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Prelims 9/9/02 12:29 pm Page i

Endocrinology in Clinical Practice

Prelims 9/9/02 12:29 pm Page ii

Philip E Harris trained in endocrinology at St Bartholomew’s

Hospital, London and The University of Newcastle upon Tyne.
In 1990-91, Dr Harris worked as an MRC Travelling Fellow at
the Endocrine and Reproductive Endocrine Units, Massachusetts
General Hospital, Boston, USA. In 1994, he was appointed
Senior Lecturer and Consultant Endocrinologist at King’s
College Hospital, London. His main clinical and research inter-
est is in the field of endocrine oncology, in particular pituitary
disease. In April 2001, Dr Harris was appointed Medical
Director in Adult Endocrinology Europe, Pharmacia
Corporation. He also holds an Honorary Consultant appoint-
ment in Endocrinology at St Bartholomew’s Hospital, London.

Pierre-Marc G Bouloux is presently Director of the Centre for

Neuroendocrinology at the Royal Free and University College
of Medicine Schools. He was an MRC Training Fellow under
Michael Besser at St Bartholomew’s Hospital, London and was
subsequently Lecturer in Medicine at the University
Department of Medicine at St Bartholomew’s. He has held his
present post since 1991 and in addition to running a clinical
service, he has a special interest in neuroendocrinology. He has
published over 150 peer-reviewed publications.

Beverly MK Biller is an associate professor of Medicine at Harvard

Medical School, Boston, Massachusetts and on the staff of
Massachusetts General Hospital, Boston. She is a supervising
physician in the multidisciplinary Neuroendocrine Clinical
Center there, and an attending physician on the Endocrinology
Consultation Service at Massachusetts General Hospital, where
she also serves as Director of the Clinical Fellowship in
Endocrinology. Dr. Biller is an associate editor of Journal of
Clinical Endocrinology and Metabolism. Research interests include
neuroendocrine causes of osteoporosis, hormone-secreting
pituitary adenomas, and growth hormone deficiency in adults.
Prelims 9/9/02 12:29 pm Page iii

Endocrinology
in Clinical Practice
Philip E Harris
Honorary Consultant Endocrinologist
St Bartholomew’s Hospital
London
UK
Pierre-Marc G Bouloux
Director, Centre for Neuroendocrinology
Royal Free and University College
of Medicine Schools
London
UK
Editors

Beverly MK Biller
Neuroendocrine Unit
Massachusetts General Hospital
Boston MA
USA
Associate Editor

Foreword by Larry Jameson

Chief, Division of Endocrinology,
Metabolism and Molecular Medicine
Northwestern University
Chicago IL
USA

iii
CRC Press
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Prelims 9/9/02 12:29 pm Page v

Contents

Contributors vii
Foreword xi
Preface xii
1. Interpretation of biochemical investigations
Joan Butler
2. Genetic mechanisms of pituitary and thyroid neoplasia
Philip E Harris
3. Neuroendocrine disease
Philip E Harris
4. Growth and growth disorders
John Miell, Annice Mukherjee
5. Carcinoma of the thyroid
Philip E Harris
6. Multiple endocrine neoplasia
Bin Tean Teh, Catharina Larsson
7. Amenorrhea and infertility
Beverley Vollenhoven, Henry Burger
8. Hypogonadism, erectile dysfunction and infertility in men
Shalender Bhasin, Atam B Singh, Charles E Fisher
9. Hirsutism and virilization
Frances J Hayes, Janet E Hall
10. Autoimmune disease
Anthony P Weetman
11. Non-autoimmune thyroid disease
Soo-Mi Park, Luca Persani, Paolo Beck-Peccoz, Krishna Chatterjee
12. Multinodular goiter, toxic adenoma and thyroiditis
Arie Berghout, Alex F Muller
Prelims 9/9/02 12:29 pm Page vi

13. Calcium disorders and bone diseases

Laura Masi, Alberto Falchetti, Luigi Gennari, Maria Luisa Brandi
14. Endocrinology and systemic disease
R Andrew James, Richard Quinton, Steven G Ball
15. Disorders of fluid and electrolytes
Pierre-Marc G Bouloux
16. Endocrine hypertension
Jennifer E Lawrence, Robert G Dluhy
17. Obesity
Pierre-Marc G Bouloux
18. Psychoneuroendocrinology
Salim Janmohamed, Ashley B Grossman
19. Endocrine emergencies
Victor HF Hung, John P Monson
Appendix 1 Pituitary function testing
William M Drake, Peter J Trainer
Appendix 2 Pharmacopoeia
Pierre-Marc G Bouloux and Jessica M Kubie
Appendix 3 Reference values
Index
Prelims 9/9/02 12:29 pm Page vii

