion with that of known concentrations and then display or tion of reagent strip results and eliminating personnel color bias
print concentration units or transmit data to a laboratory in- and timing discrepancies. Depending on the instrument and
formation system (LIS). the reagent strip used, the following tests can be performed:
Several automated instruments are available that standard- leukocyte, nitrite, protein, blood, glucose, ketone, bilirubin, uro-
ize sample processing, analyze chemistry test strips, perform bilinogen, pH, specific gravity, color, creatinine, and protein-to-
urine sediment analysis, and report results with consistent creatinine ratio. Semiautomated analyzers are well suited for
quality and reduced hands-on time. The instruments are user- small- and medium-volume laboratories and physicians’ offices
friendly and include visual and audio prompts for operation. and meet the Clinical Laboratory Improvement Amendments
The various manufacturers’ instruments include different fea- (CLIA)–waived standards.
tures and principles for testing. See Table 2-1 for a breakdown Semiautomated analyzers are self-calibrating, and some in-
of the measurement technologies used by the major urine an- struments perform automatic checks (auto-checks) to identify
alyzer manufacturers.1,2 strip type and humidity exposure. For a semiautomated instru-
Additional advantages to automation include online com- ment, the reagent strips are manually dipped into the urine and
puter capability with an LIS interface; barcoding; manual entry placed on the strip reader, the reaction pads are read at the correct
of color, clarity, and microscopic results to be included on the time, and the strip is moved to the waste container. The results

Table 2–1 Measurement Technology Methods in Automated Urinalysis1,2

Urine Measurement Technology
Manufacturer Color Clarity Specific Gravity
U.S. ARKRAY, Inc. Reflectance photometry Light scatter Refractive index
Iris Diagnostics- Beckman Light transmission/light scatter Light transmission/light scatter Refractive index
Coulter, Inc.
DIRUI Reflectance photometry Turbidity Refractometry
Roche Diagnostics Reflectance photometry Turbidity Refractometry
Siemens Healthcare Reflectance photometry Light transmission/light scatter Refractive index
Diagnostics, Inc.
Sysmex Corporation Reflectance photometry Reflectance photometry Refractometry
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Chapter 2 | Urine and Body Fluid Analysis Automation 77

Table 2–2 Urinalysis Automation

Equipment Manufacturer
Semiautomated Chemistry Instruments
Clinitek Advantus Siemens Healthineers
Clinitek Status Siemens Healthineers
Urisys 1800 system Roche Diagnostics
COBAS u411 Roche Diagnostics
AUTION Eleven AE-4022 U. S. ARKRAY Inc.
Fully Automated Chemistry Instruments
Clinitek Atlas Siemens Healthineers
Clinitek Novus Siemens Healthineers
Urisys 2400 system Roche Diagnostics
AUTION MAX AX-4030 U.S. ARKRAY, Inc.
IChemVELOCITY Iris Diagnostics-Beckman Coulter, Inc.
Cobas u 601 urine analyzer Roche Diagnostics
FUS-100 and FUS-200 DIRUI
Automated Microscopy
UF-1000i Urine Cell Analyzer Sysmex Corporation
UN-2000 Sysmex Corporation
iQ 200 Automated Urine Microscopy Iris Diagnostics-Beckman Coulter, Inc.
(IQ200SELECT, IQ200ELITE, IQ200SPRINT)
Urine Analyzer (iQ 200 Sprint) Iris Diagnostics-Beckman Coulter, Inc.
Cobus u 701 Microscopy Analyzer Roche Diagnostics
LabUMat 2 77 Elektronika
Automated Urinalysis Systems
iRICELL Urinalysis – IQ Series Urinalysis Workcell Iris Diagnostics-Beckman Coulter, Inc.
(iRICELL 3000plus, iRICELL 2000plus, iRICELL 3000pro, iRICELL 2000pro, iRICELL 1500)
CLINITEK AUWi Pro System Siemens Healthineers
Cobas 6500 Roche Diagnostics
Dirui FUS – 100/200 with H-800 DIRUI
UriSed 2 or UriSed 3 with LabUMat2 77 Elektronika
UX-2000 Sysmex Corporation
AUTION HYBRID AU-4050 U.S. ARKRAY, Inc.
Body Fluid Analyzers
ADVIA2120i with Body Fluids Software Siemens Healthcare Diagnostics, Inc.
Sysmex XN-Series using Body Fluids mode Sysmex Corporation
iQ 200 using Body Fluids Software Iris Diagnostics-Beckman Coulter, Inc.
DxH 900 Hematology Analyzer Beckman Coulter, Inc.
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78 Part One | Basic Principles

are displayed, printed, or transmitted to an LIS. Patient identifi-

cation and specimen color and clarity may be entered manually,
or a barcode reader can be used to identify samples. Positive
results are flagged to indicate a patient sample that requires
additional confirmation testing or microscopic evaluation.
The semiautomated instrument requires the operator to:
1. Dip the reagent strip into a urine sample that has been
mixed well.
2. Blot the strip to remove excess urine.
3. Place the strip onto the reagent strip platform.
4. Press the analyze/enter button.
The results are printed out, and abnormal results are
flagged automatically. As the strip moves through the instru-
ment, reflectance readings are taken at the correct time intervals.
Some manufacturers’ test strips have a color compensation pad
that adjusts results for urine color. This feature allows the in-
strument to subtract the urine color from the color developed
on the reaction pad, providing an accurate result for each pad Figure 2–2 Cobas u 411 urine chemistry analyzer. (Image courtesy
despite the interference. Then the strips are moved to the waste of Roche Diagnostics.)
container. Results are stored in the analyzer, printed, or sent to
the LIS. Examples of semiautomated instruments are shown in
Figures 2-1 through 2-5.
Daily maintenance is minimal and includes cleaning the
reagent strip platform and emptying the reagent strip waste
container.

Fully Automated Urine Chemistry

Analyzers
Fully automated instruments are designed for a high-volume
urinalysis laboratory with user walk-away capability. The various
instruments can load many labeled tubes of urine on a carousel
or rack at one time with the capability to insert a stat sample dur-
ing the run. The “start” or “analyze” button is pressed to begin
testing, and the sample moves through the instrument auto-
matically. The sample is identified, mixed, and aspirated. A sam-
ple probe aspirates an exact amount of urine and dispenses it

Figure 2–1 AUTION ELEVEN™ AE-4022. (Image courtesy of U.S. Figure 2–3 Urisys 1100 semiautomated urine chemistry analyzer.
ARKRAY, Inc.) (Image courtesy of Roche Diagnostics.)
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Chapter 2 | Urine and Body Fluid Analysis Automation 79

directly onto the reagent strip. The reagent strip advances

automatically to the reflectance photometer to measure the
color change of each reagent pad. Then the strip advances au-
tomatically to the disposal area. Tests are measured by the dry
chemistry test strip, using reflectance photometry to detect
color change and then taking readings at the appropriate time
and wavelength for each specific test. Analytes measured
vary by the instrument and may include leukocytes, ketones,
protein, glucose, nitrite, blood, urobilinogen, pH, bilirubin,
color, clarity, creatinine, and protein-low. Color is measured by
either reflectance photometry or spectrophotometry at multiple
wavelengths. Specific gravity is measured by the refractive index
methodology, and clarity is a measurement of transmitted or scat-
tered light. The instruments use integrated barcoded sample
A identification and allow abnormal ranges to be selected so that
samples that require microscopic examination or confirmatory
testing can be identified and flagged. Patient results and quality
control results and calibrations are stored for visual display, print-
out, or transmission to an LIS. Standardized controls are run as
set by laboratory protocol. Examples of fully automated urine
chemistry analyzers are shown in Figures 2-6 through 2-9.

Automated Microscopy Analyzers

In a routine urinalysis, a test strip determines the chemical ana-
lytes and the formed elements are determined by microscopy.
Manual microscopy is not easily standardized because of the
high variation among operators even in the same facility. Routine
specimen processing, such as centrifugation, can affect accuracy
because rare elements, such as casts or cells, may be lost during
handling. It has been demonstrated that recovery of formed
B elements in the sediment after centrifugation is highly variable.
Results are not quantitative because they must be reported in
Figure 2–4 Clinitek Status + Analyzer. A. Clinitek Status Connect
with barcode stand. B. Clinitek Status with test strip. (Images
courtesy of Siemens Healthcare Diagnostics, Inc.)

Figure 2–5 Clinitek Advantus semiautomated urine chemistry

analyzer. (Image courtesy of Siemens Healthcare Diagnostics, Figure 2–6 Urisys 2400 automated urine chemistry analyzer. (Image
Inc.) courtesy of Roche Diagnostics.)
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80 Part One | Basic Principles

ranges or averages. Overall, manual microscopy is not cost

effective because of the labor and time required to process and
analyze the specimen, which ultimately increases turnaround
times (TATs) for results. Automated urine cell microscopy
analyzers provide efficient standardized results in less than
1 minute compared with approximately 6 minutes using the
manual method, markedly improving TATs. A number of
automated urine cell microscopy analyzers are currently avail-
able in the United States. Two types of technology are used for
urine sediment analysis with these instruments: flow cytometry
and digital imaging techniques.

Sysmex UF-1000i Fully Automated Urine Particle

Analyzer and Sysmex UN-2000 Automated
Urinalysis System
The Sysmex UF-1000i (Sysmex Corporation, Kobe, Japan) uses
Figure 2–7 Clinitek Atlas automated urine chemistry analyzer.
fluorescent flow cytometry to measure the forward-scattered,
(Image courtesy of Siemens Healthcare Diagnostics, Inc.)
side-scattered, and fluorescence light characteristics of particles
present in urine. The information obtained from these meas-
urements is used to detect and identify stained urine sediment
particles (Fig. 2-10).
To perform an automated urine sediment analysis, 1.2 mL
of uncentrifuged urine is aspirated into the instrument and
divided into two channels: the sediment channel for urine par-
ticle analysis and the bacteria channel for bacteria staining and
detection (Fig. 2-11). Each channel has a specific stain that
targets surface and/or internal components of the cells. The
stained urine sample passes through the flow cell, where it is
hydrodynamically focused and presented to a red semiconduc-
tor laser (635 nm) (Fig. 2-12). Particles are identified by meas-
uring the height and width of the fluorescent and light scatter
signals, which are presented in scattergrams and histograms.
In the bacteria channel, the diluent stabilizes the pH and lyses
the nonbacterial particles, reducing interference from amor-
phous crystals. The stain is specific to the ribonucleic acid
(RNA) in a bacterial cell, eliminating any nonspecific staining
of debris. The width of the fluorescent signal measures cellular
inclusions, and the width of forward light scatter measures the
Figure 2–8 AUTION MAX AX-4030 fully automated urine chemistry length of cells (Fig. 2-13).
analyzer. (Image courtesy of U.S. ARKRAY, Inc.)

Figure 2–9 iChemVELOCITY automated urine chemistry analyzer. Figure 2–10 Sysmex UF 1000i urine chemistry analyzer. Image
(Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.) courtesy of Sysmex Corporation, Kobe, Japan.)
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Chapter 2 | Urine and Body Fluid Analysis Automation 81

Forward scatter confirmed by manual microscopy. A latex particle quality

signal amplifier
control system monitors performance, and quality control
records can be viewed on the instrument screen in a Levy-
Fluorescence Jennings graph. The analyzers can store up to 10,000 patient
signal amplifier
results, including scattergrams, histograms, and specimen
Red semiconductor
laser characteristics, which can be saved and viewed by the user.
A bidirectional interface is provided to download and report
results. The UF-1000i can be used independently or inte-
Side scatter
grated with an automated urine chemistry strip reader to
Sheath reagent signal amplifier create a complete urinalysis system.
Conductivity sensor
The newest Sysmex model, the UN-2000 (Fig. 2-15), is
an automated, modular, and scalable urinalysis system that
Dilution and staining Dilution and staining
for bacteria analysis for sediment analysis combines urine flow cytometry with digital image analysis
by integrating the UF-5000 Fully Automated Urine Particle
Analyzer (Fig. 2-16) and the UD-10 Fully Automated Urine
Particle Digital Imaging Device (Fig. 2-17). The UF-5000 uti-
Urine sample lizes fluorescent flow cytometry coupled with a blue semicon-
ductor laser to measure particle length, particle volume,
particle internal complexity, and total amount of nucleic
Flow cytometry acid contained within the particle (Figs. 2-18 and 2-19). The
shorter wavelength blue (488 nm) semiconductor laser offers
Figure 2–11 Diagram of urine particle analysis in the Sysmex the benefit of enhanced detection and differentiation of small
UF1000i. (Image courtesy of Sysmex Corporation, Kobe, Japan.) particles. The sediment and bacteria channels used in the
previous-generation UF-1000i have been replaced with two
Resulting values are presented in quantitative cells per new analysis channels to further enhance the sensitivity and
microliter and cells per high- or low-powered field. Thresholds specificity of particle detection in urine sediment.
to be flagged for primary elements can be established, and The core channel (CR ch) stains elements with nuclear ma-
abnormal results are flagged for confirmatory review. The main terial, such as WBCs, epithelial cells, and bacteria (Fig. 2-20).
particles enumerated are red blood cells (RBCs), white blood In regard to the bacteria analysis in the CR ch, the degree of
cells (WBCs), squamous epithelial cells, hyaline casts, and bac- staining, and thus intensity, of the side fluorescence signal is
teria. The results are displayed as scattergrams (Fig. 2-14). dependent on the bacterial cell wall structure. The surface chan-
These parameters are reportable directly without technologist nel (SF ch) stains and measures anucleate elements, such as
intervention and may be autovalidated. Flagged particles RBCs, crystals, and casts (Fig. 2-21).3
include pathological casts, crystals, small round cells (renal In addition to the two new measuring channels, the
tubular epithelial cells or transitional epithelial cells), sperm, UF-5000 has a new depolarized side scatter (DSS) detector
mucus, and yeastlike cells, and these particles must be that provides information on the particles’ ability to depolarize

UF-1000i Technology

Diluents

Sediments Bacteria

Sediments Bacteria

Incubation

Detection
unit

Figure 2–12 Staining elements for

the Sysmex UF1000i. (Image courtesy Stain
of Sysmex Corporation, Kobe,
Japan.) Sediments Bacteria
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82 Part One | Basic Principles

UF-1000i Signal Waveforms for Cells

Forward scattered Lateral fluorescent Lateral scattered

light waveform light waveform light waveform

RBC

WBC

Bacteria

Epithelial cells Figure 2–13 UF1000i signal waveform

for cells. (Image courtesy of Sysmex Corporation,
Kobe, Japan.)

Figure 2–15 UN-2000-110™ Sysmex Urinalysis System. (Image cour-

tesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–14 Scattergram showing Sysmex UF1000i microscopy

results. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

light (Fig. 2-22). Through the detection of depolarized light, the

DSS detector works to differentiate urine particles that are bire-
fringent, such as crystals. An advantage of the Sysmex UF-5000
is that it requires a minimal volume of urine for analysis.
The Sysmex UD-10 is a complementary digital imaging
system designed to capture detailed images of urine particles
in urine samples that require further review. All captured urine Figure 2–16 UF-5000™ Fully Automated Urine Particle Analyzer.
particle images are classified, based on size, into eight different (Image courtesy of Sysmex Corporation, Kobe, Japan.)
classes. Then the technologist has the opportunity to review
the captured images and identify the type(s) of urine particles with the LIS or middleware system. In the United States and
present.3 Canada, this data management system is called the Urinalysis
The UN-2000 uses a single-screen data management system Data Manager (UDM). In regions outside the United States and
for monitoring analyzer status, result reporting, and reflex rule Canada, U-WAM is the name of the data management system
setting, as well as providing the connection point for interface for UN-Series analyzers.
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Chapter 2 | Urine and Body Fluid Analysis Automation 83

Cell length

Diameter
of nucleus

Signal intensity Nucleic acid content

(fluorescence signal waveform area)

Figure 2–19 UF-5000™ measurement of the cell length, diameter,

and nucleic acid content. (Image courtesy of Sysmex Corporation,
Kobe, Japan.)

The analyzer mixes the sample and aspirates 1.3 mL of

urine. The sample is hydrodynamically focused in a planar
flow cell and presented to a digital microscope in a thin mov-
ing sheet. Then a digital camera takes 500 images as the sam-
Figure 2–17 UD-10™ Fully Automated Urine Particle Digital Imaging
Device. (Image courtesy of Sysmex Corporation, Kobe, Japan.) ple advances through the flow cell (Fig. 2-24). Individual
urine particles are extracted from the raw images and auto-
matically classified using a proprietary neural network algo-
Beckman Coulter’s iQ200 Microscopy Analyzer
rithm into 12 major classification categories: RBCs, WBCs,
The iQ200 Automated Urine Microscopy Analyzer (Iris WBC clumps, squamous epithelial cells, nonsquamous epithelial
Diagnostics-Beckman Coulter, Brea, CA) uses digital flow cells, unclassified casts, hyaline casts, unclassified crystals,
morphology (imaging) and auto particle recognition (APR) bacteria, yeast, mucus, and sperm (Figs. 2-25 and 2-26).4
to categorize and count urine particles automatically in un- Results are either reviewed by a trained operator or auto-
centrifuged urine based on size, shape, texture, and contrast. released to the LIS based on user-defined parameters. Because
The instrument also can be used for counts of body fluid the images are archived digitally, results can be reviewed easily
cells by adding the optional body fluids software module and reclassified by the operator without the need for manual
(Fig. 2-23). The microscopy unit can be integrated with an microscopy.
automated urine chemistry analyzer to provide a complete In addition to the 12 major categories, the software allows
urinalysis system. the user to subclassify particles into 27 additional categories,

Figure 2–18 Diagram of the urine particle analysis in the Sysmex UF-5000™. (Image courtesy of Sysmex Corporation, Kobe, Japan.)
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84 Part One | Basic Principles

Figure 2–20 UF-5000 core channel. The CR channel analyzes nucleic acid–containing particles such as white blood cells, bacteria, and epithe-
lial cells. Nucleic acid–containing cells are detected and classified through the use of a newly developed nucleic acid staining reagent. The
nucleic acid–containing particles are further classified based on the area under the fluorescence signal waveform, which reflects the amounts
of nucleic acid content and the known difference in nucleic acid content of different types of particles. A short-wavelength blue semiconduc-
tor laser is used to detect small particles, such as bacteria, with greater accuracy. In this channel, red blood cells are lysed and removed by a
diluent, and crystals are removed by a chelating agent, contained in the diluent. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

refined strain

Figure 2–21 UF-5000 surface channel. The SF channel analyzes urine particles that do not contain nucleic acid, such as casts, red blood cells,
and crystals. For differentiating casts from castlike elements (mucus threads, salt clumps, bacterial clumps, etc.), those particles are dispersed
by reagents, and then the analyzer uses waveform information about particles stained with a newly developed staining reagent for more pre-
cise analysis. Besides this, amorphous salts are moved by the chelating action of a reagent and heating process. (Image courtesy of Sysmex
Corporation, Kobe, Japan.)
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Chapter 2 | Urine and Body Fluid Analysis Automation 85

Figure 2–22 UF-5000 depolarized side-scattered light (DSS) detector. (Image courtesy of Sysmex Corporation, Kobe, Japan.)

Figure 2–25 Auto particle recognition (APR) process. (Image

courtesy of Iris Diagnostics-Beckman Coulter, Inc.)
Figure 2–23 iQ 200 microscopy analyzer. (Image courtesy of Iris
Diagnostics-Beckman Coulter, Inc.)

o c

c
l
Figure 2–26 iQ 200 urinalysis results display, showing particle
categories available for analysis or counting. (Image courtesy of Iris
Figure 2–24 Diagram of the iQ 200 digital flow capture process. Diagnostics-Beckman Coulter, Inc.)
(Image courtesy of Iris Diagnostics-Beckman Coulter, Inc.)

UriSed 2 requires a minimum of 2 mL of urine that is cen-

such as specific types of crystals, casts, nonsquamous epithelial
trifuged in a special cuvette to produce a monolayer of urine
cells, yeast with pseudohyphae, trichomonas, and oval fat bod-
sediment. The sediment is analyzed by a bright-field micro-
ies. Additional user observations can be added to the report in
scope and digital camera to capture and categorize 15 particle
the Edit Comment section.
images based upon size and shape using AIEM software.1,5 An
77 Electronika UriSed 2 and 3Pro Automated Urine advantage to this instrument is the zoom capability to view im-
ages, and interpretation of the images is similar to that of manual
Sediment Analyzer
microscopic smears. The UriSed 3 Pro incorporates phase-
The UriSed 2 and UriSed 3 Pro (77 Electronika, Budapest, contrast microscopy in addition to bright-field microscopy to im-
Hungry) perform automated microscopy with digital imaging prove differentiation of elements, such as hyaline casts, RBC
using auto image evaluation module (AIEM) software. The membranes, crystals, and yeast.1 Both the UriSed 2 and
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86 Part One | Basic Principles

UriSed 3 can be linked with the chemistry analyzer LabUMat 2

to make a complete automated urinalysis laboratory system.1

FUS-100 and FUS-200 Analyzers

The FUS-100 and FUS-200 urine sediment analyzers (DIRUI In-
dustrial Co., Ltd., Changchun City, Jilin Province, China) identify
cells using flat-flow cell digital imaging technology and a trained
neural network. Artificial imaging identification (AII) software is
used to classify and quantify the cells and formed particles in the
uncentrifuged urine.6 A digital camera captures up to 820 photos,
and the AII identifies and classifies 12 visible components in urine
based on shape, contrast, texture, and frequency domain
features.1,6 The FUS instruments can be integrated with the H-
800 chemistry analyzer for a total automated urinalysis system.6

Automated Urinalysis Systems

Figure 2–27 AUWi, a fully automated urinalysis system that com-
Combining automated urine chemistry analyzers and automated bines the Siemens Clinitek Atlas Chemistry analyzer and the Sysmex
urine cell analyzers to create completely automated urinalysis sys- UF-1000i particle analyzer. (Image courtesy of Siemens Healthcare
tems has improved TATs for urinalysis significantly. Technologists’ Diagnostics, Inc.)
hands-on time has been reduced significantly as well. Using sim-
ilar sample racks and moving on a conveyor system, samples are mode and 2 mL in the manual mode. Samples can be placed
easily transferred from one instrument to the next, providing on the instrument with fewer manual steps, and no centrifu-
complete walkaway capability with minimal sample handling gation is required. Autoverification of results to be reported to
from sampling through results. By interfacing with the LIS, bar- the LIS is based on laboratory protocol. In addition, the system
coded samples are identified automatically and processed accord- is capable of automatically reflexing samples requiring sedi-
ing to the requested tests. The systems can independently ment analysis based on rules defined by each laboratory.7
perform both physical and chemical testing, microscopy analysis,
and a combination of both. A complete urinalysis report can be iRICELL Urinalysis Systems
sent directly to the LIS or printed out, thereby reducing clerical The iRICELL Automated Urinalysis System (Iris Diagnostics-
error. Autoverification of results and reflex testing can be vali- Beckman Coulter, Brea, CA) consists of the iChemVELOCITY
dated according to laboratory protocol. Abnormal results are urine chemistry analyzer and the iQ200 urine microscopy ana-
flagged for manual examination by laboratory personnel. lyzer (Fig. 2-28). A minimum of 4 mL of urine is required. Bar-
coded tubes are placed into the 10-position rack and are moved
Clinitek AUWi System and AUWi Pro System to the iChemVELOCITY. Upon completion of the physical and
The Clinitek Atlas System (Siemens Healthcare Diagnostics, chemical analysis, the rack moves across the connecting bridge
Tarrytown, NY), an automated urine chemistry analyzer, and to the iQ200 for microscopy testing, where a reflex rule may be
the Sysmex UF-1000i (Sysmex Corporation, Mundelein, IL), applied based on settings. Combined chemistry and microscopy
an automated urine cell analyzer, have been integrated to de- urinalysis results are transmitted to the LIS or printed.
velop the Clinitek AUWi System (Siemens), which performs a The IQ series of urinalysis workcells (iQ2000 and iQ3000)
completely automated urinalysis (Fig. 2-27). A minimum of pairs the Iris IRICELL series of urine microscopy instrumentation
5 mL of urine is required in the automated mode. The bar- and the ARKRAY AUTION MAX AX4030 fully automated urine
coded tubes are racked and placed onto the system. The rack chemistry analyzer to provide a complete walk-away urinalysis
advances to the Atlas analyzer, where the sample is identified, system (refer back to Table 2-2).
mixed, aspirated, and tested for physical and chemical com-
ponents. Then the sample travels across a connecting bridge
to the UF-1000i for microscopic analysis. The instrument
automatically reflexes samples requiring sediment analysis,
reducing the time associated with manual microscopic analysis.
Results are verified automatically and integrated into a
complete urinalysis report to be sent to the LIS or printed.
The Clinitek AUWi PRO Automated Urinalysis System
integrates the Clinitek Novus Automated Urine Chemistry
Analyzer and the Sysmex UF-1000i Urine Particle Analyzer. Figure 2–28 iRICELL3000, a fully automated urinalysis system that
This automated system can load up to 200 sample tubes and combines the iChemVELOCITY urine chemistry analyzer and the iQ
complete up to 80 tests per hour. As with the AUWi system, 200 microscopy analyzer. (Image courtesy of Iris Diagnostics-
the sample volume requirement is 5 mL in the automated Beckman Coulter, Inc.)
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Chapter 2 | Urine and Body Fluid Analysis Automation 87

LabUMat 2 with UriSed 2 or UriSed 3 System are available—Fuchs-Rosenthal, Nageotte, and Neubauer—and
each differs in the design of the calibrated counting area etched
The chemistry analyzer LabUMat 2 (77 Electronika) can be on the slide. An exact amount of fluid fills the chamber that
integrated with the UriSed 2 or UriSed 3 (77 Electronika) provides a defined volume for cell enumeration. These proce-
microscopic urine analyzers to make a complete automated dures are labor-intensive and time-consuming, and often they
urinalysis laboratory system. The minimum volume is 3 mL of are subject to technologist variability.
uncentrifuged urine for the combined instruments, and the sys- Visit www.fadavis.com for Video 2-2 (Hemacytometer).
tem can analyze up to 240 tests per hour. The samples are loaded
into the 10-position sample racks where the physical (color, clar-
ity, and specific gravity) and chemical tests (10 parameters) are Automation brings quality control, precision, faster TATs,
performed by the LabUMat2. Then the sample racks are trans- and standardization of results10 to a method that previously was
ferred across a connecting bridge to the UriSed 2 (bright-field uncontrolled. However, automated instruments have not elimi-
microscopy) or the UriSed 3 (both bright-field and phase nated completely the use of a manual hemacytometer count.
microscopy) for the urine microscopic analysis. Body fluids with low cell counts or malignant cells still require
a manual differential using a stained cytospin smear. The labo-
cobas 6500 Urine Analyzer ratory must define the limits for the automated instrument and
The cobas 6500 Urine Analyzer (Roche Diagnostics, Indi- establish the lower limits for cell counting to determine when a
anapolis, IN) is another fully automated urine system. Its mod- manual procedure must be performed.5 For example, the iQ 200
ular design integrates the cobas u 701 microscopy analyzer and is linear down to zero (0 to 10,000 cells/L). Linearity can be
the cobas u 601 fully automated urine chemistry analyzer into extended during method comparison. In addition, the laboratory
one platform. Cassettes with urine testing strips and sediment must follow manufacturers’ recommended procedures for special
cuvettes are loaded onto the instrument, and 2.8 mL of urine treatment required for the specific body fluid analyzed, intended
is required. Urine is pipetted on the chemical strip, where use, and reportable ranges.11
12 physical and chemical urine tests are performed using the Visit www.fadavis.com for Video 2-3 (Making a cytospin
smear).
cobas u 601 urine analyzer. After chemical testing, the sample
is resuspended before pipetting and automatically centrifuged Hematology analyzers that are used to perform body fluids
at 2000 rpm for 10 seconds to a monolayer of sediment. The cell counts include the ADVIA 2120i (Siemens), the Sysmex
cobas u 701 uses digital imaging to take 15 microscopic images XN-Series analyzers (Sysmex America, Inc.), and the DxH 900
of the sediment, and the images are displayed on the result (Beckman Coulter, Inc.).
screen. Particle recognition software determines the identifica-
tion of RBCs, WBCs, bacteria, epithelial cells, casts, crystals, ADVIA 2120i
yeast, sperm, and mucus. Automated result validation and
The ADVIA 2120i uses flow cytometry, light scattering, and
automated reflex testing are available.
absorbance to count RBCs and WBCs, as well as to perform a
UX-2000 Automated Urinalysis Analyzer WBC differential that includes percentages and absolute num-
bers of mononuclear cells and polymorphonuclear cells on
The UX-2000 (Sysmex Corporation, Kobe, Japan) is a fully
specimens with more than 20 WBC/µL. The WBC differential
automated integrated urine analyzer. It consists of a chemical
includes the numbers of neutrophils, lymphocytes, monocytes,
component for analyzing the physical and chemical part of urine
and eosinophils. A specimen of cerebrospinal fluid (CSF) is
as well as a flow cytometry component for microscopic exami-
pretreated with CSF reagent to fix and spherize the cells. The
nation of sediments contained in a single instrument.8 The sys-
prepared specimen remains stable for 4 minutes to 4 hours
tem requires 5 mL of urine. For the physical examination,
when stored at 18°C to 30°C. The specimen is aspirated into
refractometry, reflectivity measurement, and light scattering are
the instrument, and cells are differentiated and enumerated by
used to measure specific gravity, turbidity, and color. The chem-
three optical measurements. The signals are digitized and used
ical examination uses a test strip that is measured by the dual-
to construct the CSF cytogram. With this system, more cells are
wavelength reflectance methods.9 The microscopic examination
counted, achieving increased accuracy and precision. The au-
uses fluorescent-flow cytometry to measure RBCs, WBCs, hya-
tomated results for RBC, WBC, polymorphonuclear, mononu-
line casts, bacteria, and epithelial cells. Crystals, yeast, small
clear, and differential are available within 1 minute of sample
round cells, spermatozoa, and casts are detected and flagged for
aspiration. In addition, the ADVIA 2120i can provide a rapid
laboratory personnel to review because this analyzer cannot dif-
automated diagnostic test for fetal lung maturity by counting
ferentiate between those types of particles in urine sediment.8
Visit www.fadavis.com for Video 2-1 (Automated urine lamellar bodies in amniotic fluid. Lamellar bodies are counted
chemical strip test). in the platelet channel using high and low laser light scattering.
The analyzer is approved for counting cells in pleural fluids,
peritoneal fluids, and peritoneal dialysates.
Body Fluid Analysis Automation
Sysmex XN-Series Analyzers
Traditionally body fluid counts for RBCs and WBCs, as well as
WBC differentials, are performed manually using a hemocy- The Sysmex XN-Series analyzer is the newest-generation hema-
tometer and optical microscopy. Three types of hemocytometers tology analyzer that includes a dedicated body fluid mode that
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88 Part One | Basic Principles

is cleared for analysis of synovial fluid, pleural fluid, and peri- Module. The Body Fluid menu includes such fluids as CSF,
toneal fluid, and the XN-10 analyzer is also cleared for CSF synovial, pleural, peritoneal, peritoneal dialysate, peritoneal
specimens in the body fluid mode.10 Bronchoalveolar lavage lavage, pericardial, and general serous. Two dilutions of the
and amniotic fluid are not cleared for analysis on the XN-Series body fluid specimen are analyzed; one tube is diluted with Iris
analyzers. Table 2-3 lists the fluid types cleared for analysis on Diluent, and the other with the iQ Body Fluids Lysing Reagent.
the XN-Series analyzers. The body fluid mode has extended The instrument provides counts for total cells, nucleated cells,
cell counting to increase precision in specimens with small and RBCs. As with urine microscopy, results are either re-
numbers of cells. Body fluids can be analyzed without speci- viewed by a trained operator or auto-released to the LIS based
men preparation or pretreatment. on user-defined-parameters.
The body fluid mode on the XN-Series reports both a body Visit www.fadavis.com for Video 2-4 (Body fluid cell
fluid white blood cell count (WBC-BF) and a total nucleated count automated analyzer).
body fluid cell count (TC-BF) as well as a reportable two-part
automated differential that differentiates mononuclear and poly-
morphonuclear cells using flow cytometry technology.10 The XN
analyzer also identifies high-fluorescing body fluid cells (HF-BF), For additional resources please visit
such as mesothelial cells, synovial cells, and malignant/tumor www.fadavis.com
cells, or, rarely, large cell clusters. Digital imaging technology also
may be used for body fluid differentials. The CellaVision DI-60
software classifies five different nucleated cell types found in body References
fluid. This software allows users to add reference cells to a digital 1. Kenyon, SM, and Cradic, KW: Automated urinalysis in the
library, enables tagging and sharing of cell images electronically, clinical lab. Clinical Issues – Urinalysis. Web site: http://www.
and offers a program to improve staff competency.10 mlo-online.com. Published October 24, 2017. Accessed
February 19, 2020.
The XN analyzer uses impedance counting principles for
2. Halasey, S: Tech guide: Urinalysis. Clinical Laboratory Products
RBC-BF enumerations and flow cytometry for performing the (CLP). April 2019.
WBC-BF counts and the two-part differential. The XN series uses 3. Godfrey, D: Advancing urinalysis. Thoughtful automation aims
Lysercell WDF as an RBC lysing reagent and Fluorocell WDF to for a new level of standardization and workflow efficiency.
stain RNA and DNA in the nucleated cells. In addition, the dif- Clinical Lab Products (CLP). Web site: http://www.clpmag.com/
2019/05/advancing-urinalysis. Published May 22, 2019.
ferential scatterplots should be inspected visually to detect non-
Accessed July 31, 2019.
cellular particulate matter, such as bacteria, Cryptococcus, and 4. Clinical Diagnostic Products and Solutions/Urinalysis/iQ200
interference from large cells (macrophages and mesothelial cells). Series. Beckman Coulter, 2019. Web site: https://www.
beckmancoulter.com/en/products/urinalysis/iq200. Accessed
GloCyte Automated Cell Counter for CSF July 29, 2019.
5. Block, DR, and Lieske, JC: Automated urinalysis in the clinical
The GloCyte analyzer (Sysmex Corporation, Kobe, Japan) com- lab. Medical Laboratory Observer. Web site: www.mlo-online.
bines the principles of both technologies to accurately enumerate com. Published October 19, 2012. Accessed February 19, 2020.
cells present in CSF, even at low numbers.10 A fluorochrome- 6. Yuksel, H, Kilic, E, Ekinci, A, and Evliyaoglu, O: Comparison
labeled antibody stains the RBCs, and a dye specific to nucleic of fully automated urine sediment analyzers H800-FUS100
and Labumat-UriSed with manual microscopy. J Clin Lab
acids in WBCs is used to treat aliquots of the CSF specimen.10 Anal. 27:312–316, 2013.
Digital imaging is used to count the cells as they are illuminated 7. AuWi Pro Automated Urinalysis System Brochure. Siemens
with a semiconductor laser. The instrument automatically enu- Diagnostics, 2015. Web site: www.usa.siemens.com/
merates each cell type and displays the stained cells on a screen.10 diagnostics. Accessed July 31, 2019.
8. Wesarachkitti, B, Khejonnit, V, Pratumvinit, B, Reesukumal, K,
Beckman Coulter’s iQ200 Meepanya, S, Pattanavin, C, and Wongkrajang, C: Performance
evaluation and comparison of the fully automated urinalysis
The iQ200 (Iris Diagnostics – Beckman Coulter, Brea, CA) can analyzers UX-2000 and cobas 6500. Lab Med 47(2):124–133,
be used for body fluid analysis using the iQ200 Body Fluids 2016. https://doi.org/10.1093/labmed/lmw002. Accessed
August 1, 2019.
9. Laiweipithaya, S, Wongkraiang, P, Reesukumal, K, Bucha, C,
Meepanva, S, Pattanavin, C, Kheionnit, V, and Chuntarut, A:
Table 2–3 Body Fluids Analyzed on the Sysmex
UriSed 3 and UX-2000 automated urine sediment analyzers vs.
XN-Series10 manual microscopic method: A comparative performance
analysis. J Clin Lab Anal. 2018 Feb;32(2). Doi: 10.1002/jcla.
XN-10 XN-11 XN-20
22249. Epub 2017 May 2. Accessed February 19, 2020.
CSF Yes No Yes 10. Sysmex: The Value-Driven Laboratory. White Paper. Automat-
ing Body Fluid Analysis for Increased Efficiency. 2019. Web
Peritoneal fluid Yes Yes Yes site: www.sysmex.com/us. Accessed April 23, 2020.
Pleural fluid Yes Yes Yes 11. Clinical and Laboratory Standards Institute: Body Fluid
Analysis for Cellular Composition: Approved Guideline.
Pericardial fluid No No No CLSI Document H-56A. CLSI, Wayne, PA, 2006.
Synovial fluid Yes Yes Yes
Additional Information Sources
CSF, Cerebrospinal fluid. U.S. ARKRAY, Inc., Minneapolis, MN: www.arkrayusa.com
DIRUI, Changchun, China: en.dirui.com.cn
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Chapter 2 | Urine and Body Fluid Analysis Automation 89

77 Elektronika Kft, Budapest, Hungary: en.e77.hu/products/ Roche Diagnostics, Indianapolis, IN: www.roche.com/products
urine-analyzer Siemens Healthcare Diagnostics Inc., Deerfield, IL: www.usa.
Iris Diagnostics – Beckman Coulter, Brea, CA: www. siemens.com/diagnostics
beckmancoulter.com Sysmex Corporation, Kobe, Japan: www.sysmex.com/usa

Study Questions
1. The principle commonly used to measure the concentra- 7. Which automated urine particle counter combines urine
tion of a particular analyte in the chemical examination of flow cytometry with digital image analysis?
urine is: A. UN-2000
A. Reflectance photometry B. iRICELL
B. Digital imaging C. UF-1000i
C. Flow cytometry D. iQ 200
D. Auto particle recognition
8. Which of the following urine sediment particles cannot
2. In automated urinalysis, the specific gravity is measured by: be autovalidated but will be flagged and must be
A. Light transmittance reviewed by laboratory personnel?
B. Light scattering A. RBCs
C. Refractometry B. WBCs
D. Turbidity C. RTEs
D. Squamous epithelial cells
3. All of the following are true concerning fully automated
urine chemistry analyzers, except: 9. Which of the automated body fluid analyzers does not
A. They are designed for a high-volume urinalysis need to dilute or pretreat body fluids before analysis?
laboratory. A. ADVIA 2120i
B. The reagent strip is dipped into the well-mixed urine. B. XN Series
C. The urine tube moves through the instrument. C. iQ 200
D. A sample probe aspirates the urine. D. None of the above
4. The advantages of an automated urine microscopy 10. What is a disadvantage of counting body fluid cells
analyzer over manual microscopy includes: using an automated instrument versus a Neubauer
A. Cost-effective hemocytometer?
B. Centrifugation not required A. Less labor-intensive and time-consuming
C. Standardized results B. More precise
D. All of the above C. Unable to count low WBC numbers and
malignant cells
5. Which of the following is a complete urinalysis auto-
D. Able to perform a WBC differential
mated urinalysis system?
A. AUTION ELEVEN AE 4022
B. Clinitek Atlas
C. iQ200 Automated Urine Microscopy
D. Clinitek AUWi Pro System
6. What two technologies are used for urine sediment
analysis?
A. Light scattering and refractometry
B. Light scattering and flow cytometry
C. Flow cytometry and digital imaging
D. Digital imaging and refractometry
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90 Part One | Basic Principles

Case Studies and Clinical Situation

1. A medical laboratory scientist student is being trained on 2. A patient in the emergency department (ED) presented
the laboratory automated urinalysis system that performs with a high temperature, headache, and stiff neck. The
both the chemical and microscopic examination of urine. doctor suspected meningitis and performed a lumbar
The student poured 12 mL of urine into the urine tube puncture to obtain a cerebrospinal fluid (CSF) specimen
that was labeled correctly and placed it on the instru- for analysis in the laboratory.
ment. The reagent strip results indicated that there was a a. What instruments would be used to count the number
large amount of blood and leukocytes; however, the of white blood cells in the CSF?
microscopic results reported 0 to 2 RBC/hpf and 0 to
b. Which instrument would require that the specimen be
2 WBC/hpf.
pretreated?
a. Do the reagent strip and microscopic results correlate?
c. How long is the prepared CSF specimen stable?
b. What mistake in the procedure may have caused these
results?
c. What should be performed to ensure that the instru-
ment was functioning properly?
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CHAPTER 3
Introduction to Urinalysis
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
3-1 List three major organic and three major inorganic 3-8 Discuss the actions of bacteria on a urine specimen
chemical constituents of urine. that is unpreserved.
3-2 Describe a method for determining whether a fluid in 3-9 Briefly discuss five methods for preserving urine speci-
question is urine. mens, including the advantages and disadvantages
of each.
3-3 Recognize normal and abnormal daily urine volumes.
3-10 Instruct a patient in the correct procedure for collect-
3-4 Describe the characteristics of the specimen containers
ing the following specimens: random, first morning,
recommended for urine.
24-hour timed, catheterized, midstream clean-catch,
3-5 Describe the correct procedure for labeling urine suprapubic aspiration, three-glass collection, four-glass
specimens. collection, and pediatric. Identify a diagnostic use for
each collection technique.
3-6 State four possible reasons why a laboratory would
reject a urine specimen. 3-11 Describe the type of specimen needed for optimal re-
sults when a specific urinalysis procedure is requested.
3-7 List the changes that may take place in a urine speci-
men that remains at room temperature for more than
2 hours.

KEY TERMS
Albuminuria Midstream clean-catch specimen Random specimen
Anuria Nocturia Suprapubic aspiration
Catheterized specimen Oliguria Timed specimen
Chain of custody (COC) Polydipsia
First morning specimen Polyuria
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92 Part One | Basic Principles

History and Importance

Analyzing urine was actually the beginning of laboratory
medicine. References to the study of urine can be found in
the drawings of cavemen and in Egyptian hieroglyphics, such
as the Edwin Smith Surgical Papyrus. Pictures of early physi-
cians commonly showed them examining a bladder-shaped
flask of urine (Fig. 3-1). Often these physicians never saw the
patient, only the patient’s urine. Although these physicians
lacked the sophisticated testing mechanisms now available,
they were able to obtain diagnostic information from such
basic observations as color, turbidity, odor, volume, viscosity,
and even sweetness (by noting that certain specimens
attracted ants or tasted sweet). These same urine characteris-
tics are still noted by laboratory personnel. However, modern
urinalysis has expanded beyond the physical examination of
urine to include chemical analysis and microscopic examina-
tion of urinary sediment.
Many well-known names in the history of medicine are
associated with the study of urine, including Hippocrates, who,
in the 5th century BCE, wrote a book on “uroscopy.” During
the Middle Ages, physicians concentrated their efforts very
intensively on the art of uroscopy, receiving instruction in urine
examination as part of their training (Fig. 3-2). By 1140 CE, color
charts had been developed that described the significance of
20 different colors (Fig. 3-3). Chemical testing progressed from
“ant testing” and “taste testing” for glucose to Frederik Dekkers’s
discovery in 1694 of albuminuria by boiling urine.1

Figure 3–2 Instruction in urine examination. (Courtesy of National

Library of Medicine.)

The credibility of urinalysis became compromised when

charlatans without medical credentials began offering their pre-
dictions to the public for a healthy fee. These charlatans, called
“pisse prophets,” became the subject of a book published by
Thomas Bryant in 1627. The revelations in this book inspired
the passing of the first medical licensure laws in England—
another contribution of urinalysis to the field of medicine.
The invention of the microscope in the 17th century led
to the examination of urinary sediment and to the development
by Thomas Addis of methods for quantitating the microscopic
sediment. Richard Bright introduced the concept of urinalysis
as part of a doctor’s routine patient examination in 1827. By
the 1930s, however, the number and complexity of the tests
performed in a urinalysis had reached a point of impracticality,
and urinalysis began to disappear from routine examinations.
Fortunately, the development of modern testing techniques res-
cued routine urinalysis, which has remained an integral part
of the patient examination.
Two unique characteristics of a urine specimen account
for this continued popularity:
Figure 3–1 Physician examines urine flask. (Courtesy of National 1. A urine specimen is readily available and easily
Library of Medicine.) collected.
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Chapter 3 | Introduction to Urinalysis 93

for nearly half of the total dissolved solids in urine. Other or-
ganic substances include primarily creatinine and uric acid.
The major inorganic solid dissolved in urine is chloride, fol-
lowed by sodium and potassium. Small or trace amounts of
many additional inorganic chemicals are also present in urine
(Table 3-1). Dietary intake greatly influences the concentra-
tions of these inorganic compounds, making it difficult to
establish normal levels. Other substances found in urine in-
clude hormones, vitamins, and medications. Although not a
part of the original plasma filtrate, the urine also may contain
formed elements, such as cells, casts, crystals, mucus, and bac-
teria. Increased amounts of these formed elements are often
indicative of disease.
Sometimes it is necessary to determine whether a fluid
is urine. The best way to do so is to consider the components
of the specimen. Creatinine, urea, sodium, and chloride are
significantly higher in urine than in other body fluids. Protein
and glucose are not present in a normal urine specimen.

Urine Volume
Urine volume depends on the amount of water that the kidneys
Figure 3–3 A chart used for urine analysis. (Courtesy of National excrete. Water is a major body constituent; therefore, the
Library of Medicine.)
amount excreted is usually determined by the body’s state of
hydration. Factors that influence urine volume include fluid
2. Urine contains information, which can be obtained by intake, fluid loss from nonrenal sources, variations in the se-
inexpensive laboratory tests, about many of the body’s cretion of antidiuretic hormone (ADH), and need to excrete
major metabolic functions. increased amounts of dissolved solids, such as glucose or salts.
Taking these factors into consideration, although the normal
These characteristics fit in well with the current trends to- daily urine output is usually 1200 to 1500 mL, a range of 600
ward preventive medicine and lower medical costs. In fact, the to 2000 mL is considered normal.
Clinical and Laboratory Standards Institute (CLSI) defines
urinalysis as “the testing of urine with procedures commonly
performed in an expeditious, reliable, accurate, safe, and cost- Technical Tip 3-1. Should it be necessary to deter-
effective manner.” The reasons for performing urinalysis identi- mine whether a particular fluid is urine, the specimen
fied by CLSI include aiding in the diagnosis of disease, screening can be tested for its urea and creatinine content.
asymptomatic populations for undetected disorders, and moni- Because both these substances are present in much
toring the progress of disease and the effectiveness of therapy.2 higher concentrations in urine than in other body
fluids, a fluid that is high in urea and creatinine
content can be identified as urine.
Urine Formation
The kidneys continuously form urine as an ultrafiltrate of Oliguria, a decrease in urine output (less than 1 mL/kg/hr
plasma. Reabsorption of water and filtered substances essential in infants, less than 0.5 mL/kg/hr in children, and less than
to body function converts approximately 170,000 mL of 400 mL/day in adults), is seen commonly when the body en-
filtered plasma to the average daily urine output of 1200 mL, ters a state of dehydration as a result of excessive water loss
depending on fluid intake. (Refer to Chapter 4.) from vomiting, diarrhea, perspiration, or severe burns.
Oliguria leading to anuria, cessation of urine flow, may
Urine Composition result from any serious damage to the kidneys or from a
decrease in the flow of blood to the kidneys.
In general, urine consists of urea and other organic and inor- The kidneys excrete two to three times more urine during
ganic chemicals dissolved in water. Urine is normally 95% the day than during the night. An increase in the nocturnal ex-
water and 5% solutes, although considerable variations in the cretion of urine is termed nocturia. Polyuria, an increase in
concentrations of these solutes can occur due to the influence daily urine volume (greater than 2.5 L/day in adults and 2.5 to
of factors such as dietary intake, physical activity, body metab- 3 mL/kg/day in children), is often associated with diabetes mel-
olism, and endocrine functions. litus and diabetes insipidus; however, it may be induced artifi-
Urea, a metabolic waste product produced in the liver cially by diuretics, caffeine, or alcohol, all of which suppress the
from the breakdown of protein and amino acids, accounts secretion of ADH.
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94 Part One | Basic Principles

Table 3–1 Primary Components in Normal Urine3 patient with diabetes mellitus has a high specific gravity
because of the increased glucose content.
Component Comment Diabetes insipidus results from a decrease in the pro-
duction or function of ADH; thus, the water necessary for
Urea Primary organic component. Product of
adequate body hydration is not reabsorbed from the plasma
metabolism of protein and amino acids
filtrate. In this condition, the urine is truly dilute and has
Creatinine Product of metabolism of creatine by a low specific gravity. Fluid loss in both diseases is
muscles compensated by increased ingestion of water (polydipsia),
Uric acid Product of breakdown of nucleic acid in producing an even greater volume of urine. Polyuria
food and cells accompanied by increased fluid intake is often the first
Chloride Primary inorganic component. Found in symptom of either disease.
combination with sodium (table salt)
and many other inorganic substances
Sodium Primarily from salt, varies by intake
Specimen Collection
Potassium Combined with chloride and other salts As discussed in Chapter 1, urine is a biohazardous substance
Phosphate Combines with sodium to buffer the whose handling requires the observance of Standard Precau-
blood tions (SP). Gloves should be worn at all times when in contact
with the urine specimen.
Ammonium Regulates blood and tissue fluid acidity
The type of urine collection container, as well as specimen
Calcium Combines with chloride, sulfate, and preservation and storage, will affect the quality of urine test
phosphate results. Written criteria for the collection, preservation, han-
dling, storage, and labeling of urine must be available.

Diabetes mellitus and diabetes insipidus produce polyuria Containers

for different reasons, and analysis of the urine is an important Specimens must be collected in clean, dry, leakproof contain-
step in the differential diagnosis (Fig. 3-4). Diabetes mellitus ers. Disposable containers should be used because they elimi-
is caused by a defect either in the pancreatic production of in- nate the chance of contamination due to improper washing.
sulin or in the function of insulin, which results in an increased These disposable containers are available in a variety of sizes
concentration of body glucose. The kidneys do not reabsorb and shapes, including bags with adhesive for the collection of
excess glucose, necessitating excretion of increased amounts pediatric specimens and large containers for 24-hour speci-
of water to remove the dissolved glucose from the body. mens. Properly applied screw-top lids are less likely to leak
Although appearing to be dilute, a urine specimen from a than are snap-on lids.
Containers for routine urinalysis should have a wide
Polydipsia mouth to facilitate collections from female patients and a wide,
flat bottom to prevent overturning. They should be made of a
clear material to allow for determination of color and clarity.
Polyuria The recommended capacity of the container is 50 mL, which
allows 12 mL of specimen needed for microscopic analysis,
additional specimen for repeat analysis, and enough room for
Specific gravity the specimen to be mixed by swirling the container.
Individually packaged sterile containers with secure
closures should be used for microbiological studies of urine.
Decreased SG Increased SG Sterile containers are also suggested if more than 2 hours elapse
between specimen collection and analysis.2
Specially designed sterile containers are available that have
Decreased production Decreased insulin
or or a screw top containing an integrated transfer device. A BD
Function of ADH Decreased function Vacutainer Urine Transfer Straw is a nonsterile, plastic holder
of insulin device that contains a needle with a straw attachment that can
be used with the collection container to fill evacuation tubes.
Diabetes insipidus Increased glucose This device allows for the sterile transfer of urine to tubes con-
taining preservatives for microbiology testing and tubes with
conical bottoms for sediment analysis or round bottoms for
Diabetes mellitus automated reagent strip testing.4 Additional information and
pictures can be found at https://www.bd.com/en-us/offerings/
Figure 3–4 Differentiation between diabetes mellitus and diabetes capabilities/specimen-collection/urine-collection/bd-
insipidus. vacutainer-collection-and-transfer-products.
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Chapter 3 | Introduction to Urinalysis 95

Labels 7. Specimens that have not been preserved correctly during

a time delay
All specimens must be labeled immediately after collection
8. Specimens for urine culture collected in a nonsterile
with the patient’s last and first name and identification number;
container
the date and time of collection; and additional information
such as the patient’s age and location, the health-care provider’s 9. Inappropriate collection for the type of testing needed
name, and the preservative used, if any, as required by facility (for example, midstream clean-catch specimen for
protocol. Labels must be attached to the container, not to bacterial culture)
the lid, and should not become detached if the container is
refrigerated or frozen. Technical Tip 3-2. Never discard a specimen before
checking with a supervisor.
Requisition Form
A requisition form (manual or computerized) must accompany
specimens delivered to the laboratory. The information on the Laboratories should have a written policy detailing their
form must match the information on the specimen label. Addi- conditions for specimen rejection.
Visit www.fadavis.com for Video 3-1 (Preparing urine for
tional information on the form can include method of collection urinalysis).
or type of specimen, possible interfering medications, and the
patient’s clinical information. The time the specimen is received
in the laboratory should be recorded on the form. Specimen Handling
The fact that a urine specimen is so readily available and easily
Specimen Rejection collected often leads to laxity in the treatment of the specimen
after its collection. Changes in urine composition take place
The laboratory should reject specimens that are improperly not only in vivo but also in vitro, requiring correct handling
labeled and/or collected, and appropriate personnel should be procedures.
notified to collect a new specimen. Situations leading to
specimens that should be rejected can include: Specimen Integrity
1. Specimens in containers that are unlabeled or After collection, specimens should be delivered to the laboratory
improperly labeled promptly and tested within 2 hours. A specimen that cannot be
2. Labels and requisition forms that do not match delivered and tested within 2 hours should be refrigerated or have
3. Specimens contaminated with feces or toilet paper an appropriate chemical preservative added. Table 3-2 describes
the most significant changes that may occur in a specimen
4. Containers with contaminated exteriors
allowed to remain unpreserved at room temperature for longer
5. Specimens of insufficient quantity than 2 hours. Notice that most of the changes are related to the
6. Specimens that have been transported improperly presence and growth of bacteria.

Table 3–2 Changes in Unpreserved Urine

Analyte Change Cause
Color Modified/darkened Oxidation or reduction of metabolites
Clarity Decreased Bacterial growth and precipitation of amorphous material
Odor Increased ammonia smell Bacterial multiplication causing breakdown of urea to ammonia
pH Increased Breakdown of urea to ammonia by urease-producing bacteria/loss
of CO2
Glucose Decreased Glycolysis and bacterial use
Ketones Decreased Volatilization and bacterial metabolism
Bilirubin Decreased Exposure to light/photo oxidation to biliverdin
Urobilinogen Decreased Oxidation to urobilin
Nitrite Increased Multiplication of nitrate-reducing bacteria
Red and white blood Decreased Disintegration/lyse in dilute alkaline urine
cells and casts
Bacteria Increased Multiplication
Trichomonas Decreased Loss of motility, death
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96 Part One | Basic Principles

These variations are discussed again under the individual Technical Tip 3-4. When preserving specimens that
test procedures. At this point, it is important to realize that im- will be transported to another laboratory, confirm that
proper preservation can seriously affect the results of a routine the appropriate preservative is used.
urinalysis.

Specimen Preservation
The method of preservation used most routinely is refrigeration
Types of Specimens
at 2°C to 8°C, which decreases bacterial growth and metabo- To obtain a specimen that is representative of a patient’s meta-
lism. If the urine is to be cultured, it should be refrigerated dur- bolic state, regulation of certain aspects of specimen collection
ing transit and kept refrigerated until cultured, up to 24 hours.2 is often necessary. These special conditions may include time,
The specimen must return to room temperature before chemical length, and method of collection, as well as the patient’s dietary
testing by reagent strips. Refrigeration also can cause precipita- and medicinal intake. It is important to instruct patients when
tion of amorphous urate and phosphate crystals. they must follow special collection procedures. Frequently
When a specimen must be transported over a long dis- encountered specimens are listed in Table 3-4.
tance and refrigeration is impossible, chemical preservatives
may be added. Commercially prepared transport tubes with a Random Specimen
lyophilized preservative are available that allow for the trans-
This is the specimen received most commonly because of
port, testing, and storage of the urine specimens. The ideal
its ease of collection and convenience for the patient. The
preservative should be bactericidal, inhibit urease, and preserve
random specimen may be collected at any time, but the actual
formed elements in the sediment. At the same time, the
time of voiding should be recorded on the container.2 The ran-
preservative should not interfere with chemical tests. Unfortu-
dom specimen is useful for routine screening tests to detect
nately, as seen in Table 3-3, the ideal preservative does not
obvious abnormalities. However, it also may show erroneous
exist; therefore, a preservative that best suits the needs of the
results resulting from dietary intake or physical activity just be-
required analysis should be chosen.
fore collection. Then the patient will be requested to collect an
additional specimen under more controlled conditions.
Technical Tip 3-3. Specimens must be returned to First Morning Specimen
room temperature before chemical testing by reagent
strips because the enzyme reactions on the strips per-
Although it may require the patient to make an additional
form best at room temperature.
trip to the laboratory, this is the ideal screening specimen. It
is also essential for preventing false-negative pregnancy tests

Table 3–3 Urine Preservatives

Preservatives Advantages Disadvantages Additional Information
Refrigeration Does not interfere with Precipitates amorphous Prevents bacterial growth
chemical tests phosphates and urates for 24 hours2
Acids (boric acid, Prevents bacterial growth Interferes with analysis of Keeps pH at about 6.0
HCL, acetic acid, and metabolism drugs and hormones Can be used for transport
tartaric acid) of urine cultures
Formalin (formaldehyde) Excellent sediment Acts as a reducing agent, Rinse specimen container
preservative interfering with chemical with formalin to pre-
tests for glucose, blood, serve cells and casts
leukocyte esterase, and
copper reduction
Sodium fluoride Good preservative for drug Inhibits reagent strip tests
analyses for glucose, blood, and
leukocytes
Commercial preservative Convenient when refrigera- Check tablet composition to
tablets tion not possible determine possible effects
Have controlled concentration on desired tests
to minimize interference
Urine Collection Kits4 Contains collection cup,
(Becton, Dickinson, transfer straw, culture and
Rutherford, NJ) sensitivity (C&S) preser-
vative tube, or UA tube
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Chapter 3 | Introduction to Urinalysis 97

Table 3–3 Urine Preservatives—cont’d

Preservatives Advantages Disadvantages Additional Information
Light gray and gray Specimen stable at room Do not use if urine is below Preservative is boric acid,
C&S tube temperature (RT) for minimum fill line sodium borate, and
48 hours; prevents sodium formate
bacterial growth and Keeps pH at about 6.0
metabolism
Yellow UA Plus tube Use on automated Must refrigerate within Round or conical bottom,
instruments 2 hours no preservative
Cherry red/yellow Specimen stable for Must be filled to minimum Preservative is sodium pro-
Preservative Plus tube 72 hours at RT; fill line pionate, ethyl paraben,
instrument-compatible Bilirubin and urobilinogen and chlorhexidine
may be decreased if speci- Round or conical bottoms
men is exposed to light
and left at RT

Table 3–4 Types of Urine Specimens HISTORICAL NOTE

Type of Specimen Purpose
Glucose Tolerance Specimens
Random Routine screening
First morning Routine screening Glucose tolerance specimens were sometimes collected to
correspond with the blood specimens drawn during an
Pregnancy tests
oral glucose tolerance test (OGTT). The number of spec-
Orthostatic protein imens varied with the length of the test. GTTs included
24-hour (or timed) Quantitative chemical tests fasting, half-hour, 1-hour, 2-hour, and 3-hour specimens
Catheterized Bacterial culture and possibly 4-hour, 5-hour, and 6-hour specimens. The
urine was tested for glucose and ketones, and the results
Midstream clean- Routine screening
were reported along with the blood test results as an aid
catch
to interpreting the patient’s ability to metabolize a meas-
Bacterial culture ured amount of glucose. The results are correlated with
Suprapubic Bladder urine for bacterial culture the renal threshold for glucose. Collection of these speci-
aspiration mens was a facility option.5
Cytology
Three-glass Prostatic infection
collection
such as catecholamines, 17-hydroxysteroids, and electrolytes
Four-glass Prostatic infection
in which the lowest concentration is in the early morning
collection
and the highest concentration occurs in the afternoon.2
When the concentration of the substance to be measured
changes with diurnal variations and with daily activities,
such as exercise, meals, and body metabolism, 24-hour col-
and for evaluating orthostatic proteinuria (see Chapter 6). lection is required. If the concentration of a particular sub-
The first morning specimen is a concentrated specimen, stance remains constant, the specimen may be collected over
thereby assuring detection of chemicals and formed elements a shorter period. Care must be taken, however, to keep the
that may not be present in a dilute random specimen. The patient adequately hydrated during short collection periods.
patient should be instructed to collect the specimen imme- Patients must be instructed on the procedure for collecting
diately on arising and to deliver it to the laboratory within a timed specimen.
2 hours or keep it refrigerated. To obtain an accurate timed specimen, the patient must
begin and end the collection period with an empty bladder. The
24-Hour (or Timed) Specimen concentration of a substance in a particular period must be
Measuring the exact amount of a urine chemical is often nec- calculated from the urine volume produced during that time.
essary instead of just reporting its presence or absence. A care- On its arrival in the laboratory, a 24-hour specimen must
fully timed specimen must be used to produce accurate be mixed thoroughly and the volume accurately measured and
quantitative results. Many solutes exhibit diurnal variations recorded. If only an aliquot is needed for testing, the amount
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98 Part One | Basic Principles

saved must be adequate to permit repeat or additional testing. Technical Tip 3-5. Adding urine formed before the start
If a specimen is collected in two containers, the contents of the of the collection period will falsely elevate the results,
containers should be combined and thoroughly mixed before and failure to include the urine produced at the end of
aliquoting. Consideration also must be given to the preservation the collection period will falsely decrease the results.
of specimens collected over extended periods. All specimens
should be refrigerated or kept on ice during the collection pe-
riod and may require addition of a chemical preservative. The
preservative chosen must be nontoxic to the patient and should
not interfere with the tests to be performed. Appropriate col- Catheterized Specimen
lection information is included with test procedures and should This specimen is collected under sterile conditions by passing
be read before issuing a container and instructions to the pa- a hollow tube (catheter) through the urethra into the bladder.
tient. Box 3-1 lists the most common errors associated with Urine passes from the bladder through the catheter into a plas-
timed urine collections. tic bag, where it accumulates. Urine specimens then can be
collected from this urine bag. The test requested most com-
monly on a catheterized specimen is a bacterial culture.
Box 3–1 Common Errors Associated With Timed
Urine Collections
Midstream Clean-Catch Specimen
• Loss of urine specimen As an alternative to the catheterized specimen, the mid-
stream clean-catch specimen provides a safer, less trau-
• Inclusion of two first morning specimens
matic method for obtaining urine for bacterial culture and
• Inaccurate measurement of total urine volume routine urinalysis. It provides a specimen that is less con-
• Inadequate urine preservation taminated by epithelial cells and bacteria and therefore is
• Transcription error more representative of the actual urine than the routinely
voided specimen. Patients must be provided with appropri-
ate cleansing materials, a sterile container, and instructions
for cleansing and voiding. Strong bacterial agents, such as
hexachlorophene or povidone-iodine, should not be used as
PROCEDURE 3-1 cleansing agents. Mild antiseptic towelettes are recom-
mended. Some urine collection transfer kits contain Castile
24-Hour (Timed) Urine Specimen Collection Soap Towelettes. Procedures 3-2 and 3-3 provide instruction
Procedure details for male and female patients.
Equipment Suprapubic Aspiration
Requisition form
Occasionally urine may be collected by external introduction of
24-hour urine specimen container with lid a needle through the abdomen into the bladder. Because the blad-
Label der is sterile under normal conditions, suprapubic aspiration
Container with ice, if required provides a specimen for bacterial culture that is completely free
of extraneous contamination, particularly in infants or children.
Preservative, if required
The specimen also can be used for cytological examination.
Procedure
1. Provide the patient with written instructions, and
Prostatitis Specimen
explain the collection procedure. Several methods are available to detect the presence of
2. Provide the patient with the collection container and prostatitis.
preservative, if required.
Three-Glass Collection
3. Day 1: 7 a.m.: Patient voids and discards specimen;
collects all urine for the next 24 hours. Before collection, the area is cleansed using the male midstream
clean-catch procedure. Then, instead of discarding the first
4. Day 2: 7 a.m.: Patient voids and adds this urine to
urine passed, it is collected in a sterile container. Next, the mid-
the previously collected urine.
stream portion is collected in another sterile container. Then
5. Specimen is transported to the laboratory, where the the prostate is massaged so that prostate fluid will be passed
entire 24-hour specimen is thoroughly mixed and the with the remaining urine into a third sterile container. Quan-
volume is measured and recorded. titative cultures are performed on all specimens, and the first
6. The required amount of urine (approximately 50 mL) and third specimens are examined microscopically. In prostatic
is aliquoted for testing. infection, the third specimen will have a white blood cell/high-
7. The remaining specimen is discarded. power field count and a bacterial count 10 times that of the
first specimen. Macrophages containing lipids also may be
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Chapter 3 | Introduction to Urinalysis 99

PROCEDURE 3-2
Clean-Catch Specimen Collection: Female 4. Cleanse from front to back on either side of the urinary
Cleansing Procedure2 opening with an antiseptic towelette, using a clean one
Equipment for each side.
Requisition form 5. Hold the skin folds apart and begin to void into the toilet.
Sterile urine container with label 6. Bring the urine container into the middle stream of
urine, and collect an adequate amount of urine. Do not
Sterile antiseptic towelettes touch the inside of the container or allow the container
Written instructions for cleansing and voiding to touch the genital area.
Procedure 7. Finish voiding into the toilet.
Instruct the patient to: 8. Cover the specimen with the lid. Touch only the out-
1. Wash her hands. side of the lid and container.
2. Remove the lid from the sterile container without 9. Confirm the container is labeled correctly with the
touching the inside of the container or lid. patient’s first and last name and time of collection, and
3. Separate the skin folds (labia). place it in the specified area, or follow facility policy.

PROCEDURE 3-3
Clean-Catch Specimen Collection: Male Cleansing 3. Cleanse the tip of the penis with antiseptic towelette
Procedure2 and let it dry. Retract the foreskin if uncircumcised.
Equipment 4. Void into the toilet. Hold back the foreskin if necessary.
Requisition form 5. Bring the sterile urine container into the middle stream
Sterile urine container with label of urine, and collect an adequate amount of urine. Do
not touch the inside of the container or allow the con-
Sterile antiseptic towelettes tainer to touch the genital area.
Written instructions for cleansing and voiding 6. Finish voiding into the toilet.
Procedure 7. Cover the specimen with the lid. Touch only the out-
Instruct the patient to: side of the lid and container.
1. Wash his hands. 8. Confirm the container is labeled correctly with the pa-
2. Remove the lid from the sterile container without tient’s first and last name and time of collection, and
touching the inside of the container or lid. place it in the specified area, or follow facility policy.

present. The second specimen is used as a control for bladder significant bacteriuria in the postmassage specimen of greater
and kidney infection. If it is positive, the results from the third than 10 times the premassage count.7
specimen are invalid because infected urine has contaminated
the specimen.6 Stamey-Meares Test for Prostatitis
The traditional four-glass urine collection technique, as de-
scribed by Meares and Stamey,7,8 includes examination of four
Technical Tip 3-6. When both a routine urinalysis urine specimens as follows:
and a culture are requested on a catheterized or
midstream collection, the culture should be per-
• The first urine specimen is voided bladder (VB1), which
formed first to prevent contamination of the speci-
is the first 10 mL of urine and represents the urethral
men. A collection transfer kit also can be used.
specimen.
• Then the patient voids another 100 to 150 mL of urine.
• The second specimen, voided bladder 2 (VB2), is col-
Pre- and Postmassage Test lected, which is another 10 mL of urine and represents
In the pre- and postmassage test (PPMT), a clean-catch mid- the bladder specimen.
stream urine specimen is collected. A second urine sample is • The third specimen is the expressed prostatic specimen
collected after the prostate is massaged. A positive result is (EPS),which is the fluid collected during prostatic massage.
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100 Part One | Basic Principles

• The fourth specimen, voided bladder 3 (VB3), consists of the specific individual submitting the specimen. The chain
of the first 10 mL of urine collected after EPS; it contains of custody (COC) is the process that provides this documen-
any EPS trapped in the prostatic urethra. tation of proper specimen identification from the time of col-
• All four specimens are sent for culture. lection to the receipt of laboratory results. The COC is a
standardized form that must document and accompany every
• The three urine specimens are centrifuged, and the
step of drug testing, from collector to courier to laboratory to
sediment is examined for white blood cells/aggregates,
medical review officer to employer.
macrophages, oval fat bodies, bacteria, and fungal hypha.
For urine specimens to withstand legal scrutiny, it is
Urethral infection or inflammation is tested for by the necessary to prove that no tampering of the specimen oc-
VB1, and the VB2 tests for urinary bladder infection. The pro- curred, such as substitution, adulteration, or dilution. All
static secretions are cultured and examined for white blood personnel handling the specimen must be noted. The spec-
cells. Having more than 10 to 20 white blood cells per high- imen must be handled securely, with a guarantee that no
power field is considered abnormal.7 unauthorized access to the specimen was possible. Proper
identification of the individual whose information is indi-
Pediatric Specimens cated on the label is required. Either photo identification or
Collection of pediatric specimens can present a challenge. Soft, positive identification by an employer representative with
clear plastic bags are available for collecting routine specimens; photo ID is acceptable.
these bags have hypoallergenic skin adhesive to attach to the Urine specimen collections may be “witnessed” or “unwit-
cleaned genital area of both boys and girls. Sterile specimens nessed.” The decision to obtain a witnessed collection is indi-
may be obtained by catheterization or by suprapubic aspira- cated when it is suspected that the donor may alter or
tion. Care must be taken not to touch the inside of the bag substitute the specimen or when it is the policy of the client
when applying it to the patient’s skin. ordering the test. If a witnessed specimen collection is ordered,
For routine specimen analysis, ensure the area is free of a same-gender collector will observe the collection of 30 to
contamination. Attach the bag firmly over the cleaned genital 45 mL of urine. Witnessed and unwitnessed collections should
area, avoiding the anus. A diaper is placed over the collection be handed to the collector immediately.
bag. When enough specimen has been collected, remove the The urine temperature must be taken within 4 minutes
bag and label it, or pour the specimen into a container and from the time of collection to confirm the specimen has not
label the container following facility policy. been adulterated. The temperature should read within the
For microbiology specimens, clean the area with soap and range of 32.5°C to 37.7°C. If the specimen temperature is not
water and sterilely dry the area, removing any residual soap within range, the temperature should be recorded and the
residue. Firmly apply a sterile bag. Sterilely transfer the collected supervisor or employer contacted immediately. Urine temper-
specimen into a sterile container and label the container.2 atures outside of the recommended range may indicate speci-
men contamination. Re-collection of a second specimen as
soon as possible will be necessary. The urine color is also
Technical Tip 3-7. Check the applied bags approxi- inspected to identify any signs of contaminants. The pH and
mately every 15 minutes until the needed amount of specific gravity of the specimen also may be tested. A urine pH
sample has been collected. of greater than 9 suggests adulteration of the urine specimen
and requires that the specimen be re-collected if clinically
necessary. A specific gravity of less than 1.005 could indicate
Drug Specimen Collection dilution of the urine specimen and requires re-collection. The
Urine specimen collection is the most vulnerable part of a specimen is labeled, packaged, and transported following
drug-testing program. Correct collection procedures and doc- laboratory-specific instructions. A typical urine drug specimen
umentation are necessary to ensure that the results are those collection is described in Procedure 3-4.

PROCEDURE 3-4
Urine Drug Specimen Collection Procedure Procedure
Equipment 1. The collector sanitizes his or her hands and wears
Requisition form gloves.
Gloves 2. The collector adds bluing agent (dye) to the toilet water
reservoir to prevent an adulterated specimen.
Bluing agent (dye)
3. The collector eliminates any source of water other than
Specimen container
toilet by taping the toilet lid and faucet handles.
Label
4. The donor provides photo identification or positive
COC form identification from the employer’s representative.
Temperature strip
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Chapter 3 | Introduction to Urinalysis 101

PROCEDURE 3-4—cont’d
5. The collector completes step 1 of the COC form and 12. The specimen must remain in the sight of the donor
has the donor sign it. and collector at all times.
6. The donor leaves his or her coat, briefcase, and/or 13. With the donor watching, the collector peels off the
purse outside the collection area to avoid the possibil- specimen identification strips from the COC form
ity of concealed substances contaminating the urine. (COC step 3) and puts them on the capped bottle,
7. The donor sanitizes his or her hands and receives a covering both sides of the cap.
specimen cup. 14. The donor initials the specimen bottle seals.
8. The collector remains in the restroom but outside the 15. The collector writes the date and time on the
stall, listening for unauthorized water use, unless a bottle seals.
witnessed collection is requested. 16. The donor completes step 4 on the COC form.
9. The donor hands the specimen cup to the collector. 17. The collector completes step 5 on the COC form.
The transfer is documented.
18. Each time the specimen is handled, transferred, or
10. The collector checks the urine for abnormal color and placed in storage, every individual must be identified
for the required amount (30 to 45 mL). and the date and purpose of the change recorded.
11. The collector checks that the temperature strip on the 19. The collector follows laboratory-specific instructions
specimen cup reads 32.5°C to 37.7°C. The collector for packaging the specimen bottles and laboratory
records the in-range temperature on the COC form copies of the COC form.
(COC step 2). If the specimen temperature is out of
20. The collector distributes the COC copies to appropri-
range or the specimen is suspected of having been
ate personnel.
diluted or adulterated, a new specimen must be
collected and a supervisor notified.

Product Circular, 2014. Becton, Dickinson and Company,

For additional resources please visit 1 Becton Drive, Franklin Lakes, NJ 07417. Web site: www.bd.
www.fadavis.com com/vacutainer/referencematerial. ©2014 BD 8606531 Mfg
by BD, USA 04/2014. Accessed April 25, 2019.
5. Baer, DM: Glucose tolerance test: Tips from the clinical experts.
Medical Laboratory Observer, Sept. 2003.
References 6. Rous, SN: The Prostate Book. Consumers Union, Mt. Vernon,
NY, 1988.
1. Herman, JR: Urology: A View Through the Retrospectroscope.
7. Stevermer, JJ, and Easley, SK: Treatment of prostatitis. Am Fam
Harper & Row, Hagerstown, MD, 1973.
Physician 61(10):2015–3022, 2000.
2. Clinical and Laboratory Standards Institute, Urinalysis:
8. Kraemer, Samantha, MD: Chronic Bacterial Prostatitis Workup.
Approved Guideline, ed 3, CLSI Document GP16-A3: Wayne,
Medscape. Web site: https://emedicine.medscape.com/article/
PA, 2009, CLSI.
458391-workup. Published January 15, 2019. Accessed
3. Torora, GJ, and Anagnostakos, NP: Principles of Anatomy and
April 22, 2019.
Physiology, ed 6, Harper & Row, New York, 1990, p. 51.
4. Becton, Dickinson and Company: BD Vacutainer Urine Products
for Collection, Storage, and Transport of Urine Specimens.

Study Questions
1. The primary inorganic substance found in urine is: 2. An unidentified fluid is received in the laboratory with a
A. Sodium request to determine whether the fluid is urine or another
body fluid. Using routine laboratory tests, which substances
B. Phosphate
would determine that the fluid is most probably urine?
C. Chloride
A. Glucose and ketones
D. Calcium
B. Urea and creatinine
C. Uric acid and amino acids
D. Protein and amino acids
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102 Part One | Basic Principles

3. The average daily output of urine is: 10. For general screening, the specimen collected most
A. 200 mL frequently is a:
B. 500 mL A. Random one
C. 1200 mL B. First morning
D. 2500 mL C. Midstream clean-catch
D. Timed
4. A patient presenting with polyuria, nocturia, polydipsia,
and a low urine specific gravity is exhibiting symptoms of: 11. The primary advantage of a first morning specimen over
A. Diabetes insipidus a random specimen is that it:
B. Diabetes mellitus A. Is less contaminated
C. Urinary tract infection B. Is more concentrated
D. Uremia C. Is less concentrated
D. Has a higher volume
5. A patient with oliguria might progress to having:
A. Nocturia 12. If a routine urinalysis and a culture are requested on a
catheterized specimen, then:
B. Polyuria
A. Two separate containers must be collected
C. Polydipsia
B. The routine urinalysis is performed first
D. Anuria
C. The patient must be recatheterized
6. All of the following are characteristics of recommended
D. The culture is performed first
urine containers except:
A. A flat bottom 13. If a patient fails to discard the first specimen when
collecting a timed specimen, then the:
B. A capacity of 50 mL
A. Specimen must be re-collected
C. A snap-on lid
B. Results will be falsely elevated
D. Are disposable
C. Results will be falsely decreased
7. Labels for urine containers are:
D. Both A and B
A. Attached to the container
14. The primary cause of unsatisfactory results in an
B. Attached to the lid
unpreserved routine specimen not tested for 8 hours is:
C. Placed on the container before collection
A. Bacterial growth
D. Not detachable
B. Glycolysis
8. A urine specimen may be rejected by the laboratory for all C. Decreased pH
of the following reasons except the fact that the:
D. Chemical oxidation
A. Requisition form states the specimen is catheterized
15. Prolonged exposure of a preserved urine specimen to
B. Specimen contains toilet paper
light will cause:
C. Label and requisition form do not match
A. Decreased glucose
D. Outside of the container has contamination from fecal
B. Increased cells and casts
material
C. Decreased bilirubin
9. A cloudy specimen received in the laboratory may have
D. Increased bacteria
been preserved using:
A. Boric acid 16. Which of the following would be least affected in a
specimen that has remained unpreserved at room
B. Chloroform
temperature for more than 2 hours?
C. Refrigeration
A. Urobilinogen
D. Formalin
B. Ketones
C. Protein
D. Nitrite
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Chapter 3 | Introduction to Urinalysis 103

17. Bacterial growth in an unpreserved specimen will: 19. Which of the following would not be given to a patient
A. Decrease clarity before the collection of a midstream clean-catch
specimen?
B. Increase bilirubin
A. Sterile container
C. Decrease pH
B. Iodine cleanser
D. Increase glucose
C. Antiseptic towelette
18. The most sterile specimen collected is a:
D. Instructions
A. Catheterized
20. Urine specimen collection for drug testing requires the
B. Midstream clean-catch
collector to do all of the following except:
C. Three-glass
A. Inspect the specimen color
D. Suprapubic aspiration
B. Perform reagent strip testing
C. Read the specimen temperature
D. Fill out a chain-of-custody form

Case Studies and Clinical Situations

1. A patient brings a first morning specimen to the labora- 4. The laboratory receives a urine preservative tube for cul-
tory at 1:00 p.m. ture containing a volume of specimen that is considerably
a. How could this affect the urinalysis results? below the minimum fill line.
b. If the patient is a known diabetic, what results would a. Could this affect the culture?
be most suspect? b. Why?
c. What is the best course of action? 5. A worker suspects that he or she will be requested to col-
d. What could the patient have said that would have lect an unwitnessed urine specimen for drug analysis. He
made the specimen satisfactory for testing? or she carries a substitute specimen in his or her pocket
for 2 days before being told to collect the specimen.
2. A patient collecting a midstream clean-catch specimen
Shortly after the worker delivers the specimen to the col-
voids immediately into the container.
lector, he or she is instructed to collect another specimen.
a. How could this affect the clarity of the specimen?
a. What test was performed on the specimen to deter-
b. How could this affect the microscopic examination? mine possible specimen manipulation?
3. A patient brings a 24-hour-timed specimen to the labora- b. How was the specimen in this situation affected?
tory and reports that he or she forgot to collect a speci- c. If a specimen for drug analysis tests positive, state a
men voided during the night. possible defense related to specimen collection and
a. How will this affect the results of a quantitative test for handling that an attorney might employ.
creatinine? d. How can this defense be avoided?
b. What should the patient be told to do?
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7582_Ch04_105-123 24/06/20 5:38 PM Page 105

CHAPTER 4
Renal Function
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
4-1 Identify the components of the nephron, kidney, and 4-10 Given hypothetical laboratory data, calculate a creati-
excretory system. nine clearance and determine whether the result is
normal.
4-2 Trace the flow of blood through the nephron, and state
the physiological functions that occur. 4-11 Discuss the clinical significance of the glomerular
filtration rate tests.
4-3 Describe the process of glomerular ultrafiltration.
4-12 Describe and contrast the Modification of Diet in Renal
4-4 Discuss the functions and regulation of the
Disease (MDRD), cystatin C, and beta2-microglobulin
renin–angiotensin–aldosterone system (RAAS).
tests for performing estimated glomerular filtration
4-5 Differentiate between active and passive transport in rates (eGFR).
relation to renal concentration.
4-13 Define osmolarity, and discuss its relationship to urine
4-6 Explain the function of antidiuretic hormone in the concentration.
concentration of urine.
4-14 Describe the basic principles of freezing-point
4-7 Describe the role of tubular secretion in maintaining osmometers.
acid–base balance.
4-15 Given hypothetic laboratory data, calculate a
4-8 Identify the laboratory procedures used to evaluate free-water clearance and interpret the result.
glomerular filtration, tubular reabsorption and secre-
4-16 Given hypothetic laboratory data, calculate a PAH
tion, and renal blood flow.
clearance and relate this result to renal blood flow.
4-9 Describe the creatinine clearance test.
4-17 Describe the relationship of urinary ammonia and
titratable acidity to the production of an acidic urine.

KEY TERMS
Active transport Creatinine clearance Glomerular filtration rate (GFR)
Afferent arteriole Cystatin C Glomerulus
Aldosterone Density Juxtaglomerular apparatus
Antidiuretic hormone (ADH) Distal convoluted tubule Loops of Henle
Beta2-microglobulin (B2M) Endogenous procedure Macula densa
Clearance tests Efferent arteriole Maximal reabsorptive capacity (Tm)
Collecting duct Exogenous procedure Metabolic acidosis
Concentration tests Fenestrated endothelium Nephron
Countercurrent mechanism Free water clearance Osmolality
Creatinine Glomerular filtration barrier Osmolar clearance
Continued
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106 Part One | Basic Principles

K E Y T E R M S —cont’d
Osmolarity Renal threshold Titratable acidity
Passive transport Renal tubular acidosis Tubular reabsorption
Peritubular capillaries Renin Tubular secretion
Podocytes Renin–angiotensin–aldosterone Vasa recta
Proximal convoluted tubule system (RAAS) Vasopressin
Renal plasma flow Shield of negativity

Introduction and flows slowly through the cortex and medulla of the kidney
close to the tubules. The peritubular capillaries surround the
This chapter reviews nephron anatomy and physiology and proximal and distal convoluted tubules, providing for the
discusses their relationship to urinalysis and renal function immediate reabsorption of essential substances from the fluid
testing. A section on laboratory assessment of renal function is in the proximal convoluted tubule and final adjustment of
included. the urinary composition in the distal convoluted tubule. The
vasa recta are located adjacent to the ascending and descending
loops of Henle in juxtamedullary nephrons. In this area, the
Renal Physiology major exchanges of water and salts take place between the
Each kidney contains approximately 1 to 1.5 million functional blood and the medullary interstitium. This exchange main-
units called nephrons. As shown in Figure 4-1, the human tains the osmotic gradient (salt concentration) in the medulla,
kidney contains two types of nephrons. Cortical nephrons, which is necessary for renal concentration. Box 4-2 outlines
which make up approximately 85% of nephrons, are situated the urinary filtrate flow.
primarily in the cortex of the kidney. They are responsible Based on an average body size of 1.73 m2 of surface, the
primarily for removal of waste products and reabsorption of total renal blood flow is approximately 1200 mL/min, and the
nutrients. Juxtamedullary nephrons have longer loops of Henle total renal plasma flow ranges from 600 to 700 mL/min. Nor-
that extend deep into the medulla of the kidney. Their primary mal values for renal blood flow and renal function tests depend
function is concentration of the urine. on body size. When dealing with body sizes that vary greatly
The ability of the kidneys to clear waste products selec- from the average 1.73 m2 of body surface, a correction must
tively from the blood and simultaneously to maintain the be calculated to determine whether the observed measure-
body’s essential water and electrolyte balances is controlled in ments represent normal function. This calculation is covered
the nephron by the following renal functions: in the discussion on tests for glomerular filtration rate (GFR)
• Renal blood flow later in this chapter. Variations in normal values have been
published for different age groups and should be considered
• Glomerular filtration
when evaluating renal function studies.
• Tubular reabsorption
• Tubular secretion Glomerular Filtration
The physiology, laboratory testing, and associated pathol- The glomerulus consists of a coil of approximately eight capil-
ogy of these four functions are discussed in this chapter. lary lobes, the walls of which are referred to as the glomerular
filtration barrier. The glomerulus is located within Bowman
Renal Blood Flow capsule, which forms the beginning of the renal tubule.
The renal artery supplies blood to the kidney. The human kid- Although the glomerulus serves as a nonselective filter of
neys receive approximately 25% of the blood pumped through plasma substances with molecular weights less than 70,000,
the heart at all times. Blood enters the capillaries of the several factors influence the actual filtration process. These
nephron through the afferent arteriole. It then flows through include the cellular structure of the capillary walls and Bowman
the glomerulus and into the efferent arteriole. The varying capsule, hydrostatic pressure and oncotic pressure, and the
sizes of these arterioles help create the hydrostatic pressure dif- feedback mechanisms of the renin–angiotensin–aldosterone
ferential that is important for glomerular filtration and to main- system (RAAS).
tain consistency of glomerular capillary pressure and renal
Cellular Structure of the Glomerulus
blood flow within the glomerulus. Notice the smaller size of
the efferent arteriole in Figure 4-2. This increases the glomeru- Plasma filtrate must pass through three glomerular filtration
lar capillary pressure. Renal blood flow is outlined in Box 4-1. barrier cellular layers: the capillary wall membrane, the base-
Before returning to the renal vein, blood from the efferent ment membrane (basal lamina), and the visceral epithelium of
arteriole enters the peritubular capillaries and the vasa recta the Bowman capsule. The endothelial cells of the capillary wall
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Chapter 4 | Renal Function 107

Glomerulus

Renal
cortex

Cortical
Renal nephron
tubule

Renal
Papilla of medulla
pyramid
Loop of
Henle Juxtamedullary
nephron
Collecting
Renal duct
artery

Calyx

Renal
vein

Cortex

Renal pelvis

Ureter

Figure 4–1 The relationship of the nephron to the Urinary bladder

kidney and excretory system. (From Scanlon, VC, and
Sanders, T: Essentials of Anatomy and Physiology,
ed 3. FA Davis, Philadelphia, PA, 1999, p 405, with
permission.) Urethra

differ from those in other capillaries by containing pores and important because it is the place where albumin (the primary
are referred to as fenestrated endothelium. The pores increase protein associated with renal disease) has a negative charge and
capillary permeability but do not allow the passage of large is repelled (Figs. 4-3B and 4-3C).
molecules and blood cells. Further restriction of large mole-
cules occurs as the filtrate passes through the basement mem-
Glomerular Pressure
brane and the thin membranes covering the filtration slits
formed by the intertwining foot processes of the podocytes of As mentioned previously, the presence of hydrostatic pressure,
the inner layer of the Bowman capsule (Fig. 4-3A). resulting from the smaller size of the efferent arteriole and the
In addition to the structure of the glomerular filtration bar- glomerular capillaries, enhances filtration. This pressure is nec-
rier that prohibits the filtration of large molecules, the barrier essary to overcome the opposition of pressures from the fluid
contains a shield of negativity that repels molecules with a within the Bowman capsule and the oncotic pressure of unfil-
negative charge even though they are small enough to pass tered plasma proteins in the glomerular capillaries. By increas-
through the three layers of the barrier. The shield is very ing or decreasing the size of the afferent and efferent arterioles,
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108 Part One | Basic Principles

Juxtaglomerular
Afferent
apparatus
arteriole
Efferent
arteriole

Bowman
capsule

Glomerulus

Distal
Cortex
convoluted
Proximal
tubule
convoluted
tubule Collecting
duct
Peritubular
capillaries
Vasa recta
Thick descending Medulla
loop of Henle
Thick ascending
Vasa recta loop of Henle

Thin descending Thin ascending

loop of Henle loop of Henle

Figure 4–2 The nephron and its component

parts.

Box 4–1 Renal Blood Flow Box 4–2 Urinary Filtrate Flow
1. Renal artery 1. Bowman capsule
2. Afferent arteriole 2. Proximal convoluted tubule
3. Glomerulus 3. Descending loop of Henle
4. Efferent arteriole 4. Ascending loop of Henle
5. Peritubular capillaries 5. Distal convoluted tubule
6. Vasa recta 6. Collecting duct
7. Renal vein 7. Renal calyces
8. Ureter
9. Bladder
an autoregulatory mechanism within the juxtaglomerular ap- 10. Urethra
paratus maintains the glomerular blood pressure at a relatively
constant rate, regardless of fluctuations in systemic blood pres-
sure. Dilation of the afferent arterioles and constriction of the
efferent arterioles when blood pressure drops prevent a marked
decrease in blood flowing through the kidney, thus preventing Renin–Angiotensin–Aldosterone System
an increase in the blood level of toxic waste products. Likewise, The RAAS regulates the flow of blood to and within the glomeru-
an increase in blood pressure results in constriction of the lus. The system responds to changes in blood pressure and
afferent arterioles to prevent overfiltration or damage to the plasma sodium content that are monitored by the juxtaglomeru-
glomerulus. lar apparatus, which consists of the juxtaglomerular cells in the
afferent arteriole and the macula densa of the distal convoluted
tubule (Fig. 4-4). Low plasma sodium content decreases water
Technical Tip 4-1. If it were not for the shield of nega-
retention within the circulatory system, resulting in a decreased
tivity, all routine urines would have positive readings
overall blood volume and subsequent decrease in blood pres-
on reagent strips for protein and albumin.
sure. When the macula densa senses such changes, a cascade of
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Chapter 4 | Renal Function 109

Afferent
arteriole
Efferent
arteriole
Hydrostatic Glomerular
pressure basement
membrane
Oncotic
Protein
pressure
(unfiltered
plasma
proteins) Glomerular
B
basement
membrane
Foot Fenestrated
processes Slit diaphragm
epithelium
of podocyte Podocyte foot
Podocyte
process
Bowman’s
space Glomerular
Proximal convoluted
basement
tubule
Blood membrane
A Fenestrated
C endothelium

Figure 4–3 Factors affecting glomerular filtration in the renal corpuscle (A). Inset B, glomerular filtration barrier. Inset C, the shield of
negativity.

reactions within the RAAS occurs (Fig. 4-5). Renin, an enzyme

produced by the juxtaglomerular cells, is secreted and reacts
with the bloodborne substrate angiotensinogen to produce the
Afferent Distal tubule inert hormone angiotensin I. As angiotensin I passes through the
arteriole alveoli of the lungs, angiotensin-converting enzyme (ACE)
changes it to the active form angiotensin II. Angiotensin II
Macula Efferent
densa
corrects renal blood flow in the following ways:
arteriole
• Causing vasodilation of the afferent arterioles and
constriction of the efferent arterioles
• Stimulating reabsorption of sodium and water in the
Juxtaglomerular proximal convoluted tubules
cells
• Triggering the release of the sodium-retaining hormone
aldosterone by the adrenal cortex and antidiuretic hor-
mone by the hypothalamus (Box 4-3).
As systemic blood pressure and plasma sodium content
increase, the secretion of renin decreases. Therefore, the actions
of angiotensin II produce a constant pressure within the
nephron.
As a result of the glomerular mechanisms just discussed,
every minute, approximately 2 to 3 million glomeruli filter ap-
proximately 120 mL of water-containing low-molecular-weight
Glomerulus substances. Because this filtration is nonselective, the only dif-
ference between the compositions of the filtrate and the plasma
is the absence of plasma protein, any protein-bound sub-
stances, and cells. Analysis of the fluid as it leaves the glomeru-
lus shows the filtrate to have a specific gravity of 1.010 and
Figure 4–4 Close contact of the distal tubule with the afferent arte- confirms that it is chemically an ultrafiltrate of plasma. This
riole, macula densa, and the juxtaglomerular cells within the juxta- information provides a useful baseline for evaluating the renal
glomerular apparatus. Note the smaller size of the afferent arteriole mechanisms involved in converting the plasma ultrafiltrate into
indicating increased blood pressure. the final urinary product.
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110 Part One | Basic Principles

Low blood pressure

Low plasma sodium

Renin secretion

Angiotensinogen

Angiotensin I
Angiotensin-
converting enzymes
Angiotensin II

Vasoconstriction Proximal convoluted tubule Aldosterone ADH

Sodium reabsorption

Distal convoluted tubule Collecting duct

Sodium reabsorption Water resorption Figure 4–5 Algorithm of the renin–
angiotensin–aldosterone system.

Box 4–3 Actions of the RAAS Table 4–1 Tubular Reabsorption

1. Dilates the afferent arteriole and constricts the efferent arteriole Substance Location
2. Stimulates sodium reabsorption in the proximal convoluted
tubule
Active Glucose, amino Proximal convo-
transport acids, salts luted tubule
3. Triggers the adrenal cortex to release the sodium-retaining hor-
mone aldosterone to cause sodium reabsorption and potassium Chloride Ascending loop of
excretion in the distal convoluted tubule and collecting duct Henle
4. Triggers the hypothalamus to release antidiuretic hormone to Sodium Proximal and distal
stimulate water reabsorption in the collecting duct convoluted
tubules
Passive Water Proximal convo-
transport luted tubule
Descending loop of
Tubular Reabsorption Henle
The body cannot lose 120 mL of water-containing essential Collecting duct
substances every minute. Therefore, when the plasma ultrafil-
Urea Proximal convo-
trate enters the proximal convoluted tubule, the nephrons,
luted tubule
through cellular transport mechanisms, begin reabsorbing
these essential substances and water (Table 4-1). Ascending loop of
Henle
Reabsorption Mechanisms Sodium Ascending loop of
The cellular mechanisms involved in tubular reabsorption are Henle
termed active transport and passive transport. For active
transport to occur, the substance to be reabsorbed must com-
bine with a carrier protein contained in the membranes of the Passive transport is the movement of molecules across a
renal tubular epithelial cells. The electrochemical energy cre- membrane as a result of differences in their concentration or
ated by this interaction transfers the substance across the cell electrical potential on opposite sides of the membrane. These
membranes and back into the bloodstream. Active transport is physical differences are called gradients. Passive reabsorption
responsible for the reabsorption of the following substances: of water takes place in all parts of the nephron except the as-
cending loop of Henle, the walls of which are impermeable to
• Glucose, amino acids, and salts in the proximal convo-
water. Urea is passively reabsorbed in the proximal convoluted
luted tubule
tubule and the ascending loop of Henle, and passive reabsorp-
• Chloride in the ascending loop of Henle tion of sodium accompanies the active transport of chloride in
• Sodium in the distal convoluted tubule the ascending loop.
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Chapter 4 | Renal Function 111

Active transport, like passive transport, can be influenced osmotic gradient in the medulla, as well as the hormone
by the concentration of the substance being transported. When vasopressin (antidiuretic hormone [ADH]) that is released
the plasma concentration of a substance that is normally com- by the posterior pituitary gland when the amount of water in
pletely reabsorbed reaches a level that is abnormally high, the the body decreases. One would expect that as the dilute filtrate
filtrate concentration exceeds the maximal reabsorptive in the collecting duct comes in contact with the higher osmotic
capacity (Tm) of the tubules, and the substance begins appear- concentration of the medullary interstitium, passive reabsorp-
ing in the urine. The plasma concentration at which active tion of water would occur. However, the process is controlled
transport stops is termed the renal threshold. For glucose, the by the presence or absence of ADH, which renders the walls
plasma renal threshold is 160 to 180 mg/dL, and glucose of the distal convoluted tubule and collecting duct permeable
appears in the urine when the plasma concentration reaches or impermeable to water. A high level of ADH increases per-
this level. Knowledge of the renal threshold and the plasma meability, resulting in increased reabsorption of water, and a
concentration can be used to distinguish between excess solute low-volume concentrated urine. Likewise, absence of ADH
filtration and renal tubular damage. Active transport of more renders the walls impermeable to water, resulting in a large
than two-thirds of the filtered sodium out of the proximal con- volume of dilute urine. Just as the production of aldosterone
voluted tubule is accompanied by the passive reabsorption of is controlled by the body’s sodium concentration, the produc-
an equal amount of water. Therefore, as seen in Figure 4-6, the tion of ADH is determined by the state of body hydration.
fluid leaving the proximal convoluted tubule still maintains the Therefore, the chemical balance in the body is actually the final
same concentration as the ultrafiltrate. determinant of urine volume and concentration. The concept
of ADH control can be summarized as follows:

↑ Body Hydration = ↓ ADH = ↑ Urine Volume

Technical Tip 4-2. Glucose appearing in the urine of a ↓ Body Hydration = ↑ ADH = ↓ Urine Volume
person with a normal blood glucose level is the result
of tubular damage and not diabetes mellitus. A non- Tubular Secretion
fasting patient with high glucose intake would not
In contrast to tubular reabsorption, in which substances are
have a normal blood glucose. The plasma glucose
removed from the glomerular filtrate and returned to the
would reach the renal threshold and appear in
blood, tubular secretion involves the passage of substances
the urine.
from the blood in the peritubular capillaries to the tubular
filtrate (Fig. 4-7). Tubular secretion serves two major functions:
eliminating waste products not filtered by the glomerulus and
Tubular Concentration regulating the acid–base balance in the body through the
Renal concentration begins in the descending and ascending secretion of hydrogen ions.
loops of Henle, where the filtrate is exposed to the high Many foreign substances, such as medications, cannot be
osmotic gradient of the renal medulla. Water is removed by filtered by the glomerulus because they are bound to plasma
osmosis in the descending loop of Henle, and sodium and proteins. When these protein-bound substances enter the per-
chloride are reabsorbed in the ascending loop. Excessive reab- itubular capillaries, they develop a stronger affinity for the tu-
sorption of water as the filtrate passes through the highly con- bular cells and dissociate from their carrier proteins, which
centrated medulla is prevented by the water-impermeable walls results in their transport into the filtrate by the tubular cells.
of the ascending loop. This selective reabsorption process is The major site for removal of these nonfiltered substances is
called the countercurrent mechanism and serves to maintain the proximal convoluted tubule.
the osmotic gradient of the medulla (see Fig. 4-6). The sodium
and chloride leaving the filtrate in the ascending loop prevent Acid–Base Balance
dilution of the medullary interstitium by the water reabsorbed To maintain the normal blood pH of 7.4, the blood must buffer
from the descending loop. Maintenance of this osmotic gradi- and eliminate the excess acid formed by dietary intake and
ent is essential for the final concentration of the filtrate when body metabolism. The buffering capacity of the blood depends
it reaches the collecting duct. on bicarbonate (HCO3–) ions, which are readily filtered by the
In Figure 4-6, the actual concentration of the filtrate leaving glomerulus and must be returned to the blood expediently to
the ascending loop is quite low due to the reabsorption of salt maintain the proper pH. As shown in Figure 4-8, the secretion
and not water in that part of the tubule. Reabsorption of sodium of hydrogen ions (H+) by the renal tubular cells into the filtrate
continues in the distal convoluted tubule, but now it is under prevents the filtered bicarbonate from being excreted in the
the control of the hormone aldosterone, which regulates reab- urine and causes the return of a bicarbonate ion to the plasma.
sorption in response to the body’s need for sodium (Fig. 4-5). This process, which occurs primarily in the proximal convo-
luted tubule, provides for almost 100% reabsorption of filtered
Collecting Duct Concentration
bicarbonate.
The final concentration of the filtrate through the reabsorption As a result of their small molecular size, hydrogen ions are
of water begins in the late distal convoluted tubule and con- readily filtered and reabsorbed. Therefore, the actual excretion
tinues in the collecting duct. Reabsorption depends on the of excess hydrogen ions also depends on tubular secretion.
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112 Part One | Basic Principles

Cortex
Blood (vein)
300 mOsm/L
Efferent
Proximal convoluted tubule
arteriole

300

Na+Cl- H2O
Glomerulus H2O 300 Na+

100
Descending Na+Cl-
limb
Thick
H2O ascending Na+
limb
600
Na+Cl-
H2O H2O

H2O
900
H2O
H2O Loop of
Henle
1200

Medulla Countercurrent mechanism

Reabsorption
Secretion
Variable
reabsorption
Figure 4–6 Renal concentration.

Tubular Renal tubular Peritubular

lumen cell plasma

Efferent HCO3– (filtered)

arteriole
– –
Afferent HCO3 + H+ H+ HCO3 HCO3
arteriole
H2CO3

H2CO3 Carbonic anhydrase

H2O + CO2 H2O + CO2 CO2

Bowman’s Final urine

capsule Reabsorption
To blood
Glomerular Figure 4–8 Reabsorption of filtered bicarbonate.
filtrate Secretion
Tubule
Figures 4-9 and 4-10 are diagrams of the two primary methods
for the excretion of hydrogen ions in the urine. In Figure 4-9
To urine
the secreted hydrogen ion combines with a filtered phosphate
ion instead of a bicarbonate ion and is excreted rather than
Figure 4–7 Summary of movement of substances in the reabsorbed. In the proximal convoluted tubule, ammonia is
nephron. produced from the breakdown of the amino acid glutamine.
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Chapter 4 | Renal Function 113

Tubular Renal tubular Peritubular PAH

lumen cell plasma
Osmolarity
HPO4– (filtered) GFR
tests
HPO4– + H+ H+ HCO3– HCO3–

H2CO3
Ammonia
H2PO4 Carbonic anhydrase

H2O + CO2 CO2

Titratable Free water
acidity clearance
Final urine

Figure 4–9 Excretion of secreted hydrogen ions combined with

phosphate.
Osmolarity

Tubular Renal tubular Peritubular

lumen cell plasma
NH3
Figure 4–11 The relationship of nephron areas to renal
– –
NH3 + H+ H+ HCO3 HCO3 function tests.
H2CO3
which the kidneys are able to remove (to clear) a filterable sub-
NH4+ Carbonic anhydrase stance from the blood. To ensure that glomerular filtration is
being measured accurately, the substance analyzed must be one
that is neither reabsorbed nor secreted by the tubules. Other
H2O + CO2 CO2 factors to consider in selecting a clearance test substance in-
clude the stability of the substance in urine during a possible
Final urine 24-hour collection period, the consistency of the plasma level,
the substance’s availability to the body, and the availability of
Figure 4–10 Excretion of secreted hydrogen ions combined with tests to analyze the substance.
ammonia produced by the tubules.
Clearance Tests
The ammonia reacts with the H+ to form the ammonium A variety of substances has been used to measure the GFR.
ion (NH4+) (Fig. 4-10). The resulting ammonium ion is ex- Newer methods that eliminate many of the problems men-
creted in the urine. Should there be additional need for the tioned previously have replaced some of these tests. They are
elimination of hydrogen ions, the distal convoluted tubule and summarized as Historical Notes.
the collecting duct are also able to produce ammonium ions. At present, creatinine, beta2-microglobulin (B2M),
All three of these processes occur simultaneously at rates cystatin C, and, possibly, radioisotopes are the primary sub-
determined by the acid–base balance in the body. A disruption stances used in clearance tests. Each procedure has its advan-
in these secretory functions can result in metabolic acidosis or tages and disadvantages.
renal tubular acidosis, the inability to produce an acid urine.

HISTORICAL NOTE
Renal Function Tests
Urea Clearance
This brief review of renal physiology shows that there are many
metabolic functions and chemical interactions to be evaluated
The earliest glomerular filtration tests measured urea
through laboratory tests of renal function. In Figure 4-11, the
because of its presence in all urine specimens, as well as
parts of the nephron are related to the laboratory tests used to
the existence of routinely used methods of chemical analy-
assess their function.
sis. Because approximately 40% of the filtered urea is
Glomerular Filtration Tests reabsorbed, normal values were adjusted to reflect the
reabsorption, and patients were hydrated to produce a
The standard tests used to measure the filtering capacity of the urine flow of 2 mL/min to ensure that no more than 40%
glomeruli are termed clearance tests. As its name implies, a of the urea was reabsorbed.
clearance test measures the rate in milliliters per minute at
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114 Part One | Basic Principles

Newer methods that do not require the collection of timed

HISTORICAL NOTE
(24-hour) urine specimens have been developed using just the
serum creatinine, cystatin C, or B2M values. The results of
Inulin Clearance
these tests are reported as estimated glomerular filtration rate
(eGFR). The traditional procedure for creatinine clearance is
Inulin, a polymer of fructose, is an extremely stable sub-
included here because it is still being performed, and its
stance that is neither reabsorbed nor secreted by the
principles apply to other clearance procedures using urine.
tubules. It is not a normal body constituent, however, and
must be infused by IV at a constant rate throughout the Procedure
testing period. Therefore, although inulin was the original By far, the greatest source of error in any clearance procedure
reference method for clearance tests, current methods are using urine is the use of urine specimens that are improperly
available that are endogenous and can provide accurate timed. The importance of using a specimen that is accurately
GFR results. timed (see Chapter 3) will become evident in the following
discussion of the calculations involved in converting isolated
laboratory measurements to the GFR. The GFR is reported in
A test that requires an infused substance is termed an milliliters cleared per minute; therefore, it is necessary to
exogenous procedure and is seldom the method of choice determine the number of milliliters of plasma from which the
if a suitable test substance is already present in the body clearance substance (creatinine) is removed completely during
(endogenous procedure). 1 minute. To calculate this information, one must know urine
volume in mL/min (V), urine creatinine concentration in
Creatinine Clearance mg/dL (U), and plasma creatinine concentration in mg/dL (P).
Creatinine is a waste product of muscle metabolism that is pro- The urine volume is calculated by dividing the number of
duced enzymatically by creatine phosphokinase from creatine, milliliters in the specimen by the number of minutes used to
which links with adenosine triphosphate (ATP) to produce collect the specimen.
adenosine diphosphate (ADP) and energy. Because creatinine
is normally found at a relatively constant level in the blood, it EXAMPLE
provides the laboratory with an endogenous procedure for
evaluating glomerular function. The use of creatinine has sev- Calculate the urine volume (V) for a 2-hour specimen measur-
eral disadvantages, and careful consideration should be given ing 240 mL:
to them: 2 hours × 60 minutes = 120 minutes
1. Some creatinine is secreted by the tubules, and secretion 240 mL/120 minutes = 2 mL/min
increases as blood levels rise. V = 2 mL/min
2. Chromogens present in human plasma react in the The concentrations of plasma and urine are determined by
chemical analysis. Their presence, however, may help chemical testing. The standard formula used to calculate the
counteract the falsely elevated rates caused by tubular milliliters of plasma cleared per minute (C) is:
secretion.
UV
3. Medications, including gentamicin, cephalosporins, and C =
P
cimetidine (Tagamet), inhibit tubular secretion of creati-
nine, thus causing serum levels that are falsely low.1 This formula is derived as follows: The milliliters of plasma
4. Bacteria will break down urinary creatinine if specimens cleared per minute (C) times the mg/dL of plasma creatinine
are kept at room temperature for extended periods.2 (P) must equal the mg/dL of urine creatinine (U) times the
urine volume in mL/min (V), because all of the filtered creati-
5. A diet heavy in meat consumed during collection of a nine will appear in the urine. Therefore:
24-hour urine specimen will influence the results if the
plasma specimen is drawn before the collection period. UV
CP = UV and C =
The increased intake of meat can raise the creatinine P
levels in urine and plasma during the 24-hour collection
period.
6. Measurement of creatinine clearance is not a reliable EXAMPLE
indicator in patients suffering from muscle-wasting Using urine creatinine of 120 mg/dL (U), plasma creatinine of
diseases or those involved in heavy exercise or athletes 1.0 mg/dL (P), and urine volume of 1440 mL obtained from a
supplementing with creatine. 24-hour specimen (V), calculate the GFR.
7. Accurate results depend on the accurate completeness of
1440 mL
a 24-hour collection. V = = 1 mL/min
60 minutes × 24 = 1440 minutes
8. Creatinine clearance values must be corrected for body
surface area, unless normal is assumed, and must always 120 mg/dL × 1 mL/min (V)
C = = 120 mL/dL
be corrected for children. 1.0 mg/dL (P)
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Chapter 4 | Renal Function 115

440 200
Plasma (1 mg/dL = 0.01 mg/mL creatinine) 420 190
400 180
380 170
360
Glomerulus 3.00 160
340
2.90 150
2.80 320
2.70 140
220 300
Plasma filtrate (120 mL/min x 0.01 mg/mL = 1.2 mg) 215 2.60 290
7' 130
10" 210 2.50 280
205 2.40 270
8" 260 120
6" 200 2.30
Reabsorption 195 250
4" 2.20 240 110
(119 mL H2O) 2" 190
2.10 230
185
6' 220 100
10" 180 2.00
1.95 210 95
175
8" 1.90 200
Urine (1 mL/min) 170 1.85 90
6" 1.80 190
Creatinine (1.2 mg/mL or 120 mg/dL) 165 1.75 85
4" 180
160 1.70 80
2" 1.65 170
155 1.60 75
Figure 4–12 Creatinine filtration and excretion. 5'
150 1.55 160
10" 1.50 70
145 150

Surface area in square meters

1.45
8" 1.40 65
140
By analyzing this calculation and referring to Figure 4-12, at a 140

Height in centimeters

Weight in kilograms
6" 1.35

Weight in pounds
135 60
concentration of 1 mg/dL, each milliliter of plasma contains 1.30 130

Height in feet
4"
130 1.25
0.01 mg creatinine. Therefore, to arrive at a urine concentration 2" 1.20 120 55
125
of 120 mg/dL (1.2 mg/mL), it is necessary to clear 120 mL of 4' 1.15 110 50
120 1.10
plasma. Although the filtrate volume is reduced, the amount of 10"
115 1.05 100 45
creatinine in the filtrate does not change because the creati- 8" 1.00
110
nine is not reabsorbed. .95 90
6" 40
Knowing that in the average person (1.73 m2 body sur- 105
.90
4"
face), the approximate amount of plasma filtrate produced 100 .85
80
35
per minute is 120 mL, it is not surprising that normal creati- 2"
95 .80
70
nine clearance values approach 120 mL/min (men, 107 to 3' .75
90 30
139 mL/min; women, 87 to 107 mL/min). The normal refer-
10" .70
ence range of plasma creatinine is 0.6 to 1.2 mg/dL. These 85 60
.65
reference values consider variations in size and muscle mass. 8" 25
80
Values are considerably lower in older people; however, an .60
6" 50
adjustment also may have to be made to the calculation 75
.55
when dealing with body sizes that deviate greatly from 20
1.73 m2 of surface, such as with children. To adjust a .50
clearance for body size, the formula is: 40

UV 1.73
C= ×
P A 15

with A being the actual body size in square meters of surface. Figure 4–13 A nomogram for determining body surface area. (From
The actual body size may be calculated as: Boothby, WM, and Sandiford, RB: Nomogram for determination of
body surface area. N Engl J Med 185:227, 1921, with permission.)
log A = (0.425 × log weight) + (0.725 × log height)
– 2.144
or it may be obtained from the nomogram shown in The formula used most frequently is called the Modifica-
Figure 4-13. tion of Diet in Renal Disease (MDRD) study. The formula has
been modified several times to make it more accurate and stan-
dardized. At present, the formula recommended by the Na-
tional Kidney Disease Education Program (NKDEP) is called
Estimated Glomerular Filtration Rates
the MDRD-IDMS-traceable formula. A primary discrepancy in
In the past years, a variety of formulas for eGFR have been the previous formulas was found to be the methods used to
used, and those formulas continue to be revised. Because the measure serum creatinine. eGFR equations are superior to
formulas can be programmed into automated instruments, es- serum creatinine alone because they include variables for race,
timated clearances can be used for routinely screening patients age, and gender.3 Current laboratory methods primarily use
as part of a metabolic profile and to monitor patients already creatinine assays, such as enzyme assays, that do not have the
diagnosed with renal disease or at risk for renal disease. In ad- same interference as the original Jaffe chemical method. These
dition, the formulas are valuable when there is a need to pre- methods correspond more closely to the isotope dilution mass
scribe medications that require adequate renal clearance. spectrophotometry (IDMS) reference method.
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116 Part One | Basic Principles

The MDRD-IDMS traceable formula is: Beta2-Microglobulin

GFR = 175 × serum creatinine–1.154
× age–0.203
× 0.742 B2M (molecular weight 11,800) dissociates from human leuko-
(if patient is female) × 1.212 (if patient is black) cyte antigens at a constant rate and is removed rapidly from the
The formula is designed to essentially equal the results plasma by glomerular filtration. The B2M may be used to dis-
that compare to the reference body size of 1.73 m2. tinguish disorders of the kidney as either glomerular or tubular.
Because eGFRs are calculated for an average body size, they Its excretion in urine is normally low, but when the renal tubules
are not accurate for pediatric patients. They also have been shown become damaged or diseased, the B2M concentration increases
to be most accurate when results are lower than 60 mL/min.4 It due to the decreased ability to reabsorb this protein. By measur-
is recommended that results be reported with numerical values ing B2M concentrations of both blood and urine to evaluate kid-
below 60 mL/min (for example, 59 mL/min) and higher values ney damage or disease, disorders that affect the glomerulus and
reported as equal to or greater than 60 mL/min (for example, renal tubules may be distinguished.8 The concentration of B2M
≥60 mL/min). in serum is normal, and its concentration in urine increases
The formula recommended for use when serum creatinine when tubular reabsorption is reduced. Increased levels of B2M
methods do not compare to the IDSM standard is provided as in the blood combined with low levels in the urine indicate that
a Historical Note. the disorder is associated with glomeruli disorders. The B2M
The eGFR is classified into the following stages based on also is used to identify end-stage renal disease and early rejection
the kidney disease. The Improving Global Outcomes (KDIGO) of a kidney transplant. Sensitive methods using enzyme im-
stages of chronic kidney disease (CKD) are3: munoassay are available for the measurement of B2M. A rise in
the plasma level of B2M has been shown to be a more sensitive
• Stage 1 GFR greater than 90 mL/min/1.73 m indicator of a decrease in GFR than creatinine clearance.
• Stage 2 GFR between 60 and 89 mL/min/1.73 m However, the test is not reliable in patients who have a history
• Stage 3 GFR between 45 and 59 mL/min/1.73 m of immunologic disorders or malignancy.9
• Stage 4 GFR between 15 and 29 mL/min/1.73 m Radionucleotides
• Stage 5 GFR less than 15 mL/min/1.73 m (end-stage
Although it is an exogenous procedure and more labor intensive
renal disease)
and costly, injecting radionucleotides such as 125I-iothalamate
provides a method for determining glomerular filtration through
Cystatin C the plasma disappearance of the radioactive material and enables
The measurement of serum cystatin C has been shown to be a visualization of the filtration in one or both kidneys.10 This
good procedure for screening and monitoring GFR. Cystatin procedure can be valuable to measure the viability of a trans-
C is a small protein (molecular weight 13,359) produced at a planted kidney. Other exogenous markers used are radioiso-
constant rate by all nucleated cells. It is filtered readily by the topes, such as chromium-51 ethylene-diamine-tetra-acetic acid
glomerulus and reabsorbed and broken down by the renal (51 Cr-EDTA), and technetium-99-labeled diethylene-triamine-
tubular cells. Therefore, no cystatin C is secreted by the pentaacetate (99-Tc-DTPA). A nonradioactive contrast agent,
tubules, and the serum concentration can be related directly iohexol, is used for children.3
to the GFR. Immunoassay procedures are available for meas- Clinical Significance
uring cystatin C.5 Monitoring levels of cystatin C is recom- When interpreting the results of a creatinine clearance test, the
mended for pediatric patients, people with diabetes, the elderly, GFR is determined not only by the number of functioning
and patients who are critically ill.6 An advantage of cystatin C nephrons but also by the functional capacity of these nephrons.
is that it is independent of muscle mass. In other words, even though half of the available nephrons may
Recent studies also have shown that measuring both be nonfunctional, a change in the GFR will not occur if the
serum or plasma cystatin C and creatinine can provide even remaining nephrons double their filtering capacity. This is
more accurate information on a patient’s GFR.7 evident in people who lead normal lives with only one kidney.
Therefore, although the GFR is a frequently requested labora-
tory procedure, its value does not lie in the detection of early
renal disease. Instead, it is used to determine the extent of
nephron damage in known cases of renal disease, to monitor
HISTORICAL NOTE the effectiveness of treatment designed to prevent further
nephron damage, and to determine the feasibility of adminis-
Original MDRD Calculation tering medications, which can build up to dangerous blood
levels if the GFR is markedly reduced.
The formula for MDRD calculation of GFR when the
serum creatinine method is not standardized to IDMS. Tubular Reabsorption Tests
GFR = 173 × serum creatinine–1.154 × age–0.203 × 0.742 As discussed, although measurement of the GFR is not a useful
(if patient is female) × 1.212 (if patient is black) indication of early renal disease, the loss of tubular reabsorption
capability is often the first function affected in renal disease.
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Chapter 4 | Renal Function 117

This is not surprising when one considers the complexity of the ratio (U:S ratio) of 3:1 or greater or a urine osmolality of
tubular reabsorption process. 800 mOsm or greater indicates normal tubular reabsorption.
Tests to determine the ability of the tubules to reabsorb If the test continues to be abnormal, additional testing is
the essential salts and water that have been nonselectively fil- performed to determine whether the failure to concentrate the
tered by the glomerulus are called concentration tests. As urine is caused by diabetes insipidus that occurs as the result
mentioned, the ultrafiltrate that enters the tubules has a specific of a problem with the production of or the response of the kid-
gravity of 1.010; therefore, after reabsorption, one would ney to ADH. The patient is injected with ADH, and specimens
expect the final urine product to be more concentrated. How- of serum and urine are collected in 2 and 4 hours. Normal test
ever, many urine specimens do not have a specific gravity results indicate that the patient is not capable of producing
higher than 1.010, yet no renal disease is present. This is ADH (neurogenic diabetes insipidus). If the test is abnormal,
because urine concentration is largely determined by the body’s then the renal tubules are not responding to ADH (nephro-
state of hydration, and the normal kidney will reabsorb only genic diabetes insipidus). See Figure 4-15.
the amount of water necessary to preserve an adequate supply
of body water. Osmolality
As seen in Figure 4-14, both urine specimens contain the
As will be discussed in Chapter 5, osmolality measures only
same amount of solute; however, the urine density (specific
the number of particles in a solution, whereas specific gravity
gravity) of patient A will be higher. Therefore, control of fluid
is influenced by both the number and density (molecular
intake must be incorporated into laboratory tests that measure
weight) of the particles. Renal concentration is concerned with
the concentrating ability of the kidney.
small particles, primarily sodium and chloride molecules.
Throughout the years, various methods have been used to
Large-molecular-weight molecules, such as glucose and urea,
produce water deprivation, including the Fishberg and Mosenthal
do not contribute to the evaluation of renal concentration.
concentration tests, which measured specific gravity. In the
Therefore, osmolality is performed for a more accurate evalu-
Fishberg test, patients were deprived of fluids for 24 hours
ation of renal concentrating ability.
before specific gravity was measured. The Mosenthal test com-
pared the volume and specific gravity of urine specimens col-
Freezing-Point Osmometers
lected during the day and at night to evaluate concentrating
ability. Neither test is used now because the information pro- Measurement of freezing-point depression was the first prin-
vided by specific gravity measurements is most useful as a ciple incorporated into clinical osmometers, and many instru-
screening procedure, and quantitative measurement of renal ments employing this technique are available now. These
concentrating ability is best assessed through osmometry. osmometers determine the freezing point of a solution by
Currently renal concentrating testing is performed after supercooling a measured amount of sample to approximately
various periods of fluid deprivation, measuring urine, and 27°C. The supercooled sample is vibrated to produce crystal-
often serum osmolality. Controlled intake procedures can in- lization of water in the solution. The heat of fusion produced
clude after-dinner overnight deprivation of fluid for 12 hours by the crystallizing water temporarily raises the temperature
followed by collection of a urine specimen. A urine osmolality of the solution to its freezing point. A temperature-sensitive
reading of 800 mOsm or higher is normal, and the test can be probe called a thermistor, in which resistance decreases as
discontinued. If the urine test is abnormal, the fluid is re- temperature increases, measures this temperature increase,
stricted for another 2 hours, and specimens of both urine and which corresponds to the freezing point of the solution, and
serum are collected for osmolality testing. A urine-to-serum the information is converted into milliosmoles. Conversion is

Patient A Patient B

Water (1 glass) Water (4 glasses)

Glomerulus Glomerulus

120 mL Water 120 mL Water

Ultrafiltrate 300 mg Solute Ultrafiltrate 300 mg Solute
119 mL Water 110 mL Water
Reabsorption Reabsorption
100 mg Solute 100 mg Solute
Figure 4–14 The effect of hydration Urine
1 mL Water
Urine
10 mL Water
200 mg Solute 200 mg Solute
on renal concentration. Notice the de-
creased specific gravity in the more-
hydrated Patient B. Specific gravity 1.015 Specific gravity 1.005
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118 Part One | Basic Principles

Polyuria • Values that are falsely elevated due to lactic acid forma-
Urine osmolality <200 mOsm, urine:serum 1:1 tion also occur with both methods if serum specimens
are not separated or refrigerated within 20 minutes.
Fluid restriction • Vapor pressure osmometers do not detect the presence
Urine osmolality <400 mOsm, urine:serum 1:1
of volatile substances, such as alcohol, as they become
part of the solvent phase; however, measurements
ADH challenge
performed on similar specimens using freezing-point
osmometers will be elevated.
Urine osmolality >800 mOsm, Urine osmolality <400 mOsm,
urine:serum 3:1 urine:serum 1:1
Clinical Significance
Major clinical uses of osmolarity include initially evaluating
renal concentrating ability, monitoring the course of renal dis-
Neurogenic diabetes Nephrogenic diabetes
insipidus insipidus
ease, monitoring fluid and electrolyte therapy, establishing the
differential diagnosis of hypernatremia and hyponatremia,
Figure 4–15 Differentiation of neurogenic and nephrogenic and evaluating the secretion of and renal response to ADH.
diabetes insipidus. These evaluations may require determination of serum in
addition to urine osmolarity.
made possible by the fact that 1 mol (1000 mOsm) of a Reference serum osmolality values are from 275 to
nonionizing substance dissolved in 1 kg of water is known to 300 mOsm. Reference values for urine osmolality are difficult
lower the freezing point 1.86°C. Therefore, by comparing the to establish because factors, such as fluid intake and exercise,
freezing-point depression of an unknown solution with that of can greatly influence the urine concentration. Values can range
a known molal solution, the osmolarity of the unknown from 50 to 1400 mOsm.2 Determining the ratio of urine to
solution can be calculated. Clinical osmometers use solutions serum osmolality can provide a more accurate evaluation.
of known NaCl concentration as their reference standards Under normal random conditions, the ratio of urine to serum
because a solution of partially ionized substances is more osmolality should be at least 1:1; after controlled fluid intake,
representative of urine and plasma composition. it should reach 3:1 (Fig. 4-15).

