Autoantibodies

Atlas ofTissue

AutoantibodiesThird Edition

R.G. HughesM.J. SurmaczA.R. KarimA.R. Bradwell

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Atlas

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Autoantibodies

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Atlas of

Tissue AutoantibodiesThird Edition

RG Hughes, BSc., DPhil.MJ Surmacz, BSc(Hons).

AR Karim, BSc(Hons)., PhD.AR Bradwell, MB ChB, FRCP, FRCPath.

A.R. Bradwell 2008All rights reserved. No part of this publication may be reproduced, stored in aretrieval system or transmitted in any form or by any means - electronic, mechanical,photocopying, recording or otherwise - without the prior permission of the publisher orin accordance with the provisions of the Copyright Act 1988

First published in the United Kingdom in 1997

Distributors:The Binding Site Ltd., P.O. Box 11712, Birmingham, B14 4ZB, UK.The Binding Site Inc., 5889 Oberlin Drive, Suite 101, San Diego, Ca 92121, USA.The Binding Site GmbH, Robert-Bosch-Str. 2A, D-68723 Schwetzingen, Germany.The Binding Site, Centre Atoll, 14 rue des Glairaux, BP 226, 38522 Saint Egrve,France.The Binding Site, Balmes 243 4o 3a, 08006 Barcelona, Spain.

Published by The Binding Site Ltd., PO Box 11712, Birmingham, B14 4ZB, UKPrinted in the UK by HSW Print, Rhondda, Wales, UKThis book was produced using QuarkXpress 7.2 and Powerpoint 97A CIP record for this book is available from the British Library.

ISBN: 070442701X9780704427013

Atlas of Tissue Autoantibodies

ii

Preface to Third Edition

It is high time for a new atlas of autoantibody patterns. It is 9 years sincethe last edition so there have been numerous clinical and technicaldevelopments. It is also appropriate to merge the original Atlas ofAutoantibody Patterns with the Advanced Atlas, into a single volume. This3rd Edition now encompasses all the tissue patterns within an expanded versionof 230 pages larger than both previous editions combined. Importantly, wehave kept the same simple format with large photographic images, andmaintained the emphasis on clinical interpretation and clinical relevance.Image numbers have increased from 76 to 140 and they are of the highestquality. Many show rare patterns, but that is the intention, so that there iscomprehensive coverage of all relevant clinical issues in a single volume.

There is extensive text on new antigens, now increased to over 120 from theoriginal 58. There are detailed descriptions of many new brain, liver and skinantigens together with numerous others. Furthermore, we have increased thenumbers of diagrams and tables from 12 to 20 in order to provide clarity inthose areas that are complex.

We believe this new edition will provide an important update to the field ofautoimmunity and allow those who perform clinical and laboratory work tobetter understand what they are undertaking.

And, a note to my Dear Lady of Immunofluorescence. You have aged well.Although 52 years old, your charms remain. Your surrogate offspring, Elisa,may have usurped your previous dominance but your vibrant beauty andcomplex personality have lost none of their splendour. And fear not the newmultiple antigen arrays; such features were always part of your character. Thisnew atlas is dedicated to those who are still captivated by your magic.

AR Bradwell July 2008

iii



iv

Can you identify these patterns?

Sera with unusual patterns will be viewed with interest.

vAtlas of Tissue Autoantibodies

Acknowledgements

We would like to thank the following:-

Mark Drayson, Senior Lecturer, and Tim Plant, Laboratory Manager, fromthe Department of Immunology, (Medical School, University of Birmingham,UK) for their assistance. Margaret Richards who constructed the diagrams.Stephanie Stump who assisted with the manuscript. All those who havegenerously donated rare sera, acknowledged under the respective photographs.Debbie Hardie from the Department of Immunology (Medical School,University of Birmingham, UK) for technical assistance with the confocalmicroscopy. Lakhvir Assi for significant contributions in writing the ANCA andAPS chapters. Graham Mead for challenging conversations, patience andcritical reviews. Simon Hendy from The Binding Site Ltd., who provided theimmunofluorescence tissues and all other materials that made this atlaspossible.

Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . x

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Detection of Autoantibodies on Tissues . . . . . . . . . . . . . . . . . . . . . . . 3Choice of Tissue Substrate and Species . . . . . . . . . . . . . . . . . . . . 3Preparation of Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . 6ABO Blood Group Reactions on Monkey Tissues . . . . . . . . . . . 6Heterophile Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Fluorescein-Conjugated Antibodies . . . . . . . . . . . . . . . . . . . . . . . 11Collection and Preparation of Patient Samples . . . . . . . . . . . . . . 11Immunofluorescence Assay Procedure . . . . . . . . . . . . . . . . . . . . 12Techniques to Increase Sensitivity of IFA . . . . . . . . . . . . . . . . . . 13Enzyme Immunohistochemistry Staining on Tissues . . . . . . . . . 14Interpretation of Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15Practical Aspects of Running a Clinical Autoantibody Service . 16Identification of Antibody Specificity . . . . . . . . . . . . . . . . . . . . . 17

3. Detection of Autoantibodies Using Enzyme Immunoassays . . . . . . 19EIA Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Commercial Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22Assay Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23Quantitation and Assay Calibration . . . . . . . . . . . . . . . . . . . . . . . 24EIAs and Autoimmune Assessment . . . . . . . . . . . . . . . . . . . . . . . 25

4. Standardisation and Quality Control . . . . . . . . . . . . . . . . . . . . . . . . 27International Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Quality Control Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Positive Control Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

5. Atlas Section Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31


vi

6. Autoimmune Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Smooth Muscle Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Mitochondrial Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Nuclear Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43Liver/Kidney Microsomal Antibodies . . . . . . . . . . . . . . . . . . . . . 45Liver Cytosol Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48Soluble Liver Antigen Antibodies (Liver Pancreas Protein) . . . . 50Asialoglycoprotein Receptor Antibodies . . . . . . . . . . . . . . . . . . . 51Glutathione S-Transferase A1-1 Antibodies . . . . . . . . . . . . . . . . 52Other Liver Autoantigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53Liver Transplant and de novo Autoimmune Hepatitis . . . . . . . . . 58

7. Gastro-intestinal Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . 59Gastric Parietal Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 60Intrinsic Factor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61Coeliac Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64Endomysial/Tissue Transglutaminase Antibodies . . . . . . . . . . . . 65Gliadin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Reticulin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70Chronic Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . 75

8. Autoimmune Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82Serum Autoantibody Patterns in Different Renal Diseases . . . . . 83Glomerular Basement Membrane Antibodies . . . . . . . . . . . . . . . 87Tubular Basement Membrane Antibodies . . . . . . . . . . . . . . . . . . 91dsDNA Antibodies and Renal Disease . . . . . . . . . . . . . . . . . . . . . 93Renal Antibodies of Unknown Significance . . . . . . . . . . . . . . . . 94

9. Autoimmune Endocrine Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97Autoantibodies Against Steroid Producing Cells . . . . . . . . . . . . . 99Diseases Related to Steroidal Cell Antibodies . . . . . . . . . . . . . . . 105The Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107Pancreatic Islet Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 108The Thyroid Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112Thyroglobulin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113Thyroid Peroxidase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 113Thyrotrophin Receptor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 114The Pituitary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116Pituitary Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

vii


Pituitary Gland Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

10. Autoimmune Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119Indirect Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121Indirect Immunofluorescence Using Salt-Split Skin . . . . . . . . . . 127Direct Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129Autoantigens in Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 131Autoimmune Skin Diseases - Brief Summaries . . . . . . . . . . . . . 134