Contributors

Steven G Ball Krishna Chatterjee

Senior Lecturer and Honorary Consultant Physician Professor, Department of Medicine
The Endocrine Unit University of Cambridge
Royal Victoria Infirmary Addenbrooke’s Hospital
Newcastle-upon-Tyne Cambridge
UK UK
Paolo Beck-Peccoz Robert G Dluhy
Institute of Endocrine Sciences Professor of Medicine, Harvard Medical School
University of Milan Brigham and Women’s Hospital
Milan Boston MA
Italy USA
Arie Berghout William M Drake
MCRZ-Zuider Department of Endocrinology
Rotterdam St Bartholomew’s and The Royal School of Medicine
The Netherlands and Dentistry
St Bartholomew’s Hospital
Shalender Bhasin London
Professor of Medicine UK
UCLA School of Medicine
Chief, Division of Endocrinology, Metabolism Alberto Falchetti
and Molecular Medicine Endocrinologist and Genetist Assistant in Research
Charles R Drew University of Medicine and Science Department of Internal Medicine
Los Angeles CA University of Florence
USA Florence
Italy
Maria Luisa Brandi
Full Professor of Endocrinology Charles E Fisher
Department of Internal Medicine Charles R Drew University of Medicine and Science
University of Florence Los Angeles CA
Florence USA
Italy
Luigi Gennari
Henry Burger Endocrinologist Assistant in Research
Director, Prince Henry’s Institute of Medical Research Department of Internal Medicine
at Monash Medical Centre University of Florence
Clayton, Victoria Florence
Australia Italy
Joan Butler Ashley B Grossman
Honorary Lecturer Professor of Neuroendocrinology
Department of Clinical Biochemistry Department of Endocrinology
Guy’s, King’s and St Thomas’ School of Medicine St Bartholomew’s Hospital
London London
UK UK
Prelims 9/9/02 12:29 pm Page viii

CONTRIBUTORS

Janet E Hall Jennifer E Lawrence

Assistant Professor in Medicine, Harvard Medical Brigham and Women’s Hospital
School and Reproductive Endocrine Unit Boston MA
National Center for Infertility Research USA
Massachusetts General Hospital
Boston MA Laura Masi
USA Department of Physiopathology
Endocrine Unit
Frances J Hayes University of Florence
Instructor in Medicine, Harvard Medical School Florence
and Reproductive Endocrine Unit Italy
National Center for Infertility Research
Massachusetts General Hospital John Miell
Boston MA Senior Lecturer & Consultant Physician
USA Guy’s, King’s and St Thomas’ School of Medicine
London
Victor HF Hung UK
Department of Endocrinology
St Bartholomew’s and The Royal School of Medicine John P Monson
and Dentistry Department of Endocrinology
St Bartholomew’s Hospital St Bartholomew’s and The Royal School of Medicine
London and Dentistry
UK St Bartholomew’s Hospital
London
R Andrew James UK
Consultant Physician & Senior Lecturer
The Endocrine Unit Annice Mukherjee
Royal Victoria Infirmary Registrar in Endocrinology
Newcastle-upon-Tyne Christie Hospital
UK Manchester
UK
Salim Janmohamed
Lecturer in Medicine Alex F Muller
Department of Endocrinology Hôpital Cantonal Universitaire
St Bartholomew’s Hospital Geneva
and Department of Medicine Switzerland
Salisbury District Hospital
Salisbury Soo-Mi Park
UK Department of Medicine
University of Cambridge
Jessica M Kubie Addenbrooke’s Hospital
University of London Cambridge UK
London
UK Luca Persani
Institute of Endocrine Sciences
Catharina Larsson University of Milan
Department of Molecular Medicine Milan
Karolinska Hospital Italy
Stockholm
Sweden
Prelims 9/9/02 12:29 pm Page ix

CONTRIBUTORS

Richard Quinton Peter J Trainer

Consultant Physician and Senior Lecturer Christie Hospital
The Endocrine Unit Manchester
Royal Victoria Infirmary UK
Newcastle-upon-Tyne
UK Beverly Vollenhoven
Senior Lecturer
Atam B Singh Department of Obstetrics & Gynaecology
Charles R Drew University of Medicine and Science Monash Medical Centre
Los Angeles CA Clayton, Victoria
USA Australia

Bin Tean Teh Anthony P Weetman

Senior Principal Investigator Section of Medicine
Laboratory of Cancer Genetics Division of Clinical Sciences (North)
Van Andel Research Institute Northern General Hospital
Grand Rapids MI Sheffield
USA UK
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Prelims 9/9/02 12:29 pm Page xi

Foreword

The practice of endocrinology is based upon a solid foundation of genetics, biochemistry, cell signaling,
and physiology. In most cases, the clinical manifestations of hormonal disorders can be explained by
understanding the physiologic role of hormones - whether deficient or excessive. The conceptual
framework for understanding hormone secretion, hormone action, and principles of feedback control
allows the practitioner to design a logical diagnostic approach using appropriate laboratory testing
and/or imaging studies. This body of knowledge arms the clinician with abundant evidence for
decision-making and makes endocrinology a rewarding field for clinical practice.