Vapor Pressure Osmometers

Technical Tip 4-3. Vapor pressure osmometers are
The other instrument used in clinical osmometry is called the used primarily to analyze serum and sweat microsam-
vapor pressure osmometer. The actual measurement performed, ples for disorders not related to renal function, such as
however, is that of the dew point (temperature at which water cystic fibrosis. They are used primarily in the chemistry
vapor condenses to a liquid). The depression of dew point tem- department.
perature by solute parallels the decrease in vapor pressure,
thereby providing a measure of this colligative property.
Samples are absorbed into small-filter paper disks that are The ratio of urine to serum osmolality, in conjunction with
placed in a sealed chamber containing a temperature-sensitive procedures such as controlled fluid intake and injection of ADH,
thermocoupler. The sample evaporates in the chamber, form- is used to differentiate whether diabetes insipidus is caused by
ing a vapor. When the temperature in the chamber is lowered, decreased ADH production or inability of the renal tubules to re-
water condenses in the chamber and on the thermocoupler. spond to ADH. Failure to achieve a ratio of 3:1 after injecting
The heat of condensation produced raises the temperature of ADH indicates that the collecting duct does not have functional
the thermocoupler to the dew point temperature. This dew ADH receptors. In contrast, if concentration does take place
point temperature is proportional to the vapor pressure from after ADH injection, an inability to produce adequate ADH is
the evaporating sample. Temperatures are compared with those indicated. Tests to measure the ADH concentration in plasma and
of the NaCl standards and converted into milliosmoles. urine directly are available for difficult diagnostic cases.11
The vapor pressure osmometer uses microsamples of less than
0.01 mL; therefore, care must be taken to prevent any evapo- Free Water Clearance
ration of the sample before testing. Correlation studies have The ratio of urine to serum osmolarity can be expanded further
shown more variation with vapor pressure osmometers, by performing the analyses using water deprivation and a timed
stressing the necessity of careful technique. urine specimen and then calculating the free water clearance.
Technical Factors The free water clearance is determined by first calculating the
Factors to consider because of their influence on true osmo- osmolar clearance using the standard clearance formula:
larity readings include lipemic serum, lactic acid, and volatile
Uosm × V
substances, such as ethanol, in the specimen. Cosm =
Posm
• In lipemic serum, the serum water displacement by in-
soluble lipids produces erroneous results with both and then subtracting the osmolar clearance value from the
vapor pressure and freezing-point osmometers. urine volume in mL/min.
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Chapter 4 | Renal Function 119

EXAMPLE Although it has the disadvantage of being exogenous, the

Using a urine osmolality of 600 mOsm (U), a urine volume of chemical PAH meets the criteria needed to measure renal blood
2 mL/min (V), and a plasma osmolality of 300 mOsm (P), cal- flow. This nontoxic substance is loosely bound to plasma pro-
culate the free water clearance: teins, which permits its complete removal as the blood passes
through the peritubular capillaries. Except for a small amount
600 (U) × 2 (V) of PAH contained in plasma that does not come in contact with
Cosm = = 4.0 mL/min
300 (P) functional renal tissue, all the plasma PAH is secreted by the
CH2O = 2 (V) – 4.0 (Cosm) = –2.0 (free water clearance) proximal convoluted tubule. Therefore, the volume of plasma
flowing through the kidneys determines the amount of PAH
excreted in the urine. The standard clearance formula can be
Calculating osmolar clearance indicates how much water used to calculate the effective renal plasma flow:
must be cleared each minute to produce a urine with the same
osmolality as the plasma. The ultrafiltrate contains the same U (mg/dL PAH) × V (mL/min urine)
CPAH (mL/min) =
osmolality as the plasma; therefore, the osmotic differences in P (mg/dL PAH)
the urine are the result of renal concentrating and diluting
mechanisms. By comparing the osmolar clearance with the Based on normal hematocrit readings, reference values for
actual urine volume excreted per minute, it can be determined the effective renal plasma flow range from 600 to 700 mL/min,
whether the water being excreted is more or less than the making the average renal blood flow about 1200 mL/min. The
amount needed to maintain an osmolality that is the same as actual measurement is renal plasma flow rather than renal
that of the ultrafiltrate. blood flow, because the PAH is contained only in the plasma
The calculation in the previous Example shows a free portion of the blood. Also, the term “effective” is included be-
water clearance of –2.0, indicating that less than the necessary cause approximately 8% of the renal blood flow does not come
amount of water is being excreted: a possible state of dehydra- into contact with the functional renal tissue.
tion. If the value had been 0, no renal concentration or dilution The amount of PAH infused by IV must be monitored
would be taking place; likewise, if the value had been +2.0, carefully to ensure accurate results; therefore, usually the test
excess water would have been excreted. The calculation of the is performed by specialized renal laboratories. Nuclear medi-
free water clearance is used to determine the ability of the cine procedures using radioactive hippurate can determine
kidney to respond to the body’s state of hydration. renal blood flow by measuring the plasma disappearance of a
single radioactive injection and, at the same time, provide
Tubular Secretion and Renal Blood visualization of the blood flowing through the kidneys.11
Flow Tests Titratable Acidity and Urinary Ammonia
Tests to measure tubular secretion of nonfiltered substances
The ability of the kidney to produce an acid urine depends on
and renal blood flow are closely related in that total renal
the tubular secretion of hydrogen ions, as well as production
blood flow through the nephron must be measured by a sub-
and secretion of ammonia by the cells of the distal convoluted
stance that is secreted rather than filtered through the
tubule. A typical person excretes approximately 70 mEq/day
glomerulus. Impaired tubular secretory ability or inadequate
of acid in the form of titratable acid (H+), hydrogen phosphate
presentation of the substance to the capillaries due to de-
ions (H2PO4–), or ammonium ions (NH4+). In typical people,
creased renal blood flow may cause an abnormal result.
a diurnal variation in urine acidity, consisting of alkaline tides,
Therefore, an understanding of the principles and limitations
appears shortly after arising and postprandially at approxi-
of the tests and correlation with other clinical data are
mately 2 p.m. and 8 p.m. The lowest pH is found at night.
important in test interpretation.
The inability to produce an acid urine in the presence of
The test most commonly associated with tubular secretion
metabolic acidosis is called renal tubular acidosis. This condition
and renal blood flow is the p-aminohippuric acid (PAH) test.
may result from impaired tubular secretion of hydrogen ions
associated with the proximal convoluted tubule or defects in
PAH Test
ammonia secretion associated with the distal convoluted tubule.
To measure the exact amount of blood flowing through the Urine pH, titratable acidity, and urinary ammonia meas-
kidney, it is necessary to use a substance that is completely re- urements can be used to determine the defective function. The
moved from the blood (plasma) each time it comes in contact tests can be run simultaneously on either fresh urine specimens
with functional renal tissue. The principle is the same as in the or those preserved with toluene and collected at 2-hour intervals
clearance test for glomerular filtration. However, to ensure from patients who have been primed with an acid load consist-
measurement of the blood flow through the entire nephron, ing of oral ammonium chloride. By titrating the amount of free
the substance must be removed from the blood primarily in H+ (titratable acidity) and then the total acidity of the specimen,
the peritubular capillaries rather than being removed when the the ammonium concentration can be calculated as the difference
blood reaches the glomerulus. between the titratable acidity and the total acidity.
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120 Part One | Basic Principles

editors: Clinical Diagnosis and Management by Laboratory

HISTORICAL NOTE Methods, ed 22, Elsevier, Philadelphia, 2011.
3. Gounden, V, and Jialad, I: Renal Function Tests. Nation Center
Phenolsulfonphthalein Test for Biotechnology Information (NCBI), U. S. National Library
of Medicine. PMID 29939598 Web site: https://www.ncbi.nlm.
Historically, excretion of the dye phenolsulfonphthalein nih.gov/books/NBK507821/ Bethesda, MD. Published April 2,
2019. Accessed May 3, 2019.
(PSP) was used to evaluate these functions. Standardiza- 4. Levey, AS, et al: A new equation to estimate glomerular
tion and interpretation of PSP results are difficult, how- filtration rate. Ann Intern Med 150(9):601–612, 2009.
ever, because of interference by medications, elevated 5. Laterza, OE, Price, CP, and Scott, MG: Cystatin C: An improved
waste products in patients’ serum, the necessity to obtain estimator of glomerular filtration rate? Clin Chem 48(5):
several very accurately timed urine specimens, and the 699–707, 2002.
6. Tan, GS, et al: Clinical usefulness of cystatin C for the estima-
possibility of producing anaphylactic shock. Therefore, tion of glomerular filtration rate in type 1 diabetes. Crit Care
the PSP test is not currently performed. 9(2):139–143, 2005.
7. Inker, LA: Estimating glomerular filtration rate from
serum creatinine and cystatin C. N Engl J Med 367:20–29,
2012.
8. Beta-2 Microglobulin Kidney Disease. Lab Tests Online.
For additional resources please visit American Association for Clinical Chemistry. Web site:
www.fadavis.com http://labtestonline.org/tests/beta-2-microglobulin-kidney-
disease. Accessed April 29, 2019.
9. Foley, K: Beta 2 microglobulin: a facultative marker. Advance
for MLP, Sept 30, 2008, p 13.
References 10. Chachati, A, et al: Rapid method for the measurement of differ-
1. Berger, A: Renal function and how to assess it. Brit J Med ential renal function: Validation. J Nucl Med 28(5): 829–836,
321:1444, 2000. 1987.
2. Pincus, MR, Bock, JL, Bluth, MH: Evaluation of renal function, 11. Davis, BB, and Zenser, TV: Evaluation of renal concentrating
water, electrolytes, and acid-base. In McPherson RA, Ben-Ezra J, and diluting ability. Clin Lab Med 13(1):131–134, 1993.

Study Questions
1. The type of nephron responsible for renal concentration 4. Filtration of protein is prevented in the glomerulus by:
is the: A. Hydrostatic pressure
A. Cortical B. Oncotic pressure
B. Juxtaglomerular C. Renin
C. Efferent D. The glomerular filtration barrier
D. Afferent
5. The renin–angiotensin–aldosterone system is responsible
2. The function of the peritubular capillaries is: for all of the following except:
A. Reabsorption A. Vasoconstriction of the afferent arteriole
B. Filtration B. Vasoconstriction of the efferent arteriole
C. Secretion C. Reabsorbing sodium
D. Both A and C D. Releasing aldosterone
3. Blood flows through the nephron in the following order: 6. The primary chemical affected by the renin–angiotensin–
A. Efferent arteriole, peritubular capillaries, vasa recta, aldosterone system is:
afferent arteriole A. Chloride
B. Peritubular capillaries, afferent arteriole, vasa recta, B. Sodium
efferent arteriole C. Potassium
C. Afferent arteriole, efferent arteriole, peritubular D. Hydrogen
capillaries, vasa recta
D. Efferent arteriole, vasa recta, peritubular capillaries,
afferent arteriole
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Chapter 4 | Renal Function 121

7. Secretion of renin is stimulated by: 14. ADH regulates the final urine concentration by
A. Juxtaglomerular cells controlling:
B. Angiotensin I and II A. Active reabsorption of sodium
C. Macula densa cells B. Tubular permeability
D. Circulating angiotensin-converting enzyme C. Passive reabsorption of urea
D. Passive reabsorption of chloride
8. The hormone aldosterone is responsible for:
A. Hydrogen ion secretion 15. Decreased production of ADH:
B. Potassium secretion A. Produces a low volume of urine
C. Chloride retention B. Produces a high volume of urine
D. Sodium retention C. Increases excretion of ammonia
D. Affects active transport of sodium
9. The fluid leaving the glomerulus has a specific gravity of:
A. 1.005 16. Bicarbonate ions filtered by the glomerulus are returned
to the blood:
B. 1.010
A. In the proximal convoluted tubule
C. 1.015
B. Combined with hydrogen ions
D. 1.020
C. By tubular secretion
10. For active transport to occur, a chemical must:
D. All of the above
A. Combine with a carrier protein to create
electrochemical energy 17. If ammonia is not produced by the distal convoluted
tubule, the urine pH will be:
B. Be filtered through the proximal convoluted tubule
A. Acidic
C. Be in higher concentration in the filtrate than in the
blood B. Basic
D. Be in higher concentration in the blood than in the C. Hypothenuric
filtrate D. Hypersthenuric
11. Which of the tubules is impermeable to water? 18. Place the appropriate letter in front of the following
A. Proximal convoluted tubule clearance substances:
B. Descending loop of Henle A. Exogenous
C. Ascending loop of Henle B. Endogenous
D. Distal convoluted tubule beta2-microglobulin
creatinine
12. Glucose will appear in the urine when the:
cystatin C
A. Blood level of glucose is 200 mg/dL
125I-iothalmate
B. Tm for glucose is reached
C. Renal threshold for glucose is exceeded 19. The largest source of error in creatinine clearance
tests is:
D. All of the above
A. Secretion of creatinine
13. Concentration of the tubular filtrate by the
B. Improperly timed urine specimens
countercurrent mechanism depends on all of the
following except: C. Refrigeration of the urine
A. High salt concentration in the medulla D. Time of collecting blood specimen
B. Water-impermeable walls of the ascending loop of 20. Given the following information, calculate the creatinine
Henle clearance:
C. Reabsorption of sodium and chloride from the 24-hour urine volume: 1000 mL; serum creatinine:
ascending loop of Henle 2.0 mg/dL; urine creatinine: 200 mg/dL
D. Reabsorption of water in the descending loop of
Henle
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122 Part One | Basic Principles

21. Clearance tests used to determine the glomerular 28. The normal serum osmolarity is:
filtration rate must measure substances that are: A. 50 to 100 mOsm
A. Not filtered by the glomerulus B. 275 to 300 mOsm
B. Completely reabsorbed by the proximal convoluted C. 400 to 500 mOsm
tubule
D. 3 times the urine osmolarity
C. Secreted in the distal convoluted tubule
29. After controlled fluid intake, the urine-to-serum
D. Neither reabsorbed nor secreted by the tubules
osmolarity ratio should be at least:
22. Performing a clearance test using radionucleotides: A. 1:1
A. Eliminates the need to collect urine B. 2:1
B. Does not require an infusion C. 3:1
C. Provides visualization of the filtration D. 4:1
D. Both A and C
30. Calculate the free water clearance from the following
23. Variables that are included in the MDRD-IDSM results:
estimated calculations of creatinine clearance include all
urine volume in 6 hours: 720 mL; urine osmolarity:
of the following except:
225 mOsm; plasma osmolarity: 300 mOsm
A. Serum creatinine
31. To provide an accurate measure of renal blood flow, a
B. Weight
test substance should be completely:
C. Age
A. Filtered by the glomerulus
D. Gender
B. Reabsorbed by the tubules
24. An advantage to using cystatin C to monitor GFR is that: C. Secreted when it reaches the distal convoluted
A. It does not require urine collection tubule
B. It is not secreted by the tubules D. Cleared on each contact with functional renal tissue
C. It can be measured by immunoassay 32. Given the following data, calculate the effective renal
D. All of the above plasma flow:
25. Solute dissolved in solvent will: urine volume in 2 hours: 240 mL; urine PAH: 150 mg/dL;
A. Raise the vapor pressure plasma PAH: 0.5 mg/dL
B. Lower the boiling point 33. Renal tubular acidosis can be caused by the:
C. Decrease the osmotic pressure A. Production of excessively acidic urine due to
D. Lower the freezing point increased filtration of hydrogen ions
B. Production of excessively acidic urine due to
26. Substances that may interfere with freezing-point
increased secretion of hydrogen ions
measurement of urine and serum osmolarity include all
of the following except: C. Inability to produce an acidic urine due to impaired
production of ammonia
A. Ethanol
D. Inability to produce an acidic urine due to increased
B. Lactic acid
production of ammonia
C. Sodium
34. Tests performed to detect renal tubular acidosis after
D. Lipids
administering an ammonium chloride load include all of
27. Clinical osmometers use NaCl as a reference solution the following except:
because: A. Urine ammonia
A. 1 g molecular weight of NaCl will lower the freezing B. Arterial pH
point 1.86°C
C. Urine pH
B. NaCl is readily frozen
D. Titratable acidity
C. NaCl is partially ionized, similar to the composition
of urine
D. 1 g equivalent weight of NaCl will raise the freezing
point 1.86°C
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Chapter 4 | Renal Function 123

Case Studies and Clinical Situations

1. A 44-year-old man diagnosed with acute tubular necrosis 4. A laboratory is obtaining erratic serum osmolarity results
has a blood urea nitrogen of 60 mg/dL and a blood on a patient who is being monitored at 6 a.m., 12 p.m.,
glucose level of 100 mg/dL. A 2+ urine glucose is also 6 p.m., and 12 a.m. Osmolarities are not performed on
reported. the night shift; therefore, the midnight specimen is run at
a. State the renal threshold for glucose. the same time as the 6 a.m. specimen.
b. What is the significance of the positive urine glucose a. What two reasons could account for these
and normal blood glucose? discrepancies?
b. What substance is causing the erratic results?
2. A patient develops a sudden drop in blood pressure.
c. If a friend were secretly bringing the patient a pint of
a. Diagram the reactions that take place to ensure
whiskey every night, would this affect the results?
adequate blood pressure within the nephrons.
Explain your answer.
b. How do these reactions increase blood volume?
5. After overnight (6 p.m. to 8 a.m.) fluid deprivation, the
c. When blood pressure returns to normal, how does the
urine-to-serum osmolarity ratio in a patient who is ex-
kidney respond?
hibiting polyuria and polydipsia is 1:1. The ratio remains
3. A physician would like to prescribe a nephrotoxic antibi- the same when a second specimen is tested at 10 a.m.
otic for a 60-year-old white man. The patient has a serum Then ADH is administered subcutaneously to the patient,
creatinine level of 1.5 mg/dL. and the fluid deprivation is continued until 2 p.m., when
a. How can the physician determine whether it is safe to another specimen is tested.
prescribe this medication before the patient leaves a. What disorder do these symptoms and initial labora-
the office? tory results indicate?
b. State two additional blood tests that the physician b. If the urine-to-serum osmolarity ratio on the 2 p.m.
could use to continue monitoring this patient. specimen is 3:1, what is the underlying cause of the
c. If the patient has a history of prostate malignancy, patient’s disorder?
would both of the methods listed in answer choice 3b c. If the urine-to-serum osmolarity ratio on the 2 p.m.
provide reliable results? Explain your answer. specimen remains 1:1, what is the underlying cause of
the patient’s disorder?
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PART TWO

Urinalysis
Chapter 5: Physical Examination of Urine
Chapter 6: Chemical Examination of Urine
Chapter 7: Microscopic Examination of Urine
Chapter 8: Renal Disease
Chapter 9: Urine Screening for Metabolic Disorders
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CHAPTER 5
Physical Examination
of Urine
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
5-1 List the common terminology used to report normal 5-10 List three pathological and four nonpathological
urine color. causes of cloudy urine.
5-2 Discuss the relationship of urochrome to normal urine 5-11 Define specific gravity, and tell why this measurement
color. can be significant in the routine analysis.
5-3 State how the presence of bilirubin, biliverdin, uroery- 5-12 Describe the principles of the refractometer, reagent
thrin, and urobilin in a specimen may be suspected. strip, and osmolality for determining specific gravity.
5-4 Discuss the significance of cloudy red urine versus 5-13 Given the concentration of glucose and protein in a
clear red urine. specimen, calculate the correction needed to compen-
sate for these high-molecular-weight substances in the
5-5 Name two pathological causes of black or brown urine.
refractometer reading of specific gravity.
5-6 Discuss the significance of phenazopyridine in a
5-14 Name two nonpathogenic causes of abnormally high
specimen.
readings of specific gravity using a refractometer.
5-7 State the clinical significance of urine clarity.
5-15 Describe the advantages of measuring specific gravity
5-8 List the common terminology used to report clarity. using a reagent strip and osmolality.
5-9 Describe the appearance and discuss the significance 5-16 State possible causes of abnormal urine odor.
of amorphous phosphates and amorphous urates in
urine that was freshly voided.

KEY TERMS
Clarity Refractive index Urochrome
Hypersthenuric Refractometry Uroerythrin
Hyposthenuric Specific gravity
Isosthenuric Urobilin
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126 Part Two | Urinalysis

Introduction noticeable change in urine color is often the reason that a patient
seeks medical advice; then it becomes the responsibility of
The physical examination of urine includes the determination the laboratory to determine whether this color change is normal
of the urine color, clarity, and specific gravity. As mentioned or pathological. The more common normal and pathological
in Chapter 3, early physicians based many medical decisions correlations of urine colors are summarized in Table 5-1.
on the color and clarity of urine. Today, observation of these
characteristics provides preliminary information concerning Normal Urine Color
disorders, such as glomerular bleeding, liver disease, inborn
errors of metabolism, and urinary tract infection. Measure- The terminology used to describe the color of normal urine
ment of specific gravity aids in the evaluation of renal tubular may differ slightly among laboratories but should be consistent
function. The results of the physical portion of the urinalysis within each laboratory. Common descriptions include pale yel-
also can be used to confirm or to explain findings in the chem- low, yellow, and dark yellow. See Figure 5-1 for examples of
ical and microscopic areas of urinalysis. normal urine color. Care should be taken to examine the spec-
imen under a good light source, looking down through the
Color container against a white background. The yellow color of
urine is caused by the presence of a pigment, which Thu-
The color of urine varies from almost colorless to black. These dichum named urochrome in 1864. Urochrome is a product
variations may be due to normal metabolic functions, physical of endogenous metabolism, and under normal conditions, the
activity, ingested materials, or pathological conditions. A body produces it at a constant rate. The actual amount of

Table 5–1 Laboratory Correlation of Urine Color1,4

Color Cause Clinical/Laboratory Correlations
Colorless Recent fluid consumption Commonly observed with random specimens
Pale yellow Polyuria or diabetes insipidus Increased 24-hour volume and low specific gravity
Diabetes mellitus Elevated specific gravity and positive glucose test result
Dilute random specimen Recent fluid consumption
Dark yellow Concentrated specimen May be normal after strenuous exercise or in first morning
specimen
B complex vitamins
Dehydration Fever or burns
Bilirubin Yellow foam when shaken and positive chemical test results
for bilirubin
Acriflavine Negative bile test results and possible green fluorescence
Nitrofurantoin Antibiotic administered for urinary tract infections
Orange-yellow Phenazopyridine (Pyridium) Drug commonly administered for urinary tract infections
Phenindione Anticoagulant, orange in alkaline urine, colorless in acid urine
Sulfasalazine (Azulfidine) Anti-inflammatory drug
Yellow-green Bilirubin oxidized to biliverdin Colored foam in acidic urine and false-negative chemical test
results for bilirubin
Green Pseudomonas infection Positive urine culture
Asparagus None
Blue-green Amitriptyline Antidepressant
Methocarbamol (Robaxin) Muscle relaxant, may be green-brown
Clorets None
Indican Bacterial infections, intestinal disorders
Methylene blue Fistulas
Phenol When oxidized
Propofol Anesthetic
Familial hypercalcemia “Blue diaper syndrome”
Indomethacin (Indocin, Tivorbex) Nonsteroidal anti-inflammatory drug
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Chapter 5 | Physical Examination of Urine 127

Table 5–1 Laboratory Correlation of Urine Color1,4—cont’d

Color Cause Clinical/Laboratory Correlations
Pink RBCs Cloudy urine with positive chemical test results for blood
Red and RBCs visible microscopically
Hemoglobin Clear urine with positive chemical test results for blood; in-
travascular hemolysis
Myoglobin Clear urine with positive chemical test results for blood;
muscle damage
Beets Alkaline urine of people who are genetically susceptible

Rifampin Tuberculosis medication

Menstrual contamination Cloudy specimen with RBCs, mucus, and clots

Port wine Porphyrins Negative test for blood, may require additional testing
Red-brown RBCs oxidized to methemoglobin Seen in acidic urine after standing; positive chemical test
result for blood
Myoglobin
Brown Homogentisic acid (alkaptonuria) Seen in alkaline urine after standing; specific tests are
Black available
Malignant melanoma Urine darkens on standing and reacts with nitroprusside
Melanin or melanogen and ferric chloride
Phenol derivatives Interfere with copper reduction tests

Argyrol (antiseptic) Color disappears with ferric chloride

Methyldopa or levodopa Antihypertensive

Metronidazole (Flagyl) Darkens on standing, intestinal and vaginal infections

Chloroquine and primaquine Antimalarial drugs

Methocarbamol Muscle relaxant

Fava beans, rhubarb, or aloe None

urochrome produced is dependent on the body’s metabolic amorphous urates in an acid urine. Uroerythrin attaches to the
state, with increased amounts produced in patients with thy- urates, giving a pink color to the sediment. Urobilin, an oxi-
roid conditions and/or those in fasting states.2 Urochrome also dation product of the normal urinary constituent urobilinogen,
increases in urine that stands at room temperature.3 imparts an orange-brown color to urine that is not fresh.
Because urochrome is excreted at a constant rate, the inten-
sity of the yellow color in a fresh urine specimen can give a rough Abnormal Urine Color
estimate of urine concentration. A dilute urine will be pale
yellow, and a concentrated specimen will be dark yellow. As seen in Table 5-1, abnormal urine colors are as numerous
Remember that due to variations in the body’s state of hydration, as their causes. Certain colors, however, are seen more fre-
these differences in the yellow color of urine can be normal. quently and have a greater clinical significance than do others.
Two additional pigments, uroerythrin and urobilin, are
Dark Yellow/Amber/Orange
also present in the urine in much smaller quantities, and they
contribute little to the color of normal, fresh urine. The presence Dark yellow or amber urine may not always signify a normal con-
of uroerythrin, a pink pigment, is most evident in specimens centrated urine but can be caused by the presence of the abnor-
that have been refrigerated, resulting in the precipitation of mal pigment bilirubin. If bilirubin is present, it will be detected
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128 Part Two | Urinalysis

Figure 5–1 Examples of normal urine color ranging from light

yellow to dark yellow.
Figure 5–2 Examples of blood in the urine.
during the chemical examination; however, its presence is
suspected if yellow foam appears when the specimen is shaken.
Normal urine produces only a small amount of rapidly disap- remaining in an acidic urine for several hours cause the urine
pearing foam when shaken, and a large amount of white foam to turn brown due to the oxidation of hemoglobin to methe-
indicates an increased concentration of protein. A urine specimen moglobin. A fresh urine containing blood that is brown also
that contains bilirubin also may contain hepatitis virus, reinforc- may indicate glomerular bleeding, resulting from the conver-
ing the need to follow standard precautions. The photo-oxidation sion of hemoglobin to methemoglobin.5
of large amounts of excreted urobilinogen to urobilin also pro- Besides RBCs, two other substances, hemoglobin and
duces a yellow-orange urine; however, yellow foam does not ap- myoglobin, produce a red urine and result in a positive chem-
pear when the specimen is shaken. Photo-oxidation of bilirubin ical test result for blood (Fig. 5-3). When intact RBCs (hema-
imparts a yellow-green color to the urine caused by the presence turia) are present, the urine is red and cloudy; however, if
of biliverdin. hemoglobin or myoglobin is present, the specimen is red and
Also frequently encountered in the urinalysis laboratory clear. Distinguishing between hemoglobinuria and myoglobin-
is the yellow-orange specimen caused by the administration of uria may be possible by examining the patient’s plasma. He-
phenazopyridine (brand name Pyridium) or azo-gantrisin moglobinuria resulting from the in vivo breakdown of RBCs is
compounds to people who have urinary tract infections. This accompanied by red plasma. Breakdown of skeletal muscle
thick, orange pigment not only obscures the natural color of produces myoglobin. Myoglobin is cleared more rapidly from
the specimen but also interferes with chemical tests that are the plasma than is hemoglobin and therefore does not affect
based on color reactions. It is important to recognize the pres- the color of the plasma. Fresh urine containing myoglobin fre-
ence of phenazopyridine in a specimen so that laboratories can quently exhibits a more reddish-brown color than does urine
use alternative testing procedures. Specimens containing containing hemoglobin. The possibility of hemoglobinuria
phenazopyridine produce a yellow foam when shaken, which being produced from the in vitro lysis of RBCs also must be
could be mistaken for bilirubin. considered. Chemical tests to distinguish between hemoglobin
Other medications that can cause an orange-colored urine and myoglobin are available (see Chapter 6).
include the anti-inflammatory drug sulfasalazine (Azulfidine), Urine specimens containing porphyrins also may appear
some laxatives, and certain chemotherapy drugs.4 red, resulting from the oxidation of porphobilinogen to
porphyrins. They are often referred to as having the color
Red/Pink/Brown
of port wine.
One of the most common causes of abnormal urine color is Nonpathogenic causes of red urine include menstrual con-
the presence of blood. Red is the usual color that blood pro- tamination or ingestion of highly pigmented foods and/or med-
duces in urine, but the color may range from pink to brown, ications. In people who are genetically susceptible, eating fresh
depending on the amount of blood, the pH of the urine, and beets causes a red color in alkaline urine.6 Ingestion of blackber-
the length of contact (Fig. 5-2). Red blood cells (RBCs) ries can produce a red color in acidic urine. Many medications,
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Chapter 5 | Physical Examination of Urine 129

Red urine found in certain IV medications produce green urine on oxida-

tion.8 A purple staining may occur in catheter bags and is caused
by indican in the urine or a bacterial infection, frequently caused
Clear Cloudy by Klebsiella or Providencia species.9

Hemoglobinuria Myoglobinuria Red blood cells present

Clarity
(hematuria)
“Clarity” is a general term that refers to the transparency or
turbidity of a urine specimen. In routine urinalysis, clarity is
Red plasma Clear plasma
determined in the same manner that ancient physicians used:
by visually examining the mixed specimen while holding it in
Figure 5–3 Differentiation of red urine testing chemically positive front of a light source. The specimen should, of course, be in
for blood. a clear container. Color and clarity are routinely determined at
the same time. Common terminology used to report clarity in-
cludes clear, hazy, cloudy, turbid, and milky. As discussed in
including rifampin, phenolphthalein, phenindione, and pheno- the previous section on urine color, terminology for clarity
thiazines, produce red urine. should be consistent within a laboratory. A description of urine
Brown/Black clarity reporting is presented in Table 5-2.

Additional testing is recommended for urine specimens that Normal Clarity

turn brown or black on standing and have negative chemical Freshly voided, normal urine is usually clear, particularly if it
test results for blood, inasmuch as they may contain melanin is a clean-catch midstream specimen. Precipitation of amor-
or homogentisic acid. Melanin is an oxidation product of the phous phosphates and carbonates may cause a white cloudi-
colorless pigment melanogen, which is produced in excess ness in an alkaline urine (Fig. 5-4).
when a malignant melanoma is present. Homogentisic acid, a
metabolite of phenylalanine, imparts a black color to alkaline Nonpathological Turbidity
urine from patients with the inborn error of metabolism, called
alkaptonuria. These conditions are discussed in Chapter 8. The presence of squamous epithelial cells and mucus, partic-
Medications producing brown/black urines include anti- ularly in specimens from women, can result in urine that is
malarial drugs (chloroquine and primaquine, levodopa, methyl- hazy but normal.
dopa, phenol derivatives, and the antibiotics metronidazole Specimens that are allowed to stand or that are refrigerated
[Flagyl] and nitrofurantoin [Furadantin]); laxatives containing also may develop turbidity that is nonpathological. As dis-
cascara or senna; and methocarbamol, a muscle relaxant.4 Liver cussed in Chapter 3, improper preservation of a specimen re-
and kidney disorders and muscle injury from extreme exercise sults in bacterial growth; this increases specimen turbidity but
can result in a dark-brown urine.4 is not representative of the actual specimen.
Nonpathogenic causes of a dark-brown (cola-colored) Refrigerated specimens frequently develop a thick turbidity
urine can include ingesting large amount of fava beans, rhubarb, caused by the precipitation of amorphous phosphates, carbon-
or aloe.4 ates, and urates. Amorphous phosphates and carbonates pro-
duce a white precipitate in urine with an alkaline pH, whereas
Blue/Green amorphous urates produce a precipitate in acidic urine that
resembles pink brick dust due to the presence of uroerythrin.
Pathogenic causes of blue/green urine are limited to bacterial Additional nonpathological causes of urine turbidity
infections, including urinary tract infection by Pseudomonas include mucus, normal urine crystals, semen, fecal contami-
species, and intestinal tract infections, resulting in increased nation, radiographic contrast media, talcum powder, and
urinary indican. A familial benign hypercalcemia, a rare inher- vaginal creams (Box 5-1).
ited disorder, is sometimes called “blue diaper syndrome”
because children with the disorder will have blue urine.4
Ingestion of breath deodorizers (Clorets) can result in a
green urine.7 Brightly colored food dyes, B vitamins, and aspara- Table 5–2 Urine Clarity
gus also can cause a green urine.4 The medications methocar-
bamol (Robaxin), methylene blue, indomethacin (Indocin, Clarity Term
Tivorbex), amitriptyline (Elavil), and propofol (Diprivan) may Clear No visible particulates, transparent
cause blue urine.4
Hazy Few particulates, print easily seen through urine
Observation of specimen collection bags from patients who
are hospitalized frequently reveals urine that is abnormally col- Cloudy Many particulates, print blurred through urine
ored. This may signify either a pathological condition that re- Turbid Print cannot be seen through urine
quires the urine to stand for a period of time before color Milky May precipitate or be clotted
development or the presence of medications. Phenol derivatives
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130 Part Two | Urinalysis

Box 5–1 Nonpathological Causes of Urine Turbidity

Squamous epithelial cells
Mucus
Amorphous phosphates, carbonates, urates
Semen, spermatozoa
Fecal contamination
Radiographic contrast media
Talcum powder
Vaginal creams

Box 5–2 Pathological Causes of Urine Turbidity

RBCs
WBCs
Bacteria
Yeast
Trichomonads
Nonsquamous epithelial cells
Abnormal crystals
Figure 5–4 Examples of urine clarity. The urine in the left tube is
clear. The urine in the right tube is turbid. Lymph fluid
Lipids

PROCEDURE 5-1
Urine Color and Clarity Procedure
Procedure
1. Evaluate an adequate volume of specimen.
2. Use a well-mixed specimen.
3. View the urine through a clear container.
4. View the urine against a white background using
adequate room lighting.
5. Maintain adequate room lighting.
6. Evaluate a consistent volume of urine:
• Determine the urine color.
• Describe the urine clarity (Table 5-2).

Pathological Turbidity
The pathological causes of turbidity in a fresh specimen that are
encountered most commonly are RBCs, white blood cells
(WBCs), and bacteria caused by infection or a systemic organ Figure 5–5 Examples of urine specimens with abnormal clarity
disorder. Other causes of pathological turbidity that are encoun- and color.
tered less frequently include abnormal amounts of nonsquamous
epithelial cells, yeast, trichomonads, abnormal crystals, lymph turbidity should correspond with the amount of material
fluid, and lipids (Box 5-2). See Figure 5-5 for examples of observed under the microscope.
abnormal urine turbidity. Clear urine is not always normal. However, with the in-
The clarity of a urine specimen certainly provides a key to creased sensitivity of the routine chemical tests, most abnormal-
the microscopic examination results because the amount of ities in clear urine will be detected before the microscopic
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Chapter 5 | Physical Examination of Urine 131

analysis. Current criteria used to determine the necessity of Table 5–3 Current Urine Specific Gravity
performing a microscopic examination on all urine specimens Measurements
include both clarity and chemical tests for RBCs, WBCs, bacteria,
and protein. Method Principle
Refractometry Refractive index
Specific Gravity Osmolality Changes in colligative properties by
particle number
As discussed in Chapter 4, the kidney’s ability to concentrate
the glomerular filtrate by selectively reabsorbing essential Reagent strip pKa changes of a polyelectrolyte by ions
chemicals and water from the glomerular filtrate is one of the present
kidney’s most important functions. The evaluation of urine
concentration is included in the routine urinalysis by measur-
ing the specific gravity of the specimen. Including specific grav- The refractometer provides the distinct advantage of deter-
ity in the routine urinalysis provides an additional function, mining specific gravity using a small volume of specimen (one
which is to determine whether specimen concentration is or two drops). Temperature corrections are not necessary because
adequate to ensure the accuracy of chemical tests. the light beam passes through a temperature-compensating liquid
The specific gravity of the plasma filtrate entering the before being directed at the specific gravity scale. Temperature is
glomerulus is 1.010. The term isosthenuric is used to describe compensated between 15°C and 38°C. Corrections for glucose
urine with a specific gravity of 1.010. Specimens below 1.010 and protein must be calculated by subtracting 0.003 for each
are hyposthenuric, and those above 1.010 are hypersthenuric. gram of protein present and 0.004 for each gram of glucose pres-
One would expect urine that has been concentrated by the ent. The amount of protein or glucose present can be determined
kidneys to be hypersthenuric, but this is not always true. Normal from the chemical reagent strip tests.
random specimens may range from approximately 1.002 to When using the refractometer, a drop of urine is placed
1.035, depending on the patient’s amount of hydration. Speci- on the prism, the instrument is focused at a good light source,
mens measuring lower than 1.002 probably are not urine. Most and the reading is taken directly from the specific gravity scale.
random specimens fall between 1.015 and 1.030. The prism and its cover should be cleaned after each specimen
Specific gravity is defined as the density of a solution com- is tested. Figure 5-6 illustrates the use of the refractometer.
pared with the density of a similar volume of distilled water Visit www.fadavis.com for Video 5-1 (Specific gravity via
(SG 1.000) at a similar temperature. Because urine is actually refractometer).
water that contains dissolved chemicals, the specific gravity of
The refractometer is calibrated using distilled water that
urine is a measure of the density of the dissolved chemicals in
should give a reading of 1.000. If necessary, the instrument
the specimen. As a measure of specimen density, specific grav-
contains a zero setscrew to adjust the reading for distilled water
ity is influenced not only by the number of particles present
(Fig. 5-7). The calibration is further checked using either
but also by their size. Therefore, large molecules contribute
5% NaCl, which, as shown in the refractometer conversion
more to the reading than do small molecules. This may require
tables, should read 1.022 ± 0.001, or 9% sucrose, which
the need to correct for the presence of substances that are
should read 1.034 ± 0.001. Urine control specimens represent-
not normally seen in urine, such as glucose and protein in the
ing low, medium, and high concentrations also should be run
specimen. Currently the only method in use in routine urinal-
at the beginning of each shift. Calibration and control results
ysis that requires correcting is the refractometer. The other two
are always recorded in the appropriate quality control records.
methods in use are chemical reagent strips and osmolality. The
principles behind current specific gravity measurement tech-
EXAMPLE
niques are presented in Table 5-3.
A specimen containing 1 g/dL protein and 1 g/dL glucose
Refractometer has a specific gravity reading of 1.030. Calculate the corrected
reading.
Refractometry determines the concentration of dissolved par-
ticles in a specimen by measuring refractive index. Refractive 1.030 – 0.003 (protein) = 1.027 – 0.004 (glucose) =
index is a comparison of the velocity of light in the air with 1.023 corrected specific gravity
the velocity of light in a solution. The concentration of dis-
solved particles present in the solution determines the velocity
and angle at which light passes through a solution. Clinical Results that are abnormally high—above 1.040—are seen
refractometers make use of these principles of light by using a in patients who have recently undergone an IV pyelogram. This
prism to direct a specific (monochromatic) wavelength of is caused by the excretion of the injected radiographic contrast
daylight against a manufacturer-calibrated scale of specific media. Patients who are receiving dextran or other high-
gravity. The concentration of the specimen determines the molecular-weight IV fluids (plasma expanders) also produce
angle at which the light beam enters the prism. Therefore, the urine with an abnormally high specific gravity. Once the for-
specific gravity scale is calibrated in terms of the angles at eign substance has been cleared from the body, the specific
which light passes through the specimen. gravity returns to normal. In these circumstances, urine
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132 Part Two | Urinalysis

O X X

1. Put one or two drops of sample 2. Close the daylight plate gently. 3. The sample must spread all over
on the prism. the prism surface.

1.355
1.050

1.040 1.350

1.030 1.345

1.020
1.340
1.010
1.335
1.000 1.333
U.G. 20˚C nD

4. Look at the scale through the 5. Read the scale where the boundary 6. Wipe the sample from the prism
eyepiece. line intercepts it. clean with a tissue paper and water.

Figure 5–6 Steps in the use of the urine specific gravity refractometer. (Courtesy of NSG Precision Cells, Inc., 195G Central Ave.,
Farmingdale, NY, 11735.)

concentration can be measured using the reagent strip chemi-

cal test or osmometry because they are not affected by these
high-molecular-weight substances.10

Osmolality
As stated previously, specific gravity depends on the number
Calibration of particles present in a solution, as well as the density of these
screw particles; in contrast, osmolality is affected only by the number
of particles present. When evaluating renal concentration
ability, the substances of interest are small molecules, primarily
sodium (molecular weight 23) and chloride (molecular
weight 35.5). However, urea (molecular weight 60), which is
of no importance to this evaluation, will contribute more to
the specific gravity than will the sodium and chloride mole-
cules. Because all three molecules contribute equally to the
osmolarity of the specimen, a more representative measure of
renal concentrating ability can be obtained by measuring
osmolarity (see Chapter 4).
An osmole is defined as 1 g molecular weight of a
substance divided by the number of particles into which
it dissociates. A nonionizing substance, such as glucose
(molecular weight, 180), contains 180 g per osmole, whereas
sodium chloride (NaCl) (molecular weight 58.5), if com-
pletely dissociated, contains 29.25 g per osmole. Just like
Figure 5–7 Calibration of the urine specific gravity refractometer. molality and molarity, there are osmolality and osmolarity.
(Courtesy of NSG Precision Cells, Inc., 195G Central Ave., Farmingdale, An osmolal solution of glucose has 180 g of glucose dis-
NY, 11735.) solved in 1 kg of solvent. An osmolar solution of glucose has
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Chapter 5 | Physical Examination of Urine 133

solvent. Solute dissolved in solvent causes the following

HISTORICAL NOTE
changes in colligative properties: lower freezing point, higher
boiling point, increased osmotic pressure, and lower vapor
Urinometry
pressure (see Table 5-4).
Because water is the solvent in urine, the number of par-
The urinometer consists of a weighted float attached to a
ticles present in a sample can be determined by comparing
scale that has been calibrated in terms of urine specific
a colligative property value of the sample with that of pure
gravity. The weighted float displaces a volume of liquid
water. To measure osmolality in the urinalysis laboratory
equal to its weight and has been designed to sink to a level
requires special equipment referred to as an osmometer
of 1.000 in distilled water. The additional mass provided
and, therefore, an additional step in the routine urinalysis
by the dissolved substances in urine causes the float to dis-
procedure.
place a volume of urine smaller than that of distilled water.
The A2O Advanced Automated Osmometer (Advanced In-
The level to which the urinometer sinks, as shown in the
struments, Inc., Two Technology Way, Norwood, MA 02062)
figure, represents the specimen’s mass or specific gravity.
uses freezing-point depression to measure osmolality, provid-
ing a more automated method for measuring both urine and
0 serum osmolality. (The principles and uses of the freezing-
10
point and vapor pressure osmometers currently in use in the
clinical laboratory are covered in Chapter 4.)
0 20

10 30

20 40
Technical Tip 5-1. The term “molality” is used most
30
commonly because both the solute and the solvent
50
are expressed in the same units of measure.
40

50
Technical Tip 5-2. Additional information on osmom-
etry can be found at https://www.aicompanies.com/
osmometers/a2o-advanced-automated-osmometer/
(accessed March 11, 2020). A video also can be
accessed there.

Reagent Strip Specific Gravity

The addition of a specific gravity testing area to urinalysis chem-
ical reagent strips has provided a convenient way to perform
the routine urinalysis by eliminating the need for an additional
procedure.
The reagent strip reaction is based on the change in pKa
(dissociation constant) of a polyelectrolyte in an alkaline
medium. The polyelectrolyte ionizes, releasing hydrogen
ions in proportion to the number of ions in the solution.
The higher the concentration of urine, the more hydrogen ions
Urinometry is less accurate than the other methods are released, thereby lowering the pH. Incorporation of the in-
currently available and is not recommended by the Clini- dicator bromothymol blue on the reagent pad measures the
cal and Laboratory Standards Institute (CLSI).11
HISTORICAL NOTE

Harmonic Oscillation Densitometry

180 g of glucose dissolved in 1 L of solvent. The unit of
measure used in the clinical laboratory is the milliosmole Harmonic oscillation densitometry is based on the principle
(mOsm), because it is not practical when dealing with body that the frequency of a sound wave entering a solution
fluids to use a measurement as large as the osmole (23 g of changes in proportion to the density of the solution. This
sodium per kilogram). technique was originally used in early automated urinalysis
The osmolarity of a solution can be determined by meas- instruments. The addition of reagent strip analysis for
uring a property that is mathematically related to the number specific gravity has replaced this technique in automated
of particles in the solution (colligative property) and com- systems.
paring this value with the value obtained from the pure
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134 Part Two | Urinalysis

Table 5–4 Particle Changes to Colligative Table 5–5 Possible Causes of Urine Odor1
Properties
Odor Cause
Normal Pure Effect of 1 Mole
Aromatic Normal
Property Water Point of Solute
Foul, ammonia- Bacterial decomposition, urinary tract
Freezing Point 0°C Lowered 1.86°C like infection
Boiling Point 100°C Raised 0.52°C Fruity, sweet Ketones (diabetes mellitus,
Vapor Pressure 2.38 mm Hg at Lowered 0.3 mm starvation, vomiting)
25°C Hg at 25°C Maple syrup Maple syrup urine disease
Osmotic 0 mm Hg Increased 1.7 × Mousy Phenylketonuria
Pressure 109 mm Hg
Rancid Tyrosinemia
Sweaty feet Isovaleric acidemia
Cabbage Methionine malabsorption
change in pH. As the specific gravity increases, the indicator
Bleach Contamination
changes from blue (1.000 [alkaline]), through shades of
green, to yellow (1.030 [acid]). Readings can be made in
0.005 intervals by careful comparison with the color chart. A
diagram of the specific gravity reaction is shown in Chapter 6,
For additional resources please visit
Figure 6-6. www.fadavis.com

Odor
References
Although it is seldom of clinical significance and is not a part 1. Henry, JB, Lauzon, RB, and Schumann, GB: Basic examina-
of the routine urinalysis, urine odor is a noticeable physical tion of urine. In Henry, JB (ed): Clinical Diagnosis and
property. Freshly voided urine has a faint aromatic odor. As the Management by Laboratory Methods. WB Saunders,
specimen stands, the odor of ammonia becomes more promi- Philadelphia, 1996.
nent. The breakdown of urea is responsible for the character- 2. Drabkin, DL: The normal pigment of urine: The relationship
of urinary pigment output to diet and metabolism. J Biol Chem
istic ammonia odor. Causes of unusual odors include bacterial 75:443–479, 1927.
infections, which cause a strong, unpleasant odor similar to 3. Ostow, M, and Philo, S: The chief urinary pigment: The
ammonia, and diabetic ketones, which produce a sweet or relationship between the rate of excretion of the yellow
fruity odor. A serious metabolic defect results in urine with a pigment and the metabolic rate. Am J Med Sci 207:507–512,
strong odor of maple syrup and is called, appropriately, maple 1944.
4. Mayo Clinic: Urine color. Symptoms and Causes. Web site.
syrup urine disease. This and other metabolic disorders with https://www.mayoclinic.org/diseases-conditions/urine-color/
characteristic urine odors are discussed in Chapter 9. Ingestion symptoms-causes/syc-20367333. Published October 27, 2017.
of certain foods, including onions, garlic, and asparagus, can Accessed May 5, 2019.
cause an unusual or pungent odor in urine. Studies have 5. Berman, L: When urine is red. JAMA 237:2753–2754,
shown that although everyone who eats asparagus produces 1977.
6. Reimann, HA: Re: Red urine. JAMA 241(22):2380, 1979.
an odor, only certain people who are genetically predisposed 7. Evans, B: The greening of urine: Still another “Cloret sign.”
can smell it.12 Common causes of urine odors are summarized N Engl J Med 300(4):202, 1979.
in Table 5-5. 8. Bowling, P, Belliveau, RR, and Butler, TJ: Intravenous medications
and green urine. JAMA 246(3):216, 1981.
9. Dealler, SF, et al: Purple urine bags. J Urol 142(3):769–770,
Technical Tip 5-3. Because ions, such as Na+, 1989.
Cl–,and NH4 are important in evaluating renal
+ 10. Smith, C, Arbogast, C, and Phillips, R: Effect of x-ray contrast
media on results for relative density of urine. Clin Chem 19(4):
concentrating ability, the reagent strip method 730–731, 1983.
provides additional information and is not affected 11. Clinical and Laboratory Standards Institute: Urinalysis; Approved
by nonionizing substances, including urea, glucose, Guideline-Third Edition. CLSI document GP16-A3. Clinical and
protein, and contaminating substances such as Laboratory Standards Institute, Wayne, PA, 2009, CLSI.
radiographic dye. 12. Mitchell, SC, et al: Odorous urine following asparagus inges-
tion in man. Experimenta 43(4):382–383, 1987.
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Chapter 5 | Physical Examination of Urine 135

Study Questions
1. The concentration of a normal urine specimen can be 8. Microscopic examination of a clear urine that produces a
estimated by which of the following? white precipitate after refrigeration will show:
A. Color A. Amorphous urates
B. Clarity B. Porphyrins
C. Foam C. Amorphous phosphates
D. Odor D. Yeast
2. The normal yellow color of urine is produced by: 9. The color of urine containing porphyrins will be:
A. Bilirubin A. Yellow-brown
B. Hemoglobin B. Green
C. Urobilinogen C. Orange
D. Urochrome D. Port wine
3. The presence of bilirubin in a urine specimen produces a: 10. Which of the following specific gravities would be most
A. Yellow foam when shaken likely to correlate with a urine that is pale yellow?
B. White foam when shaken A. 1.005
C. Cloudy specimen B. 1.010
D. Yellow-red specimen C. 1.020
D. 1.030
4. A urine specimen containing melanin will appear:
A. Pale pink 11. A urine specific gravity measured by a refractometer is
1.029, and the temperature of the urine is 14°C. The
B. Dark yellow
specific gravity should be reported as:
C. Blue-green
A. 1.023
D. Black
B. 1.027
5. Specimens that contain hemoglobin can be visually C. 1.029
distinguished from those that contain RBCs because:
D. 1.032
A. Hemoglobin produces a clear yellow specimen
12. The principle of refractive index is to compare:
B. Hemoglobin produces a cloudy pink specimen
A. Light velocity in solutions with light velocity
C. RBCs produce a cloudy red specimen
in solids
D. RBCs produce a clear red specimen
B. Light velocity in air with light velocity in solutions
6. A patient with a viscous orange specimen may have been: C. Light scattering by air with light scattering by
A. Treated for a urinary tract infection solutions
B. Taking vitamin B pills D. Light scattering by particles in solution
C. Eating fresh carrots 13. A correlation exists between a specific gravity by a
D. Taking antidepressants refractometer of 1.050 and a:
7. The presence of a pink precipitate in a refrigerated speci- A. 2+ glucose
men is caused by: B. 2+ protein
A. Hemoglobin C. First morning specimen
B. Urobilin D. Radiographic dye infusion
C. Uroerythrin
D. Beets
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136 Part Two | Urinalysis

14. A cloudy urine specimen turns black upon standing and 20. Which of the following colligative properties is not
has a specific gravity of 1.012. The major concern about stated correctly?
this specimen would be: A. The boiling point is raised by solute
A. Color B. The freezing point is raised by solute
B. Turbidity C. The vapor pressure is lowered by solute
C. Specific gravity D. The osmotic pressure is raised by solute
D. All of the above
21. An osmole contains:
15. A specimen with a specific gravity of 1.035 would be A. One gram molecular weight of solute dissolved in 1
considered: liter of solvent
A. Isosthenuric B. One gram molecular weight of solute dissolved in 1
B. Hyposthenuric kilogram of solvent
C. Hypersthenuric C. Two gram molecular weights of solute dissolved in 1
D. Not urine liter of solvent
D. Two gram molecular weights of solute dissolved in 1
16. A specimen with a specific gravity of 1.001 would be
kilogram of solvent
considered:
A. Hyposthenuric 22. The unit of osmolality measured in the clinical
laboratory is the:
B. Not urine
A. Osmole
C. Hypersthenuric
B. Milliosmole
D. Isosthenuric
C. Molecular weight
17. A strong odor of ammonia in a urine specimen could
D. Ionic charge
indicate:
A. Ketones 23. In the reagent strip specific gravity reaction, the
polyelectrolyte:
B. Normalcy
A. Combines with hydrogen ions in response to ion
C. Phenylketonuria
concentration
D. An old specimen
B. Releases hydrogen ions in response to ion
18. The microscopic examination of a clear red urine is concentration
reported as many WBCs and epithelial cells. What does C. Releases hydrogen ions in response to pH
this suggest?
D. Combines with sodium ions in response to pH
A. Urinary tract infection
24. Which of the following will react in the reagent strip
B. Dilute random specimen
specific gravity test?
C. Hematuria
A. Glucose
D. Possible mix-up of specimen and sediment
B. Radiographic dye
19. Which of the following would contribute the most to a C. Protein
urine osmolality?
D. Chloride
A. One osmole of glucose
B. One osmole of urea
C. One osmole of sodium chloride
D. All contribute equally
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Chapter 5 | Physical Examination of Urine 137

Case Studies and Clinical Situations

1. Given the following physical urinalysis results, determine 3. State two pathological causes of a clear red urine.
additional urinalysis results that may be abnormal. a. State a method that could distinguish between the two
a. A green specimen with a strong foul odor of ammonia causes that does not require laboratory testing.
b. A pale yellow urine with a specific gravity of 1.030 4. A woman frequently shops at the farmer’s market near her
c. A dark yellow specimen with yellow foam home. She notices her urine has a red color and brings a
d. A cloudy red urine specimen to her physician. The specimen tests negative
for blood.
2. The urology clinic questions a urinalysis report from the
a. What is a probable cause of the woman’s red urine?
laboratory.
b. The woman collects a specimen at the physician’s of-
The laboratory report states a reagent strip reading of a fice. The color is yellow, and the pH is 5.5. Is this con-
specific gravity of 1.020, protein 3 g/dL, and glucose sistent with the previous answer? Why or why not?
2 g/dL. The specific gravity in the urology clinic was
greater than 1.035. 5. Is a clear urine always normal? Explain your answer.
a. Correct the refractometer reading to account for the
concentrations of protein and glucose. What is the
corrected specific gravity?
b. Do the specific gravities correlate?
c. If the specific gravity was also checked using osmome-
try, should the result agree with the laboratory or the
urology clinic results? Why or why not?
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7582_Ch06_139-166 29/07/20 4:14 PM Page 139

CHAPTER 6
Chemical Examination
of Urine
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
6-1 Describe the proper technique for performing reagent 6-14 Differentiate among hematuria, hemoglobinuria, and
strip testing. myoglobinuria with regard to the appearance of urine
and serum, as well as the clinical significance of each.
6-2 List four causes of premature deterioration of reagent
strips, and describe how to avoid them. 6-15 Describe the chemical principle of the reagent strip
method for blood testing, and list possible causes of
6-3 List five quality-control procedures routinely per-
interference.
formed with reagent strip testing.
6-16 Outline the steps in the degradation of hemoglobin to
6-4 List the reasons for measuring urinary pH, and discuss
bilirubin, urobilinogen, and urobilin.
their clinical applications.
6-17 Describe the relationship of urinary bilirubin and uro-
6-5 Discuss the principle of pH testing by reagent strip.
bilinogen to each of the following diagnoses: bile duct
6-6 Differentiate among prerenal, renal, and postrenal pro- obstruction, liver disease, and hemolytic disorders.
teinuria, and give clinical examples of each.
6-18 Discuss the principle of the reagent strip test for uri-
6-7 Explain the “protein error of indicators,” and list any nary bilirubin, including possible sources of error.
sources of interference that may occur with this
6-19 State two reasons for increased urine urobilinogen and
method of protein testing.
one reason for a decreased urine urobilinogen.
6-8 Discuss microalbuminuria, including significance,
6-20 Discuss the principle of the nitrite reagent strip test for
reagent strip tests, and their principles.
bacteriuria.
6-9 Explain why glucose that is normally reabsorbed in the
6-21 List five possible causes of a false-negative result
proximal convoluted tubule may appear in the urine,
in the reagent strip test for nitrite.
and state the renal threshold levels for glucose.
6-22 State the principle of the reagent strip test for
6-10 Describe the principle of the glucose oxidase method
leukocytes.
of reagent strip testing for glucose, and name possible
causes of interference with this method. 6-23 Discuss the advantages and sources of error of the
reagent strip test for leukocytes.
6-11 Describe the copper reduction method for the detec-
tion of urinary-reducing substances, and discuss the 6-24 Explain the principle of the chemical test for specific
current use of this procedure. gravity.
6-12 Name the three “ketone bodies” appearing in urine 6-25 Compare reagent strip testing for urine specific gravity
and three causes of ketonuria. with osmolality and refractometer testing.
6-13 Discuss the principle of the sodium nitroprusside reac- 6-26 Correlate physical and chemical urinalysis results.
tion to detect ketones, including sensitivity and possi-
ble causes of interference.
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140 Part Two | Urinalysis

KEY TERMS
Ascorbic acid Greiss reaction Orthostatic proteinuria
Bacteriuria Hematuria Postrenal proteinuria
Bilirubin Hemoglobinuria Prerenal proteinuria
Diazo reaction Hemosiderin Protein error of indicators
Ehrlich reaction Jaundice Proteinuria
Fanconi syndrome Ketonuria Renal proteinuria
Ferritin Leukocyturia Stercobilinogen
Glucosuria Microalbuminuria Urobilinogen
Glycosuria Myoglobinuria Uromodulin

Introduction
Routine chemical examination of urine has changed dramati-
cally since the early days of urine testing due to the develop-
ment of the reagent strip method for chemical analysis.
Commercial reagent strips currently provide a simple, rapid
means for performing medically significant chemical analysis
of urine, including pH, protein, glucose, ketones, blood,
bilirubin, urobilinogen, nitrite, leukocytes, and specific grav-
ity. The two major types of reagent strips are manufactured
under the trade names Multistix (Siemens Healthcare Diagnos-
tics, Inc., Tarrytown, NY) and Chemstrip (Roche Diagnostics,
Indianapolis, IN). These products are available with single- or
multiple-testing areas, and the brand and number of tests used
Figure 6–1 Chemical examination of urine comparing reagent
are a matter of laboratory preference. Certain variations relating
strip color reactions using reagent strips consisting of chemical-
to chemical reactions, sensitivity, specificity, and interfering impregnated absorbent pads attached to a plastic strip. (From
substances occur among the products and are discussed in the Strasinger, SK, and DiLorenzo, MA: The Phlebotomy Textbook,
following sections. Reagent strip brands are also specified by 4th ed. FA Davis, Philadelphia, 2019.)
instrumentation manufacturers.
the edge of the strip on the container when withdrawing
it from the specimen, blotting the strip horizontally on an ab-
Reagent Strips sorbent medium, waiting the specified length of time for reactions
Chemical reagent strips consist of chemical-impregnated ab- to take place according to the manufacturer, and then comparing
sorbent pads attached to a plastic strip (Fig. 6-1). A color- the colored reactions against the manufacturer’s chart using a
producing chemical reaction takes place when the absorbent good light source.
pad comes in contact with urine. The reactions are interpreted
Errors Caused by Improper Technique
by comparing the color produced on the pad within the required
time frame with a chart supplied by the manufacturer (Fig. 6-2). 1. Formed elements, such as red and white blood cells,
Several colors or intensities of a color for each substance being sink to the bottom of the specimen and will be unde-
tested appear on the chart. By careful comparison of the colors tected in an unmixed specimen.
on the chart with the strip, a semiquantitative value of trace, 2. Allowing the strip to remain in the urine for an
1+, 2+, 3+, or 4+ can be reported. An estimate of the mil- extended period may cause leaching of reagents
ligrams per deciliter present is available for appropriate testing from the pads.
areas. Automated reagent strip readers also provide Système
3. Allowing excess urine to remain on the strip after its
International (SI) units.
removal from the specimen can produce a runover
between chemicals on adjacent pads, producing distor-
Reagent Strip Technique tion of the colors. To ensure against runover, blotting the
The testing methodology includes dipping the reagent strip edge of the strip on absorbent paper and holding the
completely, but briefly, into a well-mixed specimen at room strip horizontally while comparing it with the color
temperature, removing excess urine from the strip by running chart is recommended.
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Chapter 6 | Chemical Examination of Urine 141

5. A good light source is essential for accurate interpreta-

tion of color reactions.
6. The strip must be held close to the color chart without
actually being placed on the chart. Manufacturers vary
in the direction of the reagent strip to the color chart on
the container when reading results. Automated reagent
strip instruments standardize the color interpretation
and timing of the reaction and are not subject to room
lighting deficiencies or inconsistency among laboratory
personnel (Chapter 2).
7. Reagent strips and color charts from different manufac-
A turers are not interchangeable.
8. Specimens that have been refrigerated must be allowed
to return to room temperature before reagent strip
testing, as the enzymatic reactions on the strips are
temperature dependent.

Handling and Storing Reagent Strips

In addition to using the correct testing technique, reagent strips
must be protected from deterioration caused by moisture,
volatile chemicals, heat, and light. Reagent strips are packaged
in opaque containers with a desiccant to protect them from
light and moisture. Strips are removed just before testing, and
the bottle is tightly resealed immediately. Bottles should not be
opened in the presence of volatile fumes. Manufacturers rec-
ommend that reagent strips be stored at room temperature
below 30°C (but never refrigerated). All bottles are stamped
with an expiration date that represents the functional life ex-
pectancy of the chemical pads. Reagent strips must not be used
past the expiration date. Care must be taken not to touch the
chemical pads when removing the strips. A visual inspection
of the strip should be done each time a strip is used to detect
deterioration, even though the strips may still be within the
expiration date.

PROCEDURE 6-1
Reagent Strip Technique1,2
Visit www.fadavis.com for Video 6-1 (Chemical
testing of urine).
B
Procedure
Figure 6–2 (A) Siemens Multistix 10 SG Reagent Strips Color Chart
for Urinalysis. (B) Roche Chemstrip 10SG Reagent Strips Color Chart. 1. Dip the reagent strip briefly into a well-mixed,
(Courtesy of Roche Diagnostics Corporation.) uncentrifuged urine specimen at room temperature.
2. Remove excess urine by touching the edge of
the strip to the container as the strip is withdrawn.
3. Blot the edge of the strip on a disposable absorbent pad.
4. The timing for reactions to take place varies between
tests and manufacturers and ranges from 30 seconds to 4. Wait the specified amount of time for the reaction to
120 seconds for leukocyte esterase (LE). For the best occur.
semiquantitative results, the manufacturer’s stated time 5. Compare the color reaction of the strip pads to the
should be followed; however, when precise timing can- manufacturer’s color chart in good lighting.
not be achieved, manufacturers recommend that reac- 6. Read the results at the correct time, and record the
tions be read between 60 and 120 seconds, with the LE results.
reaction read at 120 seconds.
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142 Part Two | Urinalysis

Quality Control of Reagent Strips SUMMARY 61 Reagent Strip Testing

Reagent strips must be checked with both positive and negative
Care of Reagent Strips
controls according to the frequency established by the labora-
tory policy. Many laboratories perform this check at the begin- 1. Store with desiccant in an opaque, tightly closed
ning of each shift. Testing is also performed when a new bottle container.
of reagent strips is opened, questionable results are obtained, 2. Store below 30°C; do not freeze.
or there is concern about the integrity of the strips. All quality 3. Do not expose to volatile fumes.
control results must be recorded following laboratory protocol.
4. Do not use past the expiration date.
Several companies manufacture both positive and negative
controls. Distilled water is not recommended as a negative con- 5. Do not use if chemical pads become discolored.
trol because reagent strip chemical reactions are designed to 6. Remove strips immediately before use.
perform at ionic concentrations similar to urine. All readings of
Technique
the negative control must be negative, and positive control read-
ings should agree with the published value. Results that do not 1. Mix specimen well.
agree with the published values must be resolved through the 2. Let refrigerated specimens warm to room tempera-
testing of additional strips and controls (see Chapter 1). ture before testing.
Demonstration of chemically acceptable reagent strips 3. Dip the strip completely, but briefly, into the
does not entirely rule out the possibility of inaccurate results. specimen.
Interfering substances in the urine, technical carelessness, and
4. Remove excess urine by withdrawing the strip against
color blindness also produce errors. Reagent strip manufactur-
the rim of the container and by blotting the edge of
ers have published information concerning the limitations
the strip.
(e.g., interfering substances, sensitivities) of their chemical
reactions, and laboratory personnel should be aware of these 5. Compare reaction colors with the manufacturer’s
conditions. As mentioned in Chapter 5, a primary example of chart under a good light source at the specified time.
reagent strip interference is the masking of color reactions by 6. Perform confirmatory tests when indicated.
the orange pigment present in the urine of people taking 7. Be alert for the presence of interfering substances.
phenazopyridine compounds. If laboratory personnel do not
8. Understand the principles and significance of the test;
recognize the presence of this pigment or other pigments, they
read package inserts.
will report many erroneous results.
9. Relate chemical findings to each other and to the
Confirmatory Testing physical and microscopic urinalysis results.
Confirmatory tests are procedures using different reagents or Quality Control
methodologies to detect the same substances as detected by the 1. Test open bottles of reagent strips with known posi-
reagent strips with the same or greater sensitivity or specificity.3 tive and negative controls per facility protocol.
Nonreagent strip testing procedures using tablets and liquid
2. Resolve control results that are out of range by further
chemicals may be available when questionable results are ob-
testing.
tained or highly pigmented specimens are encountered. In the
past, many of these procedures were used routinely to confirm 3. Test reagents used in confirmatory tests with positive
positive results. Increased specificity and sensitivity of reagent and negative controls.
strips and the use of automated strip readers have reduced the 4. Perform positive and negative controls on new
need for routine use of these procedures.3 The chemical reliabil- reagents and newly opened bottles of reagent strips.
ity of these procedures also must be checked using positive and 5. Record all control results and reagent lot numbers.
negative controls.
Specific confirmatory tests are discussed in this chapter
under their specific sections or the Historical Notes devoted to
the chemical parameters for which they are used. The princi-
phosphate, and weak organic acids and by the reabsorption
ples and procedures for these tests are included to provide ad-
of bicarbonate from the filtrate in the convoluted tubules (see
ditional information on the principles of the reagent strips and
Chapter 3). A healthy individual usually produces a first
to provide the methodology to perform these tests if necessary.
morning specimen with a slightly acidic pH of 5.0 to 6.0; a
Facility protocol will determine the situations when they
more alkaline pH is found after meals (alkaline tide). The
should be performed.
pH of normal random specimens can range from 4.5 to 8.0.
Consequently, no normal values are assigned to urinary pH,
pH and it must be considered in conjunction with other patient
information, such as the acid–base content of the blood, the
Along with the lungs, the kidneys are the major regulators of patient’s renal function, the presence of a urinary tract infec-
the acid–base content in the body. They do this through the tion (UTI), the patient’s dietary intake, and the age of the
secretion of hydrogen in the form of ammonium ions, hydrogen specimen (Table 6-1).
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Chapter 6 | Chemical Examination of Urine 143

Table 6–1 Causes of Acid and Alkaline Urine formation of bicarbonate after digestion of many fruits and veg-
etables. An exception to the rule is in patients who consume
Acid Urine Alkaline Urine cranberry juice or supplements, which produce an acidic urine
and have long been used as a home remedy for minor bladder
Emphysema Hyperventilation
infections because they inhibit the colonization of certain urinary
Diabetes mellitus Vomiting pathogens. People who are prone to frequent UTIs are often ad-
Starvation Renal tubular acidosis vised to drink cranberry juice or take over-the-counter cranberry
Dehydration Presence of urease- pills. Medications prescribed for UTIs, such as methenamine
producing bacteria mandelate (Mandelamine) and fosfomycin tromethamine
(Monurol), are metabolized to produce an acidic urine.
Diarrhea Vegetarian diet
The pH of freshly excreted urine does not reach above 8.5
Presence of acid-producing Old specimens in normal or abnormal conditions. A pH above 8.5 is associ-
bacteria (Escherichia coli) ated with a specimen that has been preserved improperly and
High-protein diet indicates that a fresh specimen should be obtained to ensure
Cranberry juice the validity of the analysis.
Medications (methenamine
mandelate Technical Tip 6-1. Collecting specimens in containers
[Mandelamine], other than the single-use laboratory-supplied contain-
fosfomycin ers can produce a pH above 8.5 if alkaline detergent
tromethamine remains in the container.
[Monurol])

Reagent Strip Reactions

Clinical Significance The Multistix and Chemstrip brands of reagent strips measure
urine pH in 0.5- or 1-unit increments between pH 5 to 8.5 visu-
The importance of urinary pH is primarily as an aid in deter- ally and pH 5 to 9 instrumentally.2 To differentiate pH units
mining the existence of systemic acid–base disorders of meta- throughout this wide range, both manufacturers use a double-
bolic or respiratory origin and in the management of urinary indicator system of methyl red and bromothymol blue. Methyl
conditions that require the urine to be maintained at a specific red produces a color change from red to yellow in the pH range
pH. In respiratory or metabolic acidosis not related to renal 4 to 6, and bromothymol blue turns from yellow to blue in the
function disorders, the urine is acidic; conversely, if respiratory range of 6 to 9. Therefore, in the pH range 5 to 9 measured by
or metabolic alkalosis is present, the urine is alkaline. There- the reagent strips, colors progress from orange at pH 5 through
fore, a urinary pH that does not conform to this pattern may yellow and green to a final deep blue at pH 9.
be used to rule out the suspected condition, or, as discussed
in Chapter 4, it may indicate a disorder resulting from the Methyl red + H+ → bromothymol blue – H+
kidneys’ inability to secrete or to reabsorb acid or base. (Red-orange → yellow) (green → blue)
The precipitation of inorganic chemicals dissolved in
the urine forms urinary crystals and renal calculi. This precip- No known substances interfere with urinary pH measure-
itation depends on urinary pH and can be controlled by ments performed by reagent strips. However, bacterial growth
maintaining the urine at a pH that is incompatible with the by certain organisms in a specimen may cause a marked alka-
precipitation of the particular chemicals causing the calculi for- line shift, usually because of urea conversion to ammonia.2
mation. For example, calcium oxalate, a frequent constituent
of renal calculi, precipitates primarily in acidic and not alkaline
urine. Therefore, maintaining urine at an alkaline pH discour-
Technical Tip 6-2. Care must be taken to prevent
runover between the pH testing area and the adja-
ages formation of the calculi. Knowledge of urinary pH is im-
cent, highly acidic protein testing area on Multistix, as
portant in the identification of crystals observed during
this may produce a reading that is falsely acidic in an
microscopic examination of the urine sediment. This will be
alkaline urine.
discussed in detail in Chapter 7.
Maintaining an acidic urine can be valuable in treating
UTIs caused by urea-splitting organisms because they do not
multiply as readily in an acidic medium. These same organisms Protein
are also responsible for the highly alkaline pH found in speci-
mens that have been allowed to sit unpreserved for extended Of the routine chemical tests performed on urine, the most in-
periods. Urinary pH is controlled primarily by dietary regula- dicative of renal disease is the protein determination. Often
tion, although medications also may be used. People on high- proteinuria is associated with early renal disease, making the
protein and high-meat diets tend to produce acidic urine, urinary protein test an important part of any physical exami-
whereas urine from vegetarians is more alkaline due to the nation. Normal urine contains very little protein: usually, less
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144 Part Two | Urinalysis

of proteinuria are varied and can be grouped into three major

SUMMARY 62 Clinical Significance
categories: prerenal, renal, and postrenal, based on the origin
of Urine pH
of the protein.
• Respiratory or metabolic acidosis/ketosis
Prerenal Proteinuria
• Respiratory or metabolic alkalosis
As the name implies, prerenal proteinuria is caused by condi-
• Defects in renal tubular secretion and reabsorption of
tions affecting the plasma before it reaches the kidney and there-
acids and bases—renal tubular acidosis
fore is not indicative of actual renal disease. Often this condition
• Renal calculi formation and prevention is transient and caused by increased levels of low-molecular-
• Treatment of UTIs weight plasma proteins, such as hemoglobin, myoglobin, and
• Precipitation/identification of crystals the acute-phase reactants associated with infection and inflam-
mation. The increased filtration of these proteins exceeds the
• Determination of unsatisfactory specimens
normal reabsorptive capacity of the renal tubules, resulting in
an overflow of the proteins into the urine. Because reagent strips
detect primarily albumin, prerenal proteinuria is usually not
discovered in a routine urinalysis.
SUMMARY 63 pH Reagent Strip
Bence Jones Protein
Reagents Methyl red, bromothymol blue
Sensitivity Multistix: 5.0–8.5 in 0.5 increments A primary example of proteinuria due to increased levels of
visually; 5.0–9 instrumentally serum protein is the excretion of Bence Jones protein by peo-
ple with multiple myeloma, a proliferative disorder of the
Chemstrip: 5.0–9.0 in 1.0 increments
immunoglobulin-producing plasma cells. In multiple myeloma,
Sources of error/ No known interfering substances the serum contains levels of monoclonal immunoglobulin light
interference Runover from adjacent pads chains (Bence Jones protein) that are markedly elevated. This
Old specimens low-molecular-weight protein is filtered in quantities exceeding
the tubular reabsorption capacity and therefore is excreted in
Bacterial growth
the urine. Suspected cases of multiple myeloma must be diag-
Correlations with Nitrite nosed by performing serum electrophoresis and immunoelec-
other tests Leukocytes trophoresis. The screening test for Bence Jones protein is not
Microscopic performed routinely, as cases of multiple myeloma are easily de-
tected by chemical methods (see the Historical Note, Screening
Test for Bence Jones Protein).
than 10 mg/dL or 100 mg per 24 hours is excreted. This pro- Renal Proteinuria
tein consists primarily of low-molecular-weight serum proteins
that have been filtered by the glomerulus, as well as proteins Proteinuria associated with true renal disease may be the result
produced in the genitourinary tract. Due to its low molecular of damage to the glomerular membrane or tubular dysfunction.
weight, albumin is the major serum protein found in normal Glomerular Proteinuria
urine. Even though it is present in high concentrations in the
plasma, the normal albumin content in urine is low because the When the glomerular membrane is damaged, selective filtra-
majority of albumin presented to the glomerulus is not filtered, tion is impaired and increased amounts of serum protein and,
and much of the filtered albumin is reabsorbed by the tubules.
Other proteins include small amounts of serum and tubular
microglobulins; Tamm-Horsfall protein (THP), also known as
HISTORICAL NOTE
uromodulin, produced by the renal tubular epithelial cells;
and proteins from prostatic, seminal, and vaginal secretions.
Screening Test for Bence Jones Protein
Uromodulin is a more recent name for THP. Uromodulin, a
glycoprotein, is produced routinely in the ascending loop of
Unlike other proteins, which coagulate and remain coagu-
Henle.4 As will be discussed in Chapter 7, uromodulin forms
lated when exposed to heat, Bence Jones protein coagulates
the matrix of casts.
at temperatures between 40°C and 60°C and dissolves when
Clinical Significance the temperature reaches 100°C. Therefore, a specimen that
appears turbid between 40°C and 60°C and clear at 100°C
Demonstration of proteinuria in a routine analysis does not can be suspected of containing Bence Jones protein. Inter-
always signify renal disease; however, its presence does require ference due to other precipitated proteins can be removed
additional testing to determine whether the protein represents by filtering the specimen at 100°C and then observing the
a condition that is normal or pathological. Clinical proteinuria specimen for turbidity as it cools to between 40°C and 60°C.
is indicated at 30 mg/dL or greater (300 mg/L).2,5 The causes
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Chapter 6 | Chemical Examination of Urine 145

eventually, red and white blood cells pass through the mem- believed to account for this condition. Patients suspected of
brane and are excreted in the urine. Conditions that present orthostatic proteinuria are requested to empty the bladder
the glomerular membrane with abnormal substances (e.g., before going to bed, collect a specimen immediately upon aris-
amyloid material, toxic substances, and immune complexes ing in the morning, and collect a second specimen after
found in lupus erythematosus and streptococcal glomeru- remaining in a vertical position for several hours. Both speci-
lonephritis) are major causes of proteinuria due to glomerular mens are tested for protein, and if orthostatic proteinuria is
damage. present, a negative reading will be seen on the first morning
Increased pressure from the blood entering the glomerulus specimen, and a positive result will be found on the second
may override the selective filtration of the glomerulus, causing specimen.
increased albumin to enter the filtrate. This condition may be
reversible, such as occurs during strenuous exercise and dehy- Tubular Proteinuria
dration or is associated with hypertension. Proteinuria that Increased albumin is also present in disorders affecting tubular
occurs during the latter months of pregnancy may indicate a reabsorption because the albumin that is normally filtered can
preeclamptic state and should be considered by the physician no longer be reabsorbed. Other low-molecular-weight proteins
in conjunction with other clinical symptoms, such as hyper- that are usually reabsorbed are also present. Causes of tubular
tension, to determine whether this condition exists. dysfunction include exposure to toxic substances and heavy
The discovery of protein, particularly in a random speci- metals, severe viral infections, and Fanconi syndrome. The
men, is not always of pathological significance because several amount of protein that appears in the urine after glomerular
benign causes of renal proteinuria exist. Benign proteinuria damage ranges from slightly above normal to 4 g/day, whereas
is usually transient and can be produced by conditions such protein levels that are markedly elevated are seldom seen in
as strenuous exercise, high fever, dehydration, and exposure tubular disorders.
to cold.
Postrenal Proteinuria
Microalbuminuria
Protein can be added to a urine specimen as it passes through
The development of diabetic nephropathy leading to reduced the structures of the lower urinary tract (ureters, bladder, ure-
glomerular filtration and eventual renal failure is a common thra, prostate, and vagina). Bacterial and fungal infections and
occurrence in people with both type 1 and type 2 diabetes inflammations produce exudates containing protein from the
mellitus. Onset of renal complications can be predicted interstitial fluid. The presence of blood as the result of injury
first by detection of microalbuminuria (albumin levels in or menstrual contamination contributes protein, as does the
the urine are 20 to 200 mg/L), and the progression of renal presence of prostatic fluid and large amounts of spermatozoa.
disease can be prevented through better stabilization of blood
glucose levels and control of hypertension. The presence of
microalbuminuria also is associated with an increased risk of SUMMARY 64 Clinical Significance
cardiovascular disease.6,7 of Urine Protein
Orthostatic (Postural) Proteinuria Prerenal Tubular Disorders
A persistent benign proteinuria occurs frequently in young Intravascular hemolysis Fanconi syndrome
adults and is termed orthostatic proteinuria, or postural pro-
teinuria. It occurs after periods spent in a vertical posture and Muscle injury Toxic agents/heavy metals
disappears when a horizontal position is assumed. Increased Acute-phase reactants Severe viral infections
pressure on the renal vein when in the vertical position is Multiple myeloma
Renal Postrenal

HISTORICAL NOTE Glomerular disorders Lower urinary tract infections/

inflammation
Microalbuminuria Testing Immune complex Injury/trauma disorders
Amyloidosis Menstrual contamination
Before the development of current reagent strip methods
Toxic agents Prostatic fluid/spermatozoa
that are specific for albumin, detection of microalbumin-
uria required collection of a 24-hour urine specimen. Diabetic nephropathy Vaginal secretions
Specimens were tested using quantitative procedures for Strenuous exercise
albumin. Results were reported in mg of albumin/24 hours Dehydration
or as the albumin excretion rate (AER) in µg/min. With
Hypertension
these methods, microalbumin was considered significant
when 30 to 300 mg of albumin were excreted in 24 hours Preeclampsia
or the AER was 20 to 200 µg/min. Orthostatic or postural proteinuria
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146 Part Two | Urinalysis

Reagent Strip Reactions PROCEDURE 6-2

Traditional reagent strip testing for protein uses the principle of
Sulfosalicylic Acid Precipitation Test
the protein error of indicators to produce a visible colorimetric
reaction. Contrary to the general belief that indicators produce Procedure
specific colors in response to particular pH levels, certain indi- 1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged
cators change color in the presence of protein even though the urine.
pH of the medium remains constant. This is because protein 2. Mix by inversion, and observe for cloudiness.
(primarily albumin) accepts hydrogen ions from the indicator.
3. Grade the degree of turbidity (see the following
The test is more sensitive to albumin because albumin contains
table).
more amino groups to accept the hydrogen ions than other
proteins. Depending on the manufacturer, the protein area of
the strip contains either tetrabromophenol blue (Multistix) or
3′,3″,5′,5″-tetrachlorophenol, 3,4,5,6-tetrabromosulfonphthalein
(Chemstrip) and an acid buffer to maintain the pH at a con-
stant level. At a pH level of 3, both indicators appear yellow in
the absence of protein; however, as the protein concentration
increases, the color progresses through various shades of green
and, finally, to blue. Readings are reported in terms of negative,
trace, 1+, 2+, 3+, and 4+ or the semiquantitative values
of 30, 100, 300, or 2000 mg/dL corresponding to each color
change. Trace values are considered to be less than 30 mg/dL.
Interpretation of trace readings can be difficult. Reporting of
trace values may be a laboratory option.

pH 3.0
Indicator + protein protein + H+
(Yellow) indicator – H+
(blue-green) Table Reporting SSA Turbidity
Reaction Interference Protein Range
Grade Turbidity (mg/dL)
The major source of error with reagent strips occurs with
highly buffered alkaline urine that overrides the acid buffer sys- Negative No increase in Less than 6
tem, producing a rise in pH and a color change unrelated to turbidity
protein concentration. Likewise, a technical error of allowing Trace Noticeable turbidity 6–30
the reagent pad to remain in contact with the urine for a pro-
1+ Distinct turbidity, 30–100
longed period may remove the buffer. False-positive readings
no granulation
are obtained when the reaction does not take place under
acidic conditions. Highly pigmented urine and contamination 2+ Turbidity, granulation, 100–200
of the container with quaternary ammonium compounds, no flocculation
detergents, and antiseptics also cause false-positive readings. 3+ Turbidity, granulation, 200–400
A urine that is visibly bloody may cause results that are falsely flocculation
elevated.2 A false-positive trace reading may occur in speci- 4+ Clumps of protein Greater
mens with a high specific gravity. than 400

Sulfosalicylic Acid Precipitation Test

The sulfosalicylic acid (SSA) test is a cold precipitation test
that reacts equally with all forms of protein. Various con- Technical Tip 6-3. The specific gravity of the urine
centrations and amounts of SSA can be used to precipitate specimen should be considered in evaluating urine
protein, and methods vary greatly among laboratories. All protein, because a trace protein in a dilute specimen is
precipitation tests must be performed on centrifuged speci- more significant than in a concentrated specimen.
mens to remove any extraneous contamination. Based on the
protocol of the laboratory, an SSA test may be performed as
Testing for Microalbuminuria
a confirmatory test in certain situations. This test may not be
relevant to current laboratory practice; however, the proce- Several semiquantitative reagent strip methods have been de-
dure is included in this section to serve as a reference if veloped so that patients at risk for renal disease can be moni-
needed (Procedure 6-2).3 tored using random or first morning specimens. These methods
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Chapter 6 | Chemical Examination of Urine 147

are based on immunochemical assays for albumin or albumin-

SUMMARY 65 Protein Reagent Strip
specific reagent strips that also measure creatinine to produce
an albumin:creatinine (A:C) ratio. Reagents Multistix: Tetrabromophenol blue
Immunochemical assays include the Micral-Test (Roche
Chemstrip: 3’,3”,5’,5”-tetrachlorophenol
Diagnostics, Indianapolis, IN) and the ImmunoDip (Sekisui
Diagnostics, Framingham, MA). Both reagent strips are read 3,4,5,6-tetrabromosulfophthalein
visually, and first morning specimens are recommended. Sensitivity Multistix: 15 to 30 mg/dL albumin
Micral-Test reagent strips contain a gold-labeled antihu- Chemstrip: 6 mg/dL albumin
man albumin antibody–enzyme conjugate. Strips are dipped
Sources of error/ False positive
into the urine up to a level marked on the strip and held for
interference: Highly buffered interference alkaline
5 seconds. Albumin in the urine binds to the antibody. The
bound and unbound conjugates move up the strip by wicking urine
action. Unbound conjugates are removed in a captive zone by Pigmented specimens, phenazopyridine
combining with albumin embedded in the strip. The urine Quaternary ammonium compounds
albumin–bound conjugates continue up the strip and reach an (detergents)
area containing enzyme substrate. The conjugated enzyme
Antiseptics, chlorhexidine
reacts with the substrate, producing colors ranging from white
to red. The amount of color produced represents the amount Loss of buffer from prolonged exposure
of albumin present in the urine. The color is compared with a of the strip to the specimen reagent
chart on the reagent strip bottle after 1 minute. Results range High specific gravity
from 0 to 10 mg/dL. False negative
The ImmunoDip reagent strip uses an immunochromo-
Proteins other than albumin
graphic technique. Strips are packaged individually in spe-
cially designed containers. The container is placed in the urine Microalbuminuria
specimen for 3 minutes. A controlled amount of urine enters Correlations Blood
the container through a vent hole. The urine encounters blue with other Nitrite
latex particles coated with antihuman albumin antibody. Al- tests:
Leukocytes
bumin in the urine binds with the coated particles. The bound
and unbound particles continue to migrate up the strip. The Microscopic
migration is controlled by the size of the particles; unbound
particles do not migrate as far as the bound particles. First a
blue band is formed by the unbound particles. The bound
particles continue to migrate and form a second blue band far- Reagent Strip Reactions
ther up the strip. Therefore, the top band represents the
bound particles (urine albumin), and the bottom band repre- Albumin
sents unbound particles. The color intensity of the bands is Albumin reagent strips use the dye bis(3’,3”-diiodo-4’,
compared against the manufacturer’s color chart. A darker bot- 4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetrabromo sulphon-
tom band represents less than 1.2 mg/dL, equal band colors phthalein (DIDNTB), which has a higher sensitivity and speci-
represent 1.2 to 1.8 mg/dL, and a darker top band represents ficity for albumin. Whereas conventional protein reagent pads
2.0 to 8.0 mg/dL of albumin. A darker bottom band is nega- have a sensitivity of 30 mg/dL or greater and may include proteins
tive, equal band color is borderline, and a darker top band other than albumin, the DIDNTB strips can measure albumin be-
represents positive results. tween 8 and 15 mg/dL (80 and 150 mg/L) without inclusion of
other proteins. Reaction interference by highly buffered alkaline
Albumin:Creatinine Ratio urine (always a concern with conventional reagent strips) is con-
trolled by using paper treated with bis-(heptapropylene glycol)
The Clinitek Microalbumin reagent strips and the Multistix Pro carbonate. The addition of polymethyl vinyl ether decreases
reagent strips (Siemens Healthcare Diagnostics, Inc., Tarrytown, the nonspecific binding of polyamino acids to the albumin pad.
NY) provide simultaneous measurement of albumin/protein Colors range from pale green to aqua blue. Falsely elevated results
and creatinine that permits an estimation of the 24-hour can be caused by urine that is visibly bloody, whereas urine that
microalbumin excretion.8 As discussed in Chapter 4, creatinine is abnormally colored may interfere with the readings.2
is produced and excreted at a consistent rate for each individ-
ual. Therefore, by comparing the albumin excretion to the cre- Creatinine
atinine excretion, the albumin reading can be corrected for The principle of the reagent strip for creatinine is based on the
either overhydration or dehydration in a random specimen. In pseudoperoxidase activity of copper–creatinine complexes. The
addition to including creatinine on the reagent strip, the albu- reaction follows the same principle as the reaction for blood on
min low-test pad is changed to a dye-binding reaction that is the reagent strips discussed later in this chapter. Reagent strips
more specific for albumin than the protein error of indicators’ contain copper sulfate (CuSO4), 3,3’,5,5’-tetramethylbenzidine
reaction on strips measuring protein. (TMB), and diisopropyl benzene dihydroperoxide (DBDH).
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148 Part Two | Urinalysis

Creatinine in the urine combines with the copper sulfate to these strips. The strips can be read manually or on automated
form copper–creatinine peroxidase. This reacts with the perox- Clinitek instruments. The protein-high reaction uses the pro-
ide DBDH, releasing oxygen ions that oxidize the chromogen tein error of indicators principle, whereas the protein-low
TMB and producing a color change from orange through green reaction is the dye-binding method discussed previously.
to blue.5 Results are reported as the protein:creatinine ratio, although
the protein-low result is used in the calculation. Results from
CuSO4 + CRE → Cu(CRE) peroxidase the Clinitek are calculated automatically. Results are reported
Cu(CRE) peroxidase as normal or abnormal. A result of normal dilute indicates that
DBDH + TMB oxidized TMB + H2O the specimen should be re-collected, making sure it is a first
(peroxidase) (chromogen) (orange to blue) morning specimen.
When the reagent strip is read manually, a manufacturer-
Results are reported as 10, 50, 100, 200, or 300 mg/dL or
supplied chart is used to determine the ratio based on the results
0.9, 4.4, 8.8, 17.7, or 26.5 mmol/L of creatinine.
of the readings for protein-high, protein-low, and creatinine.
Reagent strips are unable to detect the absence of creati-
When using this chart, the higher of the protein-low or protein-
nine. Results that are falsely elevated can be caused by urine
high result is used (Fig. 6-3).9
that is visibly bloody, as well as the presence of the gastric acid–
reducing medication cimetidine (Tagamet). Urine that is
abnormally colored also may interfere with the readings. Glucose
No creatinine readings are considered abnormal, as creati-
Because of its value in the detection and monitoring of
nine is normally present in concentrations of 10 to 300 mg/dL.
diabetes mellitus, the glucose test is the chemical analysis
The purpose of the creatinine measurement is to correlate the
performed most frequently on urine. Due to the nonspecific
albumin concentration to the urine concentration, producing a
symptoms associated with the onset of diabetes, it is esti-
semiquantitative albumin:creatinine ratio (A:C) ratio.
mated that more than half of the cases in the world are undi-
Albumin/Protein:Creatinine Ratio agnosed. Therefore, blood and urine glucose tests are included
Both automated and manual methods are available for determin- in all physical examinations and are often the focus of mass
ing the A:C ratio based on the reactions discussed previously. health screening programs. Early diagnosis of diabetes melli-
The Clinitek Microalbumin reagent strips are designed for in- tus through blood and urine glucose tests provides a prog-
strumental use only. Strips are read on Clinitek Urine Chemistry nosis that is greatly improved. Using reagent strip methods
Analyzers. The strips measure only albumin and creatinine, and for both blood and urine glucose testing that are currently
the analyzer calculates the A:C ratio automatically. Results are available, patients can monitor themselves at home and can
displayed and printed out for albumin, creatinine, and the A:C detect regulatory problems before the development of serious
ratio in both conventional and SI units. Abnormal results for the complications.
A:C ratio are 30 to 300 mg/g or 3.4 to 33.9 mg/mmol.8
The Siemens Multistix Pro 10 reagent strips include reagent
Clinical Significance
pads for creatinine, protein-high, and protein-low (albumin), Under normal circumstances, almost all the glucose filtered by
along with pads for glucose, ketones, blood, nitrite, LE, pH, the glomerulus is actively reabsorbed in the proximal convoluted
bilirubin, and specific gravity. Urobilinogen is not included on tubule; therefore, urine contains only minute amounts of glucose.

SUMMARY 66 Microalbumin Testing

Immunological Tests
Albumin:Creatinine Ratio
Micral-Test ImmunoDip Clinitest Microalbumin Strips/Multistix-Pro
Principle Enzyme immunoassay Immunochromographics Sensitive albumin tests related to creatinine
concentration to correct for patient
hydration
Sensitivity 0–10 mg/dL 1.2–8.0 mg/dL Albumin: 10–150 mg/L
Creatinine: 10–300 mg/dL, 0.9–26.5 mmol/L
Reagents Gold-labeled antibody Antibody-coated blue Albumin: dye DIDNTB
B-galactosidase latex particles Creatinine: CuSO4, 3,3’,5,5’-TMB, and DBDH
Chlorophenol red
galactoside
Interference False negative: Dilute False negative: Dilute Visibly bloody or abnormally colored urine
urine urine Creatinine: cimetidine: False positive
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Chapter 6 | Chemical Examination of Urine 149

Reported Creatinine result increased levels of circulating glucose. Epinephrine is also a

protein (mg/dL) strong inhibitor of insulin secretion and is increased when the
result
(mg/dL) 10 50 100 200 300 body is subjected to severe stress, which accounts for the
Negative Recollect* No glucosuria seen in conjunction with cerebrovascular trauma
rm and myocardial infarction.
15 al
Ab Glycosuria occurs in the absence of hyperglycemia when
no the reabsorption of glucose by the renal tubules is compro-
30 rm
100, 300,
al mised. This is frequently referred to as “renal glycosuria” and
or 2000 is seen in end-stage renal disease, cystinosis, and Fanconi syn-
*Specimen is too dilute to determine ratio result accurately. Repeat test on new drome. Glycosuria not associated with gestational diabetes is
specimen, preferably a first-morning collection.
seen occasionally as a result of a temporary lowering of the
Figure 6–3 A protein-creatinine ratio determination chart. (Image renal threshold for glucose during pregnancy.
adapted from Bayer HealthCare LLC, Elkhart, IN.)
Reagent Strip (Glucose Oxidase) Reaction
The glucose oxidase procedure provides a specific test for glu-
Tubular reabsorption of glucose is by active transport in response cose. Reagent strips employ the glucose oxidase testing method
to the body’s need to maintain an adequate concentration of by impregnating the testing area with a mixture of glucose ox-
glucose. Should the blood level of glucose become elevated idase, peroxidase, chromogen, and buffer to produce a double
(hyperglycemia), as occurs in diabetes mellitus, the tubular sequential enzyme reaction. In the first step, glucose oxidase
transport of glucose has reached its renal threshold, and glucose catalyzes a reaction between glucose and room air (oxygen) to
appears in the urine. The blood level at which tubular reabsorp- produce gluconic acid and peroxide. In the second step, per-
tion stops (renal threshold) for glucose is approximately 160 to oxidase catalyzes the reaction between peroxide and chro-
180 mg/dL. Blood glucose levels fluctuate, and a typical person mogen to form an oxidized colored compound that is
who is nonfasting may have glycosuria after a meal containing a produced in direct proportion to the concentration of glucose.
high glucose content. Therefore, the most informative glucose re-
sults are obtained from specimens collected under controlled Glucose oxidase
1. Glucose + O2 (air) gluconic acid + H2O2
conditions. Fasting before the collection of specimens for screen-
ing tests is recommended. For purposes of diabetes monitoring, Peroxidase
2. H2O2 + chromogen oxidized
specimens are usually tested 2 hours after meals. A first morning
colored chromogen + H2O
specimen does not always represent a fasting specimen because
glucose from an evening meal may remain in the bladder Reagent strip manufacturers use several different chro-
overnight, and patients should be advised to empty the bladder mogens, including potassium iodide (green to brown) (Multistix)
and collect the second specimen.2 and tetramethylbenzidine (yellow to green) (Chemstrip). Urine
Hyperglycemia that occurs during pregnancy and disap- glucose may be reported in terms of negative, trace, 1+, 2+, 3+,
pears after delivery is called gestational diabetes. The onset of and 4+; however, the color charts also provide semiquantitative
hyperglycemia and glycosuria is normally around the sixth measurements ranging from 100 mg/dL to 2 g/dL or 0.1% to 2%.
month of pregnancy, although glycosuria may occur sooner. The American Diabetes Association recommends semiquantita-
Hormones secreted by the placenta block the action of insulin, tive reporting.
resulting in insulin resistance and hyperglycemia. Detection of
gestational diabetes is important to the welfare of both the
mother and baby because glucose crosses the placenta, whereas
insulin does not. The baby develops high glucose levels, causing SUMMARY 67 Clinical Significance
the baby’s pancreas to produce more insulin. The excess glucose of Urine Glucose
transferred to the baby is stored as fat, resulting in a large baby
Hyperglycemia-Associated Renal-Associated
(macrosomia) at risk for obesity and, later, type 2 diabetes.
Women who have gestational diabetes also are prone to devel- Diabetes mellitus Fanconi syndrome
oping type 2 diabetes mellitus in later years. Pancreatitis Advanced renal disease
Hyperglycemia of nondiabetic origin is seen in a variety of
Pancreatic cancer Osteomalacia
disorders and also produces glycosuria. Many of these disorders
are associated with hormonal function and include pancreatitis, Acromegaly Pregnancy
acromegaly, Cushing syndrome, hyperthyroidism, pheochromo- Cushing syndrome
cytoma, and thyrotoxicosis. The hormones glucagon, epinephrine, Hyperthyroidism
cortisol, thyroxine, and growth hormone, which are increased
Pheochromocytoma
in these disorders, work in opposition to insulin, thereby pro-
ducing hyperglycemia and glucosuria. Whereas a primary Central nervous system damage
function of insulin is to convert glucose to glycogen for storage Stress
(glycogenesis), these opposing hormones cause the break- Gestational diabetes
down of glycogen to glucose (glycogenolysis), resulting in
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150 Part Two | Urinalysis

Reaction Interference SUMMARY 68 Glucose Reagent Strip

Because the glucose oxidase method is specific for glucose,
Reagents Multistix
false-positive reactions are not obtained from other urinary con-
stituents, including reducing sugars that may be present. False- Glucose oxidase
positive reactions may occur, however, if containers become Peroxidase
contaminated with peroxide or strong oxidizing detergents from Potassium iodide
disinfectants used on laboratory instruments.
Chemstrip
Substances that interfere with the enzymatic reaction or
strong reducing agents, such as ascorbic acid, that prevent Glucose oxidase
oxidation of the chromogen may produce false-negative re- Peroxidase
sults. To minimize interference from ascorbic acid, reagent Tetramethylbenzidine
strip manufacturers are incorporating additional chemicals
Sensitivity Multistix: 75 to 125 mg/dL
into the test pads. An example is iodate that oxidizes ascorbic
acid so that it cannot interfere with the oxidation of the chro- Chemstrip: 40 mg/dL
mogen. Product literature should be reviewed carefully for Interference False positive
current information regarding all interfering substances. High Contamination by oxidizing
levels of ketones also affect glucose oxidase tests at low glucose agents and detergents
concentrations; however, because high levels of ketones are
False negative
usually accompanied by marked glycosuria, this seldom pres-
ents a problem. High specific gravity and low temperature High levels of ascorbic acid
may decrease the sensitivity of the test. By far, the greatest High levels of ketones
source of false-negative glucose results is the technical error High specific gravity
of allowing specimens to remain unpreserved at room tem-
Low temperatures
perature for extended periods, subjecting the glucose to
bacterial degradation. Improperly preserved specimens
Correlations Ketones
Copper Reduction Test (Clinitest) with other tests Protein
Measurement of glucose by the copper reduction method was
one of the earliest chemical tests performed on urine. The test
relies on the ability of glucose and other substances to reduce
copper sulfate to cuprous oxide in the presence of alkali and
gently, and the color, ranging from blue to orange/red, can be
heat. A color change progressing from a negative blue (CuSO4)
compared with the manufacturer’s color chart to determine the
through green, yellow, and orange/red (Cu2O) occurs when the
approximate amount of reducing substance.
reaction takes place.
Care must be taken to observe the reaction closely as it
is taking place because at high glucose levels, a phenomenon
Heat
CuSO4 (cupric sulfide) + reducing substance known as “pass through” may occur. When this happens,
Alkali the color produced passes through the orange/red stage and
Cu2O (cuprous oxide) + oxidized substance → color returns to a green-brown color, and if not observed, a high
(blue/green → orange/red) glucose level may be reported as negative. An alternative
method using two drops instead of five drops of urine can
The classic Benedict solution was developed in 1908 and minimize the occurrence of “pass through.” A separate color
contained copper sulfate, sodium carbonate, and sodium citrate chart must be used to interpret the reaction. This chart pro-
buffer.10 Urine was added to the solution, heat was applied, and vides values up to 5 g/dL, whereas the five-drop method is
the resulting precipitate was observed for color. A more con- limited to 2 g/dL.
venient method that employs Benedict’s principle is the Clinitest The sensitivity of Clinitest to glucose is reduced to a mini-
tablet (Siemens Healthcare Diagnostics, Inc., Tarrytown, NY). mum of 200 mg/dL, so the Clinitest cannot be used as a confir-
The tablets contain copper sulfate, sodium carbonate, sodium matory test for glucose. As a nonspecific test for reducing
citrate, and sodium hydroxide. Upon addition of the tablet to substances, Clinitest is subject to interference from other reducing
water and urine, heat is produced by the hydrolysis of sodium sugars, including galactose, lactose, fructose, maltose, pentose,
hydroxide and its reaction with sodium citrate, and carbon ascorbic acid, certain drug metabolites, and antibiotics, such as
dioxide is released from the sodium carbonate to prevent room the cephalosporins.
air from interfering with the reduction reaction. Thick-walled Clinitest tablets are very hygroscopic and should be stored
tubes should be placed in a heat-resistant rack and not held in in their original, tightly closed packages. A strong blue color
the hand because the reaction heat could cause a burn. At the in the unused tablets suggests deterioration due to moisture
conclusion of the effervescent reaction, the tube is shaken accumulation, as does vigorous tablet fizzing.
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Chapter 6 | Chemical Examination of Urine 151

Clinical Significance of Clinitest Ketones

In addition to glucose, commonly found reducing sugars The term “ketones” represents three intermediate products of
include galactose, fructose, pentose, and lactose, of which fat metabolism, namely, acetone (2%), acetoacetic acid (20%),
galactose is the most clinically significant. Galactose in the and β-hydroxybutyrate (78%). Normally, measurable amounts
urine of a newborn represents an “inborn error of metabo- of ketones do not appear in the urine because all the metabo-
lism” in which a lack of the enzyme galactose-1-phosphate lized fat is completely broken down into carbon dioxide and
uridyl transferase (GALT) prevents breakdown of ingested water. However, when the use of available carbohydrate as the
galactose and results in failure to thrive and other complica- major source of energy becomes compromised, body stores of
tions, including death. Depending on the laboratory popu- fat must be metabolized to supply energy. Then ketones are
lation, the Clinitest was performed on all pediatric specimens detected in urine.
from patients up to at least the age of 2 years. However, now
all states have incorporated screening for galactosemia into Clinical Significance
their required newborn screening programs (see Chapter 9)
because early detection followed by dietary restriction can Clinical reasons for increased fat metabolism include the
control the condition. Because of the increased sensitivity inability to metabolize carbohydrate, as occurs in diabetes
and specificity of the newborn blood screening test for the mellitus; increased loss of carbohydrate from vomiting; and
activity of GALT, the urine screening for carbohydrate me- inadequate intake of carbohydrate associated with starvation
tabolism disorders using the Clinitest reagent tablets is no and malabsorption.
longer recommended.11 The appearance of other reducing Testing for urinary ketones is most valuable in the
sugars is usually of minimal clinical significance, and lactose management and monitoring of insulin-dependent (type 1)
is frequently found in the urine of nursing mothers. diabetes mellitus. Ketonuria shows a deficiency in insulin,
indicating the need to regulate dosage. It is often an early
indicator of insufficient insulin dosage in type 1 diabetes and
in patients who experience medical problems in addition to
diabetes. Increased accumulation of ketones in the blood leads
Technical Tip 6-4. Keep in mind that table sugar is su-
to electrolyte imbalance, dehydration, and, if not corrected,
crose, a nonreducing sugar, which does not react with
acidosis and eventual diabetic coma.
Clinitest or glucose oxidase strips. Therefore, it cannot
The use of multiple-test strips in hospital laboratories
be used as a control or in preparation of a laboratory
often produces positive ketone tests unrelated to diabetes
exercise for glucose testing.
because the patient’s illness either prevents adequate intake
or absorption of carbohydrates or produces an accelerated
loss, as in the case of vomiting. Clinics for weight loss and
eating disorders can use a practical application of ketonuria
PROCEDURE 6-3 produced by avoidance of carbohydrates to monitor patients.
Frequent strenuous exercise can cause overuse of available
Clinitest Procedure carbohydrates and produce ketonuria that can be toxic to the
Procedure kidney tubules.
1. Place a thick glass test tube in a rack; add five drops
of urine.
2. Add 10 drops of distilled water to the urine in the
test tube. SUMMARY 69 Clinical Significance
3. Drop one Clinitest tablet into the test tube, and of Urine Ketones
observe the reaction until completion (cessation of
Diabetic acidosis
boiling).
Insulin dosage monitoring
CAUTION: The reaction mixture gets very hot. Do
not touch the bottom area of the test tube. Use a Starvation
thick glass test tube only. Malabsorption/pancreatic disorders
4. Wait 15 seconds after boiling has stopped, and gently Cold exposure
shake the contents of the tube. Strenuous exercise
5. Compare the color of the mixture to the Clinitest Vomiting
color chart, and record the result in mg/dL or per-
Inborn errors of amino acid metabolism (see Chapter 9)
cent. Observe for the possibility of the “pass through”
phenomenon. If present, repeat the procedure using Alcoholism
two drops of urine instead of five drops. Febrile state in children
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152 Part Two | Urinalysis

Reagent Strip Reactions SUMMARY 610 Ketone Reagent Strip

The three ketone compounds are not present in equal amounts
Reagents Sodium nitroprusside
in urine. Both acetone and β-hydroxybutyric acid are produced
from acetoacetic acid (Fig. 6-4). The proportions of 78% Glycine (Chemstrip)
β-hydroxybutyric acid, 20% acetoacetic acid, and 2% acetone Sensitivity Multistix: 5–10 mg/dL
are relatively constant in all specimens. acetoacetic acid
Reagent strip tests use the sodium nitroprusside (nitrofer- Chemstrip: 9 mg/dL ace-
ricyanide) reaction to measure ketones. In this reaction, ace- toacetic acid; 70 mg/dL
toacetic acid in an alkaline medium reacts with sodium acetone
nitroprusside to produce a purple color. The test does not
Interference False positive:
measure β-hydroxybutyrate and is only slightly sensitive to
acetone when glycine is also present; however, inasmuch as these Phthalein dyes
compounds are derived from acetoacetic acid, their presence can Highly pigmented red
be assumed, and it is not necessary to perform individual tests. urine
Results are reported qualitatively as negative, trace, small (1+), Levodopa
moderate (2+), or large (3+), or semiquantitatively as negative,
Medications containing
trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL),
free sulfhydryl groups
or large (80 to 160 mg/dL).
False negative:
Acetoacetate (and acetone) + sodium nitroprusside Improperly preserved
alkaline specimens
+ (glycine) purple color
Correlations Glucose

Reaction Interference
Large amounts of levodopa and medications containing
sulfhydryl groups, including mercaptoethane sulfonate sodium
and lactose in tablet form. The addition of lactose gives better
(MESNA) and captopril, may produce atypical color reactions.
color differentiation. Acetest tablets are hygroscopic; if the
Reactions with interfering substances frequently fade on stand-
specimen is not absorbed completely within 30 seconds, a new
ing, whereas color development from acetoacetic acid in-
tablet should be used. See Procedure 6-4.
creases, which leads to false-positive results from readings that
are improperly timed. Values that are falsely decreased due to
the volatilization of acetone and the breakdown of acetoacetic
acid by bacteria are seen in specimens that have been preserved
Blood
improperly. Blood may be present in the urine either in the form of intact
The Acetest tablet test has been used as a confirmatory red blood cells (RBCs) (hematuria) or as the product of RBC
test for questionable reagent strip results; however, it was destruction, hemoglobin (hemoglobinuria). As discussed in
primarily used for testing serum and other bodily fluids and Chapter 5, blood present in large quantities can be detected
dilutions of these fluids for severe ketosis. The Clinical and visually; hematuria produces a cloudy red urine, and hemo-
Laboratory Standards Institute (CLSI) states that the confir- globinuria appears as a clear red specimen. Because any
matory test for ketones using the Acetest may not be relevant amount of blood greater than five cells per microliter of urine
to current laboratory practice.3 Currently, new methods meas- is considered clinically significant, visual examination cannot
uring β-hydroxybutyrate using reagent strips have been de- be relied upon to detect the presence of blood. Microscopic
veloped to provide automated methods for testing serum and examination of the urinary sediment shows intact RBCs, but
other body fluids. Notice in Figure 6-4 the ketone with the free hemoglobin produced either by hemolytic disorders or
highest concentration is β-hydroxybutyrate. lysis of RBCs is not detected. Therefore, chemical tests for he-
moglobin provide the most accurate means for determining
Acetest Tablets
the presence of blood. Once blood has been detected, the mi-
Acetest (Siemens Healthcare Diagnostics, Inc., Deerfield, IL) croscopic examination can be used to differentiate between
provides sodium nitroprusside, glycine, disodium phosphate, hematuria and hemoglobinuria.

OH O O
+2H –CO2
CH3 C CH2 COOH CH3 C CH2 COOH CH3 C CH3
–2H
H Figure 6–4 Production of acetone and butyrate from
b-hydroxybutyrate Acetoacetic acid Acetone acetoacetic acid.
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Chapter 6 | Chemical Examination of Urine 153

PROCEDURE 6-4 Myoglobinuria

Myoglobin, a heme-containing protein found in muscle tissue,
Acetest Procedure not only reacts positively with the reagent strip test for blood
Procedure but also produces a clear red-brown urine. In myoglobinuria,
1. Remove the Acetest tablet from the bottle, and place the presence of myoglobin rather than hemoglobin should be
it on a clean, dry piece of white paper. suspected in patients with conditions associated with muscle
2. Place one drop of urine on top of the tablet. destruction (rhabdomyolysis). Examples of these conditions
include trauma, crush syndromes, prolonged coma, convul-
3. Wait 30 seconds.
sions, muscle-wasting diseases, alcoholism, heroin abuse, and
4. Compare the tablet color with the manufacturer- extensive exertion. The development of rhabdomyolysis has
supplied color chart. been found to be a side effect in certain patients taking the
5. A positive result is indicated by a purple color. cholesterol-lowering statin medications.12 The heme portion
Report as negative, small, moderate, or large. of myoglobin is toxic to the renal tubules, and high concen-
trations can cause acute renal failure. The massive hemoglo-
binuria seen in hemolytic transfusion reactions also is
associated with acute renal failure.

Clinical Significance Reagent Strip Reactions

The finding of a positive reagent strip test result for blood in- Chemical tests for blood use the pseudoperoxidase activity of
dicates the presence of RBCs, hemoglobin, or myoglobin. Each hemoglobin to catalyze a reaction between the heme compo-
of these has a different clinical significance. nent of both hemoglobin and myoglobin and the chromogen
tetramethylbenzidine to produce an oxidized chromogen,
Hematuria which has a green-blue color.

Hematuria is most closely related to disorders of renal or gen- Hemoglobin

itourinary origin in which bleeding is the result of trauma or H2O2 + chromogen oxidized chromogen + H2O
Peroxidase
damage to the organs of these systems. Major causes of hema-
turia include renal calculi, glomerular diseases, tumors,
trauma, pyelonephritis, exposure to toxic chemicals, and anti- Reagent strip manufacturers incorporate peroxide and
coagulant therapy. The laboratory is frequently requested to tetramethylbenzidine into the blood testing area. Two color
perform a urinalysis when patients presenting with severe back charts are provided that correspond to the reactions that occur
and abdominal pain are suspected of having renal calculi. In
such cases, hematuria is usually of a small to moderate degree,
but its presence can be essential to the diagnosis. Hematuria SUMMARY 611 Clinical Significance
of nonpathological significance is observed after strenuous of a Positive Reaction
exercise and during menstruation. for Blood
Hematuria Brown recluse spider
Hemoglobinuria bites
Renal calculi
Hemoglobinuria may result from the lysis of RBCs produced Glomerulonephritis Myoglobinuria
in the urinary tract, particularly in dilute, alkaline urine. It Muscular trauma
Pyelonephritis
also may result from intravascular hemolysis and the subse-
Tumors Crush injuries
quent filtering of hemoglobin through the glomerulus. Lysis
of RBCs in the urine usually shows a mixture of hemoglo- Trauma Prolonged coma
binuria and hematuria, whereas no RBCs are seen in cases Exposure to toxic chemicals Convulsions
of intravascular hemolysis. Under normal conditions, the Muscle-wasting diseases
Anticoagulants
formation of large hemoglobin–haptoglobin complexes in
Strenuous exercise Alcoholism/overdose
the circulation prevents the glomerular filtration of hemo-
globin. When the amount of free hemoglobin present Hemoglobinuria Drug abuse
exceeds the haptoglobin content—as occurs in hemolytic Transfusion reactions Extensive exertion
anemias, transfusion reactions, severe burns, bites from the Cholesterol-lowering
Hemolytic anemias
brown recluse spider, infections, and strenuous exercise— statin medications
hemoglobin is available for glomerular filtration. Reabsorp- Severe burns
tion of filtered hemoglobin also results in the appearance Infections/malaria
of large yellow-brown granules of denatured ferritin called Strenuous exercise/
hemosiderin in the renal tubular epithelial cells and in the RBC trauma
urine sediment.
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154 Part Two | Urinalysis

with hemoglobinuria, myoglobinuria, and hematuria (RBCs).

SUMMARY 612 Blood Reagent Strip
In the presence of free hemoglobin/myoglobin, a uniform color
ranging from a negative yellow through green to a strongly pos- Reagents Multistix: Diisopropylbenzene
itive green-blue appears on the pad. In contrast, intact RBCs dihydroperoxide and 3,3’,5,5’-
are lysed when they come in contact with the pad, and the lib- tetramethylbenzidine
erated hemoglobin produces an isolated reaction that results
Chemstrip: dimethyldihydroperoxy-
in a speckled pattern on the pad. Reagent strip tests can detect
hexane and tetramethylbenzidine
concentrations as low as five RBCs per microliter; however,
care must be taken when comparing these figures with the ac- Sensitivity Multistix: 5–20 RBCs/mL, 0.015–0.062
tual microscopic values because the absorbent nature of the mg/dL hemoglobin
pad attracts some urine. The terms trace, small, moderate, and Chemstrip: 5 RBCs/mL, hemoglobin
large or trace, 1+, 2+, and 3+ are used for reporting. corresponding to 10 RBCs/mL
Interference False positive:
Reaction Interference
Strong oxidizing agents
False-positive reactions due to menstrual contamination may
Bacterial peroxidases
be seen. They also occur if strong oxidizing detergents, such
as sodium hypochlorite (bleach), are present in the specimen Menstrual contamination
container.2 Vegetable peroxidase and bacterial enzymes, includ- False negative:
ing an Escherichia coli peroxidase, also may cause false-positive High specific gravity/crenated cells
reactions. Therefore, sediments containing bacteria should be
Formalin
checked closely for the presence of RBCs.
Traditionally, ascorbic acid (vitamin C) has been associated Captopril
with false-negative reagent strip reactions for blood. Both Mul- High concentrations of nitrite
tistix and Chemstrip have modified their reagent strips to reduce Ascorbic acid greater than 25 mg/dL
this interference to very high levels (25 mg/dL) of ascorbic acid.
Unmixed specimens
Multistix uses a peroxide that is less subject to reduction by
ascorbic acid, and Chemstrip overlays the reagent pad with an Correlations Protein
iodate-impregnated mesh that oxidizes the ascorbic acid before with other Microscopic
it reaches the reaction pad. False-negative reactions can result tests
when urine with a high specific gravity contains crenated RBCs
that do not lyse when they come in contact with the reagent pad.
Decreased reactivity also may be seen when formalin is used as
a preservative or when the hypertension medication captopril present. RBCs settle to the bottom of the specimen container,
or high concentrations of nitrite (greater than 10 mg/dL) are and failure to mix the specimen before testing causes a reading
that is falsely decreased.

HISTORICAL NOTE Bilirubin

The appearance of bilirubin in the urine can provide an early
Hemoglobinuria Versus Myoglobinuria
indication of liver disease. It is often detected long before the
patient exhibits jaundice.
Before the development of sensitive serum immunoassay
tests for myoglobin, a procedure was used to differentiate Bilirubin Production
between hemoglobin and myoglobin in the urine. A pre-
cipitation test was used to screen for the presence of myo- Bilirubin, a highly pigmented yellow compound, is a degrada-
globin; 2.8 g of ammonium sulfate are added to 5 mL of tion product of hemoglobin. Under normal conditions, the life
centrifuged urine. After mixing and allowing the specimen span of RBCs is approximately 120 days, at which time they
to sit for 5 minutes, the urine was filtered or centrifuged, are destroyed in the spleen and liver by the phagocytic cells of
and the supernatant was tested for a reaction for blood with the reticuloendothelial system. The liberated hemoglobin is
a reagent strip. The principle of this screening test was broken down into its component parts: iron, protein, and pro-
based on the fact that the larger hemoglobin molecules toporphyrin. The body reuses the iron and protein, and the
were precipitated by the ammonium sulfate and myoglobin cells of the reticuloendothelial system convert the remaining
remained in the supernatant. Therefore, when myoglobin protoporphyrin to bilirubin. Then the bilirubin is released into
was present, the supernatant retained the red color and circulation, where it binds with albumin and is transported to
gave a positive reagent strip test for blood. Conversely, he- the liver. At this point, the kidneys cannot excrete the circu-
moglobin produced a red precipitate and a supernatant lating bilirubin because not only is it bound to albumin but
that tested negative for blood. also it is water insoluble (unconjugated bilirubin). In the liver,
bilirubin is conjugated with glucuronic acid by the action of
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Chapter 6 | Chemical Examination of Urine 155

glucuronyl transferase to form water-soluble bilirubin diglu- Table 6–2 Urine Bilirubin and Urobilinogen in
curonide (conjugated bilirubin). Usually, this conjugated Jaundice
bilirubin does not appear in the urine because it is passed di-
rectly from the liver into the bile duct and on to the intestine. Urine Bilirubin Urine Urobilinogen
In the intestine, intestinal bacteria reduce bilirubin to uro- Bile duct +++ Normal
bilinogen, which is oxidized and excreted in the feces in the obstruction
form of stercobilinogen and urobilin. Figure 6-5 illustrates
bilirubin metabolism for reference with this section and the Liver damage + or – ++
subsequent discussion of urobilinogen. Hemolytic Negative +++
disease
Clinical Significance
Only conjugated bilirubin can appear in the urine when the
normal degradation cycle is disrupted by bile duct obstruc- SUMMARY 613 Clinical Significance
tion (posthepatic jaundice) (e.g., gallstones or cancer) or of Urine Bilirubin
when the integrity of the liver is damaged (hepatic jaundice),
allowing leakage of conjugated bilirubin into circulation. Hepatitis
Hepatitis and cirrhosis are common examples of conditions Cirrhosis
that produce liver damage resulting in bilirubinuria. Not
only does the detection of urinary bilirubin provide an early Other liver disorders
indication of liver disease, but also its presence or absence Biliary obstruction (gallstones, carcinoma)
can be used in determining the cause of clinical jaundice. As
shown in Table 6-2, this determination can be even more
significant when bilirubin results are combined with urinary
urobilinogen. Jaundice due to increased destruction of RBCs
Reagent Strip (Diazo) Reactions
does not produce bilirubinuria. This is because the serum Routine testing for urinary bilirubin by reagent strip uses the
bilirubin is present in the unconjugated form and the kid- diazo reaction. Bilirubin combines with 2,4-dichloroaniline di-
neys cannot excrete it. azonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate

RBC

Hemoglobin

Ferritin Iron Heme Globin Amino

acid pool Plasma
Heme
Developing Biliverdin
oxygenase Unconjugated
erythroblasts bilirubin albumin
marrow Bilirubin
(unconjugated)
CO
Bilirubin
Glucuronyl transferase + Liver
Glucuronic
Lungs acid
Kidney
Bilirubin diglucuronide Bile ducts
(conjugated)

Urobilinogen
Bacterial
Enterohepatic circulation enzymes
Urine
Urobilinogen Stercobilinogen

Fecal urobilin

Figure 6–5 Hemoglobin degradation and production of bilirubin and urobilinogen.

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156 Part Two | Urinalysis

in an acid medium to produce an azo dye, with colors ranging because those substances combine with the diazonium salt and
from increasing degrees of tan or pink to violet, respectively. prevent its reaction with bilirubin.
Qualitative results are reported as negative, small, moderate, or
large or as negative, 1+, 2+, or 3+. Reagent strip color reactions Ictotest Tablets
for bilirubin are more difficult to interpret than are other reagent A confirmatory test for bilirubin is the Ictotest (Siemens
strip reactions, and those reactions are easily influenced by other Healthcare Diagnostics, Inc., Tarrytown, NY). Ictotest kits
pigments present in the urine. Atypical color reactions are fre- consist of testing mats and tablets containing p-nitrobenzene-
quently noted on visual examination and are measured by au- diazonium-p-toluenesulfonate, SSA, sodium carbonate, and
tomated readers. Further testing should be performed on any boric acid. Ten drops of urine are added to the mat, which has
questionable results. special properties that cause bilirubin to remain on the surface
as the urine is absorbed. After the chemical reaction, a blue-to-
Acid purple color appears on the mat when bilirubin is present. The
Bilirubin glucuronide + diazonium salt azo dye
Ictotest is four times more sensitive than the reagent strip and
Reaction Interference will detect 0.05 to 0.1 mg/dL bilirubin. Colors other than blue
or purple appearing on the mat are considered to be a negative
As discussed previously, false-positive reactions are primarily result. If interference in the Ictotest is suspected, it can usually
due to urine pigments. Of particular concern are the yellow- be removed by adding water directly to the mat after the urine
orange urines from people taking phenazopyridine compounds has been added. Interfering substances are washed into the
because the thick pigment produced may be mistaken for mat, and only bilirubin remains on the surface.
bilirubin on initial examination. The presence of indican and
metabolites of the medication Lodine may cause false-positive
readings. Urobilinogen
The false-negative results caused by the testing of speci-
As shown in Figure 6-4, when conjugated bilirubin is excreted
mens that are not fresh are the most frequent errors associated
through the bile duct into the intestine, the intestinal bacteria
with bilirubin testing. Bilirubin is an unstable compound that
convert the bilirubin to a combination of urobilinogen and
is rapidly photo-oxidized to biliverdin when exposed to light.
stercobilinogen. Some of the urobilinogen is reabsorbed from
Biliverdin does not react with diazo tests. False-negative results
the intestine into the blood, recirculates to the liver, and is
also occur when hydrolysis of bilirubin diglucuronide pro-
excreted back into the intestine through the bile duct. The
duces free bilirubin because this is less reactive in the reagent
stercobilinogen cannot be reabsorbed and remains in the
strip tests. High concentrations of ascorbic acid (greater than
intestine, where it is oxidized to stercobilin. The recirculated
25 mg/dL) and nitrite may lower the sensitivity of the test
urobilinogen that reaches the intestine is also oxidized to uro-
bilin. Both stercobilin and urobilin are excreted in the feces
and are the pigments responsible for the characteristic brown
SUMMARY 614 Bilirubin Reagent Strip color of feces. Urobilinogen appears in the urine because as it

Reagents Multistix: 2,4-dichloroaniline

diazonium salt
Sensitivity Chemstrip: 2,6-dichlorobenzene-
PROCEDURE 6-5
diazonium salt Ictotest Procedure
Multistix: 0.4–0.8 mg/dL bilirubin Procedure
Chemstrip: 0.5 mg/dL bilirubin 1. Place 10 drops of urine onto one square of the
Interference False positive: absorbent test mat.
Highly pigmented urines, 2. Using forceps, remove one Ictotest reagent tablet,
phenazopyridine recap the bottle promptly, and place the tablet in
Indican (intestinal disorders) the center of the moistened area.
Metabolites of Lodine 3. Place one drop of water onto the tablet, and wait
5 seconds.
False negative:
4. Place a second drop of water onto the tablet so that
Specimen exposure to light
the water runs off the tablet onto the mat.
Ascorbic acid greater than 25 mg/dL
5. Observe the color of the mat around the tablet at the
High concentrations of nitrite end of 60 seconds. The presence of a blue-to-purple
Correlations Urobilinogen color on the mat indicates that bilirubin is present. A
with other slight pink or red color should be ignored. Report as
tests positive or negative.
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Chapter 6 | Chemical Examination of Urine 157

circulates in the blood back to the liver, it passes through the reaction using 4-methoxybenzene-diazonium-tetrafluoroborate
kidney and is filtered by the glomerulus. Therefore, a small to react with urobilinogen, producing colors ranging from
amount of urobilinogen—less than 1 mg/dL or 1 Ehrlich white to pink. This reaction is more specific for urobilinogen
unit—is normally found in the urine. than the Ehrlich reaction. Results are reported in mg/dL. Both
tests detect urobilinogen that is present in normal quantities,
Clinical Significance and color comparisons are provided for the upper limits of
Increased urine urobilinogen (greater than 1 mg/dL) is seen in normal as well as abnormal concentrations. Reagent strip
liver disease and hemolytic disorders. Measurement of urine tests cannot determine the absence of urobilinogen, which is
urobilinogen can be valuable in the detection of early liver dis- significant in biliary obstruction.
ease; however, studies have shown that when urobilinogen
Multistix:
tests are performed routinely, 1% of the nonhospitalized pop-
ulation and 9% of a hospitalized population exhibit elevated Acid
Urobilinogen + p-dimethylaminobenzaldehyde red color
results.13 This is frequently caused by constipation.
(Ehrlich’s (Ehrlich’s reagent)
Impairment of liver function decreases the ability of the
reactive
liver to process the urobilinogen recirculated from the intestine.
substances)
The excess urobilinogen remaining in the blood is filtered by
the kidneys and appears in the urine. Chemstrip:
The clinical jaundice associated with hemolytic disorders
results from the increased amount of circulating unconjugated Acid
bilirubin. This unconjugated bilirubin is presented to the liver Urobilinogen + diazonium salt red azo dye
for conjugation, resulting in a markedly increased amount of (4-methyloxybenzene-diazonium-tetrafluoroborate)
conjugated bilirubin entering the intestines. As a result, in-
creased urobilinogen is produced, and increased amounts of Reaction Interference
urobilinogen are reabsorbed into the blood and circulated The Ehrlich reaction on Multistix is subject to a variety of in-
through the kidneys, where filtration takes place. In addition, terferences, referred to as Ehrlich-reactive compounds, that
the overworked liver does not process the reabsorbed urobilino- produce false-positive reactions. These include porphobilino-
gen as efficiently, and additional urobilinogen is presented for gen, indican, p-aminosalicylic acid, sulfonamides, methyldopa,
urinary excretion. procaine, and chlorpromazine compounds. The presence of
Although it cannot be determined by reagent strip, the porphobilinogen is clinically significant; however, the reagent
absence of urobilinogen in the urine and feces is also diagnos- strip test is not considered a reliable method to screen for its
tically significant and represents an obstruction of the bile duct presence. Porphobilinogen will be discussed in Chapter 9.
that prevents the normal passage of bilirubin into the intestine. The sensitivity of the Ehrlich reaction increases with tem-
An additional observation is the production of pale stools as perature, and testing should be performed at room tempera-
the result of the lack of urobilin. Refer back to Table 6-2 for an ture. Highly pigmented urines cause atypical readings with
outline of the relationship of urine bilirubin and urine uro- both brands of reagent strips. As a result of increased excre-
bilinogen to the pathological conditions associated with them. tion of bile salts, urobilinogen results are normally highest
after a meal.
Reagent Strip Reactions and Interference False-negative results occur most frequently when speci-
The reagent strip reactions for urobilinogen differ between mens are improperly preserved, allowing urobilinogen to be
Multistix and Chemstrip much more significantly than do photo-oxidized to urobilin. High concentrations of nitrite in-
other reagent strip parameters. Multistix uses a modification terfere with the azo-coupling reaction on Chemstrip. False-
of the Ehrlich reaction, in which urobilinogen reacts with negative readings also are obtained with both strips when
p-dimethylaminobenzaldehyde (Ehrlich reagent) to produce formalin is used as a preservative.
colors ranging from light to dark pink. Results are reported as
Ehrlich units (EU), which are equal to mg/dL, ranging from
normal readings of 0.2 and 1 through abnormal readings of Technical Tip 6-5. The urobilinogen test pad on the
2, 4, and 8. Chemstrip incorporates an azo-coupling (diazo) Multistix Pro11 and Clinitek Microalbumin strips has
been replaced by the protein-low test pad.