11. Neurological and Muscle Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 139Clinical and Pathological Significance . . . . . . . . . . . . . . . . . . . . 139Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141Yo Antibodies (PCA-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144Tr Antibodies (PCA-Tr) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146PCA-2 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147Hu Antibodies (ANNA-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148Ri Antibodies (ANNA-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150Anti-Neuronal Nuclear Antibodies Type 3 . . . . . . . . . . . . . . . . . 151CV-2/CRMP5 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152Glutamic Acid Decarboxylase Antibodies . . . . . . . . . . . . . . . . . . 154Amphiphysin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156Ma Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158Metabotropic Glutamate Neurotransmitter Receptors Antibodies 159Zic4 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 160Anti-Glial Nuclear Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 161Myelin Associated Glycoprotein Antibodies . . . . . . . . . . . . . . . 161Aquaporin-4 Antibodies (NMO-IgG) . . . . . . . . . . . . . . . . . . . . . 164Myasthenia Gravis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166Acetyl Choline Receptor Antibodies . . . . . . . . . . . . . . . . . . . . . . 167Striational Muscle Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 167Muscle Specific Receptor Tyrosine Kinase Antibodies . . . . . . . . 169Voltage Gated Calcium Channel Antibodies . . . . . . . . . . . . . . . . 170Voltage Gated Potassium Channel Antibodies . . . . . . . . . . . . . . . 171Myocardial Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172

12. Anti-Phospholipid Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175Classification Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176Lupus Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177Cardiolipin and 2 Glycoprotein I Antibodies . . . . . . . . . . . . . . 179Phosphatidylserine Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 184


viii

Additional Anti-Phospholipid Antibody Specificities . . . . . . . . . 185Prothrombin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186Annexin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

13. Vasculitis and ANCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191Guidelines and Recommendations for Testing . . . . . . . . . . . . . . 192Proteinase 3 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196Myeloperoxidase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198Cathepsin G Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200Elastase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200Bactericidal Permeability Increasing Factor Antibodies . . . . . . . 201Lactoferrin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202Other Atypical ANCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

14. Miscellaneous Autoantibody Specificities . . . . . . . . . . . . . . . . . . . . 207Filaggrin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207Salivary Duct Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209Endothelial Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210Sperm Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213

ix


Abbreviations

ACA - anti-cardiolipin antibodiesAChR - acetylcholine receptorACTH - adrenocorticotrophic hormoneAECA - anti-endothelial cell antibodiesAGNA - anti-glial nuclear antibodiesAIH - autoimmune hepatitisAIRE - autoimmune regulatorAKA - anti-keratin antibodiesAMA - anti-mitochondrial antibodiesANA - anti-nuclear antibodiesANCA - anti-neutrophil cytoplasmic antibodiesANT - adenine nucleotide translocatorAnx - annexinANNA - anti-neuronal nuclear antibodiesAPA - anti-phospholipid antibodiesAPECED - autoimmune polyendocrinopathy-candidiasis-ectodermaldystrophyAPF - anti-perinuclear factor antibodiesAPS - autoimmune polyglandular syndrome or anti-phospholipid syndromeASA - anti-sperm antibodiesASCA - anti-Saccharomyces cerevisiae antibodiesASDA - anti-salivary duct antibodiesASGPR - asialoglycoprotein receptor2GPI - 2 glycoprotein IBMZ - basement membrane zoneBP180 (BPAG2) - bullous pemphigoid antigen 2BP230 (BPAG1) - bullous pemphigoid antigen 1BPI - bactericidal permeability increasing factorC - CelsiusC-ANCA - cytoplasmic ANCACCP - cyclic citrullinated peptidesCDC - Centres for Disease ControlcDNA - complementary DNACDR - cerebellar degeneration related proteinsC1q - complement component 1, q subcomponentCNS - central nervous systemCSF - cerebro spinal fluidCSS - Churg-Strauss syndrome


x

DABCO - 1,4-diazobicyclo-(2,2,2)-octaneDNA - deoxyribonucleic acidDNAH - de novo autoimmune hepatitisDsc - desmocollindsDNA - double stranded DNADsg - desmogleinEBA - epidermolysis bullosa acquisitaEIA - enzyme immunoassayEMA - endomysial antibodyENA - extractable nuclear antigenFITC - fluorescein isothiocyanateFSH - follicle stimulating hormoneGABA - gamma-amino butyric acidGAD - glutamic acid decarboxylaseGBM - glomerular basement membraneGH - growth hormoneGI - gastro-intestinalGPC - gastric parietal cellsGS-ANCA - granulocyte specific ANCAGST - glutathione S-transferaseHBV - hepatitis B virusHCV - hepatitis C virusHEp-2 - human epithelial cell line type 2HSP - heat shock proteinIA-2 - tyrosine phosphataseICA - islet cell antibodyIDCM - idiopathic dilated cardiomyopathyIDDM - insulin dependent diabetes mellitusIF - immunofluorescenceIFA - immunofluorescence assayIg - immunoglobulinIIF - indirect immunofluorescenceJDF - Juvenile Diabetes FoundationkDa - molecular weight in kilo-DaltonsLA - lupus anti-coagulantsLADA - latent autoimmune diabetes in adultsLC - liver cytosolLEMS - Lambert Eaton myasthenic syndromeLH - luteinizing hormoneLKM - liver, kidney microsomal

xi


LKS - liver, kidney and stomachLM - liver microsomalLMA - liver membrane antigenLP - liver pancreas proteinLSP - liver specific membrane lipoproteinMAG - myelin associated glycoproteinMG - myasthenia gravismGluR - metabotropic glutamate neurotransmitter receptorsMP - microscopic polyangiitisMPO - myeloperoxidasemRNA - messenger RNAMSH - melanocyte stimulating hormoneMuSK - muscle specific receptor tyrosine kinaseNCGN - pauci-immune necrotising crescentic glomerulonephritisNEQAS - National External Quality Assurance SchemeNIBSC - National Institute of Biological Standards and ControlsNMO - neuromyelitis opticaP-ANCA - perinuclear ANCAPBC - primary biliary cirrhosisPBS - phosphate buffered salinePDC - pyruvate dehydrogenase complexPEG - polyethylene glycolPGSF1a - pituitary gland specific factorPMNs - polymorphonuclear leukocytesPN - polyarteritis nodosaPNP - paraneoplastic pemphigusPNS - paraneoplastic neurological syndromes and peripheral nervous systemPOA - paraneoplastic opsoclonus ataxiaPOEMS - polyneuritis, organomegaly, endocrinopathy, monoclonalgammopathy and skin changesPOMA - paraneoplastic opsoclonus myoclonus ataxiaPR3 - proteinase 3PRL - prolactinPSC - primary sclerosing cholangitisPVA - polyvinyl alcoholR - reticulinRA - rheumatoid athritisRF - rheumatoid factorRIA - radioimmunoassayRNA - ribonucleic acid


xii

RyR - ryanodine receptorSCLC - small cell lung carcinomaSDS - sodium dodecyl sulphateSDS-PAGE - SDS polyacrylamide gel electrophoresisSLA - soluble liver antigenSLE - systemic lupus erythematosusSm - Smith ENASMA - smooth muscle antibodySNGN - pauci-immune segmental necrotising glomerulonephritisSPS - stiff person syndromessDNA - single stranded DNAT3 - triiodothyronineT4 - thyroxine/tetraiodothyronineTBM - tubular basement membraneTg - thyroglobulinTPO - thyroid peroxidaseTSH - thyroid stimulating hormonetTG - tissue transglutaminaseUC - ulcerative colitisVGCC - voltage gated calcium channelsVGKC - voltage gated potassium channelsWB - western blottingWG - Wegeners granulomatosisWHO - World Health Organisation

xiii


Identification of autoantibodies is an essential part of clinical medicine andclinical immunology. Autoantibody testing for the assessment of systemic andorgan-specific autoimmune disease has increased progressively sinceimmunofluorescence (IF) techniques were first used in 1957 to demonstrateanti-nuclear antibodies. At present, HEp-2 cells and tissue sections allow thedetection of over 100 different autoantibodies and many more have beenreported in specific immunoassays. There are many specific immunoassays forconfirmation and quantitation of these autoantibodies. However, HEp-2 cellsand tissue sections are frequently used to screen for antibodies thus allowing amore focused choice of specific tests to be used for identification of theautoantibody specificity. The purpose of this atlas, in conjunction with the Atlasof HEp-2 patterns, is to facilitate the identification of characterisedautoantibodies and provide a springboard for the reader to identify newautoantibodies in clinical immunology.

