BASIC AND CLINICAL ENDOCRINOLOGY Editors P. Reed Larsen David R. London Brigham and Women's Hospital Queen Elizabeth Hospital Harvard Medical School Edgbaston, Birmingham, Boston, Massachusetts England Peter Sφnksen St. Thomas's Hospital Medical School London, England \. Radioassay Systems in Clinical Endocrinology, edited by Guy E. Abraham 2. Congenital Hypothyroidism, by Jean H. Dussault and Peter Walker 3. Cytochemical Bioassays: Techniques and CUnical Applications, edited by J. Chayen and Lucille Bitensky Other Volumes in Preparation CYTOCHEMICAL BIOASSAYS Techniques and Clinical Applications Edited by J. CHAYEN LUCILLE BITENSKY Division of Cellular Biology Kennedy Institute of Rheumatology London, England MARCEL DEKKER, INC. New York and Basel BUTTERWORTHS London Library of Congress Cataloging in Publication Data Main entry under title: Cytochemical bioassays. (Basic and clinical endocrinology ; 3) Includes bibliographical references and indexes. 1. Hormones-Analysis-Addresses, essays, lectures. 2. Cytochemical bioassays-Addresses, essays, lectures. I. Chayen, J. (Joseph), [date]. II. Bitensky, L. (Lucille), [date]. III. Series. [DNLM: L Biological assay-Methods. 2. Cytological techniques. 3. Cyto- diagnosis-Methods. 4. Hormones-Analysis. Wl BA813T V.3/QY 330 C997]. RB48.5.C95 1983 616.07'56 83-17661 ISBN 0-8247-7001-3 COPYRIGHT ©1983 by MARCEL DEKKER, INC. ALL RIGHTS RESERVED Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system without permission in writing from the publisher. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 10016 Current printing (last digit): 10 9 8 7654321 PRINTED IN THE UNITED STATES OF AMERICA Preface In recent years there has been a resurgence of mterest m bioassays. Two factors have contributed to this interest. The first is the fact that, in a proportion of cases, hnmunoassay has yielded results that are at obvious variance with the cUnical and physiological status of the patient. The second is the development of sensitive m vitro bioassays that are at least as sensitive as the equivalent radio­ immunoassays, so permitting comparison between immunoactivity and bioac­ tivity. The most sensitive of these in vitro bioassays are the cytochemical bio­ assays that are at least one thousand times as sensitive as the equivalent radio­ hnmunoassays. They have the further advantage that the same apparatus, and the same expertise, can be used for the bioassay of any polypeptide hormone. The cytochemical bioassay system has also proved to be of special value in help- mg to elucidate the mode of action of hormones. This use of the system has dis­ closed the existence of hnmunoglobulins that can block the effect of hormones. These bioassays, and the cytochemical bioassay system, are now being used widely both for clarifymg clinical conditions, such as the role of blocking anti­ bodies m thyroid pathology, and for more academic research. Consequently we agreed with the editors of this series that the time seemed ripe to review this rapidly expanding field. We therefore mvited some of the leading investigators to describe the cytochemical bioassay relevant to theh investigations and to dis­ cuss the clinical and research implications of their findings. /. Chayen and Lucille Bitensky III Contributors J. ALAGHBAND-ZADEH. Ph.D., F.R.C. Path., Department of Chemical Path­ ology, Charing Cross Hospital Medical School, London, England DEREK R. BANGHAM, M.B.B.S., F.R.C.P., Head of Division of Hormones, National Institute for Biological Standards and Control, London, England PETER H. BAYLIS, M.D., F.R.C.P., Consultant Physician and Senior Lecturer in Medicine, Department of Medicine, Royal Victoria Infirmary and University of Newcastle Upon Tyne, Newcastle Upon Tyne, England G. M. BESSER, M.D., F.R.C. Path., F.R.C.P., Department of Endocrinology, St. Bartholomew's Hospital, London, England LUCILLE BITENSKY, D.Sc, M.R.C.P., Head of Laboratory Medical Histochem­ istry, and Deputy Head of Division of Cellular Biology, Division of Cellular Biol­ ogy, Kennedy Institute of Rheumatology, London, England GIAN FRANCO BOTTAZZO, M.D., M.R.C. Path., Senior Lecturer in Clinical Immunology, Department of Immunology, Middlesex Hospital Medical School, London, England J. CHAYEN, D.Sc, Head of Division of Cellular Biology, Division of Cellular Biology, Kennedy Institute of Rheumatology, London, England vi Contributors KLAUS-DIETER DΦHLER, Ph.D., Professor of Experimental Endocrinology, Department of Clinical Endocrinology, Medizinische Hochschule Hannover, Han­ nover, Federal Republic of Germany DEBORAH DONIACH, M.D., F.R.C.P., Emeritus Professor Chnical Immunol­ ogy, Department of Immunology, Middlesex Hospital Medical School, Authur Stanley Institute, London, England H. A. DREXHAGE, M.D.,* Senior Lecturer in Clinical Immunology, Depart­ ment of Immunology, Arthur Stanley House, Middlesex Hospital Medical School, London, England D. EMRICH, M.D., Professor of Medicine and Nuclear Medicine, Department of Nuclear Medicme, Universitδt Gφttingen, Gφttingen, Federal Repubhc of Ger­ many STEPHEN FENTON, B.Sc, Department of Medicine, Charmg Cross Hospital Medical School, London, England TAKUMA HASHIMOTO, M.D.,** Research Fellow of Alexander von Humboldt Stiftung, Department of Chnical Endocrmology, Medizmische Hochschule Han­ nover, Hannover, West Germany RONALD