SUMMARY 615 Clinical Significance

of Urine Urobilinogen Nitrite
Early detection of liver disease Clinical Significance
Liver disorders, hepatitis, cirrhosis, carcinoma The reagent strip test for nitrite provides a rapid screening test
Hemolytic disorders for the presence of UTI. The test is designed to detect cases
in which the need for a culture may not be apparent; it is not
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158 Part Two | Urinalysis

and even septicemia. Therefore, detection of bacteriuria

SUMMARY 616 Urobilinogen Reagent
through the use of the nitrite screening test and subsequent
Strip
antibiotic therapy can prevent these serious complications. The
Reagents Multistix: p-dimethylaminobenzaldehyde nitrite test also can be used to evaluate the success of antibiotic
therapy and to periodically screen people with recurrent infec-
Chemstrip: 4-methoxybenzene-
tions, patients with diabetes, and pregnant women, all of
diazonium-tetrafluoroborate
whom are considered to be at high risk for UTI. As discussed
Sensitivity Multistix: 0.2 mg/dL urobilinogen in the following section, many laboratories use the nitrite test
Chemstrip: 0.4 mg/dL urobilinogen in combination with the LE test to determine the necessity of
Interference Multistix: performing urine cultures.
False positive: Reagent Strip Reactions
Porphobilinogen
The chemical basis of the nitrite test is the ability of certain
Indican gram-negative bacteria to reduce nitrate, a normal constituent
p-aminosalicylic acid of urine, to nitrite, which normally does not appear in the
Sulfonamides urine. Nitrite is detected by the Greiss reaction, in which
nitrite at an acidic pH reacts with an aromatic amine (para-
Methyldopa
arsanilic acid or sulfanilamide) to form a diazonium compound
Procaine that then reacts with tetrahydrobenzoquinolin compounds to
Chlorpromazine produce a pink-colored azo dye. To prevent false-positive
Highly pigmented urine reactions in externally contaminated specimens, the sensitivity
of the test is standardized to correspond with a quantitative
False negative:
bacterial culture criterion of 100,000 organisms per milliliter.
Old specimens Although different shades of pink may be produced, the test
Preservation in formalin does not measure the degree of bacteriuria, and any shade of
Chemstrip: pink is considered to represent a number of bacteria that is
clinically significant. Results are reported only as negative or
False positive:
positive.
Highly pigmented urine
False negative: Acid
Para-arsanilic acid or sulfanilamide + NO2
Old specimens
diazonium salt (nitrite)
Preservation in formalin
Acid
High concentrations of nitrite Diazonium salt + tetrahydrobenzoquinolin
Correlations Bilirubin pink azo dye
with other
tests
Reaction Interference
Several major factors can influence the reliability of the nitrite
test, and tests with negative results in the presence of clinical
symptoms that are even vaguely suspicious should always be
intended to replace the urine culture as the primary test for repeated or followed by a urine culture.
diagnosing and monitoring bacterial infection. Many UTIs are 1. Bacteria that lack the enzyme reductase do not possess
believed to start in the bladder as a result of external contam- the ability to reduce nitrate to nitrite. Reductase is found
ination and, if untreated, progress upward through the ureters in the gram-negative bacteria (Enterobacteriaceae) that
to the tubules, renal pelvis, and kidney. Infections are caused
most commonly by gram-negative organisms, such as E. coli,
Proteus species, Enterobacter species, and Klebsiella species.
UTIs are eight times more common in women than in men
SUMMARY 617 Clinical Significance
due to a shorter urethra, providing a shorter distance for the
of Urine Nitrite
bacteria to travel. Patients who are catheterized also have a
higher incidence of infection. The nitrite test is valuable for de- Cystitis
tecting initial bladder infection (cystitis) because often patients
Pyelonephritis
are asymptomatic or have vague symptoms that would not lead
the physician to order a urine culture. Pyelonephritis, an Evaluation of antibiotic therapy
inflammatory process of the kidney and adjacent renal pelvis, is Monitoring of patients at high risk for UTI
a frequent complication of untreated cystitis and can lead to Screening of urine culture specimens
renal tissue damage, impairment of renal function, hypertension,
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Chapter 6 | Chemical Examination of Urine 159

large quantities of ascorbic acid interfering with

SUMMARY 618 Nitrite Reagent Strip
the diazo reaction, and decreased sensitivity in speci-
Reagents Multistix: p-arsanilic acid mens with a high specific gravity. Large amounts of
ascorbic acid compete with nitrite to combine with
Tetrahydrobenzo(h)-quinolin-3-ol
the diazonium salt, therefore preventing a true nitrite
Chemstrip: Sulfanilamide, hydroxyte- measurement.
trahydrobenzoquinoline
Sensitivity Multistix: 0.06–0.1 mg/dL nitrite ion
Chemstrip: 0.05 mg/dL nitrite ion
Leukocyte Esterase
Interference False negative: Before the development of the reagent strip test for LE, detection
Nonreductase-containing bacteria of increased urinary leukocytes required microscopic examina-
tion of the urine sediment. This can be subject to variation
Insufficient contact time between
depending on the method used to prepare the sediment and
bacteria and urinary nitrate
the technical personnel examining the sediment. Therefore, the
Lack of urinary nitrate chemical test for leukocytes offers a more standardized means
Large quantities of bacteria convert- for the detection of leukocytes. The test is not designed to meas-
ing nitrite to nitrogen ure the concentration of leukocytes, and the manufacturers rec-
Presence of antibiotics ommend that quantitation be done by microscopic examination.
An additional advantage to the chemical LE test is that it detects
High concentrations of ascorbic acid
the presence of leukocytes that have been lysed, particularly
High specific gravity in dilute alkaline urine, and that would not appear in the
False positive: microscopic examination.
Improperly preserved specimens
Clinical Significance
Highly pigmented urine
Normal values for leukocytes are based on the examination of
Correlations Protein
microscopic sediment and vary from 0 to 2 to 0 to 5 per high-
with other
power field (hpf). Women tend to have higher numbers than
tests
men as a result of vaginal contamination. Increased urinary
Leukocytes leukocytes are indicators of UTI. The LE test detects the pres-
Microscopic ence of esterase in the granulocytic white blood cells (WBCs)
(neutrophils, eosinophils, and basophils) and monocytes, but
not lymphocytes. Neutrophils are the leukocytes most fre-
quently associated with bacterial infections. Esterases also are
most frequently cause UTIs. Non–nitrate-reducing gram- present in Trichomonas and histiocytes. Lymphocytes, erythro-
positive bacteria and yeasts, however, cause a significant cytes, bacteria, and renal tissue cells do not contain esterases.
number of infections, and the nitrite test does not detect A positive LE test result is accompanied most frequently by the
the presence of these organisms. presence of bacteria, which, as discussed previously, may or
2. Bacteria capable of reducing nitrate must remain in con- may not produce a positive nitrite reaction. Infections caused
tact with the urinary nitrate long enough to produce ni- by Trichomonas, Chlamydia, yeast, and inflammation of renal
trite. Therefore, nitrite tests should be performed on first tissues (i.e., interstitial nephritis) produce leukocyturia
morning specimens or specimens collected after urine without bacteriuria.
has remained in the bladder for at least 4 hours. The Screening urine specimens using the LE and nitrite
correlation between positive cultures and positive nitrite chemical reactions to determine the necessity of performing
test results is significantly lower when testing is per- urine cultures can be a cost-effective measure.14 The LE test
formed on random specimens.2 contributes significantly more to the reliability of this practice
3. The reliability of the test depends on the presence of ad- than does the nitrite test.
equate amounts of nitrate in the urine. This is seldom a
problem in patients on a normal diet that contains green
vegetables; however, because diet usually is not con-
trolled before testing, the possibility of a false-negative
SUMMARY 619 Clinical Significance
result due to lack of dietary nitrate does exist.
of Urine Leukocytes
4. Further reduction of nitrite to nitrogen may occur when
large numbers of bacteria are present, and this causes a Bacterial and nonbacterial UTI
false-negative reaction. Inflammation of the urinary tract
5. Other causes of false-negative results include inhibition Screening of urine culture specimens
of bacterial metabolism by the presence of antibiotics,
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160 Part Two | Urinalysis

Reagent Strip Reaction SUMMARY 620 Leukocyte Esterase

The reagent strip reaction uses the action of LE to catalyze the Reagent Strip
hydrolysis of an acid ester embedded on the reagent pad to
Reagents Multistix: Derivatized pyrrole amino
produce an aromatic compound and acid. Then the aromatic
acid ester
compound combines with a diazonium salt present on the pad
to produce a purple azo dye. Diazonium salt
Chemstrip: Indoxylcarbonic acid ester
Leukocyte Diazonium salt
Indoxylcarbonic acid ester indoxyl + acid indoxyl
Esterase Sensitivity Multistix: 5–15 WBC/hpf
Acid
Chemstrip: 10–25 WBC/hpf
+ diazonium salt purple azo dye Interference False positive:
The LE reaction requires the longest time of all the reagent Strong oxidizing agents
strip reactions (2 minutes). Reactions are reported as trace, small, Formalin
moderate, and large or trace, 1+, 2+, and 3+. Trace readings may Highly pigmented urine,
not be significant and should be repeated on a fresh specimen. nitrofurantoin
Reaction Interference False negative:
High concentrations of protein,
The presence of strong oxidizing agents or formalin in the
glucose, oxalic acid, ascorbic
collection container causes false-positive reactions. Urines that
acid, gentamicin, cephalosporins,
are highly pigmented, as well as the presence of the antibiotic
tetracyclines; inaccurate timing
nitrofurantoin, may obscure the color reaction.
False-negative results may occur in the presence of high Correlations Protein
concentrations of protein (greater than 500 mg/dL), glucose with other
(greater than 3 g/dL), oxalic acid, and ascorbic acid. In this tests
reaction, ascorbic acid also combines with the diazonium salt. Nitrite
Crenation of leukocytes preventing release of esterases may Microscopic
occur in urines with a high specific gravity. The presence of the
antibiotics gentamicin, cephalexin, cephalothin, and tetracy-
cline decreases the sensitivity of the reaction.

Specific Gravity COOH COOH

COOH COOH
The reagent strip test for specific gravity is included as part of COOH COOH
the physical examination of urine in Chapter 5 and is reviewed POLYELECTROLYTE ON REAGENT STRIP
here as part of the chemical examination. A summary of the
clinical significance is included in this chapter. + + –
+ –
– – + + –
Reagent Strip Reaction + – – +
– +

The reagent strip reaction is based on the change in pKa (disso- Ions in urine with Ions in urine with
low specific gravity high specific gravity
ciation constant) of a polyelectrolyte in an alkaline medium. The
polyelectrolyte ionizes, releasing hydrogen ions in proportion to COOH COOH COO–H+ COO–H+
the number of ions in the solution. The higher the concentration COO–H+ COO–H+ COO–H+ COO–H+
COOH COOH COO–H+ COO–H+
of urine, the more hydrogen ions are released, thereby lowering
the pH. Incorporation of the indicator bromothymol blue on the INITIAL REACTION ON REAGENT STRIP
reagent pad measures the change in pH. As the specific gravity 2H+—Bromothymol blue 6H+—Bromothymol blue
increases, the indicator changes from blue (1.000 [alkaline]),
through shades of green, to yellow 1.030 [acid]). Readings can
be made in 0.005 intervals by careful comparison with the color Blue-green alkaline pH Yellow-green acid pH
chart. The specific gravity reaction is diagrammed in Figure 6-6.
SECONDARY REACTION ON REAGENT STRIP

Reaction Interference Figure 6–6 Diagram of reagent strip–specific gravity reaction.

The reagent strip specific gravity measures only ionic solutes,
thereby eliminating the interference by the large organic mol-
ecules, such as urea and glucose, and by radiographic contrast
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Chapter 6 | Chemical Examination of Urine 161

SUMMARY 621 Clinical Significance

For additional resources please visit
of Urine Specific Gravity www.fadavis.com
Monitoring patient hydration and dehydration
Loss of renal tubular concentrating ability
Diabetes insipidus References
1. Chemstrip 10UA product Insert, Roche Diagnostics, Indianapo-
Determination of unsatisfactory specimens due to low lis, IN, 2004.
concentration 2. Multistix Reagent Strips Product Insert. Siemens Healthcare
Diagnostics, Inc., Tarrytown, NY 2010-2017.
3. Clinical and Laboratory Standards Institute. Urinalysis;
Approved Guideline-Third Edition. CLSI document GP16-A3.
Clinical and Laboratory Standards Institute, Wayne, PA,
2009, CLSI.
SUMMARY 622 Urine Specific Gravity 4. Bleyer, AJ, Kmoch, S: Tamm Horsfall glycoprotein and
Reagent Strip Uromodulin: It is all about the Tubules! Clin J Am Soc Nephrol.
2016 Jan 7; 11 (1): 6-8. Doi: 10.2215/CJN.12201115. Web
Reagents Multistix: Poly (methyl vinyl ether/maleic site: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702239/.
anhydride) bromothymol blue Published December 18, 2015. Accessed May 6, 2019.
5. Pugia, MJ, and Lott, JA: New developments in urinalysis strip
Chemstrip: Ethylene glycol diaminoethyl tests for proteins. In Bayer Encyclopedia of Urinalysis. Bayer
ether tetra-acetic acid, bromothymol Diagnostics, Elkhart, IN, 2002.
blue 6. Bhuwnesh, A, et al: Microalbumin screening by reagent strip
Sensitivity 1.000–1.030 predicts cardiovascular risk in hypertension. J Hypertens 14:
223–228, 1992.
Interference False positive: High concentrations of 7. Bianchi, S, et al: Microalbuminurea in essential hypertension.
protein J Nephrol 10(4):216–219, 1997.
8. Clinitek Microalbumin Reagent Strip Product Insert. Bayer
False negative: Highly alkaline urines Diagnostics, Elkhart, IN, 2006.
(greater than 6.5) 9. Multistix Pro Reagent Strips Product Insert. Siemens Healthcare
Diagnostics, Inc. Tarrytown, NY, 2008.
10. Benedict, SR: A reagent for the detection of reducing sugars.
J Biol Chem 5:485–487, 1909.
11. College of American Pathologists. CAP Today, Q&A Column
media and plasma expanders that are included in physical 9/16. https://www.captodayonline.com/qa-column-0916/.
measurements of specific gravity. This difference must be con- Accessed May 6, 2019.
sidered when comparing specific gravity results obtained by a 12. Lane R, and Phillips, M: Rhabdomyolysis has many causes
different method. Elevated concentrations of protein slightly including statins and may be fatal. Brit J Med 327:115–116,
increase the readings as a result of protein anions. 2003.
13. Hager, CB, and Free, AH: Urine urobilinogen as a component
Specimens with a pH of 6.5 or higher have decreased read- of routine urinalysis. Am J Med Technol 36(5):227–233, 1970.
ings caused by interference with the bromothymol blue indi- 14. Wise, KA, Sagert, LA, and Grammens, GL: Urine leukocyte es-
cator (the blue-green readings associated with an alkaline pH terase and nitrite tests as an aid to predict urine culture results.
correspond to a low reading for specific gravity). Therefore, Lab Med 15(3):186–187, 1984.
manufacturers recommend adding 0.005 to specific gravity
readings when the pH is 6.5 or higher. The correction is per-
formed by automated strip readers.

Study Questions
1. Leaving excess urine on the reagent strip after removing it 2. Failure to mix a specimen before inserting the reagent
from the specimen will: strip will primarily affect the:
A. Cause runover between reagent pads A. Glucose reading
B. Alter the color of the specimen B. Blood reading
C. Cause reagents to leach from the pads C. Leukocyte reading
D. Not affect the chemical reactions D. Both B and C
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162 Part Two | Urinalysis

3. Testing a refrigerated specimen that has not warmed to 10. Indicate the source of the following proteinurias by
room temperature will adversely affect: placing a 1 for prerenal, 2 for renal, or 3 for postrenal in
A. Enzymatic reactions front of the condition.
B. Dye-binding reactions A. Microalbuminuria
C. The sodium nitroprusside reaction B. Acute-phase reactants
D. Diazo reactions C. Preeclampsia
D. Vaginal inflammation
4. The reagent strip reaction that requires the longest
reaction time is the: E. Multiple myeloma
A. Bilirubin F. Orthostatic proteinuria
B. pH G. Prostatitis
C. Leukocyte esterase 11. The principle of the protein error of indicators reaction
D. Glucose is that:
A. Protein keeps the pH of the urine constant
5. Quality control of reagent strips is performed:
B. Albumin accepts hydrogen ions from the indicator
A. Using positive and negative controls
C. The indicator accepts hydrogen ions from albumin
B. When results are questionable
D. Albumin changes the pH of the urine
C. Per laboratory policy
D. All of the above 12. All of the following will cause false-positive protein
values on a reagent strip except:
6. All of the following are important to protect the integrity
A. Microalbuminuria
of reagent strips except:
B. Highly buffered alkaline urines
A. Removing the desiccant from the bottle
C. Delay in removing the reagent strip from the
B. Storing in an opaque bottle
specimen
C. Storing at room temperature
D. Contamination by quaternary ammonium
D. Resealing the bottle after removing a strip compounds
7. The principle of the reagent strip test for pH is the: 13. A patient with a 2+ protein reading in the afternoon is
A. Protein error of indicators asked to submit a first morning specimen. The second
B. Greiss reaction specimen has a negative protein reading. This patient is:
C. Dissociation of a polyelectrolyte A. Positive for orthostatic proteinuria
D. Double indicator reaction B. Negative for orthostatic proteinuria
C. Positive for Bence Jones protein
8. A urine specimen with a pH of 9.0:
D. Negative for clinical proteinuria
A. Indicates metabolic acidosis
B. Should be re-collected 14. Testing for microalbuminuria is valuable for early detec-
tion of kidney disease and monitoring patients with:
C. May contain calcium oxalate crystals
A. Hypertension
D. Is seen after drinking cranberry juice
B. Diabetes mellitus
9. In the laboratory, a primary consideration associated
C. Cardiovascular disease risk
with pH is:
D. All of the above
A. Identifying urinary crystals
B. Monitoring vegetarian diets 15. The primary chemical on the reagent strip in the Micral-
Test for microalbumin binds to:
C. Determining specimen acceptability
A. Protein
D. Both A and C
B. Antihuman albumin antibody
C. Conjugated enzyme
D. Galactoside
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Chapter 6 | Chemical Examination of Urine 163

16. All of the following are true for the ImmunoDip test for 23. The principle of the reagent strip tests for glucose is the:
microalbumin except: A. Peroxidase activity of glucose
A. Unbound antibody migrates farther than bound B. Glucose oxidase reaction
antibody
C. Double sequential enzyme reaction
B. Blue latex particles are coated with antihuman
D. Dye-binding of glucose and chromogen
albumin antibody
C. Bound antibody migrates farther than unbound 24. All of the following may produce false-negative glucose
antibody reactions except:
D. It utilizes an immunochromographic principle A. Detergent contamination
B. Ascorbic acid
17. The principle of the protein-high pad on the Multistix
Pro reagent strip is the: C. Unpreserved specimens
A. Diazo reaction D. Low urine temperature
B. Enzymatic dye-binding reaction 25. The primary reason for performing a Clinitest is to:
C. Protein error of indicators A. Check for high ascorbic acid levels
D. Microalbumin-Micral-Test B. Confirm a positive reagent strip glucose
18. Which of the following is not tested on the Multistix Pro C. Check for newborn galactosuria
reagent strip? D. Confirm a negative glucose reading
A. Urobilinogen 26. The three intermediate products of fat metabolism
B. Specific gravity include all of the following except:
C. Creatinine A. Acetoacetic acid
D. Protein-high B. Ketoacetic acid
19. The principle of the protein-low reagent pad on the C. β-hydroxybutyric acid
Multistix Pro is the: D. Acetone
A. Binding of albumin to sulphonphthalein dye 27. The most significant reagent strip test that is associated
B. Immunological binding of albumin to antibody with a positive ketone result is:
C. Reverse protein error of indicators reaction A. Glucose
D. Enzymatic reaction between albumin and dye B. Protein
20. The principle of the creatinine reagent pad on C. pH
microalbumin reagent strips is the: D. Specific gravity
A. Double indicator reaction 28. The primary reagent in the reagent strip test for
B. Diazo reaction ketones is:
C. Pseudoperoxidase reaction A. Glycine
D. Reduction of a chromogen B. Lactose
21. The purpose of performing an albumin:creatinine ratio C. Sodium hydroxide
is to: D. Sodium nitroprusside
A. Estimate the glomerular filtration rate 29. Ketonuria may be caused by all of the following except:
B. Correct for hydration in random specimens A. Bacterial infections
C. Avoid interference for alkaline urines B. Diabetic acidosis
D. Correct for abnormally colored urines C. Starvation
22. A patient with a normal blood glucose and a positive D. Vomiting
urine glucose should be further checked for:
30. Urinalysis is frequently performed on a patient with
A. Diabetes mellitus severe back and abdominal pain to check for:
B. Renal disease A. Glucosuria
C. Gestational diabetes B. Proteinuria
D. Pancreatitis C. Hematuria
D. Hemoglobinuria
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164 Part Two | Urinalysis

31. Place the appropriate number or numbers in front of 37. The primary cause of a false-negative bilirubin reaction is:
each of the following statements. Use both numbers for A. Highly pigmented urine
an answer if needed.
B. Specimen contamination
1. Hemoglobinuria
C. Specimen exposure to light
2. Myoglobinuria
D. Excess conjugated bilirubin
A. Associated with transfusion reactions
38. The purpose of the special mat supplied with the
B. Clear red urine and pale yellow plasma
Ictotest tablets is that:
C. Clear red urine and red plasma
A. Bilirubin remains on the surface of the mat
D. Associated with rhabdomyolysis
B. It contains the dye needed to produce color
E. Produces hemosiderin granules in
C. It removes interfering substances
urinary sediments
D. Bilirubin is absorbed into the mat
F. Associated with acute renal failure
39. The reagent in the Multistix reaction for urobilinogen is:
32. The principle of the reagent strip test for blood is based
on the: A. A diazonium salt
A. Binding of heme and a chromogenic dye B. Tetramethylbenzidine
B. Peroxidase activity of heme C. p-Dimethylaminobenzaldehyde
C. Reaction of peroxide and chromogen D. Hoesch reagent
D. Diazo activity of heme 40. The primary problem with urobilinogen tests using
Ehrlich reagent is:
33. A speckled pattern on the blood pad of the reagent strip
indicates: A. Positive reactions with porphobilinogen
A. Hematuria B. Lack of specificity
B. Hemoglobinuria C. Positive reactions with Ehrlich reactive substances
C. Myoglobinuria D. All of the above
D. All of the above 41. The reagent strip test for nitrite uses the:
34. List the following products of hemoglobin degradation A. Greiss reaction
in the correct order of metabolism by placing numbers 1 B. Ehrlich reaction
to 4 in the blank, where 1 indicates the beginning and C. Peroxidase reaction
4 indicates the end product.
D. Pseudoperoxidase reaction
A. Conjugated bilirubin
42. All of the following can cause a negative nitrite reading
B. Urobilinogen and stercobilinogen
except:
C. Urobilin
A. Gram-positive bacteria
D. Unconjugated bilirubin
B. Gram-negative bacteria
35. The principle of the reagent strip test for bilirubin is the: C. Random urine specimens
A. Diazo reaction D. Heavy bacterial infections
B. Ehrlich reaction
43. A positive nitrite test and a negative leukocyte esterase
C. Greiss reaction test is an indication of a:
D. Peroxidase reaction A. Dilute random specimen
36. An elevated urine bilirubin with a normal urobilinogen B. Specimen with lysed leukocytes
is indicative of: C. Vaginal yeast infection
A. Cirrhosis of the liver D. Specimen older than 2 hours
B. Hemolytic disease
44. All of the following can be detected by the leukocyte
C. Hepatitis esterase reaction except:
D. Biliary obstruction A. Neutrophils
B. Eosinophils
C. Lymphocytes
D. Basophils
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Chapter 6 | Chemical Examination of Urine 165

45. Screening tests for urinary infection combine the 48. A specific gravity of 1.005 would produce the reagent
leukocyte esterase test with the test for: strip color:
A. pH A. Blue
B. Nitrite B. Green
C. Protein C. Yellow
D. Blood D. Red
46. The principle of the leukocyte esterase reagent strip test 49. Specific gravity readings on a reagent strip are affected by:
uses a: A. Glucose
A. Peroxidase reaction B. Radiographic dye
B. Double indicator reaction C. Alkaline urine
C. Diazo reaction D. All of the above
D. Dye-binding technique
47. The principle of the reagent strip test for specific gravity
uses the dissociation constant of a(n):
A. Diazonium salt
B. Indicator dye
C. Polyelectrolyte
D. Enzyme substrate

Case Studies and Clinical Situations

1. A patient taken to the emergency department after an pH: 6.0 UROBILINOGEN: Normal
episode of syncope has a fasting blood glucose level of PROTEIN: Negative NITRITE: Negative
450 mg/dL. Results of the routine urinalysis are as
GLUCOSE: Negative LEUKOCYTES: Negative
follows:
a. What would be observed if this specimen were shaken?
COLOR: Yellow KETONES: 2+
b. Explain the correlation between the patient’s sched-
CLARITY: Clear BLOOD: Negative
uled surgery and the normal urobilinogen.
SP. GRAVITY: 1.015 BILIRUBIN: Negative
c. If blood were drawn from this patient, how might the
pH: 5.0 PROTEIN-LOW: 15 mg/dL appearance of the serum be described?
PROTEIN-HIGH: 30 mg/dL NITRITE: Negative d. What special handling is needed for specimens of
GLUCOSE: 250 mg/dL LEUKOCYTES: Negative serum and urine from this patient?
CREATININE: 200 mg/dL 3. Results of a urinalysis on a patient who is very anemic
a. Explain the correlation between the patient’s blood and jaundiced are as follows:
and urine glucose results. COLOR: Red KETONES: Negative
b. What is the most probable metabolic disorder CLARITY: Clear BLOOD: Large
associated with this patient?
SP. GRAVITY: 1.020 BILIRUBIN: Negative
c. Considering the patient’s condition, what is the
pH: 6.0 UROBILINOGEN: 8 EU
significance of the reading of the patient’s protein-to-
creatinine ratio? PROTEIN: Negative NITRITE: Negative
d. If the patient in this study had a normal blood glucose GLUCOSE: Negative LEUKOCYTES: Negative
level, as well as normal results for protein and creati- a. Would these results be indicative of hematuria or
nine, to what would the urinary glucose level be hemoglobinuria?
attributed? b. Correlate the patient’s condition with the urobilinogen
2. Results of a urinalysis performed on a patient scheduled result.
for gallbladder surgery are as follows: c. Why is the urine bilirubin result negative in this jaun-
COLOR: Amber KETONES: Negative diced patient?
CLARITY: Hazy BLOOD: Negative d. Would this method also measure urine porphobilino-
gen? Why or why not?
SP. GRAVITY: 1.022 BILIRUBIN: Moderate
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166 Part Two | Urinalysis

4. A female patient arrives at the outpatient clinic with The physician requests that the athlete collect another
symptoms of lower back pain and urinary frequency with specimen in the morning before classes and practice.
a burning sensation. She is a firm believer in the curative a. What is the purpose of the second specimen?
powers of vitamins. She has tripled her usual dosage of
b. What changes would you expect in the second
vitamins in an effort to alleviate her symptoms; however,
specimen?
the symptoms have persisted. She is given a sterile con-
tainer and asked to collect a clean-catch midstream urine c. Is the proteinuria present in the first specimen of
specimen. Results of this routine urinalysis are as follows: prerenal, renal, or postrenal origin?
COLOR: Dark yellow KETONES: Negative 6. A construction worker is pinned under collapsed scaffold-
CLARITY: Hazy BLOOD: Negative ing for several hours before being taken to the emergency
room. His abdomen and upper legs are severely bruised,
SP. GRAVITY: 1.012 BILIRUBIN: Negative
but no fractures are detected. A specimen for urinalysis
pH: 7.0 UROBILINOGEN: Normal obtained by catheterization has the following results:
PROTEIN: Trace NITRITE: Negative COLOR: Red-brown KETONES: Negative
GLUCOSE: Negative LEUKOCYTES: 1+ CLARITY: Clear BLOOD: 4+
Microscopic SP. GRAVITY: 1.020 BILIRUBIN: Negative
8 to 12 RBC/hpf Heavy bacteria pH: 6.5 UROBILINOGEN: 0.4 EU
40 to 50 WBC/hpf Moderate squamous PROTEIN: Trace NITRITE: Negative
epithelial cells
GLUCOSE: Negative LEUKOCYTES: Negative
a. What discrepancies exist between the chemical and
a. Would hematuria be suspected in this specimen? Why
microscopic test results? State and explain a possible
or why not?
reason for each discrepancy.
b. What is the most probable cause of the positive blood
b. What additional chemical tests could be affected by
reaction?
the patient’s vitamin dosage? Explain the principle of
the interference. c. What is the source of the substance causing the posi-
tive blood reaction and the name of the condition?
c. Discuss the correlation between urine color and spe-
cific gravity results, and give a possible cause for any d. Would this patient be monitored for changes in renal
discrepancy. function? Why or why not?
d. State three additional reasons not previously given 7. Considering the correct procedures for care, technique,
for a negative nitrite test in the presence of increased and quality control for reagent strips, state a possible
bacteria. cause for each of the following scenarios.
5. Results of a urinalysis collected from a 20-year-old college a. The urinalysis supervisor notices that an unusually
athlete after practice are as follows: large number of reagent strips are becoming discol-
ored before the expiration date has been reached.
COLOR: Dark yellow KETONES: Negative
b. A physician’s office is consistently reporting positive
CLARITY: Hazy BLOOD: 1+
nitrite test results with negative LE test results.
SP. GRAVITY: 1.030 BILIRUBIN: Negative
c. A student’s results for reagent strip blood and LE are
pH: 6.5 UROBILINOGEN: 1 EU consistently lower than those of the laboratory staff.
PROTEIN: 2+ NITRITE: Negative d. One morning, the urinalysis laboratory was reporting
GLUCOSE: Negative LEUKOCYTES: Negative results that were questioned frequently by physicians.
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CHAPTER 7
Microscopic Examination
of Urine
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
7-1 List the physical and chemical parameters included 7-9 Discuss the significance of white blood cells (WBCs) in
in macroscopic urine screening, and state their urine sediment.
significance.
7-10 Name, describe, and give the origin and significance
7-2 Discuss the advantages of commercial systems over of the three types of epithelial cells found in urine
the glass-slide method for sediment examination. sediment.
7-3 Describe the recommended methods for standardizing 7-11 Discuss the significance of oval fat bodies.
specimen preparation and volume; centrifugation;
7-12 Describe the process of cast formation.
sediment preparation, volume, and examination;
and reporting results. 7-13 Describe and discuss the significance of hyaline, RBC,
WBC, bacterial, epithelial cell, granular, waxy, fatty,
7-4 State the purpose of Sternheimer-Malbin, acetic acid,
and broad casts.
toluidine blue, Sudan III, Gram, Hansel, and Prussian
blue stains. 7-14 List and identify the normal crystals found in acidic
urine.
7-5 Identify specimens that should be referred for
cytodiagnostic testing. 7-15 List and identify the normal crystals found in alkaline
urine.
7-6 Describe the basic principles of bright-field, phase-
contrast, polarizing, dark-field, fluorescence, and inter- 7-16 Describe and state the significance of cystine, cholesterol,
ference-contrast microscopy and their relationship to leucine, tyrosine, bilirubin, sulfonamide, radiographic
sediment examination. dye, and ampicillin crystals.
7-7 Differentiate between normal and abnormal sediment 7-17 Differentiate between actual sediment constituents and
constituents. artifacts.
7-8 Discuss the significance of red blood cells (RBCs) in 7-18 Correlate physical and chemical urinalysis results with
urine sediment. microscopic observations and recognize discrepancies.

KEY TERMS
Birefringent Fluorescence microscopy Polarizing microscopy
Bright-field microscopy Interference-contrast microscopy Pyuria
Casts Köhler illumination Resolution
Clue cell Lipiduria Syncytia
Cylindruria Maltese cross formation Uromodulin
Dark-field microscopy Oval fat bodies
Dysmorphic Phase-contrast microscopy
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168 Part Two | Urinalysis

Introduction Table 7–1 Macroscopic Screening and

Microscopic Correlations
The third part of routine urinalysis, after physical and chemical
examination, is the microscopic examination of the urinary sed- Screening Test Significance
iment. Its purpose is to detect and identify insoluble materials Color Blood
present in the urine. The blood, kidney, lower genitourinary
tract, and external contamination all contribute formed ele- Clarity Hematuria versus hemoglobinuria/
ments to the urine. These include red blood cells (RBCs), white myoglobinuria
blood cells (WBCs), epithelial cells, casts, bacteria, yeast, par- Confirm pathological or nonpatho-
asites, mucus, spermatozoa, crystals, and artifacts. Because logical; cause of turbidity
some of these components are of no clinical significance and Blood RBCs, RBC casts
others are considered normal unless they are present in Protein Casts, cells
increased amounts, examination of the urinary sediment must
include both identification and quantitation of the elements Nitrite Bacteria, WBCs
present. Microscopic analysis is subject to several procedural Leukocyte esterase WBCs, WBC casts, bacteria
variations, including Glucose Yeast
• The methods by which the sediment is prepared
• The volume of sediment actually examined
phosphates, as well as other nonpathological crystals that can
• The methods and equipment used to obtain visualization
obscure other elements in the urine sediment. Warming the
• The manner in which the results are reported specimen to 37°C before centrifuging may dissolve some of
Protocols have been developed to increase the standardi- these crystals.
zation and cost-effectiveness of microscopic urinalysis, and The clean-catch midstream specimen minimizes external
they are discussed in this chapter. contamination of the sediment. As with the physical and chem-
ical analyses, dilute random specimens may cause false-negative
readings.
Macroscopic Screening Care must be taken to thoroughly mix the specimen before
To enhance the cost-effectiveness of urinalysis, many labora- decanting a portion into a centrifuge tube.
tories have developed protocols whereby microscopic exami-
nation of the urine sediment is performed only on specimens Technical Tip 7-1. Warm refrigerated urine specimens
meeting specified criteria. Abnormalities in the physical and to 37°C before centrifuging to dissolve amorphous
chemical portions of the urinalysis play a primary role in the urate crystals.
decision to perform a microscopic analysis, thus the use of the
term “macroscopic screening.”
Parameters considered significant vary among laboratories Specimen Volume
but usually include color; clarity; and the presence of blood, pro-
A standard amount of urine, usually between 10 and 15 mL, is
tein, nitrite, leukocyte esterase, and, possibly, glucose. Laboratory-
centrifuged in a conical tube. This provides an adequate volume
designated criteria also can be programmed into automated
from which to obtain a representative sample of the elements
instruments. Table 7-1 illustrates the significance of these param-
present in the specimen. Frequently, a 12-mL volume is used
eters. Percentages of abnormal specimens that would go unde-
because multiparameter reagent strips are easily immersed in this
tected using these parameters differ significantly among studies.1,2
volume, and capped centrifuge tubes are often calibrated to this
The patient population also must be considered when developing
volume.
protocols for macroscopic screening. Populations that have
If obtaining a 12-mL specimen is not possible, as with
come under consideration include pregnant women, as well
pediatric patients, the volume of the specimen used should be
as pediatric, geriatric, diabetic, immunocompromised, and renal
noted on the report form. This allows the physician to correct
patients. The Clinical and Laboratory Standards Institute (CLSI)
the results, if indicated. Some laboratories choose to make this
recommends that microscopic examination be performed when
correction before reporting. For example, if only 6 mL of urine
requested by a physician, when a laboratory-specified patient
is centrifuged, the results are multiplied by 2.
population is being tested, or when any abnormal physical or
chemical result is obtained.3 Centrifugation
The speed of the centrifuge and the length of time the specimen
Specimen Preparation is centrifuged should be consistent. Centrifugation for 5 minutes
Specimens should be examined while fresh or adequately pre- at a relative centrifugal force (RCF) of 400 produces an optimum
served. Formed elements—primarily RBCs, WBCs, and hyaline amount of sediment with the least chance of damaging the ele-
casts—disintegrate rapidly, particularly in dilute alkaline urine. ments. To correct for differences in the diameter of centrifuge
Refrigeration may cause precipitation of amorphous urates and heads, RCF rather than revolutions per minute (RPM) is used.
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Chapter 7 | Microscopic Examination of Urine 169

The RPM value shown on the centrifuge tachometer can be con- the area of the field of view using a standard microscope. This
verted to RCF using nomograms available in many laboratory information, together with the sediment concentration factor, is
manuals or by using the formula: necessary to quantitate cellular elements per milliliter of urine.

RCF = 1.118 × 10–5 × radius in centimeters × RPM2 Commercial Systems

Centrifugation calibration should be performed routinely. The conventional method of placing a drop of centrifuged urine
Use of the braking mechanism to slow the centrifuge causes on a glass slide, adding a cover slip, and examining it micro-
disruption of the sediment before decantation and should be scopically has been improved substantially through the use of
avoided. commercial slide systems.4 The CLSI recommends their use to-
To prevent biohazardous aerosols, all specimens must be gether with standardization of all phases of the methodology,
centrifuged in capped tubes. including the conventional method, as discussed in the follow-
ing sections. Systems currently available include KOVA (KOVA
Sediment Preparation International, Garden Grove, CA), Urisystem (Thermo Fisher
Scientific, Waltham, MA), Count-10 System (Myers-Stevens
A uniform amount of urine and sediment should remain in the
Group, Montebello, CA), Quick-Prep Urinalysis System (Globe
tube after decantation. Volumes of 0.5 and 1.0 mL are fre-
Scientific, Paramus, NJ), CenSlide 2000 Urinalysis System
quently used. The volume of urine centrifuged divided by the
(Iris Diagnostics – Beckman Coulter, Chatsworth, CA), and RS
sediment volume equals the concentration factor, which, in the
Urine Sediment Workstation (VWR, Avantor, Radnor, PA). The
preceding examples, are 24 and 12, respectively. The sediment
systems provide a variety of options:
concentration factor relates to the probability of detecting ele-
ments present in low quantities and is used when quantitating • Capped, calibrated centrifuge tubes
the number of elements present per milliliter. • Decanting pipettes to control sediment volume
• Slides that control the amount of sediment examined,
produce a consistent monolayer of sediment for exami-
Technical Tip 7-2. Commercial systems are available nation, and provide calibrated grids for more consistent
that have tubes designed for decanting and provide a quantitation
constant volume for suspension.
The Cen-Slide and RS Urine Sediment Workstations do not
require manual loading of the centrifuged specimen onto a slide
To maintain a uniform sediment concentration factor, and are considered closed systems that minimize exposure to
urine should be aspirated off rather than poured off, unless the specimen. Cen-Slide provides a tube that is specially
otherwise specified by the commercial system in use. Some designed to permit direct reading of the urine sediment. The RS
systems provide pipettes for this purpose. The pipettes also are Urine Sediment Workstation consists of a glass flow cell into
used for sediment resuspension as well as transfer to the slide. which urine sediment is pumped, microscopically examined,
The sediment must be resuspended thoroughly by gentle and then flushed from the system.
agitation. This can be performed using a commercial-system Examining the Sediment
pipette or by repeatedly tapping the tip of the tube with the
finger. Vigorous agitation should be avoided, as it may disrupt Microscopic examination should be performed in a consistent
some cellular elements. Thorough resuspension is essential to manner and include observation of a minimum of 10 fields
provide equal distribution of elements in the microscopic under both low (10×) and high (40×) power. First, the slide is
examination fields. examined under low power to detect casts and to ascertain the
Visit www.fadavis.com for Video 7-1 (Preparation of general composition of the sediment. When elements are
urine for microscopic examination). encountered that require identification, such as casts, the setting
is changed to high power.
If the conventional glass-slide method is being used, casts
Volume of Sediment Examined tend to locate near the edges of the cover slip; therefore, low-
The volume of sediment placed on the microscope slide should power scanning of the cover-slip perimeter is recommended. This
be consistent for each specimen. When using the conventional does not occur when using standardized commercial systems.
glass-slide method, the recommended volume is 20 µL (0.02 mL) When the sediment is examined unstained, many sediment
covered by a 22 × 22 mm glass cover slip. Allowing the specimen constituents have a refractive index similar to urine. Therefore,
to flow outside of the cover slip may result in the loss of heavier it is essential that sediments be examined under reduced light
elements, such as casts. when using bright-field microscopy.
Commercial systems control the volume of sediment ex- Initial focusing can be difficult with a fluid specimen, and
amined by providing slides with chambers capable of contain- care must be taken to ensure that the examination is being per-
ing a specified volume. Care must be taken to ensure the formed in the correct plane. Often an epithelial cell will be
chambers are filled completely. The product literature supplies present to provide a point of reference. Focusing on artifacts
the chamber volume, size of the viewing area, and approximate should be avoided because they are often larger than the reg-
number of low-power and high-power viewing areas, based on ular sediment elements and cause the microscopist to examine
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170 Part Two | Urinalysis

objects in the wrong plane. Continuous focusing with the fine Provided the same microscope and volume of sediment
adjustment aids in obtaining a complete representation of examined are used, the number of lpfs and hpfs per milliliter
the sediment constituents. of urine remains the same, thereby simplifying the calculation.
Visit www.fadavis.com for Video 7-2 (Examination of Laboratories should evaluate the advantages and disad-
urine sediment). vantages of adding another calculation step to the microscopic
examination. The CLSI states that all decisions with regard to
reporting of the microscopic examination should be based on
Technical Tip 7-3. Use reduced light when bright- the needs of the individual laboratory. Procedures should be
field and phase-contrast microscopy at both low
documented completely and followed by all personnel.3
power (10×) and high power (40×) magnifications
are used for sediment examination. Correlating Results
Microscopic results should be correlated with the physical and
Reporting the Microscopic Examination chemical findings to ensure the accuracy of the report. Speci-
The terminology and methods of reporting may differ slightly mens in which the results do not correlate must be rechecked
among laboratories but must be consistent within a particular for both technical and clerical errors. Table 7-2 shows some of
laboratory system. Routinely, casts are reported as the average the more common correlations in the urinalysis; however, the
number per low-power field (lpf) after examination of 10 fields, number of formed elements or chemicals also must be consid-
and RBCs and WBCs as the average number per 10 high-power ered, as well as the possibility of interference with chemical
fields (hpf). Epithelial cells, crystals, and other elements are fre- tests and the age of the specimen.
quently reported in semiquantitative terms, such as rare, few,
moderate, and many, or as 1+, 2+, 3+, and 4+, following labo- Technical Tip 7-4. Recheck urine specimens for
ratory format as to lpf or hpf use. Laboratories must also deter- both technical and clerical errors in which the
mine their particular reference values based on the sediment physical, chemical, and microscopic results do
concentration factor in use. For example, Urisystem, with a not correlate.
concentration factor of 30, states a reference value for WBCs of
0 to 8 per hpf, as opposed to the conventional value of 0 to
5 per hpf used with a concentration factor of 12.
Sediment Examination
EXAMPLE
Techniques
Converting the average number of elements per lpf or hpf to
the number per milliliter provides standardization among the Many factors can influence the appearance of the urinary
various techniques in use. Steps include the following: sediment, including cells and casts in various stages of de-
1. Calculate the area of a lpf or hpf for the microscope velopment and degeneration, distortion of cells and crystals
in use using the manufacturer-supplied field-of-view by the chemical content of the specimen, the presence of in-
diameter and the formula πr2 = area. clusions in cells and casts, and contamination by artifacts.
Therefore, identification can be difficult even for experienced
Diameter of hpf = 0.35 mm
3.14 × 0.1752 = 0.096 mm2
2. Calculate the maximum number of lpfs or hpfs in the
viewing area. HISTORICAL NOTE
Area under a 22 mm × 22 mm cover slip = 484 mm2 Addis Count
484
= 5040 hpfs
.096 The first procedure to standardize the quantitation of
3. Calculate the number of hpfs per milliliter of urine formed elements in urine microscopic analysis was devel-
tested using the concentration factor and the volume oped by Addis in 1926. The Addis count, as it is called,
of sediment examined. used a hemocytometer to count the number of RBCs,
WBCs, casts, and epithelial cells present in a 12-hour spec-
5040 5040 imen. Normal values have a wide range and are approxi-
= = 21,000 hpfs/mL of urine
0.02 mL × 12 .24 mately 0 to 500,000 RBCs, 0 to 1,800,000 WBCs and
4. Calculate the number of formed elements per milliliter of epithelial cells, and 0 to 5000 hyaline casts.5 The Addis
urine by multiplying the number of hpfs per milliliter by count, which was used primarily to monitor the course of
the average number of formed elements per field. diagnosed cases of renal disease, has been replaced by
various standardized commercial systems for the prepara-
4 WBC/hpf × 21,000 hpfs/mL of urine = tion, examination, and quantitation of formed elements in
84,000 WBC/mL of urine nontimed specimens.
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Chapter 7 | Microscopic Examination of Urine 171

Table 7–2 Routine Urinalysis Correlations Supravital Stains

Microscopic The stain used most frequently in urinalysis is the Sternheimer-
Elements Physical Chemical Exceptions Malbin stain, which consists of crystal violet and safranin O.6 The
stain is available commercially under a variety of names, includ-
RBCs Turbidity + Blood Number ing Sedi-Stain (Becton, Dickinson, Parsippany, NJ) and KOVA
Red color + Protein Hemolysis stain (KOVA International, Garden Grove, CA). Commercial
WBCs Turbidity + Protein Number brands contain stabilizing chemicals to prevent the precipitation
that occurred with the original stain. The dye is absorbed well by
+ Nitrite Lysis
WBCs, epithelial cells, and casts, providing clearer delineation of
+ LE structure and contrasting colors of the nucleus and cytoplasm.
Epithelial Turbidity Number Table 7-4 lists the staining reactions as described in the product
cells literature.
Casts + Protein Number A 0.5% solution of toluidine blue, a metachromatic stain,
provides enhancement of nuclear detail. It can be useful in
Bacteria Turbidity ↑ pH Number
the differentiation between WBCs and renal tubular epithelial
and type
cells and also is used in the examination of cells from other
+ Nitrite body fluids.
+ Leukocytes
Acetic Acid
Crystals Turbidity pH Number
and type Nuclear detail of WBCs and epithelial cells also is enhanced
Color + Bilirubin by the addition of one to two drops of 2% acetic acid to the
urine sediment. This method cannot be used for initial sedi-
ment analysis because RBCs are lysed by the acetic acid (see
Fig. 7-16 later in this chapter).
laboratory personnel. Identification can be enhanced
through the use of sediment stains (Table 7-3) and different Lipid Stains
types of microscopy. The passage of lipids (triglycerides, neutral fats, and choles-
terol) across the glomerular membrane results in the appear-
Sediment Stains ance of free fat droplets and lipid-containing cells and casts in
Staining increases the overall visibility of sediment elements the urinary sediment. The lipid stains Oil Red O and Sudan III
being examined using bright-field microscopy by changing their and polarizing microscopy can be used to confirm the pres-
refractive index. As mentioned, elements such as hyaline casts ence of these elements. Triglycerides and neutral fats stain
have a refractive index very similar to that of urine. Staining orange-red in the presence of stain, whereas cholesterol does
also imparts identifying characteristics to cellular structures, not stain but is capable of polarization. Usually the three lipids
such as the nuclei, cytoplasm, and inclusions. are present concurrently in the sediment, thereby permitting

Table 7–3 Urine Sediment Stain Characteristics

Stain Action Function
Sternheimer-Malbin Delineates structure and contrasting Identifies WBCs, epithelial cells, and casts
colors of the nucleus and cytoplasm
Toluidine blue Enhances nuclear detail Differentiates WBCs from RTE cells
2% acetic acid Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs, yeast, oil droplets,
WBCs and crystals
Lipid stains: Oil Red O and Stain triglycerides and neutral fats Identify free fat droplets and lipid-containing cells
Sudan III orange-red; do not stain cholesterol and casts
Gram stain Differentiates gram-positive and gram- Identifies bacterial casts
negative bacteria
Hansel stain Methylene blue and eosin Y stains Identifies urinary eosinophils
eosinophilic granules
Prussian blue stain Stains structures containing iron Identifies yellow-brown granules of hemosiderin in
cells and casts
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172 Part Two | Urinalysis

Table 7–4 Expected Staining Reactions of Urine Sediment Constituents

Elements in Urinary Usual Distinguishing
Sediment Color of Stained Elements Comments
RBCs Neutral—pink to purple
Acid—pink (unstained)
Alkaline—purple
Nuclei Cytoplasm
WBCs (dark-staining cells) Purple Purple granules
Glitter cells (Sternheimer- Colorless or light blue Pale blue or gray Some glitter cells exhibit
Malbin positive cells) brownian movement
Renal tubular Dark shade of blue- Light shade of blue-purple
epithelial cells purple
Bladder tubular Blue-purple Light purple
epithelial cells
Squamous epithelial cells Dark shade of orange- Light purple or blue
purple
Inclusions and Matrix
Hyaline casts Pale pink or pale purple Very uniform color;
slightly darker than mu-
cous threads
Coarse granular Dark purple granules in
inclusion casts purple matrix
Finely granular Fine dark purple granules
inclusion casts in pale pink or pale
purple matrix
Waxy casts Pale pink or pale purple Darker than hyaline casts,
but of a pale even color;
distinct broken ends
Fat inclusion casts Fat globules unstained in Rare; presence is con-
a pink matrix firmed if examination
under polarized light in-
dicates double refraction
Red cell inclusion casts Pink to orange-red Intact cells can be seen in
matrix
Blood (hemoglobin) casts Orange-red No intact cells
Bacteria Motile: do not stain Motile organisms are not
impaired
Nonmotile: stain purple
Trichomonas vaginalis Light blue-green Motility is unimpaired in
fresh specimens when
recommended volumes
of stain are used; immo-
bile organisms also
identifiable
Mucus Pale pink or pale blue
Background Pale pink or pale purple

From Product Profile: Sedi-Stain. Clay Adams, Division of Becton, Dickinson & Company, Parsippany, NJ, 1974, with permission.
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Chapter 7 | Microscopic Examination of Urine 173

use of either staining or using polarizing microscopy for their Microscopy

confirmation.
Microscopic examination of urine is best performed by a lab-
Gram Stain oratorian who is knowledgeable about the types of microscopes
available, their primary characteristics, and the proper use and
The Gram stain is used primarily in the microbiology section
maintenance of these microscopes.
for the differentiation between gram-positive (blue) and gram-
Bright-field microscopy is the most common type of mi-
negative (red) bacteria. Its role in routine urinalysis is limited
croscopy performed in the urinalysis laboratory. Other types
to the identification of bacterial casts, which can easily be con-
of microscopy that are useful for examining the urine sediment
fused with granular casts. To perform Gram staining, a dried,
are phase contrast, polarizing, dark field, fluorescence, and in-
heat-fixed preparation of the urine sediment must be used.
terference contrast (Table 7-5). The type of microscopy used
Hansel Stain depends on the specimen type, the refractive index of the ob-
ject, and the ability to image unstained living cells. All micro-
Polynuclear WBCs seen in the urinary sediment are almost al- scopes are designed to magnify small objects to such a degree
ways neutrophils associated with microbial infection. However, that the details of their structure can be analyzed. Basically,
in cases of a drug-induced (penicillin or its analogs) allergic they do this by employing a variety of lenses and light sources,
reaction producing inflammation of the renal interstitium, as described in the following section.
eosinophils are present in the sediment. Other causes of
eosinophiluria are renal transplant rejection, pyelonephritis, The Microscope
prostatitis, and cystitis.7 The preferred stain for urinary
Essentially all types of microscopes contain a lens system; an
eosinophils is Hansel stain, consisting of methylene blue and
illumination system; and a body consisting of a base, body tube,
eosin Y in methanol; however, Wright’s stain and Giemsa stain
and nosepiece (Fig. 7-1). The components of the lens system
also can be used. Staining is performed on a dried smear of the
are the oculars, objectives, and coarse- and fine-adjustment
centrifuged specimen or a cytocentrifuged preparation of the
knobs. The illumination system contains the light source, con-
sediment.
denser, and field and iris diaphragms. Objects to be examined
Prussian Blue Stain are placed on a platform, referred to as the mechanical stage.

As discussed in Chapter 6, after episodes of hemoglobinuria,

yellow-brown granules may be seen in renal tubular epithelial
Table 7–5 Urinalysis Microscopic Techniques
cells and casts or free-floating in the urine sediment. To con-
firm that these granules are hemosiderin, the Prussian blue Technique Function
stain for iron is used and stains the hemosiderin granules a
Bright-field microscopy Used for routine urinalysis
blue color.
Phase-contrast Enhances visualization of ele-
Cytodiagnostic Urine Testing microscopy ments with low refractive
indices, such as hyaline
Although it is not a part of the routine examination of the
casts, mixed cellular casts,
urine sediment, the preparation of permanent slides using cy-
mucous threads, and
tocentrifugation followed by staining with Papanicolaou stain
Trichomonas vaginalis
provides an additional method for detecting and monitoring
renal disease. Cytodiagnostic urine testing is frequently per- Polarizing microscopy Aids in identification of cho-
formed independently of routine urinalysis for detection of lesterol in oval fat bodies,
malignancies of the lower urinary tract. A voided first morning fatty casts, and crystals
specimen is recommended for testing, which is performed by Dark-field microscopy Aids in identification of
the cytology laboratory. Cytodiagnostic urine testing also pro- Treponema pallidum
vides more definitive information about renal tubular changes Fluorescence Allows visualization of natu-
associated with transplant rejection; viral, fungal, and parasitic microscopy rally fluorescent microor-
infections; cellular inclusions; pathological casts; and inflam- ganisms or those stained by
matory conditions. The urinalysis laboratory should refer a fluorescent dye, including
specimens with unusual cellular findings to the pathologist labeled antigens and
for further examination. antibodies
Interference-contrast Produces a three-dimensional
Technical Tip 7-5. Use supravital stains to enhance microscopy image and
the nuclei, cytoplasm, and cellular inclusions to aid in layer-by-layer imaging of a
the identification of urine components. specimen
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174 Part Two | Urinalysis

Interpupillary Ocular
distance control

Body
Nosepiece
Condenser
focus knob Objective
Course
adjustment
knob Condenser aperture
Fine diaphragm control ring
adjustment Condenser
knob
Mechanical stage Centering screw
adjustment knobs
Field diaphragm
control ring
Rheostat
Figure 7–1 Parts of the binocular
microscope.

The compound bright-field microscope is used primarily in is best when the distance between the two objects is small. It
the urinalysis laboratory and consists of a two-lens system is dependent on the wavelength of light and the numerical
combined with a light source. The first lens system is located in aperture of the lens. The shorter the wavelength of light, the
the objective and is adjusted to be near the specimen. The sec- greater the resolving power of the microscope will be. Objec-
ond lens system, the ocular lens, is located in the eyepiece. The tives used routinely in the clinical laboratory have magnifica-
path of light passes through the specimen up to the eyepiece. tions of 10× (low power, dry), 40× (high power, dry), and
The oculars or eyepieces of the microscope are located at 100× (oil immersion). The objectives used for examination of
the top of the body tube. Clinical laboratory microscopes are urine sediment are 10× and 40×. The final magnification of an
binocular, allowing the examination to be performed using object is the product of the objective magnification times the
both eyes to provide more complete visualization. For optimal ocular magnification. Using a 10× ocular and a 10× objective
viewing conditions, the oculars can be adjusted horizontally provides a total magnification of 100× and in urinalysis is the
to adapt to differences in interpupillary distance between lpf observation. The 10× ocular and the 40× objective provide
operators. A diopter adjustment knob on the oculars can be a magnification of 400× for hpf observations.
rotated to compensate for variations in vision between the op-
erators’ eyes. The oculars are designed to further magnify the
object that has been enhanced by the objectives for viewing.
Laboratory microscopes normally contain oculars capable of PROCEDURE 7-1
increasing the magnification 10 times (10×). The field of view
Care of the Microscope
is determined by the eyepiece and is the diameter of the circle
of view when looking through the oculars. The field of view 1. Carry the microscope with two hands, supporting the
varies with the field number engraved on the eyepiece and the base with one hand.
magnification of the objective. The higher the magnification, 2. Always hold the microscope in a vertical position.
the smaller the field of view will be. In urinalysis microscopy, 3. Clean optical surfaces only with a high-quality lens
sediment constituents are reported as the number per micro- tissue and commercial lens cleaner.
scopic field (number per hpf or lpf).
4. Do not use the 10× and 40× objectives with oil.
Objectives are contained in the revolving nosepiece lo-
cated above the mechanical stage. Objectives are adjusted to 5. Clean the oil immersion lens after use.
be near the specimen and perform the initial magnification of 6. Always remove slides with the low-power objective
the object on the mechanical stage. Then the image passes to raised.
the oculars for further resolution (ability to visualize fine de- 7. Store the microscope with the low-power objective in
tails). Resolution is the ability of the lens to distinguish two position and the stage centered.
small objects that are a specific distance apart. Resolving power
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Chapter 7 | Microscopic Examination of Urine 175

Objectives are inscribed with information that describes the light on the specimen and controls the light for uniform il-
their characteristics and includes the type of objective (plan used lumination. The normal position of the condenser is almost
for bright field, ph for phase contrast), magnification, numerical completely up, with the front lens of the condenser near the
aperture, microscope tube length, and cover-slip thickness to be slide but not touching it. The condenser adjustment (focus)
used. The numerical aperture number represents the refractive knob moves the condenser up and down to focus light on the
index of the material between the slide and the outer lens (air object. An aperture diaphragm in the condenser controls the
or oil) and the angle of the light passing through it. The higher amount of light and the angle of light rays that pass to the spec-
the numerical aperture, the better the light-gathering capability imen and lens, which affect resolution, contrast, and depth of
of the lens will be, thus yielding greater resolving power. The field of the image. By adjusting the aperture diaphragm to 75%
length of the objectives attached to the nosepiece varies with of the numerical aperture of the objective, maximum resolution
magnification (length increases from 10× to 100× magnifica- is achieved. The aperture diaphragm should not be used to re-
tion), thereby changing the distance between the lens and the duce light intensity because it decreases resolution. The mi-
slide when they are rotated. The higher the numerical aperture, croscope lamp rheostat is used for this adjustment.
the closer the lens is to the object. Most microscopes are de-
Köhler Illumination
signed to be parfocal, indicating that they require only minimum
Two adjustments to the condenser—centering and Köhler
adjustment when switching among objectives.
illumination—provide optimal viewing of the illuminated
The distance between the slide and the objective is con-
field. They should be performed whenever an objective is
trolled by the coarse- and fine-focusing knobs located on the
changed; see Procedure 7-2.
body tube. Initial focusing is performed using the coarse knob
Additional focusing of the object should be performed
that moves the mechanical stage noticeably up and down until
using the adjustment knobs and the rheostat on the light
the object comes into view. This is followed by adjustment
source.
using the fine-focusing knob to sharpen the image. When
Routine preventive maintenance procedures on the mi-
using a parfocal microscope, only the fine knob should be used
croscope ensure good optical performance and include the
for adjustment when changing magnifications.
following:
Illumination for the modern microscope is provided by a
light source located in the base of the microscope. The light • The microscope should be covered when not in use to
source is equipped with a rheostat to regulate the intensity of protect it from dust. If any optical surface becomes
the light. Filters also may be placed on the light source to vary coated with dust, the dust should be removed carefully
the illumination and wavelengths of the emitted light. A field with a camel-hair brush.
diaphragm contained in the light source controls the diameter • Optical surfaces should be cleaned with lens paper.
of the light beam reaching the slide and is adjusted for optimal Clean any contaminated lens immediately with a com-
illumination. Then a condenser located below the stage focuses mercial lens cleaner. An oil immersion lens must be

PROCEDURE 7-2
Microscope Alignment for Kohler Illumination 5. Open the field diaphragm until its image is at the edge
Procedure of the field.
To center the condenser and obtain Köhler illumination, take 6. Remove an eyepiece and look down through the eye-
the following steps: piece tube.
1. Place a slide on the stage, and focus the object using 7. Adjust the aperture diaphragm until approximately
the low-power objective with the condenser raised. 75% of the field is visible.
2. Close the field diaphragm. Field of view Field of
3. Lower the condenser until the edges of the field view
diaphragm are focused sharply. Illuminated Illuminated
zone zone
4. Center the image of the field diaphragm with the
rcondenser-centering screws.

Field of 8. Replace the eyepiece.

view
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176 Part Two | Urinalysis

wiped free of oil and cleaned after each use. Fingerprints

PROCEDURE 7-3
and oil smears impair the sharpness of an image.
• An annual professional cleaning for the microscope is Phase-Contrast Microscope Alignment
recommended. Procedure
• Light sources are replaced as necessary. 1. Focus the microscope in bright field with a specimen
slide.
Types of Microscopy 2. Select a low-power phase condenser ring.
3. Select the corresponding ring objective.
Bright-Field Microscopy
4. Remove an ocular, insert the adjustment telescope,
Bright-field microscopy, in which objects appear dark and look through the telescope.
against a light background, is used most frequently in the clin-
5. Observe the dark and light rings (annuli).
ical laboratory. This technique employs the basic microscope
described previously with a light source emitting light in the 6. With the adjusting screw on the telescope, center the
visible wavelength range. light annulus (condenser) over the dark annulus
Use of bright-field microscopy for the examination of (objective).
urine sediment can present problems when the amount of light Condenser
reaching the specimen is not controlled properly. Sediment ring
constituents with a low refractive index will be overlooked
when subjected to light of high intensity. Therefore, sediments
must be examined using decreased light controlled by adjust-
ing the rheostat on the light source, not by lowering the con-
denser. Staining of the sediment also increases the visualization
of these elements when using bright-field microscopy.

Phase-Contrast Microscopy
As light rays pass through an object, they are slowed in com-
parison to the rays passing through air (media), thereby de-
Phase objective Alignment
creasing the intensity of the light and producing contrast. This ring
is called phase difference and is affected by the thickness of the
object, refractive index, and other light-absorbance properties. Centering phase microscope rings
The best contrast is obtained when the light that does not pass
7. Replace the ocular.
through the specimen is shifted one quarter of a wavelength
and compared with the phase difference of the specimen.
Phase-contrast microscopy provides this contrast.
Phase-contrast microscopy is accomplished by adaptation of
a bright-field microscope with a phase-contrast objective lens and particularly advantageous for identifying low-refractive hyaline
a matching condenser. Two phase rings that appear as “targets” casts or mixed cellular casts and mucous threads.
are placed in the condenser and the objective. One phase ring is
Polarizing Microscopy
placed either in the condenser or below it, permitting light to
pass through only the central clear circular area. A second phase- The use of polarized light aids in the identification of crystals
shifting ring with a central circular area that retards the light by and lipids. Both substances have the ability to rotate the path
one-quarter wavelength is placed in the objective. Phase rings of the unidirectional polarized light beam to produce charac-
must match, so it is important to check that the objective and teristic colors in crystals and Maltese cross formation in
condenser mode are the same. The diameter of the rings varies lipids. These elements seen under polarized light microscopy
with the magnification. The image has the best contrast when the are birefringent, a property indicating that the element can
background is darkest. Phase-contrast rings must be adjusted to refract light in two dimensions at 90 degrees to each other.
have maximum contrast. The two rings are adjusted to make The halogen quartz lamp in the microscope produces light
them concentric; see Procedure 7-3. rays of many different waves. Each wave has a distinct direction
Light passes to the specimen through the clear circle in and a vibration perpendicular to its direction. Normal or unpo-
the phase ring in the condenser, forming a halo of light around larized light vibrates in equal intensity in all directions. Polarized
the specimen. Then the diffracted light enters the central circle light vibrates in the same plane or direction. As the light passes
of the phase-shifting ring, and all other light is moved one through a birefringent substance, it splits into two beams, one
quarter of a wavelength out of phase. The variations of contrast beam rotated 90 degrees of the other. Isotropic substances, such
in the specimen image due to the various refractive indexes in as blood cells, do not have this refractive property, and the light
the object are observed as the light rays merge together, en- passes through unchanged. A substance that rotates the plane of
hancing visualization and detail. Phase-contrast microscopy is polarized light 90 degrees in a clockwise direction is said to have
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Chapter 7 | Microscopic Examination of Urine 177

positive birefringence. In contrast, a substance that rotates the the bright-field microscope are required to perform this tech-
plane in a counterclockwise direction has negative birefringence. nique. Therefore, it is not used routinely in the urinalysis
Polarized light is obtained by using two polarizing filters. laboratory.
The light emerging from one filter vibrates in one plane, and a Two types of interference-contrast microscopy are available:
second filter placed at a 90-degree angle blocks all incoming modulation contrast (Hoffman) and differential-interference
light, except that rotated by the birefringent substance. The fil- contrast (Nomarski). Bright-field microscopes can be adapted
ters are in opposite directions called a “crossed configuration.” for both methods.
Between cross-polarizing filters, birefringent crystals are visible
in characteristic patterns (Fig. 7-2). Modulation-Contrast Microscopy (Hoffman)
Bright-field microscopes can be adapted for polarizing In the modulation-contrast microscope, three modifications
microscopy. Two polarizing filters must be installed in a crossed can be made: (1) a split aperture that contains a polarizing filter
configuration. The first filter, the polarizing filter, is placed in is placed below the condenser; (2) a polarizer, to control con-
the condenser filter holder; the second filter, the analyzer, is trast, is placed below the split aperture; and (3) an amplitude
placed in the head between the objectives and the ocular. The filter, called a modulator, is placed in back of each objective.
polarizing filter is rotated to allow only light vibrating in one The modulator has three zones of light transmission: a dark
direction to reach the object. If the object does not have bire- zone that transmits 1% of light, a gray zone that transmits 15% of
fringent properties, no light will reach the analyzer filter and the light, and a clear zone that transmits 100% of light. The polar-
object will appear black. Refracted rays from a birefringent object ized light rays pass through a split aperture to the various areas
will reach the analyzer, causing the object to appear white or of the specimen and to the modulator, where they are con-
colored against the black background. An additional filter, called verted into the variations of light intensity to produce a three-
a red compensated polarizing filter, can be added to the micro- dimensional image.
scope. This filter divides the light entering the microscope into
slow and fast vibrations. Crystals can be identified more easily
Differential Interference-Contrast Microscopy
by aligning them with the slow vibration and observing the blue
(Nomarski)
or yellow color they produce (see Chapter 12).
The differential interference-contrast microscope uses birefrin-
Polarizing microscopy is used in urinalysis to confirm the
gent crystal prisms as beam splitters to obtain the intensity
identification of fat droplets, oval fat bodies, and fatty casts
difference in the specimen range. To convert a bright-field mi-
that produce a characteristic Maltese cross pattern. Birefringent
croscope for differential interference-contrast microscopy re-
uric acid crystals can be distinguished from cystine crystals,
quires (1) a polarizing filter to output plane-polarized light
monohydrate calcium oxalate crystals from nonpolarizing
placed between the light source and the condenser, (2) a con-
RBCs, and calcium phosphate crystals from nonpolarizing
denser containing modified Wollaston prisms for each objec-
bacteria by their polarizing characteristics.
tive, (3) a Wollaston prism placed between the objective and
Interference-Contrast Microscopy the eyepiece, and (4) a polarizing filter placed behind the Wol-
laston prism and before the eyepiece. A two-layered Nomarski-
Interference-contrast microscopy provides a three-dimensional modified Wollaston prism that separates individual rays of light
image showing very fine structural detail by splitting the into ray pairs is used. The lower Wollaston prism is built into
light ray so that the beams pass through different areas of the condenser of the microscope. The upper prism is placed
the specimen. The light interference produced by the varied between the objective and the eyepiece and recombines the
depths of the specimen is compared, and a three-dimensional rays. Above the top Wollaston prism, another polarizing filter
image is visualized. The advantage of interference-contrast is placed that causes wave interference to occur and produce
microscopy is that an object appears bright against a dark the three-dimensional image (Fig. 7-3).
background, but without the diffraction halo associated with These two types of microscopy provide layer-by-layer im-
phase-contrast microscopy. More extensive modifications to aging of a specimen and enhanced detail for specimens with a
refractive index that is either low or high.

Birefringent Rotated Dark-Field Microscopy

specimen wave
Dark-field microscopy is a technique used in the clinical lab-
oratory to enhance visualization of specimens that cannot be
seen easily when viewed with a bright-field microscope. Often
it is used for unstained specimens and, in particular, to identify
the spirochete Treponema pallidum.
A bright-field microscope is easily adapted for dark-field
microscopy by replacing the condenser with a dark-field con-
Light Polarizing Wave separated Polarizing filter
source filter 90° by specimen 90° orientation
denser that contains an opaque disk. The disk blocks the light
from entering the objective directly, and the field of view is
Figure 7–2 Diagram of polarized light. black. As the light rays pass through the specimen at oblique
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178 Part Two | Urinalysis

Three-dimensional Ocular lens

image
Analyzer
Upper Wollaston
Recombined rays prism

Back focal plane

of objective lens
Objective

Specimen
Condenser Split rays
lens Condenser focal Objective lens
plane
Lower Wollaston
prism
Polarizer
Specimen

Figure 7–3 Differential interference-contrast (Nomarski)

microscopy.

Condenser lens
angles, the light scatters, diffracts, or reflects off the specimen
and is captured by the objective lens. The specimen appears
light against the black background or dark field (Fig. 7-4).
Dark-field stop
Fluorescence Microscopy
Fluorescence microscopy is a technique whose use in the
medical field is rapidly expanding today. It is used to detect Light rays
bacteria and viruses within cells and tissues through a tech-
nique called immunofluorescence. Fluorescence is the property Figure 7–4 Dark-field microscopy.
by which some atoms absorb light at a particular wavelength
and subsequently emit light of a longer wavelength, termed
fluorescence lifetime. The practical application in the labora-
tory is that it allows the visualization of naturally fluorescent Ocular
substances or those that have been stained with a fluorochrome
or fluorophore (fluorescent dyes) to produce an image. The Barrier filter
specimen is illuminated with a light of a specific wavelength.
Fluorescent substances absorb the energy and emit a longer
wavelength of light that is visualized with the use of special fil-
ters, called the excitation filter and the emission filter. The ex- Objective
citation filter selects the excitation wavelength of light from a Specimen
light source. The emission filter selects a specific wavelength Condenser
of emitted light from the specimen to become visible. The fil-
ters are chosen to match the excitation and emission wave-
lengths of the fluorophore that are used to label the specimen.
A dichroic mirror reflects the excitation light to the specimen
and transmits the emitted light to the emission filter, which
is collected with the objective and imaged by the detector Mercury lamp Collector
(Fig. 7-5). The fluorescent substance can be observed in the
fluorescent microscope as a bright object against a dark back-
ground with high contrast when an ultraviolet light source is
used. Powerful light sources are required and are usually either
Incoming Deflecting mirror
mercury or xenon arc lamps.8 Excitation filter
light waves
Ultraviolet light
Urine Sediment Constituents Visible light

The normal urine sediment may contain a variety of formed Figure 7–5 Fluorescent microscopy.
elements. Even the appearance of small numbers of the usually
pathologically significant RBCs, WBCs, and hyaline casts can
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Chapter 7 | Microscopic Examination of Urine 179

be normal. Likewise, many routine urine specimens contain

nothing more than a rare epithelial cell or mucous strand.
Students often have difficulty adjusting to this because in the
classroom setting, urine sediments containing abnormalities
and multiple elements are usually stressed. Students must learn
to trust their observations after looking at the recommended
number of fields. Also, cellular elements are easily distorted by
concentrations that vary widely, as well as pH and the presence
of metabolites in urine, making identification more difficult.
Actual normal numerical values are not clearly defined. As
discussed previously, preparation methods for urine sediment
determine the actual concentration of the sediment and, there-
fore, the number of elements that may be present in a micro-
scopic field. Values that are listed commonly include 0 to 2 or
3 RBCs per hpf, 0 to 5 to 8 WBCs per hpf, and zero to two Figure 7–7 Microcytic and crenated RBCs (×100).
hyaline casts per lpf. Even these figures must be taken in con-
text with other factors, such as recent stress and exercise, men- air bubbles. To differentiate, yeast cells usually exhibit budding
strual contamination, and the presence of other urine sediment (Fig. 7-8). Oil droplets and air bubbles are highly refractile
constituents. To put this in better perspective, the urine sedi- when the fine adjustment is focused up and down (Fig. 7-9);
ment constituents are now discussed individually with refer- also, they may appear in a different plane than other sediment
ence to the accompanying figures. constituents (Fig. 7-10). The rough appearance of crenated
Red Blood Cells RBCs may resemble the granules seen in WBCs, but they are
much smaller than WBCs. Should the identification continue
In the urine, RBCs appear as smooth, nonnucleated, biconcave to be doubtful, adding acetic acid to a portion of the sediment
disks measuring approximately 7 mm in diameter (Fig. 7-6). will lyse the RBCs, leaving the yeast, oil droplets, and WBCs
They must be identified using high-power (40×) objective intact. Supravital staining also may be helpful.
(×400 magnification). RBCs are routinely reported as the
average number seen in 10 hpfs.
In concentrated (hypersthenuric) urine, the cells shrink Technical Tip 7-6. RBCs, particularly dysmorphic
due to loss of water and may appear crenated or irregularly RBCs, are differentiated from yeast, which show bud-
shaped (Fig. 7-7). In dilute (hyposthenuria) urine, the cells ab- ding, and from oil droplets and air bubbles, which are
sorb water, swell, and lyse rapidly, releasing their hemoglobin highly refractile, when the fine adjustment is focused
and leaving only the cell membrane. These large empty cells are up and down. They may appear in a different plane
called ghost cells and can be missed easily if specimens are not than other sediment constituents.
examined under reduced light.
Of all the urine sediment elements, RBCs are the most dif-
ficult for students to recognize. The reasons for this include Studies have focused on the morphology of urinary RBCs as
RBCs’ lack of characteristic structures, variations in size, and an aid in determining the site of renal bleeding. RBCs that vary
close resemblance to other constituents in urine sediment. in size, have cellular protrusions, or are fragmented are termed
RBCs are confused frequently with yeast cells, oil droplets, and dysmorphic (Fig. 7-11) and have been associated primarily with

Figure 7–8 Yeast. The presence of budding forms aids in distin-

Figure 7–6 Normal RBCs (×400). guishing from RBCs (×400).
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180 Part Two | Urinalysis

exercise, indicating a glomerular origin of this phenomenon.11

The dysmorphic cell associated most closely with glomerular
bleeding appears to be the acanthocyte with multiple protru-
sions, which may be difficult to observe under bright-field
microscopy.12,13 Further analysis of sediments containing dys-
morphic RBCs using Wright’s stained preparations shows the
cells to be hypochromic and better delineates the presence of
cellular blebs and protrusions.

Clinical Significance
The presence of RBCs in the urine is associated with damage to
the glomerular membrane or vascular injury within the geni-
tourinary tract. The number of cells present is indicative of the
extent of the damage or injury. Patient histories often mention
Figure 7–9 KOVA-stained squamous epithelial cells and oil droplets the presence of macroscopic versus microscopic hematuria.
(×400). Notice how the oil droplet (arrow) resembles an RBC. When macroscopic hematuria is present, the urine
appears cloudy with a red to brown color. Microscopic analysis
may be reported in terms of greater than 100 per hpf or as
specified by laboratory protocol. Frequently macroscopic
hematuria is associated with advanced glomerular damage but
also is seen with damage to the vascular integrity of the urinary
tract caused by trauma, acute infection or inflammation, and
coagulation disorders.
The observation of microscopic hematuria can be critical
to the early diagnosis of glomerular disorders and malignancy
of the urinary tract and to confirm the presence of renal calculi.
The presence of not only RBCs but also hyaline, granular, and
RBC casts may be seen after strenuous exercise. These abnor-
malities are nonpathological and disappear after rest.14 The
possibility of menstrual contamination also must be considered
in specimens from female patients.
As discussed previously, the presence or absence of RBCs
Figure 7–10 Air bubble. Notice no formed elements are in focus
in the urine sediment cannot always be correlated with speci-
(×100).
men color or a positive chemical test result for blood. The pres-
ence of hemoglobin that has been filtered by the glomerulus
produces a red urine with a positive chemical test result for

SUMMARY 7-1 Microscopic RBCs

Appearance: Nonnucleated biconcave
disks
Crenated in hypertonic urine
Ghost cells in hypotonic
urine
Dysmorphic with glomerular
membrane damage
Figure 7–11 Dysmorphic RBCs (×400). Notice the smaller size and Sources of identifi- Yeast cells
fragmentation. cation error: Oil droplets
Air bubbles
glomerular bleeding. The number and appearance of the dys-
morphic cells also must be considered because abnormal urine Reporting: Average number per 10 hpfs
concentration affects RBC appearance, and small numbers Complete urinalysis Color
of dysmorphic cells are found with nonglomerular hematuria.9,10 correlations: Reagent strip blood reaction
Dysmorphic RBCs also have been manifest after strenuous
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Chapter 7 | Microscopic Examination of Urine 181

blood in the absence of microscopic hematuria. Likewise, a

specimen appearing macroscopically normal may contain a
small but pathologically significant number of RBCs when
examined microscopically.

White Blood Cells

WBCs are larger than RBCs, measuring an average of about
12 mm in diameter (Fig. 7-12).

Neutrophils
The predominant WBC found in the urine sediment is the neu-
trophil. Neutrophils are much easier to identify than RBCs be-
cause they contain granules and multilobed nuclei (Fig. 7-13 A
and B). However, they are still identified using high-power mi-
croscopy and are reported as the average number seen in 10 hpfs.
Neutrophils lyse rapidly in dilute alkaline urine and begin to lose A
nuclear detail.
Neutrophils exposed to hypotonic urine absorb water and
swell. Brownian movement of the granules within these larger
cells produces a sparkling appearance, and they are referred to
as “glitter cells.” When stained with Sternheimer-Malbin stain,
these large cells stain light blue, as opposed to the violet color
usually seen with neutrophils. Glitter cells are of no patholog-
ical significance (Fig. 7-14).

Eosinophils
The presence of urinary eosinophils is associated primarily
with drug-induced interstitial nephritis (acute interstitial
nephritis [AIN]). Small numbers of eosinophils may be seen
B
with urinary tract infection (UTI) and renal transplant rejec-
tion. Evaluation of a concentrated, stained urine sediment is Figure 7–13 WBCs. A. One segmented and one nonsegmented
required for performing a urinary eosinophil test. Urine sedi- WBC (×400). B. Notice the multilobed nucleoli (×400).
ment may be concentrated by routine centrifugation alone or
with cytocentrifugation. The preferred eosinophil stain is
Hansel (Fig. 7-15); however, Wright’s stain or Giemsa stain also
can be used. The percentage of eosinophils in 100 to 500 cells
is determined. Eosinophils are not normally seen in the urine;
therefore, the finding of more than 1% eosinophils is consid-
ered significant.15

Figure 7–14 Glitter cells (×400). Observe the very noticeable

granules.

Figure 7–12 RBCs and one WBC (×400). Notice the larger size and
granules in the WBC.
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182 Part Two | Urinalysis

Figure 7–15 Hansel-stained eosinophils (×400). Figure 7–16 WBCs with acetic acid nuclear enhancement. Notice
the ameboid shape in some of the WBCs.

Mononuclear Cells SUMMARY 7-2 Microscopic WBCs

Lymphocytes, monocytes, macrophages, and histiocytes may
be present in small numbers and usually are not identified in Appearance: Larger than RBCs
the wet preparation urine microscopic analysis. Because lym- Granulated, multilobed
phocytes are the smallest WBCs, they may resemble RBCs. neutrophils
They may be seen in increased numbers in the early stages of Glitter cells in hypotonic
renal transplant rejection. Monocytes, macrophages, and his- urine
tiocytes are large cells and may appear vacuolated or contain Mononuclear cells with
inclusions. Specimens containing an increased number of abundant cytoplasm
mononuclear cells that cannot be identified as epithelial cells
should be referred for cytodiagnostic urine testing. Sources of identifi- Renal tubular epithelial cells
The primary concern in the identification of WBCs is the cation error:
differentiation of mononuclear cells and disintegrating neu- Reporting: Average number per 10 hpfs
trophils from round renal tubular epithelial (RTE) cells. RTE Complete urinalysis Leukocyte esterase
cells are usually larger than WBCs with an eccentrically located correlations: Nitrite
nucleus. WBCs in the process of ameboid motion may be dif-
ficult to distinguish from epithelial cells because of their irreg- Specific gravity
ular shape. Supravital staining or the addition of acetic acid pH
can be used to enhance nuclear detail (Fig. 7-16), if necessary.

Technical Tip 7-7. Use a supravital stain to differenti- Epithelial Cells

ate mononuclear cells and disintegrating neutrophils
from RTE cells, which are usually larger than WBCs and
It is not unusual to find epithelial cells in the urine because they
have an eccentrically located nucleus.
are derived from the linings of the genitourinary system. Unless
they are present in large numbers or in abnormal forms, they
represent normal sloughing of old cells. Three types of epithelial
Usually, fewer than five leukocytes per hpf are found in nor- cells are seen in urine: squamous, transitional (urothelial), and
mal urine; however, higher numbers may be present in urine renal tubular (Fig. 7-17). They are classified according to their
from females. Although leukocytes, like RBCs, may enter the site of origin within the genitourinary system.
urine through glomerular or capillary trauma, they also are ca-
Squamous Epithelial Cells
pable of ameboid migration through the tissues to sites of infec-
tion or inflammation. An increase in urinary WBCs is called Squamous cells are the largest cells found in the urine sediment.
pyuria and indicates the presence of an infection or inflammation They contain abundant, irregular cytoplasm and a prominent
in the genitourinary system. Bacterial infections, including nucleus about the size of an RBC (Fig. 7-18 A and B). Often,
pyelonephritis, cystitis, prostatitis, and urethritis, are frequent they are the first structures observed when the urine sediment
causes of pyuria. However, pyuria is also present in nonbacterial is examined under low-power magnification. Usually at least
disorders, such as glomerulonephritis, lupus erythematosus, in- a few squamous epithelial cells are present in the urine sediment
terstitial nephritis, and tumors. Reporting the presence of bacteria and can serve as a good reference for focusing of the micro-
in specimens containing leukocytes is important. scope. After examination of the appropriate number of fields,
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Chapter 7 | Microscopic Examination of Urine 183

sediments containing large amounts of squamous cells, clumps

RBC of cells may make it more difficult to enumerate smaller patho-
logical elements, such as RBCs and WBCs, and they should be
examined carefully (Figs. 7-19, 7-20, and 7-21).

Technical Tip 7-8. Epithelial cells can be misidentified

Transitional
when they are folded and look like a cast or when they
begin to disintegrate in urine that is not fresh.

Squamous WBC
Squamous epithelial cells originate from the linings of the
RTE vagina and female urethra and the lower portion of the male
urethra. They represent normal cellular sloughing and have no
pathological significance. Increased amounts are seen more
Figure 7–17 Sediment-containing squamous, caudate transitional, frequently in urine from female patients. Specimens collected
and RTE cells (×400). using the clean-catch midstream technique contain less contam-
ination from squamous cells.
A variation of the squamous epithelial cell is the clue
cell, which does have pathological significance. Clue cells are

Figure 7-19 Phenazopyridine-stained sediment showing squa-

mous epithelial cells and phenazopyridine crystals formed after
refrigeration (×400).

Figure 7–18 A. Squamous epithelial cells identifiable under low

power (×100). B. KOVA-stained squamous epithelial cells (×400).
Compare the size of the nucleus with the RBCs in Figure 7-6.

squamous epithelial cells are commonly reported in terms of

rare, few, moderate, or many. They are reported in terms of
low-power or high-power magnification based on laboratory
protocol.
Difficulty identifying squamous cells is rare. However, oc-
casionally they may appear folded, possibly resembling a cast,
and will begin to disintegrate in urine that is not fresh. In urine Figure 7–20 Clump of squamous epithelial cells (×400).
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184 Part Two | Urinalysis

Figure 7–23 KOVA-stained spherical transitional epithelial cells

Figure 7–21 Clump of squamous epithelial cells with folded forms
(×400).
(×400).

indicative of vaginal infection by the bacterium Gardnerella

vaginalis. They appear as squamous epithelial cells covered
with the Gardnerella coccobacillus. To be considered a clue cell,
the bacteria should cover most of the cell surface (70%) and
extend beyond the edges of the cell. This gives the cell a gran-
ular, irregular appearance. Routine testing for clue cells is per-
formed by examining a vaginal wet preparation for the presence
of the characteristic cells (see Chapter 17). However, small
numbers of clue cells may be present in the urinary sediment.
Microscopists should remain alert for their presence, as urinal-
ysis may be the first test performed on the patient.

Transitional Epithelial (Urothelial) Cells

Transitional epithelial cells are smaller than squamous cells and
Figure 7–24 Caudate transitional epithelial cells (×400).
appear in several forms, including spherical, polyhedral, and
caudate (Figs. 7-22, 7-23, and 7-24). These differences are
caused by the ability of transitional epithelial cells to absorb
that are distinct and centrally located. Transitional cells are
large amounts of water. Cells in direct contact with the urine
identified and enumerated using high-power magnification.
absorb water, becoming spherical in form and much larger
Like squamous cells, usually they are reported as rare, few,
than the polyhedral and caudate cells. All forms have nuclei
moderate, or many following laboratory protocol.
Spherical forms of transitional epithelial cells are sometimes
difficult to distinguish from RTE cells. The presence of a nucleus
that is centrally located rather than eccentrically placed, as well
as supravital staining, can aid in the differentiation.
Transitional epithelial cells originate from the lining of the
renal pelvis, calyces, ureters, and bladder and from the upper
portion of the male urethra. Usually they are present in small
numbers in normal urine, representing normal cellular slough-
ing. Increased numbers of transitional cells seen singly, in pairs,
or in clumps (syncytia) are present after invasive urological
procedures, such as catheterization, and are of no clinical sig-
nificance (Fig. 7-25). An increase in transitional cells exhibiting
abnormal morphology, such as vacuoles and irregular nuclei,
may be indicative of malignancy or viral infection. In such
cases, the specimen should be referred to the pathologist.

Renal Tubular Epithelial Cells

RTE cells vary in size and shape depending on the area of the
Figure 7–22 Transitional epithelial cells. renal tubules from which they originate. The cells from the
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Chapter 7 | Microscopic Examination of Urine 185

Figure 7–25 Syncytia of transitional epithelial cells from catheter- Figure 7–27 RTE cells. Oval distal convoluted tubule cells. Notice
ized specimen (×400). the eccentrically placed nuclei (×400).

proximal convoluted tubule (PCT) are larger than other RTE polyhedral transitional cells (Fig. 7-28). Because RTE cells often
cells. They tend to have a rectangular shape and are referred are present as a result of tissue destruction (necrosis), the nucleus
to as columnar or convoluted cells. The cytoplasm is coarsely is not easily visible in unstained sediment.
granular, and the RTE cells often resemble casts. They should Cells from the collecting duct that appear in groups of
be examined closely for the presence of a nucleus, as a nucleus three or more are called renal fragments. Frequently they are
would not be present in a cast. Notice the nucleus and granules seen as large sheets of cells. PCT and DCT cells are not seen in
in Figure 7-26. This is a PCT cell that has absorbed fat globules large sheets of cells (Fig. 7-29).
and could easily be mistaken for a granular or fatty cast. RTE cells must be identified and enumerated using high-
Cells from the distal convoluted tubule (DCT) are smaller power magnification. Depending on laboratory protocol, they
than those from the PCT and are round or oval. They can be may be reported as rare, few, moderate, or many or as the actual
mistaken for WBCs and spherical transitional epithelial cells. number per hpf. Classification of RTE cells as to site of origin is
Observation of the round nucleus that is eccentrically placed not considered a part of the routine sediment analysis and often
aids in differentiating them from spherical transitional cells requires special staining techniques. The presence of more than
(Fig. 7-27). two RTE cells per hpf indicates tubular injury, and such speci-
mens should be referred for cytological urine testing.16
Technical Tip 7-9. It is important to differentiate Clinical Significance
squamous and transitional epithelial cells from RTE RTE cells are the most clinically significant of the epithelial
cells, as RTE cells are a sign of severe tubular necrosis. cells. The presence of increased amounts is indicative of necro-
sis of the renal tubules, with the possibility of affecting overall
RTE cells from the collecting duct are cuboidal and are never renal function.
round. Along with the eccentrically placed nucleus, the presence A number of conditions may produce tubular necrosis:
of at least one straight edge differentiates them from spherical and • Exposure to heavy metals
• Drug-induced toxicity

Figure 7–26 RTE cell. Columnar proximal convoluted tubule cell

with granules and attached fat globules (×400). N, nucleus. Figure 7–28 RTE cells, cuboidal from the collecting duct (×400).
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186 Part Two | Urinalysis

Figure 7–29 Fragment of RTE cells from the collecting duct under
Figure 7–30 Prussian blue–stained hemosiderin granules.
phase microscopy (×400).

• Hemoglobin and myoglobin toxicity

• Viral infections (hepatitis B)
• Pyelonephritis
• Allergic reactions
• Malignant infiltrations
• Salicylate poisoning
• Acute allogenic transplant rejection
RTE cells also may be seen as secondary effects of
glomerular disorders. Renal fragments are an indication of se-
vere tubular injury with basement membrane disruption. Sin-
gle cuboidal cells are particularly noticeable in cases of
salicylate poisoning.
Because one of the functions of RTE cells is reabsorption of Figure 7–31 Oval fat body (×400).
the glomerular filtrate, it is not unusual for them to contain sub-
stances from the filtrate. RTE cells absorb bilirubin present in the
filtrate as the result of liver damage, such as occurs with viral hep- orange-red droplets (Fig. 7-32). Examination of the urine sed-
atitis, and appear a deep yellow color. As discussed in Chapter 6, iment using polarized light results in the appearance of char-
hemoglobin present in the filtrate is absorbed by the RTE acteristic Maltese cross formations in droplets containing
cells and converted to hemosiderin. Therefore, after episodes of cholesterol (Fig. 7-33). Urine sediments negative for fat after
hemoglobinuria (transfusion reactions, paroxysmal nocturnal staining still should be checked using polarized light in case
hemoglobinuria, etc.), the RTE cells may contain the character- only cholesterol is present. Likewise, staining should be per-
istic yellow-brown hemosiderin granules. The granules also may formed on urine sediments negative under polarized light.
be seen free-floating in the urine sediment. Confirmation of the Oval fat bodies are reported as the average number per hpf.
presence of hemosiderin is performed by staining the urine
sediment with Prussian blue. The iron-containing hemosiderin
granules stain blue (Fig. 7-30). Technical Tip 7-10. Check urine sediments that are
negative for fat after staining using polarized light to
Oval Fat Bodies observe the Maltese cross formation, which occurs
RTE cells absorb lipids that are present in the glomerular fil- when only cholesterol is present.
trate. Then they appear highly refractile, and the nucleus may
be more difficult to observe. These lipid-containing RTE cells
are called oval fat bodies (Fig. 7-31). Usually they are seen in Free-floating fat droplets also stain or polarize depending
conjunction with free-floating fat droplets. on their composition. They may be observed floating on the
Identification of oval fat bodies is confirmed by staining top of the specimen. Care should be taken not to confuse the
the urine sediment with Sudan III or Oil Red O fat stains and droplets with starch and crystal particles that also polarize.
examining the sediment using polarized microscopy. The Specimen contamination by vaginal preparations and lubri-
droplets are composed of triglycerides, neutral fats, and choles- cants used in specimen collection must be considered when
terol. Fat stains stain triglycerides and neutral fats, producing only free-floating fat droplets are present.
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Chapter 7 | Microscopic Examination of Urine 187

Lipiduria is associated most frequently with damage to

the glomerulus caused by nephrotic syndrome (see Chapter 8).
It is also seen with severe tubular necrosis, diabetes mellitus,
and in trauma cases that cause release of bone marrow fat from
the long bones. In lipid-storage diseases, large fat-laden histi-
ocytes also may be present. They can be differentiated from
oval fat bodies by their large size.
In cases of acute tubular necrosis, RTE cells containing
large, nonlipid-filled vacuoles may be seen along with normal
renal tubular cells and oval fat bodies. Referred to as “bubble
cells,” they appear to represent injured cells in which the
endoplasmic reticulum has dilated before cell death.17

Bacteria
Figure 7–32 Sudan III–stained oval fat body (×400).
Bacteria are not normally present in urine. However, unless
Polarized
specimens are collected under sterile conditions (catheteriza-
Bright-field
tion), a few bacteria usually are present as a result of contami-
nation from the vagina, urethra, external genitalia, or collection
container. These contaminant bacteria multiply rapidly in spec-
imens that remain at room temperature for extended periods,
but they are of no clinical significance. They may produce a
positive nitrite test result and also frequently result in a pH
above 8, indicating an unacceptable specimen.
Bacteria may be present in the form of cocci (spherical) or
bacilli (rods). Due to their small size, they must be observed
and reported using high-power magnification. They are re-
ported as few, moderate, or many per hpf. To be considered
significant for UTI, bacteria should be accompanied by WBCs.
Some laboratories report bacteria only when observed in fresh
Figure 7–33 Oval fat body under bright-field (left) and polarized specimens in conjunction with WBCs (Fig. 7-34 A and B). The
(right) microscopy. Notice the Maltese cross formation (arrow) presence of motile organisms in a drop of fresh urine collected
(×400). under sterile conditions correlates well with a positive urine

SUMMARY 7-3 Epithelial Cells

Complete
Sources of Urinalysis Clinical
Appearance Error Reporting Correlations Significance
Squamous Largest cells in Rarely en- Rare, few, mod- Clarity Increased numbers
Cells the sediment countered, erate, or many may indicate con-
with abun- folded cells per lpf tamination of the
dant, irregular may resem- urine specimen due
cytoplasm and ble casts to poor collection
prominent technique
nuclei
Transitional Spherical, Spherical Rare, few, mod- Clarity Increased numbers
Cells polyhedral, or forms erate, or many Blood, if may be seen with
caudate with resemble per hpf malignancy UTI, renal carci-
centrally RTE cells associated noma, or after
located catheterization
nucleus
Continued
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188 Part Two | Urinalysis

SUMMARY 7-3 Epithelial Cells—cont’d

Complete
Sources of Urinalysis Clinical
Appearance Error Reporting Correlations Significance
RTE Cells Rectangular, Spherical tran- Average number Leukocyte Increased in tubular
columnar, sitional cells per 10 hpfs esterase and necrosis, possibly
round, oval, or Granular casts nitrite arising from expo-
cuboidal with (pyelonephritis) sure to heavy met-
an eccentric Color als, drug-induced
nucleus possi- toxicity, hemoglo-
Clarity
bly bilirubin bin and myoglobin
stained or Protein toxicity, viral infec-
hemosiderin- Bilirubin tions, pyelonephri-
laden (hepatitis) tis, allergic
Blood reactions, malig-
nant infiltrations,
salicylate poisoning,
and acute allogenic
transplant rejection
Oval Fat Highly refractile Confirm Average number Clarity Increased in glomeru-
Bodies RTE cells with fat per hpf Blood lar damage by
stains and nephrotic syn-
Protein
polarized drome, tubular
microscopy Free fat droplets/ necrosis, diabetes
fatty casts mellitus, long bone
trauma

A B

Figure 7–34 A. Rod-shaped bacteria often seen in urinary tract infections. B. KOVA-stained bacteria and WBC (×400).

culture. Observing bacteria for motility also is useful in differ- leukocytes are routinely followed up with a specimen for quan-
entiating them from amorphous phosphates and urates that titative urine culture. The bacteria associated with UTI most fre-
have a similar appearance. The use of phase microscopy aids quently are the Enterobacteriaceae (referred to as gram-negative
in the visualization of bacteria. rods); however, the cocci-shaped Staphylococcus and Enterococcus
The presence of bacteria can be indicative of either lower are also capable of causing UTI. The actual bacteria producing a
or upper UTI. Specimens containing increased bacteria and UTI cannot be identified with the microscopic examination.
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Chapter 7 | Microscopic Examination of Urine 189

Yeast identified in wet preparations of the urine sediment by its rapid

darting movement in the microscopic field. Trichomonas is
Yeast cells appear in the urine as small, refractile oval structures usually reported as rare, few, moderate, or many per hpf.
that may or may not contain a bud. In severe infections, they When not moving, Trichomonas is more difficult to identify
may appear as branched, mycelial forms (Fig. 7-35 A and B). and may resemble a WBC, transitional, or RTE cell. Use of
Yeast cells are reported as rare, few, moderate, or many per hpf. phase microscopy may enhance visualization of the flagella or
Sometimes differentiation between yeast cells and RBCs undulating membrane.
can be difficult. Careful observation for budding yeast cells T. vaginalis is a sexually transmitted pathogen associated
should be helpful, as shown in Figure 7-8. primarily with vaginal inflammation. Infection of the male ure-
Yeast cells, primarily Candida albicans, are seen in the urine thra and prostate is asymptomatic. Often males are asympto-
of patients who are either diabetic or immunocompromised, as matic carriers (Fig. 7-36 A and B).
well as in women with vaginal moniliasis. The acidic, glucose-
containing urine of patients with diabetes provides an ideal
medium for the growth of yeast. As with bacteria, a small Technical Tip 7-11. Phase microscopy is used to
amount of yeast entering a specimen as a contaminant multiplies differentiate Trichomonas from WBC, transitional,
rapidly if the specimen is not examined while fresh. A true yeast and RTE cells.
infection should be accompanied by the presence of WBCs.

Parasites The ova of the bladder parasite Schistosoma haematobium will

appear in the urine. However, this parasite is seldom seen in the
The parasite encountered most frequently in the urine is United States. It has been associated with bladder cancer in other
Trichomonas vaginalis. The Trichomonas trophozoite is a pear- countries (Fig. 7-37). Fecal contamination of a urine specimen
shaped flagellate with an undulating membrane. It is easily also can result in the presence of ova from intestinal parasites in
the urine sediment. The most common contaminant is ova from
the pinworm Enterobius vermicularis (Fig. 7-38 A and B).

Spermatozoa
Spermatozoa are easily identified in the urine sediment by their
Bacteria oval, slightly tapered heads and long, flagella-like tails (Fig. 7-39).

WBC

Budding WBC
yeast

RBC A
A

Flagella

B
B
Figure 7–36 Trichomonas vaginalis. A. Notice the flagella and
Figure 7–35 A. Budding yeast. B. Yeast showing mycelial forms undulating membrane. (From Leventhal and Cheadle, Ed 6, p 87).
(×400). B. Trichomonas vaginalis in urine, unstained.
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190 Part Two | Urinalysis

Figure 7–37 Schistosoma haematobium ova (×400). Eggs are often

contained in the last few drops of urine expelled from the bladder. Figure 7–39 Spermatozoa (×400).
(From Leventhal, R, and Cheadle, R: Medical Parasitology, ed 6.
F. A. Davis, Philadelphia, 2012, p A19, with permission.)

which sperm is expelled into the bladder instead of the urethra.

A positive reagent strip test for protein may be seen when in-
creased amounts of semen are present.
Laboratory protocols vary with regard to reporting or not
reporting the presence of spermatozoa in a urine specimen.
Laboratories not reporting its presence cite the lack of clinical
significance and possible legal consequences. Laboratories
supporting the reporting of spermatozoa cite the possible
clinical significance and the minimal possibility of legal
consequences.18

Technical Tip 7-12. Follow clinical facility protocol

when noting the presence of spermatozoa in a
A
child’s urine.

Mucus
Mucus is a protein material produced by the glands and ep-
ithelial cells of the lower genitourinary tract and the RTE cells.
Immunologic analysis has shown that uromodulin is a major
constituent of mucus. Uromodulin is a glycoprotein excreted
by the RTE cells of the thick ascending limb of the loop of
Henle and by the distal convoluted tubules.19
Mucus appears microscopically as threadlike structures
with a low refractive index. Subdued light is required when
B
using bright-field microscopy. Care must be taken not to con-
fuse clumps of mucus with hyaline casts. Usually the differen-
Figure 7–38 A. Enterobius vermicularis ova (×100). B. Enterobius
vermicularis ova (×400). (From Leventhal, R, and Cheadle, R: Medical tiation can be made by observing the irregular appearance of
Parasitology, 6th ed. F. A. Davis, Philadelphia, 2012, p 87, with the mucous threads (Fig. 7-40 A and B).
permission.) Mucous threads are reported as rare, few, moderate, or
many per lpf.
Mucus is present more frequently in female urine speci-
mens. It has no clinical significance when present in either
Urine is toxic to spermatozoa; therefore, they rarely exhibit the female or male urine.
motility observed when examining a semen specimen.
Occasionally spermatozoa are found in the urine of both
Technical Tip 7-13. Mucous threads have irregular
men and women after sexual intercourse, masturbation, or
ends that help to distinguish clumps of mucus with
nocturnal emission. They are rarely of clinical significance ex-
hyaline casts.
cept in cases of male infertility or retrograde ejaculation in
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Chapter 7 | Microscopic Examination of Urine 191

Casts
Casts are the only elements found in the urinary sediment that
are unique to the kidney. They are formed within the lumens
of the DCTs and collecting ducts, providing a microscopic view
of conditions within the nephron. Their shape is representative
of the tubular lumen, with parallel sides and somewhat
rounded ends, and they may contain additional elements pres-
ent in the filtrate.
Examination of the sediment for the detection of casts is
performed using lower-power magnification. When the glass
cover-slip method is used, low-power scanning should be per-
A formed along the edges of the cover slip. Observation under
subdued light is essential because the cast matrix has a low re-
fractive index. Similar to many other sediment constituents,
the cast matrix dissolves quickly in dilute, alkaline urine. Once
detected, casts must be identified further as to composition
using high-power magnification. They are reported as the av-
erage number per 10 lpfs.

Cast Composition and Formation

The major constituent of casts is uromodulin. Other proteins
present in the urinary filtrate, such as albumin and im-
munoglobulins, are also incorporated into the cast matrix.
Under normal conditions, uromodulin is excreted at a rate that
is relatively constant. The rate of excretion appears to increase
under conditions of stress and exercise, which may account for
the transient appearance of hyaline casts when these conditions
are present. The protein gels more readily under conditions of
B urine-flow stasis, acidity, and the presence of sodium and cal-
cium. The extent of protein glycosylation is also important.20
Figure 7–40 A. Mucous threads (×400). B. Mucous clump Uromodulin protein is found in both normal and abnormal
(×400). urine and, as discussed previously, is a major constituent of

SUMMARY 7-4 Miscellaneous Structures

Sources of Complete Urinalysis
Appearance Error Reporting Correlations
Bacteria Small spherical and Amorphous Few, moderate, or many pH
rod-shaped structures phosphates per hpf; the presence of Nitrite
and urates WBCs may be required
Leukocyte esterase (LE)
WBCs
Yeast Small, oval, refractile RBCs Rare, few, moderate, or Glucose
structures with buds many per hpf; the pres- LE
and/or mycelia ence of WBCs may be
WBCs
required
Trichomonas Pear-shaped, motile, WBCs, RTE cells Rare, few, moderate, or LE
flagellated many per hpf WBCs
Spermatozoa Tapered oval head with None Present, based on labora- Protein
long, thin tail tory protocol
Mucus Single or clumped Hyaline casts Rare, few, moderate, or None
threads with a low many per lpf
refractive index
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192 Part Two | Urinalysis

mucus. It is not detected by reagent strip protein methods. There-

fore, the increased urinary protein frequently associated with the
presence of casts is caused by underlying renal conditions.
Scanning electron microscope studies have provided a
step-by-step analysis of the formation of the uromodulin pro-
tein matrix21:
1. Aggregation of uromodulin protein into individual
protein fibrils attached to the RTE cells
2. Interweaving of protein fibrils to form a loose fibrillar
network (urinary constituents may become enmeshed in
the network at this time)
3. Further protein fibril interweaving to form a solid
structure
4. Possible attachment of urinary constituents to the solid Figure 7–41 Hyaline casts under low power (×100).
matrix
5. Detachment of protein fibrils from the epithelial cells
6. Excretion of the cast
As the cast forms, urinary flow within the tubule decreases
as the lumen becomes blocked. The accompanying dehydration
of the protein fibrils and internal tension may account for the A
wrinkled and convoluted appearance of older hyaline casts.22
The width of the cast depends on the size of the tubule in which
it is formed. Broad casts may result from tubular distension or,
in the case of extreme urine stasis, from formation in the col-
lecting ducts. Formation of casts at the junction of the ascend-
ing loop of Henle and the DCT may produce structures with a B
tapered end. These have been referred to as cylindroids, but
they have the same significance as casts. In fact, the presence of
urinary casts is termed cylindruria. The appearance of a cast
also is influenced by the materials present in the filtrate at the Figure 7–42 Hyaline cast (A) and amorphous urates (B) attached to
time of its formation and the length of time it remains in the mucous pseudocast (×100).
tubule. Any elements present in the tubular filtrate, including
cells, bacteria, granules, pigments, and crystals, may become
embedded in or attached to the cast matrix. The types of casts or granule may also be observed (Fig. 7-45) but does not
found in the sediment represent different clinical conditions change the cast classification.
and will be discussed separately in this section.

Hyaline Casts Technical Tip 7-14. Use a lower light when using
bright-field microscopy to identify hyaline casts be-
The cast seen most frequently is the hyaline type, which con-
cause the matrix of the cast has a low refractive index,
sists almost entirely of uromodulin. The presence of zero to
which can cause hyaline casts to be missed.
two hyaline casts per lpf is considered normal, as is the finding
of increased numbers after strenuous exercise, dehydration,
heat exposure, and emotional stress.14 Pathologically, hyaline
RBC Casts
casts are increased in acute glomerulonephritis, pyelonephritis,
chronic renal disease, and congestive heart failure. Whereas the finding of RBCs in the urine indicates bleeding
Hyaline casts appear colorless in unstained sediments and from an area within the genitourinary tract, the presence of
have a refractive index similar to that of urine; thus, they can be RBC casts is much more specific, showing bleeding within the
overlooked easily if specimens are not examined under subdued nephron. RBC casts are associated primarily with damage to
light (Figs. 7-41 and 7-42 A and B). Sternheimer-Malbin stain the glomerulus (glomerulonephritis) that allows passage of the
produces a pink color in hyaline casts. Increased visualization cells through the glomerular membrane; however, any damage
can be obtained by phase microscopy (Fig. 7-43 A and B). to the nephron capillary structure can cause their formation.
The morphology of hyaline casts is varied, consisting of Usually RBC casts associated with glomerular damage are
normal parallel sides and rounded ends, cylindroid forms, and associated with proteinuria and dysmorphic erythrocytes. RBC
wrinkled or convoluted shapes that indicate aging of the cast casts also have been observed in healthy individuals after
matrix (Fig. 7-44). The presence of an occasional adhering cell participation in strenuous contact sports.14
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Chapter 7 | Microscopic Examination of Urine 193

Figure 7–45 Hyaline cast containing occasional granules

(×400).

Figure 7–43 A. Hyaline cast (×400). B. Hyaline cast under phase

microscopy (×400).

Figure 7–46 RBC cast (×400).

Figure 7–44 Convoluted hyaline cast (×400).

RBC casts are easily detected under low power by their Figure 7–47 KOVA-stained RBC cast under phase microscopy
(×400).
orange-red color. They are more fragile than other casts and may
exist as fragments or have a more irregular shape as the result of
tightly packed cells adhering to the protein matrix (Figs. 7-46 RBC casts will be present in the absence of freestanding RBCs as
and 7-47). Examination under high-power magnification should well as a positive reagent strip test for blood (Fig. 7-48).
concentrate on determining that a cast matrix is present, thereby As a RBC cast ages, cell lysis begins and the cast develops a
differentiating the structure from a clump of RBCs. Because of more homogenous appearance, but it retains the characteristic
the serious diagnostic implications of RBC casts, the actual pres- orange-red color from the released hemoglobin (Fig. 7-49).
ence of RBCs also must be verified to prevent the inaccurate re- These casts may be distinguished as blood casts, indicating
porting of nonexistent RBC casts. It is highly improbable that greater stasis of urine flow. However, because all casts containing
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194 Part Two | Urinalysis

Figure 7–48 Disintegrating RBC cast. Notice the presence of free Figure 7–50 Granular brown cast (×400).
RBCs (arrows) to confirm identification.

frequently with pyelonephritis and are a primary marker for

distinguishing pyelonephritis (upper UTI) from cystitis (lower
UTI). However, they also are present in nonbacterial inflam-
mations, such as acute interstitial nephritis, and may accom-
pany RBC casts in glomerulonephritis.
WBC casts are visible under low-power magnification but
must be identified positively using high power. Most frequently,
A WBC casts are composed of neutrophils; therefore, they may ap-
pear granular, and, unless disintegration has occurred, multi-
lobed nuclei will be present (Fig. 7-51). Supravital staining may
be necessary to demonstrate the characteristic nuclei (Fig. 7-52).
It is particularly helpful for differentiating WBC casts from RTE
B casts. Also, observation of free WBCs in the sediment is essential
(Fig. 7-53). Bacteria are present in cases of pyelonephritis, but
they are not present with acute interstitial nephritis; however,
Figure 7–49 Cast containing hemoglobin pigment. A comparison
eosinophil casts may be present in specimens stained appropri-
of RBCs (A) and yeast (B) also can be made (×400).
ately (Hansel and Wright’s stains).
Casts packed tightly with WBCs may have irregular bor-
blood have the same clinical significance, this is not considered ders. These structures should be examined carefully to deter-
necessary. Both types of casts are reported as the number of RBC mine that a cast matrix is present. WBCs frequently form
casts per lpf.
In the presence of massive hemoglobinuria or myoglobin-
uria, homogenous orange-red or red-brown casts may be
observed. Granular, dirty, brown casts representing products of
hemoglobin degradation, such as methemoglobin, may also be
present (Fig. 7-50). They are associated with the acute tubular
necrosis often caused by the toxic effects of massive hemoglobin-
uria that can lead to renal failure. These dirty, brown casts must
be present in conjunction with other pathological findings, such
as RTE cells and a positive reagent strip test for blood.

Technical Tip 7-15. RBCs in the urine sediment and a

positive reagent strip test for blood, as well as intact
RBCs in the cast matrix, must be present to identify a
RBC cast.

WBC Casts
The appearance of WBC casts in the urine signifies infection Figure 7–51 WBC cast. Notice the free WBCs to aid in
or inflammation within the nephron. They are associated most identification.
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Chapter 7 | Microscopic Examination of Urine 195

Figure 7–52 KOVA-stained WBC cast (×400). Figure 7–54 WBC clump. Notice the absence of a cast matrix.

Epithelial Cell Casts

Casts containing RTE cells represent the presence of advanced
tubular destruction, producing urinary stasis along with dis-
ruption of the tubular linings. Similar to RTE cells, they are as-
sociated with toxicity from heavy metals, chemicals, or drugs;
viral infections; and allograft rejection. They also accompany
WBC casts in cases of pyelonephritis.
As discussed previously, the fibrils of uromodulin protein
that make up the cast matrix remain attached to the RTE cells
that produce them; therefore, the observation of an occasional
tubular cell attached to a hyaline cast can be expected. When
tubular damage is present, some cells may be incorporated into
the cast matrix, but the majority will be attached very notice-
ably to the cast surface.
Figure 7–53 Disintegrating WBC cast (×400). Due to the formation of casts in the distal convoluted
tubule, the cells visible on the cast matrix are the smaller,
round, and oval cells (Fig. 7-55). They may be difficult to dif-
clumps, and these clumps do not have the same significance ferentiate from WBCs, particularly if degeneration has oc-
as casts (Fig. 7-54). curred. Staining and the use of phase microscopy can be
helpful to enhance the nuclear detail needed for identification
Technical Tip 7-16. The presence of a WBC cast is di- (Figs. 7-56 A and B and 7-57). Fragments of epithelial tissue
agnostic for pyelonephritis and would not be present also may be attached to the cast matrix. Bilirubin-stained RTE
in cystitis. cells are seen in cases of hepatitis (see Fig. 7-57).

Technical Tip 7-17. Free-floating WBCs must be in

the urine sediment and well-defined WBCs present in
the cast to classify a WBC cast.

Bacterial Casts
Bacterial casts containing bacilli both within and bound to the
protein matrix are seen in pyelonephritis.23 They may be pure
bacterial casts or mixed with WBCs.
Identification of bacterial casts can be difficult because
casts packed with bacteria can resemble granular casts. Their
presence should be considered when WBC casts and many free
WBCs and bacteria are seen in the sediment. Confirmation of
bacterial casts is best made by performing a Gram stain on the
dried or cytocentrifuged sediment. Figure 7–55 RTE cell cast (×400).
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196 Part Two | Urinalysis

Figure 7–58 Fatty cast showing adherence of fat droplets (arrows)

to cast matrix (×400).

Figure 7–56 A. KOVA-stained RTE cell cast (×400). B. KOVA-stained

RTE cell cast under phase microscopy (×400).

Figure 7–59 Fatty cast (×400).

Figure 7–57 RTE cast with bilirubin-stained cells (×400).

Fatty Casts Figure 7–60 Fatty cast under phase microscopy (×400).

Fatty casts are seen in conjunction with oval fat bodies and free
fat droplets in disorders causing lipiduria. They are associated discussed previously, cholesterol demonstrates characteristic
most frequently with nephrotic syndrome, but they also are seen Maltese cross formations under polarized light, and triglycerides
in toxic tubular necrosis, diabetes mellitus, and crush injuries. and neutral fats stain orange with fat stains. Fats do not stain with
Fatty casts are highly refractile under bright-field microscopy. Sternheimer-Malbin stains.
The cast matrix may contain few or many fat droplets, and intact
Mixed Cellular Casts
oval fat bodies may be attached to the matrix (Figs. 7-58, 7-59,
and 7-60). Confirmation of fatty casts is performed using Considering that a variety of cells may be present in the urinary
polarized microscopy and Sudan III or Oil Red O fat stains. As filtrate, it is not uncommon to observe casts containing multiple
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Chapter 7 | Microscopic Examination of Urine 197

cell types. Mixed cellular casts encountered most frequently in-

clude RBC and WBC casts in glomerulonephritis and WBC and
RTE cell casts, or WBC and bacterial casts in pyelonephritis.
The presence of mixed elements in a cast may make iden-
tification more difficult. Staining or phase microscopy aids in
the identification. When mixed casts are present, there should
be homogenous casts of at least one of the cell types also, and
they will be the primary diagnostic marker. For example, in
glomerulonephritis, the predominant casts will be RBCs, and
in pyelonephritis, the predominant casts will be WBCs. Often
bacteria are incorporated into WBC casts and provide little ad-
ditional diagnostic significance. Laboratory protocol should be
followed in the reporting of mixed cellular casts.

Granular Casts
Figure 7–62 Granular cast formed at a tubular bend (×400).
Frequently casts that are coarsely and finely granular are seen
in the urinary sediment and may be of pathological or non-
pathological significance. It is not considered necessary to dis-
tinguish between casts that are coarsely and finely granular.
The origin of the granules in nonpathological conditions
appears to be from the lysosomes excreted by RTE cells during
normal metabolism.24 It is not unusual to see hyaline casts
containing one or two of these granules. Increased cellular
metabolism occurring during periods of strenuous exercise
accounts for the transient increase of granular casts that
accompany the increased hyaline casts (Figs. 7-61 and 7-62).14
In disease states, granules may represent disintegration of
cellular casts and tubule cells or protein aggregates filtered by
the glomerulus (Figs. 7-63 and 7-64). Scanning electron mi-
croscope studies have confirmed that granular casts seen in
conjunction with WBC casts contain WBC granules of varying
sizes.25 Urinary stasis allowing the casts to remain in the tubules Figure 7–63 Granular disintegrating cellular cast (×400).
must be present for granules to result from disintegration of
cellular casts.

Technical Tip 7-18. A hyaline cast with a few

granules within the matrix does not constitute a B C
granular cast.
A

Figure 7–64 A. Squamous epithelial cell. B. Coarsely granular cast.

C. Mucus (x400).
A

Granular casts occurring as a result of cellular disintegration

may contain an occasional cell that is recognizable. Granular
casts are visualized easily under low-power microscopy. How-
ever, final identification should be performed using high power
to determine the presence of a cast matrix.
Figure 7–61 Finely granular cast (A) and uric acid crystals (B) Artifacts, such as clumps of small crystals and fecal debris,
(×400). may occur in shapes resembling casts and must be differentiated.
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198 Part Two | Urinalysis

As mentioned previously, columnar RTE cells also may resemble

granular casts, and staining for nuclear detail may be required.
When granular casts remain in the tubules for extended
periods, the granules disintegrate further, and the cast matrix
develops a waxy appearance. The structure becomes more
rigid, the ends of the casts may appear jagged or broken, and
the diameter becomes broader (Fig. 7-65).

Waxy Casts
Waxy casts are representative of extreme urine stasis, indicating
chronic renal failure. Usually they are seen in conjunction with
other types of casts associated with the condition that has
caused the renal failure, such as hyaline, granular, and cellular. A
The brittle, highly refractive cast matrix from which these
casts derive their name is believed to be caused by degenera-
tion of the hyaline cast matrix and any cellular elements or
granules contained in the matrix.22,24
Waxy casts are visualized more easily than hyaline casts
because of their higher refractive index. As a result of the brittle
consistency of the cast matrix, they often appear fragmented
with jagged ends and have notches in their sides (Figs. 7-66
and 7-67 A and B). With supravital stains, waxy casts stain a
homogenous, dark pink (Fig. 7-68).

Figure 7–67 A. KOVA-stained waxy cast (×200). B. Unstained waxy

cast.

Figure 7–65 Granular cast degenerating into waxy cast (×400).

Figure 7–68 KOVA-stained waxy cast (×400).

Broad Casts
Often referred to as renal failure casts, broad casts, like waxy
casts, represent extreme urine stasis. As a mold of the distal
convoluted tubules, the presence of broad casts indicates de-
struction (widening) of the tubular walls. Also, when the flow
of urine to the larger collecting ducts becomes severely com-
Figure 7–66 KOVA-stained waxy casts (×100). promised, casts form in this area and appear broad.
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Chapter 7 | Microscopic Examination of Urine 199

All types of casts may occur in the broad form. However,

considering the accompanying urinary stasis, the broad casts
seen most commonly are granular and waxy (Figs. 7-69 and
7-70 A and B). Broad, waxy casts that are bile stained are
seen as the result of tubular necrosis caused by viral hepatitis
(Fig. 7-71).

Figure 7–71 Broad bile-stained waxy cast (×400).

Urinary Crystals
Crystals frequently found in the urine are rarely of clinical sig-
nificance. They may appear as true geometrically formed struc-
tures or as amorphous material. The primary reason for the
identification of urinary crystals is to detect the presence of the
Figure 7–69 KOVA-stained broad waxy cast (×400). relatively few abnormal types that may represent such disor-
ders as liver disease, inborn errors of metabolism, or renal
damage caused by crystallization of medication compounds
within the tubules. Usually crystals are reported as rare, few,
moderate, or many per hpf. Abnormal crystals may be averaged
and reported per lpf.

Crystal Formation
Crystals are formed by the precipitation of urine solutes, in-
cluding inorganic salts, organic compounds, and medications
(iatrogenic compounds). Precipitation is subject to changes
in temperature, solute concentration, and pH, which affect
solubility.
Solutes precipitate more readily at low temperatures.
Therefore, the majority of crystal formation takes place in
specimens that have remained at room temperature or have
been refrigerated before testing. Crystals are extremely abun-
dant in refrigerated specimens and often present problems
because they obscure sediment constituents that are clinically
A
significant.
As the concentration of urinary solutes increases, their ability
to remain in solution decreases, resulting in crystal formation.
The presence of crystals in freshly voided urine is associated most
frequently with concentrated (high specific gravity) specimens.
A valuable aid in the identification of crystals is the pH
of the specimen because this determines the type of chemicals
precipitated. In general, organic and iatrogenic compounds
crystallize more easily in an acidic pH, whereas inorganic salts
are less soluble in neutral and alkaline solutions. An excep-
tion is calcium oxalate, which precipitates in both acidic and
neutral urine.

B
Technical Tip 7-19. The most valuable initial aid for
Figure 7–70 A. Broad granular cast. B. Broad granular cast becom-
identifying crystals in a urine specimen is the pH.
ing waxy (×400).
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200 Part Two | Urinalysis

SUMMARY 7-5 Urine Casts

Complete
Sources of Urinalysis Clinical
Appearance Error Reporting Correlations Significance
Hyaline Colorless, Mucus, fibers, Average number Protein Glomerulonephritis
homogenous hair, per lpf Blood (exercise) Pyelonephritis
matrix increased Color (exercise) Chronic renal disease
lighting
Congestive heart
failure
Stress and exercise
RBC Orange-red color, RBC clumps Average number RBCs Glomerulonephritis
cast matrix per lpf Blood Strenuous exercise
containing
Protein
RBCs
WBC Cast matrix con- WBC clumps Average number WBCs Pyelonephritis
taining WBCs per lpf Protein AIN
LE
Bacterial Bacilli bound to Granular casts Average number WBC casts Pyelonephritis
protein matrix per lpf (pyelonephritis)
WBCs
LE
Nitrite
Protein
Bacteria
Epithelial RTE cells attached WBC cast Average number Protein Renal tubular damage
Cell to protein per lpf RTE cells
matrix
Granular Coarse and fine Clumps of Average number Protein Glomerulonephritis
granules in a small per lpf Cellular casts Pyelonephritis
cast matrix crystals RBCs Stress and exercise
Columnar RTE WBCs
cells
Waxy Highly refractile Fibers and Average number Protein Stasis of urine flow
cast with fecal per lpf Cellular casts Chronic renal failure
jagged ends material Granular casts
and notches
WBCs
RBCs
Fatty Fat droplets and Fecal debris Average number Protein Nephrotic syndrome
oval fat bodies per lpf Free fat droplets Toxic tubular necrosis
attached to
Oval fat bodies Diabetes mellitus
protein matrix
Crush injuries
Broad Wider-than- Fecal material, Average number Protein Extreme urine stasis
normal cast fibers per lpf WBCs Renal failure
matrix
RBCs
Granular casts
Waxy casts
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Chapter 7 | Microscopic Examination of Urine 201

General Identification Techniques Knowledge of these solubility characteristics can be used to aid
in identification. Amorphous urates that frequently form in re-
The crystals seen most commonly have very characteristic frigerated specimens and obscure sediments may dissolve if the
shapes and colors; however, variations do occur and can pres- specimen is warmed. Amorphous phosphates require acetic acid
ent identification problems, particularly when they resemble to dissolve, and this is not practical, as formed elements, such as
abnormal crystals. As discussed previously, the first consider- RBCs, also will be destroyed. When solubility characteristics are
ation when identifying crystals is the urine pH. In fact, crystals needed for identification, the sediment should be aliquoted to
are routinely classified not only as normal and abnormal but prevent destruction of other elements. In Table 7-6, characteris-
also as to their appearance in acidic or alkaline urine. All tics for the crystals encountered most commonly are provided.
abnormal crystals are found in acidic urine.
Another aid in crystal identification is the use of polarized
Normal Crystals Seen in Acidic Urine
microscopy. The geometric shape of a crystal determines its
birefringence and, therefore, its ability to polarize light. Al- The most common crystals seen in acidic urine are urates, con-
though the size of a particular crystal may vary (slower crys- sisting of amorphous urates, uric acid, acid urates, and sodium
tallization produces larger crystals), the basic structure remains urates. Microscopically, most urate crystals appear yellow to
the same. Therefore, polarization characteristics for a particular reddish brown and are the only normal crystals found in acidic
crystal are constant for identification purposes. urine that appear colored.
Just as changes in temperature and pH contribute to crystal Amorphous urates appear microscopically as yellow-
formation, reversal of these changes can cause crystals to dissolve. brown granules (Fig. 7-72). They may occur in clumps

Table 7–6 Major Characteristics of Normal Urinary Crystals

Crystal pH Color Appearance
Uric acid Acid Yellow-brown (rosettes, wedges)

Amorphous urates Acid Brick dust or yellow brown

Sodium urates Acid Colorless

Calcium oxalate Acid/neutral (alkaline) Colorless (envelopes, oval, dumbbell)

Amorphous phosphates Alkaline/neutral White–colorless

Continued
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202 Part Two | Urinalysis

Table 7–6 Major Characteristics of Normal Urinary Crystals—cont’d

Crystal pH Color Appearance
Calcium phosphate Alkaline/neutral Colorless

Triple phosphate Alkaline Colorless (“coffin lids”)

Ammonium biurate Alkaline Yellow-brown (“thorny apples”)

Calcium carbonate Alkaline Colorless (dumbbells)

Figure 7–72 Amorphous urates (×400).

resembling granular casts and attached to other sediment

structures (Fig.7-73). Amorphous urates are encountered fre- Figure 7–73 Amorphous urates attached to a fiber.
quently in specimens that have been refrigerated but disappear
when the urine is warmed. They produce a very characteristic
pink sediment caused by accumulation of the pigment uroery- (Figs. 7-74 and 7-75). Uric acid crystals are highly birefringent
thrin on the surface of the granules. Amorphous urates are under polarized light, which aids in distinguishing them from
found in acidic urine with a pH greater than 5.5, whereas uric cystine crystals (Fig. 7-76 A and B). Increased amounts of uric
acid crystals can appear when the pH is lower. acid crystals, particularly in fresh urine, are associated with
Uric acid crystals are seen in a variety of shapes, including increased levels of purines and nucleic acids and are seen in
rhombic, four-sided flat plates (whetstones), wedges, and patients with leukemia who are receiving chemotherapy, in
rosettes. They usually appear yellow-brown but may be col- patients with Lesch-Nyhan syndrome (see Chapter 9), and
orless and have a six-sided shape, similar to cystine crystals sometimes in patients with gout.
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Chapter 7 | Microscopic Examination of Urine 203

Figure 7–74 Uric acid crystals (×400).

Figure 7–76 A. Uric acid crystals under polarized light (×100).

B. Uric acid crystals under polarized light (×400).

Figure 7–75 Clump of uric acid crystals (×400). Notice the whet-
stone, not hexagonal, shape that differentiates uric acid crystals from
cystine crystals.

Acid urates and sodium urates are rarely encountered and,

like amorphous urates, are seen in urine that is less acidic. Fre-
quently they are seen in conjunction with amorphous urates
and have little clinical significance. Acid urates appear as larger
granules and may have spicules similar to the ammonium bi-
urate crystals seen in alkaline urine. Sodium urate crystals are
needle shaped and are seen in synovial fluid during episodes
of gout, but they also may appear in the urine (Fig. 7-77).
Calcium oxalate crystals are seen frequently in acidic
urine, but they can be found in neutral urine and, even rarely,
in alkaline urine. The most common form of calcium oxalate
crystals is the dihydrate that is easily recognized as a colorless, Figure 7–77 Sodium urate crystals.
octahedral envelope or as two pyramids joined at their bases
(Figs. 7-78, 7-79, and 7-80). Less characteristic and less fre-
quently seen is the monohydrate form (Fig. 7-81). Monohy- The finding of clumps of calcium oxalate crystals in fresh
drate calcium oxalate crystals are oval or dumbbell shaped. urine may be related to the formation of renal calculi because
Both the dihydrate and monohydrate forms are birefringent the majority of renal calculi are composed of calcium oxalate.
under polarized light. This may be helpful to distinguish the Also, they are associated with foods high in oxalic acid, such
monohydrate form from nonpolarizing RBCs. Sometimes cal- as tomatoes and asparagus, and ascorbic acid because oxalic
cium oxalate crystals are seen in clumps attached to mucous acid is an end product of ascorbic acid metabolism. The pri-
strands and may resemble casts. mary pathological significance of calcium oxalate crystals is the
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204 Part Two | Urinalysis

Figure 7–81 Monohydrate calcium oxalate crystals (×400).

Figure 7–78 Classic dihydrate calcium oxalate crystals (×400).

very noticeable presence of the monohydrate form in cases of

ethylene glycol (antifreeze) poisoning. The monohydrate form
is seen most frequently in children and pets because antifreeze
tastes sweet and uncovered containers left in the garage can
be very tempting! Massive amounts of crystals are frequently
produced in these cases.