Like most fields of medicine, the knowledge base in endocrinology is changing rapidly. In addition
to the dramatic advances generated from genetics and molecular biology, the field has benefited from
the introduction of an unprecedented number of new drugs, particularly for the management of
diabetes and osteoporosis. Common diseases like diabetes, hypertension, obesity, osteoporosis, and
polycystic ovarian syndrome have also been the subject of numerous large-scale clinical trials that
provide powerful evidence for medical decision-making. These rapid changes in endocrinology
mandate that physicians continuously update their knowledge base and clinical skills.

Endocrinology in Clinical Practice is a valuable and practical resource for continuing education and
patient management. The chapters are based on common clinical presentations, such as Obesity,
Hirsutism and Virilization, Carcinoma of the Thyroid, and Endocrine Emergencies. In addition to
consultations for these and other specific endocrine diseases, we are often called upon to provide
advice about hormone aberrations or electrolyte management in complex clinical situations. The
chapters on Endocrinology and Systemic Disease, Disorders of Fluids and Electrolytes, and
Psychoneuroendocrinology exemplify how the book is in synch with clinical practice. The appendices
summarizing Pituitary Function Testing and Pharmacopoeia provide a wealth of practical information
that should be kept close at hand in the clinic. The book also places special emphasis on how new
insights into molecular medicine can enhance our understanding of disease pathogenesis. As
examples, the chapters on Genetic Mechanisms of Pituitary and Thyroid Neoplasia, Multiple
Endocrine Neoplasia, and several others are timely reviews of these rapidly developing fields.

The authors are a highly select international group of authorities, chosen for their expertise and
writing skills. The illustrations are uniformly clear and the book is greatly enriched by numerous
photomicrographs of patients and radiologic images using CT and MRI. Endocrinology in Clinical
Practice is current, authoritative, and practical. The student, general practitioner, and endocrine
specialist will each use this book to improve the care of patients with hormonal disorders.

J. Larry Jameson,
Northwestern University, Chicago, USA
Prelims 9/9/02 12:29 pm Page xii

Preface

The aim of this book is to provide cutting edge information on clinical practice in a practical format. The book is
aimed primarily at practising endocrinologists and doctors training in endocrinology. A working knowledge of the
subject both at the preclinical and clinical levels is assumed. In consequence, there is little in the way of basic
physiology and biochemistry. A chapter on the genetic mechanisms of pituitary and thyroid neoplasia is included to
provide two models of the way in which oncology is developing in endocrinology. Molecular biology is covered where
relevant to disease pathology, such as the multiple endocrine neoplasia syndromes and thyroid hormone resistance.

We have attempted to take a problem-oriented approach to endocrinology. Chapters tend to cover general topics
rather than specific disease conditions. In consequence, a number of topics are tackled from varying perspectives
in different chapters of the book. Well recognised subjects tend to be covered in a fairly brief manner, the bulk of
the emphasis being reserved for new subject areas.

The importance of the laboratory in endocrinology is emphasised by the first chapter, which concentrates on
pitfalls in the interpretation of biochemical investigations. We have included normal ranges because we believe
that these will provide a useful reference, notwithstanding the fact that these may differ from those of local
laboratories. Endocrine investigations are covered in detail in each chapter. Nevertheless, we have provided
guidelines for pituitary function testing in Appendix 1, as these tests are central to endocrine practice in most
major centres.

Details of pharmacological treatments are included in each chapter. We have, however, included a separate
pharmacopoeia as Appendix II, as we feel that this will facilitate the practical use of this book in the clinic.

We are most grateful to the many contributors to this book. We must extend our gratitude to our families for
their patience and support whilst the book has been written and edited.