Rodent liver, kidney and stomach tissues are the traditional substrates formitochondrial, gastric parietal cell and smooth muscle antibody detection.HEp-2 cells have largely replaced rodent liver as the routine substrate for ANAdetection; they are a more sensitive substrate which allows identification ofmany staining patterns. Complex tissues cannot be replaced by single cell linesand tissue sections will remain in use. Although monkey tissues have notreplaced the traditional substrates of rodent liver, kidney, and stomach they arethe substrate of choice for detecting many of the organ specific autoantibodies.Clear examples are the use of monkey oesophagus for the detection of skin andendomysial antibodies and monkey kidney for the detection of anti-glomerularbasement membrane (GBM) autoantibodies. With respect to GBM, anadditional streptavidin-biotin step increases sensitivity to match that of enzymeimmunoassays (EIA).

The debate over IF assays (IFA) versus antigen specific asssays willcontinue, each technique having its own strengths and weaknesses. IF issimple, inexpensive and allows identification of many patterns. Introduction ofinstrumentation allowing automation of IFA may have added ten years or moreto the longevity of these assays. Other techniques such as western blot and EIA

1

Introduction

Introduction

Chapter 1

will be used increasingly because of greater sensitivity and specificity but arelikely to be used predominantly as secondary, or confirmatory tests unlessquantitative antibody concentrations are considered to be clinically essential.The choice of assay may also depend on the sample throughput of laboratories;it is clearly simpler and cheaper to screen small sample numbers by IFA ratherthan EIA.

In this atlas each well-known autoantibody pattern is described along withthe clinical associations, the autoantigens involved, the original reference fortheir identification (where appropriate), plus relevant recent references. Thesereferences may be used as good starting points should the reader want moredetailed and specific information. Brief descriptions are given for the more rareor clinically irrelevant patterns. Wherever a serum has been available,photographs have been included to illustrate typical staining patterns.

Chapter 1

2

Choice of Tissue Substrate and SpeciesIn many ways detection of autoantibodies by IFA has changed very little

since the first description of anti-nuclear antibodies by Holborow, Weir andJohnson in 1957. This lack of technical change indicates the power andsimplicity of the method. What has changed over 50 years is the number ofdescribed autoantibodies and the specific requirements for detecting them. Thishas occurred through improved quality and consistency of commercial tissuesections, the availability of more tissue types and the increased specificity andavidity of the fluorescein-labelled secondary antibodies. For some diseases theincremental improvements have been of great clinical consequence. Forinstance, anti-GBM antibodies, detected on kidney sections, can now bedetected at a similar sensitivity to that of EIA. In the case of coeliac disease,reticulin autoantibodies were of modest diagnostic value 30 years ago, but nowendomysial autoantibodies assessed on monkey oesophagus provide diagnosticspecificity and sensitivity of almost 100%.

Rodent tissues are frequently utilised as substrates for IFA but fail to revealsome autoantibodies due to differences between rodent and human antigens(Figure 2.1 and 2.2). Furthermore, heterophile antibodies are commonlyobserved with rodent tissues but of no clinical relevance.

Human tissues are the ideal substrate in terms of antigen presentation,although tissues are not readily available and fresh tissue, essential for sectionpreparation, is rarely available. Of greater importance, however, is the problemof human immunoglobulins located in the vascular and extravascularcompartments in the tissues. The labelled second antibodies bind to thesemolecules in addition to the patients autoantibodies. This results in a highbackground fluorescence which reduces sensitivity of the assay (Figure 2.2).

Monkey tissues are clearly more similar to human than rodent tissues so arepreferable in terms of antigen and organ structures. They are also readilyavailable and can be prepared in perfect condition. Unfortunately, monkey andhuman immunoglobulins are also very similar, sharing around 98% of theiramino acid sequences. Thus, monkey and human tissues produce similarbackground fluorescence when conventional anti-human immunoglobulinreagents are used (Figure 2.3).

3

Detection of Autoantibodies on Tissues


Chapter 2

The problem of the secondary antibody reacting against monkeyimmunoglobulin can be overcome with an antibody that is specific to the 2-3%of unique epitopes on human immunoglobulins (Figure 2.4). Whilst this istechnically difficult to achieve, production of species-specific anti-humanimmunoglobulin antibodies of high affinity is possible. These antibodiesdramatically improve the sensitivity of autoantibody detection on many monkeytissues and should be used in preference to second antibodies that cross-reactwith monkey immunoglobulins. For steroid cell autoantibodies assessed onmonkey adrenal or ovary tissues the sensitivity of IFA using a species-specificsecond antibody is 10-20 fold greater than using a conventional second

Chapter 2

4

Figure 2.2. Indirect immunofluorescence on human tissues. Although thehuman autoantigen () will be present in human tissue, the staining pattern maybe difficult to identify due to background staining of the endogenous humanimmunoglobulin.

Figure 2.1. Indirect immunofluorescence on rodent tissues. The rodent antigen() will be similar but not identical to the human antigen and subsequentlyoccasional autoantibodies may be missed.

antibody. A similar improvement is observed with bullous pemphigoidautoantibody positive sera (Figure 2.11).

Reference

Holborow EJ, Weir DM, Johnson GD. A serum factor in lupus erythematosus with affinity fortissue nuclei. Br Med J 1957; 2: 732-734.

5


Figure 2.3. Indirect immunofluorescence on monkey tissues. The significanthomology between monkey and human proteins ensures that the autoantibodiesare detected. However, unless a human-specific secondary conjugate is used,background staining of endogenous immunoglobulin is a significant problem.

Figure 2.4. Indirect immunofluorescence on monkey tissues using a sheep anti-human IgG, species-specific, second antibody. The benefits of proteinhomology are maintained and the problem due to background staining ofendogenous immunoglobulins is removed, resulting in improved clarity and amore sensitive test system.

Preparation of Tissue SectionsTissue sections can be prepared in the laboratory or purchased from a variety

of manufacturers. High quality sections may be stored for over a year at 4C orfor several years at -40C. Whilst it may appear cheaper or more resourceful tocut ones own tissues, in practice considerable cost and expertise is required toproduce thin, uniform sections that do not deteriorate with time, particularlysince there are no standard protocols published describing ideal fixatives,drying procedures or storage conditions. Several commercial products whichfulfil all the quality requirements are available but there are no systematiccomparisons between products from different manufacturers.

ABO Blood Group Reactions on Monkey TissuesThe most commonly used laboratory monkeys are the old world Macaques

of which there are many species. The blood group carbohydrate antigens inmonkeys are identical in structure to human antigens and whilst not present onred cells, they are expressed in many monkey tissues, depending upon secretorstatus. Therefore, on occasions, blood group reactions lead to confusing IFpatterns. It is fortunate that the majority of human AB antibodies are IgM class.So these patient samples are only problematic when using mixed class anti-immunoglobulin conjugates (anti-IgGAM). IgG class blood group antibodiesare more rare, although when present they can be of high titre and avidity. Thefalse positive staining can be removed by addition of AB antigens to the samplebuffer. Occurrence of this false positive AB reaction on monkey oesophagus is~3%, although as many as 25% of samples will show an improved clarity whenusing the AB block (unpublished data). Arguably, any uncertain pattern shouldbe re-tested with an AB blocking step so that the intensity of genuine stainingcan be assessed.