Normal Crystals Seen in Alkaline Urine

Phosphates represent the majority of the crystals seen in alkaline
urine and include amorphous phosphate, triple phosphate, and
calcium phosphate. Other normal crystals associated with al-
kaline urine are calcium carbonate and ammonium biurate.
Amorphous phosphates are granular in appearance, similar to
amorphous urates (Figs. 7-82 and 7-83). When present in large
quantities after specimen refrigeration, they cause a white pre-
cipitate that does not dissolve on warming. They can be differ-
Figure 7–79 Classic dihydrate calcium oxalate crystals under phase entiated from amorphous urates by the color of the sediment
microscopy (×400). and the urine pH.
Triple phosphate (ammonium magnesium phosphate)
crystals are seen commonly in alkaline urine. In their routine
form, they are identified easily by their prism shape that fre-
quently resembles a “coffin lid” (Figs. 7-84 A and B and 7-85).
As they disintegrate, the crystals may develop a feathery
appearance. Triple phosphate crystals are birefringent under

Figure 7–80 Attached classic dihydrate calcium oxalate crystals

(×400). Figure 7–82 Amorphous phosphates (×400). Urine pH 7.0.
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Chapter 7 | Microscopic Examination of Urine 205

Figure 7–85 Triple phosphate crystals (arrow) and amorphous

phosphates (×400).

Figure 7–83 Amorphous phosphates (×400).

or thin prisms often in rosette formations. The rosette forms
may be confused with sulfonamide crystals when the urine pH
is in the neutral range. Calcium phosphate crystals dissolve in
dilute acetic acid, but sulfonamides do not. They have no clin-
ical significance, although calcium phosphate is a common
constituent of renal calculi.
Calcium carbonate crystals are small and colorless, with
dumbbell or spherical shapes (Fig. 7-86). They may occur in
clumps that resemble amorphous material, but they can be dis-
tinguished by the formation of gas after the addition of acetic
acid. They are also birefringent, which differentiates them from
bacteria. Calcium carbonate crystals have no clinical significance.
Ammonium biurate crystals exhibit the characteristic
yellow-brown color of the urate crystals seen in acidic urine.
A
Frequently they are described as “thorny apples” because of
their appearance as spicule-covered spheres (Fig. 7-87). Except
for their occurrence in alkaline urine, ammonium biurate crys-
tals resemble other urates in that they dissolve at 60°C and
convert to uric acid crystals when glacial acetic acid is added.
Ammonium biurate crystals are almost always encountered in old
specimens and may be associated with the presence of the
ammonia produced by urea-splitting bacteria (Figs. 7-88 A and B
and 7-89).

Figure 7–84 A. Triple phosphate crystal using bright-field mi-

croscopy (×400). B. Triple phosphate under polarized light.

polarized light. They have no clinical significance; however,

they are seen often in highly alkaline urine associated with the
presence of urea-splitting bacteria.
Calcium phosphate crystals are not encountered fre-
quently. They may appear as colorless, flat rectangular plates Figure 7–86 Calcium carbonate crystals (×400).
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206 Part Two | Urinalysis

Figure 7–87 Ammonium biurate crystals (×400). Notice the “thorny

apple” appearance. (Courtesy of Kenneth L. McCoy, MD.)

Figure 7–89 Ammonium biurate crystals (×400). Note thorns

(arrow).

Abnormal Urine Crystals

Abnormal urine crystals are found in acidic urine or, rarely, in
neutral urine. Most abnormal crystals have very characteristic
shapes. However, their identity can be confirmed by patient
information, including disorders and medications (Table 7-7).
Iatrogenic crystals can be caused by a variety of compounds,
particularly when they are administered in high concentra-
tions. They may be of clinical significance when they precipi-
A
tate in the renal tubules. The iatrogenic crystals that are
encountered most commonly are discussed in this section.

Cystine Crystals
Cystine crystals are found in the urine of people who inherit a
metabolic disorder that prevents reabsorption of cystine by the
renal tubules (cystinuria). People with cystinuria tend to form
renal calculi, particularly at an early age.
Cystine crystals appear as colorless, hexagonal plates and
Triple may be thick or thin (Figs. 7-90 and 7-91). Disintegrating
phosphate
crystal forms may be seen in the presence of ammonia. They may be
difficult to differentiate from colorless uric acid crystals. Uric
acid crystals are very birefringent under polarized microscopy,
whereas only thick cystine crystals have polarizing capability.
Positive confirmation of cystine crystals is made using the
cyanide–nitroprusside test (see Chapter 9).

B Cholesterol Crystals
Cholesterol crystals are rarely seen unless specimens have been
Figure 7–88 A. Ammonium biurate and triple phosphate crystals refrigerated because the lipids remain in droplet form. How-
(×100). Note thorn (arrow). B. Ammonium biurate and triple phos- ever, when observed, they have a most characteristic appear-
phate crystals (×400). ance, resembling a rectangular plate with a notch in one or
more corners (Fig. 7-92). They are associated with disorders
producing lipiduria, such as nephrotic syndrome, and are seen
in conjunction with fatty casts and oval fat bodies. Cholesterol
crystals are highly birefringent with polarized light (Fig. 7-93).
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Chapter 7 | Microscopic Examination of Urine 207

Table 7–7 Major Characteristics of Abnormal Urinary Crystals

Crystal pH Color/Form Disorders Appearance
Cystine Acid Colorless (hexagonal Inherited cystinuria
plates)

Cholesterol Acid Colorless (notched plates) Nephrotic syndrome

Leucine Acid/neutral Yellow (concentric circles) Liver disease

Tyrosine Acid/neutral Colorless–yellow (needles) Liver disease

Bilirubin Acid Yellow Liver disease

Sulfonamides Acid/neutral Varied Infection treatment

Radiographic dye Acid Colorless (flat plates) Radiographic procedure

Ampicillin Acid/neutral Colorless (needles) Infection treatment

Radiographic Dye Crystals Crystals Associated With Liver Disorders

Crystals of radiographic contrast media appear similar to cho- In the presence of severe liver disorders, three crystals that usu-
lesterol crystals and also are highly birefringent. ally are rarely seen may be found in the urine sediment. They
Differentiation is best made by comparison of the results are crystals of tyrosine, leucine, and bilirubin.
of the other urinalysis, as well as the patient history. As Tyrosine crystals appear as fine colorless to yellow needles
mentioned previously, cholesterol crystals should be accompa- that frequently form clumps or rosettes (Figs. 7-94 and 7-95).
nied by other lipid elements and heavy proteinuria. Likewise, Usually they are seen in conjunction with leucine crystals in
the specific gravity of a specimen containing radiographic specimens with positive chemical test results for bilirubin. Ty-
contrast media is markedly elevated when measured by rosine crystals also may be encountered in inherited disorders
refractometer. of amino acid metabolism (see Chapter 9).
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208 Part Two | Urinalysis

Figure 7–90 Cystine crystals (×400). Figure 7–93 Cholesterol crystals under polarized light (×400).

Figure 7–91 Clump of cystine crystals (×400). Notice the hexagonal Figure 7–94 Tyrosine crystals in fine needle clumps (×400).
shape still visible.

Figure 7–95 Tyrosine crystals in rosette forms (×400).

Figure 7–92 Cholesterol crystals. Notice the notched corners
(×400).
yellow color of bilirubin (Fig. 7-97 A and B). A positive chemical
test result for bilirubin would be expected. In disorders that pro-
Leucine crystals are yellow-brown spheres that demon- duce renal tubular damage, such as viral hepatitis, bilirubin crys-
strate concentric circles and radial striations (Fig. 7-96). They tals may be found incorporated into the matrix of casts.
are seen less frequently than tyrosine crystals and, when pres-
ent, should be accompanied by tyrosine crystals. Sulfonamide Crystals
Bilirubin crystals are present in patients with hepatic disor- Before the development of more soluble sulfonamides, the
ders, producing large amounts of bilirubin in the urine. They finding of these crystals in the urine of patients being treated
appear as clumped needles or granules with the characteristic for UTIs was common. Inadequate patient hydration was and
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Chapter 7 | Microscopic Examination of Urine 209

include needles, rhombics, whetstones, sheaves of wheat, and

rosettes with colors ranging from colorless to yellow-brown
(Figs. 7-98 and 7-99). A check of the patient’s medication his-
tory aids in the identification confirmation.

Ampicillin Crystals
Precipitation of antibiotics is not encountered frequently except
for the rare observation of ampicillin crystals after massive
doses of this penicillin compound without adequate hydration.
Ampicillin crystals appear as colorless needles that tend to form
bundles after refrigeration (Fig. 7-100 A and B). Knowledge of
the patient’s history can aid in the identification.

Urinary Sediment Artifacts

Figure 7–96 Leucine crystals (×400). Notice the concentric Contaminants of all types can be found in urine, particularly
circles. in specimens collected under improper conditions or in dirty
containers. The artifacts encountered most frequently include
starch, oil droplets, air bubbles, pollen grains, fibers, and
fecal contamination. Because artifacts frequently resemble
pathological elements, such as RBCs and casts, artifacts can
present a major problem to students. Often, they are very
highly refractile or occur in a different microscopic plane than
the true sediment constituents. The reporting of artifacts is
not necessary.

Figure 7–98 Sulfa crystals in rosette form (×400).

Figure 7–97 A and B. Bilirubin crystals. Notice the classic bright

yellow color (×400).

still is the primary cause of sulfonamide crystallization. The

appearance of sulfonamide crystals in fresh urine can suggest
the possibility of tubular damage if crystals are forming in the
nephron.
Currently a variety of sulfonamide medications is on the
market; therefore, one can expect to encounter a variety of Figure 7–99 Sulfa crystals, WBCs, and bacteria seen in UTI
crystal shapes and colors. Shapes encountered most frequently (×400).
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210 Part Two | Urinalysis

Oil droplets and air bubbles also are highly refractile and
may resemble RBCs to inexperienced laboratory personnel. Oil
droplets may result from contamination by immersion oil or
lotions and creams and may be seen with fecal contamination
(Fig. 7-102). Air bubbles occur when the specimen is placed
under a cover slip. The presence of these artifacts should be
considered in the context of the other urinalysis results.
Pollen grains are seasonal contaminants that appear
as spheres with a cell wall and occasional concentric circles
(Fig. 7-103). Like many artifacts, their large size may cause
them to be out of focus with true sediment constituents.
Hair and fibers from clothing and diapers initially may be
A mistaken for casts (Figs. 7-104, 7-106, and 7-107), though
usually they are much longer and more refractile. Examination
under polarized light frequently can differentiate between
fibers and casts (Fig. 7-105). Fibers often polarize, whereas
casts, other than fatty casts, do not polarize.

Technical Tip 7-20. Use polarized microscopy to dif-

ferentiate between fibers, which polarize, and casts,
which do not (except for fatty casts).

Figure 7–100 Ampicillin crystals. A. Nonrefrigerated ampicillin

crystals. (×400). B. Ampicillin crystals after refrigeration (×400).

Contamination from starch granules may occur when

cornstarch is the powder used in powdered gloves. The gran-
ules are highly refractile spheres, usually with a dimpled cen-
ter (Fig. 7-101). They resemble fat droplets when polarized,
producing a Maltese cross formation. Also starch granules may
occasionally be confused with RBCs. Differentiation between
starch and pathological elements can be made by considering
other urinalysis results, including chemical tests for blood or
protein and the presence of oval fat bodies or fatty casts. Figure 7–102 Fecal material and oil artifacts (×400).

Figure 7–101 Starch granules. Notice the dimpled center (×400). Figure 7–103 Pollen grain. Notice the concentric circles (×400).
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Chapter 7 | Microscopic Examination of Urine 211

Figure 7–107 Vegetable fiber on top of a hyaline cast.

Figure 7–104 Fiber and squamous epithelial cell (×400).

Figure 7–105 Fiber under polarized light (×100). Figure 7–108 Vegetable fiber resembling waxy cast (×400).

For additional resources please visit

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References
1. Mynahan, C: Evaluation of macroscopic urinalysis as a screening
procedure. Lab Med 15(3):176–179, 1984.
2. Tetrault, GA: Automated reagent strip urinalysis: Utility in re-
ducing work load of urine microscopy and culture. Lab Med
25:162–167, 1994.
3. Clinical and Laboratory Standards Institute: Urinalysis; Approved
Guideline, ed 3. CLSI document GP16-A3. Clinical and Labora-
tory Standards Institute, Wayne, PA, 2009, CLSI.
Figure 7–106 Diaper fiber resembling a cast. Notice the refractility
4. Schumann, GB, and Tebbs, RD: Comparison of slides used for
(×400). standardized routine microscopic urinalysis. J Med Technol
3(1):54–58, 1986.
Specimens that are collected improperly or, rarely, the 5. Addis, T: The number of formed elements in the urinary
presence of a fistula between the intestinal and urinary tracts sediment of normal individuals. J Clin Invest 2(5):409–415,
1926.
may produce fecal specimen contamination. Fecal artifacts may 6. Sternheimer, R, and Malbin, R: Clinical recognition of pyelonephri-
appear as plant and meat fibers or as brown amorphous mate- tis with a new stain for urinary sediments. Am J Med 11:312–313,
rial in a variety of sizes and shapes (Fig. 7-108). 1951.
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212 Part Two | Urinalysis

7. McPherson, RA, Ben-Ezra, J, Zhao S.: Basic examination of 16. Schumann, GB: Utility of urinary cytology in renal diseases.
urine. Henry’s Clinical Diagnosis and Management by Labora- Semin Nephrol 5(34) Sept, 1985.
tory Methods. Eds. McPherson, RA, Pincus, MR. 22nd Ed. 17. Graber, M, et al: Bubble cells: Renal tubular cells in the urinary
Philadelphia: Elsevier Saunders, 2011, p.465. sediment with characteristics of viability. J Am Soc Nephrol
8. Olympus Microscopy Resource Center: Specialized Microscopy 1(7):999–1004, 1991.
Techniques: Fluorescence. Web site: http://www.olympusmicro. 18. Baer, DM: Tips from clinical experts: Reporting of spermatozoa
com/primer/techniques/fluorescence/fluorhome.html. Accessed in microscopic urine exams. MLO 12:12, 1997.
May 10, 2019. 19. Bleyer, AJ, and Stanislav, K: Tamm Horsfall Glycoprotein and
9. Simpson, LO: Effects of normal and abnormal urine on red cell Uromodulin: It is All about the Tubules! Clin J Am Soc Nephrol.
shape. Nephron 60(3):383–384, 1992. 2016 Jan 7; 11(1): 6-8. Doi: 10.2215/CJN.12201115. Web site:
10. Stapleton, FB: Morphology of urinary red blood cells: A simple https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4702239.
guide in localizing the site of hematuria. Pediatr Clin North Am Accessed May 11, 2019.
34(3):561–569, 1987. 20. Kumar, S, and Muchmore, A: Tamm-Horsfall protein—
11. Fassett, EG, et al: Urinary red cell morphology during exercise. Uromodulin, 1950–1990. Kidney Int 37:1395–1399, 1990.
Am J Clin Pathol 285(6353):1455–1457, 1982. 21. Haber, MH: Urinary Sediment: A textbook Atlas. American
12. Kohler, H, Wandel, E, and Brunch, B: Acanthocyturia: A Society of Clinical Pathologists, Chicago, 1981.
characteristic marker for glomerular bleeding. Int Soc Nephrol 22. Lindner, LE, and Haber, MH: Hyaline casts in the urine:
40:115–120, 1991. Mechanism of formation and morphological transformations.
13. Tomita, M, et al: A new morphological classification of urinary Am J Clin Pathol 80(3):347–352, 1983.
erythrocytes for differential diagnosis of hematuria. Clin 23. Lindner, LE, Jones, RN, and Haber, MH: A specific cast in acute
Nephrol 37(2):84–89, 1992. pyelonephritis. Am J Clin Pathol 73(6):809–811, 1980.
14. Haber, MH, Lindner, LE, and Ciofalo, LN: Urinary casts after 24. Haber, MH, and Lindner, LE: The surface ultrastructure of
stress. Lab Med 10(6):351–355, 1979. urinary casts. Am J Clin Pathol 68(5):547–552, 1977.
15. Corwin, HL, Bray, RA, and Haber, MH: The detection and 25. Linder, LE, Vacca, D, and Haber, MF: Identification and
interpretation of urinary eosinophils. Arch Pathol Lab Med composition of types of granular urinary cast. Am J Pathol
113:1256–1258, 1989. 80(3):353–358, 1983.

Study Questions
1. Macroscopic screening of urine specimens is used to: 5. When using the glass-slide and cover-slip method,
A. Provide results as soon as possible which of the following might be missed if the cover slip
is overflowed?
B. Predict the type of urinary casts present
A. Casts
C. Increase cost-effectiveness of urinalysis
B. RBCs
D. Decrease the need for polarized microscopy
C. WBCs
2. Variations in the microscopic analysis of urine include all
D. Bacteria
of the following except:
A. Preparation of the urine sediment 6. Initial screening of the urine sediment is performed using
an objective power of:
B. Amount of sediment analyzed
A. 4×
C. Method of reporting
B. 10×
D. Identification of formed elements
C. 40×
3. All of the following can cause false-negative microscopic
D. 100×
results except:
A. Braking the centrifuge 7. Which of the following should be used to reduce light
intensity in bright-field microscopy?
B. Failing to mix the specimen
A. Centering screws
C. Diluting alkaline urine
B. Aperture diaphragm
D. Using midstream clean-catch specimens
C. Rheostat
4. The two factors that determine relative centrifugal force
D. Condenser aperture diaphragm
are:
A. Radius of rotor head and RPM 8. Which of the following are reported as number per lpf?
B. Radius of rotor head and time of centrifugation A. RBCs
C. Diameter of rotor head and RPM B. WBCs
D. RPM and time of centrifugation C. Crystals
D. Casts
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Chapter 7 | Microscopic Examination of Urine 213

9. The Sternheimer-Malbin stain is added to urine 17. Leukocytes that stain pale blue with Sternheimer-Malbin
sediments to do all of the following except: stain and exhibit brownian movement are:
A. Increase visibility of sediment constituents A. Indicative of pyelonephritis
B. Change the constituents’ refractive index B. Basophils
C. Decrease precipitation of crystals C. Mononuclear leukocytes
D. Delineate constituent structures D. Glitter cells
10. Nuclear detail can be enhanced by: 18. Sometimes mononuclear leukocytes are mistaken for:
A. Prussian blue A. Yeast cells
B. Toluidine blue B. Squamous epithelial cells
C. Acetic acid C. Pollen grains
D. Both B and C D. Renal tubular cells
11. Which of the following lipids is/are stained by Sudan III? 19. When pyuria is detected in a urine sediment, the slide
A. Cholesterol should be checked carefully for the presence of:
B. Neutral fats A. RBCs
C. Triglycerides B. Bacteria
D. Both B and C C. Hyaline casts
D. Mucus
12. Which of the following lipids is/are capable of polarizing
light? 20. Transitional epithelial cells are sloughed from the:
A. Cholesterol A. Collecting duct
B. Neutral fats B. Vagina
C. Triglycerides C. Bladder
D. Both A and B D. Proximal convoluted tubule
13. The purpose of the Hansel stain is to identify: 21. The largest cells in the urine sediment are:
A. Neutrophils A. Squamous epithelial cells
B. Renal tubular cells B. Urothelial epithelial cells
C. Eosinophils C. Cuboidal epithelial cells
D. Monocytes D. Columnar epithelial cells
14. Crenated RBCs are seen in urine that is: 22. A squamous epithelial cell that is clinically significant
A. Hyposthenuric is the:
B. Hypersthenuric A. Cuboidal cell
C. Highly acidic B. Clue cell
D. Highly alkaline C. Caudate cell
D. Columnar cell
15. Differentiation among RBCs, yeast, and oil droplets may
be accomplished by all of the following except: 23. Forms of transitional epithelial cells include all of the
A. Observation of budding in yeast cells following except:
B. Increased refractility of oil droplets A. Spherical
C. Lysis of yeast cells by acetic acid B. Caudate
D. Lysis of RBCs by acetic acid C. Convoluted
D. Polyhedral
16. A finding of dysmorphic RBCs is indicative of:
A. Glomerular bleeding 24. Increased transitional cells are indicative of:
B. Renal calculi A. Catheterization
C. Traumatic injury B. Malignancy
D. Coagulation disorders C. Pyelonephritis
D. Both A and B
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214 Part Two | Urinalysis

25. A primary characteristic used to identify renal tubular 33. A person submitting a urine specimen after a strenuous
epithelial cells is: exercise routine normally can have all of the following in
A. Elongated structure the sediment except:
B. Centrally located nucleus A. Hyaline casts
C. Spherical appearance B. Granular casts
D. Eccentrically located nucleus C. RBC casts
D. WBC casts
26. After an episode of hemoglobinuria, RTE cells may
contain: 34. Before identifying an RBC cast, all of the following
A. Bilirubin should be observed:
B. Hemosiderin granules A. Free-floating RBCs
C. Porphobilinogen B. Intact RBCs in the cast matrix
D. Myoglobin C. A positive reagent strip blood reaction
D. All of the above
27. The predecessor of the oval fat body is the:
A. Histiocyte 35. WBC casts are associated primarily with:
B. Urothelial cell A. Pyelonephritis
C. Monocyte B. Cystitis
D. Renal tubular cell C. Glomerulonephritis
D. Viral infections
28. A structure believed to be an oval fat body produced a
Maltese cross formation under polarized light but does 36. The shape of the RTE cell associated with RTE casts is
not stain with Sudan III. The structure: primarily:
A. Contains cholesterol A. Elongated
B. Is not an oval fat body B. Cuboidal
C. Contains neutral fats C. Round
D. Is contaminated with immersion oil D. Columnar
29. The finding of yeast cells in the urine is commonly 37. When observing RTE casts, the cells are primarily:
associated with: A. Embedded in a clear matrix
A. Cystitis B. Embedded in a granular matrix
B. Diabetes mellitus C. Attached to the surface of a matrix
C. Pyelonephritis D. Stained by components of the urine filtrate
D. Liver disorders
38. The presence of fatty casts is associated with:
30. The primary component of urinary mucus is: A. Nephrotic syndrome
A. Bence Jones protein B. Crush injuries
B. Microalbumin C. Diabetes mellitus
C. Uromodulin D. All of the above
D. Orthostatic protein
39. Nonpathogenic granular casts contain:
31. The majority of casts are formed in the: A. Cellular lysosomes
A. Proximal convoluted tubules B. Degenerated cells
B. Ascending loop of Henle C. Protein aggregates
C. Distal convoluted tubules D. Gram-positive cocci
D. Collecting ducts
40. All of the following are true about waxy casts except they:
32. Cylindruria refers to the presence of: A. Represent extreme urine stasis
A. Cylindrical renal tubular cells B. May have a brittle consistency
B. Mucus-resembling casts C. Require staining to be visualized
C. Hyaline and waxy casts D. Contain degenerated granules
D. All types of casts
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Chapter 7 | Microscopic Examination of Urine 215

41. Observation of broad casts represents: 48. Match the following crystals seen in alkaline urine with
A. Destruction of tubular walls their description/identifying characteristics:
B. Dehydration and high fever Triple phosphate 1. Yellow granules
C. Formation in the collecting ducts Amorphous phosphate 2. Thin prisms
D. Both A and C Calcium phosphate 3. “Coffin lids”
Ammonium biurate 4. Dumbbell shape
42. All of the following contribute to urinary crystals
formation except: Calcium carbonate 5. White precipitate
A. Protein concentration 6. Thorny apple
B. pH 49. Match the following abnormal crystals with their
C. Solute concentration description/identifying characteristics:
D. Temperature Cystine 1. Bundles after
refrigeration
43. The most valuable initial aid for identifying crystals in a
Tyrosine 2. Highly alkaline pH
urine specimen is:
Cholesterol 3. Bright yellow clumps
A. pH
Leucine 4. Hexagonal plates
B. Solubility
Ampicillin 5. Flat plates, high spe-
C. Staining
cific gravity
D. Polarized microscopy
Radiographic dye 6. Concentric circles,
44. Crystals associated with severe liver disease include all radial striations
of the following except: Bilirubin 7. Notched corners
A. Bilirubin 8. Fine needles seen in
B. Leucine liver disease
C. Cystine 50. Match the following types of microscopy with their
D. Tyrosine descriptions:
45. All of the following crystals routinely polarize except: Bright-field 1. Indirect light is reflected off
the object
A. Uric acid
Phase 2. Objects split light into two
B. Cholesterol
beams
C. Radiographic dye
Polarized 3. Low-refractive-index ob-
D. Cystine jects may be overlooked
46. Casts and fibers usually can be differentiated using: Dark-field 4. Three-dimensional images
A. Solubility characteristics Fluorescent 5. Forms halo of light around
B. Patient history object
C. Polarized light Interference 6. Detects electrons emitted
contrast from objects
D. Fluorescent light
7. Detects specific wave-
47. Match the following crystals seen in acidic urine with lengths of light emitted
their description/identifying characteristics: from objects
Amorphous urates 1. Envelopes
Uric acid 2. Thin needles
Calcium oxalate 3. Yellow-brown,
monohydrate whetstone
Calcium oxalate 4. Pink sediment
dihydrate 5. Ovoid
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216 Part Two | Urinalysis

Case Studies and Clinical Situations

1. An 85-year-old woman with diabetes and a broken hip decreased renal concentrating ability. Urinalysis results are
has been confined to bed for the past 3 months. Results as follows:
of an ancillary blood glucose test are 250 mg/dL, and COLOR: Amber KETONES: Negative
her physician orders additional blood tests and a routine
CLARITY: Hazy BLOOD: Negative
urinalysis. The urinalysis report is as follows:
SP. GRAVITY: 1.010 BILIRUBIN: Large
COLOR: Pale yellow KETONES: Negative
pH: 7.0 UROBILINOGEN: 4.0 EU
CLARITY: Hazy BLOOD: Moderate
PROTEIN: 2+ NITRITE: Negative
SP. GRAVITY: 1.020 BILIRUBIN: Negative
GLUCOSE: Negative LEUKOCYTES: Negative
pH: 5.5 UROBILINOGEN: Normal
Microscopic:
PROTEIN: Trace NITRITE: Negative
2 to 4 WBCs/hpf 1 to 2 hyaline casts/lpf
GLUCOSE: 100 mg/dL LEUKOCYTES: 2+
1 to 3 RBCs/hpf 1 to 2 granular casts/lpf
Microscopic:
2 to 4 bile-stained RTE
20 to 25 WBCs/hpf
cells/hpf
Many yeast cells and hyphae
0 to 1 RTE casts/lpf
a. Why are yeast infections common in patients with dia-
0 to 1 bile-stained waxy
betes mellitus?
casts/lpf
b. With a blood glucose level of 250 mg/dL, should glu-
a. Based on the urinalysis results, in what area of the
cose be present in the urine? Why or why not?
nephron is damage occurring?
c. Is there a discrepancy between the negative nitrite and
b. Is this consistent with the patient’s primary diagnosis?
the positive leukocyte esterase results? Explain your
Explain your answer.
answer.
c. What is causing the RTE cells to be bile stained?
d. What is the major discrepancy between the chemical
and microscopic results? d. Why is the urobilinogen level elevated?
e. Considering the patient’s history, what is the most e. State a disorder in which the urobilinogen level would
probable cause for the discrepancy? be elevated but the bilirubin result would be negative.

2. A medical laboratory science student training in a newly 4. A 30-year-old woman being treated for a UTI brings a
renovated STAT laboratory is having difficulty performing urine specimen to the employee health clinic at 4:00 p.m.
a microscopic urinalysis. Reagent strip testing indicates The nurse on duty tells her that the specimen will be re-
the presence of moderate blood and leukocytes, but also frigerated and tested by the medical laboratory scientist
the student is observing some large unusual objects re- (MLS) the next morning. The MLS has difficulty inter-
sembling crystals and possible casts. In addition, the stu- preting the color of the reagent strip tests and reports
dent is having difficulty keeping all of the constituents in only the following results:
focus at the same time. COLOR: Amber CLARITY: Slightly cloudy
a. Why is the student having difficulty focusing? Microscopic:
b. What is a possible cause of the unusual microscopic 3 to 5 RBCs/hpf
constituents? 8 to 10 WBCs/hpf
c. Should the student be concerned about the unusual Moderate bacteria
microscopic constituents? Explain your answer.
Moderate colorless crystals appearing in bundles
d. What microscopy technique could be used to aid in
a. What could have caused the technologist to have diffi-
differentiating a cast and an artifact?
culty interpreting the results on the reagent strip?
3. A prisoner sentenced to 10 years for selling illegal drugs b. Could this specimen produce a yellow foam when
develops jaundice, lethargy, and hepatomegaly. A test for shaken?
hepatitis B surface antigen is positive, and the patient is
c. What could the technologist do to aid in the identifi-
placed in the prison infirmary. When his condition ap-
cation of the crystals?
pears to worsen and a low urinary output is observed, the
patient is transferred to a local hospital. Additional testing d. What is the probable identification of the colorless
detects a superinfection with delta hepatitis virus and crystals?
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Chapter 7 | Microscopic Examination of Urine 217

5. A 2-year-old left unattended in the garage for 5 minutes is SP. GRAVITY: 1.030 BILIRUBIN: Negative
suspected of ingesting antifreeze (ethylene glycol). The pH: 5.5 UROBILINOGEN: Normal
urinalysis has a pH of 6.0 and is negative on the chemical
PROTEIN: 2+ NITRITE: Negative
examination. Two distinct forms of crystals are observed
in the microscopic examination. GLUCOSE: Negative LEUKOCYTE: Negative
a. What type of crystals would you expect to be present? Microscopic:
b. What is the other form of this crystal? 0 to 3 WBCs/hpf
c. Describe the two forms. 0 to 4 hyaline casts/lpf
d. Which form would you expect to be predominant? 0 to 3 granular casts/lpf
Few squamous epithelial cells
6. A female patient comes to the outpatient clinic with
symptoms of UTI. She brings a urine specimen with her. a. Are these results of clinical significance?
Results of the routine analysis performed on this speci- b. Explain the discrepancy between the chemical and
men are as follows: microscopic blood results.
COLOR: Yellow KETONES: Negative c. What is the probable cause of the granular casts?
CLARITY: Hazy BLOOD: Small 8. As supervisor of the urinalysis section, you are reviewing
SP. GRAVITY: 1.015 BILIRUBIN: Negative results. State why or why not each of the following results
pH: 9.0 UROBILINOGEN: Normal would concern you.
PROTEIN: Negative NITRITE: Negative a. The presence of waxy casts and a negative protein in
urine from a 6-month-old girl
GLUCOSE: Negative LEUKOCYTE: 2+
b. Increased transitional epithelial cells in a specimen
Microscopic:
obtained after cystoscopy
1 to 3 RBCs/hpf Heavy bacteria
c. Tyrosine crystals in a specimen with a negative
8 to 10 WBCs/hpf Moderate squamous bilirubin test result
epithelial cells
d. Cystine crystals in a specimen from a patient
a. What discrepancies are present between the chemical diagnosed with gout
and microscopic test results?
e. Cholesterol crystals in urine with a specific gravity
b. State a reason for the discrepancies. greater than 1.040
c. Identify a chemical result in the urinalysis that con- f. Trichomonas vaginalis in a urine specimen from a male
firms your reason for the discrepancies. patient
d. What course of action should the laboratory take to g. Amorphous urates and calcium carbonate crystals in a
obtain accurate results for this patient? specimen with a pH of 7.0
7. A high school student is taken to the emergency room
with a broken leg that occurred during a football game.
The urinalysis results are as follows:
COLOR: Dark yellow KETONES: Negative
CLARITY: Hazy BLOOD: Moderate
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7582_Ch08_219-234 30/06/20 1:16 PM Page 219

CHAPTER 8
Renal Disease
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
8-1 Differentiate among renal diseases of varying 8-9 Compare and contrast nephrotic syndrome and mini-
origins, including glomerular, tubular, interstitial, mal change disease with regard to laboratory results
and vascular. and the course of the disease.
8-2 Describe the processes by which immunologic damage 8-10 State two causes of acute tubular necrosis.
is caused to the glomerular basement membrane.
8-11 Name the constituent of urinary sediment that is most
8-3 Describe the clinical features of glomerulonephritis. indicative of renal tubular damage.
8-4 Describe the characteristic clinical symptoms, etiology, 8-12 Describe Fanconi syndrome, Alport syndrome,
and urinalysis findings in acute poststreptococcal and uromodulin-associated renal disease, and renal
rapidly progressive glomerulonephritis, Goodpasture glucosuria.
syndrome, granulomatosis with polyangiitis, and
8-13 Differentiate between diabetic nephropathy and
Henoch-Schönlein purpura.
nephrogenic diabetes insipidus.
8-5 Name three renal disorders that also involve acute
8-14 Compare and contrast the urinalysis results in patients
respiratory symptoms.
with cystitis, pyelonephritis, and acute interstitial
8-6 Differentiate between membranous and membranopro- nephritis.
liferative glomerulonephritis.
8-15 Differentiate among causes of laboratory results associ-
8-7 Discuss the clinical course and significant laboratory ated with acute renal failure at each stage: prerenal,
results associated with immunoglobulin A nephropathy. renal, and postrenal.
8-8 Relate laboratory results associated with nephrotic 8-16 Discuss the formation of renal calculi, composition of
syndrome to the disease process. renal calculi, and patient management techniques.

KEY TERMS
Acute glomerulonephritis (AGN) Glomerulonephritis Membranoproliferative
Acute interstitial nephritis (AIN) Goodpasture syndrome glomerulonephritis (MPGN)
Acute tubular necrosis (ATN) Granulomatosis with polyangiitis Minimal change disease (MCD)
Antiglomerular basement (GPA) Nephrotic syndrome (NS)
membrane antibody Henoch-Schönlein purpura Pyelonephritis
Antineutrophilic cytoplasmic IgA nephropathy Rapidly progressive (or crescentic)
antibody (ANCA) Lithiasis glomerulonephritis (RPGN)
Chronic glomerulonephritis (CGN) Lithotripsy Systemic lupus erythematosus (SLE)
Cystitis Membranous glomerulonephritis Tubulointerstitial disease
Focal segmental glomerulosclerosis (MGN) Uromodulin-associated kidney
(FSGS) disease
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220 Part Two | Urinalysis

Introduction hypertension, oliguria, proteinuria, and hematuria. Symptoms

usually occur in children and young adults after respiratory
Disorders throughout the body can affect renal function and infections caused by certain strains of group A β-hemolytic strep-
produce abnormalities in the urinalysis. Considering that the tococci that contain M protein in the cell wall. During the course
major function of the kidneys is filtration of the blood to remove of the infection, these nephrogenic strains of streptococci form
waste products, it becomes evident that the kidneys are consis- immune complexes with their corresponding circulating antibod-
tently exposed to substances that are potentially damaging. ies and become deposited on the glomerular membranes. The
Often renal disease is classified as being glomerular, tubular, accompanying inflammatory reaction affects glomerular function.
or interstitial, based on the area of the kidney primarily affected. In most cases, treatment involves successful management
In this chapter, we will cover the disorders encountered most of the secondary complications (hypertension and electrolyte
commonly in relation to the affected areas of the kidney. Basic imbalance) until the immune complexes have been cleared
knowledge of these disorders can be helpful when analyzing the from the blood and the inflammation subsides, resulting in no
results of a routine urinalysis. permanent damage to the kidney.
Primary urinalysis findings include marked hematuria,
Glomerular Disorders proteinuria, and oliguria, accompanied by red blood cell
(RBC) casts, dysmorphic RBCs, hyaline and granular casts, and
Most glomerular disorders result from immunologic disorders white blood cells (WBCs). As toxicity to the glomerular mem-
throughout the body, including the kidney. Immune complexes brane subsides, urinalysis results return to normal, with the
formed as a result of immunologic reactions and increased possible exception of microscopic hematuria that lasts until the
serum immunoglobulins, such as immunoglobulin A (IgA), cir- membrane damage has been repaired. Blood urea nitrogen
culate in the bloodstream and are deposited on the glomerular (BUN) may be elevated during the acute stages, but, like the
membranes. Then components of the immune system, includ- urinalysis, returns to normal. Demonstration of positive anti–
ing complement, neutrophils, lymphocytes, monocytes, and cy- group A streptococcal enzyme tests (antistreptolysin O [ASO]
tokines, are attracted to the deposit area, producing changes and antideoxyribonuclease-B antibody [anti-DNase B]) pro-
and damage to the membranes. Depending on the immune sys- vides evidence that the disease is of streptococcal origin.
tem mediators involved, damage may consist of cellular infil- Since the development of rapid anti–group A streptococcal
tration or proliferation, resulting in thickening of the glomerular enzyme tests that can be performed in a physician’s office, ur-
basement membrane, as well as complement-mediated damage gent care facility, or emergency department, the incidence of
to the capillaries and basement membrane. acute poststreptococcal glomerulonephritis has declined.
Nonimmunologic causes of glomerular damage include
the following:
Technical Tip 8-1. RBC casts are a hallmark character-
• Exposure to chemicals and toxins that also affect the istic of acute glomerulonephritis.
tubules
• Disruption of the electrical membrane charges as occurs
in nephrotic syndrome (NS) Rapidly Progressive (Crescentic) Glomerulonephritis
• Deposition of amyloid material from systemic disorders
A more serious form of acute glomerular disease is called rapidly
that may involve chronic inflammation and acute-phase
progressive (or crescentic) glomerulonephritis (RPGN) and
reactants
has a much poorer prognosis, often terminating in renal failure.
• Thickening of the basement membrane associated with Symptoms are initiated by deposition of immune complexes in
diabetic nephropathy the glomerulus, often as a complication of another form of
glomerulonephritis or a disorder of the immune system, such as
Glomerulonephritis systemic lupus erythematosus (SLE). Damage by macrophages
The term glomerulonephritis refers to a sterile, in- to the capillary walls releases cells and plasma into Bowman
flammatory process that affects the glomerulus and space, and the production of crescentic formations containing
is associated with the finding of blood, protein, and macrophages, fibroblasts, and polymerized fibrin causes perma-
casts in the urine.1 A variety of types of glomeru- nent damage to the capillary tufts.
lonephritis exist, and the condition also may progress from one Initial laboratory results are similar to AGN but become
form to another (i.e., rapidly progressive glomerular nephritis, more abnormal as the disease progresses, including protein lev-
to chronic glomerulonephritis, to nephrotic syndrome and els that are markedly elevated and glomerular filtration rates
eventual renal failure). that are very low. Some forms may demonstrate increased fib-
rin degradation products, cryoglobulins, and the deposition of
Acute Poststreptococcal Glomerulonephritis IgA immune complexes in the glomerulus.2
As its name implies, acute glomerulonephritis (AGN) is a
Goodpasture Syndrome
disease marked by the sudden onset of symptoms consistent
with damage to the glomerular membrane. These may include Morphological changes to the glomeruli resembling those in rap-
fever, edema (most noticeably around the eyes), fatigue, nausea, idly progressive glomerular nephritis are seen in conjunction with
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Chapter 8 | Renal Disease 221

the autoimmune disorder termed Goodpasture syndrome. A SLE, Sjögren syndrome, secondary syphilis, hepatitis B, gold
cytotoxic autoantibody can appear against the glomerular and and mercury treatments, and malignancy. In about 75% of cases,
alveolar basement membranes after viral respiratory infections. the etiology is unknown.6 As a rule, the disease progresses
Attachment of this autoantibody to the basement membrane, slowly, with possible remission; however, symptoms of nephrotic
followed by complement activation, produces the capillary de- syndrome frequently develop.7 There also may be a tendency
struction. Referred to as antiglomerular basement membrane toward thrombosis.
antibody, the autoantibody can be detected in patient serum. Laboratory findings include microscopic hematuria and
Initial pulmonary complaints are hemoptysis and dyspnea, elevated urine protein excretion that may reach concentrations
followed by the development of hematuria. Urinalysis results similar to those in nephrotic syndrome. RBC casts are rare, but
include proteinuria, hematuria, and the presence of RBC casts. microscopic hematuria is common.6 Demonstration of one of
Progression to chronic glomerulonephritis and end-stage renal the secondary disorders through blood tests can aid in the
failure is common. diagnosis.

Granulomatosis With Polyangiitis Membranoproliferative Glomerulonephritis

Granulomatosis with polyangiitis (GPA), formerly called Membranoproliferative glomerulonephritis (MPGN) is
Wegener granulomatosis, causes a granuloma-producing in- marked by different alterations in the cellularity of the
flammation of the small blood vessels, primarily of the kidney glomerulus and peripheral capillaries. Type 1 displays in-
and respiratory system.3 Key to the diagnosis of GPA is the creased cellularity in the subendothelial cells of the mesangium
demonstration of antineutrophilic cytoplasmic antibody (interstitial area of Bowman capsule), causing thickening of the
(ANCA) in the patient’s serum.3,4 Binding of these autoanti- capillary walls, whereas type 2 (dense deposit disease) displays
bodies to the neutrophils located in the vascular walls may ini- extremely dense deposits in the glomerular basement mem-
tiate the immune response and the resulting granuloma brane, tubules, and Bowman capsule. MPGN type 3 is charac-
formation. Patients usually present first with pulmonary symp- terized by both subepithelial and subendothelial deposits.8
toms and later develop renal involvement, including hema- Many of the patients are children, and the disease has a poor
turia, proteinuria, RBC casts, and elevated levels of serum prognosis: type 1 patients progress to nephrotic syndrome, and
creatinine and BUN. type 2 patients experience symptoms of chronic glomeru-
Testing for ANCA includes incubating the patient’s serum lonephritis. There is a high incidence of recurrent disease after
with either ethanol or formalin/formaldehyde-fixed neutrophils renal transplant.8 The laboratory findings vary, but hematuria,
and examining the preparation using indirect immunofixation proteinuria, and decreased serum complement levels are usual
to detect the serum antibodies attached to the neutrophils. If findings. There appears to be an association with autoimmune
the neutrophils are fixed in ethanol, the antibodies form a disorders, infections, and malignancies.9
perinuclear pattern called p-ANCA. When the neutrophils are
Chronic Glomerulonephritis
fixed with formalin/formaldehyde, the pattern is granular
throughout the cytoplasm and is referred to as c-ANCA.5 Depending on the amount and duration of the damage
to the glomerulus in the glomerular disorders discussed
Henoch-Schönlein Purpura previously, progression to chronic glomerulonephritis
(CGN) and end-stage renal disease (ESRD) may occur.
Henoch-Schönlein purpura disease occurs primarily in chil-
Gradually worsening symptoms include fatigue, anemia, hyper-
dren after upper respiratory infections. As its name implies,
tension, edema, and oliguria.
initial symptoms include the appearance of raised, red patches
Examination of the urine reveals hematuria, proteinuria,
on the skin. Respiratory and gastrointestinal symptoms, in-
glucosuria as a result of tubular dysfunction, and many vari-
cluding blood in the sputum and stools, may be present. Renal
eties of casts, including broad casts. A glomerular filtration rate
involvement is the most serious complication of the disorder
that is markedly decreased is present in conjunction with in-
and may range from mild to heavy proteinuria and hematuria
creased BUN and creatinine levels and electrolyte imbalance.
with RBC casts. Complete recovery with normal renal function
is seen in more than 50% of patients. In other patients, pro-
gression to a more serious form of glomerulonephritis and Technical Tip 8-2. The presence of broad casts
renal failure may occur. Urinalysis and renal function assess- is often seen in chronic glomerulonephritis that
ment should be used to monitor patients after recovery from progresses to ESRD.
the original symptoms.

Membranous Glomerulonephritis
Immunoglobulin A Nephropathy
The predominant characteristic of membranous glomeru-
lonephritis (MGN) is a pronounced thickening of the glomeru- Also known as Berger disease, IgA nephropathy, in which im-
lar basement membrane resulting from the deposition of mune complexes containing IgA are deposited on the glomerular
immunoglobulin G immune complexes. Disorders associated membrane, is the most common cause of glomerulonephritis.
with membranous glomerulonephritis development include Patients have increased serum levels of IgA, which may be a
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222 Part Two | Urinalysis

result of a mucosal infection. The disorder is seen most fre- results for BUN and creatinine. It is the most common cause of
quently in children and young adults. nephrotic syndrome in children (85% to 95%) but only 10% to
Patients usually present with an episode of macroscopic 15% of cases of nephrotic syndrome in adults.11 Although
hematuria after an infection or strenuous exercise. Recovery the etiology is unknown at this time, allergic reactions, recent
from the macroscopic hematuria is spontaneous; however, immunization, and possession of the human leukocyte antigen-
asymptomatic microhematuria and elevated serum levels of IgA B12 (HLA-B12) antigen have been associated with this disease.
remain.10 Except for periodic episodes of macroscopic hema- Therefore, it is postulated that MCD is a disorder of T cells,
turia, a patient with the disorder may remain essentially which release a cytokine that injures the glomerular epithelial
asymptomatic for 20 years or more; however, there is a gradual foot processes.11 The disorder responds well to corticosteroids,
progression to chronic glomerulonephritis and ESRD. and the prognosis is generally good, with frequent complete
remissions.12
Nephrotic Syndrome
Focal Segmental Glomerulosclerosis
Nephrotic syndrome is marked by massive protein-
uria (greater than 3.5 g/day), low levels of serum In contrast to the disorders discussed previously, focal segmen-
albumin, high levels of serum lipids, and pronounced tal glomerulosclerosis (FSGS) affects only certain numbers
edema.1 Acute onset of the disorder can occur in in- and areas of glomeruli and the others remain normal. FSGS is
stances of circulatory disruption, producing systemic shock that one of the most common causes of primary glomerular disease
decreases the pressure and flow of blood to the kidney. Progres- in adults. It can occur as a primary (idiopathic) glomerular dis-
sion to nephrotic syndrome also may occur as a complication ease or secondary to another disease or drug. Often FSGS is
of the forms of glomerulonephritis discussed previously. seen in association with abuse of heroin and analgesics and with
Increased permeability of the glomerular membrane is the HIV and hepatitis viruses.13 Symptoms may be similar to
attributed to damage to the shield of negativity and the nephrotic syndrome and MCD due to damaged podocytes.
podocytes that produces a barrier that is less tightly connected. Immune deposits, primarily immunoglobulin M and C3, are a
Such damage facilitates the passage of high-molecular-weight frequent finding and can be seen in undamaged glomeruli.
proteins and lipids and negatively charged albumin into the Moderate to heavy proteinuria and microscopic hematuria are
urine. Albumin is the primary protein depleted from the circu- the most consistent findings from urinalysis.13
lation. The ensuing hypoalbuminemia appears to stimulate in- Laboratory testing and clinical information for the
creased lipid production by the liver. The lower oncotic pressure glomerular disorders are summarized in Tables 8-1 and 8-2.
in the capillaries resulting from the depletion of plasma albumin
increases fluid loss into the interstitial spaces, which, accompa- Tubular Disorders
nied by sodium retention, produces the edema. Depletion of
immunoglobulins and coagulation factors places patients at an Disorders affecting the renal tubules include those in which
increased risk of infection and coagulation disorders. Both tubular function is disrupted as a result of actual damage to
tubular and glomerular damage occurs, and nephrotic syndrome the tubules, as well as those in which a metabolic or hereditary
may progress to chronic renal failure. disorder affects the intricate functions of the tubules.
Urinalysis observations include marked proteinuria; urinary
fat droplets (lipiduria); oval fat bodies; renal tubular epithelial Acute Tubular Necrosis
(RTE) cells; epithelial, fatty, and waxy casts; and microscopic The primary disorder associated with damage to the
hematuria. Absorption of the lipid-containing proteins by the renal tubules is acute tubular necrosis (ATN).
RTE cells followed by cellular sloughing produces the charac- Damage to the RTE cells may be produced by de-
teristic oval fat bodies seen in the sediment examination. creased blood flow that causes a lack of oxygen pres-
entation to the tubules (ischemia) or the presence of toxic
substances in the urinary filtrate.
Technical Tip 8-3. As discussed in Chapter 7, using Disorders causing ischemic ATN include shock, trauma
polarized light microscopy will result in the appear-
(such as crushing injuries), and surgical procedures. “Shock”
ance of the characteristic Maltese cross formation
is a general term indicating a severe condition that decreases
in the oval fat bodies and fatty casts containing
the flow of blood throughout the body. Examples of conditions
cholesterol.
that may cause shock are cardiac failures, sepsis involving tox-
igenic bacteria, anaphylaxis, massive hemorrhage, and contact
with high-voltage electricity.
Minimal Change Disease
Exposure to a variety of nephrotoxic agents can damage
As the name implies, minimal change disease (MCD) (also and affect the function of the RTE cells. Substances include
known as lipid nephrosis or nil disease) produces little cellular aminoglycoside antibiotics, the antifungal agent amphotericin
change in the glomerulus, except for some damage to the B, cyclosporine, radiographic dye, organic solvents such as eth-
podocytes and the shield of negativity, allowing for increased ylene glycol, heavy metals, and toxic mushrooms. As discussed
protein filtration. Patients are usually children who present in Chapter 6, filtration of large amounts of hemoglobin and
with edema, heavy proteinuria, transient hematuria, and normal myoglobin is also nephrotoxic.
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Chapter 8 | Renal Disease 223

Table 8–1 Laboratory Testing in Glomerular Disorders

Disorder Primary Urinalysis Result Other Significant Tests
Acute glomerulonephritis (AGN) Macroscopic hematuria Anti–group A streptococcal enzyme tests
Proteinuria
RBC casts
Granular casts
Rapidly progressive Macroscopic hematuria BUN
glomerulonephritis (RPGN)
Proteinuria Creatinine
RBC casts eGFR
Goodpasture syndrome Macroscopic hematuria Antiglomerular basement membrane antibody
Proteinuria
RBC casts
Granulomatosis with Macroscopic hematuria Antineutrophilic peripheral or cytoplasmic antibody
polyangiitis (GPA)
Proteinuria
RBC casts
Henoch-Schönlein purpura Macroscopic hematuria Stool occult blood
Proteinuria
RBC casts
Membranous Microscopic hematuria Antinuclear antibody
glomerulonephritis (MGN)
Proteinuria Hepatitis B surface antigen
Fluorescent treponemal antibody-absorption test
(FTA-ABS)
Membranoproliferative Hematuria Serum complement levels
glomerulonephritis (MPGN)
Proteinuria
Chronic Hematuria BUN
glomerulonephritis (CGN)
Proteinuria Serum creatinine
Glucosuria eGFR
Cellular and granular casts Electrolytes
Waxy and broad casts
IgA nephropathy (early stages) Macroscopic or microscopic Serum IgA
hematuria
IgA nephropathy (late stages) See Chronic glomeru-
lonephritis (CGN)
Nephrotic syndrome (NS) Heavy proteinuria Serum albumin
Microscopic hematuria Cholesterol
Renal tubular cells Triglycerides
Oval fat bodies
Fat droplets
Fatty and waxy casts
Minimal change disease (MCD) Heavy proteinuria Serum albumin
Transient hematuria Cholesterol
Continued
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224 Part Two | Urinalysis

Table 8–1 Laboratory Testing in Glomerular Disorders—cont’d

Disorder Primary Urinalysis Result Other Significant Tests
Fat droplets Triglycerides
Focal segmental Proteinuria Drugs of abuse
glomerulosclerosis (FSGS)
Microscopic hematuria HIV tests
Macroscopic or microscopic
hematuria
Alport syndrome See Nephrotic Genetic testing
syndrome (NS)
Microalbuminuria
Diabetic nephropathy (late stages) See Chronic glomeru- Blood glucose
lonephritis (CGN)

Table 8–2 Clinical Information Associated With Glomerular Disorders

Disorder Etiology Clinical Course
Acute glomerulonephritis (AGN) Deposition of immune complexes, Rapid onset of hematuria and edema;
formed in conjunction with beta- permanent renal damage seldom
hemolytic group A Streptococcus infec- occurs
tion, on the glomerular membranes
Rapidly progressive Deposition of immune complexes from Rapid onset with glomerular damage
glomerulonephritis (RPGN) systemic immune disorders on the and possible progression to end-stage
glomerular membrane renal failure
Goodpasture syndrome Attachment of a cytotoxic antibody Hemoptysis and dyspnea followed by
formed during viral respiratory infec- hematuria
tions to glomerular and alveolar base-
ment membranes
Possible progression to end-stage renal
failure
Granulomatosis with Antineutrophilic cytoplasmic autoanti- Pulmonary symptoms, including he-
polyangiitis (GPA) body (ANCA) binds to neutrophils moptysis, develop first, followed by
in vascular walls, producing damage renal involvement and possible pro-
to small vessels in the lungs and gression to end-stage renal failure
glomerulus
Henoch-Schönlein purpura Occurs primarily in children after viral Initial appearance of purpura followed
respiratory infections; a decrease in by blood in sputum and stools and
platelets disrupts vascular integrity eventual renal involvement
Complete recovery is common, but may
progress to renal failure
Membranous Thickening of the glomerular membrane Slow progression to nephrotic syndrome
glomerulonephritis (MGN) after IgG immune complex deposition or possible remission
associated with systemic disorders
Membranoproliferative Cellular proliferation affecting the capil- Slow progression to chronic glomeru-
glomerulonephritis (MPGN) lary walls or the glomerular basement lonephritis (CGN) or nephrotic syn-
membrane, possibly immune mediated drome (NS)
Chronic Marked decrease in renal function result- Noticeable decrease in renal function
glomerulonephritis (CGN) ing from glomerular damage precipi- progressing to renal failure
tated by other renal disorders
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Chapter 8 | Renal Disease 225

Table 8–2 Clinical Information Associated With Glomerular Disorders—cont’d

Disorder Etiology Clinical Course
IgA nephropathy Deposition of IgA on the glomerular Recurrent macroscopic hematuria after
membrane resulting from increased exercise with slow progression to
levels of serum IgA chronic glomerulonephritis (CGN)
Nephrotic syndrome (NS) Disruption of the shield of negativity and Acute onset after systemic shock
damage to the tightly fitting podocyte Gradual progression from other
barrier, resulting in massive loss of glomerular disorders and then to renal
protein and lipids failure
Minimal change disease (MCD) Disruption of the podocytes occurring Frequent complete remission after corti-
primarily in children after allergic reac- costeroid treatment
tions and immunizations; dysfunction
of T-cell immunity
Focal segmental Disruption of podocytes in certain areas May resemble nephrotic syndrome (NS)
glomerulosclerosis (FSGS) of glomeruli associated with heroin or minimal change disease (MCD)
and analgesic abuse and with HIV and
hepatitis viruses
Alport syndrome Genetic disorder showing lamellated Slow progression to nephrotic syndrome
and thinning glomerular basement (NS) and end-stage renal disease
membrane

The disease course of ATN varies. It may present as an convoluted tubule. Therefore, substances affected most no-
acute complication of an ischemic event or more gradually dur- ticeably include glucose, amino acids, phosphorous, sodium,
ing exposure to toxic agents. Correcting the ischemia, removing potassium, bicarbonate, and water. Tubular reabsorption may
the toxic substances, and effectively managing the accompany- be affected by dysfunction of the transport of filtered sub-
ing symptoms of acute renal failure (ARF) frequently result in stances across the tubular membranes, disruption of cellular
a complete recovery. energy needed for transport, or changes in the tubular
Urinalysis findings include mild proteinuria, microscopic membrane permeability.
hematuria, and, most noticeably, the presence of RTE cells and Fanconi syndrome may be inherited in association with
RTE cell casts containing tubular fragments consisting of three cystinosis and Hartnup disease (see Chapter 9), acquired
or more cells. As a result of the tubular damage, a variety of through exposure to toxic agents, including heavy metals and
other casts may be present, including hyaline, granular, waxy, outdated tetracycline, or seen as a complication of multiple
and broad. myeloma or renal transplant.
Urinalysis findings include glycosuria with a normal blood
Technical Tip 8-4. RTE cell casts and the presence glucose and possible mild proteinuria. Urinary pH can be very
of RTE cells in the urine sediment are characteristic low due to the failure to reabsorb bicarbonate.
for ATN.
Alport Syndrome
Alport syndrome is an inherited disorder of collagen produc-
Hereditary and Metabolic Tubular tion affecting the glomerular basement membrane. The syn-
Disorders drome can be inherited as a sex-linked or autosomal genetic
disorder. Males inheriting the X-linked gene are affected more
Disorders affecting tubular function may be caused by systemic severely than females inheriting the autosomal gene. During
conditions that affect or override the tubular reabsorptive max- respiratory infections, males younger than 6 years of age may
imum (Tm) for particular substances normally reabsorbed by exhibit macroscopic hematuria and continue to exhibit mi-
the tubules or by failure to inherit a gene or genes required for croscopic hematuria. Abnormalities in hearing and vision
tubular reabsorption. may also develop.
The glomerular basement membrane has a lamellated ap-
Fanconi Syndrome
pearance with areas of thinning. No evidence of glomerular an-
The disorder associated with tubular dysfunction most fre- tibodies is present. The prognosis ranges from mild symptoms
quently is Fanconi syndrome. The syndrome consists of a to persistent hematuria, proteinuria, and renal insufficiency in
generalized failure of tubular reabsorption in the proximal later life to nephrotic syndrome and ESRD.
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226 Part Two | Urinalysis

Uromodulin-Associated Kidney Disease Urinalysis findings associated with DI are low specific
gravity, pale yellow color, and possible false-negative results
Uromodulin is a glycoprotein and is the only protein produced for chemical tests.
by the kidney—in the proximal and distal convoluted tubules.
Although it is not measured by routine laboratory methods, Renal Glycosuria
research has shown that it is the primary protein found in In contrast to Fanconi syndrome, which exhibits a generalized
normal urine. failure to reabsorb substances from the glomerular filtrate, renal
Uromodulin-associated kidney disease is primarily an glucosuria affects only the reabsorption of glucose. The disor-
inherited disorder caused by an autosomal mutation in the der is inherited as an autosomal recessive trait.
gene that produces uromodulin. The mutation causes a de- In inherited renal glucosuria, either the number of glucose
crease in the production of normal uromodulin that is replaced transporters in the tubules is decreased or the affinity of the
by the abnormal form. The abnormal uromodulin is still pro- transporters for glucose is decreased. Under normal conditions,
duced by the tubular cells and accumulates in them, resulting glucose is not present in the urine unless the blood glucose
in their destruction, which leads to the need for renal moni- level reaches the maximal tubular reabsorption capacity for
toring and eventual renal transplantation.14 glucose (TMG), which is 160 to 180 mg/dL. Patients with renal
The mutation also causes an increase in serum uric acid, glycosuria have increased urine glucose concentrations with
resulting in people developing gout as early as the teenage normal blood glucose concentrations.
years before the onset of detectable renal disease.14 Laboratory testing and clinical information for the hered-
itary and metabolic disorders are summarized in Tables 8-3
and 8-4.
Technical Tip 8-5. As discussed in Chapter 7, uro-
modulin forms the matrix of urinary casts seen in
many renal disorders. The defective gene is not
Interstitial Disorders
associated with other renal disorders. Considering the close proximity between the renal
tubules and the renal interstitium, disorders affecting
the interstitium also affect the tubules, resulting in
Diabetic Nephropathy the condition commonly called tubulointerstitial
Diabetic nephropathy is currently the most common cause of disease. Most of these disorders involve infections and inflam-
ESRD. Damage to the glomerular membrane occurs not only as matory conditions.
a result of glomerular membrane thickening but also because
of the increased proliferation of mesangial cells and increased
Urinary Tract Infection
deposition of cellular and noncellular material within the The most common renal disease is a urinary tract infection
glomerular matrix, resulting in accumulation of solid substances (UTI). Infection may involve the lower urinary tract (urethra
around the capillary tufts. This glomerular damage is believed and bladder) or the upper urinary tract (renal pelvis, tubules,
to be associated with deposition of glycosylated proteins result- and interstitium). Most frequently encountered is infection of
ing from poorly controlled levels of blood glucose. The vascular the bladder (cystitis), which can progress to a more serious
structure of the glomerulus also develops sclerosis. upper UTI if left untreated. Cystitis is seen more often in women
As discussed in Chapter 6, early monitoring of people and children, who present with symptoms of urinary frequency
diagnosed with diabetes mellitus for the presence of microalbu- and burning. Urinalysis reveals the presence of numerous
minuria is important to detect the onset of diabetic nephropathy. WBCs and bacteria, often accompanied by mild proteinuria and
Modification of diet and strict control of hypertension can hematuria and an increased pH. The absence of pathological
decrease the progression of the renal disease. casts differentiates cystitis from pyelonephritis.

Nephrogenic Diabetes Insipidus Acute Pyelonephritis

As discussed in Chapter 4, urine concentration is regulated Infection of the upper urinary tract, including both the tubules
in the distal convoluted tubules and the collecting ducts in and interstitium, is termed pyelonephritis and can occur in
response to antidiuretic hormone (ADH) produced by the both acute and chronic forms. Acute pyelonephritis occurs
hypothalamus. When the action of ADH is disrupted either most frequently as a result of ascending movement of bacteria
by the inability of the renal tubules to respond to ADH from a lower UTI into the renal tubules and interstitium. Pa-
(nephrogenic diabetes insipidus [DI]) or failure of the hypo- tients present with rapid onset of symptoms, including urinary
thalamus to produce ADH (neurogenic DI), excessive amounts frequency, burning on urination, and lower back pain.
of urine are excreted. Differentiation between the two types The ascending movement of bacteria from the bladder is
of DI is covered in Chapter 4. enhanced with conditions that interfere with the downward flow
Nephrogenic DI can be inherited as a sex-linked recessive of urine from the ureters to the bladder or the incomplete emp-
gene or acquired from medications, including lithium and am- tying of the bladder during urination. These include obstruc-
photericin B. It also may be seen as a complication of polycystic tions, such as renal calculi, pregnancy, and reflux of urine from
kidney disease and sickle cell anemia. the bladder back into the ureters (vesicoureteral reflux [VUR]).
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Chapter 8 | Renal Disease 227

Table 8–3 Laboratory Testing in Metabolic and Hereditary Tubular Disorders

Disorder Primary Urinalysis Results Other Significant Tests
Acute tubular necrosis (ATN) Microscopic hematuria Hemoglobin
Proteinuria Hematocrit
Renal tubular epithelial (RTE) cells
RTE cell casts Cardiac enzymes
Hyaline, granular, waxy, broad casts
Fanconi syndrome Glucosuria Serum and urine electrolytes
Possible cystine crystals Amino acid chromatography
Uromodulin-associated kidney RTE cells Serum uric acid
disease (early stages)
Uromodulin-associated kidney See chronic glomerulonephritis (CGN)
disease (late stages)
Nephrogenic diabetes insipidus (DI) Low specific gravity, polyuria ADH testing
Renal glucosuria Glucosuria Blood glucose

Table 8–4 Clinical Information Associated With Metabolic and Tubular Disorders
Disorder Etiology Clinical Course
Acute tubular necrosis (ATN) Damage to renal tubular cells caused by is- Acute onset of renal dysfunction usu-
chemia or toxic agents ally resolved when underlying cause
is corrected
Fanconi syndrome Inherited in association with cystinosis and Generalized defect in renal tubular
Hartnup disease or acquired through reabsorption requiring supportive
exposure to toxic agents therapy
Uromodulin-associated Inherited defect in the production of normal Continual monitoring of renal function
kidney disease uromodulin by the renal tubules and for progression to renal failure and
increased uric acid causing gout possible kidney transplantation
Nephrogenic diabetes Inherited defect of tubular response to ADH or Requires supportive therapy to prevent
insipidus (DI) acquired from medications dehydration
Renal glucosuria Inherited autosomal recessive trait Benign disorder

With appropriate antibiotic therapy and removal of any under- structural abnormalities may cause reflux between the bladder
lying conditions, acute pyelonephritis can be resolved without and ureters or within the renal pelvis, affecting emptying of
permanent damage to the tubules. the collecting ducts. Due to its congenital origin, chronic
Urinalysis results are similar to those seen in cystitis, includ- pyelonephritis often is diagnosed in children and may not be
ing numerous leukocytes and bacteria with mild proteinuria and suspected until tubular damage has become advanced.
hematuria. The additional finding of WBC casts, signifying in- Urinalysis results are similar to those seen in acute
fection within the tubules, is of primary diagnostic value for both pyelonephritis, particularly in the early stages. As the disease
acute and chronic pyelonephritis. Sediments also should be progresses, a variety of granular, waxy, and broad casts are pres-
observed carefully for the presence of bacterial casts. ent, accompanied by increased proteinuria and hematuria, and
renal concentration is decreased.
Chronic Pyelonephritis
As its name implies, chronic pyelonephritis is a serious disor-
der that can result in permanent damage to the renal tubules
Technical Tip 8-6. The presence of WBC casts is
and possible progression to chronic renal failure. Congenital
significant for differentiating between cystitis and
urinary structural defects producing reflux nephropathy
pyelonephritis.
are the most frequent cause of chronic pyelonephritis. The
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228 Part Two | Urinalysis

Acute Interstitial Nephritis Table 8–5 Laboratory Results in Interstitial

Acute interstitial nephritis (AIN) is marked by Disorders
inflammation of the renal interstitium followed by in- Primary Other
flammation of the renal tubules. Patients present with Urinalysis Significant
a rapid onset of symptoms relating to renal dys- Disorder Results Tests
function, including oliguria, edema, decreased renal concen-
trating ability, and a possible decrease in the glomerular Cystitis Leukocyturia Urine culture
filtration rate. Fever and the presence of a skin rash are fre- Bacteriuria
quent initial symptoms. Microscopic
AIN is associated primarily with an allergic reaction to hematuria
medications that occurs within the renal interstitium, possibly
Mild proteinuria
caused by the medication binding to the interstitial protein.
Symptoms tend to develop approximately 2 weeks after admin- Increased pH
istration of medication. Medications commonly associated with Acute Leukocyturia Urine culture
AIN include penicillin, methicillin, ampicillin, cephalosporins, pyelonephritis
rifampin, sulfonamides, NSAIDs, and thiazide diuretics. Other Bacteriuria
drugs that are implicated commonly include indinavir, proton
WBC casts
pump inhibitors, allopurinol, 5-aminosalicylates, diuretics, and
cimetidine.15 Discontinuing the offending medication and Bacterial casts
administering steroids to control the inflammation frequently Microscopic
result in a return to normal renal function. However, support- hematuria
ive renal dialysis may be required to maintain patients until Proteinuria
the inflammation subsides.
Chronic Leukocyturia Urine culture
Urinalysis results include hematuria, possibly macro-
pyelonephritis
scopic; mild to moderate proteinuria; numerous WBCs; and
WBC casts without bacteria. Differential leukocyte staining for Bacteriuria BUN
the presence of increased eosinophils may be useful to confirm WBC casts
the diagnosis.16 Bacterial casts
Laboratory testing and clinical information for the inter-
Granular, waxy, Creatinine
stitial disorders are summarized in Tables 8-5 and 8-6.
broad casts
Hematuria
Technical Tip 8-7. The presence of eosinophil Proteinuria eGFR
casts and eosinophils in the urine sediment are
Acute interstitial Hematuria Urine
characteristic for AIN.
nephritis (AIN) eosinophils
Proteinuria BUN
Leukocyturia Creatinine
Renal Failure WBC casts eGFR
Renal failure exists in both acute and chronic forms.
As discussed previously in conjunction with many
of the disorders, this may be a gradual progression
ARF, in contrast to chronic renal failure, exhibits a sudden
from the original disorder to chronic renal failure or
loss of renal function and frequently is reversible. Primary
ESRD. The progression to ESRD is characterized as follows:
causes of ARF include a sudden decrease in blood flow to the
• Marked decrease in the glomerular filtration rate (less
kidney (prerenal), acute glomerular and tubular disease (renal),
than 25 mL/min)
and renal calculi or tumor obstructions (postrenal). As seen
• Steadily rising serum BUN and creatinine values from the variety of causes (Box 8-1), patients may present with
(azotemia) many different symptoms relating to the particular disorder in-
• Electrolyte imbalance volved; however, general characteristics include a decreased
• Lack of renal concentrating ability, producing an glomerular filtration rate, oliguria, edema, and azotemia.
isosthenuric urine Similar to clinical symptoms, urinalysis findings are varied,
but because they relate to the primary cause of the ARF, they
• Proteinuria
can be diagnostically valuable. For example, the presence of
• Renal glycosuria RTE cells and casts suggests ATN of prerenal origin; RBCs indi-
• Abundance of granular, waxy, and broad casts, often cate glomerular injury; WBC casts, with or without bacteria, in-
referred to as a telescoped urine sediment dicate interstitial infection or inflammation of renal origin; and
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Chapter 8 | Renal Disease 229

Table 8–6 Clinical Information Associated With Interstitial Disorders

Disorder Etiology Clinical Course
Cystitis Ascending bacterial infection of the bladder Acute onset of urinary frequency and burning;
resolved with antibiotics
Acute pyelonephritis Infection of the renal tubules and intersti- Acute onset of urinary frequency, burning, and
tium related to interference of urine flow lower back pain; resolved with antibiotics
to the bladder, reflux of urine from the
bladder, and untreated cystitis
Chronic pyelonephritis Recurrent infection of the renal tubules and Frequently diagnosed in children; requires
interstitium caused by structural abnor- correction of the underlying structural
malities affecting the flow of urine defect
Possible progression to renal failure
Acute interstitial Allergic inflammation of the renal intersti- Acute onset of renal dysfunction often
nephritis (AIN) tium in response to certain medications accompanied by a skin rash
Resolves after discontinuation of medication and
treatment with corticosteroids

postrenal obstruction may show normal- and abnormal-appearing calculi resembling the shape of the renal pelvis and smooth,
urothelial cells possibly associated with malignancy. round bladder stones with diameters of 2 or more inches. Small
calculi may be passed in the urine, subjecting the patient to severe
pain radiating from the lower back to the legs. Larger stones can-
Technical Tip 8-8. Broad casts are often referred to as not be passed and may not be detected until patients develop
renal failure casts. symptoms of urinary obstruction. Lithotripsy, a procedure using
high-energy shock waves, can be used to break stones located in
the upper urinary tract into pieces that then can be passed in the
Renal Lithiasis urine. Also, stones can be removed surgically.
Conditions favoring the formation of renal calculi are sim-
Renal calculi (kidney stones) may form in the calyces and ilar to those favoring formation of urinary crystals, including
pelvis of the kidney, ureters, and bladder. In renal lithiasis, pH, chemical concentration, and urinary stasis. Numerous cor-
the calculi vary in size from barely visible to large, staghorn relation studies between the presence of crystalluria and renal
calculi formation have been conducted with varying results.
The finding of clumps of crystals in freshly voided urine sug-
gests that conditions may be right for calculus formation. How-
Box 8–1 Causes of Acute Renal Failure ever, due to the difference in conditions that affect the urine
within the body and in a specimen container, little importance
Prerenal
can be placed on the role of crystals in predicting calculi for-
Decreased blood pressure/cardiac output mation. Increased crystalluria has been noted during the sum-
Hemorrhage mer months in people known to form renal calculi.17
Burns
Analysis of the chemical composition of renal calculi
plays an important role in patient management. Analysis can
Surgery be performed chemically, but examination using x-ray crys-
Septicemia tallography provides a more comprehensive analysis. Approx-
Renal imately 75% of the renal calculi are composed of calcium
oxalate or calcium phosphate. Magnesium ammonium phos-
Acute glomerulonephritis (AGN)
phate (struvite), uric acid, and cystine are the other primary
Acute tubular necrosis (ATN) calculi constituents. Frequently calcium calculi are associated
Acute pyelonephritis with metabolic calcium and phosphate disorders and, occa-
Acute interstitial nephritis (AIN) sionally, diet. Magnesium ammonium phosphate calculi often
are accompanied by urinary infections involving urea-splitting
Postrenal
bacteria. The urine pH is often higher than 7.0. Uric acid cal-
Renal calculi culi may be associated with increased intake of foods with
Tumors high purine content and with uromodulin-associated kidney
disease. The urine pH is acidic. Most cystine calculi are seen
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230 Part Two | Urinalysis

in conjunction with hereditary disorders of cystine metabo- StatPerarls (Internet). Web site: https://www.ncbi.nlm.nih.gov/
lism (see Chapter 9). Patient management techniques include books/NBK499865/. Published February 15, 2019. Accessed
May 19, 2019.
maintaining the urine at a pH incompatible with crystalliza-
7. Wasserstein, AG: Membranous glomerulonephritis. In
tion of the particular chemicals, maintaining adequate hydra- Jacobson, HR, et al: Principles and Practice of Nephrology.
tion to lower chemical concentration, and suggesting possible BC Decker, Philadelphia, 1991.
dietary restrictions. 8. Kathuria, P: Membranoproliferative Glomerulonephritis.
Urine specimens from patients suspected of passing or Medscape. Web site: https://emedicine.medscape.com/
article/240056-overview. Published June 23, 2016. Accessed
being in the process of passing renal calculi are received in the
May 19, 2019.
laboratory frequently. The presence of microscopic hematuria 9. Donadio, JV: Membranoproliferative glomerulonephritis. In
resulting from irritation to the tissues by the moving calculus Jacobson, HR, et al: Principles and Practice of Nephrology.
is the primary urinalysis finding. BC Decker, Philadelphia, 1991.
10. Bricker, NS, and Kirschenbaum, MA: The Kidney: Diagnosis
and Management. John Wiley, New York, 1984
11. Mansur, A: Minimal-Change Disease. Medscape. Web site:
For additional resources please visit https://emedicine.medscape.com/article/243348-overview#a6.
www.fadavis.com Published December 24, 2018. Accessed May 19, 2019.
12. Sherbotle, JR, and Hayes, JR: Idiopathic nephrotic syndrome:
Minimal change disease and focal segmental glomerulosclerosis.
In Jacobson, HR, et al: Principles and Practice of Nephrology.
References BC Decker, Philadelphia, 1991.
1. Forland, M (ed): Nephrology. Medical Examination Publishing, 13. Rao, STK: Focal Segmental Glomerulosclerosis. Medscape.
New York, 1983. Web site: https://emedicine.medscape.com/article/245915-
2. Couser, WG: Rapidly progressive glomerulonephritis. In overview#a5. Published October 2, 2018. Accessed May 19,
Jacobson, HR, et al: Principles and Practice of Nephrology. 2019.
BC Decker, Philadelphia, 1991. 14. Bleyer, AJ, Zivna, M, and Kmoch, S: Uromodulin-associated
3. Tracy, CL: Granulomatosis with Polyangiitis (Wegener Granulo- kidney disease. Nephron Clin Prac 118(1):c31–c36, 2011.
matosis). Medscape. Web site: https://emedicine.medscape.com/ 15. Finnigan, NA, Bashir, K: Allergic Interstitial Nephritis (AIN).
article/332622-overview. Published January 4, 2019. Accessed National Center for Biotechnology Information (NCBI). Stat-
May 19, 2019. Pearls [Internet]. Bookshelf ID: 4882323 PMID: 29493948.
4. Kallenberg, CG, Mulder, AH, and Tervaert, JW: Antineutrophil Web site: https://ncbi.nlm.nih.gov/books/NBK482323/.
cytoplasmic autoantibodies: A still-growing class of autoantibod- Accessed May 20, 2019.
ies in inflammatory disorders. Am J Med 93(6):675–682, 1992. 16. Bennett, WM, Elzinga, LW, and Porter, GA: Tubulointerstitial
5. Frasier, LL, and Hoag, KA: Differential diagnosis of Wegener’s disease and toxic nephropathy. In Brenner, BM, and Rector, FC:
granulomatosis from other small vessel vasculitides. LabMed The Kidney: Physiology and Pathophysiology. WB Saunders,
38(7):437–439, 2007. Philadelphia, 1991.
6. Raza, A, and Aggarwal, S: Membranous Glomerulonephritis. 17. Hallson, PC, and Rose, GA: Seasonal variations in urinary
National Center for Biotechnology Information. NCBI Resources. crystals. Br J Urol 49(4):277–284, 1977.

Study Questions
1. Most glomerular disorders are caused by: 3. Occasional episodes of macroscopic hematuria over peri-
A. Sudden drops in blood pressure ods of 20 or more years are seen in patients with:
B. Immunologic disorders A. Crescentic glomerulonephritis
C. Exposure to toxic substances B. IgA nephropathy
D. Bacterial infections C. Nephrotic syndrome
D. GPA
2. Dysmorphic RBC casts would be a significant finding
with all of the following except: 4. Antiglomerular basement membrane antibody is seen
A. Goodpasture syndrome with:
B. AGN A. GPA
C. Chronic pyelonephritis B. IgA nephropathy
D. Henoch-Schönlein purpura C. Goodpasture syndrome
D. Diabetic nephropathy
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Chapter 8 | Renal Disease 231

5. ANCA is diagnostic for: 13. The only protein produced by the kidney is:
A. IgA nephropathy A. Albumin
B. GPA B. Uromodulin
C. Henoch-Schönlein purpura C. Uroprotein
D. Goodpasture syndrome D. Globulin
6. Respiratory and renal symptoms are associated with all 14. The presence of RTE cells and casts is an indication of:
of the following except: A. AIN
A. IgA nephropathy B. CGN
B. GPA C. MCD
C. Henoch-Schönlein purpura D. ATN
D. Goodpasture syndrome
15. Differentiation between cystitis and pyelonephritis is
7. The presence of fatty casts is associated with all of the aided by the presence of:
following except: A. WBC casts
A. Nephrotic syndrome B. RBC casts
B. FSGS C. Bacteria
C. Nephrogenic DI D. Granular casts
D. MCD
16. The presence of WBCs and WBC casts with no bacteria
8. The highest levels of proteinuria are seen with: is indicative of:
A. Alport syndrome A. Chronic pyelonephritis
B. Diabetic nephropathy B. ATN
C. IgA nephropathy C. AIN
D. NS D. Both B and C
9. Ischemia frequently produces: 17. ESRD is characterized by all of the following except:
A. Acute renal tubular necrosis A. Hypersthenuria
B. MCD B. Isosthenuria
C. Renal glycosuria C. Azotemia
D. Goodpasture syndrome D. Electrolyte imbalance
10. A disorder associated with polyuria and low specific 18. Prerenal acute renal failure could be caused by:
gravity is: A. Massive hemorrhage
A. Renal glucosuria B. ATN
B. MCD C. AIN
C. Nephrogenic DI D. Malignant tumors
D. FSGS
19. The most common component of renal calculi is:
11. An inherited disorder producing a generalized defect in A. Calcium oxalate
tubular reabsorption is:
B. Magnesium ammonium phosphate
A. Alport syndrome
C. Cystine
B. AIN
D. Uric acid
C. Fanconi syndrome
20. Urinalysis on a patient with severe back pain being
D. Renal glycosuria
evaluated for renal calculi would be most beneficial if it
12. A teenage boy who develops gout in his big toe and has showed:
a high serum uric acid should be monitored for: A. Heavy proteinuria
A. Fanconi syndrome B. Low specific gravity
B. Renal calculi C. Uric acid crystals
C. Uromodulin-associated kidney disease D. Microscopic hematuria
D. Chronic interstitial nephritis
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232 Part Two | Urinalysis

Case Studies and Clinical Situations

1. A 14-year-old boy who has recently recovered from a sore Microscopic:
throat develops edema and hematuria. Significant labora- >100 RBCs/hpf 2–4 hyaline casts/lpf
tory results include a BUN of 30 mg/dL (normal 8 to
8–10 WBCs/hpf 1–5 granular casts/lpf
23 mg/dL) and a positive group A streptococcal antibody
test. Results of a urinalysis are as follows: 0–2 waxy casts/lpf 0–2 broad waxy
Color: Red Ketones: Negative a. What specific disease do the patient’s laboratory
results and history suggest?
Clarity: Cloudy Blood: Large
b. Which laboratory result is most helpful in diagnosing
Sp. gravity: 1.020 Bilirubin: Negative
this disease?
pH: 5.0 Urobilinogen: Normal
c. What additional diagnosis does his current condition
Protein: 3+ Nitrite: Negative suggest?
Glucose: Negative Leukocyte: Trace d. What is the significance of the positive result for urine
Microscopic: glucose?
100 RBCs/hpf—many dysmorphic forms e. Is the specific gravity significant? Why or why not?
5–8 WBCs/hpf f. What is the significance of the waxy casts?
0–2 granular casts/lpf 3. A 45-year-old woman is recovering from injuries received
0–1 RBC casts/lpf in an automobile accident that resulted in her being taken
a. What disorder do these results and history indicate? to the emergency department with severe hypotension.
She develops massive edema. Significant laboratory re-
b. What specific characteristic was present in the
sults include a BUN of 30 mg/dL (normal 8 to 23 mg/dL),
organism causing the sore throat?
cholesterol of 400 mg/dL (normal 150 to 240 mg/dL),
c. What is the significance of the dysmorphic RBCs? triglycerides of 840 mg/dL (normal 10 to 190 mg/dL),
d. Are the WBCs significant? Why or why not? serum protein of 4.5 mg/dL (normal 6.0 to 7.8 mg/dL),
e. What is the expected prognosis of this patient? albumin of 2.0 mg/dL (normal 3.2 to 4.5 mg/dL), and a
total urine protein of 3.8 g/d (normal 100 mg/d). Urinaly-
f. If these urinalysis results were seen in a 5-year-old boy
sis results are as follows:
who has developed a red, patchy rash after recovery
from a respiratory infection, what disorder would you Color: Yellow Ketones: Negative
suspect? Clarity: Cloudy Blood: Moderate
2. A seriously ill 40-year-old man has a history of several Sp. gravity: 1.015 Bilirubin: Negative
episodes of macroscopic hematuria in the past 20 years. pH: 6.0 Urobilinogen: Normal
The episodes were associated with exercise or stress. Protein: 4+ Nitrite: Negative
Until recently, the macroscopic hematuria had reverted
Glucose: Negative Leukocyte: Negative
spontaneously to asymptomatic microscopic hematuria.
Significant laboratory results include a BUN of 80 mg/dL Microscopic:
(normal 8 to 23 mg/dL), serum creatinine of 4.5 mg/dL 15–20 RBCs/hpf Moderate free fat droplets
(normal 0.6 to 1.2 mg/dL), creatinine clearance of 0–5 WBCs/hpf Moderate cholesterol
20 mL/min (normal 107 to 139 mL/min), serum calcium crystals
0–2 granular casts/lpf
of 8.0 mg/dL (normal 9.2 to 11.0 mg/dL), serum phos-
phorus of 6.0 mg/dL (normal 2.3 to 4.7 mg/dL), and an 0–2 fatty casts/lpf
elevated level of serum IgA. Results of a routine urinalysis 0–2 oval fat bodies/hpf
are as follows: a. What renal disorder do these results suggest?
Color: Red Ketones: Negative b. How does the patient’s history relate to this disorder?
Clarity: Slightly cloudy Blood: Large c. What physiological mechanism accounts for the
Sp. gravity: 1.010 Bilirubin: Negative massive proteinuria?
pH: 6.5 Urobilinogen: Normal d. What is the relationship of the proteinuria to the edema?
Protein: 300 mg/dL Nitrite: Negative e. What mechanism produces the oval fat bodies?
Glucose: 250 mg/dL Leukocyte: Trace
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Chapter 8 | Renal Disease 233

4. A routinely active 4-year-old boy becomes increasingly 6. A 25-year-old pregnant woman comes to the outpatient
less active after receiving several preschool immuniza- clinic with symptoms of lower back pain, urinary frequency,
tions. His pediatrician observes noticeable puffiness and a burning sensation when voiding. Her pregnancy has
around the eyes. A blood test shows normal BUN and cre- been normal up to this time. She is given a sterile container
atinine results and markedly decreased total protein and and asked to collect a midstream clean-catch urine speci-
albumin values. Urinalysis results are as follows: men. Routine urinalysis results are as follows:
Color: Yellow Ketones: Negative Color: Pale yellow Ketones: Negative
Clarity: Hazy Blood: Small Clarity: Hazy Blood: Small
Sp. gravity: 1.020 Bilirubin: Negative Sp. gravity: 1.005 Bilirubin: Negative
pH: 6.5 Urobilinogen: Normal pH: 8.0 Urobilinogen: Normal
Protein: 4+ Nitrite: Negative Protein: Trace Nitrite: Positive
Glucose: Negative Leukocyte: Negative Glucose: Negative Leukocyte: 2+
Microscopic: Microscopic:
10–15 RBCs/hpf 0–1 hyaline casts/lpf 6–10 RBCs/hpf Heavy bacteria
0–4 WBCs/hpf 0–2 granular casts/lpf 40–50 WBCs/hpf Moderate squamous
Moderate fat droplets 0–1 oval fat bodies/hpf epithelial cells
a. What disorder do the patient history, physical a. What is the most probable diagnosis for this patient?
appearance, and laboratory results suggest? b. What is the correlation between the color and the
b. What other renal disorders produce similar urinalysis specific gravity?
results? c. What is the significance of the blood and protein tests?
c. What is the expected prognosis for this patient? d. Is this specimen suitable for the appearance of glitter
cells? Explain your answer.
5. A 32-year-old construction worker experiences respira-
tory difficulty followed by the appearance of blood- e. What other population is at a high risk for developing
streaked sputum. He delays visiting a physician until this condition?
symptoms of extreme fatigue and red urine are present. f. What disorder might develop if this disorder is not
A chest radiograph shows pulmonary infiltration, and treated?
sputum culture is negative for pathogens. Blood test re-
7. A 10-year-old patient with a history of recurrent UTIs is
sults indicate anemia, increased BUN and creatinine, and
admitted to the hospital for diagnostic tests. Initial urinal-
the presence of antiglomerular basement membrane anti-
ysis results are as follows:
body. Urinalysis results are as follows:
Color: Yellow Ketone: Negative
Color: Red Ketones: Negative
Clarity: Cloudy Blood: Small
Clarity: Cloudy Blood: Large
Sp. gravity: 1.025 Bilirubin: Negative
Sp. gravity: 1.015 Bilirubin: Negative
pH: 8.0 Urobilinogen: Normal
pH: 6.0 Urobilinogen: Normal
Protein: 2+ Nitrite: Positive
Protein: 3+ Nitrite: Negative
Glucose: Negative Leukocyte: 2+
Glucose: Negative Leukocyte: Trace
Microscopic:
Microscopic:
6–10 RBCs/hpf 0–2 WBC casts/lpf
100 RBCs/hpf 0–3 hyaline casts/lpf
Many bacteria
10–15 WBCs/hpf 0–3 granular casts/lpf
>100 WBCs/hpf 0–1 bacterial casts/lpf with
0–2 RBCs casts/lpf
clumps
a. What disorder do the laboratory results suggest?
A repeat urinalysis a day later has the following results:
b. How is this disorder affecting the glomerulus?
Color: Yellow Ketones: Negative
c. If the antiglomerular membrane antibody test is nega-
Clarity: Cloudy Blood: Small
tive, what disorder might be considered?
Sp. gravity: >1.035 Bilirubin: Negative
d. What is the diagnostic test for this disorder?
pH: 7.5 Urobilinogen: Normal
e. By what mechanism does this disorder affect the
glomerulus? Protein: 2+ Nitrite: Positive
Glucose: Negative Leukocyte: 2+
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234 Part Two | Urinalysis

Microscopic: Sp. gravity: 1.010 Bilirubin: Negative

6–10 RBCs/hpf Many bacteria pH: 7.0 Urobilinogen: Normal
0–2 WBC casts/lpf Protein: 3+ Nitrite: Negative
>100 WBCs/hpf Moderate birefringent, flat Glucose: 2+ Leukocyte: Negative
crystals Microscopic:
0–1 bacterial casts/lpf 50–60 RBCs/hpf 2–3 granular casts/lpf
a. What diagnostic procedure was performed on the 3–6 WBCs/hpf 2–3 RTE cell casts/lpf
patient that could account for the differences in the
3–4 RTE cells/hpf 0–1 waxy casts/lpf
two urinalysis results?
0–1 broad granular
b. Considering the patient’s age and history, what is the
casts/lpf
most probable diagnosis?
a. What diagnosis do the patient’s history and labora-
c. What microscopic constituent is most helpful to this
tory results suggest?
diagnosis?
b. What is the most probable cause of the patient’s
d. What is the most probable cause of this disorder?
disorder? Is this considered to be of prerenal, renal,
e. How can the presence of the bacterial cast be or postrenal origin?
confirmed?
c. What is the significance of the specific gravity
f. What is the most probable source of the crystals result?
present in the sediment?
d. What is the significance of the RTE cells?
g. Without surgical intervention, what is the patient’s
e. State two possible reasons for the presence of the
prognosis?
broad casts.
8. A 35-year-old patient being treated for a sinus infection
10. A 40-year-old man develops severe back and abdomi-
with methicillin develops fever, a skin rash, and edema.
nal pain after dinner. The pain subsides during the
Urinalysis results are as follows:
night but recurs in the morning, and he visits his fam-
Color: Dark yellow Ketones: Negative ily physician. Results of a complete blood count and an
Clarity: Cloudy Blood: Moderate amylase test are normal. Results of a routine urinalysis
Sp. gravity: 1.015 Bilirubin: Negative are as follows:
pH: 6.0 Urobilinogen: Normal Color: Dark yellow Ketones: Negative
Protein: 3+ Nitrite: Negative Clarity: Hazy Blood: Moderate
Glucose: Negative Leukocyte: 2+ Sp. gravity: 1.030 Bilirubin: Negative
Microscopic: pH: 5.0 Urobilinogen: Normal
20–30 RBCs/hpf 1–2 WBC casts/lpf Protein: Trace Nitrite: Negative
>100 WBCs/hpf 1–2 granular casts/lpf Glucose: Negative Leukocytes: Negative
Microscopic
After receiving the urinalysis report, the physician orders
a test for urinary eosinophils. The urinary eosinophil 15–20 RBCs/hpf
result is 10%. 0–2 WBCs/hpf
a. Is the urinary eosinophil result normal or abnormal? Few squamous epithelial cells
b. What is the probable diagnosis for this patient? a. What condition could these urinalysis results and
c. Discuss the significance of the increased WBCs and the patient’s symptoms represent?
WBC casts in the absence of bacteria. b. What would account for the crenated RBCs?
d. How can this condition be corrected? c. Is there a correlation between the urine color and
specific gravity and the patient’s symptoms?
9. After surgery to correct a massive hemorrhage, a 55-year-
old patient exhibits oliguria and edema. Blood test results d. Based on the primary substance that causes this
indicate increasing azotemia and electrolyte imbalance. condition, what type of crystals might have been
The glomerular filtration rate is 20 mL/min. Urinalysis present?
results are as follows: e. What changes will the patient be advised to make in
Color: Yellow Ketones: Negative his lifestyle to prevent future occurrences?
Clarity: Cloudy Blood: Moderate
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CHAPTER 9
Urine Screening
for Metabolic Disorders
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
9-1 Explain abnormal accumulation of metabolites in the 9-10 State the significance of increased urinary 5-hydrox-
urine in terms of overflow and renal disorders. yindoleacetic acid.
9-2 Discuss the importance of and the MS/MS testing 9-11 Differentiate between cystinuria and cystinosis, includ-
methods for newborn screening. ing the differences found during analysis of the urine
and the disease processes.
9-3 Name the metabolic defect in phenylketonuria, and
describe the clinical manifestations it produces. 9-12 Describe the components in the heme synthesis path-
way, including the primary specimens used for their
9-4 State three causes of tyrosyluria.
analysis, and explain the cause and clinical significance
9-5 Name the abnormal urinary substance present in of major porphyrias and the appearance of porphyrins
alkaptonuria, and explain how its presence may be in urine.
suspected.
9-13 Define mucopolysaccharides, and name three syndromes
9-6 Discuss the appearance and significance of urine that in which they are involved.
contains melanin.
9-14 State the significance of increased uric acid crystals in
9-7 Describe a basic laboratory observation that has newborns’ urine.
relevance in maple syrup urine disease.
9-15 Explain the reason for performing tests for urinary-
9-8 Discuss the significance of ketonuria in a newborn. reducing substances on all newborns.
9-9 Differentiate between the presence of urinary indican
due to intestinal disorders and Hartnup disease.

KEY TERMS
Alkaptonuria Homocystinuria Melituria
Aminoaciduria Inborn error of metabolism (IEM) Ochronosis
Cystinosis Indicanuria Organic acidemias
Cystinuria Lesch-Nyhan disease Phenylketonuria (PKU)
Galactosuria Maple syrup urine disease (MSUD) Porphyrinuria
Hartnup disease Melanuria Tyrosyluria
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236 Part Two | Urinalysis

Introduction renal type. Overflow disorders result from disruption of a nor-

mal metabolic pathway that causes increased plasma concen-
As discussed in previous chapters, many of the abnormal results trations of the nonmetabolized substances. These chemicals
obtained in the routine urinalysis are related to metabolic rather either override the reabsorption ability of the renal tubules or
than renal disease. Urine as an end product of body metabolism are not normally reabsorbed from the filtrate because they are
may contain additional abnormal substances not tested for by present in only minute amounts. Abnormal accumulations of
routine urinalysis. Often these substances can be detected or the renal type are caused by malfunctions in the tubular reab-
monitored by additional screening tests that also can be per- sorption mechanism, as discussed in Chapter 8.
formed in the urinalysis laboratory. Then positive screening tests The abnormalities encountered most frequently are associ-
can be followed up with more sophisticated procedures per- ated with metabolic disturbances that produce urinary overflow
formed in other sections of the laboratory (Table 9-1). of substances involved in the metabolism of protein, fat, and car-
The need to perform additional tests may be detected by bohydrates. This is understandable when one considers the vast
the observations of alert laboratory personnel when performing number of enzymes used in the metabolic pathways of proteins,
the routine analysis or from observations of abnormal speci- fats, and carbohydrates and the fact that their function is essen-
men color and odor by nursing staff and patients (Table 9-2). tial for complete metabolism. Disruption of enzyme function can
In other instances, clinical symptoms and family histories are be caused by failure to inherit the gene to produce a particular
the determining factors. enzyme, referred to as an inborn error of metabolism (IEM),1
or by organ malfunction from disease or toxic reactions. The ab-
Overflow Versus Renal Disorders normal urinary metabolites that are encountered most frequently
are summarized in Table 9-3, and their appearance is classified
The appearance of abnormal metabolic substances in the urine according to functional defect. Table 9-3 also includes substances
can be caused by a variety of disorders that generally can be and conditions that are covered in this chapter.
grouped into two categories, termed the overflow type and the

Newborn Screening Tests

Table 9–1 Urine Screening Tests for Metabolic
Disorders Many of the urine tests discussed in this chapter traditionally
were performed primarily to detect and monitor newborns for
Test Disorder
IEMs. In recent years, the screening of newborns has increased
Benedict test Alkaptonuria to include more sensitive detection methods and ever-increasing
Ferric chloride test Alkaptonuria levels of state-mandated tests for IEMs. Many states currently
require testing for as many as 31 core conditions and 25 second-
MSUD
ary target conditions.2
Melanuria As discussed later in this chapter, because many of these
PKU disorders cause the buildup of unmetabolized toxic food ingre-
Hoesch test Porphyria dients, it is important that the defects be detected early in life.
Levels of these substances are elevated more rapidly in blood
Nitrosonaphthol test Tyrosinuria
than in urine. Therefore, blood collected by infant heel puncture
Silver nitrate test Alkaptonuria is tested initially. Testing for many substances is now performed
Watson-Schwartz test Porphyria using tandem mass spectrophotometry (MS/MS). MS/MS is ca-
pable of screening the infant blood specimen for specific sub-
stances associated with particular IEMs. Figure 9-1 shows the
standard form collected for testing using MS/MS. Methods for
Table 9–2 Abnormal Metabolic Constituents or specific gene testing also are being developed rapidly.
Conditions Detected in the Routine
Urinalysis
Color Odor Crystals
Amino Acid Disorders
Homogentisic Phenylketonuria Cystine The amino acid disorders with urinary screening tests include
acid phenylketonuria (PKU), tyrosyluria, melanuria, alkaptonuria,
maple syrup urine disease (MSUD), organic acidemias, indi-
Melanin Maple syrup urine Leucine
canuria, cystinuria, and cystinosis.
disease
Indican Isovaleric acidemia Tyrosine Phenylalanine-Tyrosine Disorders
Porphyrins Cystinuria Lesch-Nyhan Major inherited disorders include PKU, tyrosyluria, and
Cystinosis disease alkaptonuria. Metabolic defects cause overproduction of
Homocystinuria melanin. The relationship of these varied disorders is illus-
trated in Figure 9-2.
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Chapter 9 | Urine Screening for Metabolic Disorders 237

Table 9–3 Major Disorders of Protein and Carbohydrate Metabolism Associated

With Abnormal Urinary Constituents, Classified by Functional Defect
Overflow Inherited Metabolic Renal
Phenylketonuria Infantile tyrosinemia Hartnup disease
Tyrosinemia Melanuria Cystinuria
Alkaptonuria Indicanuria
Maple syrup urine disease 5-Hydroxyindoleacetic acid
Organic acidemias Porphyria
Cystinosis
Porphyria
Mucopolysaccharidoses
Galactosemia
Lesch-Nyhan disease

screening tests are available for early detection of the abnor-

mality, and all states have laws that require the screening of
newborns for PKU.2 Once discovered, dietary changes that
eliminate phenylalanine, a major constituent of milk, from the
infant’s diet can prevent excessive buildup of serum phenylala-
nine, thereby avoiding damage to the child’s mental capabilities.
Many products that contain large amounts of phenylalanine,
such as aspartame (added to medications, diet foods, and diet
sodas), now features warning labels for people with PKU.
The initial screening for PKU does not come under the
auspices of the urinalysis laboratory because increased blood
levels of phenylalanine must, of course, occur before urinary
excretion of phenylpyruvic acid, which may take 2 to 6 weeks.
State laws require that blood be collected between 24 and
Figure 9–1 Specimen collection form for MS/MS newborn screen-
48 hours after birth and before the newborn leaves the hospi-
ing test. (From Strasinger, SK, and DiLorenzo, MA: The Phlebotomy
tal. The increasing tendency to release newborns from the hos-
Textbook, 4th ed, FA Davis.)
pital as early as 24 hours after birth has caused concern about
the ability to detect increased phenylalanine levels at that early
Phenylketonuria stage. Studies have shown that in many cases phenylalanine
The most well-known of the aminoacidurias, phenylke- can be detected as early as 4 hours after birth, and if the cutoff
tonuria (PKU) is estimated to occur in 1 of every 10,000 to level for normal results is lowered from 4 mg/dL to 2 mg/dL,
20,000 births and, if undetected, results in severe intellectual then the presence of PKU should be detected. Tests may need
disability. It was first identified in Norway by Ivan Følling in to be repeated during an early visit to the pediatrician. More
1934, when a mother with other children who were intellectually girls than boys escape detection of PKU during early tests be-
delayed reported a peculiar mousy odor to her child’s urine and cause of slower rises in levels of blood phenylalanine.1
sweat. Analysis of the urine showed increased amounts of the Urine testing using ferric chloride may be used as a follow-
keto acids, including phenylpyruvate. As shown in Figure 9-2, up test to ensure proper dietary control in cases diagnosed pre-
this occurs when the normal conversion of phenylalanine to viously and as a means of monitoring the dietary intake of
tyrosine is disrupted. Interruption of the pathway also produces pregnant women known to lack phenylalanine hydroxylase.
children with fair complexions and lighter hair and eyes—even Urine tests to screen for phenylpyruvic acid are based on
in dark-skinned families—due to the decreased production of the ferric chloride reaction performed by tube test. The addi-
tyrosine and its pigmentation metabolite melanin. Seizures, tion of ferric chloride to urine containing phenylpyruvic acid
hyperactivity, developmental delay, and psychiatric disturbances produces a permanent blue-green color (see Procedure 9-1).
also may be present in some patients.
Tyrosyluria
PKU is caused by failure to inherit the gene to produce the
enzyme phenylalanine hydroxylase. The gene is inherited as Tyrosyluria, the accumulation of excess tyrosine in the plasma
an autosomal recessive trait with no noticeable characteristics (tyrosinemia) producing urinary overflow, may be due to several
or defects exhibited by heterozygous carriers. Fortunately, causes and is not well categorized. As seen in Table 9-2, disorders
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238 Part Two | Urinalysis

Normal metabolism

Metabolic disorders Phenylalanine Enzymes

Phenylketonuria Phenylalanine hydroxylase

Tyrosine
Phenylpyruvic acid
Tyrosine Melanin
aminotransferase Thyroxine Normal byproducts
Tyrosinemia Epinephrine
type 2
p-Hydroxyphenylpyruvic
acid
Tyrosyluria
p-hydroxyphenylpyruvic acid p-hydroxyphenylpyruvate
p-hydroxyphenyllactic acid oxidase

Tyrosinemia Homogentisic acid

type 3

Homogentisic acid
Alkaptonuria oxidase

Maleylacetoacetic
acid
Homogentisic
Maleylacetoacetic
acid
acid isomerase

Tyrosinemia type 1b Fumarylacetoacetic

acid

Tyrosinemia Fumarylacetoacetic
type 1 acid hydrolase

Tyrosinemia type 1a Fumaric acid and

acetoacetic acid

Figure 9–2 Phenylalanine and tyrosine metabolic pathway, including the normal pathway (blue), enzymes (yellow), and disorders caused by
failure to inherit particular enzymes (green).

and leucine crystals may be observed during microscopic exam-

PROCEDURE 9-1
ination of the urine sediment.
Ferric Chloride Tube Test Hereditary disorders in which enzymes required in the
1. Place 1 mL of urine in a tube. metabolic pathway are not produced present serious and often
fatal conditions that result in both liver and renal tubular dis-
2. Slowly add five drops of 10% ferric chloride. ease, producing a generalized aminoaciduria. Based on the en-
3. Observe color for a permanent blue-green color. zymes affected, the hereditary disorders can be classified into
three types, all producing tyrosinemia and tyrosyluria.
As shown in Figure 9-2, type 1 is caused by the deficiency
of the enzyme fumarylacetoacetate hydrolase (FAH). Type 1
of tyrosine metabolism may result from either inherited or meta- produces a generalized renal tubular disorder and progressive
bolic defects. Also, because two reactions are directly involved in liver failure in infants soon after birth. Tyrosemia type 1 is
the metabolism of tyrosine, the urine may contain excess tyrosine diagnosed by the detection of tyrosine and succinylacetone in
or its degradation products, p-hydroxyphenylpyruvic acid and the urine and blood. Tyrosine and succinylacetone can be
p-hydroxyphenyllactic acid. measured on the newborn screening by MS/MS. Molecular
Most frequently seen is a transitory tyrosinemia in prema- genetic testing for FAH gene mutations is available to confirm
ture infants, which is caused by underdevelopment of the liver the diagnosis.3
function required to produce the enzymes necessary to complete Type 2 tyrosinemia is caused by lack of the enzyme tyro-
the tyrosine metabolism. sine aminotransferase. People with type 2 tyrosinemia develop
Acquired severe liver disease also produces tyrosyluria re- corneal erosions and lesions on the palms, fingers, and soles
sembling that of the transitory newborn variety and, of course, is of the feet, believed to be caused by crystallization of tyrosine
a more serious condition. In both instances, rarely seen tyrosine in the cells. Diagnosis of type 2 tyrosinemia is made by testing
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Chapter 9 | Urine Screening for Metabolic Disorders 239

for plasma tyrosine and molecular diagnostic tests to determine darkened after becoming alkaline from standing at room tem-
genetic markers. Patients also may have elevated 4-hydroxy perature. Therefore, the term “alkali lover,” or alkaptonuria,
phenylpyruvic acid in urine. was adopted. This metabolic defect is actually the third major
Type 3 tyrosinemia is caused by lack of the enzyme defect in the phenylalanine–tyrosine pathway and occurs from
p-hydroxyphenylpyruvic acid dioxygenase. This can result in failure to inherit the gene to produce the enzyme homogentisic
intellectual disability, seizures, and intermittent ataxia if dietary acid oxidase. Without this enzyme, the phenylalanine–tyrosine
restrictions of phenylalanine and tyrosine are not implemented. pathway cannot proceed to completion, and homogentisic acid
Type 3 tyrosinemia is diagnosed by the presence of elevated accumulates in the blood, tissues, and urine. This condition
levels of plasma tyrosine and the presence of the urine metabo- usually does not manifest itself clinically in early childhood, but
lites 4-hydroxyphenylpyruvic acid,4-hydroxyphenyllactic, and observations of brown-stained or black-stained cloth diapers
4-hydroxyphenylacetic acid. Genetic studies include testing and reddish-stained disposable diapers have been reported.5
for mutations in the gene for 4-hydroxyphenylpyruvic acid In later life, the brown pigment becomes deposited in the
dioxygenase.4 body tissues, particularly noticeable in the ears (ochronosis).
Screening tests using MS/MS and molecular diagnostic Deposits in connective tissue, especially the cartilage, eventu-
tests are available for tyrosinemia types 1, 2, and 3. See Proce- ally lead to arthritis. A high percentage of people with alkap-
dure 9-2 for a qualitative urine screening test for tyrosyluria tonuria develop liver and cardiac disorders.6
using nitroso-naphthol. Homogentisic acid reacts in several of the screening tests
historically used for metabolic disorders, including the ferric
Melanuria chloride test, in which a transient deep blue color is produced
The previous discussion focused on the major phenylalanine– in the tube test. A yellow precipitate is produced in the Clinitest,
tyrosine metabolic pathway illustrated in Figure 9-2; however, indicating the presence of a reducing substance. Another screen-
as also shown in Figure 9-2 and is the case with many amino ing test for urinary homogentisic acid is to add alkali to freshly
acids, a second metabolic pathway also exists for tyrosine. This voided urine and observe for darkening of the color; however,
pathway is responsible for the production of melanin, thyroxine, large amounts of ascorbic acid interfere with this reaction.
epinephrine, protein, and tyrosine sulfate. Of these substances, Gas chromatography–mass spectrometry (GC-MS) proce-
the major concern of the urinalysis laboratory is melanin, the dures are available for quantitating homogentisic acid. Molecular
pigment responsible for the dark color of hair, skin, and eyes. genetic testing for the homogentisic acid oxidase gene is available
Deficient production of melanin results in albinism. on a clinical basis.7 The ferric chloride test and the silver nitrate
Increased urinary melanin (melanuria) darkens the urine. test for homogentisic acid, provided in Procedure 9-3, are
The darkening appears after the urine is exposed to air. Elevated screening tests for homogentisic acid.
urinary melanin is a serious finding that indicates proliferation Treatment for alkaptonuria is aimed at the specific symp-
of the normal melanin-producing cells (melanocytes), produc- toms that are present in each patient. Vitamin C has been used
ing a malignant melanoma. These tumors secrete a colorless to treat alkaptonuria because it hinders the accumulation and
precursor of melanin, 5,6-dihydroxyindole, which oxidizes to deposition of homogentisic acid.7
melanogen and then to melanin, producing the characteristic
dark urine.
Technical Tip 9-1. Careful observation during the
Alkaptonuria physical component of urinalysis is helpful in cases of
alkaptonuria. The appearance of black urine from a pa-
Alkaptonuria was one of the six original IEMs described by tient of any age should be reported to a supervisor.
Garrod in 1902. The name alkaptonuria was derived from
the observation that urine from patients with this condition
Technical Tip 9-2. Melanin may react with sodium
nitroferricyanide (acetone reagent strip), producing
a red color.
PROCEDURE 9-2
Nitroso-Naphthol Test for Tyrosine Technical Tip 9-3. It is important to differentiate be-
1. Place five drops of urine in a tube. tween the presence of homogentisic acid and melanin
when urine is observed to have turned black upon
2. Add 1 mL of 2.63N nitric acid. standing.
3. Add one drop of 21.5% sodium nitrite.
4. Add 0.1 mL 1-nitroso-2-napthol.
5. Mix. Branched-Chain Amino Acid Disorders
6. Wait 5 minutes. The branched-chain amino acids differ from other amino acids
7. Observe for an orange-red color, indicating tyrosine by having a methyl group that branches from the main
metabolites. aliphatic carbon chain (Fig. 9-3 A and B). Two major groups
of disorders are associated with errors in the metabolism of the
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240 Part Two | Urinalysis

urinalysis, notifying the physician about this unusual finding

PROCEDURE 9-3
can prevent progression to severe intellectual disability and
Homogentisic Acid Test even death. Studies have shown that if MSUD is detected by
1. Place 4 mL of 3% silver nitrate in a tube. the 11th day, dietary regulation and careful monitoring of uri-
nary concentrations of keto acid can control the disorder.8 The
2. Add 0.5 mL of urine. 2,4-dinitrophenylhydrazine (DNPH) urine screening test for
3. Mix. MSUD is provided in Procedure 9-4. Newborn screening for
4. Add 10% NH4OH by drops. MSUD is performed with MS/MS. Plasma amino acids (PAA) test-
5. Observe for black color. ing should be performed to assess for elevated levels of branched-
chain amino acids (BCAAs) and to detect alloisoleucine. A
plasma alloisoleucine level greater than 5 µmol/L is the most
specific and sensitive marker for MSUD.9 Molecular testing for
branched-chain amino acids. In one group, accumulation of the genes reported in patients with MSUD confirm the diag-
one or more of the early amino acid degradation products oc- nosis, provide information about prognosis, and provide
curs, as is seen in MSUD. Disorders in the other group are information for genetic counseling.9
termed organic acidemias and result in accumulation of organic Treatment of MSUD is the dietary restriction of foods that
acids produced further down in the amino acid metabolic contain BCAAs. Nutritional therapies are prescribed to reduce
pathway. toxic metabolites while promoting normal growth.
A significant laboratory finding in branched-chain amino
acid disorders is the presence of ketonuria in a newborn.

Maple Syrup Urine Disease Technical Tip 9-4. As discussed in Chapter 5, as part
of the physical examination of urine, the sweet odor of
Although maple syrup urine disease (MSUD) is rare, a brief urine is a clue to MSUD.
discussion is included in this chapter because the urinalysis
laboratory can provide valuable information for the essential
early detection of this disease. MSUD is also included in new-
Organic Acidemias
born screening profiles using MS/MS.
MSUD is caused by an IEM, inherited as an autosomal re- Generalized symptoms of the organic acidemias include early
cessive trait. The amino acids involved are leucine, isoleucine, severe illness, often with vomiting accompanied by metabolic
and valine. The metabolic pathway begins normally, with acidosis; hypoglycemia; ketonuria; and increased serum am-
the transamination of the three amino acids in the liver to the monia.10 The three disorders encountered most frequently are
keto acids (␣-ketoisovaleric, ␣-ketoisocaproic, and ␣-keto- isovaleric, propionic, and methylmalonic acidemia.
β-methylvaleric). Failure to inherit the gene for the enzyme Isovaleric acidemia may be suspected when urine speci-
necessary to produce oxidative decarboxylation of these keto mens, and sometimes even the patient, possess a characteristic
acids results in their accumulation in the blood and urine.1 odor of “sweaty feet.” This odor is caused by the accumulation
Newborns with MSUD begin to exhibit clinical symptoms of isovalerylglycine due to a deficiency of isovaleryl coenzyme
associated with failure to thrive after approximately l week. A in the leucine pathway.
The presence of the disease may be suspected from these clin- Propionic and methylmalonic acidemias result from errors
ical symptoms; however, many other conditions have similar in the metabolic pathway converting isoleucine, valine, threo-
symptoms. Personnel in the urinalysis laboratory, nurses, or nine, and methionine to succinyl coenzyme A. Propionic acid is
parents may detect the disease by noticing a urine specimen the immediate precursor to methylmalonic acid in this pathway.
that produces a strong odor resembling maple syrup that is The presence of isovaleric, propionic, and methylmalonic
caused by the rapid accumulation of keto acids in the urine. acidemias can be detected by newborn screening programs
Even though a report of urine odor is not a part of the routine using MS/MS.

Alpha amino group Alpha amino group Carboxyl group

H H O H H O

N C C N C C
Figure 9–3 ␣-Alpha amino acid and
H R OH H CH OH branched chain amino acid struc-
tures. A. Structure of an ␣-amino
R group H 3C CH3 acid. B. Structure of the branched
(variant)
A B Leucine group chain amino acid leucine.
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Chapter 9 | Urine Screening for Metabolic Disorders 241

Except in cases of Hartnup disease, correction of the un-

PROCEDURE 9-4
derlying intestinal disorder returns urinary indican levels to
2,4-DNPH Test for MSUD normal. The inherited defect in Hartnup disease affects not
1. Place 1 mL of urine in a tube. only the intestinal reabsorption of tryptophan but also the renal
tubular reabsorption of other amino acids, resulting in a gen-
2. Add 10 drops of 0.2% 2,4-DNPH in 2N HCl. eralized aminoaciduria (Fanconi syndrome). The defective
3. Wait 10 minutes. renal transport of amino acids does not appear to affect other
4. Observe for yellow turbidity or precipitate. renal tubular functions. Therefore, with proper dietary supple-
ments, including niacin, people with Hartnup disease have a
good prognosis.11

5-Hydroxyindoleacetic Acid
Tryptophan Disorders
As shown in Figure 9-4, a second metabolic pathway of trypto-
The major concern of the urinalysis laboratory in the metabo- phan is for the production of serotonin used in the stimulation
lism of tryptophan is the increased urinary excretion of the of smooth muscles. Serotonin is produced from tryptophan by
metabolites indican and 5-hydroxyindoleacetic acid (5-HIAA). the argentaffin cells in the intestine and is carried through the
Figure 9-4 shows a simplified diagram of the metabolic path- body primarily by the platelets. Normally, the body uses most
ways by which these substances are produced. Other metabolic of the serotonin, and only small amounts of its degradation
pathways of tryptophan are not included because they do not product, 5-HIAA, are available for excretion in the urine. How-
relate directly to the urinalysis laboratory. ever, when carcinoid tumors involving the argentaffin (ente-
rochromaffin) cells develop, excess amounts of serotonin are
Indicanuria
produced, resulting in the elevation of urinary 5-HIAA levels.
Under normal conditions, most of the tryptophan that enters Adding nitrous acid and 1-nitroso-2-naphthol to urine
the intestine is either reabsorbed for use by the body in pro- that contains 5-HIAA causes the urine to turn purple to black,
ducing protein or is converted to indole by intestinal bacteria depending on the amount of 5-HIAA present (Procedure 9-5).
and excreted in the feces. However, in certain intestinal dis- The normal daily excretion of 5-HIAA is 2 to 8 mg, and ex-
orders (including obstruction; the presence of abnormal bac- cretion of greater than 25 mg/24 h can be an indication of
teria; malabsorption syndromes; and Hartnup disease, a rare argentaffin cell tumors.12 The test can be performed on a ran-
inherited disorder) increased amounts of tryptophan are con- dom or first morning specimen; however, false-negative results
verted to indole. Then the excess indole is reabsorbed from can occur based on the specimen concentration and also be-
the intestine into the bloodstream and circulated to the liver, cause 5-HIAA may not be produced at a constant rate through-
where it is converted to indican and then excreted in the urine out the day. If a 24-hour specimen is used, it must be preserved
(indicanuria). Indican excreted in the urine is colorless until with hydrochloric or boric acid. Also, a plasma method using
oxidized to the dye indigo blue by exposure to air. Sometimes high-performance liquid chromatography with fluorescence
early diagnosis of Hartnup disease is made when a parent re- detection is available.
ports a blue staining of the infant’s diapers, referred to as the Patients must be given explicit dietary instructions before col-
“blue diaper syndrome.” lecting any specimen to be tested for 5-HIAA because serotonin

Tryptophan
(normal)
Abnormal Abnormal
Intestinal disorder Malignant tumors
Excess indole 5-hydroxytryptophan
Reabsorbed from intestine
into bloodstream
Liver

Indican Indole Serotonin

Urine

Exposure to air

Indigo blue Feces 5-hydroxyindoleacetic

acid (5-HIAA)
Figure 9–4 Tryptophan metabolism.
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242 Part Two | Urinalysis

Because cystine is much less soluble than the other three

PROCEDURE 9-5
amino acids, laboratory screening determinations are based
Silver Nitroprusside Test on observing cystine crystals in the sediment of concentrated
1. Place 1 mL of urine in a tube. or first morning specimens. Cystine is also the only amino
acid found during the analysis of calculi from these patients.
2. Add two drops concentrated NH4OH. A chemical screening test for urinary cystine can be performed
3. Add 0.5 mL 5% silver nitrate. using cyanide–nitroprusside. Reduction of cystine by sodium
4. Wait 10 minutes. cyanide followed by the addition of nitroprusside produces a
5. Add five drops sodium nitroprusside. red-purple color in a specimen that contains excess cystine
(see Procedure 9-6). False-positive reactions occur in the pres-
6. Observe for purple-black color. ence of ketones and homocysteine, and additional tests may
have to be performed. For patients with positive test results
for cyanide–nitroprusside, ion-exchange chromatographic
is a major constituent of numerous foods, such as avocados, ba- quantitative analysis of a 24-hour collected urine specimen is
nanas, pineapples, plums, kiwi fruit, eggplant, walnuts, and toma- performed.16
toes. These foods should be avoided for 3 days before and during
urine collection.13 Medications, including phenothiazines and
acetanilides, also interfere with results. Patients should be directed Technical Tip 9-6. Positive confirmation of cystine
by their physician to withhold medications for 72 hours before crystals in the urine sediment is made using the
specimen collection. cyanide–nitroprusside test and is diagnostic for
cystinuria.
Cystine Disorders
Two distinct disorders of cystine metabolism exhibit renal man-
ifestations. Confusion as to their relationship existed for many Cystinosis
years after the discovery of renal calculi consisting of cystine. Regarded as a genuine IEM, cystinosis can occur in three vari-
However, now it is known that although both disorders are in- ations, ranging from a severe fatal disorder developed in in-
herited, one is a defect in the renal tubular transport of amino fancy to a benign form appearing in adulthood. The disorder
acids (cystinuria) and the other is an IEM (cystinosis). has two general categories, termed nephropathic and non-
nephropathic. The nephropathic category is subdivided into
infantile and late-onset cystinosis.
Technical Tip 9-5. A noticeable odor of sulfur may A defect in the lysosomal membranes prevents the release
be present in the urine of people with cystine of cystine into the cellular cytoplasm for metabolism. The in-
metabolism disorders. complete metabolism of cystine results in crystalline deposits
of cystine in many areas of the body, including the cornea,
bone marrow, lymph nodes, and internal organs. A major
Cystinuria defect in the renal tubular reabsorption mechanism (Fanconi
syndrome) also occurs. The renal tubules, particularly the
As the name implies, cystinuria is marked by elevated amounts
proximal convoluted tubules, are affected by the cystine de-
of the amino acid cystine in the urine. The presence of increased
posits that interfere with reabsorption. This is not an inherited
urinary cystine is not due to a defect in the metabolism of cys-
disorder of renal tubular reabsorption, as seen in cystinuria.
tine, but rather to the inability of the renal tubules to reabsorb
Continued deposition of cystine, if untreated, results in renal
cystine filtered by the glomerulus. The demonstration that not
failure early in life. In infantile nephropathic cystinosis, there
only cystine but also lysine, arginine, and ornithine are not re-
is rapid progression to renal failure. In late-onset nephropathic
absorbed has ruled out the possibility of an error in metabolism
cystinosis, there is a gradual progression to total renal failure.
even though the condition is inherited.14 The disorder has two
Renal transplants and the use of cystine-depleting medications
modes of inheritance:
• One in which reabsorption of all four amino acids—
cystine, lysine, arginine, and ornithine—is affected
• One in which only cystine and lysine are not reabsorbed
PROCEDURE 9-6
Genetic studies have grouped cystinuria into three types Cyanide–Nitroprusside Test for Cystine
based on the two inherited genes and their heterozygous and 1. Place 3 mL of urine in a tube.
homozygous inheritance. In general, people with any form
2. Add 2 mL sodium cyanide.
of inheritance may form renal calculi, but the calculi are less
common in people in whom only lysine and cystine are af- 3. Wait 10 minutes.
fected.15 Approximately 65% of the people in whom all four 4. Add five drops 5% sodium nitroprusside.
amino acids are affected can be expected to produce calculi 5. Observe for red-purple color.
early in life.
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Chapter 9 | Urine Screening for Metabolic Disorders 243

to prevent the buildup of cystine in other tissues are extending The solubility of the porphyrin compounds varies with
lives. Nonnephropathic cystinosis is relatively benign but may their structure. ALA, porphobilinogen, and uroporphyrin are
cause some ocular disorders. the most soluble and readily appear in the urine. Copropor-
Routine laboratory findings in infantile nephropathic phyrin is less soluble but is found in the urine, whereas proto-
cystinosis include polyuria, generalized aminoaciduria, positive porphyrin is not seen in the urine. Usually fecal analysis has
Clinitest results for reducing substances, and lack of urinary been performed to detect coproporphyrin and protoporphyrin.
concentration. A diagnosis of cystinosis can be confirmed by However, to avoid false-positive interference, bile is a more
molecular genetic testing. acceptable specimen.17 The Centers for Disease Control and
Prevention (CDC) recommends analysis of whole blood for
Homocystinuria the presence of free erythrocyte protoporphyrin (FEP) as a
Defects in the metabolism of the amino acid methionine pro- screening test for lead poisoning.
duce an increase in homocysteine throughout the body. The Disorders of porphyrin metabolism are collectively termed
increased homocysteine can result in failure to thrive, cataracts, porphyrias. They can be inherited or acquired from erythro-
intellectual disability, thromboembolic problems, stroke, and cytic and hepatic malfunctions or exposure to toxic agents.
death. Therefore, screening for homocysteine is included in Common causes of acquired porphyrias include:
newborn screening programs. Newborn screening tests are per- • Lead poisoning
formed using MS/MS testing. Early detection of this disorder • Excessive alcohol intake
(homocystinuria) and a change in diet that excludes foods
• Iron deficiency
high in methionine can alleviate the metabolic problems.
As mentioned, increased urinary homocysteine gives a pos- • Chronic liver disease
itive result with the cyanide–nitroprusside test. Therefore, an • Renal disease
additional screening test for homocystinuria must be performed Inherited porphyrias are much rarer than acquired porphyr-
by following a positive cyanide–nitroprusside test result with a ias. They are caused by failure to inherit the gene that produces
silver–nitroprusside test, in which only homocysteine will react. an enzyme needed in the metabolic pathway. The enzyme defi-
The use of silver nitrate in place of sodium cyanide reduces ciency sites for some of the more common porphyrias are shown
homocysteine to its nitroprusside-reactive form but does not in Figure 9-5. Frequently the inherited porphyrias are classified
reduce cystine. Consequently, a positive reaction in the silver– by their clinical symptoms, either neurological/psychiatric or cu-
nitroprusside test confirms the presence of homocystinuria. taneous photosensitivity or a combination of both (Table 9-4).
Fresh urine should be used when testing for homocysteine An indication of the possible presence of porphyrinuria
(see Procedure 9-7). is the observation of a red or port wine color to the urine after
exposure to air. The port wine urine color is more prevalent in
Porphyrin Disorders the erythropoietic porphyrias, and staining of the teeth also
may occur. As seen with other inherited disorders, the presence
Porphyrins are the intermediate compounds in the production of congenital porphyria is suspected sometimes from a red dis-
of heme. The basic pathway for heme synthesis presented in coloration of an infant’s diapers.
Figure 9-5 shows the three primary porphyrins (uroporphyrin, The two screening tests for porphyrinuria use the Ehrlich
coproporphyrin, and protoporphyrin) and the porphyrin pre- reaction and fluorescence under ultraviolet light in the 550- to
cursors (␣-aminolevulinic acid [ALA] and porphobilinogen). 600-nm range. The Ehrlich reaction can be used only for the de-
As seen in the figure, the synthesis of heme can be blocked at tection of ALA and porphobilinogen. Acetyl acetone must be
a number of stages. Blockage of a pathway reaction results in added to the specimen to convert the ALA to porphobilinogen
the accumulation of the product formed just before the inter- before performing the Ehrlich test. The fluorescent technique
ruption. Then detection and identification of this product in must be used for the other porphyrins. The Ehrlich reaction that
the urine, bile, feces, or blood can aid in determining the cause is included in the Multistix urobilinogen pad now was originally
of a specific disorder. used for all urobilinogen testing. Variations of the Ehrlich reaction
include the Watson-Schwartz test for differentiation between
the presence of urobilinogen and porphobilinogen (see Proce-
PROCEDURE 9-7 dures 9-8 and 9-9) and the Hoesch test (see Procedure 9-10).
These procedures are performed rarely today. High-pressure liq-
Silver–Nitroprusside Test for Homocysteine uid chromatography with fluorometric detection, ion exchange
1. Place 1 mL of urine in a tube. chromatography, and spectrometry methodologies are currently
2. Add two drops concentrated NH4OH. used for the evaluation of urine porphyrins.
Testing for the presence of porphobilinogen is most useful
3. Add 0.5 mL 5% silver nitrate.
when patients exhibit symptoms of an acute attack. This can
4. Wait 10 minutes. be done with the Hoesch test. Increased porphobilinogen is
5. Add five drops sodium nitroprusside. associated with acute intermittent porphyria. A negative test
6. Observe for red-purple color. result is obtained in the presence of lead poisoning unless ALA
is first converted to porphobilinogen.
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244 Part Two | Urinalysis

HISTORICAL NOTE

Vampires in Old Europe

Did you ever wonder how the legend of vampires got Dracula is associated with Transylvania, now Roma-
started? Think about the previous discussion on the symp- nia. Porphyria was a common disease of early royalty in
toms and inheritance of porphyrias. Europe as a result of intermarriage among the royals of
Photosensitivity→Avoidance of sunlight different countries. King George III reportedly died blind,
Pale coloring→Anemia caused by heme disorder deaf, and mad from porphyria.
Port wine-colored urine, red-stained teeth→Drinking
blood
Psychiatric symptoms→Abnormal behavior
Inherited disorder→Familial association, small gene
pool

HEME SYNTHESIS

Glycine and succinyl-CoA

Aminolevulinic acid synthetase

γ-aminolevulinate (ALA)

Aminolevulinic acid synthetase Lead exposure

ALA hydratase deficiency porphyria

Porphobilinogen

Uroporphyrinogen synthase

Acute intermittent porphyria

Hydroxymethylbilane

Uroporphyrinogen cosynthase

Congenital erythropoietic porphyria

Uroporphyrinogen (UPG)

Uroporphyrinogen decarboxylase

Porphyria cutanea tarda

Coproporphyrinogen (CPG)

Coproporphyrinogen oxidase

Hereditary coproporphyria

Protoporphyrinogen

Protoporphyrinogen oxidase

Variegate porphyria

Protoporphyrin IX

Ferrochelatase Lead exposure

Erythropoietic protoporphyria
Figure 9–5 Pathway of heme formation, including normal
Heme pathway (green), enzymes (orange), and stages affected by the
major disorders (yellow) of porphyrin metabolism.
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Chapter 9 | Urine Screening for Metabolic Disorders 245

Table 9–4 Common Porphyrias

Elevated
Porphyria Compound(s) Clinical Symptoms Laboratory Testing
Acute intermittent porphyria ALA Porphobilinogen Neurological/psychiatric Urine/Ehrlich reaction
Porphyria cutanea tarda Uroporphyrin Photosensitivity Urine fluorescence
Congenital erythropoietic Uroporphyrin Photosensitivity Urine or feces fluorescence
porphyria
Coproporphyrin
Variegate porphyria Coproporphyrin Photosensitivity/neurological Bile or feces fluorescence
Erythropoietic protoporphyria Protoporphyrin Photosensitivity Blood FEP
Bile or feces fluorescence
Lead poisoning ALA Neurological Acetoacetic acid + urine/Ehrlich
reaction
Protoporphyrin Blood FEP

PROCEDURE 9-8
Watson-Schwartz Differentiation Test substances, an additional chloroform extraction should be
The classic test for differentiating among urobilinogen, por- performed on the red urine (upper) layer in Tube 1 to ensure
phobilinogen, and Ehrlich-reactive compounds is the Watson- that the red color is not due to excess urobilinogen.
Schwartz test. The test is performed as follows:
1. To each tube, add:
Tube 1 Tube 2
2 mL urine 2 mL urine
2 mL chloroform 2 mL butanol UR B UR B UR B
4 mL sodium acetate 4 mL sodium acetate
2. Observe the color of the layers. C UR C UR C UR
3. Interpretation:
The addition of chloroform to Tube 1 results in the extrac-
Urobilinogen Porphobilinogen Ehrlich-reactive
tion of urobilinogen into the chloroform (bottom) layer, pro-
ducing a colorless urine (top) layer and a red chloroform
layer on the bottom. Neither porphobilinogen nor other
Ehrlich-reactive compounds are soluble in chloroform. Por-
phobilinogen also is not soluble in butanol; however, uro-
bilinogen and other Ehrlich-reactive compounds are
extracted into butanol. Therefore, the addition of butanol to UR B UR UR
Tube 2 produces a red (upper) butanol layer if urobilinogen
or Ehrlich-reactive compounds are present and a colorless
C UR C C
butanol layer if porphobilinogen is present. As shown in the
figure, urobilinogen is soluble in both chloroform and bu-
tanol, and porphobilinogen is soluble in neither. If both uro- Urobilinogen/porphobilinogen Excess urobilinogen
bilinogen and porphobilinogen are present, both layers
appear red. Before reporting the test as positive for both
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246 Part Two | Urinalysis

PROCEDURE 9-9
Watson-Schwartz Test Bottom layer = chloroform; if red = urobilinogen.
1. Label two tubes #1 and #2. If both layers are red, re-extract the urine layer from Tube 1.
2. To each tube add: Place 2 mL of urine layer from Tube 1 and 2 mL chloro-
Tube 1 Tube 2 form and 4 mL sodium acetate into a new tube. Repeat
procedure.
2 mL urine 2 mL urine
Interpretation: Upper layer—urine colorless
2 mL chloroform 2 mL butanol
Bottom layer—chloroform—red = excess urobilinogen
4 mL sodium acetate 4 mL sodium acetate
Both layers red = porphobilinogen and urobilinogen
3. Vigorously shake both tubes.
Tube 2
4. Place in a rack for layers to settle.
Upper layer = butanol If red = urobilinogen or
5. Observe both tubes for red color in the layers.
Ehrlich-reactive compounds
Interpretation: Bottom layer = urine If colorless = porphobilinogen
Tube 1
Upper layer = urine; if colorless = porphobilinogen or
Ehrlich-reactive compounds.