Philip E Harris
Pierre MG Bouloux
CH 01 9/9/02 10:30 am Page 1

1
Interpretation of biochemical investigations
Joan Butler

Introduction Pulsatile secretion

Biochemical data in endocrinology, the results of assays Although many hormones are secreted in a pulsatile
for hormones, offer considerable problems with inter- manner, if the half-life in blood is sufficiently long,
pretation. Hormone concentrations must always be then a single blood sample may give an adequate
considered in context, i.e. with knowledge of the estimate of secretion. This is not true for all hormones;
concentrations of the other components of that for example, for growth hormone (GH), random
endocrine system, the state of other endocrine systems samples are rarely of any value. If a random sample
and the capabilities of the assays used. In particular, from a neonate gives a high level, GH deficiency
data must be interpreted in the context of the clinical might be ruled out, and similarly an undetectable
history and examination of the patient. It would be value from an adult might be considered to exclude
convenient to have strict guidelines and cutoffs for acromegaly, but this is generally an expensive and
endocrine test results, but these are neither available sometimes frankly misleading route to diagnosis. The
nor appropriate. The aim of this chapter is to consider word ‘pituitary’ in a possible diagnosis should not
the interpretation of data provided by hormone assays, generate a list of requests for every pituitary hormone.
highlighting by example circumstances in which
erroneous results might arise or erroneous conclusions
be drawn. Diurnal variation
Diurnal rhythms exist for many hormones, though the
change during the day may be small enough to be
The state of the patient ignored. Where the change is large, laboratories have
reference ranges only for specified times, for example
for cortisol only at 0800–1000 hours and at 2300–
The steady state 2400 hours; sampling at other times of day may yield a
Vital to accurate (valid) interpretation of assay results result which is not interpretable. Diurnal rhythms may
is a knowledge of the state of the patient at the time be disturbed by admission to hospital and by stress.
of blood or urine sampling. If the patient is not in a It has been known for some time that men show a
steady state, then some or all of the components of the diurnal rhythm for testosterone, but the size and vari-
feedback system being investigated may fall outside ability of the diurnal change have generally been
their respective reference ranges for the time being. ignored in clinical practice. If blood samples are taken
Obvious examples are: adjustment to thyroxine (T4) in the afternoon, values conspicuously below the
replacement therapy, when free T4 and thyroid-stimu- morning reference range may be found in the presence
lating hormone (TSH) may take days or weeks to arrive of normal gonadotropins (luteinizing hormone (LH);
at their final values for a given dose of T4; recovery follicle-stimulating hormone (FSH)), possibly leading
from surgical stress, especially cortisol and prolactin; to the false diagnosis of pituitary insufficiency.1
and intercurrent serious non-endocrine illness.

1
CH 01 9/9/02 10:30 am Page 2

INTERPRETATION OF BIOCHEMICAL INVESTIGATIONS

Menstrual cycle cortisol the hormone and hydrocortisone the pharmaceu-

tical agent are the same compound, and have attempted
Tests of reproductive hormones in premenopausal to diagnose primary adrenal failure after the patient has
women must be done at the appropriate stage of the received 100 mg of intravenous hydrocortisone.
menstrual cycle. Day 5–7, mid-follicular phase, is Apparently unrelated medication is another source
chosen for gonadotropin assay, because of the certainty of of error,2 e.g. the effect of estrogens, which raise the
this date and the clear separation from the time of ovula- concentration of cortisol-binding globulin (CBG). A
tion and the associated large changes in hormone levels. separate reference range for cortisol is required for
Day 21, i.e. 7 days after presumed ovulation, is required patients on estrogen therapy, possibly including
for progesterone assay for detection of ovulation. If there women on oral replacement therapy, because of the
is doubt about the length of the cycle, then more than first-pass effect on the liver. Some psychotropic drugs,
one sample should be sent and the timing determined including some drugs of abuse, cause elevations of
retrospectively, the chosen sample being the nearest to 7 prolactin, occasionally to very high levels; medication
days before the date of onset of the next menstrual should be the first possibility considered for the cause
period. Results from amenorrheic patients should be of raised prolactin. Heparin in vivo activates lipases,
compared to ranges for mid-follicular-phase samples. and the resulting increase in free fatty acid concentra-
tions may displace thyroid hormones from albumin
and produce a transient increase in free T4.
Table 1.1 gives some examples of commonly occur-
Medication ring interference from medication.
It might be considered obvious that medication could
invalidate testing for related substances, but requests for
cortisol assay to assess residual adrenal function in Changes in specific binding protein
patients on prednisolone are common. Most antibodies to
cortisol show cross-reaction with prednisolone, a closely
concentrations
related steroid. Moreover, generations of doctors, finding For those hormones which circulate in the blood
steroids a difficult subject, have failed to realize that largely bound to specific serum proteins, disorders

Medication Hormone Effect

Hydrocortisone Cortisol Grossly raised

Prednisolone Cortisol Apparently raised
Estrogens Cortisol Raised (CBG raised)
Testosterone (women) Raised (SHBG raised)
Total T4 Raised (TBG raised)
Oral contraceptives FSH and LH Suppressed
Dopamine antagonists Prolactin Raised
(many antiemetics,
most tranquilizers)
Some drugs of abuse Prolactin Raised
Heparin Free T4 Raised (transiently)
Salicylates Free T4 Raised (transiently)

FSH, follicle-stimulating hormone; CBG, cortisol-binding globulin; SHBG, sex hormone-binding globulin; TBG, thyroxine-binding
globulin.