Monkey tissues positive for AB antigens: Endothelium of capillaries and largerblood vessels in most tissues. Surface and glandular epithelium of the GI tract,pancreatic ducts, trachea, bronchi and uterus. Pancreatic acinar cells.Monkey tissues negative for AB antigens: Myocardium, smooth and striatedmuscle, valvular endocardium, proximal renal tubular cells, pulmonary alveolarlining cells, hepatocytes, liver sinusoidal lining cells, bile ducts and pancreaticendocrine cells.

Mimicking of steroid cell antibodies by AB reactivityThe cytoplasm of the Leydig cells of the testis stains positively with antibodiesagainst P450 enzymes. The appearance is similar to capillaries cut in transversesection and staining positively for AB antigens in capillary endothelial cells.

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Whilst confusion is unlikely on the ovary, addition of AB antigens mayfacilitate interpretation of testis staining (Figure 2.5).

Heterophile AntibodiesHeterophile antibodies, in the context of tissue autoimmunity, are human

antibodies that give false positive staining patterns on animal tissues and maymimic clinically relevant IFA patterns. Such heterophile antibodies vary acrossdifferent tissues and animal species; rat and to a lesser extent mouse tissuesshow heterophile antibody binding (Table 2.1). Most heterophile antibodies donot stain monkey tissues. When genuine autoantibodies and heterophileantibodies co-occur the staining pattern may be difficult to assess on rat tissues,especially for the inexperienced observer. Mouse tissues largely overcome suchproblems but may be more expensive. Outlined below are descriptions to beused as an aid during interpretation.

Genuine GPC on rat tissues: No heterophile staining in the kidney or liver.Genuine AMA on rat tissues: There should be GPC, liver, and kidney tubulecytoplasmic staining but no brush border staining.Genuine SMA on mouse tissues: Staining should be seen between the gastricglands, of the muscularis mucosa and the muscularis externa tissues, and theblood vessels.Genuine anti-mitochondrial/striational/myocardial on rat heart: The antibodiesdo not show inter-fibre staining.

7


Figure 2.5. ABO blood group antibodies staining blood vessels on testis (left)and ovary (right). This false positive staining can be blocked by incubating thesample with AB antigens; only the true staining pattern will remain.

References

Hawkins BR, McDonald BL, Dawkins RL. Characterisation of immunofluorescent heterophileantibodies which may be confused with autoantibodies. J Clin Pathol 1977; 30: 299-307.

Nicholson GC, Dawkins RL, McDonald BL, Wetherall JD. A classification of anti-heart antibodies:differentiation between heart-specific and heterophile antibodies. Clin Immunol Immunopathol1977; 7: 349-363.

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Figure 2.6. Heterophile antibodies, staining gastric parietal cells of rat stomach.

Tissue Pattern Rat Mouse

Stomach Gastric parietal cells Figure 2.6. -

Stomach Between gastric pits - Figure 2.7.

Kidney Brush border Figure 2.8. -

Liver Kupffer cells and sinusoids Figure 2.9. -

Heart Endomysium Figure 2.10. -

Table 2.1. Heterophile staining on rodent tissues.

9Detection of Autoantibodies on Tissues

Figure 2.8. Heterophile antibodies on rat kidney staining only the brush borderof the tubules.

Figure 2.7. Heterophile antibodies on mouse stomach showing stainingbetween the gastric glands similar to smooth muscle, but blood vessels arenegative. Endomysial antibodies may be confusingly similar but they are IgAclass and are best demonstrated on monkey oesophagus.

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Figure 2.10. Heterophile antibodies staining rat heart endomysium.

Figure 2.9. Heterophile antibodies staining rat liver sinusoids.

Fluorescein-Conjugated AntibodiesThe species of animal used for the fluorescein-conjugated anti-human

immunoglobulin antibody are usually sheep, goat or rabbit. Immunoglobulinfractions are satisfactory, however affinity purified antibodies are preferablebecause they produce lower background staining. Antibodies are labelled at afluorescein/protein molar ratio of about three and should be diluted in PBS(Tween 20 at 0.5 g/l may be added to reduce non-specific staining). Theoptimal working dilution is established by chessboard titrations. The undilutedconjugate is stable for at least a year when stored at 4C in the dark. Incommercial kits the reagent is supplied pre-diluted and matched for optimalreactivity with the respective substrate.

When investigating autoimmune disorders, IgG class autoantibodies arecommonly of most clinical relevance. However, this will depend on theparticular autoimmune disorder under investigation as there are certaininstances where IgA or IgM class autoantibodies are the major interest. Theconjugate specificity must therefore be considered in the context of the tissuesubstrate and the particular autoantibodies in question. Frequently, laboratorieswill use a non-affinity purified fluorescein-conjugated second antibody directedagainst IgG, A and M heavy chains. This will detect IgG, A and M antibodiesequally and results are reported without identifying the antibody class, hence allresults are regarded equally and this strategy can lead to over-reporting ofirrelevant antibodies. There is an additional consideration as to whether thesecond antibodies should react with the IgG (H+L) or with IgG, A and M heavychains. The former antibody will detect IgM and IgA, via light chain binding,although less well than the latter.

Many laboratories will use a second antibody directed against the specificheavy chain of interest. Examples would be the IgA chain, IgG chain or theIgM chain when investigating tissue-transglutaminase, islet cell and myelinautoantibodies, respectively. In such circumstances one must ensure that theconjugate recognises all subclasses since autoantibodies can be subclassrestricted. In conclusion, IgM, IgA and IgG class autoantibodies can bedetected separately or in combination, using the appropriate anti-immunoglobulin antibody conjugates. The argument in favour of detectingantibodies of a specific class is that there will be no confusion with irrelevantantibodies of another class.

Collection and Preparation of Patient SamplesIdeally, serum samples should be used within a few hours of collection.

Storage at 4C is satisfactory for samples analysed up to a week after collectionwhilst for longer periods (months/years) storage at -20C is essential. Frequent

11


thawing and freezing of samples may lead to reductions in antibody levels; longterm storage may lead to increases in titre due to lyophilisation. It isrecommended to add preservative to samples for any period of storage; this isachieved by addition of sodium azide (1mg/ml).

The required dilutions of the patients sera depend upon the autoantibodiesunder investigation. Some are considered to be of significance if present at adilution of 1/5 whilst others may only be significant at 1/100 or greater (Table5.1). The individual chapters provide guidelines on the clinical relevance ofantibodies in relation to their titres. Dilution of samples should be in phosphatebuffered saline pH 7.2-7.4 (PBS). Tween 20 (0.5 g/L) or bovine serum albumin(20g/L) can be added to the dilution buffer to reduce non-specific binding ofserum globulins to the tissues.

Immunofluorescence Assay ProcedureEach manufacturer will state a specific immunofluoresence procedure which

must be adhered to as this is how the slides would have been validated. Thesesteps can be automated via available instrumentation thus freeing-up laboratorytime while providing consistent and reliable results. In many respects theoutcome is an improvement over the manual procedure, as the human variableis removed. Below is an outline of the generalised manual procedure.

1. Slides should be allowed to reach room temperature before opening. Thisprevents condensation forming on the substrate which can disrupt theantigen localisation.

2. Slides should be labelled appropriately, including the date. Labellingshould be with a pencil as ink may subsequently run across the wells andresult in unwanted auto-fluorescence.

3. Place 30-50l of the diluted serum/control on the substrate well.4. Incubate the slides in a humid chamber for 30 minutes at room

temperature, ensuring no wells dry out.5. Rinse slides briefly with PBS (room temperature) and place in a wash pot

with PBS. The slides should be washed for ten minutes with agitation.For optimal wash performance it is preferable to perform one change ofPBS especially when agitation is not possible. More important is a highratio of wash volume to the number of slides/samples i.e. the use ofcoplin jars leads to insufficient washing.

