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Prelims.indd 1 06-04-2016 16:29:09
 Principles and Interpretation of
Laboratory Practices in
Surgical Pathology

Prelims.indd 1 06-04-2016 16:29:09

Prelims.indd 2 06-04-2016 16:29:09
 Principles and Interpretation of
Laboratory Practices in
surgicaL PathoLogy

Shameem Shariff md phd

Professor and Head
Department of Pathology
Head
Department of Central Research
MVJ Medical College and Research Hospital,
Bengaluru Rural, Karnataka, India

Co-author
Amrit Kaur Kaler md
Associate Professor
ACS Medical College and Hospital
Chennai, Tamil Nadu, India

The Health Sciences Publisher

New Delhi | London | Philadelphia | Panama

Prelims.indd 3 06-04-2016 16:29:09

 Jaypee Brothers Medical Publishers (P) Ltd

Headquarters
Jaypee Brothers Medical Publishers (P) Ltd
4838/24, Ansari Road, Daryaganj
New Delhi 110 002, India
Phone: +91-11-43574357
Fax: +91-11-43574314
Email: [email protected]
Overseas Offices
J.P. Medical Ltd Jaypee-Highlights Medical Publishers Inc
83 Victoria Street, London City of Knowledge, Bld. 235, 2nd Floor, Clayton
SW1H 0HW (UK) Panama City, Panama
Phone: +44 20 3170 8910 Phone: +1 507-301-0496
Fax: +44(0) 20 3008 6180 Fax: +1 507-301-0499
Email: [email protected] Email: [email protected]
JP Medical Inc Jaypee Brothers Medical Publishers (P) Ltd
325 Chestnut Street 17/1-B Babar Road, Block-B, Shaymali
Suite 412, Philadelphia Mohammadpur, Dhaka-1207
PA 19106, USA Bangladesh
Phone: +1 215-713-718 Phone: +08801912003485
Email: [email protected] Email: [email protected]
Jaypee Brothers Medical Publishers (P) Ltd
Bhotahity, Kathmandu, Nepal
Phone +977-9741283608
Email: [email protected]

Website: www.jaypeebrothers.com
Website: www.jaypeedigital.com
© 2016, Jaypee Brothers Medical Publishers
The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not
necessarily represent those of editor(s) of the book.
All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any
means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of
the publishers.
All brand names and product names used in this book are trade names, service marks, trademarks or registered
trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in
this book.
Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative
information about the subject matter in question. However, readers are advised to check the most current
information available on procedures included and check information from the manufacturer of each product to be
administered, to verify the recommended dose, formula, method and duration of administration, adverse effects
and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither
the publisher nor the author(s)/editor(s) assume any liability for any injury and/or damage to persons or property
arising from or related to use of material in this book.
This book is sold on the understanding that the publisher is not engaged in providing professional medical services.
If such advice or services are required, the services of a competent medical professional should be sought.
Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce
copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary
arrangements at the first opportunity.

Inquiries for bulk sales may be solicited at: [email protected]

Principles and Interpretation of Laboratory Practices in Surgical Pathology

First Edition: 2016
ISBN 978-93-5250-024-6
Printed at

Prelims.indd 4 06-04-2016 16:29:09

 Dedicated to
Our families, who endured with patience our time and efforts,
Our trainees who will advance knowledge further,
Seekers of expertise, whose motivation results in innovations

Prelims.indd 5 06-04-2016 16:29:10

Prelims.indd 6 06-04-2016 16:29:10
 Preface

The laboratory has been given a central and distinctive role in science
education; science educators have suggested that there are rich benefits
in learning the accrue from using laboratory activities. Tissues from the
body, taken for diagnosis of disease processes, must be processed in the
histology laboratory to produce microscopic slides that are viewed under
the microscope by pathologists. The techniques for processing the tissues,
whether biopsies, larger specimens removed at surgery, or tissues from
autopsy, are varied and the persons who do the tissue processing and make
the glass microscopic slides are histotechnologists.
While pathologists and cytologists are mainly involved in reporting and
interpretation of slides, if the standard criteria for slide preparation are not
adhered, this makes the interpretation extremely difficult. As diagnostic
personnel, pathologists and cytologists should be aware of all techniques
used in the laboratory and have a sound knowledge of the fallacies involved
in the preparation. The knowledge of the techniques, which includes
processing of specimens, fluids and preparation of these so as to enable
them to be transferred to slide material, is all part of the training program for
pathologists.
The present book deals exhaustively with methods of processing of tissue
including fixation, embedding and cutting, staining techniques and an
extensive know-how to combat fallacies in these. The recent techniques with
regard to molecular pathology are also dealt with in detail. In addition, certain
principles of interpretation are also outlined. In fact, the entire book is user-
friendly manual for pathologists, cytologists, cyto- and histotechnologists
as well as postgraduate students. It has incorporated our vast experience in
cyto- and histopathology.