ALA. Identifying specific porphyrins requires additional tech-

PROCEDURE 9-10
niques and the analysis of fecal and erythrocyte samples. In-
Hoesch Screening Test for Porphobilinogen creased protoporphyrin is best measured in whole blood.
The Hoesch test is used for rapid screening or monitoring
of urinary porphobilinogen. Mucopolysaccharide Disorders
1. Two drops of urine are added to approximately 2 mL
of Hoesch reagent (Ehrlich reagent dissolved in Mucopolysaccharides, or glycosaminoglycans (GAGs), are a
6 M HCl). group of large compounds located primarily in the connective
2. Immediately observe the top of the solution for the tissue. They consist of a protein core with numerous polysac-
appearance of a red color that indicates the presence charide branches. Inherited disorders in the metabolism of these
of porphobilinogen. compounds prevent complete breakdown of the polysaccharide
portion of the compounds, resulting in accumulation of the in-
3. Shake the tube. completely metabolized polysaccharide portions in the lyso-
Interpretation: somes of the connective tissue cells and their increased
When the tube is shaken, the red color is seen throughout excretion in the urine. The products found most frequently in
the solution. The test detects approximately 2 mg/dL of the urine are dermatan sulfate, keratan sulfate, and heparan sul-
porphobilinogen, and urobilinogen is inhibited by the fate, with the appearance of a particular substance being deter-
highly acidic pH. High concentrations of methyldopa and mined by the specific metabolic error that was inherited.18
indican, as well as highly pigmented urines, may produce Therefore, identification of the specific enzyme deficiency is
false-positive results. necessary to establish a specific diagnosis.
There are many types of mucopolysaccharidoses (MPSs),
but the best known are Hurler syndrome, Hunter syndrome,
and Sanfilippo syndrome. In both Hurler and Hunter syn-
Fluorescent screening for the other porphyrins uses their dromes, the skeletal structure is abnormal and there is severe
extraction into a mixture of glacial acetic acid and ethyl acetate. intellectual disability; in Hurler syndrome, mucopolysaccha-
Then the solvent layer is examined. Negative reactions have a rides accumulate in the cornea of the eye. Hunter syndrome is
faint blue fluorescence. Positive reactions fluoresce as violet, inherited as sex-linked recessive and is seen rarely in females.
pink, or red, depending on the concentration of porphyrins. Without treatment, both syndromes are usually fatal during
If the presence of interfering substances is suspected, the or- childhood, whereas in Sanfilippo syndrome, the only abnor-
ganic layer can be removed to a separate tube, and 0.5 mL of mality is intellectual disability. Bone marrow transplants and
hydrochloric acid should be added to the tube. Only por- gene replacement therapy are the most promising treatments
phyrins are extracted into the acid layer, which then produces for these disorders.
a bright orange-red fluorescence. The fluorescence method Urinary screening tests for mucopolysaccharides may be re-
does not distinguish among uroporphyrin, coproporphyrin, quested either as part of a routine battery of tests performed on
and protoporphyrin, but it rules out porphobilinogen and all newborns or on infants who exhibit symptoms of intellectual
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Chapter 9 | Urine Screening for Metabolic Disorders 247

disability or failure to thrive. Historically, the screening tests used Technical Tip 9-7. Lesch-Nyhan disease should
most frequently are the acid–albumin and cetyltrimethylammo- not be confused with uromodulin-associated
nium bromide (CTAB) turbidity tests and the metachromatic kidney disease, in which the uric acid crystals
staining spot tests. In both the acid–albumin and the CTAB tests, appear later in life.
a thick, white turbidity forms when these reagents are added to
urine that contains mucopolysaccharides. Turbidity is usually
graded on a scale of 0 to 4 after 30 minutes with acid–albumin
and after 5 minutes with CTAB.19 (See Procedure 9-11.) A diag-
Galactosuria can be caused by a deficiency in any of three
nostic workup for a patient with suspected MPSs includes both
enzymes, galactose-1-phosphate uridyl transferase (GALT), galac-
the quantitative analysis of urine for the total GAGs and the
tokinase (GALK), and UDP-galactose-4-epimerase (GALE).
qualitative liquid chromatography-tandem mass spectrometry
Of these enzymes, it is GALT deficiency that causes the severe,
(LC-MS/MS) analysis of the specific sulfates. Molecular analysis
possibly fatal, symptoms associated with galactosemia. Newborn
is used for confirmation.
screening protocols currently test for the presence of GALT
deficiency. The enzyme is measured in the red blood cells as
Purine Disorders part of the protocol of the newborn heel puncture. As a result,
people with deficiencies in the other two enzymes still may
A disorder of purine metabolism known as Lesch-Nyhan dis- produce galactosuria but have negative newborn screening tests.
ease that is inherited as sex-linked recessive results in massive Galactose kinase deficiency can result in cataracts in adulthood.
excretion of urinary uric acid crystals. Failure to inherit the UDP-galactose-4-epimerase deficiency may be asymptomatic or
gene to produce the enzyme hypoxanthine guanine phospho- produce mild symptoms. Molecular genetic testing can be used
ribosyltransferase is responsible for the accumulation of uric to identify mutations in the GALT gene.
acid throughout the body. Patients suffer from severe motor Other causes of melituria include lactose, fructose, and
defects, intellectual disability, a tendency toward self-destruc- pentose. Lactosuria may be seen during pregnancy and lacta-
tion, gout, and renal calculi. Usually development is normal tion. Fructosuria is associated with parenteral feeding and
for the first 6 to 8 months, and often the first sign is uric acid pentosuria with ingestion of large amounts of fruit. Additional
crystals resembling orange sand in diapers.11 Laboratorians tests, including chromatography, can be used to identify other
should be alert for the presence of increased uric acid crystals nonglucose-reducing substances.
in pediatric urine specimens.

Carbohydrate Disorders For additional resources please visit

www.fadavis.com
The presence of increased urinary sugar (melituria) is due
most frequently to an inherited disorder. In fact, pentosuria
was one of Garrod’s original six IEMs.20 Fortunately, most meli-
turias cause no disturbance to body metabolism. However, References
as discussed in Chapter 6, pediatric urine should be screened 1. Frimpton, GW: Aminoacidurias due to inherited disorders of
routinely for the presence of reducing substances using the metabolism. N Engl J Med 1289:835–901, 1973.
Clinitest procedure. The finding of a positive result on the cop- 2. National newborn screening and global resource center (NNS-
GRC). Web site: http://genes-r-us.uthscsa.edu/sites/genes-r-
per reduction test combined with a negative reagent strip result us/files/nbsdisorders.pdf. 2014. Accessed June 13, 2019.
on the glucose oxidase test is strongly suggestive of a disorder 3. National Organization for Rare Disorders (NORD). Tyrosinemia
of carbohydrate metabolism. Of primary concern is the pres- Type 1. Web site: https://rarediseases.org/rare-diseases/
ence of galactosuria, indicating the inability to properly me- tyrosinemia-type-1. Published October 12, 2005. Accessed
tabolize galactose to glucose. The resulting galactosemia with June 13, 2019.
4. U.S. Department of Health & Human Services. National
toxic intermediate metabolic products results in infant failure Institutes of Health. Tyrosinemia type 3. Web site: https://
to thrive combined with liver disorders, cataracts, and severe rarediseases.info.nih.gov/diseases/10332/tyrosinemia-type-3.
intellectual disability. Early detection of galactosuria followed Published February 3, 2010. Accessed June 13, 2019.
by removal of lactose (a disaccharide containing galactose and 5. Nyhan, WL, and Sakati, NO: Diagnostic Recognition of
glucose) from the diet can prevent these symptoms. Genetic Disease. Lea & Febiger, Philadelphia, 1987.
6. Stanbury, JB: The Metabolic Basis of Inherited Diseases.
McGraw-Hill, New York, 1983.
7. Introne, WJ: Alkaptonuria. National Organization for Rare
PROCEDURE 9-11 Disorders (NORD). Web site: https://rarediseases.rg/rare-
diseases/alkaptonuria/. 2017. Accessed June 13, 2019.
CTAB Test for Mucopolysaccharides 8. Clow, CL, Reade, TH, and Scriver, CR: Outcome of early and
1. Place 5 mL of urine in a tube. long-term management of classical maple syrup urine disease.
Pediatrics 68(6):856–862, 1981.
2. Add 1 mL 5% CTAB in citrate buffer. 9. Defendi, GL: Maple Syrup Urine Disease (MSUD) Workup.
3. Read turbidity in 5 minutes. Medscape. Web site: https://emedicine.medscape.com/article/
946234-workup. Published May 2, 2018. Accessed June 13, 2019.
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10. Goodman, SI: Disorders of organic acid metabolism. In Emery, 16. Biyani, CS: Cystinuria Work-up. Medscape. Web site:
AEH, and Rimoin, DL: Principles and Practice of Medical https://emedicinemedscape.com/article/435678-work-up.
Genetics. Churchill Livingstone, New York, 1990. Published March 19, 2019. Accessed June 13, 2019.
11. Jepson, JB: Hartnup’s disease. In Stanbury, JB, Wyngaarden, JB, 17. Nuttall, KL: Porphyrins and disorders of porphyrin metabo-
and Fredrickson, DS (eds): The Metabolic Basis of Inherited lism. In Burtis, CA, and Ashwood, ER: Tietz Fundamentals of
Diseases. McGraw-Hill, New York, 1983. Clinical Chemistry. WB Saunders, Philadelphia, 1996.
12. Van Leeuwen, AM, Poelhuis-Leth, DJ, and Bladh, ML: Davis’s 18. McKusick, VA, and Neufeld, EF: The mucopolysaccharide
Comprehensive Laboratory and Diagnostics Handbook. 6th ed. storage diseases. In Stanbury, JB, Wyngaarden, JB, and
Philadelphia, FA Davis Company; 2015. Fredrickson, DS (eds): The Metabolic Basis of Inherited
13. American Association for Clinical Chemistry (AACC). Lab Tests Diseases. McGraw-Hill, New York, 1983.
online. 5-HIAA. Web site: https://labtestsonline.org/tests/5-hiaa. 19. Kelly, S: Biochemical Methods in Medical Genetics. Charles C.
Published June 6, 2019. Accessed June 13, 2019. Thomas, Springfield, IL, 1977.
14. Nyhan, WL: Abnormalities in Amino Acid Metabolism in 20. Garrod, AE: Inborn Errors of Metabolism. Henry Froude &
Clinical Medicine. Appleton-Century-Crofts, Norwalk, CT, Hodder & Stoughton, London, 1923.
1984.
15. Dello Strolongo, L, et al: Comparison between SLC3A1 and
SLC7A9 cystinuria patients and carriers: A need for a new
classification. J Am Soc Nephrol 13:2547–2553, 2002.

Study Questions
1. Abnormal urine screening tests categorized as an overflow 6. The least serious form of tyrosylemia is:
disorder include all of the following except: A. Immature liver function
A. Alkaptonuria B. Type 1
B. Galactosemia C. Type 2
C. Melanuria D. Type 3
D. Cystinuria
7. An overflow disorder of the phenylalanine–tyrosine
2. All states require newborn screening for PKU for early: pathway that would produce a positive reaction with the
A. Modifications of the diet reagent strip test for ketones is:
B. Administration of antibiotics A. Alkaptonuria
C. Detection of diabetes B. Melanuria
D. Initiation of gene therapy C. MSUD
D. Tyrosyluria
3. All of the following disorders can be detected by newborn
screening except: 8. An overflow disorder that could produce a false-positive
A. Tyrosyluria reaction with the Clinitest procedure is:
B. MSUD A. Cystinuria
C. Melanuria B. Alkaptonuria
D. Galactosemia C. Indicanuria
D. Porphyrinuria
4. The best specimen for early newborn screening is a:
A. Timed urine specimen 9. A urine that turns black after sitting by the sink for
several hours could be indicative of:
B. Blood specimen
A. Alkaptonuria
C. First morning urine specimen
B. MSUD
D. Fecal specimen
C. Melanuria
5. Which of the following disorders is not associated with
D. Both A and C
the phenylalanine–tyrosine pathway?
A. MSUD 10. Ketonuria in a newborn is an indication of:
B. Alkaptonuria A. MSUD
C. Albinism B. Isovaleric acidemia
D. Tyrosinemia C. Methylmalonic acidemia
D. All of the above
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Chapter 9 | Urine Screening for Metabolic Disorders 249

11. Urine from a newborn with MSUD will have a 18. Homocystinuria is caused by failure to metabolize:
significant: A. Lysine
A. Pale color B. Methionine
B. Yellow precipitate C. Arginine
C. Milky appearance D. Cystine
D. Sweet odor
19. The Ehrlich reaction will detect only the presence of:
12. Hartnup disease is a disorder associated with the A. Uroporphyrin
metabolism of:
B. Porphobilinogen
A. Organic acids
C. Coproporphyrin
B. Tryptophan
D. Protoporphyrin
C. Cystine
20. Acetyl acetone is added to the urine before performing
D. Phenylalanine
the Ehrlich test when checking for:
13. 5-HIAA is a degradation product of: A. Aminolevulinic acid
A. Heme B. Porphobilinogen
B. Indole C. Uroporphyrin
C. Serotonin D. Coproporphyrin
D. Melanin
21. The classic urine color associated with porphyria is:
14. Elevated urinary levels of 5-HIAA are associated with: A. Dark yellow
A. Carcinoid tumors B. Indigo blue
B. Hartnup disease C. Pink
C. Cystinuria D. Port wine
D. Platelet disorders
22. Which of the following specimens can be used for
15. False-positive levels of 5-HIAA can be caused by a diet porphyrin testing?
high in: A. Urine
A. Meat B. Blood
B. Carbohydrates C. Feces
C. Starch D. All of the above
D. Bananas
23. The two stages of heme formation affected by lead
16. Place the appropriate letter in front of the following poisoning are:
statements. A. Porphobilinogen and uroporphyrin
A. Cystinuria B. Aminolevulinic acid and porphobilinogen
B. Cystinosis C. Coproporphyrin and protoporphyrin
IEM D. Aminolevulinic acid and protoporphyrin
Inherited disorder of tubular reabsorption
24. Hurler, Hunter, and Sanfilippo syndromes are hereditary
Fanconi syndrome disorders affecting the metabolism of:
Cystine deposits in the cornea A. Porphyrins
Early renal calculi formation B. Purines
17. Blue diaper syndrome is associated with: C. Mucopolysaccharides
A. Lesch-Nyhan syndrome D. Tryptophan
B. Phenylketonuria
C. Cystinuria
D. Hartnup disease
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250 Part Two | Urinalysis

25. Many uric acid crystals in a pediatric urine specimen 27. Match the metabolic urine disorders with their classic
may indicate: urine abnormalities.
A. Hurler syndrome PKU A. Sulfur odor
B. Lesch-Nyhan disease Indicanuria B. Sweaty feet odor
C. Melituria Cystinuria C. Orange sand in
D. Sanfilippo syndrome diaper
Alkaptonuria D. Mousy odor
26. Deficiency of the GALT enzyme will produce a:
Lesch-Nyhan disease E. Black color
A. Positive Clinitest
Isovaleric acidemia F. Blue color
B. Glycosuria
C. Galactosemia
D. Both A and C

Case Studies and Clinical Situations

1. A premature infant develops jaundice. Laboratory tests are GLUCOSE: Negative LEUKOCYTE: Negative
negative for hemolytic disease of the newborn, but the in- Microscopic:
fant’s bilirubin level continues to rise. Abnormal urinalysis
>15–20 RBCs/hpf Few squamous epithelial
results include a dark yellow color, positive bilirubin, and
cells
needle-shaped crystals seen on microscopic examination.
0–3 WBCs/hpf Many cystine crystals
a. What is the most probable cause of the infant’s
jaundice? a. What condition does the patient’s symptoms
represent?
b. Could these same urine findings be associated with an
adult? Explain your answer. b. What is the physiological abnormality causing this
condition?
c. What kinds of crystals are present? Name another type
of crystal with a spherical shape that is associated with c. If amino acid chromatography was performed on this
this condition. specimen, what additional amino acids could be
identified if present?
d. When blood is drawn from this infant, what precau-
tion should be taken to ensure the integrity of the d. Why are they not present in the microscopic
specimen? constituents?
e. Based on the family history, what genetic disorder
2. A newborn develops severe vomiting and symptoms of
should be considered?
metabolic acidosis. Urinalysis results are positive for ketones
and negative for glucose and other reducing substances. 4. An 8-month-old boy is admitted to the pediatric unit with
a. If the urine had an odor of “sweaty feet,” what meta- a general diagnosis of failure to thrive. The parents have
bolic disorder would be suspected? observed slowness in the infant’s development of motor
skills. They also mention the occasional appearance of a
b. If the newborn was producing dark brown urine with
substance resembling orange sand in the child’s diapers.
a sweet odor, what disorder would be suspected?
Urinalysis results are as follows:
c. Would an MS/MS screen be helpful for the diagnosis?
COLOR: Yellow KETONES: Negative
3. A 13-year-old boy is awakened with severe back and ab- APPEARANCE: Slightly BLOOD: Negative
dominal pain and is taken to the emergency department by hazy
his parents. A complete blood count is normal. Family his-
SP. GRAVITY: 1.025 BILIRUBIN: Negative
tory shows that both his father and uncle are chronic form-
ers of kidney stones. Results of a urinalysis are as follows: pH: 5.0 UROBILINOGEN: Normal
COLOR: Yellow KETONES: Negative PROTEIN: Negative NITRITE: Negative
APPEARANCE: Hazy BLOOD: Moderate GLUCOSE: Negative LEUKOCYTE: Negative
SP. GRAVITY: 1.025 BILIRUBIN: Negative Microscopic:
pH: 6.0 UROBILINOGEN: Normal Many uric acid crystals
PROTEIN: Negative NITRITE: Negative a. Is the urine pH consistent with the appearance of uric
acid crystals?
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Chapter 9 | Urine Screening for Metabolic Disorders 251

b. Is there any correlation between the urinalysis results successful, the patient’s recovery is slow, and the physi-
and the substance observed in the child’s diapers? cians are concerned about his health before the ruptured
Explain your answer. appendix. The patient’s mother states that he has always
c. What disorder do the patient’s history and the been noticeably underweight despite eating a balanced
urinalysis results indicate? diet and having a strong appetite and that his younger
brother exhibits similar characteristics. A note in his chart
d. Is the fact that this is a male patient of any
from the first postoperative day reports that the evening
significance? Explain your answer.
nurse noticed a blue coloration in the urinary catheter bag.
e. Name the enzyme that is missing.
a. Is the catheter bag color significant?
5. Shortly after arriving for the day shift in the urinalysis b. What condition can be suspected from this history?
laboratory, a technician notices that an undiscarded
c. What is the patient’s prognosis?
urine specimen has a black color. The report completed
previously indicates the color to be yellow. 7. An anemic patient is suspected of having lead poisoning.
a. Is this observation significant? Explain your answer. a. Historically, what urine test was requested?
b. The original urinalysis report showed the specimen to b. What should be added to the urine before testing?
be positive for ketones. Is this significant? Why or c. What element of heme synthesis would this be
why not? testing for?
c. If the ketones are negative and the pH is 8.0, is this d. Name another substance that can be tested for lead
significant? Why or why not? poisoning.
6. An 8-year-old boy is admitted through the emergency e. What element of heme synthesis would this test for?
department with a ruptured appendix. Although surgery is
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PART THREE

Other Body Fluids

Chapter 10: Cerebrospinal Fluid
Chapter 11: Semen
Chapter 12: Synovial Fluid
Chapter 13: Serous Fluid
Chapter 14: Bronchoalveolar Lavage Fluid
Chapter 15: Amniotic Fluid
Chapter 16: Fecal Analysis
Chapter 17: Vaginal Secretions
7582_Ch10_252-276 30/06/20 1:07 PM Page 253

CHAPTER 10
Cerebrospinal Fluid
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
10-1 Describe the formation of cerebrospinal fluid (CSF), 10-11 Determine whether increased CSF albumin or im-
and state the three major functions of CSF. munoglobulin is the result of damage to the blood–
brain barrier or central nervous system production.
10-2 Distribute CSF specimen tubes numbered 1, 2, 3, and
possibly 4 to their appropriate laboratory sections, 10-12 Discuss the significance of findings of CSF elec-
and correctly preserve them. trophoresis, immunophoresis, and isoelectric focus-
ing in multiple sclerosis and the identification of CSF.
10-3 Describe the appearance of normal CSF and the
causes of CSF not appearing normally. 10-13 State the reference values for CSF glucose, and name
the possible pathological significance of a decreased
10-4 Define xanthochromia, and state its significance.
CSF glucose.
10-5 Differentiate between CSF specimens caused by
10-14 Discuss the diagnostic value of CSF lactate and gluta-
intracranial hemorrhage and a traumatic tap.
mine determinations.
10-6 Calculate CSF total cell count, as well as counts for
10-15 Name the microorganism associated with a positive
white blood cells and red blood cells, when given the
India ink preparation.
number of cells seen, amount of specimen dilution,
and squares counted in the Neubauer chamber. 10-16 Discuss the diagnostic value of the bacterial and
cryptococcal antigen tests.
10-7 Describe the leukocyte content of the CSF in various
forms of meningitis, including bacterial, viral, tuber- 10-17 Explain the advantage of molecular diagnostic
cular, and fungal. methods for determining the causative organism in
meningitis.
10-8 Describe and state the significance of macrophages in
the CSF. 10-18 Determine whether a suspected case of meningitis is
of bacterial, viral, fungal, or tubercular origin when
10-9 Differentiate between the appearance of normal
presented with pertinent laboratory data.
choroidal cells and malignant cells.
10-19 Describe the Venereal Disease Research Laboratory
10-10 State the reference values for CSF total protein, and
test and the fluorescent treponemal antibody-
name three pathological conditions that produce an
absorption test for syphilis in CSF testing.
elevated CSF protein.

KEY TERMS
Arachnoid granulations Meningitis Traumatic tap
Blood–brain barrier Oligoclonal bands Xanthochromia
Choroid plexuses Pleocytosis
Meninges Subarachnoid space
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254 Part Three | Other Body Fluids

Introduction passage of soluble nutrients and wastes between the plasma

and tissues. In the choroid plexuses, the endothelial cells have
Cerebrospinal fluid (CSF) is a major fluid of the body. It pro- very tight-fitting junctures that prevent the passage of many
vides a physiological system to supply nutrients to the nervous molecules. This tight-fitting structure of the endothelial cells
tissue, remove metabolic wastes, maintain intracranial pres- in the choroid plexuses is termed the blood–brain barrier.
sure, and produce a mechanical barrier to cushion the brain Maintaining the integrity of the blood–brain barrier is
and spinal cord against trauma. essential to protect the brain from chemicals and other sub-
stances circulating in the blood that could harm the brain tis-
sue. In addition, the junctures prevent the passage of helpful
Formation and Physiology substances, including antibodies and medications. Disruption
The brain and spinal cord are lined by the meninges, which con- of the blood–brain barrier by diseases, such as meningitis and
sist of three layers: the dura mater (Latin for “hard mother”), the multiple sclerosis, allows leukocytes, proteins, and additional
arachnoid (“spiderweb-like”), and the pia mater (Latin for “gentle chemicals to enter the CSF.
mother”). The outer layer is the dura mater that lines the skull
and vertebral canal. The arachnoid is a filamentous (spiderweb-
like) inner membrane. The pia mater is a thin membrane lining
Specimen Collection
the surfaces of the brain and spinal cord (Fig. 10-1). and Handling
CSF is produced in the choroid plexuses of the two lum-
bar ventricles and the third and fourth ventricles. In adults, ap- CSF is collected routinely by lumbar puncture between the third
proximately 20 mL of fluid is produced every hour. The fluid and fourth or fourth and fifth lumbar vertebra. Although this
flows through the subarachnoid space located between the procedure is not complicated, it does require certain precautions,
arachnoid and pia mater (Fig. 10-2). To maintain a volume of including measurement of intracranial pressure and careful tech-
90 to 150 mL in adults and 10 to 60 mL in neonates, the circu- nique, to prevent infection or damage to neural tissue.
lating fluid is reabsorbed back into the blood capillaries in the The volume of CSF that can be removed is based on the
arachnoid granulations/villae at a rate equal to its production. volume available in the patient (adult vs. neonate) and the
The cells of the arachnoid granulations act as one-way valves opening pressure of the CSF, measured when the needle first
that respond to pressure within the central nervous system enters the subarachnoid space. Elevated pressure requires fluid
(CNS) and prevent reflux of the fluid.1 to be withdrawn slowly, with careful monitoring of the pres-
The choroid plexuses are capillary networks that form the sure, and may prevent collection of a large volume.
CSF from plasma by mechanisms of selective filtration under Specimens are collected in three sterile tubes, which are
hydrostatic pressure and active transport secretion. Therefore, labeled 1, 2, and 3 in the order in which they are withdrawn:
the chemical composition of the CSF does not resemble an • Tube 1 is used for chemical and serological tests because
ultrafiltrate of plasma. Capillary walls throughout the body are these tests are least affected by blood or bacteria intro-
lined with endothelial cells that are connected loosely to allow duced as a result of the tap procedure.

Central canal
Gray matter
White matter

Spinal nerve roots

Dorsal root ganglion

Pia mater
Arachnoid Superior
granulation sagittal sinus Skin Spinal nerve
Periosteum

Skull
Dura mater
Arachnoid Arachnoid Subarachnoid
Meninges membrane
mater space
Pia mater
Dura
Subarachnoid space mater

Gray matter
Brain
A White matter tissue B

Figure 10–1 The layers of the meninges. A. The layers of the meninges in the brain. B. The layers of the meninges in the spinal cord. (A and B
from Scanlon & Sanders, Essentials of Anatomy and Physiology, 6th ed., F. A. Davis Company, Philadelphia, 2011, with permission.)
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Chapter 10 | Cerebrospinal Fluid 255

Choroid plexus
of third ventricle
Cerebrum Subarachnoid
space
Arachnoid
villus/granulation

Pia mater
Choroid plexus of Arachnoid
lateral ventricle mater

Dura
mater

Cerebellum

Choroid plexus of
fourth ventricle

Figure 10–2 The flow of CSF through the Spinal cord

brain and spinal column.

• Tube 2 usually is designated for testing in the microbiol- report. The major terminology used to describe CSF appearance
ogy laboratory. includes crystal-clear, cloudy or turbid, milky, xanthochromic,
• Tube 3 is used for the cell count because it is the least and hemolyzed/bloody (Fig. 10-4). A cloudy, turbid, or milky
likely to contain cells introduced by the spinal tap specimen can be the result of an increased concentration of pro-
procedure. tein or lipids, but also it may be indicative of infection, with the
cloudiness being caused by the presence of white blood cells
A fourth tube may be drawn for the microbiology laboratory
(WBCs). All specimens should be treated with extreme care
to better exclude skin contamination or for additional serological
tests. Supernatant fluid that is left over after each section has per-
formed its tests also may be used for additional chemical or sero-
logical tests. Excess fluid should not be discarded and should be
frozen until there is no further use for it (Fig. 10-3).
Considering the discomfort to the patient and the possible
complications that can occur during specimen collection, labo- 1 2 3
ratory personnel should handle CSF specimens carefully. Ideally,
tests are performed on a STAT basis. If this is not possible, spec-
imens are maintained in the following manner:
• Hematology tubes are refrigerated up to 4 hours.
Chemistry Microbiology Hematology
• Microbiology tubes remain at room temperature. serology
• Chemistry and serology tubes are frozen.

Technical Tip 10-1. Cells must be counted within

1 hour of collection when the specimen is maintained
at room temperature.

Appearance
The initial appearance of the normally crystal-clear CSF can pro-
Additional tests
vide valuable diagnostic information. Examination of the fluid
occurs first at the bedside and also is included in the laboratory Figure 10–3 CSF specimen collection tubes.
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256 Part Three | Other Body Fluids

Table 10–1 Clinical Significance of CSF Appearance

Major
Appearance Cause Significance
Crystal clear Normal
Hazy, turbid, WBCs Meningitis
milky,
cloudy
Microorganisms Meningitis
Protein Disorders affecting
blood–brain
barrier
Production of IgG
Figure 10–4 Tubes of CSF. Appearance left to right is normal, within the CNS
xanthochromic, hemolyzed, and cloudy.
Oily Radiographic
contrast
media
because they can be highly contagious; gloves always must be
Bloody RBCs Hemorrhage
worn and face shields or splash guards should be used while
preparing specimens for testing. Fluid for centrifugation must Traumatic tap
be in capped tubes. Xanthochromic Hemoglobin Old hemorrhage
Lysed cells from
traumatic tap
Technical Tip 10-2. If only one tube can be collected,
Bilirubin RBC degradation
it must be tested first by microbiology.
Elevated serum
bilirubin level
Technical Tip 10-3. It is not unusual for cell counts
Carotene Increased serum
requested to be performed on both Tubes 1 and 4 to
levels
check for cellular contamination by the puncture.
Protein Disorders affecting
blood–brain
barrier
Xanthochromia is a term used to describe CSF super-
natant that is pink, orange, or yellow. A variety of factors can Melanin Meningeal
cause xanthochromia, the most common being the presence melanosarcoma
of red blood cell (RBC) degradation products. Depending Clotted Protein Disorders affecting
on the amount of blood and the length of time it has been blood–brain
present, the color will vary from pink (very slight amount of barrier
oxyhemoglobin) to orange (heavy hemolysis) to yellow (con- Clotting factors Introduced by
version of oxyhemoglobin to unconjugated bilirubin). Other traumatic tap
causes of xanthochromia include elevated serum bilirubin,
Pellicle Protein Disorders affecting
presence of the pigment carotene, markedly increased pro-
blood–brain
tein concentrations, and melanoma pigment. Xanthochromia
barrier
that is caused by bilirubin due to immature liver function
also is seen commonly in infants, particularly premature in- Clotting factors Tubercular
fants. The clinical significance of CSF appearance is summa- meningitis
rized in Table 10-1.

Traumatic Collection (Tap)

Grossly bloody CSF can be an indication of intracranial
Uneven Blood Distribution
hemorrhage, but also it may be due to the puncture of a Blood from a cerebral hemorrhage will be distributed evenly
blood vessel during the spinal tap procedure. Usually, three throughout the three CSF specimen tubes, whereas a traumatic
visual examinations of the collected specimens can deter- tap will leave the heaviest concentration of blood in tube 1, with
mine whether the blood is the result of hemorrhage or a amounts gradually diminishing in tubes 2 and 3. Performing
traumatic tap. RBC counts on all three tubes to measure decreasing or constant
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Chapter 10 | Cerebrospinal Fluid 257

blood is not always reliable.2 Streaks of blood also may be seen to 200 WBCs or 400 RBCs/µL may appear clear, so it is necessary
in specimens acquired after a traumatic procedure. to examine all specimens microscopically.6 An improved
Neubauer counting chamber (Fig. 10-5) is used routinely for per-
Clot Formation forming CSF cell counts. Traditionally, electronic cell counters
Fluid collected from a traumatic tap may form clots due to the have not been used for performing CSF cell counts because of
introduction of plasma fibrinogen into the specimen. Bloody the instrument’s high background counts and poor reproducibil-
CSF caused by intracranial hemorrhage does not contain ity of low counts.
enough fibrinogen to clot. Diseases in which damage to the Automation increases precision, standardization, and faster
blood–brain barrier allows increased filtration of protein and turnaround time for results. Various automated instruments,
coagulation factors also cause clot formation but usually do such as the ADVIA 2120i (Siemens Healthcare Diagnostics In-
not produce a bloody fluid. These conditions include menin- corporated, Deerfield, IL),7 Sysmex XN Series (Sysmex, Kobe,
gitis, Froin syndrome, and blocked CSF circulation through Japan), Iris iQ200 with Body Fluids Module (Iris Diagnostics-
the subarachnoid space. A classic web-like pellicle is associated Beckman Coulter, Inc., Chatsworth, CA), DxH900 (Beckman
with tubercular meningitis and can be seen after overnight Coulter, Inc.),8 and the Glocyte System (Sysmex, Kobe, Japan),9
refrigeration of the fluid.3 are available for CSF cell counts. See Chapter 2 for more infor-
mation on automated body fluid analyzers.
Xanthochromic Supernatant
Calculating CSF Cell Counts
Usually RBCs must remain in the CSF for approximately 2 hours
before noticeable hemolysis begins; therefore, a xanthochromic The standard Neubauer calculation formula used for blood cell
supernatant would be the result of blood that has been present counts is also applied to CSF cell counts to determine the num-
longer than that introduced by the traumatic tap. Care should ber of cells per microliter.
be taken, however, to consider this examination in conjunction
Number of cells counted × dilution
with those previously discussed, because a very recent hemor- = cells/µL
Number of cells counted × volume of 1 square
rhage would produce a clear supernatant, and introduction of
serum protein from a traumatic tap also could cause the fluid to This formula can be used for both diluted and undiluted
appear xanthochromic. To examine a bloody fluid for the pres- specimens and offers flexibility in the number and size of the
ence of xanthochromia, the fluid should be centrifuged in a squares counted. Many varied calculations are available, includ-
microhematocrit tube and the supernatant examined against a ing condensations of the formula to provide single factors by
white background. which to multiply the cell count. Keep in mind that the purpose
Additional testing for differentiation includes microscopic of any calculation is to convert the number of cells counted in a
examination and the D-dimer test. The microscopic finding of specific amount of fluid to the number of cells that would be
macrophages containing ingested RBCs (erythrophagocytosis)
or hemosiderin granules indicates intracranial hemorrhage.
Detection of the fibrin degradation product D-dimer by latex
agglutination immunoassay indicates fibrin formation at a
hemorrhage site.

Cell Count
1 2 3
The cell count that is performed routinely on CSF specimens is
the leukocyte (WBC) count. Normally RBCs are not present in
CSF. As discussed previously, the presence and significance of
RBCs usually can be ascertained from the appearance of the spec-
imen. Therefore, RBC counts are determined only when a trau-
matic tap has occurred and a correction for leukocytes or protein 4 5 6
is desired. The RBC count can be calculated by performing a total
cell count and a WBC count and subtracting the WBC count
from the total count, if necessary. Any cell count should be per-
formed immediately because WBCs (particularly granulocytes)
and RBCs begin to lyse within 1 hour, and 40% of the leukocytes
disintegrate after 2 hours.4 Specimens that cannot be analyzed 7 8 9
immediately should be refrigerated up to 4 hours.

Methodology
Normal adult CSF contains 0 to 5 WBCs/µL. The number is
higher in children, and as many as 30 mononuclear cells/µL can Figure 10–5 Neubauer counting chamber depicting the nine large
be considered normal in newborns.5 Specimens that contain up square counting areas.
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258 Part Three | Other Body Fluids

present in 1 µL of fluid. Therefore, a factor can be used only when

PROCEDURE 10-1
the dilution and counting area are specific for that factor.
The methodology presented in this chapter eliminates the CSF WBC Count
need to correct for the volume counted by counting the four large Equipment
corner squares (0.4 µL) and the large center square (0.1 µL) on
each side of the counting chamber.10 3% glacial acetic acid
Pasteur pipette
EXAMPLE Hemocytometer
Number of cells counted × dilution × Methylene blue stain, if necessary
1 μL
= cells/μL Procedure
1 μL (0.1 × 10)
(volume counted) To prepare a clear specimen that does not require dilution
for counting, follow the steps listed.
1. Place four drops of mixed specimen in a clean tube.
Total Cell Count 2. Rinse a Pasteur pipette with 3% glacial acetic acid,
draining thoroughly, and draw the four drops of CSF
Clear specimens may be counted undiluted, provided no over-
into the rinsed pipette.
lapping of cells is seen during the microscopic examination.
When dilutions are required, calibrated automatic pipettes, not 3. Allow the pipette to sit for 1 minute, mix the solution
mouth pipetting, are used. Dilutions for total cell counts are in the pipette, discard the first drop, and load the
made with normal saline, mixed by inversion, and loaded into hemocytometer.
the hemocytometer with a Pasteur pipette. Cells are counted 4. As in the total cell count, WBCs are counted in the
in the four corner squares and the center square on both sides four corner squares and the center square on both
of the hemocytometer. As shown in the preceding example, sides of the hemocytometer. Multiply that number by
the number of cells counted multiplied by the dilution factor the dilution factor to obtain the number of WBCs per
equals the number of cells per microliter. microliter.

WBC Count
Lysis of RBCs must be obtained before performing the WBC
count on either diluted or undiluted specimens. Specimens If nondisposable counting chambers are used, they must
requiring dilution can be diluted in the manner described pre- be soaked in a bactericidal solution for at least 15 minutes and
viously, substituting 3% glacial acetic acid to lyse the RBCs. then rinsed thoroughly with water and cleaned with isopropyl
Adding methylene blue to the diluting fluid stains the WBCs, alcohol after each use.
providing better differentiation between neutrophils and
mononuclear cells. Differential Count on a
Visit www.fadavis.com for Video 10-1 (Manual
hemocytometer test). CSF Specimen
Identifying the type or types of cells present in the CSF is a
Technical Tip 10-4. The number of squares counted valuable diagnostic aid. The differential count should be per-
for CSF varies between laboratories. If a different num- formed on a stained smear and not from the cells in the count-
ber of squares is counted, use the standard Neubauer ing chamber. Poor visualization of the cells as they appear in
formula to obtain the number of cells per microliter. the counting chamber has led to the laboratory practice of re-
porting only the percentage of mononuclear and polynuclear
cells present, which can result in overlooking abnormal cells
Quality Control of CSF and Other Body with considerable diagnostic importance. To ensure that the
maximum number of cells is available for examination, the
Fluid Cell Counts specimen should be concentrated before preparing the smear.
Liquid commercial controls are available from several manu- Methods available for specimen concentration include
facturers for spinal fluid counts of RBCs and WBCs. They can sedimentation, filtration, centrifugation, and cytocentrifugation.
be purchased at two levels of concentration. Also, in-house Sedimentation and filtration are not used routinely in the clinical
controls can be prepared. laboratory, although they do produce less cellular distortion. Most
All diluents should be checked biweekly for contamina- laboratories that do not have a cytocentrifuge concentrate speci-
tion by examining them in a counting chamber under 400× mens with routine centrifugation. The specimen is centrifuged
magnification. Contaminated diluents should be discarded and for 5 to 10 minutes, supernatant fluid is removed and saved for
new solutions prepared. The speed of the cytocentrifuge additional tests, and slides made from the suspended sediment
should be checked monthly with a tachometer, and the timing are allowed to air dry and then are stained with Wright’s stain.
should be checked with a stopwatch. When the differential count is performed, 100 cells should be
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Chapter 10 | Cerebrospinal Fluid 259

counted, classified, and reported in terms of percentage. If the Table 10-2 presents a cytocentrifuge recovery chart for com-
cell count is low and finding 100 cells is not possible, report only parison with chamber counts. The chamber count should be re-
the numbers of the cell types seen. peated if too many cells are seen on the slide, and a new slide
Visit www.fadavis.com for Video 10-2 (Body fluid should be prepared if not enough cells are seen on the slide.10
differential cytocentrifuge).
CSF Cellular Constituents
Cytocentrifugation The cells found in normal CSF are primarily lymphocytes and
monocytes (Figs. 10-7 and 10-8). Adults usually have a predom-
A diagrammatic view of the principle of cytocentrifugation is
inance of lymphocytes to monocytes (70:30), whereas the ratio
shown in Figure 10-6. Fluid is added to the conical chamber,
is essentially reversed in children.5 Improved concentration
and as the specimen is centrifuged, cells present in the fluid
methods also are showing occasional neutrophils in normal
are forced into a monolayer within a 6-mm-diameter circle on
the slide. Fluid is absorbed by the filter paper blotter, produc-
ing a more concentrated area of cells. As little as 0.1 mL of CSF
Table 10–2 Cytocentrifuge Recovery Chart10
combined with one drop of 30% albumin produces an ade-
quate cell yield when processed with the cytocentrifuge. Number of WBCs Number of Cells on
Adding albumin increases the cell yield and decreases the Counted in Chamber Cytocentrifuge Slide
cellular distortion frequently seen on cytocentrifuged speci-
0 0–40
mens. Positively charged coated slides to attract cells (Shandon,
Inc., Pittsburgh, PA) are available also. Cellular distortion may 1–5 20–100
include cytoplasmic vacuoles, nuclear clefting, prominent nu- 6–10 60–150
cleoli, indistinct nuclear and cytoplasmic borders, and cellular 11–20 150–250
clumping that resembles malignancy. Cells from both the cen-
21 251
ter and periphery of the slide should be examined because
cellular characteristics may vary between areas of the slide.
A daily control slide for bacteria also should be prepared
using 0.2 mL saline and two drops of the 30% albumin cur-
rently being used. The slide is stained and examined if bacteria
are seen on a patient’s slide.

Stainless
steel
Cytoclip™ Cytoslide™ Completed assembly
slide clip microscope Figure 10–7 Normal lymphocytes. Some cytocentrifuge distortion
slide of cytoplasm (×1000).
Cytofunnel™ disposable
sample chamber with
attached filter card

In-load position In operation

This cutaway drawing illustrates, at left, the in-load position which

shows the sample chamber assembly in a tilted-back position, so
that the sample is not absorbed by the filter card. During spinning,
centrifugal force tilts the assembly upright and forces the sample to
flow toward the microscope slide.

Figure 10–6 Cytospin 3 cytocentrifuge specimen processing as-

sembly (Courtesy of Shandon, Inc., Pittsburgh, PA). Figure 10–8 Normal lymphocytes and monocytes (×500).
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260 Part Three | Other Body Fluids

CSF.11 The presence of increased numbers of these normal cells are lost more rapidly in CSF. Neutrophils associated with bacte-
(termed pleocytosis) is considered abnormal, as is the finding rial meningitis may contain phagocytized bacteria (Figs. 10-10
of immature leukocytes, eosinophils, plasma cells, macrophages, and 10-11). Although of little clinical significance, neutrophils
increased tissue cells, and malignant cells. may be increased after hemorrhage in the CNS, repeated lumbar
When pleocytosis involving neutrophils, lymphocytes, or punctures, and injection of medications or radiographic dye.
monocytes is present, the CSF differential count is associated Neutrophils with pyknotic nuclei indicate degenerating
most frequently with its role in providing diagnostic informa- cells. They may resemble nucleated red blood cells (NRBCs) but
tion about the type of microorganism that is causing an infec- usually have multiple nuclei. When a single nucleus is present,
tion of the meninges (meningitis). A high CSF WBC count of these neutrophils can appear similar to NRBCs (Fig. 10-12).
which the majority of the cells are neutrophils is considered NRBCs are seen as a result of contamination from bone marrow
indicative of bacterial meningitis. Likewise, a CSF WBC count during the spinal tap (Figs. 10-13 and 10-14). This is found in
with a high percentage of lymphocytes and monocytes that is approximately 1% of specimens.12 Capillary structures and en-
moderately elevated suggests meningitis of viral, tubercular, dothelial cells may be seen after a traumatic tap (Fig. 10-15).
fungal, or parasitic origin.
As seen in Table 10-3, many pathological conditions other Lymphocytes and Monocytes
than meningitis can be associated with abnormal cells in the A mixture of lymphocytes and monocytes is common in cases
CSF. Cell forms differing from those found in blood include of viral, tubercular, and fungal meningitis. Reactive lymphocytes
macrophages, choroid plexus and ependymal cells, spindle- containing increased dark blue cytoplasm and clumped chro-
shaped cells, and malignant cells. matin frequently are present in conjunction with normal cells
during viral infections (Fig. 10-16). Increased lymphocytes are
Technical Tip 10-5. Because laboratory personnel seen in cases of both asymptomatic HIV infection and AIDS. A
become so accustomed to finding neutrophils, lym- moderately elevated WBC count (less than 50 WBCs/µL) with
phocytes, and monocytes, they should be careful not increased normal and reactive lymphocytes and plasma cells
to overlook other types of cells in the CSF. may indicate multiple sclerosis or other degenerative neurolog-
ical disorders.13

Neutrophils Eosinophils
In addition to bacterial meningitis, increased neutrophils are Increased eosinophils are seen in the CSF in association with par-
seen in the early stages (1 to 2 days) of viral, fungal, tubercular, asitic infections, fungal infections (primarily Coccidioides immitis),
and parasitic meningitis. Neutrophils also may contain cytoplas- and introduction of foreign material, including medications and
mic vacuoles after cytocentrifugation (Fig. 10-9). Granules also shunts, into the CNS (Fig. 10-17).

Table 10–3 Predominant Cells Seen in CSF

Type of Cell Major Clinical Significance Microscopic Findings
Lymphocytes Normal All stages of development may be found
Viral, tubercular, and fungal meningitis
HIV infection and AIDS
Multiple sclerosis
Degenerative disorders
Parasitic infections
Neutrophils Bacterial meningitis Granules may be less prominent than in
blood
Early cases of viral, tubercular, and fungal Cells disintegrate rapidly
meningitis
Cerebral hemorrhage
Cerebral abscess
CNS infarction
Injection of medications or radiographic
dye into the subarachnoid space
Metastatic tumors
Repeated lumbar punctures
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Chapter 10 | Cerebrospinal Fluid 261

Table 10–3 Predominant Cells Seen in CSF—cont’d

Type of Cell Major Clinical Significance Microscopic Findings
Monocytes Normal Found mixed with lymphocytes
Viral, tubercular, and fungal meningitis
Multiple sclerosis
Eosinophils Parasitic infections
Fungal infections
Coccidioidal meningitis
Introducing medications and shunts in
the CNS
Macrophages RBCs in spinal fluid May contain phagocytized RBCs appear-
ing as empty vacuoles or ghost cells,
hemosiderin granules, and hema-
toidin crystals
Contrast media
Blast forms Acute leukemia Lymphoblasts, myeloblasts, or
monoblasts
Lymphoma cells Disseminated lymphomas Resemble lymphocytes with cleft nuclei
Plasma cells Multiple sclerosis Traditional and classic forms seen
Guillain-Barre syndrome
Sarcoidosis
Parasitic infections
Syphilitic meningitis
Tuberculous meningitis
Lymphocyte reactions Reactive lymphocytes
Ependymal, choroidal, and spindle- Diagnostic procedures Seen in clusters with distinct nuclei and
shaped cells distinct cell walls
Malignant cells Metastatic carcinomas Seen in clusters with fusing of cell
borders and nuclei
Primary central nervous system
carcinoma

Figure 10–9 Neutrophils with cytoplasmic vacuoles resulting from

cytocentrifugation (×500). Figure 10–10 Neutrophils with intracellular bacteria (×1000).
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262 Part Three | Other Body Fluids

Figure 10–11 Neutrophils with intracellular and extracellular bacte- Figure 10–14 Bone marrow contamination (×1000). Notice the
ria (×1000). immature RBCs and granulocytes.

Figure 10–15 Capillary and tissue fragments from a traumatic tap

Figure 10–12 Neutrophils with pyknotic nuclei. Notice the cell with
(×100).
a single nucleus in the center (×1000).

Figure 10–13 Nucleated RBCs seen with bone marrow contamina- Figure 10–16 Broad spectrum of lymphocytes and monocytes in
tion (×1000). viral meningitis (×1000).
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Chapter 10 | Cerebrospinal Fluid 263

Figure 10–17 Eosinophils (×1000). Notice cytocentrifuge Figure 10–19 Macrophages showing erythrophagocytosis
distortion. (×500).

Macrophages
The purpose of macrophages in the CSF is to remove cellular
debris and foreign objects, such as RBCs. Macrophages appear
within 2 to 4 hours after RBCs enter the CSF and frequently
are seen after repeated taps. They tend to have more cytoplasm
than monocytes in the peripheral blood (PB) (Fig. 10-18).
The finding of increased macrophages indicates a previ-
ous hemorrhage (Fig. 10-19). Further degradation of the
phagocytized RBCs results in the appearance of dark blue or
black iron-containing hemosiderin granules (Figs. 10-20
through 10-23). Yellow hematoidin crystals represent further
degeneration. They are iron-free, consisting of hemoglobin
and unconjugated bilirubin (Figs. 10-24 and 10-25).
Nonpathologically Significant Cells
Figure 10–20 Macrophage with RBC remnants (×500).
Nonpathologically significant cells are seen most frequently after
diagnostic procedures, such as pneumoencephalography, and
in fluid obtained from ventricular taps or during neurosurgery.
The cells often appear in clusters and can be distinguished from
malignant cells by their uniform appearance.
Choroidal cells are from the epithelial lining of the choroid
plexus. They are seen singularly and in clumps. Usually nucleoli
are absent, and nuclei have a uniform appearance (Fig. 10-26).

Figure 10–21 Macrophage with aggregated hemosiderin granules

(×500).

Ependymal cells are from the lining of the ventricles and neu-
ral canal. They have less defined cell membranes and frequently
are seen in clusters. Often nucleoli are present (Fig. 10-27).
Spindle-shaped cells represent lining cells from the arach-
Figure 10–18 Macrophages. Notice the large amount of cytoplasm noid. Usually they are seen in clusters and may be seen with
and vacuoles (×500). systemic malignancies (Fig. 10-28).
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264 Part Three | Other Body Fluids

Figure 10–22 Macrophage containing hemosiderin stained with Figure 10–25 Macrophages with hemosiderin and hematoidin
Prussian blue (×250). (×250). Notice the bright yellow color.

Figure 10–26 Choroidal cells showing distinct cell borders and

Figure 10–23 Macrophage with coarse hemosiderin granules nuclear uniformity (×500).
(×500).

Figure 10–24 Macrophage containing hemosiderin and hema- Figure 10–27 Ependymal cells. Notice the nucleoli and less distinct
toidin crystals (500). cell borders (×1000).
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Chapter 10 | Cerebrospinal Fluid 265

Figure 10–28 Cluster of spindle-shaped cells (×500). Figure 10–31 Monoblasts and two lymphocytes (×1000). Notice
the prominent nucleoli.

Malignant Cells of Hematologic Origin

Lymphoma cells are seen in the CSF also and indicate dis-
Lymphoblasts, myeloblasts, and monoblasts (Figs. 10-29 to semination from the lymphoid tissue. They resemble large and
10-31) in the CSF are seen frequently as a serious complication small lymphocytes and usually appear in clusters of large,
of acute leukemias. Often nucleoli are more prominent than in small, or mixed cells based on the classification of the lym-
blood smears. phoma. Nuclei may appear cleaved, and prominent nucleoli
are present (Figs. 10-32 to 10-34).

Figure 10–29 Lymphoblasts from acute lymphocytic leukemia

(×500).
Figure 10–32 Cleaved and noncleaved lymphoma cells
(×1000).

Figure 10–30 Myeloblasts from acute myelocytic leukemia

(×500). Figure 10–33 Lymphoma cells with nucleoli (×500).
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266 Part Three | Other Body Fluids

chemical composition is controlled by the blood–brain barrier,

reference values for CSF chemicals are not the same as the plasma
values. Abnormal values result from alterations in the perme-
ability of the blood–brain barrier or increased production or
metabolism by the neural cells in response to a pathological
condition. They seldom have the same diagnostic significance
as plasma abnormalities. The clinically important CSF chemicals
are few; under certain conditions, it may be necessary to measure
a larger variety.

Cerebrospinal Protein
The chemical test performed most frequently on CSF is the pro-
tein determination. Normal CSF contains a very small amount
of protein. Reference values for total CSF protein usually are
Figure 10–34 Burkitt lymphoma. Notice characteristic vacuoles listed as 15 to 45 mg/dL but are somewhat method dependent,
(×500). and higher values are found in infants and people over age 40.14
This value is reported in milligrams per deciliter and not grams
per deciliter, as are plasma protein concentrations.
In general, the CSF contains protein fractions similar to
those found in serum; however, the ratio of CSF proteins to
serum proteins varies among the fractions. As in serum, albumin
makes up most of the CSF protein. But in contrast to serum,
transthyretin (previously called prealbumin) is the second most
prevalent fraction in CSF. The alpha globulins include primarily
haptoglobin and ceruloplasmin. Transferrin is the major beta
globulin present; also, a separate carbohydrate-deficient trans-
ferrin fraction, referred to as “tau,” is seen in CSF but not in
serum. CSF gamma globulin is primarily immunoglobulin G
(IgG), with only a small amount of immunoglobulin A (IgA).
Immunoglobulin M (IgM), fibrinogen, and beta lipoprotein are
not found in normal CSF.15
Figure 10–35 Medulloblastoma (×1000). Notice cellular clustering,
nuclear irregularities, and rosette formation. Clinical Significance of Elevated Protein Values
Elevated total protein values are seen most frequently in patho-
Malignant Cells of Nonhematologic Origin logical conditions. Abnormally low values are present when
fluid is leaking from the CNS. The causes of elevated CSF pro-
Metastatic carcinoma cells of nonhematologic origin are primarily tein include damage to the blood–brain barrier, immunoglob-
from malignancies in the lung, breast, renal system, and gastroin- ulin production within the CNS, decreased normal protein
testinal system. Cells from primary CNS tumors include astrocy- clearance from the fluid, and neural tissue degeneration. Menin-
tomas, retinoblastomas, and medulloblastomas (Fig. 10-35). gitis and hemorrhage conditions that damage the blood–brain
They usually appear in clusters and must be distinguished from barrier are the most common causes of elevated CSF protein.
normal clusters of ependymal, choroid plexus, lymphoma, and Many other neurological disorders can elevate the CSF protein,
leukemia cells. Fusing of cell walls and nuclear irregularities and and finding an abnormal result on clear fluid with a low cell
hyperchromatic nucleoli are seen in clusters of malignant cells. count is not unusual (Box 10-1).

Methodology
Technical Tip 10-6. Slides containing abnormal
The two techniques used most routinely for measuring total
cells must be referred to pathology following facility
CSF protein use the principles of turbidity production and dye-
protocol.
binding ability. The turbidity method has been adapted to au-
tomated instrumentation in the form of nephelometry. Methods
for measuring CSF protein are available for most automated
Chemistry Tests chemistry analyzers.
Because CSF is formed by filtration of the plasma, one would
Protein Fractions
expect to find the same low-molecular-weight chemicals in
the CSF that are found in the plasma. This is essentially true; Routine CSF protein procedures are designed to measure total
however, because the filtration process is selective and the protein concentration. However, diagnosis of neurological
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Chapter 10 | Cerebrospinal Fluid 267

Box 10–1 Clinical Causes of Abnormal CSF Protein Normal IgG index values vary slightly among laboratories;
Values* however, in general, values greater than 0.70 indicate IgG pro-
duction within the CNS.
Elevated Results Techniques for measuring CSF albumin and globulin have
• Meningitis • Myxedema been adapted to automated instrumentation.
• Hemorrhage • Cushing disease
Electrophoresis and Immunophoretic Techniques
• Primary CNS tumors • Connective tissue disease
The primary purpose for performing CSF protein electrophoresis
• Multiple sclerosis • Polyneuritis
is to detect oligoclonal bands, which represent inflammation
• Guillain-Barré syndrome • Diabetes within the CNS. The bands are located in the gamma region of
• Neurosyphilis • Uremia the protein electrophoresis, indicating immunoglobulin produc-
• Polyneuritis tion. To ensure that the oligoclonal bands are present as the result
of neurological inflammation, simultaneous serum electrophore-
Decreased Results
sis must be performed. Disorders, such as leukemia, lymphoma,
• CSF leakage/trauma • Rapid CSF production and viral infections, may produce serum banding, which can
• Recent puncture • Water intoxication appear in the CSF as a result of leakage at the blood–brain barrier
or traumatic introduction of blood into the CSF specimen. Band-
*Reference values for protein are usually 15 to 45 mg/dL, but are ing representing both systemic and neurological involvement is
method dependent, and higher values are found in infants and seen in the serum and CSF with HIV infection.17
people older than 40 years of age. The presence of two or more oligoclonal bands in the CSF
that are not present in the serum can be a valuable tool in
diagnosing multiple sclerosis, particularly when accompanied
by an increased IgG index (Fig. 10-36). Other neurological dis-
orders, including encephalitis, neurosyphilis, Guillain-Barré
disorders associated with abnormal CSF protein often re- syndrome, and neoplastic disorders, also produce oligoclonal
quires measurement of the individual protein fractions. Pro- banding that may not be present in the serum. Therefore, the
tein that appears in the CSF as a result of damage to the presence of oligoclonal banding must be considered in con-
integrity of the blood–brain barrier contains fractions pro- junction with clinical symptoms. Oligoclonal banding remains
portional to those in plasma, with albumin present in the positive during remission of multiple sclerosis but disappears
highest concentration. Diseases, including multiple sclerosis, in other disorders.13
that stimulate the immunocompetent cells in the CNS show
a higher proportion of IgG.
To determine accurately whether IgG is increased because
it is being produced within the CNS or is elevated as the result
of a defect in the blood–brain barrier, comparisons must be
made between serum and CSF levels of albumin and IgG.
Methods include the CSF/serum albumin index to evaluate the
integrity of the blood–brain barrier and the CSF IgG index to
measure IgG synthesis within the CNS.
The CSF/serum albumin index is calculated after deter-
mining the concentration of CSF albumin in milligrams per
deciliter and the serum concentration in grams per deciliter.
The formula used is as follows:

CSF albumin (mg/dL)

CSF/serum albumin index =
Serum albumin (g/dL)

An index value less than 9 represents an intact blood–

brain barrier. The index increases relative to the amount of
damage to the barrier.
Calculation of an IgG index, which is actually a comparison
of the CSF/serum albumin index with the CSF/serum IgG index,
compensates for any IgG entering the CSF via the blood–brain
barrier.16 It is performed by dividing the CSF/serum IgG index
by the CSF/serum albumin index as follows: Figure 10–36 Normal and abnormal oligoclonal banding. (Photo-
graph courtesy of the University of Pittsburgh Department of Pathol-
CSF IgG (mg/dL)/serum IgG (g/dL) ogy, Case Index files, Case 059; http://path.upmc.edu/cases/case59.
IgG index =
CSF albumin (mg/dL)/serum albumin (g/dL) html, with permission.)
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268 Part Three | Other Body Fluids

Low protein levels in the CSF make concentration of the CSF Lactate
fluid before performing electrophoresis essential for most elec-
trophoretic techniques. Better resolution can be obtained using CSF lactate levels can be a valuable aid in diagnosing and man-
CSF immunofixation electrophoresis (IFE) and isoelectric aging meningitis cases. In bacterial, tubercular, and fungal
focusing (IEF) followed by silver staining. Specimen concen- meningitis, CSF lactate levels greater than 25 mg/dL occur
tration is not required by the more sensitive IEF procedure. much more consistently than do decreased glucose and pro-
These techniques are also the method of choice when de- vide more reliable information when the initial diagnosis is dif-
termining whether a fluid is actually CSF. CSF can be identified ficult. Levels greater than 35 mg/dL are seen frequently with
based on the appearance of the previously mentioned extra iso- bacterial meningitis, whereas in viral meningitis, lactate levels
form of tau transferrin that is found only in CSF.18 remain lower than 25 mg/dL. CSF lactate levels remain ele-
vated during initial treatment but fall rapidly when treatment
Myelin Basic Protein is successful, thus offering a sensitive method for evaluating
the effectiveness of antibiotic therapy.
Myelin basic protein (MBP) is a major component of the
Tissue destruction within the CNS due to oxygen depri-
myelin nerve sheath surrounding axons of nerves in the nerv-
vation (hypoxia) increases levels of lactic acid in the CSF.
ous system. The presence of MBP in the CSF indicates recent
Therefore, elevated CSF lactate is not limited to meningitis and
destruction of the myelin sheath that protects the axons of the
can result from any condition that decreases oxygen flow to
neurons (demyelination). The course of multiple sclerosis can
the tissues. Frequently CSF lactate levels are used to monitor
be monitored by measuring the amount of MBP in the CSF.19
patients with severe head injuries. RBCs contain high concen-
MBP also may provide a valuable measure of the effectiveness
trations of lactate, and results that are falsely elevated may be
of current and future treatments. Other causes of increased
obtained on xanthochromic or hemolyzed fluid.11
MBP include CNS trauma, encephalopathies, Guillain-Barre
syndrome, lupus, and brain tumors.20 Immunoassay techniques CSF Glutamine
are used for measurement.21
Glutamine is produced from ammonia and ␣-ketoglutarate by
CSF Glucose the brain cells. This process serves to remove the toxic meta-
bolic waste product ammonia from the CNS. The normal con-
Glucose enters the CSF by selective transport across the blood–
centration of glutamine in the CSF is 8 to 18 mg/dL.23 Elevated
brain barrier, which results in a reference value that is approx-
levels are associated with liver disorders that result in in-
imately 60% to 70% that of the plasma glucose. If the plasma
creased blood and CSF ammonia. Excess ammonia in the CNS
glucose is 100 mg/dL, then a reference CSF glucose would be
increases glutamine synthesis; therefore, determining CSF glu-
approximately 65 mg/dL. For an accurate evaluation of CSF
tamine provides an indirect test for the presence of excess
glucose, a blood glucose test must be run for comparison. The
ammonia in the CSF. Several methods of assaying glutamine
blood glucose should be drawn about 2 hours before the spinal
are available and are based on the measurement of ammonia
tap to allow time for equilibration between the blood and fluid.
liberated from the glutamine. This is preferred over the direct
CSF glucose is analyzed using the same procedures employed
measurement of CSF ammonia because the glutamine concen-
for blood glucose. Specimens should be tested immediately
tration remains more stable than the volatile ammonia con-
because glycolysis occurs rapidly in CSF.
centration in the collected specimen. The CSF glutamine level
The diagnostic significance of CSF glucose is confined to
also correlates with clinical symptoms much better than does
finding values that are decreased relative to plasma values.
the blood ammonia.23
Elevated CSF glucose values are always a result of plasma ele-
As the concentration of ammonia in the CSF increases, the
vations. Low CSF glucose values can be of considerable diag-
supply of ␣-ketoglutarate becomes depleted; glutamine can no
nostic value in determining the causative agents in meningitis.
longer be produced to remove the toxic ammonia, and coma
A markedly decreased level of CSF glucose accompanied by an
ensues. Some disturbance of consciousness is almost always seen
increased WBC count and a large percentage of neutrophils
when glutamine levels are more than 35 mg/dL.15 Therefore, the
indicates bacterial meningitis. If the WBCs are lymphocytes
CSF glutamine test is requested frequently for patients with coma
instead of neutrophils, tubercular meningitis is suspected. Like-
of unknown origin. In addition, approximately 75% of children
wise, if a normal value for CSF glucose is found with an
with Reye syndrome have elevated levels of CSF glutamine.24
increased number of lymphocytes, the diagnosis would favor
A summary of CSF chemistry tests is presented in
viral meningitis. Classic laboratory patterns such as those just
Table 10-4.
described may not be found in all cases of meningitis, but they
can be helpful when they appear.
Decreased values for CSF glucose are caused primarily by Microbiology Tests
alterations in the mechanisms of glucose transport across the
blood–brain barrier and by increased use of glucose by the brain The role of the microbiology laboratory in analyzing CSF is to
cells. The common tendency to associate decreased glucose identify the causative agent in meningitis. For positive identi-
totally with its use by microorganisms and leukocytes cannot fication, the microorganism must be recovered from the fluid
account for the variations in glucose concentrations seen in dif- by growing it on the appropriate culture medium. This can
ferent types of meningitis and the decreased levels seen in other take anywhere from 24 hours in cases of bacterial meningitis
disorders producing damage to the CNS.22 to 6 weeks for tubercular meningitis. Consequently, in many
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Chapter 10 | Cerebrospinal Fluid 269

Table 10–4 CSF Chemistry Tests

Reference Concentration Significance of Increased Significance of Decreased
Chemical Substance Value, Normal CSF Concentration Concentration
Protein 15–45 mg/dL Meningitis CSF leakage
Hemorrhage
Multiple sclerosis
Glucose 60%–70% of plasma con- None Bacterial, tubercular, and fungal
centration meningitis
Lactate 10–24 mg/dL >35 mg/dL: Bacterial meningitis None
Glutamine 8–18 mg/dL >35 mg/dL: Some disturbance None
of consciousness

instances, the CSF culture is a procedure that is actually con- and cultures should be prepared from the sediment.25 Use of
firmatory rather than diagnostic. However, the microbiology the cytocentrifuge provides a highly concentrated specimen
laboratory does have several methods available to provide in- for Gram stains. Even when concentrated specimens are used,
formation for a preliminary diagnosis. These methods include at least a 10% chance exists that Gram stains and cultures will
the Gram stain, acid-fast stain, India ink preparation, immuno- be negative. Thus, blood cultures should be taken because
logic assays (latex agglutination, coagglutination, immunoassay, often the causative organism is present in both the CSF and
and counterimmunoelectrophroesis), and molecular diagnostic the blood.11 A CSF Gram stain is one of the most difficult
nucleic acid amplification tests, such as polymerase chain slides to interpret because the number of organisms present
reaction (PCR) assay. In Table 10-5, laboratory tests used in is usually small, and they can be overlooked easily, resulting
the differential diagnosis of meningitis are compared. in a false-negative report. Also, false-positive reports can occur
if precipitated stain or debris is mistaken for microorganisms.
Therefore, considerable care should be taken when interpreting
Gram Stain a Gram stain. Organisms encountered most frequently include
The Gram stain is performed routinely on CSF from all suspected Streptococcus pneumoniae (gram-positive cocci), Haemophilus
cases of meningitis, although its value lies in detecting bacte- influenzae (pleomorphic gram-negative rods), Escherichia coli
rial and fungal organisms. All smears and cultures should be (gram-negative rods), and Neisseria meningitidis (gram-negative
performed on concentrated specimens because, often, only a cocci). The gram-positive cocci Streptococcus agalactiae and the
few organisms are present at the onset of the disease. The CSF gram-positive rods Listeria monocytogenes may be encountered
should be centrifuged at 1500 g for 15 minutes, and slides in newborns.

Table 10–5 Major Laboratory Results for Differential Diagnosis of Meningitis

Bacterial Viral Tubercular Fungal
Elevated WBC count Elevated WBC count Elevated WBC count Elevated WBC count
Neutrophils present Lymphocytes present Lymphocytes and Lymphocytes and monocytes
monocytes present present
Marked protein elevation Moderate protein Moderate to marked Moderate to marked protein
elevation protein elevation elevation
Markedly decreased glucose level Normal glucose level Decreased glucose level Normal to decreased glucose level
Lactate level >35 mg/dL Normal lactate level Lactate level >25 mg/dL Lactate level >25 mg/dL
Positive India ink with
Cryptococcus neoformans
Positive Gram stain and bacterial Pellicle formation Positive immunological test
antigen tests for C. neoformans
PCR positive for Streptococcus PCR positive for PCR positive for PCR positive for Cryptococcus
pneumonia, Streptococcus enterovirus, herpes Mycobacterium neoformans
agalactiae, Neisseria meningitidis, simplex virus, tuberculosis
Haemophilus influenzae, Listeria parechovirus
monocytogenes, Escherichia coli K1
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270 Part Three | Other Body Fluids

Acid-fast or fluorescent antibody stains are not performed occur. Interference by rheumatoid factor is the most common
routinely on specimens unless tubercular meningitis is suspected. cause of false-positive reactions.26 Several commercial kits with
Considering the length of time required to culture Mycobacteria, pretreatment techniques are available and include incubation
a positive report from this smear is extremely valuable. with dithiothreitol or pronase and boiling with ethylenedi-
Specimens from possible cases of fungal meningitis are aminetetra-acetic acid.27,28 An enzyme immunoassay technique
Gram stained and often have an India ink preparation per- has been shown to produce fewer false-positive results.
formed on them to detect the presence of thickly encapsulated The lateral flow assay (LFA) can provide a rapid method for
Cryptococcus neoformans (Fig. 10-37). As one of the complica- detecting C. neoformans with a high sensitivity and specificity.
tions of AIDS that occurs more frequently, cryptococcal menin- The procedure utilizes a reagent strip coated with monoclonal
gitis is now encountered commonly in the clinical laboratory. antibodies that react with the cryptococcal polysaccharide
Particular attention should be paid to the Gram stain for the capsule.29
classic starburst pattern produced by Cryptococcus, as this may Latex agglutination and enzyme-linked immunosorbent
be seen more often than a positive India ink (Fig. 10-38).26 assay (ELISA) methods provide a rapid means for detecting and
identifying microorganisms in CSF. Test kits are available to detect
Immunologic Assays Streptococcus group B; H. influenzae type B; S. pneumoniae; N.
Latex agglutination tests to detect the presence of C. neoformans meningitidis A, B, C, Y, and W135; Mycobacterium tuberculosis; C.
antigen in serum and CSF provide a more sensitive method immitis; and E. coli K1 antigens. The bacterial antigen test (BAT)
than the India ink preparation. However, immunologic testing does not appear to be as sensitive to N. meningitidis as it is to the
results should be confirmed by culture and demonstration of other organisms. The BAT should be used in combination with
the organisms by India ink because false-positive reactions do results from the hematology and clinical chemistry laboratories
for diagnosing meningitis.30 The Gram stain is still the recom-
mended method for detecting organisms.31
The amoeba Naegleria fowleri is an opportunistic parasite
found in ponds, small lakes, and even chlorinated swimming
pools. The Naegleria enters the nasal passages and migrates
along the olfactory nerves to invade the brain. Motile tropho-
zoites can be observed microscopically by examining a wet
preparation of CSF. Nonmotile trophozoites may be seen on
cytocentrifuged stained smears accompanied by increased
WBCs and no bacteria. Figure 10-39 shows a Naegleria tropho-
zoite. Notice the elongated form with a tapered posterior.32

PCR Molecular Diagnostic Testing

Gram staining is a specific method for detecting bacteria in CSF,
but that method lacks sensitivity. Culture is more sensitive,
but detection rates depend on the number of viable bacteria.
Figure 10–37 India ink preparation of C. neoformans capsular Nucleic acid amplification tests can detect the cause of menin-
halo (×400). Notice budding yeast form. (Courtesy of Ann K. gitis with a small amount of the pathogen’s DNA. Universal PCR
Fulenwider, MD.) detects pathogens with increased sensitivity and specificity. PCR
assays are based on the amplification of regions of ribosomal
RNA (rRNA) genes to detect and differentiate causative
pathogens of meningitis.33

Figure 10–39 N. fowleri trophozoite. (From Leventhal & Cheadle,

Figure 10–38 Gram stain of C. neoformans showing starburst Medical Parasitology, 6th ed., 2012, FA Davis Company, Philadelphia,
pattern (×1000). (Courtesy of Ann K. Fulenwider, MD.) with permission.)
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Chapter 10 | Cerebrospinal Fluid 271

Serological Testing 12728. Epub September 7, 2017. Web site: https://www.ncbi.

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10. University of Virginia Health Sciences Center: Clinical Labora-
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tification of microorganisms, serological testing of the CSF is 11. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory
performed to detect the presence of neurosyphilis. The use of Examination of Amniotic, Cerebrospinal, Seminal, Serous
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available when testing blood, the procedure recommended by children: Age-related values in patients without disorders of the
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Evaluation of the automated hematology analyzer ADVIA®120 interference. J Clin Microbiol 17(5):945–947, 1983.
for cerebrospinal fluid analysis and usage of unique hemolysis 29. Knight, FR: New enzyme immunoassay for detecting crypto-
reagent. International Journal of Laboratory Hematology. Int. J. coccal antigen. J Clin Pathol 45(9):836–837, 1992.
Lab Hematol. 2014 Feb;36(1):83-91. Doi: 10.1111/ijlh.12130. 30. Klausner, JD, Vijayan, T, Chiller, T: Sensitivity and specificity of
Epub August 13, 2013. Web site: https://www.ncbi.nlm.nih. a new cryptococcal antigen lateral flow assay in serum and
gov/pubmed/23937551 Accessed June 27, 2019. cerebrospinal fluid. MLO 45(3):16,18,20, 2013.
8. CAP TODAY 2019. Beckman Coulter, Unicel DxH900 Product 31. Wojewoda, C: Bacterial Antigen Testing, The Good, the Not So
Guide. Web site: https://captodayonline.com/productguides/ Bad and the Ugly. NewsPath. http://www.cap.org/apps/docs/
beckman-coulter-unicel-dxh-900-hematology-analyzers- newspath/1102/bacterial_antigen_testing.pdf. Accessed
november-2018.html. Accessed June 27, 2019. March 31, 2020.
9. Hod EA, Brugnara C, Pilichowska, M, et al: Automated cell counts 32. Leventhal, R and Cheadle, RF: Medical Parasitology, ed.6,
on CSF samples: A multicenter performance evaluation of the FA Davis Company, Philadelphia, 2012.
GloCyte system. International Journal of Laboratory Hematology. 33. Meyer, T, Franke, G, Polywka, KA, Lutgehetmann, M,
Int J Lab Hematol. 2018 Feb;40(1):56-65. Doi: 10.1111/ijlh. Gbadamosi, J, Magnus, T, Aepfelbacher, M: Patel, R, Editor:
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272 Part Three | Other Body Fluids

Improved Detection of Bacterial Central Nervous System 35. Albright, RE, et al: Issues in cerebrospinal fluid management.
Infections by Use of Broad-Range PCR Assay. J Clin Microbiol Am J Clin Pathol 95(3):397–401, 1991.
52(5):1751–1753, 2014. DOI: 10.1128/JCM.00469-14. https:// 36. Lofsness, KG, and Jensen, TL: The preparation of simulated
jcm.asm.org/content/52/5/1751. Accessed June 29, 2019. spinal fluid for teaching purposes. Am J Med Technology
34. Davis, LE, and Schmitt, JW: Clinical significance of cere- 49(7):493–496, 1983.
brospinal fluid tests for neurosyphilis. Ann Neurol 25:50–53,
1989.

Study Questions
1. CSF is produced mainly in the: 7. Place the appropriate letter in front of the statement that
A. Bone marrow best describes CSF specimens in these two conditions:
B. Peripheral blood A. Traumatic tap
C. Choroid plexuses B. Intracranial hemorrhage
D. Subarachnoid space Even distribution of blood in all tubes
Xanthochromic supernatant
2. The functions of the CSF include all of the following
except: Concentration of blood in Tube 1 is greater
than in Tube 3
A. Removing metabolic wastes
Specimen contains clots
B. Producing an ultrafiltrate of plasma
C. Supplying nutrients to the CNS 8. The presence of xanthochromia can be caused by all of
the following except:
D. Protecting the brain and spinal cord
A. Immature liver function
3. The CSF flows through the:
B. RBC degradation
A. Choroid plexus
C. A recent hemorrhage
B. Pia mater
D. Elevated CSF protein
C. Subarachnoid space
9. A web-like pellicle in a refrigerated CSF specimen
D. Dura mater
indicates:
4. Substances present in the CSF are controlled by the: A. Tubercular meningitis
A. Arachnoid granulations B. Multiple sclerosis
B. Blood–brain barrier C. Primary CNS malignancy
C. Presence of one-way valves D. Viral meningitis
D. Blood–CSF barrier
10. Given the following information, calculate the CSF WBC
5. What department is the CSF tube labeled 3 routinely count: cells counted, 80; dilution, 1:10; large Neubauer
sent to? squares counted, 10.
A. Hematology A. 8
B. Chemistry B. 80
C. Microbiology C. 800
D. Serology D. 8000
6. The CSF tube that should be kept at room temperature is: 11. A CSF WBC count is diluted with:
A. Tube 1 A. Distilled water
B. Tube 2 B. Normal saline
C. Tube 3 C. Acetic acid
D. Tube 4 D. Hypotonic saline
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Chapter 10 | Cerebrospinal Fluid 273

12. A total CSF cell count on a clear fluid should be: 20. Hemosiderin granules and hematoidin crystals are seen in:
A. Reported as normal A. Lymphocytes
B. Not reported B. Macrophages
C. Diluted with normal saline C. Ependymal cells
D. Counted undiluted D. Neutrophils
13. The purpose of adding albumin to CSF before 21. Myeloblasts are seen in the CSF:
cytocentrifugation is to: A. In bacterial infections
A. Increase the cell yield B. In conjunction with CNS malignancy
B. Decrease the cellular distortion C. After cerebral hemorrhage
C. Improve the cellular staining D. As a complication of acute leukemia
D. Both A and B
22. Cells resembling large and small lymphocytes with
14. The primary concern when pleocytosis of neutrophils cleaved nuclei represent:
and lymphocytes is found in the CSF is: A. Lymphoma cells
A. Meningitis B. Choroid cells
B. CNS malignancy C. Melanoma cells
C. Multiple sclerosis D. Medulloblastoma cells
D. Hemorrhage
23. The reference range for CSF protein is:
15. Neutrophils with pyknotic nuclei may be mistaken for: A. 6 to 8 g/dL
A. Lymphocytes B. 15 to 45 g/dL
B. Nucleated RBCs C. 6 to 8 mg/dL
C. Malignant cells D. 15 to 45 mg/dL
D. Spindle-shaped cells
24. CSF can be differentiated from serum by the presence of:
16. The presence of which of the following cells is increased A. Albumin
in a parasitic infection?
B. Globulin
A. Neutrophils
C. Transthyretin
B. Macrophages
D. Tau transferrin
C. Eosinophils
25. In serum, the second most prevalent protein is IgG; in
D. Lymphocytes
CSF, the second most prevalent protein is:
17. Macrophages appear in the CSF after: A. Transferrin
A. Hemorrhage B. Transthyretin
B. Repeated spinal taps C. Prealbumin
C. Diagnostic procedures D. Ceruloplasmin
D. All of the above
26. Elevated values for CSF protein can be caused by all of
18. Nucleated RBCs are seen in the CSF as a result of: the following except:
A. Elevated blood RBCs A. Meningitis
B. Treatment of anemia B. Multiple sclerosis
C. Severe hemorrhage C. Fluid leakage
D. Bone marrow contamination D. CNS malignancy
19. After a CNS diagnostic procedure, which of the 27. The integrity of the blood–brain barrier is measured
following might be seen in the CSF? using the:
A. Choroidal cells A. CSF/serum albumin index
B. Ependymal cells B. CSF/serum globulin ratio
C. Spindle-shaped cells C. CSF albumin index
D. All of the above D. CSF IgG index
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274 Part Three | Other Body Fluids

28. Given the following results, calculate the IgG index: 34. Measurement of which of the following can be replaced
CSF IgG, 50 mg/dL; serum IgG, 2 g/dL; CSF albumin, by CSF glutamine analysis in children with Reye
70 mg/dL; serum albumin, 5 g/dL. syndrome?
A. 0.6 A. Ammonia
B. 6.0 B. Lactate
C. 1.8 C. Glucose
D. 2.8 D. ␣-Ketoglutarate
29. The CSF IgG index calculated in Study Question 28 35. Before performing a Gram stain on CSF, the specimen
indicates: must be:
A. Synthesis of IgG in the CNS A. Filtered
B. Damage to the blood–brain barrier B. Warmed to 37°C
C. Cerebral hemorrhage C. Centrifuged
D. Lymphoma infiltration D. Mixed
30. The finding of oligoclonal bands in the CSF and not in 36. All of the following statements are true about
the serum is seen with: cryptococcal meningitis except:
A. Multiple myeloma A. An India ink preparation is positive
B. CNS malignancy B. A starburst pattern is seen on Gram stain
C. Multiple sclerosis C. The WBC count is over 2000
D. Viral infections D. A confirmatory immunology test is available
31. Which condition is suggested by the following results: 37. The most sensitive and specific method to detect the
a CSF glucose of 15 mg/dL, WBC count of 5000, causative organism in meningitis is:
90% neutrophils, and protein of 80 mg/dL? A. Gram stain
A. Fungal meningitis B. Culture and sensitivity
B. Viral meningitis C. India ink stain
C. Tubercular meningitis D. PCR assay
D. Bacterial meningitis
38. The test of choice to detect neurosyphilis is the:
32. A patient with a blood glucose of 120 mg/dL would A. RPR
have a normal CSF glucose of:
B. VDRL
A. 20 mg/dL
C. FAB
B. 60 mg/dL
D. FTA-ABS
C. 80 mg/dL
D. 120 mg/dL
33. CSF lactate will be more consistently decreased in:
A. Bacterial meningitis
B. Viral meningitis
C. Fungal meningitis
D. Tubercular meningitis
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Chapter 10 | Cerebrospinal Fluid 275

Case Studies and Clinical Situations

1. Three tubes of CSF containing evenly distributed visible a. Name and perform the calculation to determine the
blood are drawn from a 75-year-old disoriented patient integrity of the patient’s blood–brain barrier.
and delivered to the laboratory. Initial test results are as b. Does the patient have an intact barrier?
follows:
c. Name and perform the calculation used to determine
WBC count: 250 µL Protein: 150 mg/dL whether IgG is being synthesized within the CNS.
Glucose: 70 mg/dL Gram stain: No organisms d. What does this result indicate?
seen
e. Considering the patient’s clinical symptoms and the
Differential: Neutrophils, 68%; monocytes, 3%; lympho- calculation results, what diagnosis is suggested?
cytes, 28%; eosinophils, 1%
f. If immunofixation electrophoresis is performed on
Many macrophages containing ingested RBCs the patient’s serum and CSF, what findings would be
a. What is the most probable condition indicated by expected?
these results? State two reasons for your answer. g. What substance in the CSF can be measured to
b. Are the elevated WBC count and protein significant? monitor this patient?
Explain your answer.
4. A 5-year-old female patient is admitted to the pediatric
c. Are the percentages of the cells in the differential of ward with a temperature of 105°F, lethargy, and cervical
any significance? Explain your answer. rigidity. A lumbar spinal tap is performed, and three tubes
d. What two other structures besides RBCs might be of cloudy CSF are delivered to the laboratory. Preliminary
contained in the macrophages? test results are as follows:
e. If the blood was distributed unevenly and nucleated Appearance: Cloudy
RBCs and capillary structures were seen instead of WBC count: 800 cells/␮L
macrophages, what would this indicate?
Differential: 80% lymphocytes, 15% monocytes,
2. A patient with AIDS is hospitalized with symptoms of high 5% neutrophils
fever and rigidity of the neck. Routine laboratory tests on Protein: 65 mg/dL
the CSF show a WBC count of 100/µL with a predomi-
Glucose: 70 mg/dL
nance of lymphocytes and monocytes, glucose of 55 mg/dL
(plasma: 85 mg/dL), and a protein of 70 mg/dL. The Gram Gram stain: No organisms seen
stain shows a questionable starburst pattern. a. From these results, what preliminary diagnosis could
a. What additional microscopic examination should be the physician consider?
performed? b. Is the Gram stain result of particular significance?
b. If the test is positive, what is the patient’s diagnosis? Why or why not?
c. If the results of the test are questionable, what addi- c. Are the lymphocytes significant? Why or why not?
tional testing can be performed? d. Would a CSF lactate test be of any value for the diag-
d. What could cause a false-positive reaction in this test? nosis? Why or why not?
e. If the tests named in a and c are negative, the glucose 5. State possible technical errors that could result in the fol-
level is 35 mg/dL, and a pellicle is observed in the lowing discrepancies:
fluid, what additional testing should be performed? a. An unusual number of Gram stains reported as gram-
f. If CSF and serum IEF was performed on this patient, positive cocci fail to be confirmed by positive cultures.
what unusual findings might be present? b. A physician complains that CSF differentials are being
3. A 35-year-old woman is admitted to the hospital with reported only as polynuclear and mononuclear cells.
symptoms of intermittent blurred vision, weakness, and c. Bacteria observed on the cytospin differential cannot
loss of sensation in her legs. A lumbar puncture is per- be confirmed by Gram stain or culture.
formed with the following results: d. The majority of CSF specimens sent to the laboratory
Appearance: Colorless, clear from the neurology clinic have glucose readings of less
WBC count: 35 cells/µL (90% lymphocytes) than 50% of the corresponding blood glucose results
performed in the clinic.
Glucose: 60 mg/dL (plasma: 100 mg/dL)
Protein: 60 mg/dL (serum: 8 g/dL)
Albumin: 40 mg/dL (serum: 6 g/dL)
IgG globulin: 20 mg/dL (serum: 2 g/dL)
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CHAPTER 11
Semen
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
11-1 State the structures involved in sperm production 11-10 Describe the appearance of normal sperm, including
and their function. structures and their functions.
11-2 Describe the four components of semen with regard 11-11 Differentiate between routine and strict criteria for
to source and function. evaluating sperm morphology.
11-3 Explain the procedures for collecting and handling 11-12 Given an abnormal result in a routine semen analysis,
semen specimens. determine additional tests that might be performed.
11-4 Describe the normal appearance of semen and three 11-13 Describe the two methods routinely used to detect
abnormalities in appearance. antisperm antibodies.
11-5 State two possible causes of low semen volume. 11-14 List two methods for identifying a questionable fluid
as semen.
11-6 Discuss the significance of semen liquefaction and
viscosity. 11-15 State the World Health Organization reference values
for routine and follow-up semen analysis.
11-7 Calculate a sperm concentration and count when
provided with the number of sperm counted, the di- 11-16 Discuss the types and significance of sperm function
lution, the area of the counting chamber used, and tests.
the ejaculate volume.
11-17 Describe methods of quality control appropriate for
11-8 Define round cells, and explain their significance. semen analysis.
11-9 State the two parameters to consider when evaluating
sperm motility.

KEY TERMS
Acrosomal cap Prostate gland Spermatozoa
Andrology Semen Testes
Bulbourethral gland Seminal vesicles Vasectomy
Epididymis Seminiferous tubules Viscosity
Liquefaction Spermatids
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278 Part Three | Other Body Fluids

Introduction proteolytic enzymes responsible for both the coagulation and

liquefaction of the semen after ejaculation.
Advances in the field of andrology and assisted reproductive The bulbourethral glands, located below the prostate,
technology (ART), as well as increased concern over fertility, contribute about 5% of the fluid volume in the form of a thick,
particularly by couples choosing to have children later in life, alkaline mucus that helps neutralize acidity from the prostate
have resulted in increased emphasis on semen analysis. Patients secretions and the vagina. It is important for semen to be alka-
with abnormal results on the routine semen analysis performed line to neutralize the vaginal acidity present as a result of nor-
in the clinical laboratory often are referred to specialized androl- mal bacterial vaginal flora. Without this neutralization, sperm
ogy laboratories for further testing to determine the need for motility would be diminished.
in vitro fertilization (IVF). Clinical laboratory personnel also
may be employed in andrology laboratories and perform both
routine and specialized testing.
Specimen Collection
In addition to fertility testing, the clinical laboratory per- The variety in the composition of the semen fractions makes
forms postvasectomy semen analysis and forensic analyses to proper collection of a complete specimen essential for accurate
determine the presence of semen. evaluation of male fertility. Most of the sperm are contained in
the first portion of the ejaculate, making complete collection
Physiology essential for accurate testing of both fertility and postvasectomy
specimens. When a part of the first portion of the ejaculate is
Semen is composed of four fractions that are contributed by the missing, the sperm count will be decreased, the pH will be
testes, epididymis, seminal vesicles, prostate gland, and bul- falsely increased, and the specimen will not liquefy. Likewise,
bourethral glands (Fig. 11-1). Each fraction differs in its com- when part of the last portion of ejaculate is missing, the semen
position, and the mixing of all four fractions during ejaculation volume is decreased, the sperm count is falsely increased, the
is essential for the production of a normal semen specimen pH is falsely decreased, and the specimen will not clot. Patients
(Table 11-1). should receive detailed written instructions for specimen
The testes are paired glands in the scrotum that contain the collection.
seminiferous tubules for the secretion of sperm. The external Specimens are collected after a period of sexual abstinence
location of the scrotum contributes to a lower scrotum temper- of at least 2 days to not more than 7 days.2 Specimens collected
ature that is optimal for sperm development. Germ cells for the after prolonged abstinence tend to have higher volumes and
production of spermatozoa are located in the epithelial cells of decreased motility.3 When performing fertility testing, the
the seminiferous tubules. Specialized Sertoli cells provide sup- World Health Organization (WHO) recommends that two
port and nutrients for the germ cells as they undergo mitosis and or three specimens be collected not less than 7 days or more
meiosis (spermatogenesis). When spermatogenesis is complete, than 3 weeks apart, with two abnormal specimens considered
the immature sperm (nonmotile) enter the epididymis. In the significant. The laboratory should provide the patient with
epididymis, the sperm mature and develop flagella. The entire warm sterile glass or plastic containers. Whenever possible, the
process takes approximately 90 days. The sperm remain stored specimen is collected in a room provided by the laboratory.
in the epididymis until ejaculation, at which time they are pro- However, if this is not appropriate, the specimen should be
pelled through the ductus deferens (vas deferens) to the ejacu-
latory ducts.
The ejaculatory ducts receive both the sperm from the
ductus deferens and fluid from the seminal vesicles. The sem- SUMMARY 111 Semen Production
inal vesicles produce most of the fluid present in semen (60%
Structure Function
to 70%), and this fluid is the transport medium for the sperm.
The fluid contains a high concentration of fructose and flavin. Seminiferous tubules of Spermatogenesis
Spermatozoa metabolize the fructose for the energy needed for testes
the flagella to propel them through the female reproductive Epididymis Sperm maturation
tract. In the absence of fructose, sperm do not display motility
Ductus deferens Propel sperm to ejaculatory
in the semen analysis. Flavin is responsible for the gray appear-
ducts
ance of semen, as well as the blue to yellow fluorescence when
semen is visualized under ultraviolet light (Wood’s lamp).1 Seminal vesicles Provide nutrients for sperm
Various proteins secreted by the seminal vesicles are involved and fluid
in the coagulation of the ejaculate. Prostate gland Provides enzymes and
The muscular prostate gland, located just below the blad- proteins for coagulation
der, surrounds the upper urethra and aids in propelling the and liquefaction
sperm through the urethra by contractions during ejaculation. Bulbourethral glands Add alkaline mucus to
Approximately 20% to 30% of the semen volume is acidic fluid neutralize prostatic acid
produced by the prostate gland. The milky acidic fluid contains and vaginal acidity
high concentrations of acid phosphatase, citric acid, zinc, and
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Chapter 11 | Semen 279

Seminal
vesicle

Urinary Rectum
bladder

Ejaculatory
Vas duct
deferens

Prostate
gland
Urethra

Penis

Glans Epididymis
penis Anus
Testis
Bulbourethral
Scrotum gland

Ureter
Urinary
bladder

Vas
deferens Seminal
vesicle

Prostate Bulbourethral
gland gland

Epididymis

Testis

Urethra
Penis

Figure 11–1 The male genitalia. Top, sagittal view; bottom, anterior view.

kept at room temperature and delivered to the laboratory

within 1 hour of collection. Laboratory personnel must record
the patient’s name and birth date, the period of sexual absti-
nence, the completeness of the specimen, any difficulties
Table 11–1 Semen Composition with collection, and the times of specimen collection and
specimen receipt. Specimens awaiting analysis should be kept
Spermatozoa 5%
at 37°C. Specimens should be collected by masturbation
Seminal fluid 60%–70% into the container. If this is not possible, only nonspermicidial,
Prostate fluid 20%–30% nonlubricant-containing rubber or polyurethane condoms
Bulbourethral glands 5% should be used. Ordinary condoms are not acceptable because
they contain spermicides.
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280 Part Three | Other Body Fluids

Technical Tip 11-1. Coitus interruptus is not a reliable Table 11–2 Reference Values for Semen
means of semen collection because the first portion of Analysis2,6
the ejaculate, which contains the highest number of
Reference Lower Reference
spermatozoa, may be lost and the low pH of the vagi-
Parameter Values Limit1
nal fluid may affect sperm motility.2
Volume 2–5 mL 1.5 mL
Viscosity Pours in
Specimen Handling droplets
within
All semen specimens are potential reservoirs for HIV, herpes 60 minutes
viruses, and hepatitis viruses, and standard precautions must be
pH 7.2– 8.0 >7.2
observed at all times during analysis. Specimens are discarded
as biohazardous waste. Sterile materials and techniques must be Sperm >20 million/mL 15 million/mL
used when semen culture is to be performed or when the spec- concentration
imen is to be processed for bioassay, intrauterine insemination Sperm count >40 million/ 39 million/
(IUI), or IVF.2 ejaculate ejaculate
Motility >50% within 40% within 1 hr
Semen Analysis 1 hr
Quality >2.0 or a, b, c
Semen analysis for fertility evaluation consists of both macro- in Table 11-3
scopic and microscopic examination. Parameters reported in-
Vitality (% alive) >75% 58%
clude appearance, volume, viscosity, pH, sperm concentration
and count, motility, and morphology. Reference values are Morphology >14% normal 4% normal forms
shown in Table 11-2. forms (strict
criteria)
Appearance >30% normal
Normal semen has a gray-white color, appears translucent, and forms (routine
has a characteristic musty odor. When the sperm concentration criteria)
is very low, the specimen may appear almost clear.2 Increased Round cells <1.0 million/mL
white turbidity indicates the presence of white blood cells
1Lowerreference limits recommended by the WHO (2010) for
(WBCs) and infection within the reproductive tract. If required,
semen characteristics.
specimen culturing is performed before continuing with the
semen analysis. During microscopic examination, WBCs must
be differentiated from immature sperm (spermatids). The
leukocyte esterase reagent strip test may be useful to screen for
bromelain must be accounted for when calculating sperm con-
the presence of WBCs.4 Varying amounts of red coloration are
centration.2 Jelly-like granules (gelatinous bodies) may be present
associated with the presence of red blood cells (RBCs) and are
in liquefied semen specimens and have no clinical significance.
abnormal. Yellow coloration may be caused by urine contami-
Mucous strands, if present, may interfere with semen analysis.2
nation, specimen collection after prolonged abstinence, and
medications. Urine is toxic to sperm, thereby affecting the
evaluation of motility.
Technical Tip 11-2. Delivery of the semen specimen
Liquefaction to the laboratory within 1 hour of collection and docu-
mentation of the collection time are critical to evaluate
A fresh semen specimen is clotted and should liquefy within liquefaction.
30 to 60 minutes after collection; therefore, recording the time
of collection is essential for evaluating semen liquefaction. Failure
of liquefaction to occur within 60 minutes may be caused by a
deficiency in prostatic enzymes and should be reported. Analysis
Volume
of the specimen cannot begin until liquefaction has occurred. If Normal semen volume ranges between 2 and 5 mL. It can be
after 2 hours the specimen has not liquified, an equal volume of measured by pouring the specimen into a clean graduated
Dulbecco’s phosphate-buffered saline (DPBS) (Procedure 11-1) cylinder calibrated in 0.1-mL increments. Increased volume
or proteolytic enzymes, such as alpha-chymotrypsin or bromelain may be seen after periods of extended abstinence. Decreased
(Procedure 11-2), may be added to induce liquefaction and allow volume is associated more frequently with infertility and may
the rest of the analysis to be performed. These treatments may indicate improper functioning of one of the semen-producing
affect biochemical tests, sperm motility, and sperm morphology, organs, primarily the seminal vesicles. Also, incomplete spec-
so their use must be documented. The dilution of semen with imen collection must be considered.
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Chapter 11 | Semen 281

PROCEDURE 11-1
Semen Dilution With Dulbecco’s e. 8.0 g of sodium chloride (NaCl)
Phosphate-Buffered Saline2 f. 2.16 g of disodium hydrogen phosphate heptahydrate
Prepare an equal volume of diluent and semen (one part (Na2HPO4.7H2O)
diluent and one part semen) using Dulbecco’s phosphate- g. 1.00 g of D-glucose
buffered saline. Repeated pipetting of the prepared dilution 2. In a 10-mL volumetric flask, dissolve 0.132 g of
will induce liquefaction. calcium chloride dehydrate (CaCl2.2H2O) in 10 mL
Preparation of Dulbecco’s Phosphate-Buffered Saline2 of purified water.
1. Using a 1-L volumetric flask, add the following: 3. To prevent precipitation, add calcium chloride
a. 750 mL of purified water dehydrated solution to the 1-L flask slowly, stirring
b. 0.20 g of potassium chloride (KCL) continuously.
c. 0.20 g of potassium dihydrogen phosphate (KH2PO4) 4. Adjust the pH to 7.4 with 1 mol/L sodium hydroxide
(NaOH).
d. 0.10 g of magnesium chloride hexahydrate
(MgCl2.6H2O) 5. Make up to 1000 mL with purified water.

PROCEDURE 11-2 Technical Tip 11-3. Gently mixing the specimen

container during liquefaction can help produce a
Digestion with Bromelain2 homogeneous specimen.2
1. Prepare 10 IU/mL bromelain in Dulbecco’s phos-
phate-buffered saline.
a. Into a 100-mL volumetric flask, add 1000 IU of pH
bromelain.
b. Add 60 mL Dulbecco’s phosphate-buffered saline. The pH of semen indicates the balance between the pH values
from the acidic prostatic secretion and the alkaline seminal vesi-
c. Mix to dissolve. It takes about 15 to 20 minutes. cles secretion. The pH should be measured within 1 hour of
Bring the volume to the calibration mark using ejaculation due to the loss of CO2 that occurs.2 The normal pH
buffered saline. of semen is alkaline with a range of 7.2 to 8.0. Increased pH in-
2. Dilute one part semen 1:2 with the 10 IU/mL brome- dicates infection within the reproductive tract. A decreased pH
lain (one part semen + one part bromelain solution). may be associated with increased prostatic fluid, obstruction of
3. Stir with a pipette tip. the ejaculatory duct, or poorly developed seminal vesicles.2
4. Incubate at 37°C for 10 minutes. Semen for pH testing can be applied to the pH pad of a urinal-
ysis reagent strip and the color compared with the manufac-
5. Mix the sample well before analysis. turer’s chart. Dedicated pH testing paper with a range of 4.0 to
10.0 also can be used.The results should be recorded to the
nearest 0.1 pH unit. Also, a pH meter device that provides a
numerical value can be used.

Viscosity Sperm Concentration and Sperm Count

Specimen viscosity refers to the consistency of the fluid and Even though fertilization is accomplished by one spermatozoon,
may be related to specimen liquefaction. Specimens that are the actual number of sperm present in a semen specimen is a
liquefied incompletely are clumped and highly viscous. The valid measurement of fertility. Various factors can affect sperm
normal semen specimen should be drawn into a pipette easily concentration, such as the days of sexual abstinence before the
and form small discrete droplets that do not appear clumped collection, infection, or stress; therefore, more than one semen
or stringy when falling by gravity from the pipette. Droplets specimen should be evaluated for infertility studies. Reference
that form threads longer than 2 cm are considered highly values for sperm concentration are commonly listed as greater
viscous and are recorded as abnormal. Ratings of 0 (watery) to than 20 to 250 million sperm per milliliter; concentrations be-
4 (gel-like) can be assigned to the viscosity report.5 Viscosity tween 10 and 20 million per milliliter are considered border-
also can be reported as low, normal, or high. Increased viscosity line. The total sperm count for the ejaculate can be calculated
and incomplete liquefaction impede testing for sperm motility, by multiplying the sperm concentration by the specimen vol-
sperm concentration, antisperm antibody detection, and meas- ume. Total sperm counts greater than 40 million per ejaculate
urement of biochemical markers.2 are considered normal (20 million per milliliter × 2 mL).
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282 Part Three | Other Body Fluids

In the clinical laboratory, sperm concentration is usually greater than 1 million leukocytes per milliliter is associated
performed using the Neubauer counting chamber. The sperm with inflammation or infection of the reproductive organs that
are counted in the same manner as cells in the cerebrospinal can lead to infertility.
fluid cell count, that is, by diluting the specimen and counting The presence of more than 1 million spermatids per
the cells in the Neubauer chamber. The amount of the dilution milliliter indicates disruption of spermatogenesis. This may
and the number of squares counted vary among laboratories. be caused by viral infections, exposure to toxic chemicals,
The dilution used most commonly is 1:20 prepared using and genetic disorders.
a mechanical (positive-displacement) pipette.6 Dilution of the
semen is essential because it immobilizes the sperm before Calculating Sperm Concentration and Sperm Count
counting. The traditional diluting fluid contains sodium bicar- Calculation of sperm concentration depends on the dilution
bonate and formalin, which immobilize and preserve the cells; used and the size and number of squares counted. When
however, good results also can be achieved using saline and using the 1:20 dilution and counting the five squares (RBCs)
distilled water. in the large center square, as described previously, the num-
Using the Neubauer hemocytometer, sperm usually are ber of sperm can be multiplied by 1,000,000 (add 6 zeros)
counted in the four corner and center squares of the large cen- to equal the sperm concentration per milliliter. Notice that,
ter square, similar to a manual RBC count (Fig. 11-2). Both unlike blood cell counts, the sperm concentration is reported
sides of the hemocytometer are loaded and allowed to settle in millions per milliliter rather than microliters. Sperm con-
for 3 to 5 minutes; then they are counted, and the counts centration also can be calculated using the basic formula for
should agree within 10%. An average of the two counts is used cell counts covered in Chapter 10. Because this formula pro-
in the calculation. If the counts do not agree, both the dilution vides the number of cells per microliter, the figure that is cal-
and the counts are repeated. Counts are performed using either culated must be multiplied by 1000 to calculate the number
phase or bright-field microscopy. The addition of a stain, such of sperm per milliliter. The total sperm count is calculated
as crystal violet, to the diluting fluid aids in visualization when by multiplying the number of sperm per milliliter by the
using bright-field microscopy. specimen volume.
Only fully developed sperm should be counted. Immature
sperm and WBCs, often referred to as “round” cells, must not EXAMPLES
be included. However, their presence can be significant, and 1. Using a 1:20 dilution, an average of 60 sperm are
they may need to be identified and counted separately. Stain counted in the five RBC counting squares on both sides
included in the diluting fluid aids in differentiating between of the hemocytometer. Calculate the sperm concentra-
immature sperm cells (spermatids) and leukocytes, and they tion per milliliter and the total sperm count in a speci-
can be counted in the same manner as mature sperm. A count men with a volume of 4 mL.
60 sperm counted × 1,000,000 = 60,000,000 sperm/mL
60,000,000 sperm/mL × 4 mL = 240,000,000 sperm/
ejaculate
2. In a 1:20 dilution, 600 sperm are counted in two WBC
counting squares. Calculate the sperm concentration per
W W milliliter and the total sperm count in a specimen with a
volume of 2 µL.
600 sperm counted × 20 (dilution) 60,000 sperm/µL
=
R R 2 sq mm × (squares counted) × (volume counted)
0.1 mm (depth)
R
60,000 sperm/µL × 1000 = 60,000,000 sperm/mL
60,000,000/mL × 2 mL = 120,000,000 sperm/ejaculate

R R
Several methods have been developed using specially
designed and disposable counting chambers that do not re-
quire specimen dilution. Comparison of these methods and
W W the standard Neubauer counting chamber method showed
poor correlation with the Neubauer method and also among
themselves. The WHO states that the “validity of these
alternative counting chambers must be established by
checking chamber dimensions, comparing results with the
Figure 11–2 Areas of the Neubauer counting chamber used for red improved Neubauer hemocytometer method, and obtaining
and white blood cell counts. W, typical WBC counting area; R, typical satisfactory performance as shown by an external quality
RBC counting area. control program.”2
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Chapter 11 | Semen 283

Sperm Motility Table 11–4 Alternative Sperm Motility Grading

The presence of sperm capable of forward, progressive move- Criteria2
ment is critical for fertility because once presented to the Progressive Sperm moving linearly or in a large
cervix, the sperm must propel themselves through the cervical motility (PM) circle
mucosa to the uterus, fallopian tubes, and ovum. Traditionally,
Nonprogressive Sperm moving with an absence of
clinical laboratory reporting of sperm motility has been a
motility (NP) progression
subjective evaluation performed by examining an undiluted
specimen and determining the percentage of motile sperm and Immotility (IM) No movement
the quality of the motility.
Sperm motility should be assessed using a well-mixed, liq-
uefied semen specimen within 1 hour of specimen collection. The presence of a high percentage of immobile sperm and
The practice of examining sperm motility at timed intervals clumps of sperm requires further evaluation to determine
over an extended period has been shown to serve no useful sperm vitality or the presence of sperm agglutinins.
purpose.7 To provide continuity in reporting, laboratories
should place a consistent amount of semen on a slide under Automated Semen Analysis Systems
the same size cover slip, such as 10 µL under a 22 × 22 mm
In recent years, instrumentation capable of performing com-
cover slip using a calibrated positive-displacement pipette, and
puter-assisted semen analysis (CASA) has been developed.
allow it to settle for 1 minute. This procedure should be done
CASA provides objective determination of both sperm velocity
in duplicate for accuracy. Then the percentage of sperm show-
and trajectory (direction of motion). Also, sperm concentration
ing actual forward movement can be estimated after evaluating
and morphology are included in the analysis. Currently, CASA
approximately 20 high-power fields. An alternative procedure
instrumentation is found primarily in laboratories that special-
is to examine 200 sperm per slide and count the percentages
ize in andrology and perform a high volume of semen analysis.
of the different motile categories using a manual cell counter.2
Studies comparing several automated systems with manual
Motility is evaluated by both speed and direction. Grading can
methods demonstrate a good correlation. Automated systems,
be done using a scale of 0 to 4, with 4 indicating rapid, straight-
however, provide faster results with higher precision and stan-
line movement and 0 indicating no movement (Table 11-3). A
dardization, as well as reduced potential for human error.8 Three
minimum motility of 50% with a rating of 2.0 after 1 hour is
different automated systems are the Sperm Class Analyzer (SCA,
considered normal.3
Microptic, Spain),9 the CEROS CASA systems (Hamilton Thorne,
The WHO uses a rating scale of a, b, c, and d (see
Beverly, MA), and the Automated Sperm Quality Analyzers (SQA-
Table 11-3). Interpretation states that within 1 hour, 50% or
Vision, Medical Electronic Systems, Los Angeles, CA). CASA
more of the sperm should be motile in categories a, b, and c,
systems capture and process microscopic images by computer
or 25% or more should show progressive motility (a and b).6
software to detect sperm motility. The SCA consists of a micro-
The WHO Laboratory Manual for the Examination and Pro-
scope equipped with a digital camera, motorized heating stage,
cessing of Human Semen (2010)2 currently recommends a simpler
and image analysis software with an analytical filter that elimi-
system for grading motility that does not include speed because
nates other bodies that could cause inconsistent analysis, such
of the difficulty in standardized reporting. Motility is graded as
as cell debris, isolated sperm heads, or flagella. The analyzer
progressive motility (PM), nonprogressive motility (NP), and
measures sperm concentration, motility, vitality, DNA fragmen-
immotility (IM). Motility must be specified as total motility (PM
tation, acrosome reaction, morphology, and leukocytes.9 The
and NP) or progressive motility (PM).2 (See Table 11-4.)
SQA measures sperm concentration, percent motility, percent
normal morphology, concentration of motile and functional
sperm, concentration of progressively motile sperm, velocity, and
sperm motility index using electro-optics combined with spec-
trophotometry and computer algorithms.
Table 11–3 Sperm Motility Grading
WHO Sperm Morphology
Grade Criteria Sperm Motility Action Just as the presence of a normal number of sperm that are non-
4.0 a Motile with rapid, straight-line motile produces infertility, the presence of sperm that are mor-
motility phologically incapable of fertilization also results in infertility.
Sperm morphology is evaluated with respect to the struc-
3.0 b Motile with slower speed, some
ture of the head, neckpiece, midpiece, and tail. Abnormalities
lateral movement
in head morphology are associated with poor ovum penetra-
2.0 b Motile with slow forward progres- tion, whereas abnormalities in the neckpiece, midpiece, and
sion, noticeable lateral movement tail affect motility.
1.0 c Motile without forward progression The normal sperm has an oval-shaped head approximately
0 d No movement 5 µm long and 3 µm wide with a long, flagellar tail approximately
45 µm long (Fig. 11-3). Critical to ovum penetration is the
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284 Part Three | Other Body Fluids

(Figs. 11-4 and 11-5). Abnormal sperm tails are frequently

Acrosome doubled, coiled, or bent (Figs. 11-6 and 11-7). A neckpiece
Head
Cell membrane that is abnormally long may cause the sperm head to bend
Nucleus
backward and interfere with motility (Fig. 11-8).

Midpiece

Mitochondria

Tail
(flagellum)

Figure 11–4 Spermatozoon with double head, hematoxylin–eosin

(×1000).
Figure 11–3 Normal spermatozoon structure.

enzyme-containing acrosomal cap located at the tip of the head.

The acrosomal cap should encompass approximately half of the
head and cover approximately two thirds of the sperm nucleus.6
The neckpiece attaches the head to the tail and the midpiece. The
midpiece is approximately 7 µm long and is the thickest part of
the tail because it is surrounded by a mitochondrial sheath that
produces the energy required by the tail for motility.

Technical Tip 11-4. Sperm motility can be evaluated

at room temperature or at 37°C. When assessing motil-
ity at 37°C, the specimen should be incubated at this
temperature and the preparation made with pre-
warmed slides and cover slips.2 Figure 11–5 Spermatozoon with amorphous head, hematoxylin–
eosin (×1000).

Sperm morphology is evaluated from a thinly smeared,

stained slide under oil immersion. Smears are made by placing
approximately 10 µL of semen near the frosted end of a clean
microscope slide. Place a second slide with a clean, smooth
edge in front of the semen drop at a 45-degree angle and draw
the slide back to the edge of the drop of semen, allowing the
semen to spread across the end. When the semen is evenly dis-
tributed across the spreader slide, lightly pull the spreader slide
forward with a continuous movement across the first slide to
produce a smear. Staining can be performed using Wright’s,
Giemsa, Shorr, or Papanicolaou stain; the stain used is a matter
of laboratory preference. Air-dried slides are stable for 24 hours.
At least 200 sperm should be evaluated and the percentage of
abnormal sperm reported. Abnormalities in head structure that
are identified routinely include double heads, giant and amor- Figure 11–6 Spermatozoon with double tail, hematoxylin–eosin
phous heads, pinheads, tapered heads, and constricted heads (×1000).
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Chapter 11 | Semen 285

Normal Double Giant Amorphous Pinhead

head head head

Figure 11–7 Common abnormalities of Tapered Constricted Double Coiled Spermatid

sperm heads and tails. head head tail tail

Calculating Round Cells

Differentiation and enumeration of round cells (immature
sperm and leukocytes) also can be made during the morphol-
ogy examination (Fig. 11-9). Peroxidase-positive granulocytes
are the predominant form of leukocyte in semen and can be
differentiated further from spermatogenic cells and lympho-
cytes using a peroxidase stain. By counting the number of sper-
matids or leukocytes seen in conjunction with 100 mature
sperm, the amount per milliliter can be calculated using the
following formula, where N is the number of spermatids or
neutrophils counted per 100 mature sperm, and S is the sperm
concentration in millions per milliliter:

N × S
C =
Figure 11–8 Spermatozoon with bent neck and spermatid, 100
hematoxylin–eosin (×1000).
This method can be used when counting but cannot be
Additional parameters in evaluating sperm morphology in- performed during the hemocytometer count and to verify
clude measuring head, neck, and tail size; measuring acrosome counts performed by hemocytometer.
size; and evaluating for the presence of vacuoles. Inclusion of More than 1 million WBCs per milliliter per ejaculate
these parameters is referred to as Kruger’s strict criteria.10 Strict indicates an inflammatory condition associated with infection
criteria evaluation requires the use of a stage micrometer or mor- and poor sperm quality; it may impair sperm motility and DNA
phometry.11 At present, evaluation of sperm morphology using integrity.2
strict criteria is not performed routinely in the clinical laboratory
but is recommended by the WHO.6 Strict criteria evaluation is Additional Testing
an integral part of evaluations in assisted reproduction.
Normal values for sperm morphology depend on the eval- Should abnormalities be discovered in any of these routine
uation method used and vary from greater than 30% normal parameters, additional tests may be requested (Table 11-5).
forms when using routine criteria to greater than 14% normal The most common are tests for sperm vitality, seminal fluid
forms when using strict criteria.6 fructose level, sperm agglutinins, and microbial infection.
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286 Part Three | Other Body Fluids

Figure 11–9 Immature spermatozoa, hematoxylin–eosin Figure 11–10 Nonviable spermatozoa demonstrated by the eosin–
(×1000). nigrosin stain (×1000).

Table 11–5 Additional Testing for Abnormal the motility evaluated previously. The presence of a large pro-
Semen Analysis portion of vital but immobile cells may indicate a defective fla-
gellum, whereas a high number of immotile and nonviable
Abnormal Possible cells may indicate epididymal pathology.2
Result Abnormality Test
Seminal Fluid Fructose
Decreased Vitality Eosin–nigrosin stain
motility Low sperm concentration may be caused by lack of the support
with nor- medium produced in the seminal vesicles, which can be indi-
mal count cated by a fructose level that is low to absent in the semen.
Low fructose levels are caused by abnormalities of the seminal
Decreased Lack of seminal Fructose level
vesicles, bilateral congenital absence of the vas deferens, ob-
count vesicle support
struction of the ejaculatory duct, partial retrograde ejaculation,
medium
and androgen deficiency.2 Specimens can be screened for the
Decreased Male antisperm Mixed agglutination presence of fructose using the resorcinol test that produces an
motility antibodies reaction and im- orange color when fructose is present (Procedure 11-3).
with munobead tests A normal quantitative level of fructose is equal to or
clumping greater than 13 µmol per ejaculate. This can be determined
Sperm agglutination using spectrophotometric methods. Specimens for fructose lev-
with male serum els should be tested within 2 hours of collection or frozen to
Normal Female antisperm Sperm agglutination prevent fructolysis.
analysis antibodies with female serum
with con-
Antisperm Antibodies
tinued Antisperm antibodies can be present in both men and women.
infertility They may be detected in semen, cervical mucosa, or serum and
are considered a possible cause of infertility. It is not unusual
for both partners to demonstrate antibodies, although male
antisperm antibodies are encountered more frequently.
Sperm Vitality
Decreased sperm vitality may be suspected when a specimen
has a normal sperm concentration with markedly decreased
PROCEDURE 11-3
motility. Sperm vitality should be assessed within 1 hour of Seminal Fructose Screening Test7
ejaculation. Vitality is evaluated by mixing the specimen with
1. Prepare reagent (50 mg resorcinol in 33 mL concen-
an eosin–nigrosin stain, preparing a smear, and counting the
trated HCl diluted to 100 mL with water).
number of dead cells in 100 sperm using a bright-field or
phase-contrast microscope. Living cells are not infiltrated by 2. Mix 1 mL of semen with 9 mL of reagent.
the dye and remain bluish white, whereas dead cells stain red 3. Boil.
against the purple background (Fig. 11-10). Normal vitality 4. Observe for orange-red color.
requires 50% or more living cells and should correspond to
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Chapter 11 | Semen 287

Under normal conditions, the blood–testes barrier sepa- transpeptidase, and prostatic acid phosphatase. Just as
rates sperm from the male immune system. When this barrier decreased fructose levels are associated with a lack of seminal
is disrupted, as can occur after surgery, vasectomy reversal fluid, decreased neutral α-glucosidase, glycerophospho-
(vasovasostomy), trauma, and infection, the antigens on the choline, and L-carnitine suggest a disorder of the epididymis.
sperm produce an immune response that damages the sperm. Decreased zinc, citric acid, glutamyl transpeptidase, and acid
The damaged sperm may cause the production of antibodies phosphatase indicate a lack of prostatic fluid (Table 11-6).
in the female partner.12 Spectrophotometric methods are used to quantitate citric
The presence of antibodies in a male subject can be sus- acid and zinc.
pected when clumps of sperm are observed during a routine On certain occasions, the laboratory may be called on to
semen analysis. Sperm-agglutinating antibodies cause sperm determine whether semen is actually present in a specimen.
to stick to each other in a head-to-head, head-to-tail, or tail- A primary example is in cases of alleged rape or sexual as-
to-tail pattern.2 The agglutination is graded as “few,” “moder- sault. Microscopically examining the vaginal fluid specimen
ate,” or “many” on microscopic examination. for the presence of sperm may be possible, with the best re-
The presence of antisperm antibodies in a female subject sults being obtained by enhancing the specimen with xylene
results in a normal semen analysis accompanied by continued and examining under phase microscopy.14 Motile sperm can
infertility. The presence of antisperm antibodies in women be detected for up to 24 hours after intercourse, whereas non-
may be demonstrated by mixing the semen with the female motile sperm can persist for 3 days. As the sperm die off, only
cervical mucosa or serum and observing for agglutination. A the heads remain and may be present for 7 days after inter-
variety of immunoassay kits are available for both semen and course. Seminal fluid contains a high concentration of pro-
serum testing. static acid phosphatase, so detecting this enzyme can aid in
Two tests used frequently to detect the presence of anti- determining the presence of semen in a specimen. A more
body-coated sperm are the mixed agglutination reaction (MAR) specific method is the detection of seminal glycoprotein p30
test and the immunobead test. The MAR test is a screening pro- (prostatic specific antigen [PSA]), which is present even in
cedure used primarily to detect the presence of immunoglobu- the absence of sperm.15 Often, further information can be ob-
lin G (IgG) antibodies. The semen sample containing motile tained by performing ABO blood grouping and DNA analysis
sperm is incubated with IgG antihuman globulin (AHG) and a on the specimen.
suspension of latex particles or treated RBCs coated with IgG.
The bivalent AHG binds simultaneously to both the antibody Postvasectomy Semen Analysis
on the sperm and the antibody on the latex particles or RBCs, Postvasectomy semen analysis is a much less involved procedure
forming microscopically visible clumps of sperm and particles compared with infertility analysis because the only concern is the
or cells. A finding of less than 10% of the motile sperm attached presence or absence of spermatozoa. The length of time required
to the particles is considered normal. for complete sterilization can vary greatly among patients and de-
The immunobead test is a more specific procedure in that pends on both time and number of ejaculations. Therefore, find-
it can be used to detect the presence of IgG, IgM, and IgA ing viable sperm in a postvasectomy patient is not uncommon,
antibodies and demonstrates the area of the sperm (head, neck- and care should be taken not to overlook even a single sperm.
piece, midpiece, or tail) being affected by the autoantibodies. Specimens are tested routinely at monthly intervals, beginning at
Head-directed antibodies can interfere with penetration into 2 months postvasectomy and continuing until two consecutive
the cervical mucosa or ovum, whereas tail-directed antibodies monthly specimens show no spermatozoa.
affect movement through the cervical mucosa.13 In the Recommended testing includes examining a wet prepara-
immunobead test, sperm are mixed with polyacrylamide beads tion using phase microscopy for the presence of motile and
known to be coated with either anti-IgG, anti-IgM, or anti-IgA. nonmotile sperm. A negative wet preparation is followed by
Microscopic examination of the sperm shows the beads specimen centrifugation for 10 minutes and examination of the
attached to sperm at particular areas. Depending on the type sediment.7
of beads used, the test could be reported as “IgM tail antibod-
ies,” “IgG head antibodies,” and so forth. The presence of beads
on less than 50% of the sperm is considered normal as defined Technical Tip 11-5. A single “motile” sperm on a
by the WHO.2 wet preparation is an indication of an unsuccessful
vasectomy.
Microbial and Chemical Testing
The presence of more than 1 million leukocytes per millimeter
indicates infection within the reproductive system, frequently Table 11–6 Reference Semen Chemical Values2
the prostate. Routine aerobic and anaerobic cultures and tests
Neutral α-glucosidase ≥20 mU/ejaculate
for Chlamydia trachomatis, Mycoplasma hominis, and Ureaplasma
urealyticum are performed most frequently. Zinc ≥2.4 µmol/ejaculate
Additional chemical testing performed on semen may Citric acid ≥52 µmol/ejaculate
include determining the levels of neutral α-glucosidase, free Acid phosphatase ≥200 units/ejaculate
L-carnitine, glycerophosphocholine, zinc, citric acid, glutamyl
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288 Part Three | Other Body Fluids

Sperm Function Tests

For additional resources please visit
Advances in assisted reproduction and IVF have resulted in a www.fadavis.com
need for more sophisticated semen analysis to assess not only
the characteristics of sperm but also the functional ability. The
tests are performed most commonly in specialized andrology
laboratories and include the hamster egg penetration assay, cer- References
vical mucus penetration test, hypo-osmotic swelling test, and 1. Noureddine, M: Forensic Tests for Semen: What you should
know. Bodily Fluids and Forensics. Web site: https://ncforensics.
the in vitro acrosome reaction (Table 11-7).16 wordpress.com/2011/10/19 forensic-tests-for-semen-what-you-
should-know/. Accessed July 1, 2019.
Semen Analysis Quality Control 2. WHO Laboratory Manual for the Examination and Processing
of Human Semen. World Health Organization. WHO Press,
Traditionally, routine semen analysis has been subject to very little Geneva, Switzerland, 2010.
quality control,17 a situation that has resulted from a lack of ap- 3. Sarhar, S, and Henry, JB: Andrology laboratory and fertility
propriate control materials and the subjectivity of the motility assessment. In Henry, JB (ed): Clinical Diagnosis and Manage-
and morphology analyses. The analysis is rated as a high com- ment by Laboratory Methods. WB Saunders, Philadelphia,
plexity test under the Clinical Laboratory Improvement Amend- 1996.
4. Lopez, A, et al: Suitability of solid-phase chemistry for quantifi-
ments, and standards for testing personnel must be observed. cation of leukocytes in cerebrospinal, seminal and peritoneal
Increased interest in fertility testing has promoted the de- fluid. Clin Chem 33(8):1475–1476, 1987.
velopment of quality control materials and in-depth training 5. Overstreet, JW, and Katz, DF: Semen analysis. Urol Clin North
programs. The standardized procedures developed by the Am 14(3):441–449, 1987.
WHO have provided a basis for laboratory testing and report- 6. World Health Organization: WHO Laboratory Manual for the
Examination of Human Semen and Sperm-Cervical Interaction.
ing. The use of CASA has aided in reducing the subjectivity of Cambridge University Press, London, 1999.
the analysis. However, even computerized analysis has been 7. Sampson, JH, and Alexander, NJ: Semen analysis: A laboratory
shown to vary among operators.18 approach. Lab Med 13(4):218–223, 1982.
Now laboratories can participate in proficiency testing 8. Lammers, J, Splingart, C, Barriere, P, Jean, M, and Freour, T:
programs offered by the College of American Pathologists and Double-blind prospective study comparing two automated
sperm analyzers versus manual semen assessment. J Assist
the American Association of Bioanalysts that include sperm Reprod Genet 31:35, 2014. doi: 10.1007/s10815-013-0139-2.
concentration, vitality, and morphology. Commercial quality- Web site: https://www.ncbi.nlm.nih.gov/pmc/articles/
control materials and training aids are available and should be PMC3909144/. Accessed July 1, 2019.
incorporated into laboratory protocols. 9. Sperm Class Analyzer® CASA System Product Brochure.
Microptic Automatic Diagnostic Systems.https://www.
micropticsl.com/products/sperm-class-analyzer-casa-system/.
Accessed March 31, 2020.
Table 11–7 Sperm Function Tests 10. Kruger, T, et al: Predictive value of sperm morphology in IVF.
FertilSteril 112–117, 1988.
Test Description 11. Harr, R: Characterization of spermatozoa by planar morphome-
try. Clin Lab Sci 10(4):190–196, 1997.
Hamster egg penetration Sperm are incubated with 12. Cearlock, DM: Autoimmune antispermatozoa antibodies in men:
species-nonspecific ham- Clinical detection and role in infertility. Clin Lab Sci 2(3):
ster eggs, and penetration 165–168, 1989.
is observed microscopically 13. Marshburn, PB, and Kutteh, WH: The role of antisperm
antibodies in infertility. Fertil Steril 61:799–811, 1994.
Cervical mucus Observation of sperm’s abil- 14. Fraysier, HD: A rapid screening technique for the detection of
penetration ity to penetrate partner’s spermatozoa. J Forensic Sci 32(2):527–528, 1987.
midcycle cervical mucus 15. Graves, HC, Sensabaugh, CF, and Blake, ET: Postcoital
detection of a male-specific semen protein, application to
Hypo-osmotic swelling Sperm exposed to low- the investigation of rape. N Engl J Med 312(6):338–343,
sodium concentrations are 1985.
evaluated for membrane in- 16. Yablonsky, T: Male fertility testing. Lab Med 27(6):378–383,
tegrity and sperm viability 1996.
17. Baker, DJ, et al: Semen evaluations in the clinical laboratory.
In vitro acrosome Evaluation of the acrosome Lab Med 25(8):509–514, 1994.
reaction to produce enzymes essen- 18. Krause, W, and Viethen, G: Quality assessment of computer-
tial for ovum penetration assisted semen analysis (CASA) in the andrology laboratory.
Andrologia 31(3):125–129, 1999.
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Chapter 11 | Semen 289

Study Questions
1. Maturation of spermatozoa takes place in the: 8. An increased semen pH may be caused by:
A. Sertoli cells A. Poorly developed seminal vesicles
B. Seminiferous tubules B. Increased prostatic secretions
C. Epididymis C. Obstruction of the ejaculation duct
D. Seminal vesicles D. Prostatic infection
2. Enzymes for the coagulation and liquefaction of semen 9. Proteolytic enzymes may be added to semen specimens to:
are produced by the: A. Increase the viscosity
A. Seminal vesicles B. Dilute the specimen
B. Bulbourethral glands C. Decrease the viscosity
C. Ductus deferens D. Neutralize the specimen
D. Prostate gland
10. The normal sperm concentration is:
3. The major component of seminal fluid is: A. Less than 20 million/µL
A. Glucose B. More than 20 million/mL
B. Fructose C. Less than 20 million/mL
C. Acid phosphatase D. More than 20 million/µL
D. Citric acid
11. Given the following information, calculate the sperm
4. If the first portion of a semen specimen is not collected, concentration: dilution, 1:20; sperm counted in
the semen analysis will have which of the following? five RBC squares on each side of the hemocytometer,
A. Decreased pH 80 and 86; volume, 3 mL.
B. Increased viscosity A. 80 million/mL
C. Decreased sperm count B. 83 million/mL
D. Decreased sperm motility C. 86 million/mL
D. 169 million/µL
5. Failure of laboratory personnel to document the time a
semen specimen is collected primarily affects the interpre- 12. Using the information from question 11, calculate the
tation of semen: sperm concentration when 80 sperm are counted in
A. Appearance 1 WBC square and 86 sperm are counted in another
WBC square.
B. Volume
A. 83 million/mL
C. pH
B. 166 million per ejaculate
D. Viscosity
C. 16.6 million/mL
6. Liquefaction of a semen specimen should take place
D. 50 million per ejaculate
within:
A. 1 hour 13. The primary reason to dilute a semen specimen before
performing a sperm concentration is to:
B. 2 hours
A. Immobilize the sperm
C. 3 hours
B. Facilitate the chamber count
D. 4 hours
C. Decrease the viscosity
7. A semen specimen delivered to the laboratory in a con-
D. Stain the sperm
dom has a normal sperm count and markedly decreased
sperm motility. This indicates:
A. Decreased fructose
B. Antispermicide in the condom
C. Increased semen viscosity
D. Increased semen alkalinity
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290 Part Three | Other Body Fluids

14. When performing a sperm concentration, 60 sperm 21. Normal sperm morphology when using the WHO
are counted in the RBC squares on one side of the criteria is:
hemocytometer and 90 sperm are counted in the A. >30% normal forms
RBC squares on the other side. The specimen is
B. <30% normal forms
diluted 1:20. The:
C. >15% abnormal forms
A. Specimen should be rediluted and counted
D. <15% normal forms
B. Sperm count is 75 million/mL
C. Sperm count is greater than 5 million/mL 22. Additional parameters measured by Kruger’s strict
morphology include all of the following except:
D. Sperm concentration is abnormal
A. Vitality
15. Sperm motility evaluations are performed:
B. Presence of vacuoles
A. Immediately after the specimen is collected
C. Acrosome size
B. Within 1 hour of collection
D. Tail length
C. After 3 hours of incubation
23. Round cells that are of concern and may be included in
D. At 6-hour intervals for 1 day
sperm counts and morphology analysis are:
16. The percentage of sperm showing average motility that A. Leukocytes
is considered normal is:
B. Spermatids
A. 25%
C. RBCs
B. 50%
D. Both A and B
C. 60%
24. If 5 round cells per 100 sperm are counted in a sperm
D. 75%
morphology smear and the sperm concentration is
17. The purpose of the acrosomal cap is to: 30 million, the concentration of round cells is:
A. Penetrate the ovum A. 150,000
B. Protect the nucleus B. 1.5 million
C. Create energy for tail movement C. 300,000
D. Protect the neckpiece D. 15 million
18. The sperm part containing a mitochondrial sheath is the: 25. After an abnormal sperm motility test with a normal
A. Head sperm count, what additional test might be ordered?
B. Neckpiece A. Fructose level
C. Midpiece B. Zinc level
D. Tail C. MAR test
D. Eosin–nigrosin stain
19. All of the following are associated with sperm motility
except the: 26. Follow-up testing for a low sperm concentration would
A. Head include testing for:
B. Neckpiece A. Antisperm antibodies
C. Midpiece B. Seminal fluid fructose
D. Tail C. Sperm vitality
D. Prostatic acid phosphatase
20. The morphological shape of a normal sperm head is:
A. Round 27. The immunobead test for antisperm antibodies:
B. Tapered A. Detects the presence of male antibodies
C. Oval B. Determines the presence of IgG, IgM, and IgA
antibodies
D. Amorphous
C. Determines the location of antisperm antibodies
D. All of the above
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Chapter 11 | Semen 291

28. Measurement of α-glucosidase is performed to detect a 30. After a negative postvasectomy wet preparation, the
disorder of the: specimen should be:
A. Seminiferous tubules A. Centrifuged and reexamined
B. Epididymis B. Stained and reexamined
C. Prostate gland C. Reported as no sperm seen
D. Bulbourethral glands D. Both A and B
29. A specimen delivered to the laboratory with a request 31. Standardization of procedures and reference values for
for prostatic acid phosphatase and glycoprotein p30 was semen analysis is provided primarily by the:
collected to determine: A. Manufacturers of instrumentation
A. Prostatic infection B. WHO
B. Presence of antisperm antibodies C. Manufacturers of control samples
C. A possible rape D. Clinical Laboratory Improvement Amendments
D. Successful vasectomy

Case Studies and Clinical Situations

1. A repeat semen analysis for fertility testing is reported as 3. A yellow-colored semen specimen is received in the labo-
follows: ratory. The analysis is normal except for decreased sperm
VOLUME: 3.5 mL SPERM COUNT: 6 million/mL motility. Explain the possible connection between the two
abnormal findings.
VISCOSITY: Normal SPERM MOTILITY: 30%—
grade 1.0 4. Abnormal results of a semen analysis are volume = 1.0 mL
pH: 7.5 MORPHOLOGY: <30% normal and sperm concentration = 1 million/mL. State a non-
forms—30 spermatids/ pathological cause of these abnormal results.
100 sperm 5. A semen specimen with normal initial appearance fails to
The results correspond with the first analysis. liquefy after 60 minutes.
a. List three abnormal parameters. a. Would a specimen pH of 9.0 be consistent with this
b. What is the sperm concentration? Is this normal? observation? Why or why not?
c. What is the spermatid count? Is this normal? b. State three chemical tests that would be of value in
this analysis.
d. Could the sperm concentration and the spermatid
count be related to the infertility? Explain your c. How does this abnormality affect fertility?
answer. 6. A specimen is delivered to the laboratory with a request
2. A semen analysis on a vasovasostomy patient has a nor- to determine whether semen is present.
mal sperm concentration; however, motility is decreased, a. What two chemical tests could be performed on the
and clumping is observed on the wet preparation. specimen?
a. Explain the possible connection between these obser- b. What additional examination could be performed on
vations and the patient’s recent surgery. the specimen?
b. What tests could be performed to further evaluate the
patient’s infertility?
c. Briefly explain the different interpretations offered by
these two tests.
d. State three ways in which a positive result on these
tests could be affecting male fertility.
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CHAPTER 12
Synovial Fluid
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
12-1 Describe the formation and function of synovial fluid. 12-7 List and describe six crystals found in synovial fluid.
12-2 Relate laboratory test results to the four common 12-8 Explain the differentiation of monosodium urate and
classifications of joint disorders. calcium pyrophosphate crystals using polarized and
compensated polarized light.
12-3 State the five diagnostic tests performed most rou-
tinely on synovial fluid. 12-9 State the clinical significance of glucose and lactate
tests on synovial fluid.
12-4 Determine the appropriate collection tubes for re-
quested laboratory tests on synovial fluid. 12-10 List four genera of bacteria found most frequently in
synovial fluid.
12-5 Describe the appearance of synovial fluid in normal
and abnormal states. 12-11 Describe the relationship of serological serum testing
to joint disorders.
12-6 Discuss the normal and abnormal cellular composi-
tion of synovial fluid.