6. The slides should be removed from the wash; excess fluid knocked offand if necessary, blotted briefly with absorbent paper. The fluorescein-labelled conjugate (30-50l) is then added onto each well. Slides areincubated for a further 30 minutes at room temperature in a light

Chapter 2

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protected humid chamber.7. Rinse and wash the slides again, as in step 5.8. Place a drop of mounting medium on each well and cover with a

coverslip carefully ensuring there are no trapped air bubbles. Traditionalmounting media are glycerol based (70-90% in PBS pH 8.6), however,for most substrates a polyvinyl alcohol (PVA) based mounting media willbetter maintain staining and improve clarity.*

9. Observe the wells through an epi-fluorescence microscope with filtersoptimal for fluorescein detection. Magnification is dependent on thetissue; when a 50x magnification or above is used an immersion lens ispreferable. Properly prepared fluorescein labelled sections are stable forseveral weeks when stored at 4C.

Counterstain: The use of counterstain is very much a matter of personalpreference. Some manufacturers provide Evans Blue pre-diluted in the kitconjugate. Alternatively, a minute amount can be added to the final wash. Caremust be taken as excessive amounts can lead to a loss in sensitivity.

* There are various recipes for mounting medium. We recommend either aglycerol based mounting medium (90% glycerol in PBS pH 8.6) or a polyvinyl-alcohol (PVA) based mounting medium. Chemicals can be added to themounting medium to reduce fading of fluorescence during illumination. Werecommend DABCO (1,4-diazobicyclo-[2,2,2]-octane, Sigma-AldrichCompany Ltd.) 2.5g per 100ml of mounting medium.

Johnson GD, Davidson RS, McNamee KC, Russell G, Goodwin D, Holborow EJ. Fading ofimmunofluorescence during microscopy: a study of the phenomenon and its remedy. J ImmunolMethods 1982; 55: 231-242.

Techniques to Increase Sensitivity of IFASensitivity of IFA is sufficient to detect the majority of clinically significant

autoantibodies. Indeed, ANA detection using HEp-2 cells is arguably toosensitive at sample dilutions of 1/40 to 1/80 and should preferably be performedat 1/160. The argument becomes stronger in the case of samples from theelderly; the frequency of autoantibodies, especially ANA increases with age.

The quality and proper maintenance of the epi-fluorescence microscope is asignificant factor affecting detection of autoantibodies. Mercury bulbs have alimited life span and must be regularly replaced as their intensity willdeteriorate with age. Centring of the arc should be assessed weekly to guaranteea consistent level of sensitivity. Lenses and filters must be checked and cleanedon a regular basis. Modern epi-fluorescence microscopes will provide a very

13


strong level of excitation and in some cases it may be advisable to employ filtersto moderate this intensity. Some autoantibody specificities, such as anti-GBMare often screened by EIA because it is considered more sensitive but somesimple procedures can increase IFA sensitivity by up to 10 times.Longer incubation times: The traditional method of increasing sensitivity is toincrease incubation times of the patients sera by up to 18 hours. Sensitivity isincreased approximately four-fold but background fluorescence also increasesand the tissues become progressively more fragile with long exposure to liquidreagents. Typically, pancreatic islet cell antibodies have been detected in thismanner.Anti-human, species-specific, second antibodies: As mentioned earlier thesereagents enhance sensitivity by reducing background staining (Figure 2.11).This gain can be further utilised by reducing the sample dilution factor. Forexample, adrenal antibodies are normally screened using a 1/5 dilution becausea more concentrated sample would result in far too high background staining.A species-specific secondary antibody allows a dilution of 1/2 to be used thusallowing weaker autoantibodies to be detected. The clinical utility of detectingweaker autoantibodies would need evaluating in the respective clinicalenvironment.

Enzyme Immunohistochemistry Staining on TissuesAutoantibody staining patterns can also be visualised on tissue sections using

enzyme-conjugated second antibodies. The immunoperoxidase technique with3,3-diaminobenzidine (DAB) or 3-amino-9-ethyl-carbazole (EAC) as substrateproduces excellent, permanent staining. This procedure has several advantages:

1. Conventional light microscopy can be used.2. Previous samples can be checked as staining is permanent.3. Contrasting histochemistry stains such as haemotoxylin allow simple

localisation of the autoantibody target.4. There is no photo-bleaching which makes photography simpler.5. There is limited impact of microscope variability and slides can be

assessed by different laboratories.

However, this procedure does have certain disadvantages which must beconsidered:

1. Sensitivity is lower, particularly for antigens that are sparsely distributedsuch as mitotic spindle antigens in HEp-2 cells.

2. The procedure is more time consuming due to the extra incubation step.

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Interpretation of ResultsThe intensity and patterns of fluorescence should be assessed and recorded.

The intensity can be expressed according to a scale of values compatible withthe guidelines established by the reference centres for established control sera(e.g. CDC, Atlanta) as negative or + to ++++. A semi-quantitative evaluationcan be obtained by performing serial dilutions of the test serum to endpointfluorescence. Such slides should be read from the most dilute sample upwardsand the end point is considered the first sample where a discernable positivepattern is observed. For confidence in reporting results, the positive andnegative controls must have performed to expectations.

Considerations during interpretationAutoantibodies occur in both physiological and pathological conditions. In

general, high titres (>1/20) are significant disease indicators but low or absenttitres do not exclude disease. The lack of detection of circulating autoantibodies

15


Figure 2.11. Monkey oesophagus stained with pemphigoid antibodies andspecies-specific (left) and non species-specific (right) secondary antibodies.Endogenous monkey IgG in the highly vascular lamina propria is notrecognised using the species-specific antibody.

may be because the antibodies are absent, poor presentation of target antigensor unsatisfactory assay technique. A further possibility is that the level ofactivity of the disease results in adsorption of the autoantibodies by antigensreleased into the circulation. In contrast, low titre antibodies may be found innormal people, relatives of patients with autoimmune conditions and a varietyof diseases such as inflammation and cancer with no autoimmune basis.Autoantibody levels also increase with age, particularly in women, withoutnecessarily being harmful. Interpretation must be made with reference to theindividuals medical history, age and existing conditions. Of equal importanceis awareness of the limitations of the tests and quality control of substrates andreagents.

The majority of IF patterns are only indicative of autoantibody specificityand exact specificity must be confirmed by other techniques, such asimmunoblotting or EIA. Where the antigens are known, such specific assaysprovide quantitative and definitive results. However, frequently theautoantigens are not fully characterised and IF is necessary. In many clinicallaboratories EIA and other methods are likely to be secondary, or confirmatorytests unless quantitative antibody concentrations are clinically essential.

Mixed patterns are a frequent occurrence on tissue sections. These have tobe distinguished by using combinations of tissue such as liver, kidney andstomach. They also have to be distinguished from heterophile antibodies. ForHEp-2 cells, diluting test sera may help to resolve the titre and specificity ofdifferent autoantibody combinations and hence their clinical relevance.

Practical Aspects of Running a Clinical Autoantibody ServiceIdentification of immunofluorescent staining is predominantly subjective so

there may be considerable variation in interpretation of results from day to dayand between different observers. Should discrepancies arise, particularlybetween past and present specimens, it is important to be able to identify oldslides and compare them with the new slides. For this reason we wouldrecommend including the following procedures.

Preparation1. Only use slides that have numerically identified wells.2. Label each slide with the date that the test was carried out.3. Lay out the slides in order of use and number sequentially.4. Transcribe the plan of each slide into the dated pages of a work book

while leaving space for results to be recorded alongside each specimennumber.

5. Follow a standardised assay protocol

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Interpretation (dependent upon specificity, see Table 5.1)1. Results should be scored from weak to strong positive by two

experienced personnel and reported as 1/20 (weak), 1/100 (+), 1/400(++), or 1/1600 (+++). Follow-up specimens should be tested within 28days.