Shameem Shariff
Amrit Kaur Kaler

Prelims.indd 7 06-04-2016 16:29:10

Prelims.indd 8 06-04-2016 16:29:10
 Acknowledgments

Our sincere thanks to Dr MJ Mohan, Chairman, MVJ Medical College &

Research Hospital, Bengaluru Rural, Karnataka, India, for providing the
facilities, atmosphere and environment at the institution, which has enabled
us to work towards the completion of this project.
Our sincere thanks to our families whose patience has enabled this book
to see the light of the day.
Our thanks are also due to our trainees Dr Pritha Aggarwal, Dr Hasrath
Mohammedi and Dr Rajeshwari G, whose efforts in the completion of the
compilation are acknowledged.
We are also thankful to Shri Jitendar P Vij (Group Chairman), Mr Ankit Vij
(Group President), Mr Tarun Duneja (Director–Publishing) and staff of
Bengaluru Production Unit/Bengaluru Branch of M/s Jaypee Brothers
Medical Publishers (P) Ltd, New Delhi, for their support.

Prelims.indd 9 06-04-2016 16:29:10

Prelims.indd 10 06-04-2016 16:29:10
 Contents

1. Fixation 1
Aims 1
Qualities of a Good Fixative 1
Classification of Fixatives 2
Fixation Methods 3
Types of Fixatives 8
Mailing of Unstained Smears 15
Prefixation of Cytological Material 15
Rehydration of Air-dried Smears 15
Factors Affecting Fixation 15
Fixation of Individual Tissues 16
Softening Hard Tissues 17
2. Decalcification 18
Types of Bone and Decalcification 18
Bone Techniques 18
Methods of Decalcification 20
Endpoint of Decalcification 23
Factors Affecting the Rate of Decalcification 25
Morphometry of Bone 25
3. Tissue Processing 27
Tissue Sampling 27
Principles of Tissue Processing 27
Initial Precautions Before Processing 28
Stages of Tissue Processing 28
Automated Tissue Processing Methods 35
General Considerations During Processing 39
Tissue Recovery Procedures 39
Tissue Processing Methods 40
4. Tissue Embedding 45
Embedding Tissues in Paraffin Wax 45
Technique of Embedding 46
Different Methods of Embedding 46
Paraffin Oven or Incubators 48
Orientation of Specimens, While Embedding 48
Tissue Marking Substances 48
Alternative Embedding Media 49

Prelims.indd 11 06-04-2016 16:29:10

 xii Principles and Interpretation of Laboratory Practices in Surgical Pathology

Embedding Technique 52
Double Embedding and Double Infiltration Methods 53
5. Microtomy 55
Historical Aspects 55
Principles of Microtomy 55
Classes of Microtomes 56
Care of a Microtome 62
Microtome Knives: Types and Parts 63
Care of a Microtome Knife 67
6. Section Cutting 72
General Principles of Microtomy 72
Paraffin Section Cutting 72
Frozen Section Cutting 77
Procedure for Ultramicrotome 78
7. Introduction to Staining and Principles of Staining 79
Types of Staining 79
Classification of Stains 79
Methods of Staining 81
Metachromatic Staining 81
Theories of Staining 82
Principle of Staining Methods 83
Types of Differentiators 83
Hematoxylin and Eosin Stain 84
8. Special Histochemical Stains 96
Histochemical Staining 96
Mucicarmine Stain 99
Alcian Dyes 100
Periodic Acid-Schiff’s Reaction (Schiff’s Leucofuchsin) 103
Combined Alcian Blue and PAS Reagent 106
Stains for Glycogen 107
Stains for Sulfated Acid Mucins 109
Other Stains to Detect PAS-positive Acid Mucins 110
Use of Metachromasia in Mucin Stains 112
Connective Tissue Stains 112
Special Stains for Microorganisms 126
Stains for Pigments and Minerals 137
Practical Applications of Histochemical Techniques 145
9. Special Biopsies 149
Kidney Biopsy 149
Liver Biopsy 151
Muscle Biopsy 153
Nerve Biopsy 155
Brain Biopsy 158
Endomyocardial Biopsy 160
Bone Marrow Trephine Biopsy 162
Testicular Biopsy 163