KEY TERMS
Arthritis Hyaluronic acid Synovial fluid
Arthrocentesis Pseudogout Synoviocytes
Gout
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294 Part Three | Other Body Fluids

Physiology synovial fluid. Damage to the articular membranes produces

pain and stiffness in the joints, collectively referred to as arthritis.
Synovial fluid, often referred to as “joint fluid,” is a viscous Laboratory results of synovial fluid analysis can be used to de-
liquid found in the cavities of the movable joints (diarthroses) termine the pathological origin of arthritis. The beneficial tests
or synovial joints. As shown in Figure 12-1, the bones in the performed most frequently on synovial fluid are the white blood
synovial joints are lined with smooth articular cartilage and cell (WBC) count, differential, Gram stain, culture, and crystal
separated by a cavity containing the synovial fluid. The joint is examination.1 Reference values are shown in Table 12-1.2
enclosed in a fibrous joint capsule lined by the synovial mem- A variety of conditions, including infection, inflammation,
brane that is lubricated by synovial fluid. The synovial membrane metabolic disorders, trauma, physical stress, and advanced age,
contains specialized cells called synoviocytes. Two types of syn- are associated with arthritis. Frequently disorders are classified
oviocytes are present in the synovial membrane.Type A cells are into four groups, as shown in Table 12-2. Some overlap of test
macrophage-like cells located in the superficial layer of the syn- results among the groups may occur (Table 12-3)3; the patient’s
ovial membrane and play an important role in phagocytosis. clinical history also must be considered when assigning a
Type B cells are fibroblast-like cells with prominent endoplasmic category.
reticulum located in a deeper layer of the synovial membrane
and produce hyaluronic acid, fibronectin, and collagen to pro-
duce synovial fluid. The smooth articular cartilage and synovial
Specimen Collection
fluid together reduce friction between the bones during joint and Handling
movement. In addition to providing lubrication in the joints,
synovial fluid provides nutrients to the articular cartilage and Synovial fluid is collected from a joint by needle aspiration
lessens the shock of joint compression that occurs during activ- called arthrocentesis. The amount of fluid present varies with
ities such as walking and jogging. the size of the joint and the extent of fluid buildup in the joint.
Synovial fluid is formed as an ultrafiltrate of plasma across For example, the normal amount of fluid in the adult knee
the synovial membrane. The filtration is nonselective, except for cavity is less than 3.5 mL, but the fluid level can increase to
the exclusion of high-molecular-weight proteins. Therefore, most greater than 25 mL with inflammation. In some instances, only
of the chemical constituents, although seldom of clinical signif- a few drops of fluid are obtained, but these still can be used
icance, have concentrations similar to plasma values. They do, for microscopic analysis or culturing. The volume of fluid
however, provide nutrients for the vascular-deficient cartilage. collected should be recorded.
The synoviocytes secrete a mucopolysaccharide containing Normal synovial fluid does not clot; however, fluid from a
hyaluronic acid and a small amount of protein (approximately diseased joint may contain fibrinogen and will clot. Therefore,
one fourth of the plasma concentration) into the fluid. The large fluid is collected in a syringe that has been moistened with he-
hyaluronate molecules contribute the noticeable viscosity to parin. When sufficient fluid is collected, it should be distributed
into specific tubes based on the required tests, as presented in
Table 12-4. The Clinical Laboratory Standards Institute recom-
mends that synovial fluid should be collected as follows2:
• Tube 1: The first 4 to 5 mL of the synovial fluid obtained
should be placed into a plain, nonanticoagulated red
stopper tube and observed for clotting. The tube is
Bone

Bursa
Synovial
membrane Table 12–1 Normal Synovial Fluid Values2
Volume <3.5 mL
Joint Articular Color Colorless to pale yellow
capsule cartilage
Clarity Clear
Joint cavity Viscosity High; Able to form a string
(synovial fluid) 4—6 cm long
Tendon
Leukocyte count <200 cells/µL
Neutrophils <25% of the differential
Bone Crystals None present
Glucose:plasma <10 mg/dL lower than the blood
difference glucose level
Figure 12–1 Structure of a synovial joint. (From Scanlon & Sanders, Total protein <3 g/dL
Essentials of Anatomy and Physiology, 6th ed., F. A. Davis Company, Lactate <25.0 mg/dL
Philadelphia, 2019, with permission.)
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Chapter 12 | Synovial Fluid 295

Table 12–2 Classification and Pathological Table 12–3 Laboratory Findings in Joint
Significance of Joint Disorders Disorders3
Group Classification Pathological Significance Group Classification Laboratory Findings
I. Noninflammatory Degenerative joint disorders, I. Noninflammatory Clear, yellow fluid
osteoarthritis Good viscosity
II. Inflammatory Immunologic disorders, rheuma- WBCs <1000 µL
toid arthritis, systemic lupus
Neutrophils <30%
erythematosus, scleroderma,
polymyositis, ankylosing Similar to blood glucose
spondylitis, rheumatic fever, II. Inflammatory
Lyme arthritis Immunologic Cloudy, yellow fluid
Crystal-induced gout, origin Poor viscosity
pseudogout
WBCs 2,000—75,000 µL
III. Septic Microbial infection
Neutrophils >50%
IV. Hemorrhagic Traumatic injury, tumors,
Decreased glucose level
hemophilia, other coagulation
disorders Possible autoantibodies present
Anticoagulant overdose Crystal-induced Cloudy or milky fluid
origin Low viscosity
WBCs up to 100,000 µL
Neutrophils <70%
centrifuged to remove cellular and other components.
The supernatant is used for chemical or immunologic Decreased glucose level
analysis. Crystals present
• Tube 2: The next 4 to 5 mL is collected into a tube to III. Septic Cloudy, yellow-green fluid
which 25 units (U) of sodium heparin per mL (green Variable viscosity
stopper) is added or to a ethylenediaminetetraacetic
WBCs 50,000 to 100,000 µL
acid (EDTA) tube (lavender stopper) for cell count,
differential count, and crystal identification. Neutrophils >75%
• Tube 3: The last 4 to 5 mL is placed into a sterile tube to Decreased glucose level
which 25 U per mL heparin is added (green stopper) or Positive culture and Gram stain
to a sodium polyanethol sulfonate (yellow stopper) tube IV. Hemorrhagic Cloudy, red fluid
for microbiological studies.
Low viscosity
Powdered anticoagulants should not be used because they
WBCs equal to blood
may produce artifacts that interfere with crystal analysis. The
nonanticoagulated tube for other tests must be centrifuged and Neutrophils equal to blood
separated to prevent cellular elements from interfering with Normal glucose level
chemical and serological analyses.Ideally, all testing should be
done as soon as possible to prevent cellular lysis and possible
changes in crystals.
Table 12–4 Required Tube Types for Synovial
Technical Tip 12-1. Specimens for crystal analysis Fluid Tests
should not be refrigerated because refrigeration can
cause them to produce additional crystals that can in- Synovial Fluid Test Required Tube Type
terfere with the identification of significant crystals. Gram stain and Sterile sodium heparin or sodium
culture polyanethol sulfonate
Cell counts Sodium heparin or liquid ethyl-
enediaminetetraacetic acid
Color and Clarity (EDTA)
A report of the gross appearance is an essential part of the Glucose analysis Sodium fluoride or
analysis of synovial fluid. Normal synovial fluid appears col- nonanticoagulated
orless to pale yellow. The word “synovial” comes from the Latin All other tests Nonanticoagulated
word for egg, ovum. Normal viscous synovial fluid resembles
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296 Part Three | Other Body Fluids

egg white. The color becomes a deeper yellow in the presence contains saponin is a suitable diluent. Methylene blue added
of noninflammatory and inflammatory effusions and may have to the normal saline stains the WBC nuclei, permitting
a greenish tinge with bacterial infection. As with cerebrospinal separation of the RBCs and WBCs during counts performed
fluid, in synovial fluid, the presence of blood from a hemor- on mixed specimens.
rhagic arthritis must be distinguished from blood from a trau-
matic aspiration. This is accomplished primarily by observing
the uneven distribution of blood or even a single blood streak
Technical Tip 12-2. Do not use fluids that contain
acetic acid to dilute synovial fluids because the
in the specimens obtained from a traumatic aspiration.
acetic acid can cause mucin clot formation and cell
Clarity is determined by the presence of WBCs, red blood
clumping.
cells (RBCs), synoviocytes, crystals, fat droplets, fibrin, and
cellular debris in the synovial fluid. Turbidity is associated
frequently with the presence of WBCs; however, synovial cell The recommended technique is to line a petri dish with
debris and fibrin also produce turbidity. The fluid may appear moist paper and place the hemocytometer on two small sticks
milky when crystals are present. to elevate it above the moist paper. Fill and count both sides
of the hemocytometer for compatibility. Acceptable ranges are
Viscosity determined by the laboratory.
Counting procedure:
Synovial fluid viscosity comes from polymerization of the • For counts less than 200 WBCs/µL, count all nine large
hyaluronic acid and is essential for the proper lubrication of squares.
joints. Arthritis affects both the production of hyaluronate and • For counts greater than 200 WBCs/µL in the previous
its ability to polymerize, thus decreasing the fluid viscosity. count, count the four corner squares.
Several methods are available to measure the synovial fluid
viscosity, the simplest being to observe the fluid’s ability to • For counts greater than 200 WBCs/µL in the previous
form a string from the tip of a syringe—a test that can be done count, count the five small squares used for a RBC
easily at the bedside. A string measuring 4 to 6 cm is consid- count.2
ered normal. Automated cell counters can be used for synovial fluid
Hyaluronate polymerization can be measured using a counts; however, highly viscous fluid may block the apertures,
Ropes, or mucin clot, test. When added to a solution of 2% to and the presence of debris and tissue cells may elevate counts
5% acetic acid, normal synovial fluid forms a solid clot sur- falsely. As described previously, incubating the fluid with
rounded by clear fluid. As the ability of the hyaluronate to hyaluronidase decreases specimen viscosity. Analyzing scatter-
polymerize decreases, the clot becomes less firm and the sur- grams can aid in detecting tissue cells and debris. Automated
rounding fluid increases in turbidity. The mucin clot test is re- counts that are properly controlled provide higher precision
ported in terms of good (solid clot), fair (soft clot), low (friable than manual counts.4 (See Chapter 2.)
clot), and poor (no clot). The mucin clot test is not performed WBC counts less than 200 cells/µL are considered normal
routinely because all forms of arthritis decrease viscosity and and may reach 100,000 cells/µL or higher in severe infections.5
little diagnostic information is obtained. Formation of a mucin There is, however, considerable overlap of elevated leukocyte
clot after adding acetic acid can be used to identify a question- counts between septic and inflammatory forms of arthritis.
able fluid as synovial fluid. Pathogenicity of the infecting organisms also produces varying
results in septic arthritis, as does antibiotic administration.
Cell Counts
Differential Count
The total leukocyte count is the cell count performed most fre-
quently on synovial fluid. RBC counts are seldom requested. Differential counts should be performed on cytocentrifuged
To prevent cellular disintegration, counts should be performed preparations or on thinly smeared slides. Fluid should be in-
as soon as possible, or the specimen should be refrigerated. cubated with hyaluronidase before slide preparation. Mononu-
Very viscous fluid may need to be pretreated by adding one clear cells, including monocytes, macrophages, and synovial
drop of 0.05% hyaluronidase in phosphate buffer per milliliter tissue cells, are the primary cells seen in normal synovial fluid.
of fluid and incubating at 37°C for 5 minutes. Neutrophils should account for less than 25% of the differential
Manual counts on specimens that have been mixed thor- count and lymphocytes less than 15%. Increased neutrophils
oughly are done using the Neubauer counting chamber. Usu- indicate a septic condition, whereas an elevated cell count with
ally, clear fluids can be counted undiluted, but dilutions are a predominance of lymphocytes suggests a nonseptic inflam-
necessary when fluids are turbid or bloody. Dilutions can be mation. In both normal and abnormal specimens, cells may ap-
made using the procedure presented in Chapter 10; however, pear more vacuolated than they do on a blood smear.3 Besides
traditional WBC diluting fluid cannot be used because it increased numbers of these usually normal cells, other cell ab-
contains acetic acid that causes the formation of mucin clots. normalities include the presence of eosinophils, lupus erythe-
Normal saline can be used as a diluent. If it is necessary matous (LE) cells, Reiter cells (or neutrophages, vacuolated
to lyse the RBCs, hypotonic saline (0.3%) or saline that macrophages with ingested neutrophils), and rheumatoid
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Chapter 12 | Synovial Fluid 297

arthritis (RA) cells (or ragocytes, neutrophils with small, dark • Chemotherapy treatment of leukemias
cytoplasmic granules consisting of precipitated rheumatoid • Decreased renal excretion of uric acid5
factor). Lipid droplets may be present after crush injuries, and
Pseudogout is associated most often with degenerative
hemosiderin granules are seen in cases of pigmented villon-
arthritis, producing cartilage calcification, and endocrine dis-
odular synovitis. The cells and inclusions encountered most
orders that produce elevated serum calcium levels.
frequently in synovial fluid are summarized in Table 12-5.
Additional crystals that may be present include hydro-
xyapatite (basic calcium phosphate) associated with calcified
Crystal Identification cartilage degeneration; cholesterol crystals associated with
chronic inflammation, such as in cases of RA; corticosteroids
Microscopic examination of synovial fluid for the presence of after injections; and calcium oxalate crystals in patients on
crystals is an important diagnostic test in evaluating arthritis. renal dialysis. Patient history must be considered always.
Crystal formation in a joint frequently results in an acute, painful Characteristics and significance of the crystals encountered
inflammation. Also, it can become a chronic condition. Causes commonly are presented in Table 12-6. Artifacts present may
of crystal formation include metabolic disorders and decreased include talcum powder and starch from gloves, precipitated
renal excretion that produce elevated blood levels of crystallizing anticoagulants, dust, and scratches on slides and cover slips.
chemicals, degeneration of cartilage and bone, and injection of Slides and cover slips should be examined and, if necessary,
medications, such as corticosteroids, into a joint. cleaned again before use.
Types of Crystals
Technical Tip 12-3. Specimens must be collected
The primary crystals seen in synovial fluid are monosodium only in tubes containing sodium heparin or EDTA
urate (uric acid) (MSU), found in cases of gout, and calcium tubes to avoid artifact crystallization.
pyrophosphate dihydrate (CPPD), seen with pseudogout. The
most frequent causes of gout include the following:
• Increased serum uric acid resulting from impaired Slide Preparation
metabolism of purines Ideally, crystal examination should be performed soon after
• Increased consumption of high-purine-content foods, fluid collection to ensure that crystals are not affected by
alcohol, and fructose changes in temperature and pH. Both MSU and CPPD crystals

Table 12–5 Cells and Inclusions Seen in Synovial Fluid

Cell/Inclusion Description Significance
Neutrophil Polymorphonuclear leukocyte Bacterial sepsis
Crystal-induced inflammation
Lymphocyte Mononuclear leukocyte Nonseptic inflammation
Macrophage (monocyte) Large mononuclear leukocyte, may be vacuolated Normal
Viral infections
Synovial lining cell Similar to macrophage, but may be multinucle- Normal
ated, resembling a mesothelial cell Disruption from arthrocentesis
LE cell Neutrophil containing characteristic ingested Lupus erythematosus
“round body”
Reiter cell Vacuolated macrophage with ingested Reactive arthritis (infection in another part
neutrophils of the body)
RA cell (ragocyte) Neutrophil with dark cytoplasmic granules Rheumatoid arthritis
containing immune complexes Immunologic inflammation
Cartilage cells Large, multinucleated cells Osteoarthritis
Rice bodies Macroscopically resemble polished rice Tuberculosis
Microscopically show collagen and fibrin Septic and rheumatoid arthritis
Fat droplets Refractile intracellular and extracellular globules Traumatic injury
Stain with Sudan dyes Chronic inflammation
Hemosiderin Inclusions within clusters of synovial cells Pigmented villonodular synovitis
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298 Part Three | Other Body Fluids

Table 12–6 Characteristics of Synovial Fluid Crystals

Compensated
Crystal Shape Image Polarized Light Significance
Monosodium urate Needles Negative birefringence Gout

Calcium pyrophosphate Rhomboid square, rods Positive birefringence Pseudogout

Cholesterol Notched, rhomboid plates Negative birefringence Extracellular

Corticosteroid Flat, variable-shaped plates Positive and negative Injections

birefringence

Calcium oxalate Envelopes Negative birefringence Renal dialysis

Apatite (calcium Small particles No birefringence Osteoarthritis

phosphate) Require electron microscopy

are reported as being located extracellularly and intracellularly Technical Tip 12-4. To avoid misidentification of
(within neutrophils); therefore, fluid must be examined before CPPD crystals, the classic rhomboid shape should
WBC disintegration. be observed and confirmed with red-compensated
Fluid is examined as an unstained wet preparation. One polarized microscopy.
drop of fluid is placed on a precleaned glass slide and cover-
slipped. The slide can be examined initially under low and
high power using a regular light microscope (Fig. 12-2). Crys-
tals may be observed in Wright’s-stained smears (Fig. 12-3);
Crystal Polarization
however, this should not replace the wet prep examination and Once the presence of the crystals has been determined using
the use of polarized and red-compensated polarized light for direct polarization, positive identification is made using first-
identification. order red-compensated polarized light. A control slide for
MSU crystals are seen routinely as needle-shaped crystals. the polarization properties of MSU can be prepared using
They may be extracellular or located within the cytoplasm of betamethasone acetate corticosteroid.
neutrophils. Frequently they are seen sticking through the Both MSU and CPPD crystals have the ability to polarize
cytoplasm of the cell. light, as discussed in Chapter 7; however, MSU is more highly
CPPD crystals usually appear rhomboid-shaped or square birefringent and appears brighter against the dark background
but may appear as short rods. Usually they are located within (Figs. 12-4 and 12-5).
vacuoles of the neutrophils, as shown in Figure 12-3. MSU When compensated polarized light is used, a red com-
crystals lyse phagosome membranes and therefore do not pensator is placed in the microscope between the crystal and
appear in vacuoles.6 the analyzer. The compensator separates the light ray into
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Chapter 12 | Synovial Fluid 299

Figure 12–2 Unstained wet prep of MSU crystals (×400). Notice the Figure 12–5 Weakly birefringent CPPD crystals under polarized
characteristic yellow-brown of the urate crystals. light (×1000).

Figure 12–3 Wright’s-stained neutrophils containing CPPD crystals Figure 12–6 Extracellular MSU crystals under compensated polar-
(×1000). ized light. Notice the change in color with crystal alignment
(×100).

to identify it. The molecules in MSU crystals run parallel to the

long axis of the crystal, and when aligned with the slow vibra-
tion, the velocity of the slow light passing through the crystal
is not impeded as much as the fast light, which runs against
the grain and produces a yellow color. This is considered neg-
ative birefringence (subtraction of velocity from the fast ray).
In contrast, the molecules in CPPD crystals run perpendicular
to the long axis of the crystal; when aligned with the slow axis
of the compensator, the velocity of the fast light passing
through the crystal is much quicker, producing a blue color
and positive birefringence.7 When the crystals are aligned per-
pendicular to the slow vibration, the color is reversed, as
shown in Figure 12-6. Care must be taken to ensure crystals
Figure 12–4 Strongly birefringent MSU crystals under polarized being analyzed are aligned in accordance with the compensator
light (×500). axis. Notice how the colors of the MSU crystals in Figure 12-6
vary with the alignment. Figures 12-7 and 12-8 illustrate the
slow-moving and fast-moving vibrations and produces a red characteristics of MSU and CPPD crystals under compensated
background (Fig. 12-6). polarized light.
Because of differences in the linear structure of the mole- Crystal shapes and patterns of birefringence that vary
cules in MSU and CPPD crystals, the color produced by each from the standard MSU and CPPD patterns may indicate the
crystal when it is aligned with the slow vibration can be used presence of one of the crystals encountered less commonly,
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300 Part Three | Other Body Fluids

n)
gr y
in ra
ai
ga low
st
S
gr ay
n)
ith r
ai
(w Slow

(a
A

Figure 12–7 MSU crystals under compensated polarized light. The

yellow crystal is aligned with the slow vibration (×500).

gr y
)
ith ra
ain
(w low
in)
st y

S
ain w ra
gra
(ag Slo
B

Figure 12–9 Negative and positive birefringence in MSU and CPPD

crystals. A. MSU crystal with grain running parallel to the long axis.
The slow ray passes with the grain, producing negative (yellow)
birefringence. B. CPPD crystal with grain running perpendicular to
the long axis. The slow ray passes against the grain and is retarded,
producing positive (blue) birefringence.

as osteoarthritis, pigmented villonodular synovitis, trauma, or he-

mangioma, have synovial fluid glucose levels of 10 and 20 mg/dL
or less than plasma levels measured simultaneously. Inflammatory
Figure 12–8 CPPD crystals under compensated polarized light. The disorders are 0 to 40 mg/dL below plasma level, and infectious
blue crystal is aligned with the slow vibration (×1000). and crystal-induced levels are 20 to 100 mg/dL and 0 to
80 mg/dL less than the plasma level.2
requiring further investigation (Fig. 12-9 A and B). Cholesterol,
oxalate, and corticosteroid crystals exhibit birefringence, as do Technical Tip 12-5. To prevent falsely decreased
many contaminants. Apatite crystals are not birefringent.5 values caused by glycolysis, specimens should be
analyzed within 1 hour or preserved with sodium
fluoride (gray stopper tube).
Chemistry Tests
Because synovial fluid is chemically an ultrafiltrate of plasma,
chemistry test values are approximately the same as serum
values. Therefore, few chemistry tests are considered clinically Total Protein
important. The test requested most frequently is the glucose de- The total protein level commonly exceeds 3.0 g/dL in all but
termination because markedly decreased glucose values indicate noninflammatory synovial effusions; therefore, it has little
inflammatory (group II) or septic (group III) disorders. diagnostic significance.2
Because the large protein molecules are not filtered
Glucose through the synovial membranes, normal synovial fluid con-
Because normal glucose values in synovial fluid are based on tains less than 3 g/dL protein (approximately one third of the
the blood glucose level, simultaneous blood and synovial fluid serum value). Increased levels are found in patients with in-
specimens should be obtained, preferably after the patient has flammatory and hemorrhagic disorders; however, synovial
fasted for 8 hours to allow equilibration between the two fluids. fluid protein measurement does not contribute greatly to the
Under these conditions, normal synovial fluid glucose should classification of these disorders. When requested, the analysis
not be more than 10 mg/dL lower than the blood value. Patients is performed using the same methods used for serum protein
with noninflammatory and hemorrhagic joint disorders, such determinations.
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Chapter 12 | Synovial Fluid 301

Uric Acid can cause osteoarticular tuberculosis; and Chlamydia trachomatis

and N. gonorrhoeae, which can cause venereal arthritis.8
The elevation of serum uric acid in cases of gout is well known;
therefore, demonstration of an elevated uric acid level in the
synovial fluid may be used to confirm the diagnosis when the Serological Tests
presence of crystals cannot be demonstrated in the fluid. Often
Because of the association of the immune system with the in-
serum uric acid is measured as a first evaluation in suspected
flammation process, serological testing plays an important role
cases of gout. Frequently fluid analysis for MSU crystals is still
in the diagnosis of joint disorders. However, most of these tests
required.
are performed on serum, and synovial fluid analysis actually
Lactate serves as a confirmatory measure in cases that are difficult to
diagnose. The autoimmune diseases RA and systemic lupus
Synovial fluid lactate levels are increased in septic arthritis caused erythematosus cause very serious joint inflammation and are
by gram-positive cocci and gram-negative bacilli, except in arthri- diagnosed in the serology laboratory by demonstrating the
tis caused by Neisseria gonorrhoeae, where lactate values are nor- presence of their particular autoantibodies in the patient’s
mal or low.2 Levels greater than 9 mmol/L (81 mg/dL) indicate serum. These same antibodies also can be demonstrated in syn-
bacterial arthritis and indicate an immediate onset of treatment.2 ovial fluid, if necessary. Arthritis is a frequent complication
of Lyme disease. Therefore, demonstrating antibodies to the
Enzymes causative agent B. burgdorferi in the patient’s serum can confirm
Several enzymes, including acid and alkaline phosphatase, the cause of the arthritis.
gamma-glutamyltransferase, adenosine deaminase, murami-
dase, cytidine deaminase, lactate dehydrogenase, and aspartate
aminotransferase, may be tested in synovial fluid to monitor For additional resources please visit
the severity and prognosis of RA.2 www.fadavis.com

Microbiological Tests
An infection may occur as a secondary complication of References
inflammation caused by trauma or through dissemination of 1. Shmerling, RH: Synovial fluid analysis. A critical reappraisal.
Rheum Dis Clin North Am 20(2):503–512, 1994.
a systemic infection; therefore, Gram stains and cultures are 2. Clinical and Laboratory Science Institute: Analysis of Body
two of the most important tests performed on synovial fluid. Fluids in Clinical Chemistry, Approved Guideline, C49-A,
Both tests must be performed on all specimens, as organisms Wayne, PA, 2007, CLSI.
often are missed on Gram stain. Bacterial infections are seen 3. Smith, GP, and Kjeldsberg, CR: Cerebrospinal, synovial,
most frequently; however, fungal, tubercular, and viral in- and serous body fluids. In Henry, JB (ed): Clinical Diagnosis
and Management by Laboratory Methods. WB Saunders,
fections also can occur. When they are suspected, special Philadelphia, 2001.
culturing procedures should be used. Patient history and 4. Brown, W, et al: Validation of body fluid analysis on the Coulter
other symptoms can aid in requests for additional testing. LH 750. Lab Hem 9(3):155–159, 2004.
Routine bacterial cultures should include an enrichment 5. Schumacher, HD, Clayburne, G, and Chen, L: Synovial fluid
medium, such as chocolate agar, because in addition to aspiration and analysis in evaluation of gout and other crystal-
induced diseases. Arthritis Foundation: Bulletin on the
Staphylococcus and Streptococcus, the common organisms that Rheumatic Diseases 53(3), 2004.
infect synovial fluid are the fastidious Haemophilus species 6. Harris, MD, Siegel, LB, and Alloway, JA: Gout and hyperuricemia.
and N. gonorrhoeae. Am Fam Physician 59(4):925–934, 1999.
Molecular methods using the polymerase chain reaction 7. Cornbleet, PJ: Synovial fluid crystal analysis. Lab Med 28(12):
(PCR) are available for the detection of microorganisms. This 774–779, 1997.
8. Jalava, J, Skurnik, M, Toivanen, A, Toivanen, P, and Eerola, E:
testing is particularly beneficial for the organisms that are hard Bacterial PCR in the diagnosis of joint infection. Annals of the
to detect and culture, such as Borrelia burgdorferi, which can rheumatic diseases. 60. 287–289, 2001. DOI:10.1136/ard.
cause Lyme disease arthritis; Mycobacterium tuberculosis, which 60.3.287.
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302 Part Three | Other Body Fluids

Study Questions
1. The functions of synovial fluid include all of the following 7. Before testing, very viscous synovial fluid should be
except: treated with:
A. Lubrication for the joints A. Normal saline
B. Removal of cartilage debris B. Hyaluronidase
C. Cushioning joints during jogging C. Distilled water
D. Providing nutrients for cartilage D. Hypotonic saline
2. The primary function of synoviocytes is to: 8. The color of the synovial fluid from a patient with a
A. Provide nutrients for the joints bacterial infection may be:
B. Secrete protein A. Yellow tinged
C. Regulate glucose filtration B. Green tinged
D. Prevent crystal formation C. Red streaked
D. Opalescent
3. Which of the following tests is not performed frequently
on synovial fluid? 9. Which of the following could be affected most
A. Uric acid significantly if a synovial fluid is refrigerated before
testing?
B. WBC count
A. Glucose
C. Crystal examination
B. Crystal examination
D. Gram stain
C. Mucin clot test
4. The procedure for collecting synovial fluid is called:
D. Differential
A. Synovialcentesis
10. The highest WBC count can be expected to be seen in
B. Arthrocentesis
patients with:
C. Joint puncture
A. Noninflammatory arthritis
D. Arteriocentesis
B. Inflammatory arthritis
5. Match the following disorders with their appropriate C. Septic arthritis
group:
D. Hemorrhagic arthritis
A. Noninflammatory
11. When diluting a synovial fluid WBC count, all of the
B. Inflammatory
following are acceptable except:
C. Septic
A. Acetic acid
D. Hemorrhagic
B. Isotonic saline
Gout
C. Hypotonic saline
Neisseria gonorrhoeae infection
D. Saline with saponin
Systemic lupus erythematosus
12. The lowest percentage of neutrophils would be seen in
Osteoarthritis
patients with:
Hemophilia
A. Noninflammatory arthritis
Rheumatoid arthritis
B. Inflammatory arthritis
Heparin overdose
C. Septic arthritis
6. Normal synovial fluid resembles: D. Hemorrhagic arthritis
A. Egg white
13. All of the following are abnormal when seen in synovial
B. Normal serum fluid except:
C. Dilute urine A. Neutrophages
D. Lipemic serum B. Ragocytes
C. Synovial lining cells
D. Lipid droplets
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Chapter 12 | Synovial Fluid 303

14. Synovial fluid crystals that occur as a result of purine 20. If crystals shaped like needles are aligned perpendicular
metabolism or chemotherapy for leukemia are: to the slow vibration of compensated polarized light,
A. Monosodium urate what color are they?
B. Cholesterol A. White
C. Calcium pyrophosphate B. Yellow
D. Apatite C. Blue
D. Red
15. Synovial fluid crystals associated with inflammation in
patients on dialysis are: 21. Negative birefringence occurs under red-compensated
A. Calcium pyrophosphate dihydrate polarized light when:
B. Calcium oxalate A. Slow light is impeded more than fast light
C. Corticosteroid B. Slow light is impeded less than fast light
D. Monosodium urate C. Fast light runs against the molecular grain of the
crystal
16. Crystals associated with pseudogout are:
D. Both B and C
A. Monosodium urate
22. Often synovial fluid cultures are plated on chocolate
B. Calcium pyrophosphate dihydrate
agar to detect the presence of:
C. Apatite
A. Neisseria gonorrhoeae
D. Corticosteroid
B. Staphylococcus agalactiae
17. Synovial fluid for crystal examination should be C. Streptococcus viridans
examined as a/an:
D. Enterococcus faecalis
A. Wet preparation
23. The chemical test performed most frequently on
B. Wright’s stain
synovial fluid is:
C. Gram stain
A. Total protein
D. Acid-fast stain
B. Uric acid
18. Crystals that have the ability to polarize light are: C. Calcium
A. Corticosteroid D. Glucose
B. Monosodium urate
24. Which of the following chemistry tests can be performed
C. Calcium oxalate on synovial fluid to determine the severity of RA?
D. All of the above A. Glucose
19. In an examination of synovial fluid under compensated B. Protein
polarized light, rhomboid-shaped crystals are observed. C. Acid phosphatase
What color would these crystals be when aligned
D. Uric acid
parallel to the slow vibration?
A. White 25. Serological tests on patients’ serum may be performed to
detect antibodies causing arthritis for all of the following
B. Yellow
disorders except:
C. Blue
A. Pseudogout
D. Red
B. Rheumatoid arthritis
C. Systemic lupus erythematosus
D. Lyme arthritis
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304 Part Three | Other Body Fluids

Case Studies and Clinical Situations

1. A 50-year-old man presents in the emergency department 3. Fluid obtained from the knee of an obese 65-year-old
with severe pain and swelling in the right knee. Arthro- woman being evaluated for a possible knee replacement
centesis is performed, and 20 mL of milky synovial fluid has the following results:
is collected. The physician orders a Gram stain, culture, APPEARANCE: Pale yellow and hazy
and crystal examination of the fluid, as well as a serum
WBC COUNT: 500 cells/µL
uric acid. She requests that the synovial fluid be saved for
possible additional tests. GRAM STAIN: Negative
a. Describe the tubes into which the fluid would be GLUCOSE: 110 mg/dL (serum glucose: 115 mg/dL)
placed. a. What classification of joint disorder do these results
b. If the patient’s serum uric acid level is elevated, what suggest?
type of crystals and disorder are probable? b. Under electron microscopy, what crystals might be
c. Describe the appearance of these crystals under direct detected?
and compensated polarized light. c. How does the glucose result aid in the disorder
d. Why were the Gram stain and culture ordered? classification?

2. A medical laboratory science student dilutes a synovial 4. A synovial fluid specimen delivered to the laboratory for a
fluid specimen before performing a WBC count. The fluid cell count is clotted.
forms a clot. a. What abnormal constituent is present in the fluid?
a. Why did the clot form? b. What type of tube should be sent to the laboratory for
b. How can the student perform a correct dilution of the a cell count?
fluid? c. Could the original tube be used for a Gram stain and
c. After the correct dilution is made, the WBC count is culture? Why or why not?
100,000/µL. State two arthritis classifications that
could be considered.
d. State two additional tests that could be run to deter-
mine the classification.
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CHAPTER 13
Serous Fluid
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
13-1 Describe the normal formation of serous fluid. 13-8 List three common chemistry tests performed on
pleural fluid, and state their significance.
13-2 Describe four primary causes of serous effusions.
13-9 State the common etiologies of pericardial effusions.
13-3 Differentiate between a transudate and an exudate,
including etiology, appearance, and laboratory tests. 13-10 Discuss the diagnostic significance of peritoneal
lavage.
13-4 Differentiate between a hemothorax and a hemor-
rhagic exudate. 13-11 Calculate a serum–ascites gradient, and state its
significance.
13-5 Differentiate between a chylous and a pseudochylous
exudate. 13-12 Differentiate between ascitic effusions of hepatic and
peritoneal origin.
13-6 State the significance of increased neutrophils,
lymphocytes, eosinophils, and plasma cells in 13-13 State the clinical significance of the carcinoembryonic
pleural fluid. antigen and CA 125 tests.
13-7 Describe the morphological characteristics of 13-14 List four chemical tests performed on ascitic fluid,
mesothelial cells and malignant cells. and state their significance.

KEY TERMS
Ascites Oncotic pressure Serous fluid
Ascitic fluid Paracentesis Serum-ascites albumin gradient
Chylous effusion Parietal membrane (SAAG)
Effusion Pericardiocentesis Thoracentesis
Exudate Pericarditis Transudate
Hydrostatic pressure Peritonitis Visceral membrane
Mesothelial cell Pseudochylous effusion
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306 Part Three | Other Body Fluids

Introduction amount of positive pressure in the parietal and visceral capil-

laries creates a small excess of fluid that is reabsorbed by the
The closed cavities of the body—namely, the pleural, pericardial, lymphatic capillaries located in the membranes. In Figure 13-2,
and peritoneal cavities—are lined by two membranes referred the normal formation and absorption of pleural fluid are
to as the serous membranes. One membrane lines the cavity wall demonstrated.
(parietal membrane), and the other covers the organs within Disruption of the mechanisms of formation and reabsorp-
the cavity (visceral membrane) (Fig. 13-1). The fluid between tion of serous fluid causes an increase in fluid between the
the membranes is called serous fluid, and it provides lubrication membranes. This increase is termed an effusion. Primary
between the parietal and visceral membranes. Lubrication is nec- causes of effusions include increased hydrostatic pressure (con-
essary to prevent the friction between the two membranes that gestive heart failure), decreased oncotic pressure (hypopro-
occurs as a result of movement of the enclosed organs, such as teinemia), increased capillary permeability (inflammation and
in the expansion and contraction of the lungs. Normally, only a infection), and lymphatic obstruction (tumors) (Box 13-1).
small amount of serous fluid is present because production and
reabsorption take place at constant rates.
Specimen Collection
Formation and Handling
Serous fluids are formed as ultrafiltrates of plasma; no addi- Fluids for laboratory examination are collected by needle aspi-
tional material is contributed by the mesothelial cells that line ration from the respective cavities. These aspiration procedures
the membranes. Production and reabsorption are subject to are referred to as thoracentesis (pleural), pericardiocentesis
hydrostatic pressure and colloidal pressure (oncotic pres- (pericardial), and paracentesis (peritoneal). Usually, abundant
sure) from the capillaries that serve the cavities and the capil- fluid (>100 mL) is collected; therefore, suitable specimens are
lary permeability. Under normal conditions, colloidal pressure available for each section of the laboratory.
from serum proteins is the same in the capillaries on both sides An ethylenediaminetetraacetic acid (EDTA) tube is used
of the membrane. Therefore, hydrostatic pressure in the pari- for cell counts and the differential. Sterile heparinized or
etal and visceral capillaries causes fluid to enter between the sodium polyanethol sulfonate (SPS) evacuated tubes are used
membranes. Filtration of the plasma ultrafiltrate results in in- for microbiology and cytology. For better recovery of microor-
creased oncotic pressure in the capillaries that favors reabsorp- ganisms and abnormal cells, concentration of large amounts of
tion of fluid back into the capillaries. This action produces a fluid is performed by centrifugation. Chemistry tests can be
continuous exchange of serous fluid and maintains the normal run on clotted specimens in plain tubes or in heparin tubes.
volume of fluid between the membranes. The slightly different Specimens for pH must be maintained anaerobically in ice.1

Parietal pleura
Pleural cavity
Parietal
Visceral pleura
pericardium
Pericardial Lung
cavity
Body wall
Visceral
pericardium

Liver

Stomach
Parietal
peritoneum
Visceral
peritoneum Intestine
Peritoneal
cavity

Figure 13–1 The body areas and membranes where

serous fluid is produced.
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Chapter 13 | Serous Fluid 307

Parietal Visceral Chemical tests performed on serous fluids are compared fre-
pleura pleura quently with plasma chemical concentrations because the fluids
Chest Pleural Lung are essentially plasma ultrafiltrates. Therefore, blood specimens
wall space should be obtained at the time of collection.

Transudates and Exudates

Systemic Pulmonary
capillary capillary A general classification of the cause of an effusion can be
accomplished by categorizing the fluid as either transudate or
exudate. Effusions that form because of a systemic disorder
that disrupts the balance in the regulation of fluid filtration and
Oncotic reabsorption—such as the changes in hydrostatic pressure cre-
Oncotic pressure ated by congestive heart failure or the hypoproteinemia asso-
pressure
(mm Hg) ciated with nephrotic syndrome—are called transudates.
Exudates are produced by conditions that directly involve the
membranes of the particular cavity, including infections and
– 26 26 + malignancies. Classifying a serous fluid as a transudate or ex-
udate can provide a valuable initial diagnostic step and aid in
Hydrostatic Hydrostatic the course of further laboratory testing because usually it is not
pressure pressure
(mm Hg)
necessary to test transudate fluids.2 Traditionally, a variety of
laboratory tests have been used to differentiate between tran-
+30 –11 sudates and exudates, including appearance, total protein, lac-
+5 –5
Intrapleural Intrapleural tic dehydrogenase, cell counts, and spontaneous clotting.
pressure pressure However, the most reliable differentiation is obtained by
+9 +10 Lymphatic system
determining the fluid:blood ratios for protein and lactic dehy-
drogenase.3 Differential values for these parameters are shown
Figure 13–2 The normal formation and absorption of pleural in Table 13-1.4 Additional tests are available for specific fluids
fluid. and will be discussed in the following sections.

General Laboratory Procedures

Serous fluid examination—including classification as a transu-
Box 13–1 Pathological Causes of Effusions
date or exudate; appearance; cell count and differential; and
Increased capillary hydrostatic pressure chemistry, microbiology, and cytology procedures—is per-
Congestive heart failure formed in the same manner on all serous fluids. However, the
Salt and fluid retention
significance of the test results and the need for specialized tests
vary among fluids. Therefore, the interpretation of routine and
Decreased oncotic pressure special procedures will be discussed individually for each of
Nephrotic syndrome the three serous fluids.
Hepatic cirrhosis Tests that usually are performed on all serous fluids in-
clude evaluation of the appearance and differentiation between
Malnutrition
a transudate and an exudate. Then effusions of exudative origin
Protein-losing enteropathy are examined for the presence of microbiological and cytolog-
Increased capillary permeability ical abnormalities. Additional tests are ordered based on spe-
cific clinical symptoms. Red blood cell (RBC) and white
Microbial infections
blood cell (WBC) counts are not performed routinely on
Membrane inflammations serous fluids because they provide little diagnostic informa-
Malignancy tion.5 In general, WBC counts greater than 1000/µL and RBC
Lymphatic obstruction counts greater than 100,000/µL indicate an exudate. Serous
fluid cell counts can be performed manually by using a
Malignant tumors, lymphomas Neubauer counting chamber and the methods discussed in
Infection and inflammation Chapter 10 or by electronic cell counters (see Chapter 2).
Thoracic duct injury When manual cell counts are performed, they frequently in-
clude a count of all nucleated cells.1 Inclusion of tissue cells
and debris in the count must be considered when electronic
counters are used, and care must be taken to prevent the
blocking of tubing with debris.
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308 Part Three | Other Body Fluids

Table 13–1 Laboratory Differentiation of may be either transudative or exudative. In addition to the tests
Transudates and Exudates3,4 routinely performed to differentiate between transudates and
exudates, two more procedures are helpful when analyzing
Transudate Exudate pleural fluid: the ratio of pleural fluid cholesterol and
fluid:serum cholesterol and the ratio of pleural fluid:serum total
Appearance Clear, pale yellow Cloudy, color
bilirubin. A pleural fluid cholesterol >60 mg/dL or a pleural
variable
fluid:serum cholesterol ratio >0.3 provides reliable information
WBC count <1000/µL (pleural, >1000/µL that the fluid is an exudate.6 A fluid:serum total bilirubin ratio
pericardial) of 0.6 or more also indicates the presence of an exudate. Meas-
<500/µL urement of glucose is recommended only in effusions suspected
(peritoneal) of being due to rheumatoid arthritis.4
Spontaneous No Possible
clotting
Appearance
Fluid total 30g /L or less >30g/L Considerable diagnostic information concerning the etiology
protein of a pleural effusion can be learned from the specimen appear-
<0.5 >0.5 ance (Table 13-2). Normal and transudate pleural fluids are clear
Fluid:serum
and pale yellow. Usually turbidity is related to the presence of
protein ratio
WBCs and indicates bacterial infection, tuberculosis, or an im-
Fluid:serum <0.6 >0.6 munologic disorder, such as rheumatoid arthritis. The presence
LD ratio of blood in the pleural fluid can signify a hemothorax (trau-
Fluid LD <0.67 × ULN >0.67 × ULN matic injury), membrane damage such as occurs in malignancy,
serum serum or a traumatic aspiration. As seen with other fluids, blood from
Pleural fluid <45–60 mg/dL >45–60 mg/dL a traumatic tap appears streaked and uneven.
cholesterol To differentiate between a hemothorax and hemorrhagic
<0.3 >0.3 exudate, a hematocrit can be run on the fluid. If the blood is
Pleural fluid:
from a hemothorax, the fluid hematocrit is more than 50% of
serum
the whole blood hematocrit because the effusion comes from
cholesterol
the inpouring of blood from the injury.7 An effusion from a
ratio
chronic membrane disease contains both blood and increased
Pleural fluid: <0.6 >0.6 pleural fluid, resulting in a much lower hematocrit.
bilirubin ratio The appearance of a milky pleural fluid may be due to the
Serum-ascites >1.1 <1.1 presence of chylous material from thoracic duct leakage or to
albumin pseudochylous material produced in chronic inflammatory
gradient conditions. Chylous material contains a high concentration of
Glucose Equal to serum Less than or triglycerides, whereas pseudochylous material has a higher
equal to
serum
Specific <1.015 >1.015 Table 13–2 Correlation of Pleural Fluid
Gravity Appearance and Disease6

LD, Lactate dehydrogenase; ULN, upper limit of normal; WBC, white Appearance Disorder
blood cell count.
Clear, pale yellow Normal
Turbid, white Microbial infection (tuberculosis)
Differential cell counts are performed routinely on serous Bloody Hemothorax
fluids, preferably on Wright’s-stained, cytocentrifuged speci- Hemorrhagic effusion, pulmonary
mens or on slides prepared from the sediment of centrifuged embolus, tuberculosis, malignancy
specimens. Smears must be examined not only for WBCs but Milky Chylous material from thoracic
also for normal and malignant tissue cells. Any suspicious cells duct leakage
seen on the differential are referred to the cytology laboratory Pseudochylous material from
or the pathologist. chronic inflammation
Brown Rupture of amoebic liver abscess
Pleural Fluid
Black Aspergillus
Pleural fluid is obtained from the pleural cavity, located between Viscous Malignant mesothelioma (increased
the parietal pleural membrane lining the chest wall and the vis- hyaluronic acid)
ceral pleural membrane covering the lungs. Pleural effusions
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Chapter 13 | Serous Fluid 309

concentration of cholesterol. Therefore, Sudan III staining is Table 13–4 Significance of Cells Seen in Pleural
strongly positive with materials in chylous effusions. In con- Fluid
trast, pseudochylous effusions contain cholesterol crystals.6
Differentiation between chylous and pseudochylous effusions Cells Significance
is summarized in Table 13-3. Pancreatitis
Neutrophils
Hematology Tests Pulmonary infarction
As mentioned previously, the differential cell count is the most Lymphocytes Tuberculosis
diagnostically significant hematology test performed on serous Viral infection
fluids. Primary cells associated with pleural fluid include Autoimmune disorders
macrophages, neutrophils, lymphocytes, eosinophils, mesothe- Malignancy
lial cells, plasma cells, and malignant cells. Macrophages nor-
mally account for 64% to 80% of a nucleated cell count, Lymphoma
followed by lymphocytes (18% to 30%) and neutrophils (1% Sarcoidosis
to 2%) (Table 13-4). These same cells also are found in pericar- Mesothelial cells Normal and reactive forms have no
dial and peritoneal fluids. clinical significance
Similar to other body fluids, an increase in pleural fluid Decreased mesothelial cells are as-
neutrophils indicates a bacterial infection, such as pneumonia. sociated with tuberculosis
Also, neutrophils are increased in effusions resulting from pan-
creatitis and pulmonary infarction. Plasma cells Tuberculosis
Lymphocytes are noticeably present normally in both tran- Malignant cells Primary adenocarcinoma and small-
sudates and exudates in a variety of forms, including small, cell carcinoma
large, and reactive. More prominent nucleoli and cleaved nu- Metastatic carcinoma
clei may be present. Elevated lymphocyte counts are seen in
effusions resulting from tuberculosis, viral infections, malig-
nancy, and autoimmune disorders, such as rheumatoid arthritis
and systemic lupus erythematosus. Systemic lupus erythemato-
sus cells may be seen (Fig. 13-3).
Increased eosinophil levels (>10%) may be associated with
trauma resulting in the presence of air or blood (pneumothorax
and hemothorax) in the pleural cavity. They also are seen in
patients with allergic reactions and parasitic infections.
The membranes lining the serous cavities contain a single
layer of mesothelial cells, so it is not unusual to find these cells

Table 13–3 Differentiation Between Chylous and

Pseudochylous Pleural Effusions
Chylous Pseudochylous
Effusion Effusion Figure 13–3 Systemic lupus erythematosus cell in pleural fluid.
Notice the ingested “round body” (×1000).
Cause Thoracic duct Chronic
damage inflammation
Lymphatic in the serous fluids. Mesothelial cells are pleomorphic; they
obstruction resemble lymphocytes, plasma cells, and malignant cells, fre-
Appearance Milky/white Milky/green tinge quently making identification difficult. They often appear as sin-
gle small or large round cells with abundant blue cytoplasm and
round nuclei with uniform dark purple cytoplasm and may be
Leukocytes Predominantly Mixed cells
referred to as “normal” mesothelial cells (Figs. 13-4 and 13-5).
lymphocytes
In contrast, “reactive” mesothelial cells may appear in clusters;
Cholesterol Absent Present have varying amounts of cytoplasm, eccentric nuclei, and
crystals prominent nucleoli; and be multinucleated, thus more closely
Triglycerides >110 mg/dL <50 mg/dL resembling malignant cells (Figs. 13-6 and 13-7). An increase
Sudan III Strongly positive Negative/weakly in mesothelial cells is not a finding that is diagnostically sig-
staining positive nificant, but they may be increased in cases of pneumonia and
malignancy. Of more significance is the noticeable lack of
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310 Part Three | Other Body Fluids

B
A

A
B

Figure 13–4 Normal pleural fluid mesothelial cells (A), lymphocytes Figure 13–7 One normal (A) and two reactive (B) mesothelial cells
(B), and monocytes (C) (×250). with a multinucleated form (×500).

Plasma
cells

Plasma cells

Figure 13–5 Normal mesothelial cell (×500). Figure 13–8 Pleural fluid plasma cells seen in a case of tuberculosis.
Notice the absence of mesothelial cells (×1000).

cells and other tissue cells from malignant cells is difficult. Dis-
tinguishing characteristics of malignant cells may include nu-
clear and cytoplasmic irregularities, hyperchromatic nucleoli,
cellular clumps with cytoplasmic molding (community bor-
ders), and abnormal nucleus:cytoplasm ratios (Figs. 13-9 to
13-11). Malignant pleural effusions most frequently contain
large, irregular adenocarcinoma cells, small or oat cell carci-
noma cells resembling large lymphocytes, and clumps of
metastatic breast carcinoma cells (Figs. 13-12 to 13-14). Spe-
cial staining techniques and flow cytometry may be used for
positive identification of tumor cells. Box 13-2 describes the
primary characteristics of malignant serous fluid cells.

Figure 13–6 Reactive mesothelial cells showing eccentric nuclei

Chemistry Tests
and vacuolated cytoplasm (×500). In addition to the chemical tests performed to differentiate be-
tween a pleural transudate and a pleural exudate, the most
mesothelial cells associated with tuberculosis, which results common chemical tests performed on pleural fluid are glucose,
from exudate covering the pleural membranes. Also associated pH, adenosine deaminase (ADA), and amylase (Table 13-5).
with tuberculosis is an increase in the presence of pleural fluid Triglyceride levels also may be measured to confirm the pres-
plasma cells (Fig. 13-8). ence of a chylous effusion.
A primary concern in examining all serous effusions is de- Decreased glucose levels are seen with cases of tuberculo-
tecting malignant cells. Often differentiating among mesothelial sis, rheumatoid inflammation, malignant effusion, esophageal
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Chapter 13 | Serous Fluid 311

Figure 13–9 Pleural fluid adenocarcinoma showing cytoplasmic Figure 13–12 Poorly differentiated pleural fluid adenocarcinoma
molding (×250). showing nuclear irregularities and cytoplasmic vacuoles (×500).

Figure 13–10 Pleural fluid adenocarcinoma showing nuclear and Figure 13–13 Pleural fluid small-cell carcinoma showing nuclear
cytoplasmic molding and vacuolated cytoplasm (×1000). molding (×250).

Figure 13–11 Enhancement of nuclear irregularities (×250). Figure 13–14 Metastatic breast carcinoma cells in pleural fluid.
Notice the hyperchromatic nucleoli (×1000).
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312 Part Three | Other Body Fluids

Box 13–2 Characteristics of Malignant Cells As with serum, elevated amylase levels are associated with
pancreatitis, and often amylase is elevated first in the pleural
Increased nucleus:cytoplasm (N:C) ratio: the higher the ratio, the fluid. Pleural fluid amylase, including salivary amylase, also
more poorly differentiated are the cells
may be elevated in cases of esophageal rupture and malignancy.
Irregularly distributed nuclear chromatin
Variation in size and shape of nuclei Microbiological and Serological Tests
Increased number and size of nucleoli Microorganisms primarily associated with pleural effusions in-
Hyperchromatic nucleoli clude Staphylococcus aureus, Enterobacteriaceae, anaerobes, and
Mycobacterium tuberculosis. Gram stains, cultures (both aerobic
Giant cells and multinucleation
and anaerobic), acid-fast stains, and mycobacteria cultures are
Nuclear molding performed on pleural fluid when clinically indicated. Molecu-
Cytoplasmic molding (community borders) lar testing using polymerase chain reaction (PCR) is a more
Vacuolated cytoplasm, mucin production sensitive test to detect M. tuberculosis.11
Serological testing of pleural fluid is used to differentiate
Cellular crowding, phagocytosis
effusions of immunologic origin from noninflammatory processes.
Tests for antinuclear antibody (ANA) and rheumatoid factor (RF)
are performed most frequently.
Detection of the tumor markers carcinoembryonic antigen
(CEA), CA 125 (metastatic uterine cancer), CA 15.3 and CA 549
Table 13–5 Significance of Chemical Testing
(breast cancer), and CYFRA 21-1 (lung cancer) provide valuable
of Pleural Fluid
diagnostic information in effusions of malignant origin.12
Test Significance Pleural fluid testing and its significance are summarized
in Figure 13-15.
Glucose Decreased in rheumatoid inflammation
Decreased in purulent infection
Lactate Elevated in bacterial infection Pericardial Fluid
Triglyceride Elevated in chylous effusions
Normally, only a small amount (10 to 50 mL) of fluid is found
pH Decreased in pneumonia not responding between the pericardial serous membranes. Pericardial effu-
to antibiotics sions are primarily the result of changes in the membrane
Markedly decreased with esophageal permeability due to infection (pericarditis), malignancy, and
rupture trauma-producing exudates. Metabolic disorders, such as ure-
ADA Elevated in tuberculosis and malignancy mia or hypothyroidism, as well as autoimmune disorders, are
the primary causes of transudates. An effusion is suspected
Amylase Elevated in pancreatitis, esophageal
when cardiac compression (tamponade) is noted during the
rupture, and malignancy
physician’s examination. Table 13-6 summarizes the signifi-
cance of pericardial fluid testing.

Appearance
rupture, lupus pleuritis, and purulent infections.8 As an ultra-
filtrate of plasma, pleural fluid glucose levels parallel plasma Normal and transudate pericardial fluid appear clear and pale
levels, and values less than 60 mg/dL are considered decreased. yellow. Effusions resulting from infection and malignancy are
Fluid values should be compared with plasma values. Pleural turbid, and malignant effusions are frequently blood streaked.
fluid lactate levels are elevated in bacterial infections and can Grossly bloody effusions are associated with accidental cardiac
be considered in addition to the glucose level. puncture and misuse of anticoagulant medications. Milky flu-
Pleural fluid pH lower than 7.3 may indicate the need for ids representing chylous and pseudochylous effusions also may
chest-tube drainage in addition to administration of antibiotics be present.
in cases of pneumonia. In cases of acidosis, the pleural fluid
pH should be compared with the blood pH. Pleural fluid pH
Laboratory Tests
at least 0.30 degrees lower than the blood pH is considered Tests performed on pericardial fluid are directed primarily at
significant.9 A pH value as low as 6.0 indicates an esophageal determining whether the fluid is a transudate or an exudate
rupture that is allowing the influx of gastric fluid. Collection and include the ratios for fluid:serum protein and lactic dehy-
of specimens for pH should be taken and analyzed in a manner drogenase (LD). Like pleural fluid, WBC counts are of little
identical to that for arterial blood gas specimens. The fluid clinical value, although a count of >1000 WBCs/µL with a high
should be collected anaerobically using a heparinized syringe percentage of neutrophils can indicate bacterial endocarditis.
and measured in a blood gas analyzer at 37°C.10 Cytological examination of pericardial exudates for the
ADA levels higher than 40 U/L are highly indicative of tu- presence of malignant cells is an important part of the fluid
berculosis. Also, they are elevated frequently with malignancy. analysis. Cells encountered most frequently are the result of
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Chapter 13 | Serous Fluid 313

PLEURAL FLUID TESTING ALGORITHM

Fluid protein and LD

Fluid:serum protein >0.5 Appearance

Fluid:serum LD >0.6
Fluid LD >2/3 upper normal serum value
Bloody Milky
Any positive
No Yes Hematocrit Triglycerides

Transudate Exudate

Glucose
ADA
WBC/Diff
Amylase
pH

ADA >40 U/L WBC/Diff Amylase pH

Lymphocytosis >300/μL Elevated <7.2 <6.0

Granulocytosis

Tuberculosis Bacterial infection Pancreatitis Possible Esophageal

need for rupture
chest tubes
Figure 13–15 Algorithm of pleural fluid testing.

Table 13–6 Significance of Pericardial Fluid metastatic lung or breast carcinoma and resemble those found
Testing in pleural fluid. Figure 13-16 shows a metastatic giant
mesothelioma cell that is seen frequently in pleural fluid and
Test Significance is associated with asbestos contact. Marker levels for a pericar-
dial fluid tumor correlate well with cytological studies.12
Appearance
Bacterial cultures and Gram stains are performed on con-
Clear, pale yellow Normal, transudate centrated fluids when endocarditis is suspected. Frequently in-
Blood-streaked Infection, malignancy fections are caused by previous respiratory infections, including
Grossly bloody Cardiac puncture, anticoagulant
medications
Milky Chylous and pseudochylous
material
Additional testing
Increased Bacterial endocarditis
neutrophils
Malignant cells Metastatic carcinoma
Carcinoembryonic Metastatic carcinoma
antigen
Gram stain and Bacterial endocarditis
culture
Acid-fast stain Tubercular effusion
Adenosine Tubercular effusion Figure 13–16 Malignant pericardial effusion showing giant
deaminase mesothelioma cell with cytoplasmic molding and hyperchromatic
nucleoli (×1000).
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314 Part Three | Other Body Fluids

Streptococcus, Staphylococcus, adenovirus, and coxsackievirus. Table 13–7 Significance of Peritoneal Fluid
Effusions of tubercular origin are increasing as a result of AIDS. Testing
Therefore, acid-fast stains and chemical tests for adenosine deam-
inase are requested often on pericardial effusions. Molecular Test Significance
analysis using PCR is a more sensitive technique for diagnosing
Appearance
infections caused by organisms that are viral, bacterial, or fungal.
Clear, pale yellow Normal
Peritoneal Fluid Turbid Microbial infection
Green Bile, gallbladder, pancreatic
Accumulation of fluid between the peritoneal membranes is disorders
called ascites, and the fluid is referred to commonly as ascitic
Blood-streaked Trauma, infection, or
fluid rather than peritoneal fluid. In addition to the causes of
malignancy
transudative effusions discussed previously, hepatic disorders,
such as cirrhosis, are frequent causes of ascitic transudates. Milky Lymphatic trauma and blockage
Bacterial infections (peritonitis)—often as a result of intestinal Peritoneal lavage >100,000 RBCs/µL indicates
perforation or a ruptured appendix—and malignancy are the blunt trauma injury
most frequent causes of exudative fluids (Table 13-7). WBC count
Sometimes normal saline is introduced into the peritoneal
cavity as a lavage to detect abdominal injuries that have not yet <500 cells/µL Normal
resulted in fluid accumulation. Peritoneal lavage is a diagnostic >500 cells/µL Bacterial peritonitis, cirrhosis
procedure used to detect intra-abdominal bleeding in blunt Differential Bacterial peritonitis, malignancy
trauma cases, and results of the RBC count can be used along
Other Tests
with radiographic procedures to aid in determining the need
for surgery. RBC counts greater than 100,000/µL are indicative Carcinoembryonic Malignancy of gastrointestinal
of blunt trauma injuries. However, this test has been largely re- antigen origin
placed by focused assessment with sonography for trauma CA 125 Malignancy of ovarian origin
(FAST) and computed tomography (CT) with comparable
Glucose Decreased in tubercular
sensitivity and superior specificity.13
peritonitis, malignancy
Cell counts and differentials also may be requested on
fluid from peritoneal dialysis to detect infection, as well as Amylase Increased in pancreatitis,
eosinophil counts to detect allergic reactions to the equipment gastrointestinal perforation
or introduction of air into the peritoneal cavity.1 Alkaline phosphatase Increased in gastrointestinal
perforation
Transudates Versus Exudates Blood urea nitrogen/ Ruptured or punctured bladder
Differentiation between ascitic fluid transudates and exudates creatinine
is more difficult than for pleural and pericardial effusions. The Gram stain and Bacterial peritonitis
serum–ascites albumin gradient (SAAG) is recommended culture
over the fluid:serum total protein and LD ratios to detect tran-
Acid-fast stain Tubercular peritonitis
sudates of hepatic origin.14 Levels of fluid and serum albumin
are measured concurrently, and then the fluid albumin level is Adenosine deaminase Tubercular peritonitis
subtracted from the serum albumin level. A difference (gradi-
ent) of 1.1 or greater suggests a transudate effusion of hepatic
origin, and lower gradients are associated with exudative
effusions, as shown in the following example. of bile, which can be confirmed using standard chemical tests
for bilirubin. Blood-streaked fluid is seen after trauma and
EXAMPLE with cases of tuberculosis, intestinal disorders, and malignancy.
Chylous or pseudochylous material may be present with cases
Serum albumin of 3.8 mg/dL – fluid albumin of
of trauma or blockage of lymphatic vessels.
1.2 mg/dL = gradient of 2.6 = transudate effusion
Serum albumin of 3.8 mg/dL – fluid albumin of Laboratory Tests
3.0 mg/dL = gradient of 0.8 = exudate effusion
Normal WBC counts are less than 500 cells/µL, and the count
increases with bacterial peritonitis and cirrhosis.4 To distin-
guish between those two conditions, an absolute neutrophil
Appearance count should be performed. An absolute neutrophil count
Like pleural and pericardial fluids, normal peritoneal fluid is >250 cells/µL or >50% of the total WBC count indicates infec-
clear and pale yellow. Exudates are turbid with bacterial or fun- tion. Lymphocytes are the predominant cell in tuberculosis and
gal infections. Green or dark-brown color indicates the presence peritoneal carcinomatosis.
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Chapter 13 | Serous Fluid 315

Cellular Examination
Examination of ascitic exudates for the presence of malignant
cells is important for detecting tumors of primary and metastatic
origin. Malignancies are most frequently of gastrointestinal,
prostate, or ovarian origin. Other cells present in ascitic fluid in-
clude leukocytes, abundant mesothelial cells, and macrophages,
including lipophages (Fig. 13-17). Microorganisms, including
bacteria, yeast, and Toxoplasma gondii, may also be present
(Fig. 13-18). Malignant cells of ovarian, prostatic, and colonic
origin, often containing mucin-filled vacuoles, are seen fre-
quently (Figs. 13-19 to 13-22). Psammoma bodies containing
concentric striations of collagen-like material can be seen in be-
nign conditions and also are associated with ovarian and thyroid
malignancies (Fig. 13-23).
Figure 13–19 Ovarian carcinoma showing community borders,
Chemical Testing nuclear irregularity, and hyperchromatic nucleoli (×500).

Chemical examination of ascitic fluid consists primarily of glu-

cose, amylase, and alkaline phosphatase determinations. Glucose
is decreased below serum levels in bacterial and tubercular
peritonitis and malignancy. Amylase is determined on ascitic
fluid to ascertain cases of pancreatitis, and it may be elevated

Figure 13–20 Ovarian carcinoma cells with large mucin-containing

vacuoles (×500).

Figure 13–17 Lipophages (macrophages containing fat droplets) in

peritoneal fluid (×500).

Figure 13–21 Adenocarcinoma of the prostate showing cytoplas-

mic vacuoles, community borders, and hyperchromatic nucleoli
Figure 13–18 Budding yeast in peritoneal fluid (×400). (×500).
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316 Part Three | Other Body Fluids

sensitive and specific method for the diagnosis of bacterial in-

fections, including M. tuberculosis.15

Serological Tests
Measurement of the tumor markers CEA and CA 125 is a valu-
able procedure for identifying the primary source of tumors
producing ascitic exudates. The presence of CA 125 antigen
with a negative CEA suggests the source is from the ovaries,
fallopian tubes, or endometrium.9

For additional resources please visit

www.fadavis.com

Figure 13–22 Colon carcinoma cells containing mucin vacuoles

and nuclear irregularities (×400).
References
1. Clinical and Laboratory Standards Institute: Body Fluid Analy-
sis for Cellular Composition, Approved Guideline H56-A,
Wayne, PA, 2006, CLSI.
2. Jay, SJ: Pleural effusions: Definitive evaluation of the exudate.
Postgrad Med 80(5):180–188, 1986.
3. Light, RW: Clinical practice: Pleural effusion. N Engl J Med
346:1971–1977, 2002.
4. Cooper, A: Blood Sciences Test. Transudate or Exudate. Exeter
Clinical Laboratory International. Web site: www.exeterlabora-
tory.com/test/transudate-or-exudate/. Published March 19,
2019. Accessed July 10, 2019.
5. Kjeldsberg, CR, and Knight, JA: Body Fluids: Laboratory
Examination of Amniotic, Cerebrospinal, Seminal, Serous and
Synovial Fluids. A Textbook Atlas. ASCP, Chicago, 1993.
6. Valdez, L, et al: Cholesterol: A useful parameter for distinguish-
ing between pleural exudates and transudates. Chest 99(5):
1097–1102, 1991.
7. Porcel, JM, and Light, RW: Diagnostic approach to pleural fluid
effusion in adults. Am Fam Physician 73:1211–1220, 2006.
8. Boka, K: Pleural Effusion Workup. Medscape. Web site:
Figure 13–23 Psammoma bodies exhibiting concentric striations https://emedicine.medscape.com/article/299959-workup#c9.
(×500). Published December 28, 2018. Accessed July 10, 2019.
9. Colice, GL, et al: Medical and surgical treatment of parapneu-
monic effusions: An evidence-based guideline. Chest 118:
1158–1171, 2000.
in patients with gastrointestinal perforations. An elevated al- 10. Clinical and Laboratory Science Institute: Analysis of Body
kaline phosphatase level is also highly diagnostic of intestinal Fluids in Clinical Chemistry, Approved Guideline, C49-A,
perforation. Wayne, PA, 2007, CLSI.
Measurements of blood urea nitrogen and creatinine in the 11. Na, MJ: Diagnostic Tools of Pleural Effusion. NCBI. 76(5):
199–210, 2014 DOI: 10.4046/trd.2014.76.5.199. Web site:
fluid are requested when a ruptured bladder or accidental
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050067/.
puncture of the bladder during the paracentesis is of concern. Published May 29, 2014. Accessed July 10, 2019.
Bilirubin is measured when leakage of bile into the peri- 12. Porcel, JM, et al: Use of a panel of tumor markers (carcinoem-
toneum is suspected after trauma or surgery. Bile contains pri- bryonic antigen, cancer antigen 125, carbohydrate antigen 15-3
marily conjugated bilirubin; therefore, a test for total bilirubin and cytokeratin 19 fragments) in pleural fluid for differential
diagnosis of benign and malignant effusions. Chest 126:
is acceptable.10
1757–1763, 2004.
Amylase or lipase can be measured to determine whether 13. Jagminas, L: Diagnostic Peritoneal Lavage. Medscape. Web site:
the pancreatitis or damage to the pancreas is accounting for the https:/emedicine.medscape.com/article/82888-overview#a1.
accumulation of these pancreatic enzymes in the ascitic fluid.10 Published October 20, 2017. Accessed July 10, 2019.
14. Runyan, BA, et al: The serum-ascites albumin gradient is
Microbiology Tests superior to the exudate transudate concept in the differential
diagnosis of ascites. Ann Intern Med 117:215–218, 1992.
Gram stains and bacterial cultures for both aerobes and anaer- 15. Suceveanu, AI, Todescu, D, Mazilu, L, Manousos, FG, Julea, R,
obes are performed when bacterial peritonitis is suspected. In- Voinea, F, Dumitru, E, and Suceveanu, AP: Modern Tools for
oculation of fluid into blood culture bottles immediately at the Diagnosis in Tuberculous Ascites. DOI: 10.5772/intechopen.
70417. Web site: https://www.intechopen.com/books/ascites-
bedside increases the recovery of anaerobic organisms. Acid- physiopathology-treatment-complications-and-prognosis/
fast stains, adenosine deaminase, and cultures for tuberculosis modern-tools-for-diagnosis-in-tuberculous-ascites. Published
may also be requested. Molecular testing using PCR is a more November, 29, 2017. Accessed July 10, 2019.
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Chapter 13 | Serous Fluid 317

Study Questions
1. The primary purpose of serous fluid is to: 8. Fluid: serum protein and lactic dehydrogenase ratios are
A. Remove waste products performed on serous fluids:
B. Lower capillary pressure A. When malignancy is suspected
C. Lubricate serous membranes B. To classify transudates and exudates
D. Nourish serous membranes C. To determine the type of serous fluid
D. When a traumatic tap has occurred
2. The membrane that lines the wall of a cavity is the:
A. Visceral 9. Which of the following requires the most additional
testing?
B. Peritoneal
A. Transudate
C. Pleural
B. Exudate
D. Parietal
10. An additional test performed on pleural fluid to classify
3. During normal production of serous fluid, the slight ex-
the fluid as a transudate or exudate is the:
cess of fluid is:
A. WBC count
A. Absorbed by the lymphatic system
B. RBC count
B. Absorbed through the visceral capillaries
C. Fluid:cholesterol ratio
C. Stored in the mesothelial cells
D. Fluid-to-serum protein gradient
D. Metabolized by the mesothelial cells
11. A milky-appearing pleural fluid indicates:
4. Production of serous fluid is controlled by:
A. Thoracic duct leakage
A. Capillary oncotic pressure
B. Chronic inflammation
B. Capillary hydrostatic pressure
C. Microbial infection
C. Capillary permeability
D. Both A and B
D. All of the above
12. Which of the following best represents a hemothorax?
5. An increase in the amount of serous fluid is called a/an:
A. Blood HCT: 42 Fluid HCT: 15
A. Exudate
B. Blood HCT: 42 Fluid HCT: 10
B. Transudate
C. Blood HCT: 30 Fluid HCT: 10
C. Effusion
D. Blood HCT: 30 Fluid HCT: 20
D. Malignancy
13. All of the following are normal cells seen in pleural fluid
6. Pleural fluid is collected by:
except:
A. Pleurocentesis
A. Mesothelial cells
B. Paracentesis
B. Neutrophils
C. Pericentesis
C. Lymphocytes
D. Thoracentesis
D. Mesothelioma cells
7. Place the appropriate letter in front of the following state-
14. A differential observation of pleural fluid associated with
ments describing transudates and exudates.
tuberculosis is:
A. Transudate
A. Increased neutrophils
B. Exudate
B. Decreased lymphocytes
Caused by increased hydrostatic pressure
C. Decreased mesothelial cells
Caused by increased capillary permeability
D. Increased mesothelial cells
Caused by decreased oncotic pressure
Caused by congestive heart failure
Malignancy related
Tuberculosis related
Endocarditis related
Clear appearance
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318 Part Three | Other Body Fluids

15. All of the following are characteristics of malignant cells 21. The recommended test for determining whether
except: peritoneal fluid is a transudate or an exudate is the:
A. Cytoplasmic molding A. Fluid:serum albumin ratio
B. Absence of nucleoli B. Serum ascites albumin gradient
C. Mucin-containing vacuoles C. Fluid:serum lactic dehydrogenase ratio
D. Increased nucleus:cytoplasm ratio D. Absolute neutrophil count
16. A pleural fluid pH of 6.0 indicates: 22. Given the following results, classify this peritoneal fluid:
A. Esophageal rupture serum albumin, 2.2 g/dL; serum protein, 6.0 g/dL; fluid
albumin, 1.6 g/dL.
B. Mesothelioma
A. Transudate
C. Malignancy
B. Exudate
D. Rheumatoid effusion
23. Differentiation between bacterial peritonitis and
17. Plasma cells seen in pleural fluid indicate:
cirrhosis is done by performing a/an:
A. Bacterial endocarditis
A. WBC count
B. Primary malignancy
B. Differential
C. Metastatic lung malignancy
C. Absolute neutrophil count
D. Tuberculosis infection
D. Absolute lymphocyte count
18. A significant cell found in pericardial or pleural fluid
24. Detection of the CA 125 tumor marker in peritoneal
that should be referred to cytology is a:
fluid indicates:
A. Reactive lymphocyte
A. Colon cancer
B. Mesothelioma cell
B. Ovarian cancer
C. Monocyte
C. Gastric malignancy
D. Mesothelial cell
D. Prostate cancer
19. Another name for a peritoneal effusion is:
25. Chemical tests primarily performed on peritoneal fluid
A. Peritonitis include all of the following except:
B. Lavage A. Amylase
C. Ascites B. Glucose
D. Cirrhosis C. Alkaline phosphatase
20. A test performed primarily on peritoneal lavage fluid D. Calcium
is a/an:
26. Cultures of peritoneal fluid are incubated:
A. WBC count
A. Aerobically
B. RBC count
B. Anaerobically
C. Absolute neutrophil count
C. At 37°C and 42°C
D. Amylase
D. Both A and B
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Chapter 13 | Serous Fluid 319

Case Studies and Clinical Situations

1. Fluid from a patient with congestive heart failure is 4. Paracentesis is performed on a patient with ascites. The
collected by thoracentesis and sent to the laboratory for fluid appears turbid and has an elevated WBC count.
testing. It appears clear and pale yellow and has a WBC Additional tests ordered include an absolute granulocyte
count of 450/µL, fluid:serum protein ratio of 0.35, and count, amylase, creatinine, CEA, and CA 125.
fluid:serum LD ratio of 0.46. a. What is the purpose for the absolute granulocyte
a. What type of fluid was collected? count? If it is less than 250 cells/µL, what condition is
b. Based on the laboratory results, would this fluid be indicated?
considered a transudate or an exudate? Why? b. If the amylase level is elevated, what is its significance?
c. List two other tests that could be performed to aid in State an additional test that might be ordered.
classifying this fluid. c. Explain the significance of an elevated creatinine level.
2. A cloudy pleural fluid has a glucose level of 30 mg/dL d. What is the purpose of the CEA and CA 125 tests?
(serum glucose level is 100 mg/dL) and a pH of 6.8. 5. Describe a situation in which paracentesis might be per-
a. What condition do these results indicate? formed on a patient who does not have ascites. If the RBC
b. What additional treatment might the patient receive, count is 300,000/µL, what does this indicate?
based on these results? 6. Microscopic examination of an ascitic fluid shows many
3. The following results were obtained on a peritoneal fluid: cells with nuclear and cytoplasmic irregularities contain-
serum albumin, 2.8 g/dL; fluid albumin, 1.2 g/dL. ing psammoma bodies. The CEA test result is normal.
What additional test would be helpful?
a. Calculate the SAAG.
b. Is this a transudate or an exudate? Why?
c. What is the most probable cause of the effusion?
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7582_Ch14_321-326 30/06/20 1:05 PM Page 321

CHAPTER 14
Bronchoalveolar Lavage
Fluid
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
14-1 State the indications for performing a bronchoalveolar 14-4 Describe the appearance of BAL fluid in normal and
lavage (BAL). abnormal conditions.
14-2 Describe the procedure for performing a BAL. 14-5 Discuss the normal and abnormal cellular composition
of BAL fluid.
14-3 Explain the procedures for the collecting, handling,
and transport of specimens for BAL.

KEY TERMS
Bronchoalveolar lavage (BAL) Bronchoscopy Flow cytometry
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322 Part Three | Other Body Fluids

Clinical Significance of the specimen. The appearance of the BAL fluid can provide
valuable diagnostic information. The color of BAL fluid can be
Analyzing specimens obtained by bronchoalveolar lavage described as:
(BAL) is a method for obtaining cellular, immunologic, and • Colorless
microbiological information from the lining fluid of the lower
• Milky white
respiratory tract. BAL is particularly useful in evaluating pa-
tients who are immunocompromised or patients with any • Light brown-beige
number of possible diagnoses, including interstitial lung dis- • Gray-beige (typical for smokers)
ease (infectious, noninfectious, immunologic, or malignant), • Red
airway diseases, suspected alveolar hemorrhage, pulmonary
The clarity refers to the transparency or turbidity of the
alveolar proteinosis, and Langerhans cell histiocytosis, as well as
BAL fluid. It is described as:
exposure to dust and asbestos. Often, BAL is used in conjunction
with high-resolution computerized tomography (HRCT), med- • Clear
ical history, and physical examination to determine the need • Hazy
for a surgical biopsy. • Cloudy
• Turbid
Specimen Collection
A bloody BAL fluid with increasing intensities during
During bronchoscopy, a fiber-optic bronchoscope is guided into the sequential aliquots indicates acute diffuse alveolar hem-
a selected bronchopulmonary segment, usually the right middle orrhage, whereas orange-red BAL fluid is the result of an
or lingular lobe of the lung; however, target areas are better de- older hemorrhagic syndrome and would be evaluated for
fined using HRCT before the procedure. Optimal targets are areas intracellular iron content by cytochemistry.2 A milky or light
of alveolar ground-glass opacity, more prominent nodular profu- brown-beige–colored BAL fluid indicates an accumulation of
sion, or fine reticulation.1 The segment to be lavaged should be phospholipid–protein complexes derived from pulmonary
recorded on the requisition form. Aliquots of sterile normal saline surfactant in the lung alveoli and strongly suggests pul-
are instilled into the alveolar spaces through the bronchoscope monary alveolar proteinosis. The BAL fluid should be cen-
to mix with the bronchial contents and are aspirated for cellular trifuged if it looks milky. If typical lipid–protein complexes
examination and culture. In this manner, cells and organisms in exist, a creamy layer forms on top of the rest of the BAL after
the alveoli distant to the bronchoscope can be sampled.2 The in- centrifugation.2 The presence of clots should be noted. The
stillation volume is between 100 and 300 mL of sterile saline in fluid volume is measured, and cell counts and differential
20- to 50-mL aliquots.2 The first aliquot is discarded; the remain- counts are performed.
ing aliquots are sent either individually or pooled for further
analysis.2 The desired fluid volume for analysis is 10 to 20 mL Cell Counts
(minimal volume is 5 mL). Optimal sampling retrieves greater
than 30% with a typical recovery range of 50% to 70%. Low-vol- Counts for white blood cells (WBCs) and red blood cells
ume recovery (less than 25%) caused by fluid retention in the (RBCs) are performed on BAL and may be diluted to facilitate
lung may appear in cases of chronic obstructive lung diseases and counting using a hemocytometer. Cell viability can be deter-
should be noted on the requisition form. mined by adding Trypan blue. Counts also can be performed
with certain automated cell counters as designated by the
Specimen Handling and Transport manufacturer. When cell concentration is less than the auto-
mated instrument’s linearity specifications, hemocytometry
Specimens should be kept at room temperature during trans- should be used.2
port to the laboratory and processed immediately. When de- If a hemocytometer is used, WBC counts may be diluted
livery to the laboratory is delayed for longer than 30 minutes, using the BMP LeukoChek system to facilitate counting. A
specimens should be transported on ice (4°C).1 Specimens that BMP LeukoChek system is available with a 1 to 100 dilution
will not be analyzed immediately should be centrifuged, the of ammonium oxalate to lyse the RBCs. When the RBCs have
cells resuspended in a nutrient-supplemented medium, and lysed and the solution is clear, plate the fluid on a hemocy-
refrigerated at 4°C for up to 24 hours.1 Cell counts should be tometer and allow the cells to settle for 5 minutes. Count all
performed within 1 hour, but are stable for up to 3 hours if cells in the 18 squares on both sides of the hemocytometer
the fluid is in a nutrient-supplemented medium.2 Specimens and calculate the average of the two sides. Using the follow-
can be filtered through loose gauze (50- to 70-µm nylon filter) ing formula, calculate the WBC count:3
to remove mucus, phlegm, and dust.
average number of cells × dilution factor × 10
WBC/µL =
Color and Clarity 9 squares

Diagnostic tests on BAL fluid include a cell count with differ- RBC counts may be diluted with isotonic saline using
ential, microbiological studies, and cytopathology. A macro- an MLA pipette. Plate the fluid on a hemocytometer and
scopic observation is recorded describing the color and clarity allow it to settle for 5 minutes. Count both sides of the
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Chapter 14 | Bronchoalveolar Lavage Fluid 323

hemocytometer and use the following formula to calculate

the RBC/µL3:

number of cells × dilution factor × 10

RBC/µL =
Number of squares counted

Cells must be distributed evenly over the hemocytometer

surface. For a leukocyte count within the reference range, there
should be no more than a 15-cell difference between the high-
est and lowest total number of cells found among the squares
counted. For a RBC count, there should be no more than a
30-cell difference between the highest and lowest total number
of cells found among the squares counted. Total cells counted
on each side of the counting chamber should agree within 10%
of each other. Counts that do not meet this standard should
not be reported. Mix the specimen well and repeat the count. Figure 14–1 Bronchoalveolar lavage: Normal macrophages and
If clumps of cells or clots are present, note on the requisition lymphocytes (×1000).
form that the “cell count may be inaccurate due to clumps of
cells and/or clots.”
A lymphocyte differential count equal to or greater than 25%
suggests granulomatous lung disease, whereas a lymphocyte
differential count greater than 50% suggests hypersensitivity
Technical Tip 14-1. BAL body fluids may not be
pneumonitis or nonspecific interstitial pneumonia.1 The ratio
counted on the Sysmex instrumentation because of
of CD4 to CD8 lymphocytes further defines the disease process
the varied types of cells present.3
(Table 14-1). Immunologic analysis is performed by flow
cytometry.
Leukocytes Neutrophils
Evaluating the predominant inflammatory cellular pattern provides Neutrophils are the primary granulocyte seen, with a normal
valuable information to the clinician in determining a differential value of less than 3%. They are elevated in patients who are cig-
diagnosis. The morphological appearance of cells and particles, arette smokers and in cases of bronchopneumonia, toxin expo-
such as the morphology of macrophages in extrinsic allergic sure, and diffuse alveolar damage. A neutrophil count equal to
alveolitis and sarcoidosis, or the detection of dust particles in oc- or greater than 50% strongly suggests acute lung injury, aspira-
cupational exposure conditions provides diagnostic information.2 tion pneumonia, or suppurative infection.1
Visit www.fadavis.com for Video 14-1 (Preparation of a
cytocentrifuge slide). Eosinophils
Differential slides are prepared by cytocentrifugation Eosinophils, usually less than 1% to 2% of the total cells, are el-
using routine procedures with staining (Wright-Giemsa or May evated in cases of asthma, drug-induced lung disease, infections
Grunwald–Giemsa), and at least 300 cells, but often 500 to (parasitic, mycobacterial, or fungal), hypersensitivity, pneumoni-
1000 cells, are counted and classified.4 Cells seen in BAL fluid tis, and eosinophilic pneumonia. An eosinophil differential
include macrophages, lymphocytes, ratio of CD4+ and CD8+
lymphocytes (CD4/CD8 ratio), neutrophils, eosinophils, ciliated
columnar bronchial epithelial cells, and squamous epithelial cells.
Table 14–1 CD4/CD8 T Cell Findings Associated
Macrophages With Disease Conditions
Macrophages, often containing a variety of phagocytized ma- CD4/CD8 Ratio Conditions
terial, are the cells seen most frequently, in numbers ranging
from 56% to 80% (Fig. 14-1). Phagocytized material includes he- Low Hypersensitivity pneumonitis
mosiderin; golden, brown, or black pigment inclusions; or foamy Silicosis
cells. A predominance of macrophages containing smoking- Drug-induced disease
related inclusions suggests smoking-related interstitial lung dis-
HIV infection
ease or pulmonary Langerhans cell histiocytosis.1
Normal Tuberculosis
Lymphocytes Malignancies
Lymphocytes, normally constituting 1% to 15% of the cell pop- High Sarcoidosis
ulation, are increased in cases of interstitial lung disease, drug Connective tissue disorder
reactions, pulmonary lymphoma, and nonbacterial infections.
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324 Part Three | Other Body Fluids

count greater than or equal to 25% diagnoses eosinophilic lung Box 14–1 Microorganisms Isolated From
disease.1 Bronchoalveolar Lavage Fluid
Mast Cells Bacteria

A mast cell differential count greater than 1% combined with a Staphylococcus aureus (methicillin-sensitive S. aureus [MSSA]) and
lymphocyte count greater than 50% and a neutrophil count methicillin-resistant S. aureus (MRSA)
greater than 3% strongly suggests hypersensitivity pneumonitis.1 Streptococcus pneumoniae
Mycoplasma pneumoniae
Erythrocytes
Chlamydia
The presence of erythrocytes indicates an acute alveolar hem-
Legionella pneumophila
orrhage. Phagocytosed erythrocytes suggest that an alveolar
hemorrhage has occurred within the past 48 hours, whereas Strongyloides stercoralis
hemosiderin-laden macrophages indicate an alveolar hemor- Actinomyces
rhage older than 48 hours. Mycobacterium tuberculosis

Epithelial Cells Klebsiella pneumoniae

Escherichia coli
Ciliated columnar bronchial epithelial cells are more numerous
in bronchial wash specimens than in bronchial lavage speci- Viruses
mens because of the more vigorous washing technique. In a Cytomegalovirus (CMV)
lavage specimen, the presence of these cells normally ranges Herpes simplex virus (HSV)
from 4% to 17% (Fig. 14-2).
Varicella-zoster virus (VZV)
Adenoviruses
Microbiological Tests
Measles virus
Fungal elements, bacteria, and viral inclusions also may be ob- Influenza A and B viruses
served in respiratory specimens. Box 14-1 lists the organisms Respiratory syncytial virus
that may be identified.
Quantitative or semiquantitative cultures are useful for Fungal
diagnosis in cases of ventilator-associated pneumonia and can Aspergillus
diagnose the infection if the organism is identified. Newer mo- Pneumocystis jirovecii (formerly P. carinii)
lecular techniques have allowed for a rapid diagnosis of organ-
Histoplasma capsulatum
isms in BAL fluid.5 With the increasing concern about health
care–acquired infections and antibiotic-resistant microorganisms, Blastomyces dermatitidis
BAL is performed more frequently on patients using ventilators Coccidioides immitis
to detect infection and monitor antibiotic therapy. Cryptococcus neoformans
BAL is becoming an important diagnostic test for Pneu-
Mucor
mocystis jirovecii5 (previously Pneumocystis carinii) in patients
who are immunocompromised. With P. jirovecii, characteristic Candida

amorphous material is seen microscopically under low power,

and organisms are visible under high power (Figs. 14-3 and
14-4).6
Cryptococcus neoformans has become a significant oppor-
tunistic pathogen in patients with AIDS. A diagnosis of pul-
monary cryptococcosis can be made by demonstrating a
positive cryptococcal antigen in respiratory specimens exhibit-
ing yeast cells that morphologically resemble C. neoformans.
The extent of the cryptococcal infection correlates with the
antigen titer.

Cytology
Cytological studies include observing sulfur granules (actino-
Figure 14–2 Bronchoalveolar lavage: Ciliated bronchial epithelial mycetes), hemosiderin-laden macrophages, Langerhans cells,
cells; notice the eosinophilic bar (×1000). cytomegalic cells, fat droplets seen in fat embolism with an Oil
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Chapter 14 | Bronchoalveolar Lavage Fluid 325

Red O stain, and lipid-laden alveolar macrophages using a

Sudan III stain. Periodic acid–Schiff staining or Oil Red O stain-
ing may be useful in diagnosing cases of pulmonary alveolar
proteinosis or aspiration.1 Dust particle inclusions indicate
pneumoconioses or asbestos exposure. Specimens are evaluated
by a pathologist in cytology whenever malignancy is suspected.

For additional resources please visit

www.fadavis.com

References
1. Meyer, KC, Raghu, G, Baughman, RP, et al: An official American
Figure 14–3 Bronchoalveolar lavage: Amorphous material associ- Thoracic Society clinical practice guideline: The clinical utility of
ated with P. carinii when examined under low power (×100). bronchoalveolar lavage cellular analysis in interstitial lung disease.
Am J Respir Crit Care Med 185(9):1004–1014. DOI:10.1164/
rccm.201202-0320ST. https://www.atsjournals.org/doi/full/
10.1164/rccm.201202-0320ST. Published May 1, 2012.
Accessed: October 29, 2019.
2. Clinical and Laboratory Standards Institute: Body Fluid Analysis
for Cellular Composition; Approved Guideline. CLSI document
H56-A. Clinical and Laboratory Standards Institute. Wayne, PA
2006, CLSI.
3. Bronchoalveolar Lavage, BAL Cell Count and Differential.
Methodist Hospital: Clinical Laboratory Procedure Manual.
Omaha, NE, January 5, 2012.
4. Jacobs, JA, DeBrauwer, EI, et al: Accuracy and precision of quan-
titative calibrated loops in transfer of bronchoalveolar lavage
fluid. J Clin Micro 38(6):2117–2121, 2000.
5. Baldassarri, RJ, Rebecca, Kumar, D, Baldassarri, S, and Cai, G:
Diagnosis of Infectious Diseases in the Lower Respiratory Tract.
A Cytopathologist’s Perspective. Arch Pathol Lab Med. 2019;143:
683–694; DOI: 10.5858/ arpa.2017-0573-RA. Web site: https://
www.archivesofpathology.org/doi/pdf/10.5858/arpa.2017-0573-
Figure 14–4 Bronchoalveolar lavage: Characteristic cup-shaped RA. Accessed September 10, 2019.
organisms indicating P. carinii (×1000). 6. Linder, J: Bronchoalveolar Lavage. ASCP, Chicago, 1988.

Study Questions
1. All of the following could be diagnosed by collecting and 3. In bronchoalveolar lavage, the targeted area of the lung is:
analyzing a BAL except: A. Flushed with antibiotics
A. Asbestos-related pulmonary disease (dust particles) B. Rinsed with sterile saline
B. Interstitial lung disease C. Rinsed with water
C. Alveolar hemorrhage D. Flushed with a fluorometric stain
D. Meningitis
4. A BAL fluid that appears orange-red is an indication of
2. What procedure is used for bronchoalveolar lavage? which of the following:
A. Bronchoscopy A. Acute diffuse alveolar hemorrhage
B. Arthrocentesis B. Alveolar proteinosis
C. Colonoscopy C. Patient who is a heavy smoker
D. Thoracentesis D. Older hemorrhage syndrome
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326 Part Three | Other Body Fluids

5. Cell counts from a BAL fluid must be performed within: 9. What is an opportunistic pathogen in patients with
A. 1 hour AIDS that can be recovered in BAL fluid?
B. 3 hours A. Toxoplasma gondii
C. 24 hours B. Legionella pneumophila
D. 36 hours C. Cryptococcus neoformans
D. Mycobacterium tuberculosis
6. An elevated CD4/CD8 lymphocyte ratio indicates:
A. Sarcoidosis 10. The stain used in cytology for the diagnosis of lipid-
laden alveolar macrophages is:
B. Tuberculosis
A. Periodic acid stain
C. HIV infection
B. Oil Red O stain
D. Silicosis
C. Sudan III stain
7. Immunological study of cells is typically performed by:
D. Iron stain
A. Cytocentrifugation
B. Flow cytometry
C. Differential count
D. Hemocytometer cell count
8. The cell in a BAL fluid seen most frequently is:
A. Eosinophil
B. Neutrophil
C. Lymphocyte
D. Macrophage

Case Studies and Clinical Situations

1. A patient presented in the emergency department with a 2. A 35-year-old male with a history of HIV infection is ad-
cough, shortness of breath, and hemoptysis (coughing up mitted to the hospital with fever, dyspnea, and a cough.
blood). A bronchoscopy with bronchoalveolar lavage was BAL is performed.
performed. a. Which of the following findings from the BAL may be
a. What would the specimen possibly look like indicative of his condition?
macroscopically? 1. Presence of lymphocytes with increased ratio of
b. How would an acute diffuse alveolar hemorrhage CD4 and CD8
appear? 2. Presence of lymphocytes with decreased ratio of
c. What cellular element would suggest that the alveolar CD4 and CD8
hemorrhage occurred within the past 48 hours? 3. Increase in eosinophils
d. How would an older hemorrhagic syndrome appear? 4. >5% of epithelial cell counts
e. What intracellular content would be present in the b. What opportunistic organism is often seen in
case of an older hemorrhagic syndrome? HIV/AIDS patients?
f. What procedure would be used to identify the c. What is a confirmatory test?
intracellular content?
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CHAPTER 15
Amniotic Fluid
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
15-1 State the functions of amniotic fluid. 15-7 Interpret a Liley graph.
15-2 Describe the formation and composition of amniotic 15-8 Describe the analysis of amniotic fluid for the detec-
fluid. tion of neural tube disorders.
15-3 Differentiate maternal urine from amniotic fluid. 15-9 Explain the physiological significance of the lecithin-
sphingomyelin (L/S) ratio.
15-4 State indications for performing an amniocentesis.
15-10 State the relationship of phosphatidyl glycerol to FLM.
15-5 Describe the specimen-handling and processing pro-
cedures for testing amniotic fluid for bilirubin, fetal 15-11 Define lamellar bodies and describe their significance
lung maturity (FLM), and cytogenetic analysis. to FLM.
15-6 Discuss the principle of the spectrophotometric 15-12 Discuss the principle of and sources of error for the
analysis for evaluation of hemolytic disease of the L/S ratio, Amniostat-FLM, lamellar body count, and
fetus and newborn. Foam Stability Index for FLM.

KEY TERMS
Amniocentesis Hemolytic disease of the fetus and Meconium
Amnion newborn (HDFN) Oligohydramnios
Amniotic fluid Lamellar bodies Polyhydramnios
Cytogenetic analysis Lecithin-sphingomyelin ratio (L/S Respiratory distress syndrome (RDS)
ratio)
Fetal lung maturity (FLM) Surfactants
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328 Part Three | Other Body Fluids

Introduction Umbilical
cord

Although the testing of amniotic fluid is associated frequently

with cytogenetic analysis, the clinical laboratory also Chorion
performs several significant tests on amniotic fluid. Because
amniotic fluid is a product of fetal metabolism, the constituents
that are present in the fluid provide information about the
metabolic processes taking place during—as well as the
Placenta
progress of—fetal maturation. When conditions arise that Amnion
adversely affect the fetus, the danger to the fetus must be meas-
ured against the ability of the fetus to survive an early delivery. Amniotic Myometrium
The tests covered in this chapter are used to determine the cavity with
amniotic
extent of fetal distress and fetal maturity (Table 15-1). fluid Cervical canal
Advances in prenatal screening using a blood specimen from
a pregnant woman for fragments of cell-free DNA produced by Figure 15–1 Fetus in amniotic sac. (From Thompson, Understand-
the placenta to determine chromosomal abnormalities have led ing Anatomy and Physiology, 3rd Ed. F.A. Davis Co., Philadelphia,
to a decrease in testing amniotic fluid obtained by amniocen- PA.)
tesis. However, often confirmatory testing is necessary after a
positive prenatal blood screening test or to test for conditions
not covered by the screening test. involved in the exchanges of water and chemicals between the
fluid, the fetus, and the maternal circulation and produces pep-
tides, growth factors, and cytokines. The primary functions of
Physiology amniotic fluid are to provide a protective cushion for the fetus,
Function allow fetal movement, stabilize the temperature to protect the
fetus from extreme temperature changes, and permit proper
Amniotic fluid is present in the amnion, a membranous sac lung development.
that surrounds the fetus (Fig. 15-1). The amnion, a membrane
composed of cuboidal cells, is metabolically active and is Volume
Amniotic fluid volume is regulated by a balance between the
production of fetal urine and lung fluid and the absorption
from fetal swallowing and intramembranous flow. Intramem-
Table 15–1 Tests for Fetal Well-Being and branous flow is the absorption of amniotic fluid water and
Maturity solutes into the fetal vascular system.1 The amount of amniotic
Reference Values fluid increases in quantity throughout pregnancy, from approx-
Test at Term14 Significance imately 60 mL at 12 weeks’ gestation and reaching a peak of
approximately 800 to 1200 mL during the third trimester, and
Bilirubin scan ΔA450 >0.025 Hemolytic dis- then it gradually decreases before delivery. A volume of amni-
ease of the otic fluid greater than 1200 mL is called polyhydramnios,
fetus and whereas the volume of amniotic fluid less than 800 mL is
newborn termed oligohydramnios. In either case, some analytes will be
Alpha- <2.0 MoM Neural tube falsely low or falsely high.2 During the first trimester, the ap-
fetoprotein disorders proximately 35 mL of amniotic fluid is derived primarily from
Lecithin- ≥2.0 Fetal lung the maternal circulation. During the latter third to half of preg-
sphingomyelin maturity nancy, the fetus secretes a volume of lung liquid necessary to
ratio expand the lungs with growth. During each episode of fetal
respiratory movement, secreted lung liquid enters the amniotic
Amniostat-FLM Positive Fetal lung
fluid, bathing the lungs and washing pulmonary and alveolar
maturity/
contents, such as lecithin, sphingomyelin, and phosphatidyl
phosphatidyl
glycerol, into the amniotic fluid surrounding the fetus.2 These
glycerol
lung surfactants serve as an index of fetal lung maturity.1 After
Foam Stability ≥47 Fetal lung the first trimester, fetal urine is the major contributor to the
Index maturity amniotic fluid volume. At the time that fetal urine production
Optical density ≥0.150 Fetal lung occurs, fetal swallowing of the amniotic fluid begins and regu-
650 nm maturity lates the increase in fluid from the fetal urine. The fetus swallows
Lamellar body ≥32,000/mL Fetal lung amniotic fluid, which is absorbed through the gastrointestinal
count maturity tract and then reexcreted by the kidneys from the blood into
fetal urine and back into amniotic fluid.2
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Chapter 15 | Amniotic Fluid 329

Failure of the fetus to begin swallowing results in excessive The fern test also can differentiate amniotic fluid from
accumulation of amniotic fluid (polyhydramnios) and is an urine and other body fluids. It is a test used to evaluate pre-
indication of fetal distress, often associated with neural tube mature rupture of the membranes. In the fern test, a vaginal
disorders. Polyhydramnios may be associated secondarily with fluid specimen is spread on a glass slide and allowed to
fetal structural anomalies, cardiac arrhythmias, congenital infec- completely air dry at room temperature; then it is observed
tions, or chromosomal abnormalities.1 Increased fetal swallowing, microscopically. The presence of “fern-like” crystals due to the
urinary tract deformities, and membrane leakage are possible protein and sodium chloride content is a positive screen for
causes of decreased amniotic fluid (oligohydramnios). Oligohy- amniotic fluid.5
dramnios may be associated with congenital malformations, pre- Other tests used to diagnose premature rupture of mem-
mature rupture of amniotic membranes, and umbilical cord branes (PROM) include the pH test, nitrazine test, and biomarker
compression, resulting in decelerated heart rate and fetal death.1 testing (see Chapter 17). Normal vaginal pH is between 4.5
and 6.0. Amniotic fluid has a pH of 7.1 to 7.3; therefore, the pH
Chemical Composition of the specimen of vaginal fluid will be higher than normal if the
membranes have ruptured. The nitrazine test is a screening test
The placenta is the ultimate source of amniotic fluid water and used to determine the presence of amniotic fluid in vaginal se-
solutes. Amniotic fluid has a composition similar to that of cretions. A drop of fluid obtained from the vagina is placed onto
maternal plasma and contains a small amount of sloughed fetal a strip containing nitrazine dye. The strip will turn blue if the pH
cells from the skin, digestive system, and urinary tract. These is greater than 6.0, suggesting that the membranes have ruptured.
cells provide the basis for cytogenetic analysis. The fluid also However, nitrazine testing is unreliable, as substances such as
contains biochemical substances that are produced by the urine, blood, and semen can cause false positives.6
fetus, such as bilirubin, lipids, enzymes, electrolytes, urea, cre- Biomarkers in amniotic fluid have been identified to better
atinine, uric acid, proteins, and hormones that can be tested diagnose rupture of the fetal membranes. They are used in con-
to determine the health or maturity of the fetus. Neural tube junction with the patient examination, visual pooling of fluid,
defects (NTDs) allow fetal cerebrospinal fluid to enter the am- pH testing, and the fern test for the diagnosis of membrane rup-
niotic fluid directly. Alpha-fetoprotein and acetylcholinesterase ture. The AmniSure ROM test (Qiagen, Germantown, MD) is
are two biochemical markers tested to detect these defects.2
an immunochromatographic test that detects the presence of
A portion of the fluid arises from the fetal respiratory tract,
the placental alpha macroglobulin (PAMG-1) protein in vaginal
fetal urine, the amniotic membrane, and the umbilical cord.
discharge.7,8 PAMG-1 in amniotic fluid is 1,000 to 10,000 times
As would be expected, the chemical composition of the amni- higher than in vaginal fluid. High concentrations of this protein
otic fluid changes when production of fetal urine begins. The in vaginal fluid are diagnostic of a rupture of the fetal mem-
concentrations of creatinine, urea, and uric acid increase, brane.9 Actim PROM (CooperSurgical, Trumbull, CT) is a rapid
whereas the concentrations of glucose and protein decrease. immunoassay point-of-care test that detects insulin-like growth
Concentrations of electrolytes, enzymes, hormones, and meta- factor binding protein-1 (IGFBP-1), also referred to as placental
bolic end products also vary but are of little clinical signifi- protein 12 (PP12), in vaginal fluid for suspected cases of
cance. Measurement of amniotic fluid creatinine has been used PROM.10 The advantage of this test is that it is not affected by
to determine fetal age. Before 36 weeks’ gestation, the amniotic contaminants, such as blood, urine, or semen. Another im-
fluid creatinine level ranges between 1.5 and 2.0 mg/dL. It then munoassay test for the detection of PROM is ROM Plus (Clinical
rises above 2.0 mg/dL, thereby providing a means of determin- Innovations, Murray, UT), which is unique in that it detects
ing fetal age greater than 36 weeks.3 both alpha-fetoprotein (AFP) and IGFBP-1 in the vaginal dis-
charge of pregnant women.11 A comparison between the ROM
Differentiating Maternal Urine Plus and the Actim PROM test is shown in Table 15-2.
From Amniotic Fluid
Differentiation between amniotic fluid and maternal urine
may be necessary to determine possible premature membrane
Specimen Collection
rupture or accidental puncture of the maternal bladder dur-
ing specimen collection. Chemical analysis of creatinine,
Indications for Amniocentesis
urea, glucose, and protein aids in the differentiation. Levels Amniocentesis is recommended for cases of NTDs when
of creatinine and urea are much lower in amniotic fluid than screening blood tests, such as the maternal serum AFP test, are
in urine. Creatinine does not exceed 3.5 mg/dL and urea does abnormal or to detect genetic disorders or to evaluate the
not exceed 30 mg/dL in amniotic fluid, whereas values as health of the fetus. Fetal body measurements taken with ultra-
high as 10 mg/dL for creatinine and 300 mg/dL for urea may sonography accurately estimate the gestational age of the fetus
be found in urine.4 Measurement of glucose and protein by a and provide an assessment of the size and growth of the fetus
reagent strip is a less reliable indicator because glucose and throughout pregnancy to diagnose and manage intrauterine
protein are not uncommon urine constituents during preg- growth retardation. Finding an abnormality on the ultrasound
nancy. However, under normal circumstances, the presence could indicate potential problems with fetal development and
of glucose, protein, or both is associated more closely with indicate the need for an amniocentesis and laboratory meas-
amniotic fluid. urements of fetal lung maturity.
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330 Part Three | Other Body Fluids

Table 15–2 Comparison Between the Clinical Box 15–1 Indications for Performing
Innovations ROM and the Actim PROM Amniocentesis
Clinical Amniocentesis may be indicated at 15 to 18 weeks’ gestation
Innovations ROM Actim PROM for the following conditions to determine early treatment
or intervention:
Protein IGFBP-1 and IGFBP-1 • Mother’s age of 35 or older at delivery
Detection AFP
• Family history of chromosome abnormalities, such as trisomy
Detection 5 ng/mL 25 ng/mL IGFBP-1 21 (Down syndrome)
Limit IGFBP-1
• Parents carry an abnormal chromosome rearrangement
150 ng/mL AFP
• Earlier pregnancy or child with birth defect
Time to 5–20 min 5 min
• Parent is a carrier of a metabolic disorder
Result
• Family history of genetic diseases, such as sickle cell disease,
Specimen 6 hours at RT 4 hours RT or Tay-Sachs disease, hemophilia, muscular dystrophy, sickle cell
Stability frozen anemia, Huntington chorea, and cystic fibrosis
Reagent RT Frozen then RT for • Elevated maternal serum alpha-fetoprotein
Storage 2 months
• Abnormal triple-marker screening test
Sensitivity 99% 95.5%
• Previous child with a neural tube disorder, such as spina bifida, or
Specificity 75% 86%–91% ventral wall defects (gastroschisis)
Control 2 levels 3 levels • Three or more miscarriages
(30 days, lot/
Amniocentesis is indicated later in the pregnancy (20–42 weeks) to
shipment) evaluate:
Interference Reject bloody Bloody specimens • Fetal lung maturity
specimens acceptable
(reduced • Fetal distress
interference) • HDFN caused by Rh blood type incompatibility
• Infection

Fetal epithelial cells in amniotic fluid indicate the genetic

material of the fetus and the biochemical substances that the
fetus has produced. These cells can be separated from the fluid,
A maximum of 30 mL of amniotic fluid is collected in ster-
cultured, and examined for chromosome abnormalities by
ile syringes. The first 2 or 3 mL collected can be contaminated
karyotyping, fluorescence in situ hybridization (FISH), fluo-
by maternal blood, tissue fluid, and cells and are discarded.
rescent mapping spectral karyotyping (SKY), and DNA testing.
Specimens should be transferred to sterile plastic containers and
Biochemical substances produced by the fetus can be analyzed
taken immediately to the laboratory. Fluid for bilirubin analysis
by thin-layer chromatography (TLC) to evaluate the health of
in cases of hemolytic disease of the fetus and newborn
the fetus (Box 15-1).
(HDFN) must be protected from light at all times.
In general, amniocentesis is a safe procedure, particularly
when performed after the 14th week of gestation. Fluid for
chromosome analysis usually is collected at approximately Specimen Handling
16 weeks’ gestation, and tests for intrauterine growth retarda-
tion are performed near the end of the second trimester,
and Processing
whereas tests for fetal distress and maturity are performed later Procedures for handling and processing of amniotic fluid vary
in the third trimester.2 with the tests requested and with the methodology used by the
laboratory performing the test. However, in all circumstances,
Collection special handling procedures should be performed immediately
Amniotic fluid is obtained by needle aspiration into the amni- and the specimen delivered promptly to the laboratory. Fluid
otic sac, a procedure called amniocentesis. The procedure per- for fetal lung maturity (FLM) tests should be placed in ice for
formed most frequently is a transabdominal amniocentesis. delivery to the laboratory and kept refrigerated. Specimens
Using continuous ultrasound for guidance, the physician lo- for bilirubin testing must be protected from light immediately.
cates the fetus and placenta to perform the procedure safely. A This can be accomplished by placing the specimens in amber-
thin, hollow needle is inserted through the mother’s abdomen colored tubes, wrapping the collection tube in foil, or by use
into the uterus and into the amniotic sac to aspirate the amni- of a black plastic cover for the specimen container. Specimens
otic fluid. Vaginal amniocentesis also may be performed; how- for cytogenetic studies or microbial studies must be processed
ever, this method carries a greater risk of infection. aseptically and maintained at room temperature or body
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Chapter 15 | Amniotic Fluid 331

temperature (37°C incubation) before analysis to prolong the Table 15–3 Amniotic Fluid Color
life of the cells needed for analysis.
All fluid for chemical testing should be separated from Color Significance
cellular elements and debris as soon as possible to prevent
Colorless Normal
distortion of chemical constituents by cellular metabolism or
disintegration. This can be performed using centrifugation or Blood-streaked Traumatic tap, abdominal trauma,
filtration. The time and speed of centrifugation also depend on intra-amniotic hemorrhage
the test and laboratory protocol. Yellow Hemolytic disease of the fetus and
newborn (bilirubin)
Dark green Meconium
Technical Tip 15-1. Handling and processing of Dark red-brown Fetal death
amniotic fluid vary with the test requested. Refer to
the laboratory procedure manual for guidance. For
example, specimens for bilirubin must be protected
from light. of fetal red blood cells results in the appearance of the red
blood cell degradation product—unconjugated bilirubin—in
the amniotic fluid. By measuring the amount of bilirubin in
Color and Appearance the fluid, the extent of hemolysis taking place may be deter-
mined and the danger this anemia presents to the fetus may
Normal amniotic fluid is colorless and may exhibit slight to be assessed (Fig. 15-2).
moderate turbidity from cellular debris, particularly in later Amniotic fluid bilirubin is measured by spectrophotomet-
stages of fetal development. Blood-streaked fluid may be pres- ric analysis using serial dilutions. As illustrated in Figure 15-3,
ent as the result of a traumatic tap, abdominal trauma, or intra- the optical density (OD) of the fluid is measured in intervals
amniotic hemorrhage. The source of the blood (maternal or between 365 nm and 550 nm and the readings plotted on semi-
fetal) can be determined using the Kleihauer-Betke test for fetal logarithmic graph paper. In normal fluid, the OD is highest at
hemoglobin and is important for further case management. 365 nm and decreases linearly to 550 nm, illustrated by a
The presence of bilirubin gives the fluid a yellow color and straight line. When bilirubin is present, a rise in OD is seen at
is indicative of red blood cell destruction resulting from HDFN. 450 nm because this is the wavelength of maximum bilirubin
Meconium, which is usually defined as a newborn’s first bowel absorption. The difference between the OD of the theoretic
movement, is formed in the intestine from fetal intestinal se- baseline and the OD at 450 nm represents the amniotic fluid
cretions and swallowed amniotic fluid. It is a dark green, bilirubin concentration. Then this difference in OD, referred to
mucus-like material. It may be present in the amniotic fluid as as the absorbance difference at 450 nm (ΔA450), is plotted on
a result of fetal distress. Fetal aspiration of meconium during a Liley graph to determine the severity of the hemolytic disease
fetal swallowing is a concern when increased amounts are pres- (Fig. 15-4).12
ent in the fluid. A very dark red-brown fluid is associated with Notice that the Liley graph plots the ΔA450 against gesta-
fetal death (Table 15-3). tional age and is divided into three zones that represent the ex-
tent of hemolytic severity. Values falling in zone I indicate no
Tests for Fetal Distress more than a mild effect on the fetus; those in zone II indicate
moderate hemolysis and require careful monitoring, anticipat-
Hemolytic Disease of the Fetus ing an early delivery or exchange transfusion upon delivery;
and a value in zone III indicates severe hemolysis and suggests
and Newborn a severe effect on the fetus. Intervention through induction of
The oldest laboratory test performed routinely on amniotic labor or intrauterine exchange transfusion must be considered
fluid evaluates the severity of the fetal anemia produced by when a ΔA450 is plotted in zone III.
HDFN. The incidence of this disease has been decreasing The Queenan curve is a modified Liley curve that shows
rapidly since the development of methods to prevent anti-Rh ΔA450 values from 14 to 40 weeks’ gestation, providing an
antibody production in postpartum mothers. However, anti- earlier prediction of a possible hemolytic crisis. Using the
bodies against other red blood cell antigens also are capable of Queenan chart, ΔA450 values in the lowest zone indicate a Rh-
producing HDFN, and immunization of Rh-negative mothers negative (unaffected) fetus. The indeterminate and Rh positive
may not be effective or even performed in all cases. Initial ex- (affected) zones indicate increasing hemolytic severity. Values
posure to foreign red blood cell antigens occurs during gesta- in the intrauterine death risk zone indicate a high risk of mor-
tion, delivery of the placenta, or a previous pregnancy when tality for the fetus (Fig. 15-5).13
fetal red blood cells enter into the maternal circulation and As mentioned, specimens must be protected from light at
stimulate the mother to produce antibodies to the antigen. all times. Markedly decreased values will be obtained with as
When these antibodies present in the maternal circulation little as 30 minutes of exposure to light. Contamination of the
cross the placenta into the fetal circulation and bind to the anti- fluid by cells, hemoglobin, meconium, or other debris will in-
gen on the fetal cells, the cells are destroyed. The destruction terfere with the spectrophotometric analysis. Specimens should
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332 Part Three | Other Body Fluids

Rh– Rh– Rh–

Baby 1 Rh Baby 2 Rh+

Rh+ antibody

1st Pregnancy 2nd Pregnancy

Rh+ fetus Rh+ fetus

Fetal blood enters IgG antibodies

maternal circulation cross placenta
via placenta to destroy
fetal Rh+ RBCs
IgG antibodies remain
in mother’s circulation Figure 15–2 Rh antibodies crossing the placenta.