2. All new positive samples should be routinely titrated.3. Store slides for a minimum of 28 days, preferably at 4C in the dark.

ReportingCarefully transcribe all results to computer-generated worksheets. To avoid

mistakes it is imperative that transcription is carried out by the person whointerpreted the fluorescence. When results have been verified, with particularreference to the age, sex and diagnosis of the patient, they are authorised forreporting. The clinical immunologist adds appropriate comments and signs theresult form before dispatch. Autoantibodies are more common in patients over50 years of age and therefore weaker samples for certain specificities may havelittle clinical relevance. This must be aluded to in the report. If a discrepancyarises between assay runs, the appropriate historical slide may be readilylocated by diary date, slide number, and well number and the sample checked.

Identification of Antibody SpecificityIdentification of autoantibody specificity is generally of clinical interest once

a patient serum has been shown to contain autoantibodies. In the majority ofcases the staining pattern is no more than suggestive of antibody specificity andprecise specificity must be confirmed by other means.Immunoblotting: The western blot assay is an essential tool for thecharacterisation of many autoantibodies. The following is a brief descriptionbut details should be sought in the relevant publications. Proteins from anextract of cultured cells are separated by polyacrylamide gel electrophoresisunder denaturing and reducing conditions with sodium dodecyl-sulphate (SDS-PAGE). Proteins in the gel are then transferred to nitrocellulose paper, whichprovides a solid support for the antigens. The nitrocellulose paper is cut intostrips and each is incubated with a serum to be tested. Several methods are inuse for detecting a specific antigen-antibody reaction: enzymes such ashorseradish peroxidase on radiolabelled probes, followed bychemiluminescence reactions, and autoradiography. Molecular mass markersand positive and negative controls are run together in each assay. They providethe necessary comparison bands alongside the patient sera. The main problemwith these assays is that the antigens are not in their native structure but as linearpeptides. This inevitably distorts non-linear epitopes so that results are not

17


always accurate. The assay is also very sensitive and produces interpretationproblems with weakly positive samples.EIA: the status of EIAs has grown significantly as more autoantigens have beencharacterised in detail. The following chapter is dedicated to their descriptionand utility.

General Reference

Storch WB. Immunofluorescence in Clinical Immunology. A Primer and Atlas. Birkhuser Verlag;2000.

Karim AR. Immunofluorescence Image Library:http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/

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Enzyme immunoassays (EIA) are widely used in clinical laboratories forquantifying thyroid, neutrophil, anti-dsDNA and other autoantibodies. Briefcomments are made on the clinical role of these assays in the relevant chapters,methods and other technical considerations are discussed here. The concept ofan EIA is similar to that of the immunofluorescent assay, where the conjugatedfluorochrome of the secondary antibody is replaced with an enzyme. There are,however, marked differences between these two methods which confer certainpractical advantages. The use of specifically selected purified antigens in EIAsensures greater assay specificity compared to IFA, where an array of antigensare presented. EIAs are also more sensitive and give quantifiable results,although autoantibody concentrations do not necessarily correlate with diseaseactivity and so this is not always advantageous. EIAs are easily automated,making them the method of choice for autoantibody monitoring in manyclinical laboratories. This is especially the case when there is a high samplethroughput. This popularity has led to the commercial availability of EIAs forthe detection of most autoantibody specificities where the clinically relevantantigen has been identified.

EIA ProtocolsThere are several variants of EIA employed in the clinical laboratory,

including indirect, sandwich, and competitive assays. Indirect EIAs are themost commonly used for the detection of autoantibodies and so these are thefocus here. Briefly, autoantigen is adsorbed onto a solid support, mostcommonly a 96 well, polystyrene microtitre plate. Any remaining proteinbinding sites are blocked to prevent non-specific adsorption of serumimmunoglobulins. The plate is then ready for the determination of autoantibodylevels (Figure 3.1). Alternatively, the plate can be dried and stored in an airtightcontainer. Here the block will also act as a stabiliser and, when storedappropriately, the plate should be stable for more than 12 months. It is essentialto ensure that all components are given sufficient time to reach roomtemperature prior to commencing the assay.

19

Enzyme Immunoassays

Detection of Autoantibodies UsingEnzyme Immunoassays

Chapter 3

Test Protocol

1. Sample dilution: 1:100 is commonly employed as a starting dilution. This isa greater screening dilution than generally used in IFA, a reflection of thegreater sensitivity of the assay system.

2. Sample addition: 100l of diluted sample is added to each microtitre well.Separate wells are used for positive, negative, cut-off controls and calibrators,wherever relevant. The plate is incubated at room temperature for therecommended period of time (e.g. 30 minutes).

3. Wash step: After sample incubation, adequate washing must be carried out toremove any non-specifically bound antibodies and other serum components, (3-5 washes using 300l of wash buffer). Detergents (e.g. Tween-20) are oftenemployed at a low concentration to maximise the efficiency of this step.

4. Conjugate addition: 100l of diluted enzyme-conjugated anti-immunoglobulin is added to the wells and the plate is incubated at roomtemperature (e.g. 30 minutes). Horse-radish peroxidase (HRP) and alkalinephosphatase (AP) are the most commonly employed enzymes.

5. Wash step: The wells are washed again, removing unbound conjugate.

6. Substrate: 100l of substrate is added. The plate is incubated for a furtherperiod to allow the coloured product to form (e.g. 30 minutes). There are manyenzyme substrates available. For HRP 3,35,5Tetramethylbenzidene (TMB)solution can be used, this forms a soluble blue reaction product when oxidised,which turns yellow after addition of stop solution. In the case of AP, p-Nitrophenyl Phosphate is commonly used and this produces a soluble colouredend product (p-nitrophenyl).

7. After the last incubation, the reaction is stopped by the addition of 100l ofstop solution. For HRP the stop solution is a strong acid, e.g. 1M HCl, H2SO4or H3PO4 and in the case of AP 1M NaOH is used.

8. The wells are then read within 30 minutes (preferably immediately) bymeasuring the absorbance of the coloured end product using aspectrophotometer. The wavelength examined depends on the enzyme substrateutilised; TMB 450nm and p-Nitrophenyl phosphate 405/650nm.

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Practical ConsiderationsIn an EIA, all components and conditions have to be considered carefully,

both individually and as a complete process (some major considerations areoutlined below):

Antigen source: The source and purity of the antigen has a fundamental effecton the assay performance; several factors should be taken into account.Evaluation of antigen from different sources is recommended as large variationsin performance may be seen. It is not unknown for the same grade of antigenfrom different suppliers to perform differently: phospholipids are a clearexample. To reduce non-specific binding the antigen should be as pure aspossible. The choice between recombinant and native antigen rests on twoconsiderations. Native antigen of human origin is not always available and sothere remains a question of homology between the human autoantigen and theantigen from the chosen species. Recombinant antigen is usually derived fromthe human sequence and so the amino acid sequence homology will be exact.However, the recombinant antigen may not be folded correctly nor have hadpost translational modifications such as glycosylation. The final choice is oftendown to price and availability and is resolved by trial and error.

Choice of surfaces: Polystyrene microtitre plates are most frequently the surfaceof choice for EIAs. These can have different binding capacities for protein,

21

Enzyme Immunoassays

Figure 3.1. Schematic illustrating the individual steps of an indirect EIA.

often referred to as high- or low-bind plates: where plates have been irradiatedor are non-irradiated respectively. For optimisation of the assay, testing of thesystem with a range of surfaces can prove beneficial.

Coating conditions: There are two main considerations here. The first is tochoose a suitable buffer system for the antigen in question. Secondly, anoptimal antigen concentration is determined from chessboard titrations.Generally, 100l of antigen is dispensed at an optimised antigen concentrationand incubated at 4C for a period of 12-18 hours, in a moist, sealed container.