Prelims.indd 12 06-04-2016 16:29:10

 Contents xiii
10. Immunohistochemistry and Immunofluorescence 165
Immunohistochemistry 165
Antigen Retrieval 175
Blocking the Endogenous Activity 176
Types of Primary Antibodies 177
Chromogens Used for Visualization of Reactions 179
Immunofluorescence 183
11. Cytopreparatory Techniques 187
Body Cavities in Disease 187
Diagnostic Cytopathology 187
Collection/Preservation/Examination of Body Fluids 190
Method of Processing 192
Hydrocele Fluid 200
Synovial Fluid 200
Cerebrospinal Fluid 206
Urine 209
Cytology of the Gastrointestinal Tract 218
Cytology of Respiratory Tract 220
Intraoperative Cytology 224
Conjunctival Impression Cytology 226
Female Genital Tract 228
Fine Needle Aspiration Cytology 238
Stains Specific in Cytology 244
Cytology and Indexing 248
Systematized Nomenclature of Pathology 248
Organization of Cytopathology Laboratory 255
12. Quality Control in Cytopathology 261
Terminologies 261
Maintaining Quality Control 262
13. Quality Control in Histopathology 269
Quality Control/Quality Assurance 269
Diagnostic Standards 269
Phases of Quality Control in Histopathology 272
14. Telepathology and Telecytology 275
Telepathology 275
Telecytology 279
15. Molecular Diagnostic Pathology 281
In Situ Hybridization 281
Fluorescence In Situ Hybridization 285
Tissue Microarray 287
Flow Cytometry 289
Polymerase Chain Reaction 291

Prelims.indd 13 06-04-2016 16:29:10

 xiv Principles and Interpretation of Laboratory Practices in Surgical Pathology

16. Laboratory Waste Management 294

Impact of Improper Waste Management 294
Principles and Objectives of Waste Management 294
Healthcare Waste Generation 295
Categories of Waste 295
Hospital Waste Disposal 295
17. Principles in the Functioning of Microscopes 300
Historical Aspects 300
Lenses 300
Compound Microscope 302
Fluorescence Microscope 309
Electron Microscope 314
Confocal Microscopy 315
Index 319

Prelims.indd 14 06-04-2016 16:29:10

 Chapter

1 Fixation

DEFINITION
Fixation is the process by which the constituents of the cells are fixed in a
physical and partly chemical state so that they will withstand subsequent
treatment with various reagents with minimal loss of significant distortion or
decomposition and keep the tissue in as life-like manner as possible.

AIMS
1. Should stop bacterial degeneration, autolysis and putrefaction.
2. Should not distort the cellular constituents by swelling or shrinkage, and
maintain as close a resemblance as possible to the natural structure of
tissue components.
3. The tissue should withstand the chemicals used at various stages of
processing.
4. Allow clear staining of the sections.
5. To increase tissue consistency to permit the cutting of thin slices of
tissue at varying microns.
6. To increase optical differentiation of cellular structures.

QUALITIES OF A GOOD FIXATIVE

An ideal fixative is one, which provides:
1. Good tissue penetration.
2. Stabilizes the tissue, preserving the character and distribution of cellular
components.
3. Prevents fixation artifacts.
4. Prevents structure deformation, maintaining shape and volume (is
isotonic).
5. Preserves cellular constituents.
6. Safe to handle—nontoxic and nonallergenic.
An easy way to remember is ‘PRISM’, Penetrate tissue easily, Rapid
in action, Isotonic, Stable and safe to handle, Minimal loss or damage to
physical and chemical composition of tissue and its components. Effects of
fixation on tissues include:
 2 Principles and Interpretation of Laboratory Practices in Surgical Pathology

1. Hardening of tissues.
2. Rendering the cells insensitive to hypotonic and hypertonic solutions.
3. Aiding or inhibiting staining.