0.6 Oxyhemoglobin Bilirubin Neural Tube Defects

peak at 410 peak at 450
NTDs are one of the most common birth defects in the United
0.5
States. They can be detected by the maternal serum alpha-
fetoprotein (MSAFP) blood test, high-resolution ultrasound,
Absorbance

0.4
and amniocentesis. Increased levels of AFP in both the mater-
0.3 No
rm nal circulation and the amniotic fluid can be indicative of fetal
sca al
0.2 n NTDs, such as anencephaly and spina bifida. AFP is the major
protein produced by the fetal liver during early gestation
0.1
(before 18 weeks). It is found in the maternal serum due to the
combined fetal-maternal circulations and in the amniotic fluid
365 410 450 500 550 from diffusion and excretion of fetal urine. Increased levels are
Wavelength (nm) found in the maternal serum and amniotic fluid when the skin
fails to close over the neural tissue, as occurs in anencephaly
Figure 15–3 Spectrophotometric bilirubin scan showing bilirubin and spina bifida.
and oxyhemoglobin peaks. Measurement of amniotic fluid AFP levels is indicated
when maternal serum levels are elevated or a family history of
previous NTDs exists. The possibility of a multiple pregnancy
be centrifuged immediately to remove particulate interference. also must be investigated when serum levels are elevated. Normal
Specimens contaminated with meconium will cause falsely low values are based on the week of gestational age, as the fetus
ΔA450 values and are not acceptable for spectrophotometric produces maximal AFP between 12 and 15 weeks’ gestation,
analysis. Specimens that are contaminated with blood are gen- after which levels in amniotic fluid begin to decline. Both
erally unacceptable because maximum absorbance of oxyhe- serum and amniotic fluid AFP levels are reported in terms of
moglobin occurs at 410 nm and can interfere with the bilirubin multiples of the median (MoM). The median is the laboratory’s
absorption peak (refer back to Fig. 15-3). This interference can reference level for a given week of gestation. A value two times
be removed by extraction with chloroform if necessary.14 A the median value is considered abnormal (greater than 2 MoM)
control may be prepared by diluting commercial chemistry for both maternal serum and amniotic fluid. Elevated amniotic
control sera 1 to 10 with normal saline and treating it in the fluid AFP levels are followed by measurement of amniotic
same manner as the patient specimen. Bilirubin and protein acetylcholinesterase (AChE). The test is more specific for
levels approximate those in amniotic fluid and can be varied NTDs than AFP, provided it is not performed on a bloody
by using low or high control sera.15 specimen, because blood contains AChE.4
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Chapter 15 | Amniotic Fluid 333

1.00
0.90
0.80
0.70
0.60
0.50
0.40
Severely affected fetus
Zone III intervention required
0.30

Change in optical density (ΔOD)

0.20

Zone II
0.10
0.09
0.08 Moderately affected fetus
0.07 requiring close monitoring
0.06
0.05
0.04

0.03
Zone I
0.02
Nonaffected or mildly
affected fetus
0.01
20 22 24 26 28 30 32 34 36 38 40
Figure 15–4 Example of a Liley graph. Gestational age in weeks

0.20
Intrauterine Death Risk
cycle of inhalation and exhalation. Surfactant keeps the alveoli
0.18 from collapsing by decreasing surface tension and allows them
Rh positive
0.16
(affected)
to inflate with air more easily. If the surfactant concentrations
0.14 are too low, then the alveoli will collapse, causing RDS. The
OD 450nm

0.12 incidence of RDS decreases with increasing gestational age and

Indeterminate
0.10 lung maturity.16 Therefore, laboratory tests must be performed
0.08 to determine the maturity of the fetal lungs. The amount of
0.06 surfactant in fetal lungs can be estimated by measuring the
Rh negative
0.04
(unaffected) amount of surfactant in amniotic fluid.16 Several laboratory
0.02
tests are available to measure FLM.
0.00
14 16 18 20 22 24 26 28 30 32 34 36 38 40 Lecithin-Sphingomyelin Ratio
Weeks’ gestation
The reference method to which tests of FLM are compared is
Figure 15–5 Queenan curve for amniotic fluid ΔA450 values the lecithin-sphingomyelin (L/S) ratio. Lecithin is the pri-
at 14 to 40 weeks’ gestation mary component of the surfactants (phospholipids, neutral
lipids, and proteins) that make up the alveolar lining and
account for alveolar stability.
Tests for Fetal Maturity Lecithin is produced at a relatively low and constant rate
until the 35th week of gestation, at which time a noticeable in-
Fetal distress, whether caused by HDFN or other conditions, crease in its production occurs, resulting in the stabilization of
forces the obstetrician to consider a preterm delivery. At this the fetal lung alveoli. Sphingomyelin is a lipid that is produced
point, fetal maturity must be assessed. at a constant rate after about 26 weeks’ gestation; therefore, it
can serve as a control on which to base the rise in lecithin. Both
Fetal Lung Maturity lecithin and sphingomyelin appear in the amniotic fluid in
Respiratory distress syndrome (RDS) is the most frequent amounts proportional to their concentrations in the fetus.17
complication of early delivery and is the seventh most common Before 35 weeks’ gestation, the L/S ratio is usually less than 1.6
cause of morbidity and mortality in the premature infant.16 because large amounts of lecithin are not being produced at
This disease is caused by an insufficiency of lung surfactant this time. After 35 weeks’ gestation, the lecithin concentration
production and structural immaturity of the fetal lungs. Sur- increases while the sphingomyelin concentration remains
factant normally appears in mature lungs and allows the alveoli constant. The L/S ratio will rise to 2.0 or higher as the lecithin
(air sacs of the lung) to remain open throughout the normal production increases to prevent alveolar collapse. Therefore,
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334 Part Three | Other Body Fluids

when the L/S ratio reaches 2.0, usually a preterm delivery is

PROCEDURE 15-1
considered to be a relatively safe procedure. Falsely elevated
results are encountered in fluid contaminated with blood or “Foam Test” or “Shake Test”
meconium, because both these substances contain lecithin and 1. Mix equal parts of amniotic fluid with 95% ethanol.
sphingomyelin.
Quantitative measurement of lecithin and sphingomyelin 2. Vigorously shake for 15 seconds.
is performed using TLC. Because the procedure is labor inten- 3. Allow to sit undisturbed for 15 minutes.
sive and subject to high coefficients of variation, many labora- 4. Observe for the presence of a continuous line of
tories have replaced the L/S ratio with the quantitative bubbles around the outside edge.
phosphatidyl glycerol (PG) immunoassays and lamellar body
density procedures.18

Phosphatidyl Glycerol PROCEDURE 15-2

The presence of another lung surface lipid, PG, is also essential
for adequate lung maturity and can be detected after 35 weeks’ Foam Stability Index
gestation. The production of PG normally parallels that of 1. Add 0.5 mL of amniotic fluid to tubes containing
lecithin, but its production is delayed in cases of maternal di- increasing amounts of 95% ethanol ranging from
abetes. In this circumstance, respiratory distress occurs in the 0.42 to 0.55 mL in 0.01-mL increments.
presence of an L/S ratio of 2.0. Therefore, a TLC lung profile 2. Vigorously shake for 15 seconds.
must include lecithin, sphingomyelin, and PG to provide an
3. Allow to sit undisturbed for 15 minutes.
accurate measurement of FLM.19
Development of an immunologic agglutination test for PG 4. Observe for the presence of a continuous line of bub-
has provided a method for assessment of fetal maturity that is bles around the outside edge.
more rapid and easy to perform and that does not require a 5. Values ≥47 indicate fetal lung maturity.
laboratory to be equipped to perform TLC. The Amniostat-
FLM (Irving Scientific, Santa Ana, CA) uses antisera containing
polyclonal anti-PG antibodies that are specific for PG-contain- for PG. The test cannot be used with contaminated amniotic
ing lamellar bodies in the amniotic fluid. The size of the ag- fluid because blood and meconium also reduce surface tension,
glutinates is read macroscopically, and the results are reported yielding a result of a falsely mature index.
as negative, indicating pulmonary immaturity, or low positive
or high positive, indicating pulmonary maturity. The test is not Lamellar Bodies
affected by specimen contamination with blood and meco- Surfactant is composed of approximately 90% phospholipid and
nium.20 Studies have shown good correlation with TLC but 10% protein and is packaged into layered storage granules called
with a slightly higher incidence of false-negative results that lamellar bodies.16,23 Lamellar bodies are densely packed layers
may need to be followed up with further testing.21,22 of phospholipids that represent a storage form of pulmonary
surfactant. They are secreted by the type II pneumocytes of the
Foam Stability Index fetal lung at about 24 weeks of gestation and are absorbed into
Until the development of biochemical techniques to measure the alveolar spaces to provide surfactant. They enter the amniotic
the individual lung-surface lipid concentrations, a mechanical fluid at about 26 weeks of gestation and increase in concentra-
screening test, called the “foam” or “shake” test, was used to tion from 50,000 to 200,000 per microliter by the end of the
determine their presence. Because it can be performed at the third trimester. As the fetal lung matures, increased production
bedside or in the laboratory, the test is still in use. Amniotic of lamellar bodies is reflected by an increase in amniotic fluid
fluid is mixed with 95% ethanol, shaken for 15 seconds, and phospholipids and the L/S ratio.24 Therefore, the number of
allowed to sit undisturbed for 15 minutes. At the end of this lamellar bodies present in the amniotic fluid correlates with the
time, the surface of the fluid is observed for the presence of a amount of phospholipid present in the fetal lungs.
continuous line of bubbles around the outside edge. The pres- The presence of lamellar bodies increases the OD of
ence of bubbles indicates that a sufficient amount of phospho- the amniotic fluid. Specimens are centrifuged at 2000 g for
lipid is available to reduce the surface tension of the fluid even 10 minutes and examined using a wavelength of 650 nm,
in the presence of alcohol, an antifoaming agent. which rules out interference from hemoglobin, but not other
A modification of the foam test uses 0.5 mL of amniotic fluid contaminants, such as meconium. An OD of 0.150 has been
added to increasing amounts of 95% ethanol, providing a gradi- shown to correlate well with an L/S ratio of greater than or
ent of ethanol/fluid ratios ranging from 0.42 mL to 0.55 mL equal to 2.0 and the presence of PG.25
in increments of 0.01 mL, which can be used to provide a
Lamellar Body Count
semiquantitative measure of the amount of surfactant present.
A value of 47 or higher indicates FLM. The Foam Stability Lamellar body diameter is similar to that of small platelets, rang-
Index has shown good correlation with the L/S ratio and tests ing in size from 1.7 to 7.3 fL, or 1 to 5µm; therefore, lamellar
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Chapter 15 | Amniotic Fluid 335

body counts (LBCs) can be obtained using the platelet channel

of automated hematology analyzers using either optical or im- For additional resources please visit
pedance methods for counting.23 Some automated cell counters www.fadavis.com
use both impedance and optical methods for counting. Because
the various hematology analyzers count lamellar bodies differ-
ently and require different specimen preparation, cutoff values
vary for FLM, making it necessary to establish analyzer-specific References
LBC clinical decision limits.23 The advantages of lamellar body 1. Ross, MG, Brace, RA, and the NIH Workshop Participants,
counting include the following: National Institute of Child Health and Development Confer-
ence summary: Amniotic fluid biology—basic and clinical
1. Rapid turnaround time aspects. J Maternal-Fetal Med 10:2–19, 2001.
2. Low reagent cost 2. Clinical and Laboratory Standards Institute: Analysis of Body
Fluids in Clinical Chemistry: approved guideline, CLSI
3. Wide availability Document C49-A, Wayne, PA, 2007, CLSI.
4. Low degree of technical difficulty 3. Weiss, RR, et al: Amniotic fluid uric acid and creatinine as
measures of fetal maturity. Obstet Gynecol 44(2):208–214,
5. Low volume of amniotic fluid required 1974.
6. Excellent clinical performance23 4. Wenk, RE, and Rosenbaum, JM: Examination of amniotic fluid.
In Henry, JB (ed): Clinical Diagnosis and Management by
Amniotic fluid specimens containing whole blood, meco- Laboratory Methods. WB Saunders, Philadelphia, 1996.
nium, and mucus should not be used. Blood initially raises the 5. Heron, HJ: The use of the Fern test to differentiate amniotic
LBC because of the presence of platelets and then can lower the fluid from urine. Triangle Jul:20:60–62, 1963.
LBC as lamellar bodies are trapped in the fibrin strands.23 Meco- 6. Galan, N: Tests for Premature Rupture of Membranes. Health-
line. Web site: https://www.healthline.com/health/pregnancy/
nium and mucus cause a false increase in the LBC. Specimens premature-rupture-test. Published January 8, 2016. Accessed
may be stored at 2°C to 8°C but never frozen. A consensus pro- July 11, 2019.
tocol for performing LBC has been published by the Clinical and 7. Caughey, AM, et al: Contemporary Diagnosis and management
Laboratory Standards Institute (CLSI) and is described in Proce- of Preterm Premature Rupture of Membranes. Rev. Obstet.
dure 15-3. Results are reported in units of lamellar bodies per Gynecol. 2008;1(1):11–22.
8. AmniSure®ROM Test Package Insert. Web site: www.amnisure.
microliter and should be accompanied by the laboratory’s estab- com/resource/prom-amniotic-fluid-testing. Published 2019.
lished values for maturity, as well as the instrument that was used. Accessed July 11, 2019.
A consensus protocol for noncentrifuged specimens considers 9. Lee, SM, Lee, JH, Seong, HS, et al: The Clinical Significance of
LBCs greater than 50,000/µL an indication of FLM and values a Positive Amnisure Test in Women with Term Labor with
below 15,000/µL as immature. Results in between these two val- Intact Membranes. J. Matern Fetal Neonatal Med. 22(4):
305–310, 2009. DOI: 10:1080/14767050902801694. Web
ues are considered indeterminate, and further testing using alter- site: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2744034.
native methods is recommended.26 Accessed July 11, 2019.
10. Abdelazim IA: Insulin-like growth factor binding protein-1
(Actim PROM test) for the detection of premature rupture of
fetal membranes. J Obstet Gynaecol Res. 40(4),961–967,
2014. DOI: 10.1111/jog. 12296 Epub Feb 6, 2014. Web site:
PROCEDURE 15-3 https://www.ncbi.nlm.nih.gov/pubmed/24612210. Accessed
July 11, 2019.
Lamellar Body Count23 11. ROM plus +. Clinical Innovations. Package Insert. Web site:
https://clinicalinnvations.com/wp-content/uploads/sites/21/
1. Mix the amniotic fluid specimen by inverting the 2015/12/050-0642-Rev-D.pdf. Published 2016. Accessed
capped specimen container five times. July 11, 2019.
2. Transfer the fluid to a clear test tube to allow for 12. Liley, AW: Liquor amnii analysis in the management of the
pregnancy complicated by Rhesus sensitization. Am J Obstet
visual inspection.
Gynecol 82:1359, 1961.
3. Visually inspect the specimen. Fluids containing 13. Wagle, S: Hemolytic Disease of the Newborn Clinical Presenta-
obvious mucus, whole blood, or meconium should tion. Medscape. Web site: https://emedicine.medscape.com/
not be processed for an LBC. article/974349-clinical. Published December 28, 2017.
Accessed July 11, 2019.
4. Cap the tube and mix the specimen by gentle inver- 14. Spinnato, JA, et al: Amniotic bilirubin and fetal hemolytic
sion or by placing the test tube on a tube rocker disease. Am J Obstet Gynecol 165(4):1030–1035, 1991.
for 2 minutes. 15. McDonald, OL, and Watts, MT: Use of commercially prepared
control sera as quality control materials for spectrophotometric
5. Flush the platelet channel; analyze the instrument’s bilirubin determinations in amniotic fluid. Am J Clin Pathol
diluents buffer until a background count deemed 84(4):513–517, 1985.
acceptable by the laboratory is obtained in two 16. Grenache, D: Fetal lung maturity testing: what labs need to
consecutive analyses. know now. Medical Lab Observer (MLO), February 2012.
17. Gluck, L, et al: Diagnosis of the respiratory distress syndrome
6. Process the sample through the cell counter and by amniocentesis. Am J Obstet Gynecol 109(3):440–445, 1971.
record the platelet channel as the LBC. 18. Dubin, SB: Assessment of FLM: Practice parameter. Am J Clin
Pathol 110:723–732, 1998.
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336 Part Three | Other Body Fluids

19. Kulovich, MV, Hallman, MB, and Gluck, L: The lung profile: 24. Khazardoost, S, et al: Amniotic fluid lamellar body count and
Normal pregnancy. Am J Obstet Gynecol 135:57–60, 1979. its sensitivity and specificity in evaluating of fetal lung maturity.
20. Eisenbrey, AB, et al: Phosphatidyl glycerol in amniotic fluid: J Obstet Gynaecol 25(3):257–259, 2005.
Comparison of an “ultrasensitive” immunologic assay with TLC 25. Sbarra, AJ, et al: Correlation of amniotic fluid optical density at
and enzymatic assay. Am J Clin Pathol 91(3):293–297, 1989. 650 nm and lecithin/sphingomyelin ratios. Obstet Gynecol 48:
21. Chapman, JF: Current methods for evaluating FLM. Lab Med 613, 1976.
17(10):597–602, 1986. 26. Lu Ji, Gronowski, AM, Eby, C: Lamellar Body Counts Performed
22. Saad, SA, et al: The reliability and clinical use of a rapid phos- on Automated Hematology Analyzers to Assess Fetal Lung
phatidyl glycerol assay in normal and diabetic pregnancies. Am Maturity. LabMedicine 39(7): 419–423, 2008.
J Obstet Gynecol 157(6):1516–1520, 1987.
23. Clinical and Laboratory Standards Institute. Assessment of fetal
lung maturity by the lamellar body count; approved guideline,
CLSI document C58-A. Wayne, PA, 2011, CLSI.

Study Questions
1. Which of the following is not a function of amniotic fluid? 6. How are specimens for FLM testing delivered to and
A. Allows movement of the fetus stored in the laboratory?
B. Allows exchange of carbon dioxide and oxygen A. Delivered on ice and refrigerated
C. Protects the fetus from extreme temperature changes B. Immediately centrifuged
D. Acts as a protective cushion for the fetus C. Kept at room temperature
D. Delivered in a vacuum tube
2. What is the primary cause of the normal increase in am-
niotic fluid as a pregnancy progresses? 7. Why are amniotic specimens for cytogenetic analysis
A. Fetal cell metabolism incubated at 37°C before analysis?
B. Fetal swallowing A. To detect the presence of meconium
C. Fetal urine B. To differentiate amniotic fluid from urine
D. Transfer of water across the placenta C. To prevent photo-oxidation of bilirubin to biliverdin
D. To prolong fetal cell viability and integrity
3. Which of the following is not a reason for decreased
amounts of amniotic fluid? 8. Match the following colors in amniotic fluid with their
A. Fetal failure to begin swallowing significance.
B. Increased fetal swallowing A. Colorless 1. Fetal death
C. Membrane leakage B. Dark green 2. Normal
D. Urinary tract defects C. Red-brown 3. Presence of bilirubin
D. Yellow 4. Presence of meconium
4. Why might a creatinine level be requested on an amniotic
fluid? 9. A significant rise in the OD of amniotic fluid at 450 nm
A. Detect oligohydramnios indicates the presence of which analyte?
B. Detect polyhydramnios A. Bilirubin
C. Differentiate amniotic fluid from maternal urine B. Lecithin
D. Evaluate lung maturity C. Oxyhemoglobin
D. Sphingomyelin
5. Amniotic fluid specimens are placed in amber-colored
tubes before sending them to the laboratory to prevent 10. Plotting the amniotic fluid OD on a Liley graph represents
the destruction of: the severity of hemolytic disease of the fetus and newborn.
A. Alpha-fetoprotein A value that is plotted in zone II indicates what condition
of the fetus?
B. Bilirubin
A. No hemolysis
C. Cells for cytogenetics
B. Mildly affected fetus
D. Lecithin
C. Moderately affected fetus that requires close
monitoring
D. Severely affected fetus that requires intervention
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Chapter 15 | Amniotic Fluid 337

11. The presence of a fetal neural tube disorder may be 16. True or False: Phosphatidyl glycerol is present with an
detected by: L/S ratio of 1.1.
A. Increased amniotic fluid bilirubin 17. A rapid immunologic test for FLM that does not require
B. Increased maternal serum alpha-fetoprotein performance of thin-layer chromatography is:
C. Decreased amniotic fluid phosphatidyl glycerol A. AFP levels
D. Decreased maternal serum acetylcholinesterase B. Amniotic acetylcholinesterase
12. True or False: An AFP MoM value greater than two times C. Amniostat-FLM
the median value is considered an indication of a neural D. Bilirubin scan
tube disorder.
18. Does the failure to produce bubbles in the Foam
13. When severe HDFN is present, which of the following Stability Index indicate increased or decreased lecithin?
tests on the amniotic fluid would the physician not order A. Increased
to determine whether the fetal lungs are mature enough
B. Decreased
to withstand a premature delivery?
A. AFP levels 19. The presence of phosphatidyl glycerol in amniotic fluid
fetal lung maturity tests must be confirmed when:
B. Foam stability index
A. Hemolytic disease of the fetus and newborn is
C. Lecithin/sphingomyelin ratio
present
D. Phosphatidyl glycerol detection
B. The mother has maternal diabetes
14. True or False: Before 35 weeks’ gestation, the normal L/S C. Amniotic fluid is contaminated by hemoglobin
ratio is less than 1.6.
D. A neural tube disorder is suspected
15. When performing an L/S ratio by thin-layer
20. A lamellar body count of 50,000 correlates with:
chromatography, a mature fetal lung will show:
A. Absent phosphatidyl glycerol and L/S ratio of 1.0
A. Sphingomyelin twice as concentrated as lecithin
B. L/S ratio of 1.5 and absent phosphatidyl glycerol
B. No sphingomyelin
C. OD at 650 nm of 1.010 and an L/S ratio of 1.1
C. Lecithin twice as concentrated as sphingomyelin
D. OD at 650 nm of 0.150 and an L/S ratio of 2.0
D. Equal concentrations of lecithin and sphingomyelin

Case Studies and Clinical Situations

1. Amniocentesis is performed on a woman believed to be in 2. Amniocentesis is performed after a maternal serum AFP
approximately the 31st week of gestation. This is the sec- level of 2.2 MoM at 15 weeks’ gestation.
ond pregnancy for this Rh-negative woman with diabetes. a. What fetal condition is suspected?
Spectrophotometric analysis of the fluid shows a ΔA450
b. If the amniotic fluid AFP is 2.5 MoM, what additional
of 0.3.
test could be performed?
a. Based on the Liley graph, should the physician
c. In what situation would this additional test not be
consider inducing labor?
performed?
b. What else must the physician consider before
inducing labor? 3. How might a dark green amniotic fluid affect the results
of the following tests?
The physician decides to induce labor based on a
positive Amniostat-FLM. a. Foam Stability Index
c. What information did this test provide for the physician? b. L/S ratio
d. Why did the physician prefer an Amniostat-FLM to an c. Amniostat-FLM
L/S ratio in this situation? d. OD650
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338 Part Three | Other Body Fluids

4. How might a blood-streaked amniotic fluid affect the re- c. Specimen was exposed to light
sults of the following tests? d. Specimen reached the reference laboratory within
a. L/S ratio 30 minutes
b. AChE 6. A woman was in the 35th week of pregnancy and had a
c. Bilirubin analysis feeling like she could not stop urinating. She noticed a
d. Amniostat-FLM vaginal discharge or wetness that was more than she
thought normal. She immediately called her obstetrician
5. Amniocentesis is performed on a woman whose last two and was told to come into the clinic.
pregnancies resulted in stillbirths due to hemolytic dis-
a. What could possibly have occurred?
ease of the fetus and newborn. A screening test performed
at the hospital is positive for bilirubin, and the specimen b. Name five tests that could be performed to confirm
is sent to a reference laboratory for a bilirubin scan. this condition.
Physicians are concerned when the report comes back c. What biomarkers have been identified that can con-
negative. What factors would be considered in evaluating firm this condition?
this result? d. Which test is not affected by the presence of blood?
a. Incorrect specimen was sent
b. Specimen was refrigerated
7582_Ch16_339-354 13/07/20 5:47 PM Page 339

CHAPTER 16
Fecal Analysis
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
16-1 Describe the normal composition and formation of 16-11 Describe a positive microscopic examination for
feces. muscle fibers.
16-2 Differentiate between secretory and osmotic diarrhea 16-12 Name the fecal fats stained by Sudan III, and give the
using fecal electrolytes, fecal osmolality, and stool pH. conditions under which they will stain.
16-3 List three causes of secretory and osmotic diarrhea. 16-13 Describe and interpret the microscopic results
that are seen when a specimen from a patient with
16-4 Describe the mechanism of altered motility and at
steatorrhea is stained with Sudan III.
least three conditions that can cause it.
16-14 Discuss the collection procedure for a quantitative
16-5 List three causes of steatorrhea.
fecal fat, as well as methods for analysis.
16-6 Differentiate malabsorption from maldigestion syn-
16-15 Explain the methods used to detect fecal occult
dromes, and name a test that distinguishes the two
blood.
conditions.
16-16 Instruct a patient in the collection of specimens for
16-7 Instruct patients in the collection of random and
occult blood, including an explanation of dietary
quantitative stool specimens.
restrictions for the guaiac test.
16-8 State a pathogenic and a nonpathogenic cause for
16-17 Briefly describe a chemical screening test performed
stools that are red, black, and pale yellow.
on feces for each of the following: fetal hemoglobin,
16-9 State the significance of stools that are bulky, ribbon- pancreatic insufficiency, and carbohydrate intolerance.
like, or contain mucus.
16-10 State the significance of increased neutrophils in a
stool specimen.

KEY TERMS
Acholic stools Malabsorption Secretory diarrhea
Constipation Maldigestion Steatorrhea
Diarrhea Occult blood
Dysentery Osmotic diarrhea
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340 Part Three | Other Body Fluids

Introduction intestinal gas (flatus). Carbohydrates, especially oligosaccha-

rides, that are resistant to digestion pass through the upper in-
In the minds of most laboratory personnel, fecal specimen testine unchanged but are metabolized by bacteria in the lower
analysis fits into the category of a “necessary evil.” However, intestine, producing large amounts of flatus. Excessive gas pro-
as an end product of body metabolism, feces do provide valu- duction also occurs in people who are lactose intolerant when
able diagnostic information. Routine fecal examination in- the intestinal bacteria metabolize the lactose from consumed
cludes macroscopic, microscopic, and chemical analyses for milk or lactose-containing substances.
the early detection of gastrointestinal (GI) bleeding, liver and Although digestion of ingested proteins, carbohydrates,
biliary duct disorders, maldigestion/malabsorption syndromes, and fats takes place throughout the alimentary tract, the small
pancreatic diseases, inflammation, and causes of diarrhea and intestine is the primary site for the final breakdown and reab-
steatorrhea. Of equal diagnostic value is the detection and sorption of these compounds. Digestive enzymes secreted into
identification of pathogenic bacteria, viruses, and parasites; the small intestine by the pancreas include trypsin, chy-
however, these procedures are best covered in a microbiology motrypsin, amino peptidase, and lipase. Bile salts provided by
textbook and are not discussed here. the liver aid in the digestion of fats. A deficiency in any of these
substances creates an inability to digest and, therefore, to re-
Physiology absorb certain foods. Excess undigested or unreabsorbed ma-
terials then appear in the feces, and the patient exhibits
The normal fecal specimen contains bacteria, cellulose, undi- symptoms of maldigestion and malabsorption. As shown in
gested foodstuffs, GI secretions, bile pigments, cells from the Figure 16-1, approximately 9,000 mL of ingested fluid and
intestinal walls, electrolytes, and water. Approximately 100 to saliva, as well as secretions of gastric, liver, pancreatic, and in-
200 g of feces is excreted in a 24-hour period. Many species of testinal origins, enter the digestive tract each day. Under normal
bacteria make up the normal flora of the intestines. Bacterial conditions, only between 500 and 1,500 mL of this fluid
metabolism produces the strong odor associated with feces and reaches the large intestine, and only about 150 mL is excreted

Food and drink

2000 mL

Saliva
1500 mL

Gastric
secretions
1,500 mL

Bile from liver

1,000 mL

Pancreas
1,000 mL
Intestinal
secretions
2,000 mL
Mucous
(water and
secretions
electrolytes)
200 mL

Feces Colon
150 mL (up to 3,000 mL of Figure 16–1 Fluid regulation in the gastrointestinal
water reabsorbed) tract.
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Chapter 16 | Fecal Analysis 341

in the feces. Water and electrolytes are readily absorbed in both hormones, inflammatory bowel disease (Crohn disease, ulcer-
the small and large intestines, resulting in a fecal electrolyte ative colitis, lymphocytic colitis, diverticulitis), endocrine dis-
content that is similar to that of plasma. orders (hyperthyroidism, Zollinger-Ellison syndrome, VIPoma),
The large intestine is capable of absorbing approxi- neoplasms, and collagen vascular disease.
mately 3,000 mL of water. When the amount of water reach-
ing the large intestine exceeds this amount, that water is Technical Tip 16-1. Process specimens for osmolality
excreted with the solid fecal material, producing diarrhea. testing immediately. Specimens that are stored for
Constipation, on the other hand, provides time for addi- hours may have a markedly increased osmolality due
tional water to be reabsorbed from the fecal material, pro- to the increased degradation of carbohydrates.
ducing small, hard stools.

Osmotic Diarrhea
Diarrhea and Steatorrhea
Osmotic diarrhea is caused by poor absorption that exerts os-
Diarrhea motic pressure across the intestinal mucosa. Incomplete break-
down or reabsorption of food presents increased fecal material
Diarrhea is defined as an increase in daily stool weight above to the large intestine, resulting in retention of water and elec-
200 g, increased liquidity of stools, and frequency of more trolytes in the large intestine (osmotic diarrhea), which, in turn,
than three times per day. Diarrhea classification is based on results in stool that is excessively watery. Maldigestion (impaired
four factors: food digestion) and malabsorption (impaired nutrient absorp-
• Illness duration tion by the intestine) contribute to osmotic diarrhea. The pres-
• Mechanism ence of unabsorbable solute increases the stool osmolality and
the concentration of electrolytes is lower, resulting in an in-
• Severity
creased osmotic gap. Causes of osmotic diarrhea include disac-
• Stool characteristics charidase deficiency (lactose intolerance), malabsorption (celiac
Diarrhea lasting less than 4 weeks is defined as acute, sprue), poorly absorbed sugars (lactose, sorbitol, mannitol), lax-
whereas diarrhea persisting for more than 4 weeks is termed atives, magnesium-containing antacids, amebiasis, and antibiotic
chronic diarrhea. administration. Laboratory testing of feces is performed fre-
The major mechanisms that cause diarrhea are secretory, quently to aid in determining the cause of diarrhea (Table 16-1).
osmotic, and intestinal hypermotility. Watery diarrhea is di- Table 16-2 differentiates the features of osmotic diarrhea and
vided into secretory or osmotic types by measuring fecal elec- secretory diarrhea.
trolytes and the osmotic gap.1 The laboratory tests used to
differentiate these mechanisms include fecal electrolytes (fecal Altered Motility
sodium, fecal potassium), fecal osmolality, and stool pH. The Altered motility describes conditions of either enhanced motil-
normal total fecal osmolarity is close to the serum osmolality ity (hypermotility) or slow motility (constipation). Both can be
(290 mOsm/kg), normal fecal sodium is 30 mmol/L, and fecal
potassium is 75 mmol/L. The fecal sodium and fecal potassium
results are used to calculate the fecal osmotic gap. The fecal
osmotic gap is calculated as follows: Table 16–1 Common Fecal Tests for Diarrhea

Osmotic gap = 290 – [2 (fecal sodium + fecal potassium)] Secretory Osmotic

Stool cultures Microscopic fecal fats
A fecal osmotic gap of <50 mOsm/Kg suggests secretory
diarrhea and >75 mOsm/kg suggests osmotic diarrhea.1 Elec- Ova and parasite examinations Muscle fiber detection
trolytes are increased in secretory diarrhea and negligible in Qualitative fecal fats
osmotic diarrhea. A fecal fluid pH of less than 5.6 indicates a Rotavirus immunoassay Trypsin screening
malabsorption of sugars, causing an osmotic diarrhea. Fecal leukocytes Microscopic fecal fats
Secretory Diarrhea Muscle fiber detection
Secretory diarrhea is caused by increased secretion of water. Quantitative fecal fats
Bacterial, viral, and protozoan infections produce increased se- Clinitest
cretion of water and electrolytes, which override the reabsorp- D-xylose tolerance test
tive ability of the large intestine, leading to secretory diarrhea.
Lactose tolerance test
Enterotoxin-producing organisms, such as Escherichia coli,
Clostridium, Vibrio cholerae, Salmonella, Shigella, Staphylococcus, and Fecal electrolytes
Campylobacter; protozoa; and parasites, such as Cryptosporidium Stool pH
can stimulate these secretions of water and electrolytes. Other Fecal osmolality
causes of secretory diarrhea are drugs, stimulant laxatives,
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342 Part Three | Other Body Fluids

Table 16–2 Differential Features for Diarrhea1 in the breakdown and subsequent reabsorption of dietary fat
(primarily triglycerides) produces an increase in stool fat (steat-
Laboratory Osmotic Secretory orrhea) that exceeds 6 g per day. Likewise, pancreatic disorders,
Test Diarrhea Diarrhea including cystic fibrosis, chronic pancreatitis, and carcinoma,
that decrease the production of pancreatic enzymes also are as-
Osmotic gap >75 Osm/kg <50 Osm/kg
sociated with steatorrhea. Steatorrhea may be present in those
Stool Na <60 mmol/L >90 mmol/L with either maldigestion or malabsorption conditions and can
Stool output in <200 g >200 g be distinguished by the D-xylose test. D-xylose is a sugar that
24 hr does not need to be digested but does need to be absorbed to
pH <5.3 >5.6 be present in the urine. If urine D-xylose is low, the resulting
steatorrhea indicates a malabsorption condition. Malabsorption
Reducing Positive Negative
causes include bacterial overgrowth, intestinal resection, celiac
substances
disease, tropical sprue, lymphoma, Whipple disease, Giardia
lamblia infestation, Crohn disease, and intestinal ischemia. A
normal D-xylose test indicates pancreatitis.
seen in patients with irritable bowel syndrome (IBS), a func-
tional disorder in which the nerves and muscles of the bowel Specimen Collection
are extra sensitive, causing cramping, bloating, flatus, diarrhea,
and constipation. IBS can be triggered by food, chemicals, Collection of a fecal specimen, frequently called a stool speci-
emotional stress, and exercise. men, is not an easy task for patients. Detailed instructions and
Intestinal hypermotility is the excessive movement of in- appropriate containers, depending on the type of test and
testinal contents through the GI tract that can cause diarrhea amount of feces required, should be provided. For certain tests,
because normal absorption of intestinal contents and nutrients dietary restrictions are required before fecal specimen collection.
cannot occur. It can be caused by enteritis, the use of parasym- Patients should be instructed to collect the specimen in
pathetic drugs, or with complications of malabsorption. Rapid a clean container, such as a bedpan or disposable container,
gastric emptying (RGE) dumping syndrome describes hyper- and transfer the specimen to the laboratory container.
motility of the stomach and the shortened gastric emptying Patients should understand that the specimen must not be
half-time, which causes the small intestine to fill too quickly contaminated with urine or toilet water, which may contain
with undigested food from the stomach. It is the hallmark of chemical disinfectants or deodorizers that can interfere with
early dumping syndrome (EDS).2 Healthy people have a gas- chemical testing. Containers that have preservatives for ova
tric emptying half-time range of 35 to 100 minutes, which and parasites must not be used to collect specimens for
varies with age and gender. A gastric emptying time of less than other tests.
35 minutes is considered RGE.2 RGE can be caused by distur- Random specimens suitable for qualitative testing for
bances in the gastric reservoir or in the transporting function. blood and microscopic examination for leukocytes, muscle
Alterations in the motor functions of the stomach result in fibers, and fecal fats usually are collected in plastic or glass
accumulation of large amounts of osmotically active solids and containers with screw-tops similar to those used for urine
liquids to be transported into the small intestine. Normal specimens. Material collected on a physician’s glove and
gastric emptying is controlled by fundic tone, duodenal feed- samples applied to filter paper in occult blood testing kits
back, and GI hormones. These are altered after gastric surgery, also are received.
resulting in clinically significant dumping syndrome in approx- For quantitative testing, such as for fecal fats, timed speci-
imately 10% of patients.3 mens are required. Because of the variability of bowel habits and
RGE can be divided into early dumping and late dumping, the transit time required for food to pass through the digestive
depending on how soon after a meal the symptoms occur. EDS tract, the most representative specimen is a 3-day collection. Fre-
symptoms begin 10 to 30 minutes after meal ingestion.3 Symp- quently, these specimens are collected in large containers to ac-
toms include nausea, vomiting, bloating, cramping, diarrhea, commodate the specimen quantity and facilitate emulsification
dizziness, and fatigue. Late dumping occurs 2 to 3 hours after a before testing. Care must be taken when opening any fecal spec-
meal and is characterized by weakness, sweating, and dizziness.2 imen to release gas that has accumulated within the container
Hypoglycemia is often a complication of dumping syndrome. The slowly. Patients must be cautioned not to contaminate the outside
main causes of dumping syndrome include gastrectomy, gastric of the container.
bypass surgery, post-vagotomy status, Zollinger-Ellison syn-
drome, duodenal ulcer disease, and diabetes mellitus.2
Macroscopic Screening
Steatorrhea Often the first indication of GI disturbances can be changes
Detection of steatorrhea (fecal fat) is useful in diagnosing in the brown color and formed consistency of the normal
pancreatic insufficiency and small-bowel disorders that cause stool. Of course, the appearance of abnormal fecal color also
malabsorption. Absence of bile salts that assist pancreatic lipase may be caused by ingestion of highly pigmented foods and
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Chapter 16 | Fecal Analysis 343

medications, so a differentiation must be made between this Table 16–3 Macroscopic Stool Characteristics14,30
and a possible pathological cause.
Color/Appearance Possible Cause
Color
Black Upper GI bleeding
The brown color of the feces results from intestinal oxidation
Iron therapy
of stercobilinogen to urobilin. As discussed in Chapter 6, con-
jugated bilirubin formed in the degradation of hemoglobin Charcoal
passes through the bile duct to the small intestine, where in- Bismuth (antacids)
testinal bacteria convert it to urobilinogen and stercobilinogen. Red Lower GI bleeding
Therefore, stools that appear pale (acholic stools) may signify
Beets and food coloring
a blockage of the bile duct. Also, pale stools are associated with
diagnostic procedures that use barium sulfate. Rifampin
A primary concern is the presence of blood in a stool spec- Pale yellow, white, gray Bile-duct obstruction
imen. Depending on the area of the intestinal tract from which Barium sulfate
bleeding occurs, the color can range from bright red to dark red
Green Biliverdin/oral antibiotics
to black. Blood that originates from the esophagus, stomach, or
duodenum takes approximately 3 days to appear in the stool; Green vegetables
during this time, degradation of hemoglobin produces the char- Bulky/frothy Bile duct obstruction
acteristic black, tarry stool. Likewise, blood from the lower GI Pancreatic disorders
tract requires less time to appear and retains its original red color.
Ribbon-like Intestinal constriction
Both black and red stools should be tested chemically for the
presence of blood because ingestion of iron, charcoal, or bismuth Mucus- or blood-streaked Colitis
often produces a black stool, and some medications, as well as Dysentery
some foods, including beets, produce a red stool. Malignancy
Green stools may be observed in patients taking oral an-
Constipation
tibiotics because of the oxidation of fecal bilirubin to biliverdin.
Ingestion of increased amounts of green vegetables or food Small, hard Constipation
coloring also produces green stools. Watery Diarrhea

Appearance
Besides variations in color, additional abnormalities may be ob- Fecal Leukocytes
served during macroscopic examination. Table 16-3 lists com-
mon abnormalities seen in macroscopic evaluation. Examples Leukocytes, primarily neutrophils, are seen in the feces in pa-
include the watery consistency present in diarrhea; small, hard tients with conditions that affect the intestinal mucosa, such as
stools seen with constipation; and slender, ribbon-like stools, ulcerative colitis and bacterial dysentery. Microscopic screening
which suggest obstruction of the normal passage of material is performed as a preliminary test to determine whether diar-
through the intestine. rhea is being caused by invasive bacterial pathogens, including
Pale stools associated with biliary obstruction and steat- Salmonella, Shigella, Campylobacter, Yersinia, and enteroinvasive
orrhea appear bulky and frothy and frequently have a foul E. coli. Bacteria that cause diarrhea by toxin production, such
odor. Stools also may appear greasy and may float. as Staphylococcus aureus and Vibrio spp.; viruses; and parasites
The presence of mucus-coated stools indicates intestinal usually do not cause the appearance of fecal leukocytes.4 There-
inflammation or irritation. Mucus-coated stools may be caused fore, the presence or absence of fecal neutrophils can provide
by pathological colitis, Crohn disease, colon tumors, or exces- the physician with diagnostic information before receiving the
sive straining during elimination. Blood-streaked mucus sug- culture report.
gests damage to the intestinal walls, possibly caused by bacterial Specimens can be examined as wet preparations stained
or amebic dysentery or malignancy. The presence of mucus is with methylene blue or as dried smears stained with Wright’s or
abnormal and should be reported. Gram stain. Methylene blue staining is the faster procedure but
may be more difficult to interpret (see Procedure 16-1). Dried
preparations stained with either Wright’s or Gram stain provide
Microscopic Examination permanent slides for evaluation. An additional advantage of
of Feces the Gram stain is the observation of gram-positive and gram-
negative bacteria, which could aid in the initial treatment.5 All
Microscopic screening of fecal smears is performed to detect slide preparations must be performed on fresh specimens. In
the presence of leukocytes associated with microbial diarrhea, an examination of preparations under high power, as few as
as well as undigested muscle fibers and fats associated with three neutrophils per high-power field can be indicative of an
steatorrhea. invasive condition.6 Using oil immersion, the finding of any
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344 Part Three | Other Body Fluids

PROCEDURE 16-1
Methylene Blue Stain for Fecal Leukocytes
1. Place mucus or a drop of liquid stool on a slide.
2. Add two drops of Löffler methylene blue.
3. Mix with a wooden applicator stick.
4. Allow to stand for 2 to 3 minutes.
5. Examine for neutrophils under high power.

neutrophils has approximately 70% sensitivity for the presence

of invasive bacteria.7
A lactoferrin latex agglutination test is available for detect-
ing fecal leukocytes and remains sensitive in refrigerated and
Figure 16–2 Meat fibers present in fecal emulsion specimen using
frozen specimens. The presence of lactoferrin, a component of
bright-field microscopy examination (×400).
granulocyte secondary granules, indicates an invasive bacterial
pathogen.8
Qualitative Fecal Fats
Muscle Fibers
Specimens from suspected cases of steatorrhea can be screened
Microscopic examination of the feces for undigested striated microscopically for the presence of excess fecal fat (steator-
muscle fibers can be helpful in diagnosing and monitoring pa- rhea). Also, the procedure can be used to monitor patients
tients with pancreatic insufficiency, such as in cases of cystic undergoing treatment for malabsorption disorders.9 In general,
fibrosis. It is ordered frequently in conjunction with micro- correlation between the qualitative and quantitative fecal fat
scopic examinations for fecal fats. Increased amounts of striated procedures is good; however, additional unstained phospho-
fibers also may be seen in patients with biliary obstruction, lipids and cholesterol esters are measured by the quantitative
malabsorption syndromes, and gastrocolic fistulas. procedure.10,11 Lipids included in the microscopic examination
Slides for detection of muscle fiber (see Procedure 16-2) of feces are neutral fats (triglycerides), fatty acid salts (soaps),
are prepared by emulsifying a small amount of stool in 10% fatty acids, and cholesterol. Their presence can be observed
alcoholic eosin, which enhances the muscle fiber striations. microscopically by staining with the dyes Sudan III, Sudan IV,
The entire slide is examined for exactly 5 minutes, and the or oil red O; Sudan III is the one used most routinely. The
number of red-stained fibers with well-preserved striations is staining procedure consists of two parts: the neutral fat stain
counted (Fig. 16-2). Care must be taken to classify the fibers and the split fat stain.
observed correctly. Undigested fibers have visible striations Neutral fats are readily stained by Sudan III and appear as
running both vertically and horizontally. Partially digested large orange-red droplets, often located near the edge of the
fibers exhibit striations in only one direction, and digested
fibers have no visible striations. Only undigested fibers are
counted, and the presence of more than 10 is reported as in-
creased (Fig. 16-3).
To produce a representative specimen, patients should be
instructed to include red meat in their diet before collecting
the specimen. Specimens should be examined within 24 hours
of collection.

PROCEDURE 16-2
Muscle Fibers
1. Emulsify a small amount of stool in two drops of
10% eosin in alcohol.
2. Apply cover slip, and let stand 3 minutes.
3. Examine under high power for 5 minutes.
4. Count the number of undigested fibers. Figure 16–3 Note striations on meat fiber present in a fecal emul-
sion specimen (×1000).
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Chapter 16 | Fecal Analysis 345

cover slip (see Procedure 16-3).12 Observation of more than

PROCEDURE 16-4
60 droplets/high-power field can indicate steatorrhea; however,
the split fat stain representing total fat content can provide a Split Fat Stain
better indication.13 Breakdown of neutral fats by bacterial li- 1. Mix emulsified stool with one drop of 36% acetic
pase and spontaneous hydrolysis of neutral fats may lower the acid.
neutral fat count, which precludes comparison of the two slide
tests to determine whether maldigestion or malabsorption is 2. Add two drops of saturated Sudan III.
causing steatorrhea. 3. Mix and apply the cover slip.
Soaps and fatty acids do not stain directly with Sudan III, 4. Heat gently almost to boiling.
so a second slide must be examined after the specimen has 5. Examine under high power.
been mixed with acetic acid and heated. Examining this slide
reveals stained droplets that represent not only the free fatty 6. Count and measure the orange droplets per high-
acids but also the fatty acids produced by hydrolysis of the power field.
soaps and the neutral fats (Fig. 16-4). In an examination of this
split fat slide (Procedure 16-4), both the number and size of
the fat droplets must be considered. Normal specimens may Cholesterol is stained by Sudan III after heating and,
contain fewer than 100 small droplets, less than 4 µm in di- as the specimen cools, forms crystals that can be identified
ameter, per high-power field. The same number of droplets microscopically.
measuring 1 to 8 µm is considered slightly increased, whereas
100 droplets measuring 6 to 75 µm is considered increased Chemical Testing of Feces
and commonly seen in steatorrhea.14 An increased amount of
total fat on the second slide with normal fat content on the first Occult Blood
slide is an indication of malabsorption, whereas maldigestion
is indicated by increased neutral fat on the first slide. By far, the fecal analysis performed most frequently is the
detection of occult blood (hidden blood). As discussed previ-
ously, bleeding in the upper GI tract may produce a black, tarry
PROCEDURE 16-3 stool, and bleeding in the lower GI tract may result in an
overtly bloody stool. However, because any bleeding in excess
Neutral Fat Stain of 2.5 mL/150 g of stool is considered pathologically significant
1. Homogenize one part stool with two parts water. and no visible signs of bleeding may be present with this
2. Mix emulsified stool with one drop of 95% ethyl amount of blood, fecal occult blood testing (FOBT) is neces-
alcohol on the slide. sary. Annual testing for occult blood has a high positive pre-
dictive value for detecting colorectal cancer in the early stages
3. Add two drops of saturated Sudan III in 95% and is recommended by the American Cancer Society, partic-
ethanol. ularly for people over 50 years of age.15 The test also may be
4. Mix and apply the cover slip. performed to evaluate possible causes of unexplained anemia.
5. Examine under high power. Methods for detecting fecal occult blood include the guaiac,
6. Count the orange droplets per high-power field. immunochemical, and fluorometric porphyrin quantification
tests. Immunochemical tests and fecal porphyrin quantification
tests are more sensitive and specific methods than the guaiac-
based fecal occult blood tests.
Guaiac-Based Fecal Occult Blood Tests
The screening test used most frequently for fecal blood is the
guaiac-based test for occult blood (gFOBT) based on detect-
ing the pseudoperoxidase activity of hemoglobin. This is the
same principle as the reagent strip test for urinary blood but
uses a different indicator chromogen. The reaction uses the
pseudoperoxidase activity of hemoglobin reacting with hydro-
gen peroxide to oxidize a colorless compound to a colored
compound.

Pseudoperoxidase
Hemoglobin + H2O2 + guaiac oxidized guaiac + H2O
(colorless) (blue color)

Figure 16–4 Several orange-red neutral fat globules present in a Several different indicator chromogens have been used to
fecal suspension stained with Sudan III (×400). detect occult blood. All react in the same chemical manner but
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346 Part Three | Other Body Fluids

vary in their sensitivity. Contrary to most chemical testing, the should be allowed to dry before testing. The specimens should
least sensitive reagent, guaiac, is preferred for routine testing. be tested within 7 days of collection (see Procedure 16-5). Two
Considering that a normal stool can contain up to 2.5 mL of samples from three different stools should be tested before a
blood, a less sensitive chemical reactant is, understandably, negative result is confirmed. To prevent the presence of dietary
more desirable. In addition, pseudoperoxidase activity is pres- pseudoperoxidases in the stool, patients should be instructed to
ent from hemoglobin and myoglobin in ingested meat and fish, avoid eating red meats, horseradish, melons, raw broccoli,
certain vegetables and fruits, and some intestinal bacteria. cauliflower, radishes, and turnips for 3 days before specimen
Therefore, to prevent false-positive reactions, test sensitivity collection. Aspirin and NSAIDs other than acetaminophen
must be decreased, which can be accomplished by varying the should not be taken for 7 days before specimen collection to pre-
amount and purity of the guaiac reagent used in the test. vent possible GI irritation. Vitamin C and iron supplements con-
Many commercial testing kits are available for occult taining vitamin C should be avoided for 3 days before collection
blood testing with guaiac reagent. The kits contain guaiac- because ascorbic acid is a strong reducing agent that interferes
impregnated filter paper enclosed in a cardboard slide, to with the peroxidase reaction, causing a false-negative result.16
which the fecal specimen and hydrogen peroxide are added. Visit www.fadavis.com for Video 16-1 (Fecal occult blood
Two or three filter paper areas are provided for application of procedure).
material taken from different areas of the stool, and positive
and negative controls are included. Obtaining samples from
the center of the stool avoids false-positive reactions from ex- Technical Tip 16-2. Applying a thick smear of stool
ternal contamination. The patient is asked to collect samples sample on the test card can cause a false-negative re-
from stool specimens collected on 3 consecutive days. The sult or make a positive result hard to read.
sample is placed on the front side of the slide with an appli-
cator stick, and the slide is closed. Adding hydrogen peroxide Technical Tip 16-3. Failure to allow stool samples to
to the back of the filter paper slide that contains stool pro- soak into the filter paper slide for 3 to 5 minutes be-
duces a blue color with guaiac reagent when pseudoperoxi- fore adding developer may result in a false-negative
dase activity is present (Fig. 16-5A and B). result. The red blood cells (hemoglobin) must be ab-
Packaging of the guaiac-impregnated filter paper in indi- sorbed onto the test card completely before a positive
vidually sealed containers has facilitated colorectal cancer reaction can occur when the developer is added.
screening by allowing patients at home to place a specimen on
a filter paper slide and bring or mail it to the laboratory for
testing. To prevent false-positive reactions, specimens mailed
Immunochemical Fecal Occult Blood Test
to the laboratory should not be rehydrated before adding the
hydrogen peroxide unless specifically instructed by the kit The immunochemical fecal occult blood test (iFOBT) is spe-
manufacturer. Specimens applied to the paper in the laboratory cific for the globin portion of human hemoglobin and uses

A B

Figure 16–5 Fecal occult blood reaction. A. Negative result for occult blood indicated by the absence of blue color. B. Positive result for
occult blood indicated by the blue color around the edge of the stool sample. Note the control area inside the orange rectangle. The positive
control appears blue, and the negative control shows no color.
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Chapter 16 | Fecal Analysis 347

PROCEDURE 16-5 SUMMARY 161 gFOBT Interference

Guaiac-Based Fecal Occult Blood Test False-Positive
1. Open the front flap of the guaiac-impregnated filter Aspirin and anti-inflammatory medications
paper slide.
Red meat
2. Using an applicator stick, take a sample from the
Horseradish
center of the stool specimen.
Raw broccoli, cauliflower, radishes, turnips
3. Apply a thin smear of stool on the filter paper inside
the box marked “A” on the slide. Melons
4. Using the same applicator stick, take a second sam- Menstrual and hemorrhoid contamination
ple from a different part of the center of the stool. False-Negative
5. Apply a thin smear of the second sample inside the Vitamin C >250 mg/d
box marked “B.”
Iron supplements containing vitamin C
6. Close the cover of the filter paper slide.
Failure to wait the specified time after sample is applied
7. Allow the stool samples to soak into the filter paper to add the developer reagent
slide for 3 to 5 minutes.
8. Open the back of the slide.
9. Add two drops of hydrogen peroxidase developer to been converted to porphyrins. As hemoglobin progresses
the boxes marked “A” and “B.” through the intestinal tract, bacterial actions degrade it to
porphyrin that the gFOBT cannot detect, thereby making the
10. Read results within 60 seconds. Any trace of blue
HemoQuant test more sensitive to upper GI bleeding. False-
on or at the edge of the smear is positive for occult
negative results from upper GI bleeding can be seen with the
blood.
gFOBT. Additionally, the porphyrin-based test is not affected
11. Add 1 drop of hydrogen peroxidase developer be- by the presence of reducing or oxidizing substances or the
tween the positive and negative internal control. water content of the fecal specimen. False-positive results can
12. Read quality control results within 10 seconds. The occur with the porphyrin-based test when nonhuman sources
positive control will appear blue, and no color will of blood (red meat) are present; therefore, patients should be
be present in the negative control. instructed to avoid red meat for 3 days before the test. This
test is performed primarily by reference laboratories and not
performed routinely in hospital laboratories.

polyclonal antihuman hemoglobin antibodies. Because this Quantitative Fecal Fat Testing
method is specific for human blood in feces, it does not require Quantitative fecal fat analysis is used as a confirmatory test for
dietary or drug restrictions. It is more sensitive to lower GI steatorrhea. As discussed, quantitative fecal analysis requires
bleeding that could be an indicator of colon cancer or other the collection of at least a 3-day specimen. The patient must
GI disease and can be used for patients who are taking aspirin maintain a regulated intake of fat (50 to 150 g/d) for 2 to 3 days
and other anti-inflammatory medications. The iFOBT tests do before and during the stool collection period. The specimen is
not detect bleeding from other sources, such as a bleeding collected in a large, preweighed container. Before analysis, the
ulcer, thus decreasing the chance for false-positive reactions. specimen is weighed and homogenized. Refrigerating the spec-
Hemoglobin from upper GI bleeding is degraded by bacterial imen prevents any bacterial degradation. The method used rou-
and digestive enzymes before reaching the large intestine and tinely for fecal fat measurement is the Van de Kamer titration,
is immunochemically nonreactive. In contrast, there is little although gravimetric, near-infrared reflectance spectroscopy
hemoglobin degradation in lower GI bleeding, so the blood is (NIRS), and nuclear magnetic resonance spectroscopy methods
immunochemically active.17 Collection kits are similar to those are available.12 In the titration method, fecal lipids are con-
used for guaiac testing, such as the Hemoccult ICT test (Beckman verted to fatty acids and titrated to a neutral endpoint with
Coulter, Inc., Fullerton, CA), and can be provided to patients sodium hydroxide. Approximately 80% of the total fat content
for home collection. Depending on the method used, the is measured by titration, whereas the gravimetric method
results may be read visually or by an automated photometric measures all fecal fat. A drawback of the titration/gravimetric
instrument. method is that it is time consuming and uses solvents that are
corrosive and flammable. A rapid (5 minutes) and safe proce-
Porphyrin-Based Fecal Occult Blood Test
dure for analyzing quantitative fecal fat is the hydrogen nuclear
HemoQuant (SmithKline Diagnostics, Sunnyvale, CA) offers a magnetic resonance spectroscopy (1H NMR) method. In this
porphyrin-based FOBT fluorometric test for hemoglobin based method, the homogenized specimen is microwaved-dried and
on the conversion of heme to fluorescent porphyrins. The test analyzed. The results correlate well with the gravimetric
measures both intact hemoglobin and the hemoglobin that has method, and it is widely used in reference laboratories.18
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348 Part Three | Other Body Fluids

The fat content is reported as grams of fat or the coefficient microhematocrit test and is more convenient than a 72-hour
of fat retention per 24 hours. Reference values based on a stool collection. The acid steatocrit is a reliable tool to monitor
100 g/d intake are 1 to 6 g/d, or a coefficient of fat retention a patient’s response to therapy and screen for steatorrhea in
of at least 95%. The coefficient of fat retention is calculated as pediatric populations.20,21
follows: NIRS is a rapid procedure for fecal fat that requires less stool
handling by laboratory personnel. The test requires a 48- to
(dietary fat – fecal fat)
× 100 72-hour stool collection to exclude day-to-day variability, but
(dietary fat)
it does not require reagents after homogenization of the spec-
Although the Van de Kamer titration is the gold standard imen. The result is based on the measurement and computed
for fecal fat, the acid steatocrit (see Procedure 16-6) is a rapid processing of signal data from reflectance of the fecal surface,
test to estimate the amount of fat excretion.19 It is similar to the which is scanned with infrared light between 1,400 nM and
2,600 nM wavelength. The results are calculated from calibra-
tion derived from known samples. The technique quantitates
water, fat, and nitrogen in grams per 24 hours.24 A summary
PROCEDURE 16-6
of tests and current instrumentation for fecal fat analysis is pre-
Acid Steatocrit sented in Table 16-4.
1. Dilute 0.5 g of feces from a spot collection 1 to APT Test (Fetal Hemoglobin)
4 with deionized water.
2. Vortex for 2 minutes to homogenize the specimen. Grossly bloody stools and vomitus are seen sometimes in
neonates as the result of swallowing maternal blood during de-
3. Add a volume of 5 N perchloric acid equal to 20% of livery. Should it be necessary to distinguish between the pres-
the homogenate, and then vortex the mixture for ence of fetal blood or maternal blood in an infant’s stool or
30 seconds. Confirm the pH to be <1. vomitus, the APT test may be requested (see Procedure 16-7).
4. Place the acid–homogenate mixture in a 75-µL plain The material to be tested is emulsified in water to release
hematocrit capillary tube. Seal the end with wax. hemoglobin (Hb) and, after centrifugation, 1% sodium hy-
5. Centrifuge the capillary tube horizontally at droxide is added to the pink hemoglobin-containing super-
13,000 rpm for 15 minutes in a microhematocrit natant. In the presence of alkali-resistant fetal hemoglobin,
centrifuge. This separates fat as an upper layer the solution remains pink (HbF), whereas denaturation of the
overlying a solid fecal layer. maternal hemoglobin (HbA) produces a yellow-brown super-
6. Measure the length of the fat and solid layers using a natant after standing for 2 minutes. The APT test distinguishes
magnifying lens. between not only HbA and HbF but also between maternal he-
moglobins AS, CS, and SS and HbF. The presence of maternal
7. Calculate the acid steatocrit as a percentage. thalassemia major would produce erroneous results due to the
8. Calculate the fecal fat in grams per 24 hours. high concentration of HbF. Stool specimens should be tested
when fresh. They may appear bloody but should not be black
The acid steatocrit as a percentage = (fatty layer
length in cm)/[(fatty layer length in cm) + (solid
layer length)] × 100

The fecal fat for adults is quantitated as follows: Table 16–4 Tests, Materials, and Instrumentation
for Fecal Fat Analysis22
Fecal fat in grams per 24 hours = [0.45 × (acid
Procedure Materials, Instrumentation
steatocrit in percent as a whole number)] – 0.43
Sudan III Sudan stain, microscopy
An acid steatocrit value <31% is considered normal,
Steatocrit and acid Hematocrit centrifuge, gravimetric
whereas a value >31% indicates steatorrhea in adults.
steatocrit assay
The fecal fat for children up to the age of 15 years of age
is as follows: Fecal elastase I Immunoassay ELISA technique
Near-infrared NIRS spectrophotometer with spe-
Fecal fat in grams per 24 hours = [0.1939 × (acid reflectance cialized computer software
steatocrit in percent as a whole number)] – 0.2174 spectroscopy
Acid steatocrit is higher in infants and lowers with age.22 Van de Kamer Fecal fat extraction and titration of
For infants older than 6 months of age, a value of <10% is titration long-chain fatty acid by sodium
normal, 10% to 20% is equivocal, and >20% is abnormal, hydroxide
indicating steatorrhea. Infants younger than 6 months of Nuclear magnetic Microwaved-dried specimen;
age show high and widely variable steatocrit values (phys- resonance hydrogen nuclear magnetic
iological steatorrhea).23 spectroscopy spectrophotometer
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Chapter 16 | Fecal Analysis 349

PROCEDURE 16-7 HISTORICAL NOTE

APT Test Screening Test for Fecal Trypsin
1. Emulsify specimen in water.
2. Centrifuge. Historically, the absence of trypsin had been screened for
by exposing x-ray paper to stool emulsified in water. When
3. Divide pink supernatant into two tubes.
trypsin was present in the stool, it digested the gelatin on
4. Add 1% sodium hydroxide to one tube. the paper, leaving a clear area. Inability to digest the gelatin
5. Wait 2 minutes. indicated a deficiency in trypsin production. The gelatin
6. Compare color with that in the control tube. test was an insensitive procedure that detected only severe
cases of pancreatic insufficiency. In addition, false-negative
7. Prepare controls using cord blood and adult blood.
results could occur as the result of intestinal degradation
of trypsin and the possible presence of trypsin inhibitors
in the feces. The proteolytic activity of bacteria enzymes
could produce false-positive results in old specimens.
and tarry because this would indicate already denatured
hemoglobin.25

Fecal Enzymes
seen in patients with celiac disease, or lack of digestive
Enzymes supplied to the GI tract by the pancreas are essential enzymes, such as lactase, resulting in lactose intolerance.
for digesting dietary proteins, carbohydrates, and fats. Decreased Idiopathic lactase deficiency is common, predominantly
production of these enzymes (pancreatic insufficiency) is asso- occurring in the African, Asian, and southern European
ciated with disorders such as chronic pancreatitis and cystic Greek populations. Carbohydrate malabsorption or intoler-
fibrosis. Steatorrhea occurs, and undigested food appears in ance (maldigestion) is analyzed primarily by serum and
the feces. urine tests; however, an increased concentration of carbohy-
Analysis of the feces focuses primarily on the proteolytic drate can be detected by performing a copper reduction test
enzymes trypsin, chymotrypsin, and elastase I. on the fecal specimen. Testing for fecal-reducing substances
Fecal chymotrypsin is more resistant to intestinal degra- detects congenital disaccharidase deficiencies, as well as
dation than trypsin and is a more sensitive indicator of less enzyme deficiencies due to nonspecific mucosal injury. Fecal
severe cases of pancreatic insufficiency. It also remains stable carbohydrate testing is most valuable in assessing cases
in fecal specimens for up to 10 days at room temperature. of infant diarrhea and may be accompanied by a pH deter-
Chymotrypsin is capable of gelatin hydrolysis but is measured mination. Normal stool pH is between 7 and 8; however,
most frequently by spectrophotometric methods. increased use of carbohydrates by intestinal bacterial fermen-
Elastase I is an isoenzyme of the enzyme elastase and is tation increases the lactic acid level and lowers the pH to
the enzyme form produced by the pancreas. It is present in below 5.5 in cases of carbohydrate disorders.
high concentrations in pancreatic secretions and is strongly The copper reduction test is performed using a Clinitest
resistant to degradation. It accounts for about 6% of all tablet (Siemens Healthcare Diagnostics, Inc., Deerfield, IL)
secreted pancreatic enzymes.26 Fecal elastase I is pancreas and one part stool emulsified in two parts water. A result of
specific, and its concentration is about five times higher than 0.5 g/dL is considered indicative of carbohydrate intolerance.
in pancreatic juice. It is not affected by motility disorders or The Clinitest on stools can distinguish between diarrhea caused
mucosal defects.27 Elastase I can be measured by immunoas- by abnormal excretion of reducing sugars and those caused by
say using the enzyme-linked immunosorbent assay (ELISA) various viruses and parasites. Sucrose is not detected by the
kit and provides a very sensitive indicator of exocrine pan- Clinitest method because it is not a reducing sugar. In prema-
creatic insufficiency.28,29 It is easy to perform and requires ture infants, there is a correlation between a positive Clinitest
only a single stool specimen. The ELISA test uses monoclonal and inflammatory necrotizing enterocolitis. As discussed in
antibodies against human pancreatic elastase-1; therefore, the Chapter 6, this is a general test for the presence of reducing
result is specific for human enzyme and not affected by pan- substances, and a positive result would be followed by more
creatic enzyme replacement therapy.26 The test is specific in specific serum carbohydrate tolerance tests, the most common
differentiating pancreatic from nonpancreatic causes in being the D-xylose test for malabsorption and the lactose tol-
patients with steatorrhea.27 erance test for maldigestion. Stool chromatography to identify
the malabsorbed carbohydrate is available but rarely necessary
Carbohydrates to diagnose sugar intolerance. Small-bowel biopsy specimens
The presence of increased carbohydrates in the stool pro- for histological examination and the assay of disaccharidase
duces osmotic diarrhea from the osmotic pressure of the un- enzyme activity differentiate primary from secondary disaccha-
absorbed sugar in the intestine, drawing in fluid and ridase intolerance.30
electrolytes. Carbohydrates in the feces may be present as a A summary of fecal screening tests is presented in
result of intestinal inability to reabsorb carbohydrates, as Table 16-5.
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350 Part Three | Other Body Fluids

Table 16–5 Fecal Screening Tests

Test Methodology/Principle Interpretation
Examination for Microscopic count of neutrophils in smear stained with Three per high-power field indicates con-
neutrophils methylene blue, Gram stain, or Wright’s stain dition affecting intestinal wall
Qualitative Microscopic examination of direct smear stained with 60 large orange-red droplets indicate mal-
fecal fats Sudan III absorption
Microscopic examination of smear heated with acetic 100 orange-red droplets measuring 6 to
acid and Sudan III 75 µm indicate malabsorption
gFOBT Pseudoperoxidase activity of hemoglobin liberates oxygen Blue color indicates gastrointestinal
from hydrogen peroxide to oxidize guaiac reagent bleeding
iFOBT Uses polyclonal antihuman antibodies specific for the Positive test and control lines indicate GI
globin portion of human hemoglobin bleeding
APT test Addition of sodium hydroxide to hemoglobin-containing Pink color indicates presence of fetal
emulsion determines presence of maternal or fetal blood
blood
Trypsin Emulsified specimen placed on x-ray paper determines Inability to digest gelatin indicates lack of
ability to digest gelatin trypsin
Elastase 1 Immunoassay using an ELISA test Sensitive indicator of exocrine pancreatic
insufficiency
Clinitest Addition of Clinitest tablet to emulsified stool detects Reaction of 0.5 g/dL reducing substances
presence of reducing substances suggests carbohydrate intolerance

12. Van de Kamer, JH, et al: A rapid method for determination of

For additional resources please visit fat in feces. J Biol Chem 177:347–355, 1949.
www.fadavis.com 13. Freeman, JA, and Beeler, MF: Laboratory Medicine: Urinalysis
and Medical Microscopy. Lea & Febiger, Philadelphia, 1983.
14. Drummey, GD, Benson, JA, and Jones, CM: Microscopic examina-
tion of the stool for steatorrhea. N Engl J Med 264:85–87, 1961.
15. Mandel, JS, et al: The effect of fecal occult-blood screening on
References the incidence of colorectal cancer. N Engl J Med 343(22):
1. Parakh, R, Greene, DN, Mathias, PC, Block, DR, Ranjitkar, P: 1603–1607, 2000.
Laboratory Utilization of Analytical Validation of Fecal Elec- 16. Knight, KK, Fielding, JE, and Battista, RN: Occult blood
trolyte Tests. The Journal of Applied Laboratory Medicine, An screening for colorectal cancer. JAMA 261:587–590, 1989.
AACC Publication. DOI: 10.1373/jalm.2016 022590. Web site: 17. Hemoccult ICT immunochemical fecal occult blood test pack-
www.jalm.aaccjnls.org/content/1/6/668. Published 2017. age insert. Beckman Coulter Inc., Fullerton, CA, May 2008.
Accessed July 14, 2019. 18. Korpi-Steiner, N, Ward, JN, Kumar, V, and McConnell, JP:
2. Singh, A, Gull, H, and Singh, R: Clinical significance of rapid Comparative analysis of fecal fat quantitation via nuclear mag-
(accelerated) gastric emptying. Clin Nucl Med 28(8):652–658, netic resonance spectroscopy (1H NMR) and gravimetry. Clin
2003. Chim Acta 400:33, 2009.
3. Ukleja, A: Dumping syndrome: Pathophysiology and treatment. 19. Zimmerman, RL: Q & A Column. Fecal Fat Testing. CAP
Nutr Clin Pract 20(5):517–525, 2005. TODAY August, 2018. Web site: https://www.captodayon-
4. Guandalini, S: Diarrhea Workup. Medscape. Web site: https:// linecom/qa-0818/. Accessed July 15, 2019.
emedicine.medscape/com/article/928598-workup. Published 20. Bijoor, AR, Geetha, S, and Venkateash, T: Faecal fat content in
October 31, 2018. Accessed July 14, 2019. healthy adults by the “acid steatocrit method.” Indian J Clin
5. Koepke, JA: Tips from the clinical experts; Fecal Leukocytes, Biochem 19(2):20–22, 2004.
Medical Laboratory Observer, (MLO), p. 15, September, 1995. 21. Van den Neucker, AM, et al: Acid steatocrit: a reliable screening
6. Bradley, GM: Fecal analysis: Much more than an unpleasant tool for steatorrhoea. Acta Paediatr 90:873–875, 2001.
necessity. Diagn Med 3(2):64–75, 1980. 22. Guarino, A, et al: Reference values of the steatocrit and its
7. Novak, R, et al: How useful are fecal neutrophil determina- modifications in diarrheal diseases. J Pediatr Gastroenterol
tions? Lab Med 26(11):433, 1995. Nutr 14:268–274, 1992.
8. McCray, WH, and Krevsky, B: Diagnosing diarrhea in adults: A 23. Steatocrit: Test Guide. Web site: https://testguide.adhb.govt.nz/
practical approach. Hosp Med 34(4):27–36, 1998. EGuide/?elv=1&name=Steatocrit&pn=1767&mn=881&sd=
9. Walters, MP, et al: Clinical monitoring of steatorrhea in cystic 3&ts=14ce36a9094. Published February 5, 2018. Accessed
fibrosis. Arch Dis Child 65:99–102, 1990. July 15, 2019.
10. Khouri, MR, Huang, G, and Shiau, YF: Sudan stain of fecal fat: 24. Serrano, PL, Navarro, JLL, and Fernandez-Rodriguez, CM:
New insight into an old test. Gastroenterology 96(2 Pt 1): Laboratory tests and equipment for diagnostic work-up for
421–427, 1990. malabsorption syndrome. LABTECH 2004.
11. Simko, V: Fecal fat microscopy. Am J Gastroenterol 75(3): 25. Croak, M: Haemoglobin in stools from neonates: Measurement
204–208, 1981. by a modified APT test. Med Lab Sci 48(4):346–350, 1991.
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26. Elphick, DA, and Kapur, K: Comparing the urinary pancreolau- 29. Thorne, D, and O’Brien, C: Diagnosing chronic pancreatitis.
ryl ratio and faecal elastase-1 as indicators of pancreatic insuffi- Advance 12(14):8–12, 2000.
ciency in clinical practice. Pancreatology 5:196–200, 2005. 30. Robayo-Torres, CC, Quezada-Calvillo, R, and Nichols, BL:
27. Symersky, T, et al: Faecal elastase-I: Helpful in analysing Disaccharide digestion: Clinical and molecular aspects. Clin
steatorrhoea? Neth J Med 62(8):286–289, 2004. Gastroenterol Hepatol 4(3):276–287, 2006.
28. Phillips, IJ, et al: Faecal elastase I: A marker of exocrine
pancreatic insufficiency in cystic fibrosis. Ann Clin Chem
36:739–742, 1999.

Study Questions
1. In what part of the digestive tract do pancreatic enzymes 7. Diarrhea can result from all of the following except:
and bile salts contribute to digestion? A. Addition of pathogenic organisms to the normal
A. Large intestine intestinal flora
B. Liver B. Disruption of the normal intestinal bacterial flora
C. Small intestine C. Increased concentration of fecal electrolytes
D. Stomach D. Increased reabsorption of intestinal water and
electrolytes
2. Where does the reabsorption of water take place in the
primary digestive process? 8. Stools from people with steatorrhea will contain excess
A. Large intestine amounts of:
B. Pancreas A. Barium sulfate
C. Small intestine B. Blood
D. Stomach C. Fat
D. Mucus
3. Which of the following tests is not performed to detect
osmotic diarrhea? 9. Which of the following pairings of stool appearance and
A. Clinitest cause do not match?
B. Fecal fats A. Black, tarry: blood
C. Fecal neutrophils B. Pale, frothy: steatorrhea
D. Muscle fibers C. Yellow-gray: bile duct obstruction
D. Yellow-green: barium sulfate
4. The normal composition of feces includes all of the
following except: 10. Stool specimens that appear ribbon-like are indicative of
A. Bacteria which condition?
B. Blood A. Bile duct obstruction
C. Electrolytes B. Colitis
D. Water C. Intestinal constriction
D. Malignancy
5. What is the fecal test that requires a 3-day specimen?
A. Fecal occult blood 11. A black tarry stool is indicative of:
B. APT test A. Upper GI bleeding
C. Elastase I B. Lower GI bleeding
D. Quantitative fecal fat testing C. Excess fat
D. Excess carbohydrates
6. The normal brown color of the feces is produced by:
A. Cellulose 12. Chemical screening tests performed on feces include all
of the following except:
B. Pancreatic enzymes
A. APT test
C. Undigested foodstuffs
B. Clinitest
D. Urobilin
C. Pilocarpine iontophoresis
D. Quantitative fecal fats
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352 Part Three | Other Body Fluids

13. Secretory diarrhea is caused by: 21. Which of the following tests would not be indicative of
A. Antibiotic administration steatorrhea?
B. Lactose intolerance A. Fecal elastase I
C. Celiac sprue B. Fecal occult blood
D. Vibrio cholerae C. Sudan III
D. Van de Kamer
14. The fecal osmotic gap is elevated in which disorder?
A. Dumping syndrome 22. The term “occult” blood describes blood that:
B. Osmotic diarrhea A. Is produced in the lower GI tract
C. Secretory diarrhea B. Is produced in the upper GI tract
D. Steatorrhea C. Is not visibly apparent in the stool specimen
D. Produces a black, tarry stool
15. Microscopic examination of stools provides preliminary
information as to the cause of diarrhea because: 23. What is the recommended number of specimens that
A. Neutrophils are present in conditions caused by should be tested to confirm a negative occult blood
toxin-producing bacteria result?
B. Neutrophils are present in conditions that affect the A. One random specimen
intestinal wall B. Two samples taken from different parts of three stool
C. Red and white blood cells are present if the cause is specimens
bacterial C. Three samples taken from the outermost portion of
D. Neutrophils are present if the condition is of the stool specimen
nonbacterial etiology D. Three samples taken from different parts of two stool
specimens
16. True or False: The presence of fecal neutrophils would be
expected with diarrhea caused by a rotavirus. 24. The immunochemical tests for occult blood:
17. Large orange-red droplets seen on direct microscopic A. Test for human globulin
examination of stools mixed with Sudan III represent: B. Give false-positive reactions with meat hemoglobin
A. Cholesterol C. Can give false-positive reactions with aspirin
B. Fatty acids D. Are inhibited by porphyrin
C. Neutral fats 25. Guaiac tests for detecting occult blood rely on the:
D. Soaps A. Reaction of hemoglobin with hydrogen peroxide
18. Microscopic examination of stools mixed with Sudan III B. Pseudoperoxidase activity of hemoglobin
and glacial acetic acid and then heated will show small C. Reaction of hemoglobin with ortho-toluidine
orange-red droplets that represent:
D. Pseudoperoxidase activity of hydrogen peroxide
A. Fatty acids and soaps
26. What is the significance of an APT test that remains pink
B. Fatty acids and neutral fats
after the addition of sodium hydroxide?
C. Fatty acids, soaps, and neutral fats
A. Fecal fat is present.
D. Soaps
B. Fetal hemoglobin is present.
19. When performing a microscopic stool examination for C. Fecal trypsin is present.
muscle fibers, the structures that should be counted:
D. Vitamin C is present.
A. Are coiled and stain blue
27. In the Van de Kamer method for quantitative fecal fat
B. Contain no visible striations
determinations, fecal lipids are:
C. Have two-dimensional striations
A. Converted to fatty acids before titrating with sodium
D. Have vertical striations and stain red hydroxide
20. A value of 85% fat retention would indicate: B. Homogenized and titrated to a neutral endpoint with
A. Dumping syndrome sodium hydroxide
B. Osmotic diarrhea C. Measured gravimetrically after washing
C. Secretory diarrhea D. Measured by spectrophotometer after addition of
Sudan III
D. Steatorrhea
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Chapter 16 | Fecal Analysis 353

28. A patient whose stool exhibits increased fats, undigested 30. Which of the following tests differentiates a malabsorption
muscle fibers, and the inability to digest gelatin may cause from a maldigestion cause in steatorrhea?
have: A. APT test
A. Bacterial dysentery B. D-xylose test
B. A duodenal ulcer C. Lactose tolerance test
C. Cystic fibrosis D. Occult blood test
D. Lactose intolerance
29. A stool specimen collected from an infant with diarrhea
has a pH of 5.0. This result correlates with a:
A. Positive APT test
B. Negative trypsin test
C. Positive Clinitest
D. Negative occult blood test

Case Studies and Clinical Situations

1. Microscopic screening of a stool from a patient exhibiting 3. A physician’s office laboratory is experiencing inconsisten-
prolonged diarrhea shows increased fecal neutrophils and cies in the results of patient-collected specimens for FOBT.
normal qualitative fecal fats and meat fibers. Patients are instructed to submit samples from two areas of
a. What type of diarrhea do these results suggest? three different stool specimens. Positive and negative con-
trols are producing satisfactory results. Patient #1 is a
b. Name an additional test that could provide more diag-
30-year-old woman taking over-the-counter medications
nostic information.
for gastric reflux who has reported passing frequent, black
c. Name one probable result for this test and one im- stools. The results of all three specimens are negative for
probable result. occult blood. Patient #2 is a 70-year-old woman suffering
d. If the test for fecal neutrophils were negative and the from arthritis. She is taking the test as part of a routine
fecal fat concentration increased, what type of diarrhea physical. The results of all three specimens are positive for
would be suggested? occult blood. Patient #3 is a 50-year-old man advised by
the doctor to lose 30 lb. He has been doing well on a high-
2. Laboratory studies are being performed on a 5-year-old
protein, low-carbohydrate diet. Two of his three specimens
boy to determine whether there is a metabolic reason for
are positive for occult blood.
his continued failure to gain weight. In addition to having
blood drawn, the patient has a sweat chloride collected, a. What is the possible nonpathological cause of the un-
provides a random stool specimen, and is asked to collect expected results for Patient #1? Patient #2? Patient #3?
a 72-hour stool specimen. b. How could the physician’s office staff avoid these
a. How can the presence of steatorrhea be screened for discrepancies?
by testing the random stool specimen? c. What testing methodology could be used for Patients
b. How does this test distinguish among neutral fats, #2 and #3?
soaps, and fatty acids? 4. A watery black stool specimen from a neonate is received
c. What confirmatory test should be performed? in the laboratory with requests for an APT test, fecal pH,
d. Describe the appearance of the stool specimens if and a Clinitest.
steatorrhea is present. a. Can all three tests be performed on this specimen?
e. If a diagnosis of cystic fibrosis is suspected, state the Why?
screening test that could be performed on a stool b. If the Clinitest is positive, what pH reading can be
specimen to aid in the diagnosis. expected? Why?
f. State a possible reason for a false-negative reaction in c. The infant’s hemoglobin remains constant at 18 g/dL.
this test. What was the significance of the black stool?
g. What confirmatory test could be performed? d. Would this infant be expected to have ketonuria? Why
or why not?
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CHAPTER 17
Vaginal Secretions
LEARNING OUTCOMES
Upon completing this chapter, the reader will be able to:
17-1 State the indications for collecting vaginal specimens. 17-6 Describe the microscopic constituents for the common
syndromes associated with vaginitis.
17-2 Describe the procedures for specimen collection and
handling for vaginal specimens, and explain how devi- 17-7 Identify the most common causes of vaginitis, includ-
ations from the correct practice will affect test results. ing the cause, clinical signs and symptoms, laboratory
tests, and treatment.
17-3 Describe the appearance of normal and abnormal vagi-
nal secretions. 17-8 Describe tests that can be performed on vaginal secre-
tions to predict conditions of premature delivery and
17-4 Explain the significance of vaginal pH values.
rupture of fetal membranes.
17-5 List the diagnostic tests performed on vaginal secre-
tions, and explain the appropriate use for each.

KEY TERMS
Atrophic vaginitis Dysuria Trichomonas vaginalis
Bacterial vaginosis (BV) Gardnerella vaginalis Trichomoniasis
Basal cells Lactobacilli Vaginal pool
Clue cells Mobiluncus spp. Vaginitis
Desquamative inflammatory Parabasal cells Vulvovaginal candidiasis
vaginitis (DIV) Pruritus Yeast
Dyspareunia
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356 Part Three | Other Body Fluids

Introduction fresh vaginal secretions is necessary to determine the causative

agent for each syndrome and to provide the appropriate treat-
Vaginal secretions are examined in the clinical laboratory to ment for the patient and, in some cases, the sexual partner, to
diagnose infections and complications of pregnancy, as well avoid reinfection. Microscopic methods include a saline wet
as for forensic testing (see Chapter 11) in sexual assault mount examination, potassium hydroxide (KOH) examina-
patients. This chapter will discuss the disorders encountered tion, and the Gram stain, which is considered the gold stan-
most commonly and the diagnostic laboratory tests used to dard. Other tests used for differential diagnosis include litmus
evaluate vaginal secretions. pH levels, DNA probe testing, culture, and point-of-care test
Vaginitis is one of the most common conditions diagnosed kits. The clinical and microscopic features of the common
by health-care providers for female patients, particularly women syndromes are summarized in Table 17-1.
of childbearing age. It is characterized by abnormal vaginal dis-
charge or odor, pruritus, vaginal irritation, dysuria, and dys-
pareunia. Most often, vaginitis is secondary to bacterial
Technical Tip 17-1. Clinical laboratory personnel
vaginosis (BV), trichomoniasis, or vulvovaginal candidiasis;
performing urine microscopic examinations should
however, vaginitis also can occur with noninfectious conditions,
be aware that microscopic constituents observed in
such as vaginal atrophy, allergies, and chemical irritation.1
vaginal fluid also may be seen in urine specimens
Although the symptoms for the various syndromes of
when the urine specimen is contaminated with
vaginitis are similar, the effective treatment for each depends
vaginal secretions.
on an accurate diagnosis. Careful microscopic examination of

Table 17–1 Clinical Features and Laboratory Findings in Vaginitis2

Desquamative
Bacterial Inflammatory Atrophic
Findings Vaginosis Candidiasis Trichomoniasis Vaginitis Vaginitis
Appearance Thin, homoge- White, curd-like Yellow-green frothy Excessive puru- Excessive purulent
neous, white-to- vaginal adherent vaginal lent vaginal dis- vaginal dis-
gray vaginal discharge discharge charge, vaginal charge, vaginal
discharge increased in erythema erythema
volume
pH >4.5 3.8–4.5 >4.5 >4.5 >4.5
WBCs Rare or absent 3+ to 4+ 2+ to 4+ 3+ to 4+ 3+ to 4+
Lactobacilli Rare or absent Present Absent or present Absent or reduced Decreased
Clue cells >20% Absent Absent or present
Other cells Large clumps of Occasional Occasional
epithelial cells parabasal or parabasal or
basal cells basal cells
>1+ RBCs >1+ RBCs
Other Increase in small Budding yeast Trichomonas 2+ gram-positive Increased gram-
organism curved bacilli, cells and frequently cocci positive cocci and
coccobacilli, pseudohyphae associated with gram-negative
and pleomor- other organisms rods; decreased
phic bacilli large rods
Amine (whiff) Positive Negative Positive Negative Negative
test
Other tests Confirmatory tests: Confirmatory Confirmatory tests:
DNA probe, tests: DNA DNA probe or
proline amino probe, OSOM culture, OSOM
peptidase, BVBLUE Trichomonas
OSOM BVBLUE Rapid Test Rapid Test
Rapid Test
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Chapter 17 | Vaginal Secretions 357

In addition to evaluating vaginal secretions for infections, Technical Tip 17-2. Keep specimens for suspected
tests are performed on vaginal secretions to detect the placen- T. vaginalis at room temperature and examine within
tal α1-microglobulin (PAMG-1) protein, insulin-like growth 2 hours of collection to visualize movement of the fla-
factor binding protein-1 (IGFBP-1) to diagnose ruptured fetal gella or undulating membrane on a wet prep. When
membranes, or fetal fibronectin enzyme to assess the risk of not moving, Trichomonas may resemble a white blood
preterm delivery. The fern test (see Chapter 15) is used to cell (WBC), transitional, or renal tubular epithelial
identify amniotic fluid that may be present when the amniotic (RTE) cell.
sac has ruptured.2

Specimen Collection
Color and Appearance
and Handling
Normal vaginal fluid appears white with a flocculent discharge.
The health-care provider collects vaginal secretions during Microscopically, normal vaginal flora includes a predominance
a pelvic examination. Detailed instructions and the specific of large, rod-shaped, gram-positive lactobacilli and squamous
manufacturer’s collection and transport devices must be pro- epithelial cells. WBCs may be present, and red blood cells (RBCs)
vided and are specific to the organism sought. Correct speci- will be present if the patient is menstruating3 (Table 17-2).
men handling and timely transport to the laboratory are Abnormal vaginal secretions may appear as an increased
important for optimal detection of the responsible pathogen. thin, homogeneous, white-to-gray discharge often seen in BV;
A speculum moistened with warm water is used to visual- as a white “cottage cheese–like” discharge particular for Candida
ize the vaginal fornices. Lubricants may contain antibacterial infections; or as an increased yellow-green, frothy, adherent dis-
agents and must not be used. The specimen is collected by charge associated with T. vaginalis.3 C. trachomatis may present
swabbing the vaginal walls and vaginal pool to collect epithelial with a yellow, opaque cervical discharge.6
cells along with the vaginal secretions using one or more sterile,
polyester-tipped swabs on a plastic shaft or swabs specifically
designated by the manufacturer.3 Cotton swabs should not be Diagnostic Tests
used because cotton is toxic to Neisseria gonorrhoeae, the wood
in a wooden shaft may be toxic to Chlamydia trachomatis, and pH
calcium alginate can inactivate herpes simplex virus (HSV) for The health-care provider can perform a vaginal pH test when
viral cultures.4 performing a pelvic examination. The test should be performed
The health-care provider performs a gross examination of before placing the swab into saline or KOH solutions. Commer-
the vaginal secretions and then places the swab in a tube con- cial pH test paper with a narrow pH range is recommended to
taining 0.5 to 1.0 mL of sterile physiological saline. The tube evaluate pH values in the 4.5 range more accurately. The test
is sealed for transport to the laboratory, where the specimen is paper is placed in the pooled vaginal secretion, and the color
processed for microscopic analysis. The swab should be twirled change is compared with a chart with corresponding pH values.
in the saline vigorously to dislodge particulates from the swab. Factors that can interfere with the pH test include contamination
Failure to dislodge particles may lead to erroneous results. of the vaginal secretions with cervical mucus, semen, and blood.6
Specimens should be tested with pH paper before being placed The pH test helps to differentiate the causes of vaginitis,
in saline.2 An alternative method of specimen preparation is to as shown in Table 17-1. The vaginal pH is usually about 4.5
dilute a sample of vaginal discharge in one to two drops of in women with vulvovaginal candidiasis but is above 4.5 in
normal saline solution directly on a microscope slide. Then a women with BV, trichomoniasis, desquamative inflammatory
second sample is placed in 10% KOH solution in the same vaginitis (DIV), and atrophic vaginitis.1,2,4
manner. Cover slips are placed over both slides for microscopic
examination.5
Properly labeled specimens should be placed in a biohaz- Table 17–2 Normal Findings in Vaginal Secretions
ard bag with the requisition form and transported to the labo-
Appearance White, flocculent discharge
ratory as soon as possible. The requisition form must include
the patient’s name and unique identifier, as well as a patient pH 3.8–4.2
medical history that should include menstrual status; use of Amine (whiff) test Negative
vaginal creams, lubricants, and douches; and recent exposure WBCs Rare to 2+
to sexually transmitted diseases.3 Specimens should be analyzed
Lactobacilli Predominant
immediately, but if a delay in transport or analysis is necessary,
specimen handling is based on the suspected pathogen. Speci- Clue cells Absent
mens must be kept at room temperature to preserve the motility Other cells Absent (except RBCs during
of Trichomonas vaginalis and the recovery of N. gonorrhoeae, menses)
whereas specimens for C. trachomatis and HSV must be refrig- Other organisms Other lactobacilli subgroups,
erated to prevent overgrowth of normal flora.4 Specimens for occasional yeast
T. vaginalis should be examined within 2 hours of collection.2
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358 Part Three | Other Body Fluids

As described previously, normal vaginal flora includes a dry power (40×) objective with a bright-field microscope.
predominance of the bacteria lactobacilli, which produce the Using the low power objective (100× magnification), the slide
end-product lactic acid from glycogen metabolism. Lactic acid is scanned for an even distribution of cellular components,
provides an acidic vaginal environment with a pH value types and numbers of epithelial cells, clumping of epithelial
between 3.8 and 4.5. This acidity suppresses the overgrowth cells, and the presence of budding yeast or pseudohyphae.
of infectious organisms such as Mobiluncus, Prevotella, and Then the slide is examined using the high power objective
Gardnerella vaginalis, and therefore maintains the balance of (400× magnification), and the organisms and cells are counted
normal vaginal bacteria flora.7 Some lactobacilli subgroups also and reported per hpf using the criteria in Table 17-3. Typical
produce hydrogen peroxide, which is toxic to pathogens and constituents found in vaginal fluid wet mounts include squa-
helps keep the vaginal pH acidic to provide protection from uro- mous epithelial cells, WBCs, RBCs, clue cells, parabasal cells,
genital infections. BV has been associated with the absence of basal cells, bacteria, motile T. vaginalis, yeast, and hyphae/
hydrogen peroxide–producing lactobacilli.3 Estrogen production pseudohyphae.
also is necessary to preserve an acidic vaginal environment.6 Intravaginal medications might leave oil droplets that can
interfere with the interpretation of wet mounts. In this case, a
Microscopic Procedures Gram stain is useful to detect yeast or BV.2
Usually vaginal infections are diagnosed from microscopic ex-
Squamous Epithelial Cells
amination. Saline wet mounts and KOH mounts are the initial
Squamous epithelial cells measure 25 to 70 µm in diameter
screening tests, and the Gram stain is used as a confirmatory
and exhibit a polygonal “flagstone” appearance. They contain
examination for yeast or BV.2 Slides are prepared from the
a prominent nucleus that is centrally located and about the size
saline specimen solution that was made from the vaginal swab
of a RBC, as well as a large amount of irregular cytoplasm, lack-
immediately after collection. Three clean glass slides (if a Gram
ing granularity, with distinct cell margins (Fig. 17-1). These
stain is requested) are labeled with the patient’s name and a
large, flat cells originate from the linings of the vagina and
unique identifier. A drop of specimen is placed on each slide
female urethra and are present in significant numbers in the
using a disposable transfer pipette. An alternative method is to
vaginal secretions of a healthy female. Clumps of epithelial cells,
press the swab against the slide and then roll the swab over the
as observed in Figure 17-2, are an indication of the presence of
slide. The slide for Gram stain is allowed to dry and then is
increased numbers of yeast.3
heat-fixed for the Gram stain procedure and examination per-
formed in the microbiology section of the laboratory. For wet Clue Cells
mount examinations, cells and organisms are quantified per Clue cells are an abnormal variation of the squamous epithelial
high power field (hpf) (40×); for Gram stains, cells and organ- cell and are distinguished by coccobacillus bacteria attached in
isms are reported per oil immersion field (100×). clusters on the cell surface, spreading past the edges of the cell
and making the border appear indistinct or stippled. Bacteria
Technical Tip 17-3. The Seattle STD/HIV Prevention should cover at least 75% of the epithelial cell. This gives the
Training Center has developed the YouTube video cell a granular, irregular appearance sometimes described as
titled Examination of Vaginal Wet Preps, an excellent “shaggy.” Clue cells are diagnostic of BV caused by G. vaginalis
online overview of performing the microscopic (Fig. 17-3). The presence of clue cells also can be found in urine
examination of vaginal secretion. It is available at sediment and should be confirmed by the procedures already
https://www.youtube.com/watch?v=8dgeOPGx6YI.8 described.

White Blood Cells

Wet Mount Examination WBCs measure 14 to 16 µm in diameter and exhibit a granular
cytoplasm. Often they are described as polymorphonuclear
For the saline wet mount examination, a cover slip is placed (PMN) WBCs because of their characteristic multilobed nu-
on the specimen carefully to exclude air bubbles. The slide is cleus (Fig. 17-4). Normally, WBCs are present in rare to scanty
examined microscopically using the low power (10×) and high numbers in vaginal secretions. More than 3+ WBCs in vaginal

PROCEDURE 17-1
Table 17–3 Quantitation Scheme for Microscopic
pH Test Examinations2
1. Using a circular motion, gently apply the vaginal
Rare Fewer than 10 organisms or cells/slide
secretion over the surface of the pH test paper.
1+ Fewer than 1 organism or cell/hpf
2. Immediately observe the color reaction on the paper,
and compare it to a color comparison chart to 2+ 1–5 organisms or cells/hpf
determine the pH of the sample. 3+ 6–30 organisms or cells/hpf
3. Record the results. 4+ >30 organisms or cells/hpf
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Chapter 17 | Vaginal Secretions 359

Figure 17–1 Squamous epithelial cells identifiable under low

power (×100).

Figure 17–4 A. White blood cells. Notice the multilobed nucleoli

(×400). B. Stained smear.
Figure 17–2 Clump of squamous epithelial cells (×400).

Figure 17–5 Normal red blood cells (×400).

Figure 17–3 Clue cells (×400).

secretions suggest vaginal candidiasis; atrophic vaginitis; or in- can be somewhat distorted in vaginal specimens. Usually RBCs
fections with Trichomonas, Chlamydia, N. gonorrhoeae, or HSV.3 are not seen in vaginal secretions, but they might be present
during menstruation or due to a desquamative inflammatory
Red Blood Cells process.2 RBCs can be confused with yeast cells and are dis-
RBCs appear as smooth, nonnucleated, biconcave disks meas- tinguished from yeast cells by KOH, which will lyse the RBCs
uring approximately 7 to 8 µm in diameter (Fig. 17-5). RBCs but allow the yeast cells to remain intact.
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360 Part Three | Other Body Fluids

Parabasal Cells suggests an alteration in the normal flora. Often the lactobacilli
Parabasal cells are round to oval-shaped and measure 16 to are replaced by increased numbers of any of the following:
40 µm in diameter. The ratio of nucleus to cytoplasm is 1:1 to • Mobiluncus spp. (thin, curved, gram-negative,
1:2, with marked basophilic granulation or amorphic basophilic motile rods)
structures (“blue blobs”) in the surrounding cytoplasm. They
• Prevotella spp.
are located in the luminal squamous epithelium of the vaginal
mucosa. Although it is rare to find parabasal cells in vaginal • Porphyromonas spp.
secretions, less mature cells may be found if the patient is • Bacteroides spp. (anaerobic gram-negative rods)
menstruating or if the patient is postmenopausal.2 Increased • Gardnerella vaginalis (short, gram-variable coccobacilli)
numbers of parabasal cells, if present with large numbers of
• Peptostreptococcus spp. (gram-positive cocci)
WBCs, can indicate DIV2 (Fig. 17-6).
• Enterococcus spp. (gram-negative cocci)
Basal Cells • Mycoplasma hominis
Basal cells are located deep in the basal layer of the vaginal
• Ureaplasma urealyticum
stratified epithelium. These cells are round and measure 10 to
16 µm in diameter. They have a ratio of nucleus to cytoplasm
Trichomonas vaginalis
of 1:2. Basal cells are distinguished from WBCs, which are sim-
T. vaginalis is an atrial flagellated protozoan that can cause vaginal
ilar in size, by their round rather than lobed nucleus. They are
inflammation and infection in women. The organism is oval
not normally seen in vaginal fluid and, if present and accom-
shaped, measures 5 to 18 µm in diameter, and has four anterior
panied by large numbers of WBCs and altered vaginal flora,
flagella and an undulating membrane that extends half the length
can suggest DIV.2
of the body.9 An axostyle bisects the trophozoite longitudinally
Bacteria
The vagina is a nonsterile environment with complex endoge-
nous bacterial flora that vary with the age and hormonal status
of the patient. Lactobacillus spp. normally comprise the largest
portion of vaginal bacteria.7 They appear as large, gram-positive,
nonmotile rods and produce lactic acid, which maintains the
vaginal pH at 3.8 to 4.5 (Fig. 17-7 A and B). Hydrogen peroxide,
produced by lactobacilli subgroups, also can help to suppress
the overgrowth of other organisms. Other bacteria commonly
present include anaerobic streptococci, diphtheroids, coagulase-
negative staphylococci, and α-hemolytic streptococci. When
conditions are present that cause an imbalance in the normal
flora, vaginitis can occur. Absent or decreased numbers of lac-
tobacilli relative to the number of squamous epithelial cells

Figure 17–7 Bacteria. A. Large rods characteristic of Lactobacilli, the

Figure 17–6 Parabasal cell surrounded by epithelial cells predominant bacteria in normal vaginal secretions (×400). B. Bacteria
(×400). with white blood cells (×400).
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Chapter 17 | Vaginal Secretions 361

and protrudes from the posterior end, which enables the organ-
PROCEDURE 17-2
ism to attach to the vaginal mucosal and cause tissue damage
(Fig. 17-8). The “jerky” motion of the flagella and undulating Saline Wet Mount2
membrane characteristic of T. vaginalis can be observed in a wet 1. Prepare a clean glass slide labeled with the patient’s
mount. Care must be taken not to confuse T. vaginalis with name and unique identifier.
sperm, which have only a single tail, a much smaller head (ap-
proximately one-half the diameter of a RBC), and no axostyle. 2. Place one drop of vaginal specimen on the slide.
In addition, nonmotile trichomonads can be mistaken for 3. Cover the slide with a cover slip, removing any air
WBCs (Fig. 17-9). bubbles.
T. vaginalis organisms quickly lose their viability after col- 4. Examine the slide with the 10× objective for epithelial
lection. Specimens must be examined as soon as possible or, if cells and any budding yeast cells or pseudohyphae.
necessary, maintained at room temperature for a maximum of 5. Examine the slide with the 40× objective, and quan-
2 hours before preparing the wet mount to observe the organ- tify organisms and cells per hpf.
ism’s motility. Trichomonas also can be seen in a urinary micro-
scopic sample but cannot be reported unless motility is 6. Record the results.
observed, either in movement across the slide or just in the
tail. A dead trichomonad tends to appear oval and slightly
larger than a WBC.
difficult to distinguish yeast cells from RBCs on a wet mount
Yeast Cells because both measure about 7 to 8 µm in diameter; however,
Candida albicans and non-Candida spp. cause most fungal in- differentiation can be made using the KOH test. Yeast cells stain
fections, but an occasional yeast in vaginal secretions is con- gram positive.
sidered part of the normal flora. Yeast cells appear on a wet
KOH Preparation and Amine Test
mount as both budding yeast cells (blastospores) (Fig. 17-10)
or as hyphae, which are long filaments that grow and form a The KOH slide is prepared and the amine (whiff) test is per-
mycelium (Fig. 17-11). Pseudohyphae—multiple buds that formed by placing a drop of the saline specimen prepared from
do not detach and form chains—also can be seen. It can be the collection swab onto a clean slide that is labeled properly

P A

Figure 17–8 Trichomonas vaginalis. (From Leventhal and

Cheadle, Medical Parasitology, 6th edition, 2012, F.A. Davis
Company, Philadelphia, with permission.)
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362 Part Three | Other Body Fluids

and adding one drop of 10% KOH solution. The slide is

checked immediately for a “fishy” amine odor. The result is re-
ported as positive (presence of fishy odor) or negative (absence
of fishy odor). Increased numbers of anaerobic bacteria in the
vagina produce polyamines that are released into the vaginal
fluid. The odor results from trimethylamine, a volatilization prod-
uct of amines, when the KOH is added. Because volatile amines
are not present in normal vaginal secretion, a positive test result
suggests BV caused by increased numbers of G. vaginalis in
conjunction with Mobiluncus spp. and with T. vaginalis.
After the amine test has been performed, place a cover slip
over the specimen, taking care to exclude air bubbles. Allow
the KOH preparation to rest for 5 minutes to dissolve epithelial
and blood cells. Heat may be applied to speed the dissolving
process. One drop of 10% glycerin may be added after

PROCEDURE 17-3
Figure 17–9 Trichomonas vaginalis in wet mount. (Courtesy of the
U.S. Department of Health and Human Services, via the CDC.) KOH Preparation2
1. Prepare a clean glass slide labeled with the patient’s
name and unique identifier.
2. Place one drop of vaginal specimen on the slide.
3. Add one drop of 10% KOH to the slide.
4. Allow the KOH slide preparation to rest for up to
5 minutes to allow cellular tissue and other debris to
dissolve. Gentle heating may speed the dissolving
process.
5. Cover the specimen with a cover slip, removing any
air bubbles.
6. Examine the slide under the 10× objective for overall
assessment and for yeast pseudohyphae.
7. Switch to the 40× objective to examine for budding
yeast cells (smaller blastopore blastospore).
8. Record the results.
Figure 17–10 Budding yeast cells (×400).

PROCEDURE 17-4
Amine (Whiff) Test
1. Apply one drop of the saline vaginal fluid suspension
to the surface of a clean glass slide.
2. Add one drop of 10% KOH directly to the vaginal
sample.
3. Holding the slide in one hand, gently fan above the
surface of the slide with the other hand and assess for
the presence of a fishy amine odor.
4. Report as positive or negative.
Positive: The presence of a fishy odor after adding KOH.
Negative: The absence of a fishy odor after adding KOH.
Figure 17–11 Yeast cells showing mycelial forms (×400).
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Chapter 17 | Vaginal Secretions 363

the KOH to prevent specimen deterioration.2 The slide is Special media called Diamond medium is required for
examined under low power (100× magnification) for the pres- determining the presence of T. vaginalis. A commercial trans-
ence of yeast pseudohyphae and under high dry power (400× port and culture pouch system for the detection of Trichomonas
magnification) to identify smaller blastospores (yeast cells).2 is now available (InPouch TV, Biomed Diagnostics, White City,
OR). The specimen must be inoculated into the pouch within
Other Diagnostic Tests 30 minutes of collection and then is incubated for 5 days at
Although the wet mount and KOH slide examinations and the 37°C in a CO2 atmosphere. The pouch is examined microscop-
amine test are used commonly to diagnose BV, other tests may ically daily for motile trichomonads.
be required for a confirmatory diagnosis. These include Gram
DNA Testing
stain, specimen culture, DNA probe testing, and point-of-care
test kits. DNA hybridization probe methods have been developed to
identify the specific causative pathogen for vaginitis. A DNA
Gram Stain probe testing system, Affirm VPIII (Becton, Dickinson, Franklin
The Gram stain is considered the gold standard in identifying Lakes, NJ), is available for differential diagnosis of G. vaginalis,
the causative organisms for BV. It also provides a permanent Candida spp., and T. vaginalis. It is easy to perform, and results
record of the patient specimen. A scored Gram stain system are available in 1 hour, with a sensitivity of 95%. This test is
is a weighted combination of the following morphotypes: significantly more sensitive than wet mount microscopy and
Lactobacillus acidophilus (large gram-positive rods), G. vaginalis is less subjective to personal bias compared with traditional
and Bacteroides spp. (small gram-variable or gram-negative microscopic tests. New molecular assays are being developed
rods), and Mobiluncus spp. (curved gram-variable rods). The continually for the diagnosis of vaginitis.
types of bacterial morphophytes are evaluated and scored. For Trichomonas also can be detected by DNA probes amplified
example, Lactobacillus morphophytes are the predominant by polymerase chain reaction (PCR). Enzymes are added to
bacteria in normal vaginal flora; therefore, if 4+ Lactobacillus the specimen that amplifies specific regions of the DNA of
morphophytes are present on Gram stain, and Gardnerella and T. vaginalis by PCR. Then the number of DNA fragments is cal-
Bacteroides spp. morphophytes and curved gram-variable rods culated. This is the most accurate diagnostic method, and it
are absent, the score is 0. As indicated in Table 17-4, a has the advantage of detecting nonviable organisms.10
Nugent score of 0 to 3 is considered normal vaginal flora,
Point-of-Care Tests
whereas a score of 4 to 6 is reported as intermediate, and a
score of 7 or more is diagnostic of BV. Various rapid diagnostic tests are available to quickly screen
for the causative agents of vaginitis, and they provide a higher
Culture sensitivity and specificity for the organism sought. For exam-
Culture, using various types of media, is the gold standard test ple, proline aminopeptidase activity in vaginal secretions can
for detecting yeast and Trichomonas; however, it is more time be detected by rapid antigen tests to identify G. vaginalis.6
consuming and requires up to 2 days for a result. Culture for The OSOM Trichomonas Rapid Test (Genzyme Diagnos-
G. vaginalis is not diagnostic for BV because it is part of the tics, Cambridge, MA) is an immunochromatographic strip
normal flora in 50% of healthy women. test that detects T. vaginalis antigen from vaginal swabs in
10 minutes. The test is performed by placing the vaginal
swab in the kit’s sample buffer. The Trichomonas proteins are
solubilized into the buffer. The test stick coated with anti-
Table 17–4 Nugent Gram Stain Criteria to Trichomonas antibodies is placed into the sample mixture.
Diagnose Bacterial Vaginosis The solution migrates up the stick and, if Trichomonas anti-
Curved gens are present, they will react with the antibodies on the
Gardnerella and Gram- stick. A visible blue line and a red internal control line
Bacteroides spp. Variable indicate a positive result.
Lactobacillus Morphophytes Rods Points The OSOM BVBLUE test (Genzyme Diagnostics,
Cambridge, MA) detects vaginal fluid sialidase, an enzyme pro-
4+ 0 0 0 duced by the bacterial pathogens associated with BV, such as
3+ 1+ 1+ or 2+ 1 Gardnerella, Bacteroides, Prevotella, and Mobiluncus. The test
2+ 2+ 3+ or 4+ 2 takes 1 minute to perform and is read by examining the change
in the color of the solution: blue or green is positive, yellow is
1+ 3+ 3
negative.
0 4+ 4 Commercial tests to measure an elevated vaginal pH (VS-
Note: Points are added according to the morphotypes seen. Add
Sense Pro Swab) and the presence of amines (FemExam pH
the points for all three columns for a final sum. A score of 7 or and Amines TestCard, Litmus Concepts, Santa Clara, CA) use
higher indicates bacterial vaginosis.6 pH indicators and an amine test system that is read visually to
identify BV and Trichomonas.
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364 Part Three | Other Body Fluids

Vaginal Disorders Trichomoniasis

Trichomoniasis is caused by the parasitic protozoon T. vaginalis.
Bacterial Vaginosis The infection is transmitted by sexual intercourse, and it causes
BV is the most common cause of vaginitis, affecting 40% to vaginitis in women and, sometimes, urethritis in men. The
50% of women of childbearing age.1 It occurs when there is infection is classified as an STI, frequently occurs with infec-
an imbalance in the ratio of normal vaginal bacterial flora. The tions of gonorrhea or Chlamydia, and has been associated with
predominant organism in the vaginal flora is lactobacilli, which enhanced transmission rates of HIV.5,7,11 In pregnant women,
produce lactic acid that maintains the vaginal pH between 3.8 a correlation has been found between a T. vaginalis infection
and 4.5. Additionally, certain subsets of lactobacilli produce and low birth weight, premature rupture of membranes, and
hydrogen peroxide, which prevents the overgrowth of normal preterm delivery.11
vaginal flora. As the vaginal pH becomes alkaline, lactobacilli Trichomoniasis is characterized by a green-to-yellow
are replaced by an overgrowth of G. vaginalis, Mobiluncus spp., frothy vaginal discharge, malodor, pruritus, irritation, dysuria,
Prevotella spp., Porphyromonas, Peptostreptococcus, Mycoplasma dyspareunia, and vaginal mucosa erythema, although some
hominis, and Ureaplasma spp. The malodor and increased ab- patients are asymptomatic. Patients may present with a “straw-
normal vaginal discharge result from this mix of organisms and berry cervix” because of punctuate hemorrhages.11 Usually
is more apparent after intercourse.11 males are asymptomatic or may present with urethritis.
BV is associated with new or multiple sex partners, fre- Trichomoniasis can be diagnosed with the wet mount and
quent douching, use of intrauterine devices, pregnancy, and a microscopic examinations, visualizing the motile trichomonads
lack of the protective lactobacilli.1 There is evidence that BV is in a fresh specimen; however, this method has a sensitivity of
a risk factor for the premature rupture of membranes and only 60% to 70%.5 The test must be performed within 2 hours
preterm labor for pregnant women. Additional complications of specimen collection to preserve the viability of the organism.
include pelvic inflammatory disease and endometritis, as well WBCs and lactobacilli bacteria also are present with a T. vaginalis
as an increased risk for acquiring some sexually transmitted infection. The vaginal pH is greater than 4.5, and the amine
infections (STIs) such as HIV, N. gonorrhoeae, C. trachomatis, test from the KOH preparation will be positive.
and HSV-2.1,5 If the wet mount is negative for motile trichomonads, a
BV is diagnosed by examining the vaginal secretions for culture using Diamond medium or the commercially avail-
abnormal appearance or quantity, performing the pH and able pouch system (InPouch TV, Biomed Diagnostics, White
amine tests, and microscopically observing the wet mount for City, OR) is recommended for detection of T. vaginalis. The
the presence of clue cells and the absence of WBCs and lacto- DNA probe test system, Affirm VPIII (Becton, Dickinson,
bacilli morphotypes. According to Amsel’s Diagnostic Criteria, Franklin Lakes, NJ), and the point-of-care rapid antigen
three of the following four features must be present for the di- detection test, OSOM Trichomonas Rapid Test (Genzyme
agnosis of BV: Diagnostics, Cambridge, MA), are available with increased
1. Thin, white, homogeneous discharge sensitivity and specificity for T. vaginalis.
The recommended treatment for trichomoniasis is
2. Vaginal fluid pH greater than 4.5 metronidazole. For patients who develop an allergy to metron-
3. A positive amine (whiff) test idazole or for whom treatment is not effective, a newer drug,
4. Presence of clue cells on microscopic examination5 tinidazole, is available. All sexual partners of patients, even if
The Gram stain is the gold standard for determining the asymptomatic, should be treated to avoid reinfection.
ratio of each bacterial morphotype and offers a definitive diag-
nosis. Other tests used to diagnose BV include the DNA Candidiasis
hybridization probe test; Affirm VPIII (Becton, Dickinson), Vulvovaginal candidiasis is caused by an infection with the
which detects G. vaginalis; a proline aminopeptidase test (Pip yeast Candida. It is a common cause of vaginitis, and nearly
Activity TestCard, Quidel, San Diego, CA); and the OSOM 75% of adult women have at least one yeast infection in their
BVBLUE test (Genzyme Diagnostics, Cambridge, MA). lifetime.11 Most yeast infections are caused by C. albicans, but
Treatment is recommended for women to relieve vaginal other nonalbican species, such as C. glabrata, C. parapsilosis, C.
symptoms and reduce the risk of infection. In addition, bene- tropicalis, and C. krusei, have been isolated as the cause.
fits of treatment include reducing the risk of acquiring an STI Candida is part of the normal vaginal flora, but an infection
and reducing the risks associated with pregnancy. The recom- occurs when there is a change in the vaginal environment that
mended treatments are metronidazole (Flagyl), metronidazole permits the overgrowth of Candida and symptoms of the infec-
gel, or clindamycin cream.5 tion to occur. Conditions that can cause a change in the vaginal
environment include the use of broad-spectrum antibiotics,
oral contraceptives, or estrogen replacement therapy; hormonal
Technical Tip 17-4. The detection of clue cells
changes that occur with pregnancy; ovulation; and menopause.
in the wet mount examination of vaginal discharge
Increased infection rates occur in patients who are immuno-
is the most useful single indicator for the diagnosis
compromised or those with conditions such as diabetes melli-
of BV.
tus, iron deficiency, and HIV infection. The infection is found
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Chapter 17 | Vaginal Secretions 365

predominantly in women of childbearing age, who are produc- parabasal and basal cells, squamous epithelial cells, and decreased
ing large amounts of estrogen. Estrogen causes the vagina to numbers of lactobacilli that have been replaced by gram-positive
mature and produce glycogen, which facilitates the growth and cocci and gram-negative rods.3
adherence of C. albicans.11 Treatment of atrophic vaginitis is estrogen replacement.
Typical clinical symptoms of vulvovaginal candidiasis in- Topical vaginal ointments are used initially; however, for fre-
clude genital itching or burning, dyspareunia; dysuria; and the quent, recurrent episodes of atrophic vaginitis, oral or transcu-
presence of an abnormal thick, white, curd-like vaginal dis- taneous (patch) modes are more effective.
charge. The pH of the vaginal fluid remains normal (3.8 to 4.5),
and the amine test is negative. In vulvovaginal candidiasis, the Additional Procedures
microscopic examination of the saline and KOH wet prep and
Gram stain will reveal budding yeast and pseudohyphae forms, for Vaginal Secretion
large numbers of WBCs, lactobacilli, and large clumps of ep- Associated With Pregnancy
ithelial cells. A culture and DNA hybridization probe (Affirm
VPIII Microbial Identification System; Becton, Dickinson, As noted previously in this chapter, complications from vagini-
Franklin Lakes, NJ) analysis can be performed to confirm the tis syndromes can include premature rupture of fetal mem-
clinical diagnosis and to identify the species of Candida. branes and a high risk of preterm labor. The following tests are
Yeast infections are treated with over-the-counter or pre- used to evaluate these conditions:
scription azole antifungal agents. They may be intravaginal, • Fetal fibronectin (fFN) enzyme test
suppository, or oral agents, and the regimen depends on the • AmniSure (AmniSure International) test to detect the
medication. Over-the-counter intravaginal medications include amniotic fluid protein PAMG-1
butoconazole, clotrimazole, tioconazole, and miconazole. For
patients with recurrent infections (four or more episodes per • Actim PROM (Cooper Surgical Medical Devices) test to
year), a prescription medication using a longer treatment reg- detect the amniotic fluid protein IGFBP-1, also referred
imen with either oral fluconazole or intravaginal butoconazole, to as placental protein 12 (PP12)
nystatin, and terconazole may be more effective.5 Vulvovaginal • ROM Plus (Clinical Innovations) to detect the amniotic
candidiasis is not acquired through sexual intercourse, so treat- fluid proteins alpha-fetoprotein (AFP) and IGFBP-1
ment of sexual partners is not indicated. The fern test, described in Chapter 15, is another test that
determines the presence of amniotic fluid in vaginal secretions.
Desquamative Inflammatory Vaginitis Patient history, amniotic pooling in the fornix of the vagina, a
DIV is a syndrome characterized by profuse purulent vaginal vaginal pH greater than 7.0, and a positive fern test are strong
discharge, vaginal erythema, and dyspareunia.3 There is a het- indicators of amniotic sac rupture.
erogeneous group of causes of DIV; however, β-hemolytic
gram-positive streptococci can be cultured from most patients.3 Fetal Fibronectin Test
The syndrome also can occur secondary to atrophic vaginitis Preterm delivery, defined as delivery before the completion of
in postmenopausal women as a result of decreased estrogen. 37 weeks’ gestation, is the leading cause of neonatal mortality
The vaginal secretion pH is greater than 4.5, and the amine and morbidity in the United States.12 fFN is an adhesive gly-
test is negative. coprotein in the extracellular matrix at the maternal and fetal
Wet mount and Gram stain microscopic examination of interface within the uterus. It is elevated during the first
the vaginal secretions reveal large numbers of WBCs, RBCs, 24 weeks of pregnancy but then diminishes. The presence of
occasional parabasal and basal cells, squamous epithelial cells, fFN in vaginal secretions between 24 and 34 weeks’ gestation
and reduced or absent lactobacilli that have been replaced by is associated with preterm delivery. The test can be used by
gram-positive cocci (refer back to Table 17-1). health-care providers as a means to better manage patient care,
DIV is treated with 2% clindamycin.3 Hormone replace- and it can be performed routinely as part of a prenatal visit for
ment therapy is effective for patients with DIV secondary to asymptomatic women between 22 and 30 weeks’ gestation or
atrophic vaginitis. in symptomatic pregnant women between 24 and 34 weeks.
Symptoms of preterm delivery include a change in vaginal
Atrophic Vaginitis secretions, vaginal bleeding, uterine contractions, abdominal
Atrophic vaginitis is a syndrome found in postmenopausal or back discomfort, pelvic pressure, and cramping.
women. This syndrome is caused by thinning of the vaginal The specimen is obtained by rotating the swab provided
mucosa because of reduced production of both estrogen and in the specimen collection kit across the posterior fornix of the
glycogen. As a result, the vaginal environment changes, and vagina for 10 seconds to absorb the vaginal secretions. The
the balance of normal flora is altered. Clinical symptoms in- swab must not be contaminated with lubricants, creams, soaps,
clude vaginal dryness and soreness, dyspareunia, inflamed or disinfectants that may interfere with the antibody–antigen
vaginal mucosa, and purulent discharge. The vaginal secretion reaction in the test system.
pH is greater than 4.5, and the amine test is negative. The methods for detection of the fFN enzyme immunoas-
Microscopic evaluation is similar to that for DIV and includes say are solid-phase enzyme-linked immunosorbent assay
large numbers of WBCs and the presence of RBCs, occasional (ELISA) or lateral flow, solid-phase immunochromatographic
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366 Part Three | Other Body Fluids

assay using the Rapid fFN cassette. In the fFN enzyme im- patient sample, it will bind with antibodies in the test region
munoassay, the vaginal sample is incubated with FDC-6, a and produce a line. This line is produced by gold dye attached
monoclonal antibody specific for fFN, and the presence or to conjugated antibodies and indicates a rupture of fetal mem-
absence of the fFN is determined spectrophotometrically at a branes. The second control line is designed to indicate that the
wavelength of 550 nm. test is functioning well. The test is read immediately or within
The rapid fFN assay is a qualitative test for the detection 10 minutes. A positive test result indicates a membrane rupture
of fFN that uses a Rapid fFN cassette kit and a TLiIQ analyzer. and is indicated by the presence of two lines. When only the
The specimen swab is placed into an extraction buffer and fil- control line is present, it is reported as negative for membrane
tered with a plunger filter. The filtered sample is dispensed rupture. The test should be performed immediately after col-
onto the sample application well of the Rapid fFN cassette. The lection, but if there is a delay in testing, the specimen can be
sample flows from an absorbent pad across a nitrocellulose maintained in a closed sample vial and refrigerated for 6 hours.
membrane by capillary action through a reaction zone contain- False positives can be caused by the presence of large amounts
ing murine monoclonal anti-fFN antibody conjugated to blue of blood in the vaginal sample.
microspheres. The monoclonal antibody FDC-6 is specific for
fFN. The conjugate, embedded in the membrane, is mobilized Actim PROM
by the flow of the sample. Then the sample flows through a The Actim PROM (CooperSurgical, Trumbull, CT) is a
zone containing goat polyclonal antihuman fibronectin anti- 5-minute test that can be used bedside to diagnose patients
body that captures the fibronectin–conjugate complexes. The with PROM.15 It is a rapid immunoassay test that specifically
remaining sample flows through a zone containing goat poly- detects IGFBP-1 in the vaginal fluid. IGFBP-1 accumulates at
clonal antimouse IgG antibodies, which captures unbound high concentrations in amniotic fluid. When the membranes
conjugate, resulting in a control line. After 20 minutes, the are ruptured, IGFBP-1 is detectable in a vaginal swab sample.
intensities of the test line and control line are interpreted with The swab is held in the vaginal fornix for 10 to 15 seconds.
the TLiIQ analyzer. The results are reported as positive or neg- Then the swab is placed in the specimen extraction solution
ative.13 Symptomatic pregnant women with a positive fFN test and swirled vigorously for 10 to 15 seconds. The yellow
are at increased risk for delivery in less than or equal to 7 to area of the dipstick is placed into the extraction solution and
14 days from specimen collection, and asymptomatic pregnant held there until the liquid front reaches the result area, at
women are at increased risk for delivery in less than or equal which point the dipstick is removed. The test is interpreted in
to 34 weeks and 6 days of gestation.13 5 minutes. A positive result will show two lines on the dipstick,
indicating that the membranes have ruptured. A negative result
AmniSure Test will show only one control line, indicating that the membranes
The risk of premature delivery also may be caused by the are intact. Blood, urine, semen, bath and odor products, com-
premature rupture of fetal membranes (PROM). In addition, mon vaginal infections, and medications will not interfere with
rupture of fetal membranes can cause infection, fetal distress, the testing. The test has been designed to minimize interference
prolapse of the umbilical cord, postnatal endometritis, and pla- from bleeding, but in cases of heavy bleeding, the blood pres-
cental abruption. A symptom of fetal membrane rupture is ent may have a higher concentration of IGFBP-1 protein.16
leakage of amniotic fluid. PAMG-1 is present in high levels in
amniotic fluid and low levels in blood; therefore, it is a reliable
ROM Plus
marker of fetal membrane rupture. A normal level of PAMG-1 The ROM Plus fetal membranes rupture test is a qualitative
in pregnant women ranges from 0.05 to 0.22 mg/mL, which immunochromatographic test for the detection of amniotic
might increase to 3 mg/mL when vaginitis is present. Fetal fluid in vaginal secretions of pregnant women. This test is
membrane rupture causes increased concentrations of amniotic unique in that it detects both AFP and IGFBP-1 protein, also
fluid in the vaginal secretions and can raise the PAMG-1 levels known as PP12, from amniotic fluid in vaginal secretions.
to 2,000 to 25,000 mg/mL.14 The AmniSure test quickly iden- The ROM Plus test strip is a lateral flow device. The sample
tifies patients with PROM, and appropriate intervention can is collected by placing the swab on the vaginal mucosal lining
take place. for 15 seconds. Then the swab is mixed into a vial containing
The AmniSure ROM test (AmniSure International) is a 400 µL of buffer solution, and the diluted sample is applied
qualitative rapid test that uses an immunochromatographic de- to the sample pad of the test strip via the sample well on the
vice. A sample of vaginal secretions collected with a swab is cassette. The liquid moves chromatographically and unidi-
placed into a vial with solvent. The swab is rotated for 1 minute rectionally toward the absorbent pad. During migration, the
to enable the solvent to extract the sample from the swab, and sample reacts with monoclonal and polyclonal antibodies
then the swab is discarded. The AmniSure test strip is placed bound to the test strip membrane. These antibodies are im-
into the vial. Monoclonal antibodies with colloidal gold parti- munoreactive to a combination of AFP and IGFBP-1. If the
cles attached are located on the pad region of the test strip. The sample contains AFP and IGFBP-1, it binds to the antibody
antibodies attach to the PAMG-1 in the sample and transport test line, causing the test line to appear and indicate a positive
it to the test region. The solution flows from the pad region result. Two lines will be visible. If the sample does not contain
of the strip to the test region. The test region of the test strip the amniotic markers, only the control line will be visible, in-
has antibodies immobilized on it. If PAMG-1 is present in the dicating a negative result.17
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Chapter 17 | Vaginal Secretions 367

IGFBP-1 concentration in amniotic fluid is between 10,500 Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO,
and 350,000 ng/mL. AFP concentration in amniotic fluid is from 2011.
8. Seattle STD/HIV Prevention Training Center. Examination of
2,800 to 26,000 ng/mL. Serum IGFBP-1 concentration is from
vaginal wet preps (video). Web site: https://www.youtube.com/
55 to 242 U/mL (equivalent to 33 to 290 ng/mL). Concentra- watch?v=8dgeOPGx6YI. Accessed July 27, 2019.
tions of IGFBP-1 in amniotic fluid can be 100 to 1,000 times 9. Smith, LA: Diagnostic Parasitology. In Mahon, CR, Lehman,
higher than that in maternal serum.17 DC, Maneselis, and Maneselis, G: Textbook of Diagnostic
Microbiology, ed 4. Saunders Elsevier, Maryland Heights, MO,
2011.
10. Patil, MJ, Magamoti, JM, and Metgud, SC: Diagnosis of
For additional resources please visit Trichomonas vaginalis from vaginal specimens by wet mount
www.fadavis.com microscopy, in pouch TV culture system, and PCR. J. Global
Infect Dis [serial online] [cited 2012 Jul 6] 4:22–25, 2012.
Web site: http://www.jgid.org/text.asp?2012/4/1/22/ 93756.
Accessed July 23, 2019.
11. Keen, EF, and Aldous, WK: Genial infections and sexually
References transmitted diseases. In Mahon, CR, Lehman, DC, Maneselis, G:
1. Egan, MA, and Lipsky, MS: Diagnosis of vaginitis, Am Fam Textbook of Diagnostic Microbiology, ed 4, Saunders Elsevier,
Physician 62(5):1095–1104, 2000. Web site: http://www.aafp. Maryland Heights, MO, 2011.
org/afp/2000/0901/p1095.html. Accessed October 4, 2019. 12. Lockwood, CJ, Senyei, AE, Dische, MR, Casal, DC, et al:
2. Clinical and Laboratory Standards Institute. Provider-Performed Fetal fibronectin in cervical and vaginal secretions as a
Microscopy Testing: Approved Guideline, ed. 2. CLSI document predictor of preterm delivery. New Engl J Med 325:669–674,
POCT10-A2. CLSI, Wayne, PA, 2011, CLSI. 1991.
3. Metzger, GD: Laboratory diagnosis of vaginal infections. Clin 13. Fetal Fibronectin Enzyme Immunoassay and Rapid fFN for the
Lab Sci 11:47–52, 1998. TLiIQ System. AW-04196-002 Rev.002, Hologic, Inc. Web site:
4. Woods, GL, and Croft, AC: Specimen collection and handling http://www.ffntest.com/;hcp/science_fetal.html. Accessed
for diagnosis of infectious diseases. In Henry, JB (ed): Clinical July 23, 2019.
Diagnosis and Management by Laboratory Methods, ed 22. 14. Cousins, LM, et al: AmniSure Placental Alpha Microglobulin-1
Elsevier Saunders, Philadelphia, 2011. Rapid Immunoassay versus standard diagnostic methods for
5. Centers for Disease Control and Prevention: Diseases character- detection of rupture of membranes. Am J Perinatol 22(6):
ized by vaginal discharge. Sexually Transmitted Diseases 317–320, Aug 2005.
Treatment Guidelines, 2010. Web site: http://www.cdc.gov/std/ 15. Abdelazim, IA: Insulin-like growth factor binding protein-1
treatment/2010/vaginal-discharge.htm. Accessed April 14, (Actim PROM test) for detection of premature rupture of fetal
2020. membranes. J. Obstet Gynaecol Res. 2014 Apr;40(4):961–967.
6. French, L, Horton, J, and Matousek, M: Abnormal vaginal Doi: 10.1111/jog.12296. Epub 2014 Feb 26. https://www.
discharge: Using office diagnostic testing more effectively, ncbi.nlm.nih.gov/pubmed/24612210. Accessed July 23,
J Fam Practice 53(10):805–814, 2004. Web site: https://www. 2019.
mdedge.com/familymedicine/article/60266/womens-health/ 16. Actim PROM brochure. Cooper Surgical. www.coopersurgical.
abnormal-vaginal-discharge-using-office-diagnostic. Accessed com. 2014. Accessed July 23, 2019.
July 23, 2019. 17. ROM Plus Fetal Membranes Rupture Test Instructions for
7. Fader, RC: Anaerobes of clinical importance. In Mahon, CR, Use brochure. (Package insert). Clinical Innovations. www.
Lehman, DC, and Maneselis, G: Textbook of Diagnostic clinicalinnovations.com. Accessed July 23, 2019.