Block: Any uncoated regions of the polystyrene surface must be blocked toprevent serum components from sticking non-specifically to its surface. Toensure efficient blocking, the volume used must be greater than both the volumeof original antigen coat and that of the diluted sample. This step is generallycarried out at room temperature for a minimum of 30 minutes. Sugars, proteinsand proprietary blocks are all used for this purpose; ultimately the block ofchoice is dependent on the antigen being coated. The block may alsoincorporate a stabiliser thus allowing longer term storage of the coated plates.

Conjugate specificity: Clinically significant autoantibodies are frequently ofIgG class and in such cases it is important to use an IgG specific secondaryantibody. There are, however, occasions when other classes of autoantibodiesare of interest, such as IgA in coeliac disease. Whether to use a conjugate ofmixed specificity, detecting all classes, or a secondary antibody which isspecific for a particular antibody class depends on clinical relevance.

Incubation temperature: For a consistent inter-assay performance it is importantto ensure that the running conditions are kept constant. Both antibody bindingkinetics and enzyme activity can be affected by fluctuations in temperature.Ideally, when running an assay, all the components must be maintained at a settemperature and the incubations should be performed within a temperaturecontrolled environment.

Commercial AssaysGood quality commercial EIAs are available from a number of

immunodiagnostic companies. Such assays will be designed to be user-friendly and considerations are made to ensure that they are easily automated.Some companies will also recommend validated robotic instrumentation for therunning of their EIAs. Colour coding of reagents is a distinct advantage for theuser, as is a common buffer system and identical dilution steps for assays within

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a complementary range. The assays should be optimised, taking many of thepreviously mentioned points into consideration. They will also have beenvalidated for expected performance and have a generous shelf life. As with allimmunodiagnostic assays, to achieve optimum performance, allrecommendations from the manufacturer must be adhered to when running theassays.

Assay ValidationThe end user will base their confidence in the assay results on available

performance data. It is therefore necessary to establish performancecharacteristics with respect to precision, linearity, sensitivity, stability and theeffect of potentially interfering substances. Such data should be considered forin-house assays and is generated as a matter of course for commercial assays.

Linearity, precision and stability: Linearity of the assay can be shown byrunning a dilution series of several samples followed by calculating the %recovery. Intra-assay and inter-assay reproducibility can be determined bytesting a number of different samples in multiple repeats within the same assayrun and on separate occasions, respectively. It is also helpful to establish theperformance across separate production batches. Stability is assessed byrepeated testing over a period of time. It is also important to challenge the assaywith extreme temperature conditions and mimic any transient fluctuations ofstorage conditions.

23

Enzyme Immunoassays

Figure 3.2. Photograph showing a commercial kit including colour codedreagents.

Sensitivity and specificity The diagnostic sensitivity of an assay can bedetermined by studying a suitable number of patients with known disease states.Within the test group it is important to include both the disease group inquestion and a significant number of samples from patients with disease statesfor which the differential diagnosis is carried out. It is also advisable to usenormal blood donors as another control group, this may highlight unexpectedobservations for the disease state controls. The positive and negative predictivevalues of a clinical assay are dependent on the sample population underinvestigation; there will be a marked difference in calculated performancebetween screening the general population and when testing a group of patientswhich have been selected on grounds of clinical symptoms.

Effect of interfering substances There are a number of substances that arerecognised to interfere with certain immunodiagnostic assays. It is thereforenecessary to establish whether there is interference for any assay and, ifidentified, to be aware of the degree of such interference. Substances thatshould be taken into consideration include: rheumatoid factor, elevatedimmunoglobulin levels, lipids, haemolysed samples and bilirubin. Otherpotential interfering substances may be relevant when considering particularassays.

Quantitation and Assay CalibrationIn order to allow quantitative and comparable reporting of results, EIAs

should be calibrated against international standards, and units quoted in eitherinternational units or micrograms per millilitre. This is predominantly the casewhere recognised standards are available; however, this is more often theexception than the rule. Where no standard is available, one of two options isimplemented: a cut-off control is used or, alternatively, a calibration curve isgenerated against an internal reference. One must consider that the returnedvalue is not a direct measure of concentration; it is rather a reflection of thecombination of autoantibody avidity and titre. Autoantibodies are often oflower avidity, but this will differ from sample to sample. There is also thepossibility that a sample has mixed populations of autoantibodies covering arange of avidities.

Cut-off If a simple indication of negative, low, medium and high values isadequate, then results may be reported as a multiple of an established cut-offcontrol. This is a crude form of calibration and does not take into account thesigmoidal nature of the true calibration curve. An advantage of this simplecalibration is that this takes up only one test well on the microtitre plate and so

Chapter 3

24

allows more room for test samples. Also, it is an appropriate method wherethere is no evidence that the titre is a reflection of disease activity.

Calibration curve Where there is benefit in returning a quantitative result, aprecise calibration curve needs to be established. An EIA calibration curvecovering the whole range of autoantibody concentrations is expected to besigmoidal in shape. In order to establish an equation to describe such a curve a4 parameter logit fit is performed; this requires a minimum of 5 points,preferably 7, across the whole curve. To ensure reproducibility and preventcalibration drift across multiple batches it is necessary to create an internalreference against which all calibration material is standardised. Whereinternational standards are available, the internal reference should be calibratedagainst this, otherwise a suitable serum should be chosen; due to the variablenature of autoantibodies, a pool of sera is preferable. When a calibration curveis set, special emphasis should be given to the region where a differentialdiagnosis could be the outcome, this is usually at the lower end of the curve.Accurate quantification of high autoantibody values is generally of lessimportance, although reproducibility remains essential.

EIAs and Autoimmune AssessmentEIAs are available for the determination of most autoantibody specificities

where the autoantigen has been clearly described. However, the choice toperform an EIA as opposed to another immunodiagnostic technique will dependon several factors including cost and throughput. A quantitative result does notalways provide additional value when identifying autoantibodies. In manycases, the distinction between presence or absence of autoantibody is helpfuland in other cases an indication of low-, medium- and high-titres can besufficient. In such circumstances, the decision to use an EIA for autoantibodydetermination will be for purposes other than a clinical requirement, e.g. theremay be a cost advantage due to a high sample throughput. Reporting thenumerical result of an EIA does not demand the same skill and experiencewhich is necessary to interpret IFA staining patterns.

Interestingly, a number of laboratories choose to use EIAs as an initial screenfor ANA determination. This may appear counter-intuitive as HEp-2 IFA testsare generally cheaper. Apart from the benefits of automation, the reasoning infavour of this approach is that an ANA EIA has specifically selectedautoantigens, those of most clinical relevance. As the antigens on the plate areknown, one can further identify the specificity of positive samples by usingsingle-specificity EIAs. This approach will detect the majority of ANAs but aproportion of ANA samples will be missed. Any unidentified autoantibodies

25

Enzyme Immunoassays

will probably be of limited clinical importance: specificities will be for minorantigens of little known significance.

Selective screening for autoantibodies is most efficiently performed using asystem where the relevant autoantigens are all present, such as cerebellum whenlooking for paraneoplastic antibodies, monkey oesophagus when testing forautoimmune skin diseases or HEp-2 cells when investigating a connectivetissue disease query. Once positive samples are identified then more specificassays can be employed to confirm the autoantibody specificity inferred fromthe immunofluorescent staining pattern. However, when a clinician has a strongsuspicion of a particular condition and there are known autoantibody profiles,with both high sensitivity and specificity, a direct request may be made for aparticular marker, which will be determined by EIA. Tissue transglutaminaseand the M2 mitochondrial antigen are clear examples for coeliac disease andprimary biliary cirrhosis, respectively, as are anti-CCP antibodies with respectto rheumatoid arthritis. Occasionally, there are conditions which are defined bythe autoantibody profile, such as anti-phospholipid syndrome (Chapter 12).Other than the lupus anticoagulant test, there is no alternative to using EIAs fordetermination of these antibodies.