W. HOILE, M.S., F.R.CS., Lecturer in Surgery, Surgical Unit, St. Stephens Hospital, London, England JULIA C. JONES, M.Sc, Department of Medicine, Charing Cross Hospital Med­ ical School, London, England G. NEIL KENT, B.Sc, Ph.D.,t Division of Inherited Metabolic Diseases, M.R.C. Chnical Research Centre, Harrow, Middlesex, England; Division of CeUular Biology, Kennedy Institute of Rheumatology, London, England; and Depart­ ment of Endocrine Physiology and Pharmacology, National Institute for Medi­ cal Research, London, England NIGEL LOVERIDGE, Ph.D., Deputy Head WHO CoUaborating Centre for Cyto­ chemical Bioassays, Division of CeUular Biology, Kennedy Institute of Rheuma­ tology, London, England CHRISTOPH LάCKE, Μ.D.,+Department of CUnical Endocrinology, Medi­ zinische Hochscule Hannover, Hannover, Federal Repubhc of Germany Present affiliations •Department of Pathology, Free University Hospital, Amsterdam, The Netherlands **Assistant Professor of Medicine, The Central Clinical Laboratory, Kanazawa University Hospital, Kanazawa, Japan tDepartment of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Queen Elizabeth Π Medical Centre, Nedlands, Western Australia +Professor and Head Physician, Hagenhoff-Klinik, Langenhagen, Federal Republic of Germany Contributors vii GRAHAM A. MacGREGOR, F.R.C.P., Senior Lecturer/Honorary Consultant Physician, Department of Medicine, Charing Cross Hospital Medical School, London, England J. MAXWELL MCKENZIE, M.D., Professor and Chairman, Department of Medi­ cine, University of Miami School of Medicine, Miami, Florida ALEXANDER von zur MάHLEN, M.D., Professor of Internal Medicine, Chair­ man of the Department of Clinical Endocrinology, Medizinische Hochschule Hannover, Hannover, Federal Republic of Germany THOMAS O. F. WAGNER, M.D., Department of Clinical Endocrinology, Center of Internal Medicine and Dermatology, Medizinische Hochschule Hannover, Han­ nover, Federal Republic of Germany W. H. C. WALKER, F.R.C. Pathol., F.R.C.P.(C)., Professor of Pathology, McMaster University Medical Centre, Hamilton, Ontario, Canada H. E. de WARDEN ER, M.D., F.R.C. P., Emeritus Professor of Medicine, Re­ search Laboratories, Charing Cross Hospital Medical School, London, England I. A. H. WASS, M.D., M.R.C.P., Senior Lecturer and Honorary Consultant Phys­ ician, Department of Endocrinology, St. Bartholomew's Hospital, London, England JUDITH WEISZ, M.B., B.Chir., Chief, Division of Reproductive Biology, Depart­ ment of Obstetrics and Gynecology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania HANS K. WEITZEL, M.D.,* Department of Clinical Endocrinology, Medizin- ische Hochschule Hannover, Hannover, Federal Republic of Germany MARGITA ZAKARIJA, M.D., Professor of Medicine, Department of Medicine, University of Miami School of Medicine, Miami, Florida JOAN M. ZANELLI, Ph.D., Senior Scientific Staff, Hormones Division, National Institute for Biological Standards and Control, London, England Present affiliation •Professor and Chairman, Department of Obstetrics and Gynecology, Steglitz Clinic, Free University of Berlin, Berlin, Federal Republic of Germany 1 General Introduction to Cytochemical Bioassays J. Chayen and Lucille Bitensky / Kennedy Institute of Rheumatology, London, England ORIGINS Quantitative cytochemistry, as it is used in the cytochemical bioassays, was developed over many years as a form of truly cellular biochemistry, that is, the measurement of metabolic activity or of active moieties in individual cells within a histologically complex tissue. These developments have been discussed elsewhere (Chayen and Bitensky, 1968; Chayen, 1978a; 1980). To achieve this histological specificity, the sensitivity of measurement had to be increased so that the activity of one cell could be measured, as contrasted with the mean ac­ tivity of one million cells that is used in conventional biochemistry. This was done by the use of scanning and integrating microdensi tome try, which had been developed earlier (Deeley, 1955) for measuring the amount of Feulgen stain (for DNA) in individual nuclei. It is now clear that microdensitometry of individual cells yields results that are quantitatively comparable to those obtained by more conventional procedures, done on aliquots of 10^ cells (Chayen, 1978b; Olsen et al., 1981). The methods of quantitative cytochemistry depend on chilling the tissue, and sectioning it at low temperature, without producing any observable ice artifact. Perhaps the best validation of these techniques (Chayen and Bitensky, 1968; Chayen, 1978a) is the fact that sections, prepared by the techniques described in Chapter 3, respond to the relevant polypeptide hormone with the same sensi­ tivity as do the segments of the target organ. Methods were then devised for 2 Chayen and Bitensky retaining the integrity pf the undenatured sections during the cytochemical re­ action designed for disclosing the required enzymatic or other activity (as dis­ cussed in Chapter 3). The aim of quantitative cytochemistry is to precipitate the colored reaction product in the cell in which the chemical activity resides. The section is then inspected in the microdensitometer to determine the histol­ ogy and to identify the target cells; the instrument can then measure the amount of reaction product specifically in these cells. At the same time as the methods of quantitative