Table 1.1
Some commonly occurring effects of medication on hormone levels.

2
CH 01 9/9/02 10:30 am Page 3

THE STATE OF THE PATIENT

which affect the concentration of the binding protein as albumin are also abnormal. Opinion is divided on
will affect the total concentration of hormone, even the utility of measuring SHBG and calculation of the
though the free or unbound hormone level (thought to testosterone/SHBG ratio, but unsuspected changes in
be the biologically active fraction) may be unaffected. SHBG can be revealed, e.g. low values in patients with
The principal hormones in this category are thyroid pituitary tumors. In women, increased testosterone
hormones, cortisol and testosterone. It is difficult to secretion produces a decrease in SHBG concentration,
remove the protein-bound fraction, or to assay the free and these two effects can balance out to give normal
fraction in the presence of the protein-bound fraction, total testosterone concentrations. In the absence of
without disturbing the equilibrium, especially if the methods for free hormone estimation, rigorous valida-
binding is not tight, and methods for measurement tion of these correction techniques is not possible.
of the free hormone are not available at present for Table 1.2 lists some factors which affect the concen-
cortisol and testosterone. Although methods for free trations of serum binding proteins.
thyroid hormones are routinely used, they are prone to
technical difficulties (see below).
Correction for changes in binding protein by
measurement of the concentration of the binding
Intercurrent illness
protein and calculation of the hormone/protein ratio Severe intercurrent illness may affect endocrine systems
has been used for T4 and thyroxine-binding globulin and also the tests used to assess such systems, the most
(TBG) and for testosterone and sex hormone-binding important example of which is the effect of non-
globulin (SHBG). For T4, the so-called free thyroxine thyroidal illness (NTI), otherwise known as the sick
index can be calculated from the total T4 concentra- euthyroid syndrome, on thyroid function testing. In
tion and the uptake test which measures available the absence of pre-existing thyroid dysfunction, major
protein-binding sites. Such corrections are likely to be abnormalities of thyroid function tests are seen in
invalid when the concentrations of specific binding serious non-thyroidal illness and in the recovery phase.
proteins are very different from normal, and in the case Some of the difficulties of interpretation of thyroid
of the T4/TBG ratio, when other binding proteins such function tests result from the fact that the patient is
not in a steady state, e.g. acute caloric deprivation and
increases in serum free fatty acids, introduction of
drugs which affect T4 metabolism or displace T4 from
Increased binding protein concentrations its binding sites on serum proteins, a catabolic state
Genetic and decreases in concentrations of serum proteins. In
Estrogen (endogenous or exogenous) patients given dopamine or high doses of exogenous
Pregnancy and hydatidiform mole corticosteroids, TSH may be suppressed. In some
Thyrotoxicosis (SHBG) studies, the changes seen in the levels of free thyroid
hormones may have been assay-dependent artefacts,
Decreased binding protein concentrations and this has tended to hinder understanding of the
Genetic underlying pathophysiological mechanisms.3,4
Androgen
Liver disease Immunoassay problems in NTI
Protein-losing states The earliest immunoassays for measurement of free T4
Severe illness were the labeled analog methods. The analog is a
Malnutrition molecule closely related to T4 which binds to the
Malabsorption reagent antibody but which does not bind to the
Acromegaly specific binding protein TBG. The first analogs were
Cushing’s disease found to bind to albumin, and the result obtained was
High-dose corticosteroid therapy therefore in part a function of the concentration of
albumin in the sample, which is likely to be low in
NTI. The same problem was present in assays for free
Table 1.2 triiodothyronine (T3). More recent methods use
Factors which affect the concentrations of specific analogs which do not bind to albumin, notably
binding proteins. enzyme-labeled derivatives, or completely different