CLASSIFICATION OF FIXATIVES
Based on Components
Fixatives are classified based on the components present as follows:
1. Simple fixative: Contains a single chemical, e.g. formaldehyde (10%
formalin), glutaraldehyde, ethyl alcohol, etc.
2. Compound fixative: Contains more than one chemical and used as
mixtures. Advantage is due to the unequal affinity of each substance for
various structural elements:
a. Formalin based: 10% neutral buffered formalin, 10% neutral buffered
formol saline and formol calcium.
b. Mercurial fixatives: Zenker’s solution, Helly’s solution and B5
fixative reagent.
c. Dichromate fixatives: Regaud’s solution, Möller’s solution and
Orth’s solution.
d. Picric acid fixatives: Bouin’s solution and Gendre’s fluid.
e. Alcohol-containing fixatives: Carnoy’s and acetic alcohol formalin
(AAF).

Based on the Action on Tissues

Fixatives are classified based on the action on tissues or cells as follows:
1. Fixatives used in histopathology: They are of two types:
a. Microanatomical fixatives: Preserves the microscopic structure of
the tissue, e.g. formol saline, formol calcium, Zenker’s fluid, etc.
b. Histochemical fixatives: To demonstrate enzymes, e.g. buffered
neutral formalin absolute alcohol.
2. Fixatives used in cytopathology: These are used to preserve
intracellular structures. They are divided into two types:
a. Nuclear fixatives: Carnoy’s fluid, Clarke’s fluid and Flemming’s fluid.
b. Cytoplasmic fixatives: Champy’s fluid and alcohol fixatives.

Based on Their Mode of Action

Fixatives are classified based on their mode of action:
1. Physical methods, e.g. heating, microwaving and freeze drying.
2. Chemical methods, examples are as follows:
a. Aldehydes (cross-linking): Formaldehyde, glutaraldehyde and
acrolein mercurial fixatives.
b. Oxidizing agents: Osmium tetroxide and chromate-containing
fixatives.
c. Protein denaturation (coagulants/dehydrants): Acetic acid, methyl
alcohol and ethyl alcohol.
 Chapter 1: Fixation 3
FIXATION METHODS
Heat Fixation
Heat fixation is the usual mode of preparing bacteriological smears. It
generally preserves overall morphology, but not internal structures. After a
smear has dried at room temperature, the slide is gripped by tongs and passed
through the flame of a Bunsen burner several times, to ‘heat kill’ and adhere
the organism to the slide. This is routinely used with bacteria. Heat denatures
the proteolytic enzyme and prevents autolysis. Heat fixation cannot be used
in the capsular stain method as heat fixation shrinks or destroys the capsule
(glycocalyx), which cannot be seen in stains. It is also used in combination
with formal saline.

Heat Fixation on Tissues

The tissue is placed in 20–40 mL of fluid (10% formal saline) and heated
below the boiling point over the spirit flame for 1 minute or until the tissue
floats on the surface. Then it is cooled immediately and the tissue is taken
for processing. This method is generally employed fixation before frozen
section.

Microwave Fixation
Microwave fixation is a well-established technique. It provides better fixation
than direct heating. In heat fixation, the energy is absorbed from the outer
layer and transferred slowly to the rest of the substance, while microwave
provides a homogeneous rise in temperature (controlled heating) and all
the molecules take up energy simultaneously by a diffusion process. This
overcomes the problem of erratic heating by the direct flame.

Principle
Microwave energy interacts with the bipolar molecules resulting in heat
denaturation and disulfide bond formation. This process is called microwave
stabilization. There is a significant cross-linking of protein molecules with
subsequent chemical fixation.