cytochemistry were being developed, recourse was being made to the system of nonproliferative organ maintenance culture that had been developed by Trowell (1959). Thus, for ex­ ample, samples of human synovial tissue were maintained in vitro, with no ap­ parent change either in histology or in biochemical activity (Chayen and Biten­ sky, 1982), in order to test the effect of anti-infiammatory agents. These methods, both of maintenance culture and of quantitative cytochem­ istry, found use in many diverse appHcations (Pattison et al., 1979). About 1970, the late Professor John Daly suggested that, because of their sensitivity and because they could measure changes solely in the target cells, they ought to be applicable to the development of very sensitive bioassays of polypeptide hormones. At that time, his interest in adrenocorticotropic hormone (ACTH) coincided with our rheumatological interest in this hormone. The first demon­ stration of the feasibihty of this project was given in 1971 (Chayen et al., 1971). The first cytochemical bioassay, which was for ACTH, followed shortly (Chayen et al., 1972). THE NEED FOR SENSITIVE BIOASSAYS OF POLYPEPTIDE HORMONES For polypeptide hormones at least, there can be no doubt that "the hormone" is a biological concept and must be measured by the biological activity which it evinces. Consequently, for many years, such hormones were detected, defined, and measured by in vivo bioassay. However, these generally proved to be too insensitive for measuring normal circulating levels in humans or in animals; the best that they could achieve was to demonstrate excessively high circulating levels. [It may be remarked that, according to some authorities, such as Orth (1977), this is the most clinically useful purpose of assays.] The advent of radioimmunoassay, and now of the other types of immuno­ logical assay, produced profound changes in the assaying of polypeptide (and other) hormones. The much improved sensitivity over the older in vivo bio­ assays, made it possible, in most cases, to measure the normal circulating levels of these hormones and to define conditions in which there was excessive, or too little, secretion of the hormone. The fact that these immunoassays could be automated gained them ready acceptance in routine clinical chemistry. General Introduction to Cytochemical Bioassays 3 These immunoassays were able to become firmly established because of the great advances that had been, and were being, made in polypeptide chemistry. It was now possible, apparently, to isolate a "pure" peptide and say, for ex­ ample, that "this" is the adrenocorticotropic hormone. There is no intention in this volume to denigrate immunoassays. They have greatly advanced both the study of endocrinology and clinical chemistry; their place is inviolate. But it soon became clear that, while they gave an invaluable, rapid, first approximation to the endocrine status of patients, they occasionally gave results that conflicted with the clinical condition. Moreover, it seems that anomalous results were also found with some preparations that were candidates for designation as international standards because, as early as 1967, a special committee convened by the World Health Organization was concerned that the results of immunoassay should be capable of being checked by suitably sensitive bioassays (WHO Report, 1975). This concern was finally expressed by the WHO Expert Committee on Biological Standardization (WHO Report, 1975), which is responsible for international standards of polypeptide hormones. That commit­ tee recognized that "a limitation on the use of radioimmunoassays for evaluating hormonal bioactivity is that the methods measure a composite of antigenic ac­ tivity, which is not necessarily related to the bioactivity of the hormone," and called for "the development of biological microassays, which should preferably have a sensitivity comparable with radioimmunoassays, with which they should be run in parallel." FUNCTIONAL VERSUS ANALYTICAL ASSAYS The need to be able to check the results of radioimmunoassay by bioassay is inherent in the different conceptual bases of these forms of assay. Radioim­ munoassay is a good example of what have been called analytical assays (Chayen et al, 1976; Chayen, 1980). Fundamentally, these assays use methods of ana­ lytical or physical chemistry to determine the number of molecules of a more or less specific type or, more precisely, the number of antigenic determinants of a specific type, present in the sample. The basic assumption is that a particular molecule will produce the relevant hormonal activity, e.g., the 1-84 peptide of parathyroid hormone will have the biological activity of parathyroid hormone. The analytical, or physicochemical, procedures are considered to be adequate to determine the nature of the molecule; then it is assumed that such a molecule must assert its known biological properties. However, it is now apparent that this assumption is too facile. First, it seems clear that radioimmunoassay, in general, will not detect small changes in the intact molecule, such as oxidation of the methionine residues of parathyroid hormone (PTH) or of minor deletions from the N-terminus of PTH, both of which remove biological activity (Chapter 11). Second, radioimmunoassay can measure biologically inactive fragments of