3
CH 01 9/9/02 10:30 am Page 4

INTERPRETATION OF BIOCHEMICAL INVESTIGATIONS

assay designs. Some manufacturers include albumin in Timing and stress have been considered above. Visible
the reagents, to take up possible inhibitors of T4 lipid or hemolysis may interfere in the assays. The type
binding in the sample. This and other procedures may of blood collection tube (volume and anticoagulant) to
in some cases amount to tailoring the result to suit be used will depend on the assay in use in the local
present conceptions of the ‘true’ values to be found in laboratory and on the analyte to be measured. Heparin
NTI. interferes in some immunoassays, and EDTA plasma
Equilibrium dialysis, considered by many to be the may be incompatible with enzyme-labeled immuno-
most reliable method available for free thyroid assays, because of chelation of metal ion cofactors.
hormones, is also prone to problems in NTI. Fragile analytes like adrenocorticotropic hormone
Incubation of the sample at 37°C during the dialysis (ACTH) require special handling; EDTA limits in vitro
procedure, especially after heparin treatment, may degradation by inhibiting plasma proteases, but rapid
release fatty acids from lipids in the sample, which will separation of the sample and low temperature are also
displace thyroid hormone from serum proteins and very important. Specific advice about each analyte
artefactually elevate the result. If inhibitors of binding should be sought from the laboratory before beginning
are present, their effect may be diminished if they pass blood sampling.
into the dialysate.
Because of the very high affinity of TBG for T4, in
theory the free T4 concentration should change very
little on dilution of the serum. In practice, the serum
Urine
has already been diluted manyfold in the assay, but Similar considerations apply to urine samples.
limited further dilution of normal serum should give Preservatives and/or cold storage may be required for
only a slight drop in free T4. In samples from NTI some analytes. Duration of collection is an important
patients, however, large falls may be seen. This may be matter, some hormones being satisfactorily assessed in
a way of assessing whether the free T4 value is a overnight samples, and some requiring 24 h collections.
reliable index of thyroid function in a sick patient. Completeness of collection is, of course, a major source
There are many recent reviews of thyroid function of error.
in NTI, a sure sign that the problems have not been
resolved.5–7
Testing for thyroid dysfunction in the presence of
NTI or in the short-term recovery phase is not advised
Assay methods
unless there are clinical signs of such dysfunction. The use of antibodies or binding proteins as reagents,
Thyroid function testing in the sick thyrotoxic patient stemming from the work of Yalow and Berson and of
can be problematic, as the degree of suppression of Ekins in the 1950s,8,9 is the foundation of biochemical
TSH in thyrotoxicosis is not distinguishable from that endocrinology. At the present time, almost all
in NTI, and total T3 and free T3 may be suppressed endocrine assays are immunoassays, with the exception
into the normal range by NTI, rising sharply when the of methods for small molecules such as steroids and
intercurrent illness has been alleviated. catecholamines. For most clinical purposes, steroids
In patients with chronic renal failure, low albumin in blood are also measured by immunoassay.
and high lipids, assay problems may occur similar to Catecholamines and, in some circumstances, steroids
those seen in acute NTI. are measured by methods involving high-performance
liquid chromatography (HPLC) or gas–liquid
chromatography (GLC); these techniques are time-
The sample consuming and have their own problems of impre-
cision and interferences. Bioassay is seldom used
routinely.
Blood
The taking of a blood sample, the type of anticoagu-
lant used, if any, and the transport and storage of the
sample are practical matters which may have consider-
Immunoassay: assay design
able effects on assay results, and these factors must not There are two basic designs of immunoassay, though
be ignored. many variations exist. In the competitive or limited

4
CH 01 9/9/02 10:30 am Page 5

ASSAY METHODS

antibody method, generally termed immunoassay,

antigen (the analyte of interest) from the sample (or
calibrator) competes with antigen labeled with a tracer
or reporter molecule for a limited amount of antibody.

Label bound
The amount of label bound will be inversely related to
the amount of antigen in the sample. Separation of the
bound and free fractions and quantitation of the label in
either (though usually the bound fraction) and compar-
ison with a calibration curve permits quantitation
(Figure 1.1). Since binding is maximal in the absence of
analyte, anything which inhibits binding will give a
falsely high value for the analyte concentration. Concentration of analyte (units)
In non-competitive or labeled antibody methods,
generally termed immunometric assay, the analyte is Figure 1.2
reacted with an excess of labeled antibody. The bound Typical standard curve for an immunometric assay.
and free forms are separated, and the bound labeled
antibody measured; the amount bound will be directly
related to the concentration of analyte (Figure 1.2). For particles. Polyclonal antisera can be used for immuno-
small analytes, the excess labeled antibody may be metric assays, but this type of assay came into general
removed by the addition of analyte coupled to a solid use only with the advent of monoclonal antibodies.
phase; such coupling reduces reactivity, and the coupled In general, immunometric assays are faster, more
analyte does not compete with free analyte from the sensitive and have a wider working range than compet-
sample. For larger analytes, another antibody to the itive immunoassays.
analyte may be used, directed against a different The matrix of the sample, protein content, pH and
epitope. This antibody is termed the capture antibody ionic composition may affect the assay. Assays designed
and is usually coupled to a solid phase. This format is for serum may not be usable for urine or other fluids.
termed the two-site or sandwich assay, and is much The type of label used has relatively little effect on the
used for the assay of polypeptide hormones. Inhibition quality of the result, though it may affect the speed or
of binding will result in a falsely low value for the convenience of the assay. Radioactive iodine was the first
analyte concentration. Only substances capable of label to be used, and as iodination is relatively simple to
crosslinking the two antibodies will give positive inter- do, it is often used in setting up new assays. However,
ference. Solid phases may be the wall of a tube, the well the products are not very stable, and radioisotopes are
of a microtiter plate or the surface of a bead or small increasingly regarded as hazardous to health. Some of
the alternatives, e.g. time-resolved fluorescence, chemi-
luminescence or electrochemiluminescence, offer inher-
ently better sensitivity, either because more label can be
incorporated or because the label is more easily and
quickly measured. Because the equipment required for
measurement varies with the label used, changing to
Label bound