Study Questions
1. Which of the following would not be a reason to collect a 3. The appearance of the vaginal discharge in vulvovaginal
vaginal fluid for analysis? candidiasis is described as:
A. Vaginitis A. Clear and colorless
B. Complications of pregnancy resulting in preterm B. Thin, homogeneous, white-to-gray discharge
delivery C. White, curd-like
C. Forensic testing in a sexual assault D. Yellow-green and frothy
D. Pregnancy testing
4. A normal range for a vaginal pH is:
2. Which of the following organisms might not be detected A. 3.8 to 4.5
if the specimen for vaginal secretion analysis had been
B. 5.0 to 6.0
refrigerated?
C. 6.0 to 7.0
A. Prevotella bivia
D. 7.0 to 7.4
B. Lactobacillus acidophilus
C. Trichomonas vaginalis
D. Candida albicans
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368 Part Three | Other Body Fluids

5. Which of the following tests differentiates budding yeast 11. Which of the following organisms produces lactic acid
cells from RBCs? and hydrogen peroxide to maintain an acidic vaginal
A. pH environment?
B. Saline wet mount A. Gardnerella vaginalis
C. KOH prep B. Mobiluncus spp.
D. Whiff test C. Lactobacilli spp.
D. β-Hemolytic streptococci
6. Which of the following constituents is normal in healthy
vaginal fluid secretions? 12. All of the following are diagnostic of vulvovaginal
A. Lactobacilli candidiasis except:
B. Basal cells A. Large numbers of WBCs
C. Trichomonas vaginalis B. Presence of clue cells
D. Pseudohyphae C. Positive KOH test
D. Vaginal pH of 4.0
7. Vaginal specimens collected for a saline wet prep
should be: 13. All of the following are diagnostic of trichomoniasis
A. Refrigerated to preserve motility except:
B. Prepared as soon as possible A. Vaginal pH of 6.0
C. Mailed to a reference laboratory B. Positive amine test
D. Preserved with potassium hydroxide C. Positive KOH test
D. Motile trichomonads present
8. A positive amine (whiff) test is observed in which of the
following syndromes? 14. The bacteria associated with desquamative inflammatory
A. Bacterial vaginosis vaginitis is:
B. Vulvovaginal candidiasis A. β-Hemolytic streptococci
C. Atrophic vaginitis B. Trichomonas vaginalis
D. Desquamative inflammatory vaginitis C. Gardnerella vaginalis
D. Mycoplasma hominis
9. A squamous epithelial cell covered with coccobacilli that
extends beyond the cytoplasm margin is a: 15. The protein present in vaginal secretions that can identify
A. Basal cell patients who are at risk for preterm delivery is:
B. Parabasal cell A. Human chorionic gonadotropin
C. Clue cell B. Estrogen
D. Blastospore C. PAMG-1
D. Fetal fibronectin
10. All of the following are diagnostic of bacterial vaginosis
except: 16. Which of the following immunochromatographic tests
A. Vaginal pH of 3.8 detects both AFP and IGFBP-1 proteins to diagnose
PROM?
B. Presence of clue cells
A. AmniSure ROM test
C. Positive amine (whiff) test
B. Actim PROM
D. Thin, homogeneous, white-to-gray vaginal
discharge C. ROM Plus
D. Fetal fibronectin
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Chapter 17 | Vaginal Secretions 369

Case Studies and Clinical Situations

1. A 30-year-old woman has symptoms of dysuria, vaginal c. What is the best course of treatment?
itching, and a white, curd-like discharge. During her visit d. Should her sexual partner be treated?
at the women’s clinic, the patient revealed that she had re-
e. List three complications that can occur with this
cently completed a regimen of broad-spectrum antibiotics
disorder.
as treatment for a urinary tract infection. Her health-care
provider takes a swab of the vaginal secretions for analysis. 3. During a routine visit with the gynecologist, a 60-year-old
a. What tests will be performed on the vaginal specimen? woman complained of vaginal dryness and soreness.
During the examination, the health-care provider noted
b. Based on the patient history and observation of the
erythema of the vaginal mucosa. The pH of the vaginal
vaginal secretion, which test will be diagnostic for the
secretions was 6.0. The KOH and amine (whiff) tests were
probable diagnosis?
negative. The microscopic examination revealed epithelial
c. What confirmatory test can be performed? cells, basal cells, decreased lactobacilli, and increased
d. What is the probable diagnosis? gram-positive cocci and gram-negative rods.
e. What is the first choice of treatment? a. What is the name of this condition?
2. A sexually active teenager visited the women’s clinic com- b. Explain how this condition can occur.
plaining of vaginal itching and soreness. She indicated c. What is the treatment for this condition?
that she was experiencing increased vaginal secretions
that were frothy and yellow to green. Upon examination,
the health-care provider noted a strawberry-like cervix
and performed a pH test on the secretions. The pH
was 5.5, and the wet prep demonstrated “swimming”
organisms.
a. What is the probable diagnosis?
b. What other tests can be performed to confirm this
diagnosis?
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Answers to Study Questions,

Case Studies, and Clinical Situations
Chapter 1 c. At the beginning of each shift, when a new bottle of
reagent is opened, or as stated in the procedure
Study Questions manual.
d. Refer to the procedure manual. Check expiration
1. C 14. A 27. C dates of controls and reagents. Open and test a new
2. C 15. B 28. D bottle of control or reagents.
3. C 16. D 29. D e. When the controls are within range.
4. A 17. A 30. D 4. a. Delay in testing the specimen.
5. D 18. B 31. B b. Procedure manual.
6. A 19. C 32. 2, 1, 2, 3, c. Delta check.
7. B 20. C 2, 1 d. Incident report.
8. A 21. A 33. D e. Treatment of the patient will be delayed because the
9. C 22. B 34. C specimen will need to be recollected and tested. Extra
10. D 23. D 35. D expense incurred.
11. D 24. B 36. C f. Errors should be corrected as soon as possible
37. D following the facility’s policy. The original result must
12. C 25. B not be erased.
13. B 26. A

Case Studies and Clinical Situations

Chapter 2
1. a. Review of the procedure by a designated authority Study Questions
has not been documented.
1. A 5. D 9. B
b. Instructions and training are not being provided to
2. C 6. C 10. C
personnel performing collections.
3. B 7. A
c. A safety statement about the heat produced by the
reaction is not in the procedure manual. 4. D 8. C
d. The bottles have not been dated and initialed.
Case Studies and Clinical Situations
2. a. Corrective action; proficiency survey tests should be
rotated among personnel performing the tests. 1. a. No. The large amount of blood and leukocytes on the
b. Accept; quality control (QC) on the Clinitest tablets chemical reagent strip does not correlate with 0 to
must be performed only when they are used to 2 RBCs/hpf and the 0 to 2 WBCs/hpf for the
perform a test. microscopic result.
c. Corrective action; documentation of technical b. The MLS student may not have mixed the specimen
competency should be performed on all personnel before pouring the urine into the aliquot tube,
working in the section and educational qualifications causing the cellular constituents to reside at the
assessed. bottom of the original urinalysis container.
3. a. The procedure was being performed incorrectly. The c. The quality control specimens should be tested for
correct timing of the glucose reaction was not being precision and accuracy before patient specimens are
done. tested and the results reported.
b. The technologist performing the test. QC ensures that 2. a. Sysmex XN, Glocyte, iQ200, and Advia 2120i.
the reagents and instrument are working properly b. Advia 2120i.
and that the technologist is performing the test c. Up to 4 hours.
correctly.

371
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372 Answers to Study Questions, Case Studies, and Clinical Situations

Chapter 3 21. D 26. C 31. D

22. D 27. C 32. 600 mL/min
Study Questions 23. B 28. B 33. C
1. C 8. A 15. C 24. D 29. A 34. B
2. B 9. C 16. C 25. D 30. +0.5
3. C 10. A 17. A
4. A 11. B 18. D Case Studies and Clinical Situations
5. D 12. D 19. B 1. a. 160 mg/dL to 180 mg/dL.
6. C 13. D 20. B b. Renal tubular reabsorption is impaired.
7. C 14. A 2. a. Juxtaglomerular apparatus → Angiotensinogen →
Renin → Angiotensin I → Angiotensin II.
Case Studies and Clinical Situations b. Vasoconstriction, increased sodium reabsorption, and
increased aldosterone to retain sodium.
1. a. It could have increased pH, nitrite, and bacteria and
decreased clarity, glucose, ketones, bilirubin, c. Production of renin decreases and, therefore, the
urobilinogen and WBCs, RBCs, and casts. actions of the RAAS.
b. Decreased glucose and decreased ketones. 3. a. The physician can calculate the approximate creatinine
clearance using the MDRD-IDMS-traceable formula.
c. Reject the specimen and recollect.
b. The cystatin C test and the beta2-microglobulin test
d. The specimen was refrigerated and was brought serum tests.
immediately to the laboratory.
c. No. The beta2-microglobulin test requires a normal
2. a. It would be less clear. immune system, and malignancies can affect the
b. Additional epithelial cells and bacteria (making it not immune system; therefore, the test cannot be reliable
acceptable for a culture). in patients with immunologic disorders and
3. a. The results would be decreased falsely. malignancies.
b. The patient needs to collect another 24-hour 4. a. Yes. Serum from the midnight specimen is not being
specimen. separated from the clot and refrigerated in a timely
4. a. Yes. manner.
b. The correct ratio of additive to urine must be b. Lactic acid will be present in serum that is not
maintained. The excess preservative can be toxic to separated from the clot and will affect the freezing-
bacteria, causing false-negative results. point osmolarity readings.
5. a. Specimen temperature. c. If the laboratory is using a freezing-point osmometer,
results will be affected by alcohol ingestion; vapor
b. The temperature would be lower than body pressure results would not be affected and could be
temperature. used as a comparison.
c. The specimen tested was not from the defendant. 5. a. Diabetes insipidus.
d. A chain-of-custody form (COC) that is filled out b. Neurogenic diabetes insipidus.
accurately. The specimen could be collected as a
“witnessed” collection. c. Nephrogenic diabetes insipidus.

Chapter 4 Chapter 5
Study Questions Study Questions
1. B 9. B 17. B 1. A 9. D 17. D
2. D 10. A 18. B: Beta2- 2. D 10. A 18. D
3. C 11. C microglobulin; 3. A 11. C 19. C
B: Creatinine; 4. D 12. B 20. B
4. D 12. D
B: Cystatin C; 5. C 13. D 21. B
5. A 13. D A: 125I-
6. B 14. B iodothalamate 6. A 14. A 22. A
7. C 15. B 19. B 7. C 15. C 23. B
8. D 16. D 20. 69 mL/min 8. C 16. B 24. D
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Answers to Study Questions, Case Studies, and Clinical Situations 373

Case Studies and Clinical Situations Case Studies and Clinical Situations
1. a. An elevated pH and a positive reagent strip reaction 1. a. The blood glucose is elevated and has exceeded the
for nitrite. renal tubular maximum (Tm) for glucose.
b. The reagent strip specific gravity would be much b. Diabetes mellitus.
lower if the patient had been given radiographic dye. c. It indicates diabetes mellitus–related renal disease.
c. The reagent strip test for bilirubin would be positive. d. Renal tubular reabsorption disorders.
d. The reagent strip reaction for blood would be 2. a. Yellow foam.
positive, and RBCs would be seen in the microscopic
b. Possible biliary duct obstruction preventing bilirubin
examination.
from entering the intestine.
2. a. 1.018.
c. Icteric.
b. Yes.
d. Protection from light.
c. It would agree with the reagent strip reading because,
3. a. Hemoglobinuria.
like the osmometer, the reagent strip is not affected
by high-molecular-weight substances. b. Increased hemoglobin presented to the liver results
in increased bilirubin entering the intestine for
3. Hemoglobin and myoglobin.
conversion to urobilinogen.
a. Examine the patient’s plasma/serum. The breakdown
c. The circulating bilirubin is unconjugated.
of red blood cells to hemoglobin produces a red
serum/plasma. Myoglobin is produced from skeletal d. It would if a Multistix reagent strip is used and would
muscle and is rapidly cleared from the plasma/serum. not if a Chemstrip is used. A Watson-Schwartz test is
The serum/plasma color would not be affected. more specific for porphobilinogen.
4. a. The woman has been eating fresh beets. 4. a. Negative chemical reactions for blood and nitrite.
Ascorbic acid interference for both reactions. A
b. Yes. The pH of the woman’s urine is acidic or she has
random specimen or further reduction of nitrite
not recently consumed fresh beets.
could cause the negative nitrite.
5. No. The urine can contain increased pH, glucose,
b. Glucose, bilirubin, LE. Ascorbic acid is a strong
ketones, bilirubin, urobilinogen, nitrite, and small
reducing agent that interferes with the oxidation
amounts of cellular structures.
reaction in the glucose test. Ascorbic acid combines
with the diazo reagent in the bilirubin and LE tests,
Chapter 6 lowering the sensitivity.
c. The dark yellow color may be caused by beta-
Study Questions carotene and vitamin A, and some B vitamins also
1. A 17. C 33. A produce yellow urine.
2. D 18. A 34. 1, 3, 4, 2 d. Nonnitrite–reducing microorganisms; lack of dietary
nitrate; antibiotic administration.
3. A 19. A 35. A
5. a. To check for possible exercise-induced abnormal
4. C 20. C 36. D
results.
5. D 21. A 37. C
b. Negative protein and blood, possible changes in color
6. A 22. B 38. A and specific gravity.
7. D 23. C 39. C c. Renal.
8. B 24. A 40. D 6. a. No, the specimen is clear.
9. D 25. C 41. A b. Myoglobinuria.
10. 2, 1, 2, 3, 1, 26. B 42. B c. Muscle damage from the accident (rhabdomyolysis).
2, 3 27. A 43. D d. Yes. Myoglobin is toxic to the renal tubules.
11. B 28. D 44. C 7. a. Laboratory personnel are not capping the reagent
12. A 29. A 45. B strip containers tightly in a timely manner.
13. A 30. C 46. C b. Personnel performing the CLIA-waived reagent strip
14. D 31. 1, 2, 1, 2, 47. C test are not waiting 2 minutes to read the LE reaction.
15. B 1, 2 48. A c. The student is not mixing the specimen.
16. A 32. B 49. C d. The reagent strips have deteriorated, and the quality
control on the strips was not performed before
reporting the results.
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374 Answers to Study Questions, Case Studies, and Clinical Situations

Chapter 7 5. a. Calcium oxalate.

b. Monohydrate and dihydrate calcium oxalate.
Study Questions c. Oval: monohydrate; envelope: dihydrate.
1. C 19. B 37. C d. Monohydrate.
2. D 20. C 38. D 6. a. Microscopic results do not match the chemical tests
3. D 21. A 39. A for blood, nitrite, and leukocyte esterase.
4. C 22. B 40. C b. The specimen has been unpreserved at room
temperature for too long, the cells have disintegrated,
5. A 23. C 41. D and the bacteria have converted the nitrite to
6. B 24. D 42. A nitrogen.
7. C 25. D 43. A c. The pH.
8. D 26. B 44. C d. Ask the clinic personnel to instruct the patient to
9. C 27. D 45. D collect a midstream clean-catch specimen and have
10. D 28. A 46. C the specimen delivered to the laboratory immediately.
11. D 29. B 47. 4, 3, 5, 1 7. a. No, because they are associated with strenuous
exercise.
12. A 30. C 48. 3, 5, 2, 6, 4
b. The positive blood reaction is from hemoglobinuria
13. C 31. C 49. 4, 8, 7, 6, 1, or myoglobinuria resulting from participating in a
14. B 32. D 5, 3 contact sport. The protein is orthostatic.
15. C 33. D 50. 3, 5, 2, 1, c. Increased excretion of RTE cell lysosomes in the
7, 4 presence of dehydration.
16. A 34. D
17. D 35. A 8. a. Yes, the waxy casts are probably an artifact, such as a
18. D 36. C diaper fiber. Waxy casts are not associated with
negative urine protein.
Case Studies and Clinical Situations b. No, this is normal after an invasive procedure.
c. Yes, tyrosine crystals are seen in patients with severe
1. a. Yeast grows best at a low pH with an increased liver disease; therefore, the bilirubin should be
concentration of glucose. positive. The crystals may be an artifact or from a
b. Yes, this exceeds the renal threshold. medication.
c. No, yeast is not capable of reducing nitrate to nitrite. d. Yes, uric acid crystals may be mistaken for cystine
d. Moderate blood with no RBCs. crystals.
e. Myoglobin is the cause of the positive chemical test e. Yes, radiographic dye crystals associated with a high
result for blood. The patient has been bedridden for specific gravity resemble cholesterol crystals.
an extended period, causing muscle destruction. f. No, Trichomonas is carried asymptomatically by men.
2. a. The large objects are in a different plane from that of g. No, calcium carbonate crystals are found in alkaline
the urinary constituents. urine; therefore, clumps of amorphous phosphates
b. Contamination by artifacts. may be present.
c. No, because they are in a different plane.
d. Polarizing microscopy. Chapter 8
3. a. Renal tubules.
Study Questions
b. Yes, viral infections can cause tubular damage.
c. RTE cells absorb the bilirubin-containing urinary 1. B 8. D 15. A
filtrate. 2. C 9. A 16. C
d. Liver damage inhibits processing of reabsorbed 3. B 10. C 17. A
urobilinogen. 4. C 11. C 18. A
e. Hemolytic anemia. 5. B 12. C 19. A
4. a. The patient is taking a pigmented medication, such as 6. A 13. B 20. D
phenazopyridine.
7. C 14. D
b. Yes.
c. Ask what medications the patient is taking.
d. Ampicillin.
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Answers to Study Questions, Case Studies, and Clinical Situations 375

Case Studies and Clinical Situations e. Female children.

f. Pyelonephritis.
1. a. Acute glomerulonephritis.
7. a. Intravenous pyelogram.
b. M protein in the cell wall of the group A
streptococcus. b. Chronic pyelonephritis.
c. Glomerular bleeding. c. WBC cast.
d. No, they are also passing through the damaged d. Reflux nephropathy.
glomerulus. e. Performing a Gram stain.
e. Good prognosis with appropriate management of f. Radiographic dye.
secondary complications. g. Permanent tubular damage and progression to
f. Henoch-Schönlein purpura. chronic, end-stage renal disease.
2. a. IgA nephropathy. 8. a. Abnormal.
b. Serum IgA level. b. Acute interstitial nephritis.
c. Chronic glomerulonephritis/end-stage renal disease. c. This disorder is an inflammation, not an infection.
d. Impaired renal tubular reabsorption associated with d. Discontinue the medication because it is causing the
end-stage renal disease. allergic reaction.
e. The specific gravity is the same as that of the 9. a. Acute renal failure.
ultrafiltrate, indicating a lack of tubular b. The prerenal sudden decrease in blood flow to the
concentration. kidneys.
f. The presence of extreme urinary stasis. c. Lack of renal concentrating ability.
3. a. Nephrotic syndrome. d. Tubular damage.
b. Nephrotic syndrome may be caused by sudden, e. The increased diameter of the damaged distal
severe hypotension. convoluted tubule and extreme urinary stasis,
c. Changes in the electrical charges of the shield of allowing casts to form in the collecting ducts.
negativity produce increased membrane permeability. 10. a. Renal lithiasis.
d. Decreased plasma albumin lowers the capillary b. The high specific gravity.
oncotic pressure, causing fluid to enter the interstitial
c. Yes, the dark yellow color and high specific gravity
tissue.
indicate a concentrated urine, which induces the
e. Reabsorption of filtered lipids by the RTE cells. formation of renal calculi.
4. a. Minimal change disease. d. Calcium oxalate.
b. Nephrotic syndrome, focal segmental e. Increased hydration and dietary changes.
glomerulosclerosis.
c. Good prognosis with complete remission.
Chapter 9
5. a. Goodpasture syndrome.
b. The autoantibody attaches to the glomerular Study Questions
capillaries, causing complement activation and
1. C 11. D 21. D
destruction of the capillaries.
2. A 12. B 22. D
c. Granulomatosis with polyangiitis.
3. C 13. C 23. D
d. Antineutrophilic cytoplasmic antibody.
4. B 14. A 24. C
e. Granuloma formation resulting from autoantibodies
binding to neutrophils in the vascular walls and 5. A 15. D 25. B
initiating an immune response. 6. A 16. B, A, B, B, A 26. D
6. a. Cystitis, UTI. 7. C 17. D 27. D, F, A, E,
b. The specimen is very dilute. 8. B 18. B C, B
c. Irritation of the urinary tract will cause a small 9. D 19. B
amount of bleeding. The cells and bacteria may cause 10. D 20. B
a trace protein, or it may be a false-positive due to the
high pH.
d. Yes, glitter cells are seen in hypotonic urine.
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376 Answers to Study Questions, Case Studies, and Clinical Situations

Case Studies and Clinical Situations 25. C 30. C 35. C

26. C 31. D 36. C
1. a. Underdevelopment of the liver.
27. A 32. C 37. D
b. Yes, with severe acquired liver disease.
28. C 33. B 38. B
c. Tyrosine crystals; leucine crystals, bilirubin crystals.
29. A 34. A
d. Protect the specimen from light.
2. a. Isovaleric acidemia.
Case Studies and Clinical Situations
b. Maple syrup urine disease.
1. a. Cerebral hemorrhage because of the presence of
c. Yes, the MS/MS screen would be positive.
erythrophagocytosis, even distribution of blood,
3. a. Renal lithiasis. and patient’s history.
b. Impaired renal tubular reabsorption of cystine. b. No, they would be consistent with peripheral blood
c. Lysine, arginine, ornithine. entering the CSF.
d. They are more soluble than is cystine. c. No, they are consistent with the percentages seen in
e. The disorder is inherited cystinuria. peripheral blood.
4. a. Yes. d. Hemosiderin granules and hematoidin crystals.
b. Yes, uric acid crystals accumulating on the surface of e. A traumatic tap.
the diaper could have an orange color. 2. a. An India ink preparation.
c. Lesch-Nyhan disease. b. Cryptococcus meningitis.
d. Yes, the disease is inherited as sex-linked recessive. c. Immunologic testing for Cryptococcus.
e. Hypoxanthine guanine phosphoribosyltransferase. d. Rheumatoid factor.
5. a. Yes. The urine may contain melanin or homogentisic e. Acid-fast staining and culture.
acid. f. Noticeable oligoclonal bands in both the CSF and
b. Yes. Melanin will react with sodium nitroprusside, the serum.
reagent used on reagent strips for the detection of 3. a. CSF/serum albumin index = 6.7.
ketones.
b. Yes.
c. Yes. Homogentisic acid turns black in alkaline urine.
c. IgG index = 1.5.
6. a. Yes, the blue color could indicate the presence of
d. Immunoglobulin synthesis within the CNS.
indican in the urine.
e. Multiple sclerosis.
b. Hartnup disease.
f. Oligoclonal banding only in the CSF.
c. Good with proper dietary supplements.
g. Myelin basic protein.
7. a. The Ehrlich reaction.
4. a. Viral, tubercular, or fungal meningitis.
b. Acetylacetone.
b. No, the Gram stain would be negative in viral and
c. Porphobilinogen.
tubercular and not always positive in fungal
d. Blood. meningitis.
e. Free erythrocyte protoporphyrin (FEP). c. Yes. Lymphocytes are very predominant in viral
meningitis.
Chapter 10 d. Yes, a CSF lactate level of 25 mg/dL or less would aid
in confirming viral meningitis. The lactate level
Study Questions would be higher in cases of bacterial, tubercular, and
fungal meningitis.
1. C 9. A 17. D
5. a. Stain precipitate is being confused with gram-positive
2. B 10. C 18. D
cocci.
3. C 11. C 19. D
b. Differentials are being reported from the counting
4. B 12. D 20. B chamber.
5. A 13. D 21. D c. The albumin is contaminated.
6. B 14. A 22. A d. The specimens are not being delivered promptly to
7. B, B, A, A 15. B 23. B the laboratory.
8. C 16. C 24. D
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Answers to Study Questions, Case Studies, and Clinical Situations 377

Chapter 11 9. B 15. B 21. C

10. C 16. B 22. A
Study Questions 11. A 17. A 23. D
1. C 12. C 23. D 12. A 18. D 24. C
2. D 13. A 24. B 13. C 19. B 25. A
3. B 14. A 25. D 14. A 20. C
4. C 15. B 26. B
5. D 16. B 27. D Case Studies and Clinical Situations
6. A 17. A 28. B 1. a. Sterile, heparinized tube, liquid EDTA tube,
7. B 18. C 29. C nonanticoagulated tube.
8. D 19. A 30. A b. MSU crystals are seen in gout.
9. C 20. C 31. B c. Highly birefringent, needle-shaped crystals under
polarized light that turn yellow when aligned with the
10. B 21. A
slow vibration of red compensated polarized light.
11. B 22. A
d. Infection is frequently a complication of severe
inflammation.
Case Studies and Clinical Situations 2. a. WBC diluting fluid containing acetic acid was used.
1. a. Sperm concentration, motility, and morphology. b. Normal, hypotonic, or saponin-containing saline
b. 21,000,000; no. should be used.
c. 1,800,000; no. c. Crystal-induced inflammatory and septic.
d. Yes. The normal sperm concentration is 20 to 60 d. Gram stain and culture, crystal examination.
million/mL. Spermatid counts over 1 million are 3. a. Noninflammatory.
considered abnormal. Both of these abnormal results, b. Hydroxyapatite crystals.
as well as the abnormal motility, are related to defects
in sperm maturation. c. Glucose. A normal result is consistent with
noninflammatory arthritis.
2. a. Male antisperm antibodies may form after
vasovasostomy procedures. 4. a. Fibrinogen.
b. The MAR test and the immunobead test. b. EDTA or heparinized tube.
c. The MAR test detects the presence of IgG male sperm c. No, the bacteria will be trapped in the clot.
antibodies. The immunobead test delineates the areas
of the sperm (head, tail, neck) that are affected by the Chapter 13
antibodies.
d. Clumping, ovum penetration, and motility. Study Questions
3. The specimen contains urine, which is toxic to sperm, 1. C 9. B 18. B
therefore decreasing viability.
2. D 10. C 19. C
4. The specimen was collected improperly, and the first
3. A 11. D 20. B
part of the ejaculation was lost.
4. D 12. D 21. B
5. a. Yes, insufficient prostatic fluid is present.
5. C 13. D 22. B
b. Zinc, citrate, and acid phosphatase.
6. D 14. C 23. C
c. Sperm motility is affected severely.
7. B, A, A, A, B, 15. B 24. B
6. a. Acid phosphatase and seminal glycoprotein p30 tests.
B, B, A 16. A 25. D
b. Microscopic examination for the presence of sperm.
8. B 17. D 26. D

Chapter 12 Case Studies and Clinical Situations

Study Questions 1. a. Pleural fluid.
1. B 4. B 6. A b. Transudate because all the test results are consistent
with those of a transudate.
2. A 5. B, C, B, A, D, 7. B
B, D c. Pleural fluid-to-serum ratios of cholesterol and
3. A 8. B
bilirubin.
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378 Answers to Study Questions, Case Studies, and Clinical Situations

2. a. Pneumonia. 13. A 16. True 19. B

b. Chest tube drainage. 14. True 17. C 20. D
3. a. 1.6. 15. C 18. B
b. Transudate. The SAAG is above 1.1.
c. Hepatic disorder. Case Studies and Clinical Situations
4. a. To differentiate between cirrhosis and peritonitis; 1. a. Yes.
cirrhosis. b. FLM.
b. Pancreatitis or gastrointestinal perforation; alkaline c. The level of phosphatidylglycerol present in the fetal
phosphatase. lungs.
c. Rupture or accidental puncture of the bladder. d. Phosphatidylglycerol is essential for FLM, and levels
d. To detect the presence of gastrointestinal (CEA) and do not always parallel lecithin levels in fetuses of
ovarian (CA 125) cancers. diabetic mothers.
5. The patient has been a victim of blunt trauma, and the 2. a. A neural tube disorder, such as spina bifida or
physician wants to determine whether abdominal anencephaly.
bleeding is occurring; abdominal bleeding. b. An acetylcholinesterase level.
6. Thyroid profile; CA 125. c. The amniotic fluid specimen contains blood.
3. a. False-positive result.
Chapter 14 b. False-positive result.
c. No effect.
Study Questions
d. False-positive result.
1. D 5. A 9. C
4. a. False-positive result.
2. A 6. A 10. C
b. False-positive result.
3. B 7. B
c. False-positive or test interference.
4. D 8. D
d. No effect.
5. Both a and c are correct. The incorrect specimen was
Case Studies and Clinical Situations sent, or the specimen was exposed to light.
1. a. Red and cloudy. 6. a. Premature rupture of the membranes.
b. Bloody fluid with increasing intensities during b. Fern test, pH test, nitrazine test, AmniSure ROM test,
sequential aliquots. Actim PROM test, and ROM+ test.
c. Phagocytosed erythrocytes. c. PAMG-1, IGFBP-1, AFP.
d. Orange-red fluid. d. Actim PROM test.
e. Hemosiderin-laden macrophages.
f. Prussian blue for hemosiderin-laden macrophages; Chapter 16
Sudan III stain for lipid-laden alveolar macrophages.
2. a. Presence of lymphocytes with decreased ratio of CD4 Study Questions
and CD8.
1. C 11. A 21. B
b. Cryptococcus neoformans.
2. A 12. C 22. C
c. Demonstration of a positive cryptococcal antigen in
3. C 13. D 23. B
the BAL specimen. The extent of the cryptococcal
infection correlates with the antigen titer. 4. B 14. B 24. A
5. D 15. B 25. B
Chapter 15 6. D 16. False 26. B
7. D 17. C 27. A
Study Questions 8. C 18. C 28. C
1. B 5. B 9. A 9. D 19. C 29. C
2. C 6. A 10. C 10. C 20. D 30. B
3. A 7. D 11. B
4. C 8. 2, 4, 1, 3 12. True
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Answers to Study Questions, Case Studies, and Clinical Situations 379

Case Studies and Clinical Situations Chapter 17

1. a. Secretory diarrhea.
Study Questions
b. Stool culture.
c. Probable: Salmonella, Shigella, Campylobacter, Yersinia, 1. D 7. B 13. C
Escherichia coli; Improbable: Staphylococcus, Vibrio. 2. C 8. A 14. A
d. Osmotic diarrhea. 3. C 9. C 15. D
2. a. Microscopic examination for fecal fats. 4. A 10. A 16. C
b. Neutral fats stain directly and appear as large, orange- 5. C 11. C
red droplets; soaps and fatty acids appear as smaller 6. A 12. B
orange-red droplets after pretreatment of the
specimen with heat and acetic acid. Case Studies and Clinical Situations
c. Quantitative fecal fat test.
1. a. Vaginal pH, saline and KOH wet preps, Gram stain.
d. Bulky and frothy.
b. KOH will reveal budding yeast.
e. Muscle fiber screening and the gelatin test for trypsin.
c. Culture and DNA direct hybridization probe (Affirm
f. Muscle fiber: failure to include red meat in the diet. VPIII).
g. Chymotrypsin or elastase I. d. Vulvovaginal candidiasis caused by Candida albicans.
3. a. Patient #1: gastric reflux medication containing e. Antifungal agents.
bismuth may produce black stools. Patient #2:
medications, such as aspirin and other NSAIDs, may 2. a. Trichomoniasis caused by Trichomonas vaginalis.
cause gastric bleeding. Patient #3: red meat was not b. Wet mount, vaginal pH, amine test from KOH prep,
avoided for 3 days before specimen collection. DNA probe (Affirm VPIII), OSOM Trichomonas
b. Provide dietary and medication instructions to Rapid Test.
patients. c. Metronidazole.
c. The Hemoccult ICT immunochemical test. d. Yes.
4. a. The APT test cannot be performed because the e. Complications include low birth rate, premature
hemoglobin is already denatured. It must be red rupture of membranes, and preterm delivery during
blood. pregnancy.
b. The pH will be low because increased carbohydrates 3. a. Desquamative inflammatory vaginitis secondary to
are available for bacterial metabolism. atrophic vaginitis.
c. The infant had ingested maternal blood. b. Reduced estrogen production in postmenopausal
d. Yes, adequate carbohydrates are not present, and fats women.
are being metabolized for energy. c. Hormone replacement therapy (estrogen).
7582_Ans_371-380 14/07/20 9:20 AM Page 380
7582_Abbreviations_381-384 13/07/20 5:52 PM Page 381

Abbreviations

A2LA American Association for Laboratory CTAB cetyltrimethylammonium bromide

Accreditation Cu2O cuprous oxide
AABB American Association of Blood Banks CuSO4 copper sulfate
A:C albumin:creatinine [ratio] CV coefficient of variation
ACE angiotensin-converting enzyme DBDH diisopropyl benzene dihydroperoxide
AChE acetylcholinesterase DCT distal convoluted tubule
ADA adenosine deaminase DI diabetes insipidus
ADH antidiuretic hormone DIDNTB (3⬘,3⬙, diodo 4⬘,4⬙-dihydroxy-5⬘,5⬙-
AER albumin excretion rate dinitrophenyl)-3,4,5,6-tetra-bromo-
AFP alpha-fetoprotein sulphonphtalein
AGN acute glomerulonephritis DIV desquamative inflammatory vaginitis
AHG antihuman globulin DNPH 2,4-dinitrophenylhydrazine
AIDS acquired immunodeficiency syndrome DOT Department of Transportation
AIN acute interstitial nephritis EDS early dumping syndrome
ALA α-aminolevulinic acid EDTA ethylenediaminetetraacetic acid
ANA antinuclear antibody eGFR estimated glomerular filtration rate
ANCA antineutrophilic cytoplasmic antibody ELISA enzyme-linked immunosorbent assay
AOA American Osteopathic Association EPS expressed prostatic specimen
APR auto particle recognition EQA external quality assessment
ARF acute renal failure ESRD end-stage renal disease
ART assisted reproductive technology EU Ehrlich unit
ASHI American Society of Histocompatibility and FAH fumarylacetoacetate hydrolase
Immunogenetics FAST focused assessment with sonography for
ASO antistreptolysin O trauma
ATN acute tubular necrosis FDA Food and Drug Administration
BAL bronchoalveolar lavage FEP free erythrocyte protoporphyrin
BAT bacterial antigen test fFN fetal fibronectin
B2M beta2 microglobulin FISH fluorescence in situ hybridization
BCAAs branched-chain amino acids FLM fetal lung maturity
BSI body substance isolation FOBT fecal occult blood testing
BUN blood urea nitrogen FSGS focal segmental glomerulosclerosis
BV bacterial vaginosis FTA-ABS fluorescent treponemal antibody-absorption
CAP College of American Pathologists GAGs glycosaminoglycans
CASA computer-assisted semen analysis GALE galactose-4-epimerase
CDC Centers for Disease Control and Prevention GALK galactokinase
CEA carcinoembryonic antigen GALT galactose-1-phosphate uridyl transferase
CGN chronic glomerulonephritis GC-MS gas chromatography–mass spectrometry
CHP chemical hygiene plan gFOBT guaiac-based fecal occult blood test
CKD chronic kidney disease GFR glomerular filtration rate
CLIA Clinical Laboratory Improvement GHS Globally Harmonized System
Amendments GI gastrointestinal
CLRW clinical laboratory reagent water GPA granulomatosis with polyangiitis
CLSI Clinical and Laboratory Standards Institute 1H NMR hydrogen nuclear magnetic resonance
CNS central nervous system spectroscopy
COC chain of custody H+ titratable acid/hydrogen ion
COLA Commission on Laboratory Assessment H2PO4 hydrogen phosphate ion
CPPD calcium pyrophosphate dihydrate Hb hemoglobin
CPR cardiopulmonary resuscitation HbA hemoglobin A
CR ch core channel HbF hemoglobin F
CSF cerebrospinal fluid HBV hepatitis B virus
CT computed tomography hCG human chorionic gonadotropin

381
7582_Abbreviations_381-384 13/07/20 5:52 PM Page 382

382 Abbreviations

HCV hepatitis C virus NKDEP National Kidney Disease Education Program

HCO3 bicarbonate ion NRBC nucleated red blood cell
HDFN hemolytic disease of the fetus and newborn NS nephrotic syndrome
HF-BF high-fluorescing body fluid NTD neural tube defect
HICPAC Healthcare Infection Control Practices OD optical density
Advisory Committee OGTT oral glucose tolerance test
5-HIAA 5-hydroxyindoleacetic acid OSHA Occupational Safety and Health
HIV human immunodeficiency virus Administration
HLA-B12 human leukocyte antigen-B12 PAA plasma amino acids
hpf high-power field PAH p-aminohippuric acid
HRCT high-resolution computed tomography PAMG-1 placental alpha microglobulin
HSV herpes simplex virus PB peripheral blood
IBS irritable bowel syndrome PCR polymerase chain reaction
IDMS isotope dilution mass spectrophotometry PCT proximal convoluted tubule
IEF isoelectric focusing PEP postexposure prophylaxis
IEM inborn error of metabolism PG phosphatidyl glycerol
IFE immunofixation electrophoresis pKa dissociation constant
iFOBT immunochemical fecal occult blood test PKU phenylketonuria
IgA immunoglobulin A PM preventive maintenance
IgG immunoglobulin G PMNs polymorphonuclear white blood cells
IgM immunoglobulin M POC point of care
IGFBP-1 insulin-like growth factor binding protein-1 PP12 placental protein 12
IQCP individualized quality control plan PPE personal protective equipment
ISO International Organization for Standardization PPMT pre- and postmassage test
IUI intrauterine insemination PROM premature rupture of membranes
IVF in vitro fertilization PSA prostate-specific antigen
KDIGO Kidney Disease: Improving Global Outcomes PSP phenolsulfonphthalein
KOH potassium hydroxide PT proficiency testing
LBC lamellar body count QA quality assessment
LC-MS/MS liquid chromatography–tandem mass QC quality control
spectrometry QM quality management
LD lactic dehydrogenase QMS quality management system
LE leukocyte esterase RA rheumatoid arthritis
LED light-emitting diode RAAS renin–angiotensin–aldosterone system
LFA lateral flow assay RBC red blood cell
LIS laboratory information system RCA root cause analysis
lpf low-power field RCF relative centrifugal force
L/S lecithin-sphingomyelin ratio RDS respiratory distress syndrome
MAR mixed agglutination reaction RF rheumatoid factor
MBP myelin basic protein RGE rapid gastric emptying
MCD minimal change disease RNA ribonucleic acid
MDRD modification of diet in renal disease RPGN rapidly progressive (or crescentic)
MESNA mercaptoethane sulfonate sodium glomerulonephritis
MGN membranous glomerulonephritis RPM revolutions per minute
MoM multiples of the median RPR rapid plasma reagin
mOsm milliosmole RTE renal tubular epithelial (cells)
MPGN membranoproliferative glomerulonephritis SAAG serum-ascites albumin gradient
MPSs mucopolysaccharidoses SDS Safety Data Sheet
MSAFP maternal serum alpha-fetoprotein SD standard deviation
MS/MS tandem mass spectrophotometry SF ch surface channel
MSU monosodium urate (uric acid) SG specific gravity
MSUD maple syrup urine disease SKY fluorescent mapping spectral karyotyping
NaCl sodium chloride SLE systemic lupus erythematosus
NaOH sodium hydroxide SP Standard Precautions
NFPA National Fire Protection Association SPS sodium polyanethol sulfonate
NH4+ ammonium ion SSA sulfosalicylic acid
NIRS near-infrared spectroscopy STI sexually transmitted infection
7582_Abbreviations_381-384 13/07/20 5:52 PM Page 383

Abbreviations 383

TAT turnaround time UP Universal Precautions

TC-BF total nucleated body fluid UTI urinary tract infection
THP Tamm-Horsfall protein VB1 voided bladder 1
TJC The Joint Commission VB2 voided bladder 2
TLC thin-layer chromatography VB3 voided bladder 3
Tm maximal reabsorptive capacity/tubular VDRL Venereal Disease Research Laboratory
reabsorptive maximum VUR vesicoureteral reflux
TMB 3,3⬘,5,5⬘-tetramethylbenzidine WBC white blood cell
TMG maximal tubular reabsorption capacity for WBC-BF white blood cell body fluid
glucose WHO World Health Organization
7582_Abbreviations_381-384 13/07/20 5:52 PM Page 384
7582_Glossary_385-392 13/07/20 5:44 PM Page 385

Glossary

accreditation The process by which a program or institution antiglomerular basement membrane antibody Autoanti-
documents meeting established guidelines body against alveolar and glomerular capillary basement
accuracy Closeness of the measured result to the true value membranes found in patients with Goodpasture syndrome
acholic stools Pale-colored stools antineutrophilic cytoplasmic antibody (ANCA) Autoanti-
acrosomal cap Tip of a spermatozoa head, which contains body that reacts with neutrophils in the renal tubules; pro-
enzymes for entry into an ovum vides a laboratory test for granulomatosis with polyangiitis
active transport Movement of a substance across cell mem- anuria Complete stoppage of urine flow
branes into the bloodstream by electrochemical energy arachnoid granulations Projections on the arachnoid mem-
acute glomerulonephritis (AGN) Disease marked by the brane of the brain through which cerebrospinal fluid is
sudden onset of symptoms consistent with damage to the reabsorbed
glomerular membrane arthritis Inflammation of the synovial joints
acute interstitial nephritis (AIN) Inflammation of the renal arthrocentesis The puncture of a joint to obtain synovial
interstitium and tubules, frequently caused by a reaction fluid
to a medication ascites Abnormal accumulation of peritoneal fluid
acute-phase reactants Low-molecular-weight plasma pro- ascitic fluid Watery fluid that accumulates in the peritoneal
teins associated with infection and inflammation cavity in certain disease conditions
acute tubular necrosis (ATN) Disorder affecting the renal ascorbic acid A strong reducing agent that prevents oxida-
tubular cells caused by decreased renal blood flow or toxic tion of the chromogen and may produce false-negative
substances results on some tests
aerosol Fine suspension of particles in air astrocytomas Tumors of the brain and spinal cord
afferent arteriole A small branch of the renal artery through atrophic vaginitis Syndrome in postmenopausal women
which blood flows to the glomerulus of the kidney caused by reduced estrogen production
albinism An inherited condition marked by decreased produc- auto-checks Automatic checks performed by semiautomated
tion of melanin analyzers to identify strip type and humidity exposure
albuminuria Protein (albumin) in the urine auto particle recognition (APR) Capability of some equip-
aldosterone A hormone that regulates reabsorption of ment to categorize and count urine particles automatically
sodium in the distal convoluted tubule in uncentrifuged urine based on size, shape, texture, and
alimentary tract The digestive tract, including structures contrast
between the mouth and the anus autovalidated Automatic release of test results into the
alkaptonuria Homogentisic acid in the urine caused by a patient record
failure to inherit the gene responsible for the production autoverification An automated comparison of patient results
of homogentisic acid oxidase within laboratory analyzers with predetermined, preap-
aminoaciduria Disorder in which increased amino acids are proved criteria programmed into them
present in the urine azotemia Increased nitrogenous waste products in the blood
amniocentesis Transabdominal puncture of the uterus and bacterial endocarditis Inflammation of the endocardial
amnion to obtain amniotic fluid membrane of the heart caused by bacterial infection
amnion The membranous sac that contains the fetus and bacterial vaginosis (BV) Inflammation of the vagina most
amniotic fluid often caused by Gardnerella vaginalis
amniotic fluid Liquid that surrounds the fetus during bacteriuria Bacteria in the urine
gestation basal cells Cells located in the basal layer of the vaginal
amyloid material A starch-like protein–carbohydrate com- stratified epithelium
plex that is deposited abnormally in tissue in some patients beta2 microglobulin A subunit of the class I major compat-
in chronic disease states ibility antigens that enters the blood at a constant rate
anaphylaxis Severe reaction caused by an autoimmune bilirubin A bright yellow pigment produced in the degrada-
reaction to certain antigenic compounds tion of heme
andrology The study of diseases of the male reproductive biliverdin Green pigment formed by the oxidation of
organs bilirubin
antidiuretic hormone (ADH) Hormone produced by the biohazardous Pertaining to a hazard caused by infectious
hypothalamus to regulate water reabsorption in the organisms
collecting duct birefringent The ability to refract light in two directions

385
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blood–brain barrier The barrier between the brain tissue coefficient of variation (CV) Standard deviation expressed
and capillary blood that controls the passage of substances as a percentage of the mean
in the blood to the brain and cerebrospinal fluid collecting duct Part of the nephron where the final concen-
body substance isolation A guideline stating that all moist tration of urine takes place through the reabsorption of
body substances are capable of transmitting disease water
Bowman capsule Part of the nephron that contains the colligative property Freezing point, boiling point, vapor
glomerulus pressure, or osmotic pressure property that is mathemat-
bright-field microscopy A procedure by which magnified ically proportional to the concentration of a solution
images appear dark against a bright background concentration tests Tests to determine the ability of the
bronchoalveolar lavage (BAL) Diagnostic procedure by tubules to reabsorb the essential salts and water that have
which cells and other components from bronchial and been nonselectively filtered by the glomerulus
alveolar spaces are obtained for various studies constipation Infrequent production of feces that results in
bronchoscopy Procedure during which a fiber-optic bron- small, hard stools
choscope is guided into a selected bronchopulmonary control mean Average of all data points
segment, usually the right middle or lingular lobe of the control range Limit within which expected control values
lung; however, target areas are better defined using high- lie, usually plus or minus two standard deviations from
resolution computed tomography (HRCT) before the the mean
procedure countercurrent mechanism A selective urine concentration
bulbourethral glands Two small glands located on each side process in the ascending and descending loops of Henle
of the prostate gland creatinine A substance formed by the breakdown of creatine
carcinogenic Capable of causing cancer during muscle metabolism
casts Elements excreted in the urine in the shape of renal creatinine clearance A test used to measure the glomerular
tubules filtration rate
catheterized specimen A urine specimen collected by pass- crenated Shrunken and irregularly shaped or notched
ing a sterile tube into the bladder cylindroids Formation of casts at the junction of the ascend-
chain of custody (COC) Step-by-step documentation of the ing loop of Henle and the distal convoluted tubule may
handling and testing of legal specimens produce structures with a tapered end
chain of infection A continuous link in the transmission of cylindruria The presence of urinary casts
harmful microorganisms between a source and a suscep- cystatin C Small protein produced at a constant rate by all
tible host nucleated cells
chemical hygiene plan (CHP) Protocol established for the cystinosis An inherited recessive disorder that disrupts the
identification, handling, storage, and disposal of all haz- metabolism of cystine
ardous chemicals cystinuria Cystine in the urine that occurs as a result of a
choroid plexuses A network of capillaries in the ventricle of defect in the renal tubular reabsorption of amino acids
the brain that produces cerebrospinal fluid cystitis An inflammation of the bladder
chronic glomerulonephritis (CGN) Kidney disorder cytogenetic analysis An analysis of cellular chromosomes
caused by slow, cumulative damage and scarring of the dark-field microscopy Microscopic technique by which
glomeruli; may lead to kidney failure and/or end-stage magnified images appear bright against a dark background
renal disease delta check A quality management (QM) procedure that
chylous effusion Occurs when chylous material accumu- compares a patient’s test results with the previous results
lates in the pleural space, usually secondary to disruption demyelination The destruction of the myelin sheath that
of thoracic lymphatics protects a nerve
chylous material A milky lymphatic fluid that contains density Concentration of solutes present per volume of
triglycerides and chylomicrons solution
cirrhosis Chronic liver disease that results in loss of liver cell desquamative inflammatory vaginitis (DIV) Syndrome
function characterized by purulent vaginal discharge, vaginal
clarity Transparency of urine, ranging from clear to turbid erythema, and dyspareunia
clearance tests Standard tests used to measure the filtering diarrhea Watery stools
capacity of the glomeruli diarthroses Freely movable joints
Clinical Laboratory Improvement Amendments (CLIA) Diazo reaction A reaction in various diseases consisting of a
Governmental regulatory agency that establishes quality red discoloration of the urine on addition of diazoben-
standards for laboratory testing zenesulfonic acid
Clinical and Laboratory Standards Institute (CLSI) Non- digital imaging Capability of some equipment to capture
profit organization that publishes recommendations for detailed images of urine particles in urine samples that
laboratory tests require further review
clue cells Squamous epithelial cells covered with the gram- disinfectant A substance that destroys microorganisms that
negative bacteria Gardnerella vaginalis is used on surfaces rather than the skin
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Glossary 387

distal convoluted tubule Part of the nephron between the stained by a fluorescent dye produce an image when illu-
ascending loop of Henle and the collecting duct where the minated with a light of a specific wavelength
final concentration of urinary filtrate begins focal segmental glomerulosclerosis (FSGS) Disorder that
dysentery An inflammation of the intestines that is caused affects only certain areas of the glomerulus and produces
by microorganisms and results in diarrhea heavy proteinuria
dysmorphic Irregularly shaped fomite Inanimate object that can serve as a reservoir for path-
dyspareunia Abnormal pain during sexual intercourse ogenic organisms
dyspnea Difficulty breathing free water clearance A test to determine the ability of the
dysuria Painful urination kidney to respond to the state of body hydration
edema An accumulation of fluid in the tissues fructosuria The presence of fructose in the urine
efferent arteriole The small renal artery branch through galactosuria The presence of galactose in the urine
which blood flows away from the glomerulus Gardnerella vaginalis Rod-shaped bacteria that causes bac-
effusion An accumulation of fluid between the serous terial vaginitis
membranes gastrocolic fistula Abnormal passageway between the stom-
Ehrlich reaction Urobilinogen reacts with p-dimethy- ach and the colon
laminobenzaldehyde (Ehrlich reagent) to produce colors gestational diabetes Diabetes that some women get during
ranging from light to dark pink pregnancy in which the body has too much glucose in the
electronic quality control Mechanical or electrical sample blood; glucose crosses the placenta but insulin does not,
used in place of a liquid control to verify reliability of test so the baby develops high glucose levels
results ghost cells Red blood cells that have lost their hemoglobin,
endogenous procedure A test that uses a substance origi- leaving only the cell membrane; appear in hyposthenuric
nating within the body urine
epididymis Small structure that forms the first part of the Globally Harmonized System (GHS) An international effort
secretory duct of the testes to standardize both the classification of hazardous chemi-
erythrophagocytosis Engulfment of red blood cells by cals and the symbols used to communicate these hazards
macrophages on labels and in Safety Data Sheet (SDS) documentation
examination variable Processes that occur during testing of glomerular filtration barrier Structure of the walls of the
a sample glomerular capillaries that prevents the filtration of large
exogenous procedure A test that requires a substance to be molecules from the blood into the urine filtrate
infused into the body glomerular filtration rate (GFR) The volume of plasma that
external quality assessment (EQA) Testing of unknown is filtered by the glomerulus in a specified time
samples received from an outside accrediting agency to glomerulonephritis An inflammation of the glomerulus that
provide validation of the quality of patient test results results in impaired glomerular filtration
external quality control Commercial controls used to verify glomerulus Tuft of capillary blood vessels located in Bow-
accuracy and reliability of patient test results man capsule where filtration occurs
exudate Serous fluid effusion caused by conditions produc- glucosuria The amount of glucose in the glomerular filtrate
ing damage to the serous membranes exceeds the capacity of the renal tubule to reabsorb it
Fanconi syndrome A group of disorders marked by renal glycogenesis The conversion of glucose to glycogen
tubular dysfunction associated with some inherited and glycogenolysis The conversion of glycogen to glucose
acquired conditions glycosaminoglycans A group of large compounds located
fenestrated endothelium The endothelial cells of the capil- primarily in the connective tissue; also known as
lary wall that differ from those in other capillaries by con- mucopolysaccharides
taining pores glycosuria Glucose in the urine (glucosuria)
ferritin A major storage form of iron found in the liver, Goodpasture syndrome An autoimmune disorder charac-
spleen, and bone marrow terized by morphological changes to the glomeruli
fetal lung maturity (FLM) The presence of a sufficient resembling those in rapidly progressive glomerular
amount of surfactant lipoproteins to maintain alveolar nephritis
stability gout Disorder related to elevated serum uric acid that results
first morning specimen The first voided urine specimen in the accumulation of uric acid crystals in a moveable
collected immediately upon arising; recommended screen- joint
ing specimen granuloma Modular accumulation of inflammatory cells
flatus Gas expelled from the anus granulomatosis with polyangiitis (GPA) Disorder that causes
flow cytometry Used to measure the forward scattered, side a granuloma-producing inflammation of the small blood
scattered, and fluorescence light characteristics of particles vessels primarily of the kidney and respiratory system;
present in urine formerly called Wegener granulomatosis
fluorescence microscopy Microscopic technique by which Greiss reaction Nitrite at an acidic pH reacts with an aro-
naturally fluorescent substances or those that have been matic amine (para-arsanilic acid or sulfanilamide) to form
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388 Glossary

a diazonium compound that then reacts with tetrahy- interstitial Pertaining to spaces between tissue cells
drobenzoquinolin compounds to produce a pink-colored ischemia Deficiency of blood to a body area
azo dye isosthenuric Pertaining to urine specific gravity the same as
Guillain-Barré syndrome Autoimmune disorder that causes the 1.010 of the glomerular filtrate
destruction of the myelin sheath that surrounds the jaundice Yellow appearance of skin, mucous membranes,
peripheral nerves, resulting in loss of motor function and eye sclera due to increased amounts of bilirubin in
Hartnup disease A recessive inherited disorder marked by in- the blood
testinal absorption abnormalities and renal aminoaciduria juxtaglomerular apparatus Specialized cells located on the
hematoidin Yellow, crystalline substance that results from afferent arteriole that regulate secretion of renin
the destruction of red blood cells ketonuria Ketones in the urine
hematuria Blood in the urine Köhler illumination Adjustments made to the microscope
hemoglobinuria Hemoglobin in the urine condenser when objectives are changed
hemolytic disease of the fetus and newborn (HDFN) Rh labia The outer folds of the vagina
incompatibility between mother and fetus that can cause lactobacilli A group of gram-positive rod-shaped bacteria
hemolysis of the fetal red blood cells lactosuria The presence of lactose in the urine
hemoptysis Blood in the sputum lamellar bodies Organelles produced by type II pneumono-
hemosiderin An insoluble form of storage iron; a product of cytes in the fetal lung that contain lung surfactants
red blood cell hemolysis Langerhans cells Pancreatic cells
hemothorax The accumulation of blood in the pleural cavity lecithin Phospholipid that forms part of the cell wall used
Henoch-Schönlein purpura Disease involving inflammation to determine fetal lung maturity
of small blood vessels, causing them to start leaking; lecithin-sphingomyelin ratio (L/S ratio) A comparison of
occurs primarily in children after upper respiratory lung surfactants that is performed to determine fetal lung
infections maturity
histogram A graphical display of data using bars of different Lesch-Nyhan disease An inherited sex-linked recessive
heights purine metabolism disorder marked by excess uric acid
homocystinuria The presence of homocysteine in the urine crystals in the urine
caused by an inherited autosomal recessive disorder leukocyturia Leukocytes (white blood cells) in the urine
hyaluronic acid Glycosaminoglycan found in synovial fluid light-emitting diode (LED) A semiconductor diode that
that provides lubrication to the joints emits light when conducting current
hydrostatic pressure Pressure exerted by a liquid lipiduria The presence of lipids in the urine
hyperglycemia Elevated glucose levels in the blood lipophages Macrophages that have ingested fat globules seen
hypernatremia Elevated blood sodium levels in peritoneal fluid
hypersthenuric Pertaining to urine specific gravity greater liquefaction The conversion of solid or coagulated material
than the 1.010 of the glomerular filtrate to a liquid form
hyponatremia Decreased blood sodium levels lithiasis The formation of renal calculi (kidney stones)
hyposthenuric Pertaining to urine specific gravity lower lithotripsy A procedure that uses ultrasonic waves to crush
than the 1.010 of the glomerular filtrate renal calculi
hypoxia Lack of oxygen loops of Henle The U-shaped part of the renal tubule that con-
iatrogenic Pertaining to a condition caused by treatment, sists of a thin descending limb and a thick ascending limb
medications, or diagnostic procedures lysosomes Cellular organelles that contain digestive enzymes
IgA nephropathy Damage to the glomerular membrane macula densa Specialized cells located on the distal convo-
caused the deposition of IgA immune complexes on the luted tubule that interact with the juxtaglomerular cells
membrane malabsorption Impaired absorption of nutrients by the
immune complexes Antigen–antibody combinations intestine
in vitro fertilization (IVF) Fertilization between an ovum maldigestion Impaired digestion of foodstuffs
and a sperm performed in the laboratory Maltese cross formation Cross-shaped inclusions in the uri-
inborn error of metabolism (IEM) Failure to inherit the nary sediment of patients with nephrotic syndrome seen
gene to produce a particular enzyme under polarized light due to the birefringence of lipid
indicanuria The presence of indican in the urine droplets
infection control Procedures to control and monitor infec- maple syrup urine disease (MSUD) An autosomal recessive
tions within an institution trait that causes increased levels of the branched-chain
infertility The inability to conceive amino acids leucine, isoleucine, and valine and their
interference-contrast microscopy A procedure by which ketone acids in the urine
three-dimensional images of a specimen are obtained maximal reabsorptive capacity (Tm) The maximum reab-
internal quality control Electronic, internal, and procedural sorption ability for a solute by renal tubules
controls contained within the test system that ensure its meconium The dark-green mucus-containing stool formed
reliability by a fetus
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Glossary 389

medullary interstitium Spaces between the cells in the occult blood Blood that is not visible to the naked eye
medulla of the kidney that contain highly concentrated Occupational Safety and Health Administration (OSHA)
fluid The government agency created to protect employees from
medulloblastomas Malignant tumor of the fourth ventricle potential health hazards in the workplace through the
and cerebellum development and monitoring of regulations
melanoma A tumor of the melanogen-producing cells, ochronosis Deposit of brown pigment in the body tissues,
which frequently is malignant particularly noticeable in the ears
melanuria Increased melanin in the urine oligoclonal bands Electrophoretic bands migrating in the
melituria Increased urinary sugar gamma region that are present in cerebrospinal fluid and
membranous glomerulonephritis (MGN) Type of glomeru- serum
lonephritis that develops when inflammation of kidney oligohydramnios Decreased amniotic fluid
structures causes problems with kidney function oliguria A marked decrease in urine flow
membranoproliferative glomerulonephritis (MPGN) Spe- oncotic pressure The osmotic pressure of a substance in
cific type of glomerular disease that is characterized by solution caused by the presence of colloids
immune complex deposits in the kidneys glomerular organic acidemias The accumulation of organic acids in the
mesangium and a thickening of the basement membrane; blood, mainly isovaleric, propionic, and methylmalonic
occurs when the body’s immune system functions acids
abnormally orthostatic proteinuria Increased protein in urine only
meninges Protective membranes around the brain and spinal when an individual is in an upright position
cord osmolality The osmotic pressure of a solution expressed in
meningitis Inflammation of the meninges, frequently caused milliosmoles per liter; it is affected only by the number of
by microbial infection particles present
mesothelial cells Cells that line the serous membranes osmolar clearance The amount of plasma filtered each
metabolic acidosis A decrease in the blood pH caused by a minute to produce a urine with the same osmolarity as
metabolic increase in acidic elements plasma
microalbuminuria Low levels of urine protein that are not osmolarity The osmotic pressure of a solution expressed in
detected by routine reagent strips milliosmoles per kilogram; it is affected only by the
midstream clean-catch specimen Specimen collected in a number of particles present
sterile container after cleansing the glans penis or urinary osmotic diarrhea An increased retention of water and
meatus; the first portion of urine is voided into the toilet, solutes in the large intestine associated with malabsorption
the midportion is collected, and the remaining portion is and maldigestion
voided into the toilet osmotic gradient The difference in the concentration of
minimal change disease (MCD) Disease that produces little substances on either side of a membrane
cellular change in the glomerulus, except for some damage oval fat bodies Lipid-containing renal tubular epithelial
to the podocytes and the shield of negativity, allowing for (RTE) cells
increased protein filtration; also known as lipid nephrosis pancreatic insufficiency The decreased ability of the
or nil disease pancreas to secrete digestive enzymes
Mobiluncus spp. Curved, gram-variable, rod-shaped bacteria parabasal cell Cells located in the luminal squamous epithe-
mucin Glycoprotein found in mucus and in the skin, connec- lium of the vaginal mucosa
tive tissues, tendons, and cartilage paracentesis Surgical puncture into the abdominal cavity to
mucopolysaccharides (MPS) Glycosaminoglycans that consist obtain peritoneal fluid
of a protein core with polysaccharide branches parietal membrane Serous membrane that lines the walls of
mucopolysaccharidoses (MPSs) A group of genetic disor- the pleural, pericardial, and peritoneal cavities
ders marked by excess mucopolysaccharides in blood and passive transport Movement of molecules across a mem-
urine brane by diffusion because of a physical gradient
multiple myeloma Malignant disorder that results in infil- pentosuria The presence of pentose sugars in the urine
tration of the bone marrow by plasma cells pericardiocentesis Surgical puncture into the pericardial
myoglobin Iron-containing protein found in muscle tissue cavity to obtain pericardial fluid
myoglobinuria Myoglobin in the urine pericarditis An inflammation of the membranes enclosing
nephron A functional unit of the kidney that forms urine the heart
nephropathy Disease of the kidneys peritoneal lavage Introduction and subsequent removal of
nephrotic syndrome (NS) A renal disorder marked by mas- fluid into the peritoneal cavity to detect the presence of
sive proteinuria, lipiduria, and edema caused by disruption abnormal substances
of the glomerular membrane peritonitis An inflammation of the membranes that line the
neutrophages Vacuolated macrophages containing phago- peritoneal cavity
cytized neutrophils; formerly called Reiter cells peritubular capillaries The capillaries that surround the
nocturia Excessive urination during the night renal tubules
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390 Glossary

personal protective equipment (PPE) Items used to protect pseudochylous effusion Cloudy to opalescent effusions that
the body from infectious agents are not of primary lymphatic origin but arise from the
phase-contrast microscopy Procedure in which magnified breakdown products of any chronic effusion
images show varied intensities of light and dark and are pseudochylous material Milky effusion that does not
surrounded by halos contain chylomicrons
phenazopyridine Medication for urinary tract infection that pulmonary infarction Blockage of the pulmonary artery
produces a thick, orange urine specimen resulting in destruction of lung tissue
phenylketonuria (PKU) The presence of abnormal pheny- pseudogout Arthritic disorder caused by the accumulation
lalanine metabolites in the urine of calcium pyrophosphate crystals in a moveable joint
phosphatidyl glycerol Phospholipid found in amniotic fluid purpura Small capillary hemorrhages
that is used to confirm fetal lung maturity pyelonephritis Infection of the renal tubules
pigmented villonodular synovitis Proliferation of synovial pyknotic Referring to a dense, round nucleus
cells forming brown nodules, resulting in inflammation, pyuria The presence of white blood cells (pus) in the
pain, and hemorrhagic effusions urine
pleocytosis Increased numbers of normal cells in the quality assessment (QA) Methods used to measure quality
cerebrospinal fluid patient care
podocytes Epithelial cells of the inner lining of Bowman quality control (QC) Methods used to monitor the accuracy
capsule that contain foot-like processes of procedures
polarizing microscopy A procedure in which magnified quality management (QM) The overall process of guaran-
birefringent images appear bright or colored against a teeing quality patient care, regulated throughout the total
black background testing system
polydipsia Excessive thirst quality management system (QMS) All of the laboratory’s
polyhydramnios Excessive amniotic fluid policies, processes, procedures, and resources needed to
polyuria Marked increase in urine flow achieve quality testing
porphobilinogen Immediate precursor of the porphyrins quality system The overall laboratory policies, procedures,
involved in the synthesis of heme processes, and resources to achieve quality test results
porphyrias Disorders of porphyrin metabolism that are radioisotope A substance that emits radiant energy
inherited or acquired ragocytes Neutrophils that contain ingested clumps of IgG
porphyrins Intermediate compounds in the synthesis of random specimen Urine collected at any time without prior
heme patient preparation
porphyrinuria The presence of porphyrins in the urine rapidly progressive (or crescentic) glomerulonephritis
postexamination variable Process that affects the reporting (RPGN) A more serious form of acute glomerular
and interpretation of test results disease that often terminates in renal failure
postexposure prophylaxis (PEP) Preventative treatment reflectance photometry A lamp and monochromator pro-
provided after exposure to a potentially harmful agent vide an incident light beam that is directed to a colored
postrenal proteinuria Increased protein in the urine caused reaction product
by infections/inflammation that add protein to the urine refractive index A comparison of the velocity of light in the
after its formation air with the velocity of light in a solution
precision Reproducibility of a test result refractometry Measurement of the light-bending capability
preexamination variable Process that occurs before collec- of solutions
tion of a sample Reiter cells Vacuolated macrophages containing ingested
prerenal proteinuria Increased protein in the urine caused neutrophils associated with nonspecific arthritic inflam-
by factors affecting the plasma before it reaches the mation; see also neutrophages
kidney reliability The ability to maintain both precision and
preventive maintenance (PM) Checks on instruments and accuracy
equipment on a regular schedule renal plasma flow The volume of plasma passing through
proficiency testing (PT) Performance of tests on specimens the kidneys per minute
provided by an external monitoring agency renal proteinuria Protein in the urine caused by impaired
prostate gland Muscular gland surrounding the male renal function
urethra renal threshold Plasma concentration of a substance at
protein error of indicators Indicators change color in the which active transport stops and increased amounts are
presence of protein at a constant pH excreted in the urine
proteinuria Protein in the urine (albuminuria) renal tubular acidosis The inability to produce an acidic
proximal convoluted tubule The nearest tubule to the urine in the presence of metabolic acidosis
glomerulus where reabsorption of essential substances renin Proteolytic enzyme produced by the kidney that reacts
begins with angiotensinogen to produce angiotensin to increase
pruritus Symptom of itching blood pressure
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Glossary 391

renin–angiotensin–aldosterone system (RAAS) Regulates Standard Precautions (SP) Guidelines recommended by the
flow of blood to and within the kidneys by responding to Centers for Disease Control (CDC) describing personnel
changes in blood pressure and plasma sodium content protective practices
resolution The ability to separate fine structures for visuali- steatorrhea Excess fat in the feces
zation of detail stercobilinogen Substance derived from urobilinogen that
respiratory distress syndrome (RDS) Disease caused by is found in the feces and is oxidized to form urobilin,
lack of lung surfactant forming the brown color of feces
retinoblastomas Malignant glioma of the retina seen in stools Fecal material discharged from the large intestine
young children subarachnoid space The area between the arachnoid and
rhabdomyolysis Muscle destruction pia mater membranes
rheumatoid factor Immunoglobulin associated with suprapubic aspiration The technique used to obtain sterile
rheumatoid arthritis urine specimens for bacterial culture or cytological exam-
root cause analysis (RCA) A systematic process for identi- ination in which a sterile needle is introduced through the
fying “root causes” of problems or events and an approach abdomen into the bladder
for responding to them surfactants Phospholipids secreted by type II pneumocytes
sarcoidosis Multisystem disease caused by infiltration of the to maintain alveolar integrity
organs by T lymphocytes and phagocytes that form syncytia A group of cells with continuous adjoining
granulomas in the tissues cell walls
Safety Data Sheet (SDS) A document provided by the ven- synovial fluid Plasma ultrafiltrate that contains hyaluronic
dor or manufacturer of a chemical substance that describes acid and provides lubrication of the joints
the chemical’s characteristics synoviocytes Cells in the synovial membrane that secrete
scattergram A graphic representation of points referencing hyaluronic acid
two variables systemic lupus erythematosus (SLE) Autoimmune disor-
secretory diarrhea The increased secretion of water and der that affects the connective tissue and results in damage
electrolytes into the large intestine caused by bacterial to organs, particularly the kidneys and joints
enterotoxins Tamm-Horsfall protein (THP) Mucoprotein found in the
semen Fluid containing spermatozoa matrix of renal tubular casts (see uromodulin)
seminal vesicles Two sac-like structures close to the prostate tamponade Buildup of pericardial fluid affecting the
that produce the majority of the seminal fluid heart
seminiferous tubules Tubules that produce or conduct test utilization Correct procedures for ordering a test
semen thoracentesis Surgical puncture into the thoracic cavity to
sensitivity The lowest level of an analyte that a test can collect pleural fluid
detect thrombosis Formation of a blood clot
serous fluid Fluid formed as a plasma ultrafiltrate that pro- timed specimen Urine specimen collected over an interval
vides lubrication between the parietal and visceral serous of time for a quantitative analysis of a urine chemical,
membranes usually a 24-hour collection
serum-ascites albumin gradient (SAAG) Calculation used titratable acidity Hydrogen ions in the urine that can be
to distinguish if a peritoneal effusion is a transudate or an quantitated by titration with a base to a pH of 7.4
exudate transudate Serous effusion produced as a result of disruption
shield of negativity Negative ions in the glomerular filtra- of fluid production and regulation between the serous
tion barrier that prevent small proteins, such as albumin, membranes
from entering the urine filtrate traumatic tap Surgical puncture contaminated with capillary
shift Abrupt change in the mean of a series of results blood
Sjögren syndrome An autoimmune disorder in which au- trend Gradual change in one direction of the mean of a
toimmune white blood cells (WBCs) attack the moisture- control substance
producing glands Trichomonas vaginalis Flagellated protozoan that infects the
specific gravity The density of a solution compared with vagina
that of a similar volume of distilled water, influenced by trichomoniasis Vaginal infection caused by Trichomonas
both the number and size of the particles present vaginalis
specificity The likelihood of measuring the analyte desired tubular reabsorption Substances moved from the tubular
spermatids Immature spermatozoa filtrate into the blood by active or passive transport
spermatozoa Sperm cells tubular secretion The passage of substances from the blood
sphingomyelin Phospholipid found in amniotic fluid used in the peritubular capillaries to the tubular filtrate
to determine fetal lung maturity tubulointerstitial disease Renal disease that affects both the
standard deviation (SD) Measurement statistic that renal tubules and renal interstitium
indicates the average distance each data point is from turnaround time (TAT) Time from ordering a test through
the mean analysis in the laboratory to the charting of the report
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392 Glossary

tyrosyluria The presence of tyrosine in the urine vaginitis Inflammation of the vagina
Universal Precautions (UP) Centers for Disease Control vasa recta A network of capillaries that surrounds the loop
(CDC) guideline stating that all patients are capable of of Henle
transmitting bloodborne disease vasectomy Surgical removal of all or part of the vas deferens
urobilin The oxidized form of urobilinogen that provides the for the purpose of male sterilization
brown color to feces vasopressin Antidiuretic hormone that regulates reabsorp-
urobilinogen A compound formed in the intestines by the tion of water by the collecting ducts
bacterial reduction of bilirubin vasovasostomy Repair of a severed vas deferens to restore
urochrome Yellow pigment produced by endogenous fertility
metabolism that imparts the yellow color to urine vesicoureteral reflux (VUR) Urine in the bladder passing
uroerythrin Pink pigment in urine derived from melanin back into the ureters
metabolism that attaches to urates in the sediment visceral membrane The serous membrane covering the
uromodulin Glycoprotein that is the only protein produced organs contained within a cavity
by the kidney tubules; forms the matrix of casts (formerly viscosity The amount of resistance to flow in a liquid
called Tamm-Horsfall protein) vulvovaginal candidiasis Inflammation of the vulva, vagina,
uromodulin-associated kidney disease Autosomal muta- or vulvovaginal glands caused by Candida albicans
tion of the gene producing uromodulin that results in the xanthochromia Yellowish discoloration of the cerebrospinal
destruction of the renal tubular cells fluid
vaginal pool Mucus and cells in the posterior fornix of the yeast Fungus that reproduces by budding
vagina
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Index
Page numbers follow by f indicate figures; page numbers followed by t indicate tables.

A American Society of Histocompatibility and

Immunogenetics (ASHI), 62
AUTION Eleven AE-4022, 77t, 78f
AUTION HYBRID AU-4050, 77t
Abnormal urine color, 127–129, 128f, 129f
Amino acid disorders, 236–243 AUTION MAX AX-4030, 77t, 80f
Abnormal urine crystals, 34–37, 206–209, 207t,
branched-chain, 239–240, 240f Auto image evaluation module (AIEM) software, 85
208–209f
phenylalanine-tyrosine disorders, 236–239, 238f Automated body fluid analysis, 77t, 87–88, 88t
Accreditation agencies, 62
Ammonia, 113 semen analysis, 283
Accuracy, 66
measurement, 119 Automated microscopy, 77t, 79–86, 80–85f
ACE. see angiotensin-converting enzyme (ACE)
Ammonium, 94t Automated urinalysis, 76–88
Acetest tablets, 152
Ammonium biurate, 33, 202t, 205, 206f automated microscopy, 77t, 79–86, 80–85f
Acetic acid stain, 171, 171t
Amniocentesis, 329–330, 330b combination systems, 86–87, 86f
Acetylcholinesterase (AChE), 331
Amnion, 328, 328f fully automated chemistry analyzers, 77t,
Acholic stools, 343
Amniostat-FLM, 328t 78–79
Acid-albumin test, 247
Amniotic fluid, 328–335 reflectance photometry, 76, 76t
Acid-base balance, 111–113
chemical composition, 329 semi-automated urine chemistry analyzers,
Acidic urine, crystals in, 26–29, 201–204,
color and appearance, 331, 331t 76–78, 77t, 78–79f
202–204f
cytogenetic analysis of, 328, 328t Autovalidated results, 81
Acidosis
differentiating maternal urine from, 329 Autoverification, 69
metabolic, 113
function, 328, 328f
renal tubular, 113
physiology, 328–329, 328f
Acrosomal cap, 284
Actim PROM test, 366
specimen collection, 329–330, 330b B
specimen handling and processing, 330–331 Bacteria, 11, 42, 171t, 187–188, 188f
Active transport, 110, 110t
tests for fetal distress, 331–332, 332–333f bronchoalveolar lavage (BAL) fluid, 324, 324b
Acute glomerulonephritis (AGN), 220,
tests for fetal maturity, 333–335 casts, 20, 195
223t, 224t
volume, 328–329 diarrhea and, 341–342
Acute intermittent porphyria, 245t
AmniSure test, 366 fecal, 343–344
Acute interstitial nephritis (AIN), 228,
Amorphous phosphates, 30, 201t, 204, 204f pleural fluid testing for, 312
228t, 229b
Amorphous urates, 27, 201–202, 201t, 202f vaginal secretions, 360–361, 360–362f
Acute-phase reactants, 144
Ampicillin crystals, 37, 207t, 209, 210f Bacterial endocarditis, 312
Acute pyelonephritis, 226–227, 228t, 229b
Amyloid material, 145 Bacterial vaginosis (BV), 356, 356t, 364
Acute renal failure (ARF), 225
Anaphylaxis, 222 Bacteroides, 360, 363
Acute tubular necrosis (ATN), 222, 227t
ANCA. see antineutrophilic cytoplasmic antibody BAL. see bronchoalveolar lavage (BAL)
Addis, Thomas, 92
(ANCA) Basal cells, 358, 360
Addis count, 170
Andrology, 278 Bence Jones protein, 144
ADH. see antidiuretic hormone (ADH)
Angiotensin-converting enzyme (ACE), 109 Berger disease, 221–222
ADVIA2120i with Body Fluids Software,
Antidiuretic hormone (ADH), 93, 118 Beta2-microglobulin (B2M), 113, 116
77t, 87
Antiglomerular basement membrane antibody, Bilirubin, 127–128
Afferent arteriole, 106, 109f
221 amniotic fluid, 331, 332f
AGN. see acute glomerulonephritis (AGN)
Antihuman globulin (AHG), 287 in amniotic fluid, 328t
AHG. see antihuman globulin (AHG)
Antineutrophilic cytoplasmic antibody (ANCA), clinical significance of, 155, 155t
AIN. see acute interstitial nephritis (AIN)
221 crystals, 36, 207t, 208, 209f
Air bubble artifacts in urinary sediment,
Antinuclear antibody (ANA), 312 in jaundice, 155t
40, 210
Antisperm antibodies, 286–287 in peritoneal fluid, 316
Albinism, 239
Anuria, 93 production of, 154–155, 155f
Albumin, reagent strips reaction to, 147
APT test (fetal hemoglobin), 348–349 reagent strip reactions, 155–156
Albumin:creatinine ratio, 147
Arachnoid, 254, 254f interference, 156
Albumin/protein:creatinine ratio, 148
Arachnoid granulations, 254 Biliverdin, 128
Albuminuria, 92
ARF. see acute renal failure (ARF) Biohazardous material, 51, 52f
Alcohol-based cleansers, 54
ART. see assisted reproductive technology (ART) decontamination, 54–56
Aldosterone, 109
Arthritis. see synovial fluid symbol for, 56f
Alimentary tract, 340
Arthrocentesis, 294 Biological hazards, 50–56, 50t, 52f
Alkaline urine, crystals in, 30–33, 204–205,
Artifacts, urinary sediment, 38–40, 209–211, infectious agent, 50
204–206f
210–211f reservoir, 50–51
Alkaptonuria, 129, 236, 239
Artificial imaging identification (AII) software, 86 Standard Precautions, 51–54
Alpha-fetoprotein (AFP), 328t, 329, 332
Ascites, 314 Birefringent, 176
Alport syndrome, 224t, 225, 225t
Ascitic fluid, 314 Black urine, 129
Altered motility, 341–342
Ascorbic acid, 150, 154 Blast forms in cerebrospinal fluid (CSF), 261t
Amber urine, 127–128
Assisted reproductive technology (ART), 278, 288 Blood
American Association for Laboratory Accreditation
Astrocytomas, 266 casts, 172t
(A2LA), 62
ATN. See acute tubular necrosis (ATN) in cerebrospinal fluid (CSF), 256, 258, 262f,
American Association of Blood Banks (AABB), 62
Atrophic vaginitis, 356t, 357, 365 263
American Osteopathic Association (AOA), 62

393
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394 Index

in feces, 345–347, 346f broad, 25, 198–199, 199f Chain of infection, 50–51, 52f
traumatic tap, 256–257 coarse granular inclusion, 172t Chemical hazards, 50t, 56–58, 58f
in urine, 128, 128f, 129f, 152–154 composition and formation, 191–192 Globally Harmonized System (GHS), 58
clinical significance, 153 detection of, 169–170 handling, 57
reagent strip reactions, 153–154 epithelial cell, 20, 195, 195–196f hygiene plan, 57
interference, 154 fatty, 23, 172t, 177, 196, 196f, 210 labeling, 57
Blood-brain barrier, 254 gram stain, 173 Safety Data Sheets, 57–58
Blood flow, renal, 106, 108b granular, 21–22, 197–198, 197–198f spills and exposure, 56–57
tests, 119 hyaline, 17–18, 171, 172t, 178–179, 190, 192, symbols, 59f
Blood urea nitrogen (BUN), 220 192f waste disposal, 58
Blue urine, 129 lipid stains, 171 Chemical hygiene plan (CHP), 57, 58f
Body fluid analyzers, 77t, 87–88, 88t mixed cellular, 23, 196–197 Chemistry tests
Body fluids, 41–46 Prussian blue stain, 173 cerebrospinal fluid (CSF), 266–268, 267b,
cerebrospinal fluid, 41–42 RBC, 18, 180, 192–194, 192–194f 267f, 269t
peritoneal fluid, 43 red cell inclusion, 172t feces, 345–349, 350t
pleural fluid, 44–45 renal tubular epithelial cells, 185 peritoneal fluid, 315–316, 315–316f
synovial fluid, 46 waxy, 24, 172t, 198, 198f pleural fluid, 310–312, 312t
Body fluid white blood cell count (WBC-BF), 88 WBC, 19, 194–195, 194–195f semen, 287, 287t
Body surface area, 115, 115f Catheterized specimen, 97t, 98 synovial fluid, 300–301
Bowman capsule, 107, 221 CD4/CD8 T cells, 323, 323t Chlamydia trachomatis, 357, 364
Brain, 254–255f. see also cerebrospinal fluid (CSF) Cell counts Chloride, 94t
Branched-chain amino acids disorders, 239–240, bronchoalveolar lavage (BAL) fluid, 322–323 Cholesterol crystals, 35, 206, 207t, 208f
240f cerebrospinal fluid (CSF), 257–258, 257f Choroidal cells, 261t
Bright, Richard, 92 synovial fluid, 296 Choroid plexuses, 254
Bright-field microscopy, 174f, 176 Cells, urinalysis, 2–10 Chronic glomerulonephritis (CGN), 221, 223t, 224t
Broad casts, 25, 198–199, 199f epithelial cells, 5–6 Chronic pyelonephritis, 227, 228t, 229b
Bronchoalveolar lavage (BAL) fluid, 322–325 oval fat bodies, 10 Chylous material, 308–309, 309t
cell counts, 322–323 red blood cells, 2–3 Clarity and appearance
clinical significance, 322 renal tubular epithelial cells, 8–9 bronchoalveolar lavage fluid, 322
color and clarity, 322 transitional epithelial (urothelial) cells, 7–8 cerebrospinal fluid (CSF), 255–256, 256t
cytology studies, 324–325 white blood cells, 3–5 feces, 343
eosinophils in, 323–324 Cellular structure of glomerulus, 106–107, 109f pericardial fluid, 312
epithelial cells in, 324, 324f Centers for Disease Control and Prevention peritoneal fluid, 314
erythrocytes in, 324 (CDC), 50 pleural fluid, 308–309, 308t
leukocytes in, 323 on bloodborne pathogens exposure, 53 semen, 280
lymphocytes in, 323, 323t on hand washing, 54 synovial fluid, 295–296
macrophages in, 323, 323f on porphyrin disorders, 243 urine, 129–131, 129t, 130b
mast cells in, 324 Central nervous system (CNS), 254 vaginal secretions, 357
microbiological tests, 324, 324b, 325f tumors of, 266 Clearance tests, 113–115
neutrophils in, 323 Centrifugation, macroscopic screening, 168–169 Clinical and Laboratory Standards Institute
specimen collection, 322 Cerebrospinal fluid (CSF), 41–42, 254–271 (CLSI), 50, 93
specimen handling and transport, 322 appearance of, 255–256, 256f, 256t Clinical Laboratory Improvement Amendments
Bronchoscopy, 322 cell count, 257–258, 257f (CLIA), 67, 76
Brown urine, 128–129 chemistry tests, 266–268, 267b, 267f, 269t Clinical laboratory reagent water (CLRW), 64
Bryant, Thomas, 92 differential count, 258–266 Clinitek Advantus, 77t, 79f
Bulbourethral glands, 278 cellular constituents, 259–266, 260–261t, Clinitek Atlas, 77t, 80f, 86, 86f
Burkitt lymphoma, 266f 261–266f CLINITEK AUWi Pro System, 77t
cytocentrifugation, 259, 259f, 259t Clinitek Microalbumin reagent strips, 147, 148
neutrophils, 260, 261–262f Clinitek Novus, 77t
C formation and physiology, 254, 254–255f Clinitek Status, 77t, 79f
Calcium, 94t glucose, 268 Clinitest, 150–151
Calcium carbonate, 32, 202t, 205, 205f glutamine, 268 Clostridium, 341
Calcium oxalate, 28–29, 201t, 203–204, 204f lactate, 268 Clots, blood, 257
Calcium phosphate, 32, 202t, 205 in malignancies, 265, 265–266f Clue cells, 5, 358
Calibration/calibration verification, 62 microbiology tests, 268–270, 270f Coarse granular inclusion casts, 172t
Campylobacter, 341, 343 gram stain, 269–270 Cobas 6500 Urine Analyzer, 77t, 87
Candidiasis, 364–365 immunologic assays, 270 Cobas u 411 Urine Analyzer, 77t, 78f
Candida albicans, 12 PCR molecular diagnostic testing, 270 Cobas u 601 Urine Analyzer, 77t
Carbohydrate disorders, 247 nonpathologically significant cells in, 263, Cobus u 701 Microscopy Analyzer, 77t
Carbohydrates in feces, 349 264–265f Coefficient of variation (CV), 67
Carcinoembryonic antigen (CEA), 312 serological testing, 271 Collecting duct, 111
Cardiac tamponade, 312 specimen collection and handling, 254–255, Collecting duct cells, 8
Cardiopulmonary resuscitation (CPR), 60 255f College of American Pathologists (CAP), 62
CASA. See Computer-assisted semen analysis traumatic collection (tap), 256–257 Colligative properties, 133, 134t
(CASA) Cerebrospinal protein, 266–268, 267b, 267f Color
Casts, 17–25, 168, 171t Cetyltrimethylammonium bromide (CTAB) amniotic fluid, 331, 331t
bacterial, 20, 195 turbidity test, 247 bronchoalveolar lavage fluid, 322
blood (hemoglobin), 172t Chain of custody (COC), 100 feces, 343
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peritoneal fluid, 314 Diabetes mellitus, 94, 94t Examination variables, 61, 64–67, 66–68f
pleural fluid, 308–309 Diabetic nephropathy, 145, 224t, 226 Exogenous procedures, 114
semen, 280 Diarrhea, 341–342, 341–342t External quality assessment (EQA), 62, 67
synovial fluid, 295–296 Differential count External quality control, 62, 66–67, 68f
urine, 126–127t, 126–129 cerebrospinal fluid (CSF), 258–266 Exudates, 307, 308t, 314
vaginal secretions, 357 synovial fluid, 296–297 Eye wash station, 58f
Commercial systems, macroscopic screening, 169 Differential interference-contrast microscopy,
Commission on Laboratory Assessment 177–178, 178f
(COLA), 62 Digital imaging, 82 F
Composition, urine, 93, 94t Dirui FUS – 100/200 with H-800, 77t Face shields, 54
Computed tomography (CT), 314 Disinfectants, 55 Fanconi syndrome, 145, 225, 227t
Computer-assisted semen analysis (CASA), 283 Disposal of biological waste, 54, 56–57 Fat droplets
Concentration tests, 117 Distal convoluted tubule, 106, 109f in feces, 344–345, 344–345f, 348
Confirmatory tests, reagent strip, 142 Distal tubular cells, 8 in synovial fluid, 296
Congenital erythropoietic porphyria, 245t DNA testing, vaginal secretions, 363 Fatty casts, 23, 172t, 177, 196, 196f, 210
Constipation, 341 Documentation on bloodborne pathogens, 53 Fecal analysis, 340–350
Containers, specimen, 94 Drug specimen collection, 100–101 chemical testing, 345–349, 350t
Copper reduction test, 150 Dura mater, 254, 254f diarrhea and steatorrhea, 341–342, 341–342t
Core channel (CR ch) stains, 81 DxH 900 Hematology Analyzer, 77t macroscopic screening, 342–343, 343t
Cough etiquette, 53 Dysentery, 343 microscopic examination, 343–345, 344–345f
Countercurrent mechanism, 111 Dysmorphic red blood cells, 2, 179, 180f physiology of, 340–341, 340f
CPR. see Cardiopulmonary resuscitation (CPR) Dyspareunia, 356 specimen collection, 342
Creatinine, 94t Dysuria, 356 usefulness of, 340
in amniotic fluid versus maternal urine, 329 Fecal enzymes, 349
clearance tests, 113, 114–115, 115f Fecal material artifacts, 210, 210f
reagent strips reaction to, 147–148 E Fecal occult blood testing (FOBT), 345–347, 346f
Crenated cells, 179, 179f Early dumping syndrome (EDS), 342 Fecal trypsin, 349
Critical results, 69, 70f Education and competency assessment of Fenestrated endothelium, 107
Cryptosporidium, 341 personnel, 62 Fern test, 329
Crystals, synovial fluid, 296 Efferent arteriole, 106 Fetal fibronectin test (fFN), 365–366
types of, 297, 298t Effusions, 306, 307b Fetal lung maturity (FLM), 330, 333
Crystals, urinary, 171t chylous, 309, 309t Fetus
abnormal, 34–37, 206–209, 207t, 208–209f pseudochylous, 309, 309t in amniotic sac, 328, 328f
formation, 199 synovial fluid, 296 AmniSure test and, 366
general identification techniques, 201, 201–202t Ehrlich test, 243 cytogenetic testing of, 328, 328t
normal Electrical hazards, 50t, 59–60 fetal fibronectin test (fFN) and, 365–366
acidic, 26–29, 201–204, 202–204f Electronic quality control, 62, 67 hemolytic disease of the fetus and newborn
alkaline, 30–33, 204–205, 204–206f Emergency shower, 58f (HDFN), 330, 331–332, 332–333f
CSF. see Cerebrospinal fluid (CSF) Endogenous procedures, 114 lung maturity, 330, 333
Culture, vaginal secretions, 363 End-stage renal disease (ESRD), 221 neural tube defects (NTDs) in, 329, 332
Cryptococcus neoformans, 270, 270f Engineering controls for bloodborne pathogens, 53 ROM/PROM tests, 366–367
Cystatin C, 113, 116 Enterococcus, 360 Fibers, 39–40
Cystine Environmental control, 53 Fibrin, 296
crystals, 34, 206, 207t, 208f Environmental Protection Agency (EPA), 58 Fire/explosive hazards, 50t, 60–61, 61t
disorders, 242–243 Enzyme-linked immunosorbent assay (ELISA), First morning specimen, 96–97
Cystinosis, 34, 236, 242–243 270, 365–366 5-hydroyindoleacetic acid, 241–242
Cystinuria, 34, 236, 242 Enzymes Flatus, 340
Cystitis, 226, 228t, 229b in feces, 349 Flow cytometry, 80, 323
Cytocentrifugation, 259, 259f, 259t in synovial fluid, 301 Fluorescence in situ hybridization (FISH), 330
Cytodiagnostic urine testing, 173 Eosinophils, 3, 44, 46, 181, 182f Fluorescence microscopy, 174f, 178, 178f
Cytology studies in bronchoalveolar lavage (BAL) fluid, 323–324 Fluorescent mapping spectral karyotyping (SKY),
bronchoalveolar lavage (BAL) fluid, 324–325 in cerebrospinal fluid (CSF), 260, 261t, 263f 330
pericardial fluid, 312–314, 313f in pleural fluid, 309 Fluorescent treponemal antibody-absorption
Ependymal cells, 261t (FTA-ABS), 271
Epithelial cells, 5–6, 171t, 182–187, 183f Foam Stability Index, 328t, 334
D in bronchoalveolar lavage (BAL) fluid, 324, Focal segmental glomerulosclerosis (FSGS), 222,
Dark-field microscopy, 174f, 177–178, 178f 324f 224t, 225t
Dark yellow urine, 127–128 casts, 20, 195, 195–196f Focused assessment with sonography for trauma
D-dimer test, 257 oval fat bodies, 186–187, 186–187f (FAST), 314
Dekkers, Frederik, 92 renal tubular, 8–9, 184–186, 185–186f, 222 Food and Drug Administration (FDA), 67
Delta check, 69 squamous, 182–184, 183–184f Formation, urine, 93
Density, 117 transitional, 7–8, 184, 184f Fomites, 50
Depolarized side scatter (DSS) detector, 81 Equipment maintenance, 62 Four-glass collection, 97t
Desquamative inflammatory vaginitis (DIV), 356t, Erythrocytes in bronchoalveolar lavage (BAL) Free erythrocyte protoporphyrin (FEP), 243
357, 365 fluid, 324 Free water clearance, 118–119
Diabetes insipidus, 94, 94t Erythropoietic protoporphyria, 245t Freezing-point osmometers, 117–118
nephrogenic, 226, 227t Escherichia coli, 341, 343 Fructosuria, 247
osmolality in, 117, 118f Ethylenediaminetetraacetic acid (EDTA), 295 Fully automated chemistry analyzers, 77t, 78–79
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396 Index

Fungi clinical significance, 148–149, 149f Hyperglycemia, 149

bronchoalveolar lavage (BAL) fluid, Clinitest, 151 Hypernatremia, 118
322, 324b copper reduction test, 150 Hypersthenuric urine, 131
FUS-100 system, 77t, 86 reaction interference, 150 Hypertonic urine, 2, 3
FUS-200 system, 77t, 86 reagent strip (glucose oxidase) reaction, 149 Hyponatremia, 118
in synovial fluid, 300 Hyposthenuric urine, 131
Glucose tolerance specimens, 97 Hypoxia, 268
G Glucosuria, 149
Galactokinase (GALK), 247 Glutamine, cerebrospinal fluid (CSF), 268
Galactose, 151 Glycosaminoglycans (GAGs), 246 I
Galactose-1-phosphate uridyl transferase (GALT), Glycosuria, 149 IChemVELOCITY, 77t, 80f
247 Goggles, 54 Ictotest tablets, 156
Galactosuria, 247 Gold and mercury treatments, 221 Immunochemical fecal occult blood test (iFOBT),
Gardnerella vaginalis, 358, 360, 363, 364 Goodpasture syndrome, 220–221, 223t, 224t 346–347
Gas chromatography-mass spectrometry (GC-MS), Gout, 297 ImmunoDip reagent strips, 147
239 Gowns, 52 Immunofixation electrophoresis (IFE), 268
Gastrocolic fistulas, 344 Gram stain, 171t, 173 Immunoglobulin A (IgA), 220
Ghost cells, 179 cerebrospinal fluid (CSF), 269–270 nephropathy, 221–222, 223t, 225t
Globally Harmonized System (GHS), 58, 60f, 61b feces, 343 Immunoglobulin G (IgG)
GloCyte Automated Cell Counter, 88 vaginal secretions, 356, 363, 363t antihuman globulin (AHG), 287
Glomerular disorders, 220–222 Granular casts, 21–22, 197–198, 197–198f cerebrospinal fluid (CSF), 266
clinical information associated with, 224–225t Granulomatosis with polyangiitis (GPA), 221, Immunoglobulin M (IgM), 266
glomerulonephritis, 220–222 223t, 224t Inborn error of metabolism (IEM), 236
laboratory testing in, 223–224t Green urine, 129 Indicanuria, 236, 241
Glomerular filtration, 106–109, 109f Group A streptococcal enzyme tests, 220 Individualized quality control plan (IQCP),
renin-angiotensin-aldosterone system (RAAS), Guaiac-based fecal occult blood tests (gFOBT), 68–69, 69f, 70f
106, 108–109, 110b, 110f 345–346, 346f Infection control, 50, 52f
tests, 113–116 Infectious agents, 50, 52f
Glomerular filtration barrier, 106, 109f reservoir, 50–51
Glomerular filtration rate (GFR), 106 H Instrumentation and equipment, 64, 66, 66f
estimated, 115–116 Hair and fiber artifacts, 210, 211f Insulin-like growth factor binding protein-1
Glomerular pressure, 107–108 Hand hygiene, 51, 52f, 54, 55–56 (IGFBP-1), 329, 357
Glomerular proteinuria, 144–145 Hansel stain, 171t, 173 Integrity, specimen, 95–96, 95t
Glomerulonephritis, 220–222 Harmonic oscillation densitometry, 133 Interference-contrast microscopy, 174f, 177
acute glomerulonephritis (AGN), 220, 223t, Healthcare Infection Control Practices Advisory Internal quality control, 62, 67
224t Committee (HICPAC), 51 International Organization for Standardization
Alport syndrome, 224t, 225, 225t Hematoidin crystals, 263 (ISO), 61
chronic glomerulonephritis (CGN), 221, 223t, Hematuria, 152, 153 Interpreting results, 69
224t Heme formation, 244f Interstitial disorders, 226–228, 228t, 229b
diabetic nephropathy, 145, 224t, 226 Hemocytometer, 322 Inulin clearance, 114
Fanconi syndrome, 225 Hemoglobinuria, 152, 153, 154 In vitro fertilization (IVF), 278, 288
focal segmental glomerulosclerosis (FSGS), 222, Hemolytic disease of the fetus and newborn IQ 200 Automated Urine Microscopy, 77t, 83,
224t, 225t (HDFN), 330, 331–332, 332–333f 85, 85f
Goodpasture syndrome, 220–221, 223t, 224t Hemothorax, 308 IQ200 body fluid analysis, 88
granulomatosis with polyangiitis (GPA), 221, Henoch-Schönlein purpura, 221, 223t, 224t IQ 200 Sprint, 77t
223t, 224t Hepatic disease, 36 IQ 200 using Body Fluids Software, 77t
Henoch-Schönlein purpura, 221, 223t, 224t Hepatitis B, 221 IRICELL Urinalysis – IQ Series Urinalysis
immunoglobulin A nephropathy, 221–222, Hereditary and metabolic tubular disorders, Workcell, 77t, 86, 86f
223t, 225t 225–226, 227t Irritable bowel syndrome (IBS), 342
membranoproliferative glomerulonephritis Herpes simplex virus (HSV), 357, 364 Isoelectric focusing (IEF), 268
(MPGN), 221, 223t, 224t High-fluorescing body fluid cells (HF-BF), 88 Isosthenuric urine, 131
membranous glomerulonephritis (MGN), 221, Hippocrates, 92 Isotope dilution mass spectrophotometry (IDMS)
223t, 224t Histograms, 80, 81 reference method, 115–116
minimal change disease (MCD), 222, 223t, History of urinalysis, 92–93, 92–93f IVF. see in vitro fertilization (IVF)
225t Hoesch test, 243, 246
nephrogenic diabetes insipidus, 226 Hoffman microscopy, 177
nephrotic syndrome (NS), 220, 222, 223t, 225t Homocystinuria, 243 J
rapidly progressive (crescentic) Human gonadotropin (hCG), 66 Jaundice, 154
glomerulonephritis (RPGN), 220, 223t, Human immunodeficiency virus (HIV), 364 The Joint Commission, 60, 62
224t Human leukocyte antigen B12 (HLA-B12), Joint disorders. see synovial fluid
renal glycosuria, 226 222 Juxtaglomerular apparatus, 108, 109f
uromodulin-associated kidney disease, 226 Hunter syndrome, 246
Glomerulus, 106 Hurler syndrome, 246
Gloves, 51–52 Hyaline casts, 17–18, 171, 172t, 178–179, 190, K
starch granules from, 210 192, 192f Ketones
Glucose, 148–151 Hydrogen nuclear magnetic resonance clinical significance, 151
in amniotic fluid versus maternal urine, 329 spectroscopy (H NMR) method, 347 reagent strip reactions, 152
cerebrospinal fluid (CSF), 268 Hydrostatic pressure, 106, 306 interference, 152
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Ketonuria, 151 reporting microscopic examination, 170 microscope, 173–176, 174f

Köhler illumination, 175–176 sediment examination, 169–170 modulation-contrast, 177
sediment preparation, 169 red blood cells, 179–180f, 179–181
specimen preparation, 168 sediment examination techniques, 170–178
L specimen volume, 168 sediment stains, 171–172t, 171–173
Labeling volume of sediment, 169 types of, 174f, 176–177
chemical, 57, 59f Macula densa, 108, 109f vaginal secretions, 358–363, 358t, 359–362f
specimen, 64, 64b, 95 Malabsorption, 341 white blood cells, 181–182, 181–182f
Laboratory coats, 54 Maldigestion, 341 Midstream clean-catch specimen, 97t, 98
Laboratory information system (LIS), 76 Malignancies Minimal change disease (MCD), 222, 223t, 225t
LabUMat 2 system, 77t, 87 cerebrospinal fluid (CSF) in, 261t, 265, Mixed agglutination reaction (MAR), 287
Lactate 265–266f Mixed cellular casts, 23, 196–197
cerebrospinal fluid (CSF), 268 feces appearance with, 343 Mobiluncus, 358, 360, 363, 364
synovial fluid, 301 peritoneal fluid in, 315, 315–316f Modification of Diet in Renal Disease (MDRD)
Lactobacilli, 360, 360f, 363 pleural fluid in, 309, 310, 310f, 311f, 312b study, 115–116
Lactoferrin latex agglutination test, 344 Malignant melanoma, 129 Modulation-contrast microscopy, 177
Lactosuria, 247 Maltese cross formation, 176, 210 Monocytes
Lamellar body Maple syrup urine disease (MSUD), 236, 240 in cerebrospinal fluid (CSF), 41, 260, 261t,
count, 328t, 334–335 Masks, goggles, and face shields, 54 262f
density procedures, 334 Mass spectrophotometry, tandem (MS/MS), 236, in peritoneal fluid, 43
Latex agglutination tests, 270 237f, 243 in pleural fluid, 44
Lead poisoning, 245t Mast cells in bronchoalveolar lavage (BAL) Mononuclear cells, 182, 182f
Lecithin-sphingomyelin (L/S) ratio, 328t, fluid, 324 Morphology, sperm, 283–285, 284–285f
287–334 Maximal reabsorptive capacity, 111 Motility, sperm, 283, 283t
Lesch-Nyhan disease, 247 Means of transmission, 51, 52f Mouth, nose, and eye protection, 52
Leucine crystals, 34, 207t, 208, 209f Meat fibers in feces, 344, 344f MSUD. see maple syrup urine disease (MSUD)
Leukocyte esterase, 11, 23 Meconium, 331 Mucin clot, 296
clinical significance, 159 Medical controls for bloodborne pathogens, 53 Mucopolysaccharide disorders, 246–247
reagent strip reaction, 160 Medullary interstitium, 106 Mucopolysaccharidoses (MPSs), 246
interference, 160 Medulloblastomas, 266, 266f Mucus, 16, 190, 191f
Leukocytes Melanuria, 236, 239 Multiple myeloma, 144
in bronchoalveolar lavage (BAL) fluid, 323 Melituria, 247 Multiple sclerosis, 254
fecal, 343–344 Membranoproliferative glomerulonephritis Multiples of the median (MoM), 332
Levy-Jennings control charts, 67, 67f, 81 (MPGN), 221, 223t, 224t Muscle fibers in feces, 344, 344f
Light-emitting diode (LED), 76 Membranous glomerulonephritis (MGN), 221, Mycoplasma hominis, 360, 364
Liley graph, 331, 333f 223t, 224t Myelin basic protein (MBP), 268
Linens, handling of, 53 Meninges, 254, 254f Myoglobinuria, 153–154
Lipid stains, 171, 171t, 173 Meningitis, 254, 260, 269t
Lipiduria, 187 Mercaptoethane sulfonate sodium (MESNA), 152
Lipophages, 315, 315f Mesothelial cells, 43, 44, 306 N
Liquid chromatography-tandem mass in pleural fluid, 309, 310f N95 respirators, 52
spectrometry (LC-MS/MS), 247 Metabolic acidosis, 113 Naegleria fowleri, 270, 270f
Liquefaction, semen, 278, 280 Metabolic disorders, urine screening for, 236t National Kidney Disease Education Program
Lithotripsy, 229 Micral-Test reagent strips, 147 (NKDEP), 115–116
Liver disease, 34, 35 Microalbuminuria, 145 Near-infrared reflectance spectroscopy (NIRS),
Liver disorders, crystals associated with, 207–208, testing for, 146–147 347
208f Microbiological tests Neisseria gonorrhoeae, 357, 364
Loops of Henle, 106 bronchoalveolar lavage (BAL) fluid, 324, 324b, Nephrogenic diabetes insipidus, 226, 227t
Lymphocytes, 3 325f Nephrons, 106, 107f, 108f
in bronchoalveolar lavage (BAL) fluid, 323, cerebrospinal fluid (CSF), 268–270, 270f Nephrotic syndrome (NS), 220, 222, 223t, 225t
323t peritoneal fluid, 316 Neubauer hemocytometer, 257, 257f, 282, 282f
in cerebrospinal fluid (CSF), 41, 260, 260t, 262f pleural fluid, 312 Neural tube defects (NTDs), 329, 332
in peritoneal fluid, 43 semen, 287, 287t Neutrophages, 296–297
in pleural fluid, 44–45, 309 synovial fluid, 301 Neutrophils
Lymphoma cells, 261t, 265–266f Microorganisms, 11–13 in bronchoalveolar lavage (BAL) fluid, 323
Microscopes, 173–176, 174f in cerebrospinal fluid (CSF), 41–42, 260, 260t,
Microscopy, 168–211 261–262f
M automated, 77t, 79–86, 80–85f in pleural fluid, 44–45, 309
Macrophages bacteria, 187–188, 188f in synovial fluid, 46
in bronchoalveolar lavage (BAL) fluid, 323, cytodiagnostic urine testing, 173 Newborn urine screening. see urine screening
323f differential interference-contrast, 177–178, Nitrite
in cerebrospinal fluid (CSF), 261t, 263, 263f 178f clinical significance, 157–158
in pleural fluid, 309 epithelial cells, 182–187, 183–187f reagent strip reactions, 158
Macroscopic screening, 168t expected staining reactions of, 172t interference, 158–159
centrifugation, 168–169 feces, 343–345, 344–345f Nocturia, 93
commercial systems, 169 fluorescence, 174f, 178, 178f Nomarski microscopy, 177–178, 178f
correlating results, 170, 171t Köhler illumination, 175–176 Nonpathologically significant cells in cerebrospinal
fecal analysis, 342–343, 343t macroscopic screening, 168–170 fluid (CSF), 263, 264–265f
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398 Index

Nonpathological turbidity of urine, 129, 130b PH Precision, 66

Normal urine color, 126–127, 128f semen, 281 Preexamination variables, 61, 62–64, 63f, 64b, 65f
Normal urine crystals urine, 142–143, 143t Pregnancy-associated vaginal secretions, 365–367
acidic, 201–204, 202–204f vaginal secretions, 357–358 Premature rupture of membranes (PROM), 329,
alkaline, 204–205, 204–206f Phase-contrast microscopy, 174f, 176 330t, 366–367
Nucleated red blood cells (NRBCs), 260 Phenazopyridine, 128 Prerenal proteinuria, 144
Phenolsulfonphthalein test, 120 Preservation, specimen, 96, 96t
Phenylalanine-tyrosine disorders, Preventive maintenance (PM), 64
O 236–239, 238f Prevotella, 358, 360, 364
Occult blood in feces, 345–347, 346f Phenylketonuria (PKU), 236, 237 Proficiency testing (PT), 62, 67
Occupational Exposure to Bloodborne Pathogens Phosphate, 30–31, 94t Prostate gland, 278
Standard, 53 amorphous, 30 Prostatitis specimen, 97t, 98–100
Occupational health and bloodborne pathogens, 53 calcium, 32 Protein, 23, 25, 143–148
Occupational Safety and Health Administration Phosphatidyl glycerol (PG), 334 in amniotic fluid versus maternal urine, 329
(OSHA), 50 Physical hazards, 50t, 61 cerebrospinal fluid (CSF), 266–268, 267b, 267f
Occupational Exposure to Bloodborne Physiology reagent strip reactions, 146–148, 146t
Pathogens Standard, 53 amniotic sac, 328, 328f in synovial fluid, 300
Ochronosis, 239 cerebrospinal fluid (CSF), 254, 254–255f Protein error of indicators, 146
Odor fecal analysis, 340–341, 340f Proteinuria, 143–148
semen, 280 renal, 106–113 clinical significance of, 144
urine, 134, 134t semen, 278, 279f, 279t postrenal, 145
Oil droplet artifacts, 40, 210, 210f synovial fluid, 294, 294f, 294t prerenal, 144
Oligoclonal bands, 267, 267f Pia mater, 254, 254f renal, 144–145
Oligohydramnios, 328 Pigmented villonodular synovitis, 297 Protozoa, 341
Oliguria, 93 Pink urine, 128–129 Provider-performed microscopy procedures
Oncotic pressure, 106, 306 Pinworm, 14 (PPMP) tests, 68
Optical density, 328t, 331 PKU. see phenylketonuria (PKU) Proximal convoluted tubule, 106
Oral glucose tolerance test (OGTT), 97 Placental alpha macroglobulin (PAMG-1), Proximal tubular cells, 8
Orange urine, 127–128 329, 357 Pruritus, vaginal, 356
Organic acidemias, 236, 240 Placental protein 12 (PP12), 329 Prussian blue stain, 171t, 173
Orthostatic (postural) proteinuria, 145 Plasma cells Pseudochylous material, 308–309, 309t
Osmolality, 117, 118f, 132–133, 134t in cerebrospinal fluid (CSF), 261t Pseudogout, 297
Osmolar clearance, 118–119 in pleural fluid, 309 Pulmonary infarction, 309
Osmolarity, 118 Pleocytosis, 260 Purine disorders, 247
Osmotic diarrhea, 341 Pleural fluid, 44–45, 308–312 Pyelonephritis
Osmotic gradient, 106 appearance, 308–309, 308t acute, 226–227, 228t, 229b
OSOM BVBLUE test, 363 chemistry tests, 310–312, 312t chronic, 227, 228t, 229b
OSOM Trichomonas Rapid Test, 363 hematology tests, 309–310, 309–311f, 309t
“Out-of-control” procedures, 67, 68f microbiological and serological tests, 312
Oval fat bodies, 10, 186–187, 186–187f, 210 Podocytes, 107 Q
Overflow disorders, 236, 237t Point-of-care (POC) instrument, 67 Qualitative fecal fats, 344–345, 344–345f
Point-of-care tests, vaginal secretions, 363 Quality assessment (QA), 68
Polarization, synovial fluid crystal, 298–300, Quality control (QC), 61, 66–67
P 299–300f cerebrospinal fluid (CSF) cell counts, 258
P-aminohippuric acid (PAH) test, 119 Polarizing microscopy, 171, 174f, 176–177, 177f individualized plan, 68–69, 69f, 70f
Pancreatic insufficiency, 344 Pollen grain artifacts, 210, 210f reagent strips, 142
Pancreatitis, 309 Polydipsia, 94 semen analysis, 288
Parabasal cells, 358, 360, 360f Polyhydramnios, 328 Quality control plan (QCP), 68
Parasites, 14–15, 189, 189–190f, 341 Polymerase chain reaction (PCR), 269, 270, 312 Quality management (QM), 61–62
Parietal membrane, 306 Polymorphonuclear (PMN) white blood cells, 358 errors in, 70
Passive transport, 110, 110t Polyuria, 93, 94, 94t Quality management system (QMS), 61
Pathogens, 50 Porphobilinogen, 128 Quantitative fecal fat testing, 347–348
Pathological turbidity of urine, 130–131, 130b Porphyria cutanea tarda, 245t Queenan curve, 331, 333f
Patient care equipment, 53 Porphyrin-based fecal occult blood test, 347
Patient placement, 53 Porphyrin disorders, 243–246, 244f, 245t
Pediatric specimens, 100 Porphyrins, 128 R
Pentosuria, 247 Porphyrinuria, 243 RAAS. see renin-angiotensin-aldosterone system
Peptostreptococcus, 360, 364 Porphyromonas, 360, 364 (RAAS)
Pericardial fluid, 312–314, 313f, 313t Portal of entry, 51, 52f Radioactive hazards, 50t, 58–59
Pericarditis, 312 Portal of exit, 51, 52f Radiographic dye crystals, 38, 207, 207t
Peripheral blood (PB), 263 Postexamination variables, 61, 68–69 Radioisotopes, 58
Peritoneal fluid, 43, 314–316, 314t, 315–316f Postexposure prophylaxis (PEP), 53 Radionucleotides, 116
Peritoneal lavage, 314 Postrenal proteinuria, 145 Ragocytes, 297
Peritonitis, 314 Postvasectomy semen analysis, 287 Random specimen, 96
Peritubular capillaries, 106 Potassium, 94t Rapid gastric emptying (RGE), 342
Personal protective equipment (PPE), 51, 52f, 54 Potassium hydroxide (KOH) examination, 356, Rapidly progressive (crescentic)
for bloodborne pathogens, 53 357, 361–362 glomerulonephritis (RPGN), 220,
Personnel assessment, 68 Pre- and postmassage test (PPMT), 99 223t, 224t
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Rapid plasma reagin (RPR) test, 271 tubular reabsorption, 106, 110–111, 110t spermatozoa, 189–190, 190f
Reagents, 64 tubular secretion, 106, 111–113, 112–113f white blood cells, 181–182, 181–182f
Reagent strips, 140–141f, 140–142 Renal plasma flow, 106 yeast, 168, 179, 179f, 188, 189f
confirmatory testing, 142 Renal proteinuria, 144–145 Sediment examination techniques, 169–178
handling and storing, 141 Renal threshold, 111 preparation, 169
quality control of, 142 Renal tubular acidosis, 113 volume, 169
reactions, 143, 146–148, 146t Renal tubular epithelial (RTE) cells, 8–9, Sediment stains, 171–173
albumin, 147 184–186, 185–186f, 222 characteristics of, 171t
blood, 153–154 Renin, 109 expected reactions of sediment constituents,
glucose oxidase, 149 Renin-angiotensin-aldosterone system (RAAS), 172t
ketones, 152 106, 108–109, 110b, 110f supravital stains, 171, 172t
leukocyte esterase, 160 Requisition form, specimen, 63, 95 Semen, 278–288. See also spermatozoa
nitrite, 158–159 Reservoir, 50–51, 52f analysis, 280–285
specific gravity, 160–161, 160f Resolution, 174 additional testing for abnormalities,
urinary bilirubin, 155–156 Respiratory distress syndrome (RDS), 333 285–288, 286–288t, 286f
urobilinogen, 157 Respiratory hygiene, 53 appearance, 280
technique, 140–141 Respiratory tract automated, 283
Reagent strip specific gravity, 133 amniotic fluid from, 329 liquefaction, 280
Record-keeping, 62 bronchoalveolar lavage of, 322–325 microbial and chemical testing, 287, 287t
Red blood cells (RBCs), 2–3, 81, 168, 171t Results pH, 281
in acute glomerulonephritis (AGN), 220 critical, 69, 70f postvasectomy, 287
bronchoalveolar lavage (BAL) fluid, errors in, 69 quality control, 288
322–323 interpreting, 69 reference values for, 280t
casts, 18, 180, 192–194, 192–194f Retinoblastomas, 266 sperm concentration and count, 281–282,
in cerebrospinal fluid (CSF), 256, 258, Revolutions per minute (RPM), 169 282f
262f, 263 Rhabdomyolysis, 153 sperm morphology, 283–285, 284–285f
in pleural fluid, 44–45 Rheumatoid factor (RF), 270, 312 sperm motility, 283, 283t
in semen, 280 Ribonucleic acid (RNA), 80 viscosity, 281
serous fluid, 307 Risk assessment (RA), 68 volume, 280
in synovial fluid, 296 ROM Plus test, 366–367 composition, 279t
in urine, 128–129, 152, 179–180f, 179–181 Root cause analysis (RCA), 64 physiology, 278, 279f, 279t
vaginal secretions, 359, 359f Ropes test, 296 specimen collection, 278–280
in vaginal secretions, 357 Round renal tubular epithelial (RTE) cells, 182 specimen handling, 280
Red cell inclusion casts, 172t RPGN. see rapidly progressive (crescentic) Semi-automated urine chemistry analyzers,
Red O stain, 171, 171t glomerulonephritis (RPGN) 76–78, 77t, 78–79f
Red urine, 128–129 RTE. see renal tubular epithelial (RTE) cells Seminal fluid fructose, 286
Reflectance photometry, 76, 76t Rupture of membranes (ROM), 330t, 366–367 Seminal vesicles, 278
Refractometry, 131–132, 132f Seminiferous tubules, 278
Reiter cells, 296–297 Sensitivity of testing, 69
Rejection, specimen, 64, 64b, 95 S Serological tests
Relative centrifugal force (RCF), Safety Data Sheets (SDS), 57–58, 61b cerebrospinal fluid (CSF), 271
168–169 Safety hazards, 50–62 peritoneal fluid, 316
Reliability, 66 biological, 50–56, 50t, 52f pleural fluid, 312
Renal blood flow, 106, 108b chemical, 50t, 56–58, 58f synovial fluid, 301
tests, 119 electrical, 50t, 59–60 Serous fluid, 306–316, 306f
Renal disease, 220–230 fire/explosive, 50t, 60–61, 61t formation, 306, 307f
glomerular disorders, 220–222, 223–224t physical, 50t, 61 general laboratory procedures, 307–308
interstitial disorders, 226–228, 228t quality management (QM) for, 61–62 pericardial fluid, 312–314, 313f, 313t
laboratory testing in, 223–224t radioactive, 50t, 58–59 peritoneal fluid, 314–316, 314t, 315–316f
renal failure, 228–229, 229b sharps, 50t, 53, 56 pleural fluid, 308–309t, 308–312, 309–311f,
renal lithiasis, 229–230 Standard Precautions (SP) against, 51–54 312b, 312t
tubular disorders, 222, 225–226 types of, 50, 50t specimen collection and handling, 306–307
urine screening for, 236, 237t Safety programs, 62 transudates and exudates, 307, 308t
Renal failure, 228–229, 229b Salmonella, 341, 343 Sexually transmitted infections (STIs), 364
Renal function, 105–120 Scattergrams, 80, 81, 82f Sharps hazards, 50t, 53, 56
renal disease and, 220–230 Schistosoma haematoblum, 15 Shield of negativity, 107, 109f
renal physiology and, 106–113 Secretory diarrhea, 341 Shigella, 341, 343
tests, 113–119 Sediment constituents, 178–179 Siemens Multistix Pro 10 reagent strips, 148
glomerular filtration, 113–116 artifacts, 209–211, 210–211f Sjögren syndrome, 221
tubular reabsorption, 116–119, 117–118f bacteria, 187–188, 188f SLE. see systemic lupus erythematosus (SLE)
tubular secretion and renal blood flow, 119 casts, 168–170, 171–172t, 191–199, 192–199f Sodium, 94t
Renal glycosuria, 226, 227t crystals, 171t, 199, 201–202t, 201–209, Sodium urates, 29, 201t
Renal lithiasis, 229–230 202–209f Specific gravity, 131–133, 131t
Renal physiology, 106–113, 107f epithelial cells, 182–187, 183–187f osmolality, 132–133, 134t
blood flow, 106, 108b expected staining reactions of, 172t reagent strip reaction, 133, 160–161, 160f
glomerular filtration, 106–109, 109f mucus, 190, 191f interference, 160–161
renin-angiotensin-aldosterone system (RAAS), parasites, 189, 189–190f refractometry, 131–132, 132f
106, 108–109, 110b, 110f red blood cells, 179–180f, 179–181 tubular reabsorption tests, 117, 117f
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400 Index

Specificity of testing, 69 Sternheimer-Malbin stain, 171, 171t Tubular concentration, 111

Specimen collection Subarachnoid space, 254 Tubular disorders, 222, 225–226
amniotic fluid, 329–330, 330b Sudan III stain, 171, 171t hereditary and metabolic, 225–226, 227t
bronchoalveolar lavage fluid, 322 Sulfonamide crystals, 37, 207t, 208–209, 209f Tubular proteinuria, 145
cerebrospinal fluid (CSF), 254–255, 255f Sulfosalicylic acid precipitation (SSA) test, 146, Tubular reabsorption, 106, 110–111, 110t
fecal, 342 146f renal glycosuria and, 226
semen, 278–280 Suprapubic aspiration, 97t, 98 tests, 116–119, 117–118f
serous fluid, 306–307 Supravital stains, 171, 172t Tubular secretion, 106, 111–113, 112–113f
urine Surface channel (SF ch) stains, 81 tests, 119
containers, 94 Surfactants, 333 Tubulointerstitial disease, 226
labels, 64, 64b, 95 Susceptible host, 51, 52f Turnaround time (TAT), 62–63
requisition form, 63, 95 Syncytia, 184 24-hour (or timed) specimen, 97–98, 97t, 98b
vaginal secretions, 357 Synovial fluid, 46, 294–301 2% acetic acid stain, 171, 171t
Specimen handling cell counts, 296 Tyrosine
amniotic fluid, 330–331 chemistry tests, 300–301 crystals, 34, 35, 207t, 208f
bronchoalveolar lavage fluid, 322 color and clarity, 295–296 metabolism, 237–239, 238f
cerebrospinal fluid (CSF), 254–255, 255f crystals, 296 Tyrosyluria, 236, 237–239
semen, 280 polarization, 298–300, 299–300f
synovial fluid, 294–295, 295t slide preparation, 297–298
urine types of, 297, 298t U
integrity, 95–96, 95t differential count, 296–297, 297t UDP-galactose-4-epimerase (GALE), 247
preservation, 96, 96t microbiological tests, 301 Universal Precautions (UP), 51
vaginal secretions, 357 physiology, 294, 294f, 294t Urea, 93, 94t
Specimen rejection, urine, 64, 64b, 95 serological tests, 301 clearance, 113
Specimens, urine specimen collection and handling, 294–295, Ureaplasma urealyticum, 360, 364
collection and handling, 63–64, 64b, 94–96 295t Uric acid, 26–27, 94t, 201t, 202–203, 203f
criteria for rejection of, 64, 64b viscosity, 296 in synovial fluid, 301
policy for handling mislabeled, 64, 64b Synoviocytes, 296 Urinalysis, 2–40, 76–88
preservation of, 96, 96t Syphilis artifacts, 38–40
rejection of, 64, 64b cerebrospinal fluid (CSF) testing for, 271 automation, 76–88
types of, 96–100, 97t secondary, 221 casts, 17–25
24-hour, 97–98, 97t, 98b Sysmex UD-10 Fully Automated Urine Particle cause-and-effect diagram for, 63f
catheterized, 97t, 98 Digital Imaging Device, 81, 82, 83f cells, 2–10
drug specimen collection, 100–101 Sysmex UF 1000i system, 77t, 80–82, 80–82f chemical examination, 140–161
first morning, 96–97, 97t Sysmex UF-5000 Fully Automated Urine Particle crystals, 26–37
four-glass collection, 97t Analyzer, 81, 82–85f history and importance, 92–93, 92–93f
midstream clean-catch, 97t, 98 Sysmex UN-2000 system, 77t, 81, 82, 82f microorganisms, 11–13
pediatric, 100 Sysmex XN-Series using Body Fluids mode, 77t, microscopic examination, 168–211
pre- and postmassage test (PPMT), 99 87–88, 88t miscellaneous structures, 16
prostatitis specimen, 97t, 98–100 Systemic lupus erythematosus (SLE), 220, parasites, 14–15
random, 96, 97t 221, 309f physical examination, 126–134
suprapubic aspiration, 97t, 98 Urinalysis Data Manager (UDM), 82
three-glass collection, 97t, 98–99 Urinalysis procedure manual, 62–70
Sperm, 16 T components of, 62
Spermatids, 280 Tamm-Horsfall protein (THP), 144 critical results, 69, 70f
Spermatozoa. see also semen Testes, 278 electronic controls, 67
antisperm antibodies, 286–287 Testing procedure, 66 examination variables, 64–67, 66–68f
concentration and count, 281–282, 282f Test utilization, 62 example of, 63f
function tests, 288, 288t Thin-layer chromatography (TLC), 330 external quality control, 66–67, 67f, 68f
morphology, 283–285, 284–285f Three-glass collection, 97t, 98–99 individualized quality control plan, 68–69,
motility, 283, 283t Thrombosis, 221 69f, 70f
production of, 278 Timed specimen, 97–98, 97t, 98b instrumentation and equipment, 64, 66, 66f
seminal fluid fructose, 286 Titratable acidity, 119 internal quality control, 67
in urine, 189–190, 190f Toluidine blue stain, 171t interpreting results, 69
vitality, 286 Total nucleated body fluid cell count (TC-BF), 88 personnel and facilities, 68
Spills, 54–57 Training, 62, 68 postexamination variables, 68–69, 69f
Spinal cord, 254–255f. see also cerebrospinal fluid Transitional epithelial (urothelial) cells, 7–8, 184, preexamination variables, 62–64, 63f, 64b, 65f
(CSF) 184f proficiency testing, 67
Spindle-shaped cells, 261t Transudates, 307, 308t, 314 quality control, 66
Squamous epithelial cells, 182–184, 183–184f Traumatic collection of cerebrospinal fluid (CSF), reagents, 64
vaginal secretions, 358, 359f 256–257 result errors, 69
Stamey-Meares test for prostatitis, 99–100 Trichomonads, 14 specimen collection and handling, 63–64, 64b
Standardization, 62 Trichomonas vaginalis, 14, 357, 360–361, testing procedure, 66
Standard Precautions (SP), 51–54, 52f, 94 361–362f, 363 Urinary ammonia, 119
Staphylococcus, 341, 343 Trichomoniasis, 356, 356t, 357, 360–361, Urinary tract infection (UTI), 11, 19, 20, 36,
Starch granules artifacts, 38, 210, 210f 315–362f, 363, 364 188, 226
Steatorrhea, 342 Triple phosphate, 202t, 204–205, 205f Urine
Stercobilinogen, 156 Tryptophan disorders, 241–242, 241f acidic, 201–204, 202–204f
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alkaline, 204–205, 204–206f UriSed 2 system, 77t, 85–86, 87 Vesicoureteral reflux (VUR), 226
bilirubin in, 154–156, 155f, 155t UriSed 3 system, 77t, 85–86, 87 Vibrio cholerae, 341, 343
blood in, 128, 128f, 129f, 152–154 Urisys 1100 system, 78f Viruses
chemical examination of, 140–161 Urisys 1800 system, 77t bronchoalveolar lavage (BAL) fluid,
clarity of, 129–131, 129t, 130b Urisys 2400 system, 77t, 79f 324, 324b
color of, 126–127t, 126–129 Urobilin, 127 Visceral membrane, 306
abnormal, 127–129, 128f, 129f Urobilinogen, 156–157 Viscosity
normal, 126–127, 128f clinical significance, 157 semen, 281
composition of, 93, 94t in jaundice, 155t synovial fluid, 296
crystals, 171t, 199, 201–202t, 201–209, production of, 155f Volume analysis
202–209f reaction strip reactions, 157 semen, 280
differentiation of amniotic fluid from maternal, interference, 157 urine, 93–94
329 Urochrome, 126–127 Volume of sediment, macroscopic screening, 169
disposal of, 54, 56–57 Uroerythrin, 127 Vulvovaginal candidiasis, 356, 356t
formation of, 93 Uromodulin, 144, 190
glucose in, 148–151 Uromodulin-associated kidney disease,
ketones in, 151–152 226, 227t W
leukocyte esterase in, 159–160 UTI. see urinary tract infection (UTI) Waste disposal
nitrite in, 157–159 UX-2000 system, 77t, 87 biological, 54, 56–57
odor of, 134, 134t chemical, 58
pH of, 142–143, 143t Watson-Schwartz test, 243, 245–246
physical examination of, 126–134 V Waxy casts, 24, 172t, 198, 198f
protein in, 143–148 Vaginal disorders, 364–365 Wet mount examination, vaginal secretions, 358,
sediment constituents (see sediment atrophic vaginitis, 356t, 357, 365 358t
constituents, urine) bacterial vaginosis (BV), 356, 356t, 364 White blood cells (WBCs), 3–5, 81, 168, 171t
specific gravity of, 117, 117f, 131–133, 131t candidiasis, 364–365 in acute glomerulonephritis (AGN), 220
specimen collection and handling, 63–64, 64b, desquamative inflammatory vaginitis (DIV), body fluid count, 88
65f, 94–96, 95t 356t, 357, 365 bronchoalveolar lavage (BAL) fluid,
urobilinogen in, 155f, 155t, 156–157 trichomoniasis, 356, 356t, 357, 360–361, 322–323
Urine chemistry analyzers, 76–88 361–362f, 363, 364 casts, 19, 194–195, 194–195f
fully automated, 77t, 78–79 Vaginal pool, 357 cerebrospinal fluid (CSF), 255f
semi-automated, 76–78, 77t, 78–79f Vaginal secretions, 356–367 in cerebrospinal fluid (CSF), 257–258, 257f
Urine screening, 236–247 color and appearance, 357, 357t esterase in, 159
alkaptonuria, 129, 236, 239 diagnostic tests, 357–363 peritoneal fluid, 314–315, 315–316f
amino acid disorders, 236–243, 238f, 240–241f microscopic procedures, 358–363, 358t, in semen, 280
branched-chain, 239–240, 240f 359–362f serous fluid, 307
carbohydrate disorders, 247 pH, 357–358 in synovial fluid, 296
cystine disorders, 236, 242–243 disorders and laboratory findings associated in urine, 181–182, 181–182f
melanuria, 236, 239 with, 356–357, 356t vaginal secretions, 358–359, 359f
metabolic disorders, 236t pregnancy-associated, 365–367 Work practice controls for bloodborne
mucopolysaccharide disorders, 246–247 specimen collection and handling, 357 pathogens, 53
newborn, 236, 237f Vaginitis World Health Organization (WHO), 278, 283
organic acidemias, 236, 240 atrophic, 356t, 357, 365
overflow versus renal disorders, 236, 237t desquamative inflammatory vaginitis (DIV),
phenylketonuria (PKU), 236, 237 356t, 357, 365 X
porphyrin disorders, 243–246, 244f, 245t pH and, 357 Xanthrochromia, 256
purine disorders, 247 Van de Kamer titration, 347–348 Xanthrochromic supernatant, 257
tryptophan disorders, 241–242, 241f Vapor pressure osmometers, 118 X-ray procedures, 38
tyrosyluria, 236, 237–239 Variegate porphyria, 245t
Urine sediment constituents. see sediment Vasa recta, 106
constituents, urine Vasectomy, 287 Y
Urine specimens. see specimens, urine Vasopressin (antidiuretic hormone [ADH]), 111 Yeast, 12–13, 168, 179, 179f, 188, 189f
Urine volume, 93–94 Vasovasostomy, 287 vaginal secretions, 358, 361, 362f
Urinometry, 133 Venereal Disease Research Laboratory (VDRL), 271 Yersinia, 343