Applications
1. For routine histopathology light microscopy techniques includes
staining (standard and special), mucous substance histochemistry,
enzyme histochemistry and immunocytochemistry (ICC).
2. For electron microscopy.
3. For rapid fixation of routine surgical specimens and especially for
processing urgent cardiac and renal biopsies.
4. For the preparation of botanical and insect material.
 4 Principles and Interpretation of Laboratory Practices in Surgical Pathology

Procedure
The tissue is irradiated directly or irradiated after immersing in formalin
solution for a period of 4 minutes, followed by irradiation in buffered formalin
for another 4 minutes. Alternatively, the tissue is immersed in formalin for
4 hours, followed by microwave irradiation for 1½ minutes in saline, which
gives superior results. The optimal temperature required is 45–55°C. Less
heating causes poor sectioning quality, while overheating causes vacuolation
of the cytoplasm and nuclear pyknosis.

Advantages
1. It reduces the time for fixation from 12 hours to less than 20 minutes.
2. There is little difference in the volume changes in tissues fixed by
microwaves compared with conventional formaldehyde-fixed material.
3. Microwave accelerates staining and has no deleterious effect on special
staining.
4. Microwave treated tissues (50°C) postfixed in osmium tetroxide gives
good results in electron microscopy.
5. It is a prerequisite for immunohistochemical staining methods as it
stops the extraction of proteins from tissue.
6. Tissue antigens are better preserved.

Freeze Drying and Fixation

Fresh tissues can be frozen with the following:
• Liquid nitrogen (–190°C)
• Isopentane cooled by liquid nitrogen (–150°C)
• Dry ice [solid carbon dioxide (–70°C)]
• Carbon dioxide gas (–70°C)
• Aerosol sprays (–50°C).

Procedure
The common procedures used in most laboratories are:
1. Cryostat sections: In this procedure, tissues are hardened by freezing to
temperature as low as –30 to –50°C using a coolant in the cryostat, e.g.
Freon 22. Small pieces of tissue sent for rapid diagnosis are transferred
onto a chuck of the cryostat in a few drops of optimum cutting
temperature (OCT) medium. The metal weight in the cryostat is placed
on this for a couple of minutes. This helps in fixation. The chuck is then
transferred to the cryotome and serial sections are cut and stained.
2. Quenching procedure: Detailed as follows:
a. The tissue is cut into thin sections (1 mm thick) and placed in a beaker
of isopentane suspended and snap frozen in a flask of liquid nitrogen
gas at –150°C (isopentane is an extremely volatile and extremely
flammable liquid, at room temperature and pressure). This process
is known as quenching. This rapid freezing prevents the formation of
 Chapter 1: Fixation 5
ice crystals and preserves the tissue. If only liquid nitrogen is used,
it forms vapor bubbles around the tissue, thus producing artifacts
around the tissue.
b. The tissue is then transferred to the drying chamber, which is under
vacuum and at a higher temperature of –30°C. The ice (tissue water) is
removed by sublimation, the water vapor being absorbed by a drying
agent such as phosphorus pentoxide. The tissue is impregnated in the
embedding medium under reduced pressure as detailed below:
i. Transfer the dried tissue quickly to a vacuum-embedding
medium, i.e. oven containing molten wax.
ii. On sinking to the bottom of the bath, the tissue will be impregnated
with wax and takes approximately 10 minutes for complete
impregnation.

Advantages
1. Give better preservation of antigenicity by either ICC or
immunohistochemistry (IHC).
2. Not exposed to the organic solvents and therefore minimal chemical
alteration of proteins.
3. Minimizes the denaturation of proteins or inactivation of enzymes and
particularly useful for enzyme studies in neuropathology as tissues are
processed fresh.
4. Little shrinkage of tissue.

Disadvantages
• Lacks precise morphological detail
• Presents a potential biohazard
• Restricted to research laboratory
• Formation of ice crystal artifacts.

Chemical Fixation
Formaldehyde (Aldehydes)
Characteristics
Formaldehyde is commercially available as formalin (40% formaldehyde
dissolved in water) and the same is taken as 100% formalin. It is also available
as a stable solid form known as paraformaldehyde. Various forms in use are:
1. 10% formalin: This is most commonly used form in laboratories,
which contains:
a Formalin (40% of formaldehyde dissolved in water): 10 mL.
b Water: 90 mL.
This in essence is 4% formaldehyde.
2. 10% formol saline:
a. Distilled water: 90 mL.
b. Formalin: 10 mL.
c. Sodium chloride: 0.9 g.
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