alternative methods may involve major capital expendi-

ture, and this will limit the choice of methods.

Immunoassay: specificity and

cross-reactivity
Concentration of analyte (units) Antibodies are highly specific reagents and can recog-
nize short amino acid sequences or clusters, or parts of
Figure 1.1 steroid or thyronine structures, with a specificity
Typical standard curve for a competitive largely denied to chemical or physical methods.
immunoassay. Depending upon the epitope to which an antibody was

5
CH 01 9/9/02 10:30 am Page 6

INTERPRETATION OF BIOCHEMICAL INVESTIGATIONS

raised, it may or may not be able to distinguish a active in vivo. There will be marked numerical differ-
hormone molecule from its isoforms, precursors, ences between the results of an immunoassay measur-
metabolites, fragments and other related molecules. In ing both forms and the results of one which measures
the case of small molecules such as steroids, which are only the 22-kDa form.
made antigenic by attachment to a large molecule, the Cross-reactivity, unwanted reaction of another
part of the molecule close to the attachment may be molecule in an assay, occurs often, especially in the
‘invisible’ and may not contribute to the specificity of steroid field. For example, prednisolone is measured in
the antibodies. A polyclonal antiserum will contain a most if not all assays for cortisol. There are several
large number of antibodies of differing affinities and quantitative definitions of cross-reactivity, but they are
specificities, whereas a monoclonal antibody has a all arbitrary and are used only to indicate the potential
single set of readily characterizable attributes. The seriousness of such cross-reactivity. The contribution of
two-site assay for large molecules increases the speci- the cross-reactant to the measured concentration will
ficity by requiring two connected epitopes, and this depend on the concentrations of both the target analyte
can be further modified by the use of several and the cross-reactant, and calculation of the amount of
monoclonal antibodies in an oligoclonal system. either the analyte or the cross-reactant in a mixture is
Some immunoassays have suffered from serious not possible.
problems of specificity, due to the antibody being It will be clear that assays based on different anti-
directed to an epitope not unique to the biologically bodies or antisera may not necessarily give the same
active hormone; for example, early assays for para- results, given the molecular heterogeneity of many
thyroid hormone measured inactive metabolites polypeptide hormones and the number of potential
together with the active molecule, giving information reactants in body fluids. Since there are no absolute or
about the pathophysiology of this hormone which was reference methods for endocrine analytes, with the
subsequently found by the use of more specific assays possible exception of some steroids, thyroid hormones
to be incorrect. This kind of difficulty should be borne and catecholamines, it is difficult to know which assays
in mind when reviewing early literature. Structural are ‘right’, although there are various tests of validity
similarities between hormones cause similar problems, which can be applied. These include the investigation
for example: FSH and LH; the inhibins; and LH and of potential cross-reactions and interferences and of
chorionic gonadotropin (hCG). Many current assays for linearity on dilution. If the calibrant and the analyte
LH show some ‘cross-reactivity’ with hCG, which can are identical, then dilution of the sample should give a
be useful in the detection of pregnancy. With such (calculated) value the same as that found for the
an LH assay, in any patient the combination of undiluted sample, a situation often described as
undetectable FSH and normal or raised LH should dilution parallel to the standard. Non-linearity demon-
prompt an hCG determination. strates a problem of non-identity of some kind, includ-
The expected problem of hyperspecificity of ing the presence of cross-reactants or interferents, but
monoclonal antibodies, i.e. the antibody failing to the finding of linearity on dilution does not rule out
recognize some of the biologically active forms of a the presence of such substances.
hormone, has rarely been seen. An extreme example is
that of an LH genetic variant common in Nordic
populations in which a portion of the molecule close to
the junction of the subunits is deleted, but biological Immunoassay: standardization
activity is retained though possibly diminished,
subjects with this variant having proven fertility.10
(calibration)
One commercially available immunoassay, now The preparation of large quantities of pure hormones,
withdrawn, did not recognize the variant and gave especially the polypeptide hormones, is a formidable
undetectable values for LH in homozygotes. Possibly task. In most cases, it has not been possible to prepare
more misleading was the half-normal value obtained in enough pure material by extraction from tissues for
heterozygotes. In the case of GH, which has two major measurement by physical methods to provide calibrants
forms co-secreted in all subjects, the principal 22-kDa for immunoassays. Because of this, the existing
form and a 20-kDa splice variant, it is probably not International Standards (IS), prepared for use in bio-
necessary to measure both forms but only the 22-kDa assays, were adopted for use with immunoassays. This
form.11 Both forms are thought to be biologically did provide a common reference material with interna-

6
CH 01 9/9/02 10:30 am Page 7

ASSAY METHODS

tionally accepted, though arbitrary, International Units minimize all forms of error, from blunders to the
(IU), but produced a number of problems. imprecision of each step of the assay.
Bioassays measure function, and immunoassays Precision, the reproducibility of the values obtained,
quantitate the amount (of a particular epitope). Where is assessed via internal quality control. Because of the
isoforms or variants exist, the estimates by the two complexity of the technique, immunoassay is generally
types of assay may differ between preparations, as strik- less precise than many of the other methods used in
ingly shown for the IS 83/575 for FSH.12 This was clinical biochemistry. Precision, however, improved
prepared to have a high content of the more biologi- sharply with the introduction of automation in the early
cally active isoforms, and the bioassay/immunoassay 1990s. Precision will vary with the concentration of the
ratio was therefore very much higher than for the analyte, the coefficient of variation (CV, the standard
previous Standard. This IS has not been adopted for use deviation (SD) as a percentage of the mean value) usually
in immunoassay, as normal values for serum FSH would being highest at very low levels and sometimes increas-
have gone up by a factor of 4! The existence of isoforms, ing again at high levels (Figure 1.3). Inter-assay preci-
such as the glycosylation isoforms of FSH, brings into sion will be worse than intra-assay precision, because
question the whole concept of a pure hormone. of the error of recalibration. The detection limit,
Because the IS are presently calibrated by bioassays sometimes called the sensitivity, of an assay, i.e. the
in arbitrary IU, and the mass of hormone in the prepa- smallest amount which can be reliably distinguished
ration is not accurately known, the IU–mass conver- from zero, can be defined in several ways, all of which
sion factors are nominal.13 Many authorities have are dependent on the precision at low levels. Analytical
ignored this fact and have quoted assay results inappro- sensitivity is usually defined as the concentration which
priately in mass terms, e.g. prolactin or GH in the is 2 (or 2.5) SD above zero, the SD being calculated from
USA. Unit–mass conversion factors for commercial 20 replicates of the zero calibrator. Functional sensitiv-
calibrants will be specific to those calibrants. ity, a somewhat more realistic parameter, is the concen-
It has also been shown, and is evident from UK tration at which the CV (ideally between-assay) is 20%,
National External Quality Assessment Schemes data determined using sera with very low analyte content.
(see below), that the hormone in an IS may not be Although assays of the immunometric design usually
exactly the same as the form(s) existing in serum. This have working ranges of about three orders of magnitude,
may be because the material was derived from tissues, it is unusual for an assay to be able to measure with
e.g. pituitary glands, or because purification induced adequate precision all concentrations encountered in
alterations in the molecule or in the pattern of isoforms clinical samples, and many assays are set up for specific
or other components. For some important analytes clinical purposes. For example, an assay optimized for
such as ACTH, there is presently no suitable material
with which to make an IS.
For the new IS prepared by recombinant technology,
Coefficient of variation (%)

the mass of hormone can be measured by physico-

chemical means, and the content in IU determined
by immunoassay to maintain continuity of unitage. A
conversion factor or specific activity (IU/mg) can then
be set, but this value may still be a compromise 20
because of the differences between immunoassays.13,14
The ultimate aim is calibration in mass or molar units.
Some of the differences between immunoassays can
be ascribed to errors of calibration and some to the
fundamental difficulties described above. For a clear
and authoritative account of standardization and its
current controversies, see Bristow.12 x Concentration of analyte

Immunoassay: precision Figure 1.3

A typical precision profile. The concentration at
In order that a result be as close as possible to the true which the CV is 20%, given by x, is the functional
value for the sample, laboratories must attempt to sensitivity.

7
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