Chapter 3

26

Recognising IF patterns is relatively straightforward but assessing IFintensity and its clinical significance requires considerable experience.Definitive decisions on sensitivity can only be made from practice and byregularly viewing batches of normal, or allegedly normal, sera in order to obtainthe picture of a negative background. When there is doubt, a clinician shouldbe involved and the final judgement made in the context of the patients clinicalsymptoms.

Organ-specific autoantibodies are relatively easy to identify and interpret.After only a short training period most members of staff are able to recognisedifferent patterns of the more common anti-nuclear antibody types and todistinguish positive staining of cells of the islets of Langerhans, zonaglomerulosa of adrenal, theca cell layer of developing follicles etc. However,in composite blocks of liver, kidney and stomach, autoantibody patterns are lessclear because the observer is searching for patterns in three tissues, frequentlyagainst a background of other staining. There are also a huge variety ofstaining patterns which may be new even to someone with many yearsexperience. The problems are enhanced when employing rodent tissues due toheterophile antibodies which often titrate out to dilutions in excess of 1/1,000.

There is also the question of chance findings. A policy of reporting somepatterns that do not relate to the patients diagnosis may be of interest. Theclinicians can choose to ignore the results or one may indicate that they are ofno known significance. Such results may be reviewed from time-to-time toassess their importance.

27

Standardisation and Quality Control


Chapter 4

International StandardsThere are few human autoantibody standards relative to the number of

known specificities for immunofluorescence assays, apart from anti-nuclearantibodies. It is not apparent why there has been such little progress. Thefollowing is a list of the materials that have been prepared in stable form forgeneral use:

The following are available from NIBSC (National Institute for BiologicalStandards & Control, PO BOX 1193, Blanche Lane, South Mimms, Potters Bar,Herts, EN6 3QG, UK). It should be noted that the WHO standards are alsoavailable from NIBSC.

1. Human primary biliary cirrhosis serum: 67/183MRC Research Standard A. 100 units per ampoule.

2. Human anti-thyroid microsome serum: 66/387No official status. 1,000 units per ampoule.

3. Human anti-thyroglobulin serum: 65/931st International Reference Preparation 1978. 1,000 International Unitsper vial.

4. Human rheumatoid arthritis serum: 64/2 (WHO 1066)1st British Standard, 100 International Units per ampoule.

5. Human anti-islet cell serum: 97/5501st WHO Reference Reagent 1999, 20 units per ampoule (also 100 unitsof anti-GAD65 and anti-IA-2).

6. Native (ds)DNA antibody: WHO Wo801st WHO Reference Reagent 1985. 100 International Units per ampoule

7. Human anti-smooth muscle (anti-actin) serum: W1062WHO International Reference Reagent.

Cardiolipin Antibody Standards- Sapporo monoclonal antibodies (HCAL for IgG and EY2C9 for IgM).Available from: Centres for Disease Control and Prevention (CDC), 1600Clifton Road, Atlanta, GA 30333.

Chapter 4

28

- Cardiolipin IgG and IgM antibodies: NEQAS 97/656.

- Louisville (Harris) calibrators for the measurement of anti-cardiolipin IgGand IgM antibodies. Available from: Louisville APL Diagnostics, Inc., 2622NASA Pwky STE G2 Seabrook, TX 77586 USA.

Quality Control SchemesThere are several national quality control schemes of which the best known

are indicated below. These are widely used and cover the common antibodies.

1. UK NEQAS for Immunology, Department of Immunology, P.O. Box 894,Sheffield, S5 7YT, UK. Samples are distributed every six to eight weeks andcomprise normal or pathological human sera. Results are reported as U/ml ortitre and for qualitative responses are positive or negative.

- General Autoimmune Serology (rheumatoid factor, thyroid peroxidaseantibody, mitochondrial antibody, liver-kidney microsome antibody, smoothmuscle antibody and gastric parietal cell antibody).- Antibodies to Nuclear and Related Antigens (anti-nuclear antibody, dsDNAantibody, ENA antibodies, HEp-2 cell nuclear substrate and centromereantibodies).- Phospholipid Antibodies (cardiolipin antibody, 2 glycoprotein I antibodyand phosphatidylserine antibody).- ANCA and GBM Antibodies (neutrophil cytoplasmic antibody, proteinase 3antibody, myeloperoxidase antibody and glomerular basement membraneantibody).- Acetylcholine Receptor Antibodies (AChR antibody).- Bullous Dermatosis and Coeliac Disease Antibodies (skin basementmembrane antibody, desmosome antibody, gliadin antibody, endomysialantibody and tissue transglutaminase antibody).- Andrology (sperm antibodies).

2. The College of American Pathologists provides a scheme for autoimmuneserology which is available from The College of American Pathologists, 325Waukgan Road, Northfield, Illinois 60093-2750, USA. Samples are distributedfrom one to three times per year.- Special Immunology (S2) including: mitochondrial, smooth muscle andglomerular basement membrane antibodies.- Coeliac Disease (CES) including IgA endomysium antibodies.

29


- Gastrointestinal (GIH) including mitochondrial and gastric parietal cellantibodies.

3. INSTAND e.v., Ubierstr 20, sseldorf, D-40223, Germany.- Special Autoimmune including mitochondrial, smooth muscle, liver-kidneymicrosome, gastric parietal cell, endomysium and Purkinje antibodies.

Several commercial companies provide quality control schemes forautoimmune testing including The Binding Site Ltd., P.O. Box 11712,Birmingham, B14 4ZB, U.K.

Positive Control SeraMany are available from commercial sources including The Binding Site

Ltd., which has antibodies against the following antigens:

Reference

Ward AM, Sheldon J, Wild GD Editors. PRU Handbook of Autoimmunity. 3rd Edition. PRUPublications; 2004.

Chapter 4

30

Adrenal/ovary/testis steroidal cellANAC-ANCA (proteinase 3)P-ANCA (myeloperoxidase)dsDNAENAEndomysium (IgA)Gastric parietal cellsGliadinGlomerular basement membrane(GBM)Glutamic acid decarboxylase (GAD)Hu (ANNA-1)

Liver/kidney microsomal (LKM)MitochondrialMyelin associated glycoprotein (MAG)Pancreatic islet cellsPemphigoid (bullous)Pemphigus vulgarisParaneoplastic pemphigus (PNP)Reticulin 1 (IgA)Skeletal muscle fibres (striational)Smooth muscle (SMA)Yo (PCA-1)

The remaining chapters in this book describe the immunofluorescencestaining patterns of autoantibodies. Each chapter is self contained and basedupon autoimmune disease groups, rather than tissue substrate usage. Asummary table is to be found at the beginning of each chapter, this highlightsthe major staining patterns, common specificities and related disease diagnosis.The animal species of the tissue substrates are indicated for each image. Onmonkey tissues the second antibody is sheep anti-human species-specifc IgGFITC unless otherwise stated.

The figures showing immunofluorescent patterns are taken at variousmagnifications to optimally identify the autoantibodies and demonstrateconfusing patterns. Photographs were taken over a period of time and severalimage capture devices were used. A variety of objectives were employed, witha x50 water immersion lens for the higher magnifications. Subsequentphotographic enlargement was usually employed in order to optimally highlightthe relevant features.

The summary table overleaf is designed to allow rapid identification ofantibody patterns, associated diseases and vice versa. Also, the lowestsignificant titre is highlighted for each specificity. Awkward telephoneconversations can be turned into authoritative accounts of target autoantigens,disease associations and other test requirements with a quick scan of this tableand those at the beginning of each respective chapter.

31

Atlas Section

Atlas Section Introduction

Chapter 5

Chapter 5

32

Clin

ical

Diagn

osis

Autoantigens

PreferredTissue

Low

est

Sign

ificant

Titre

Other

Relevan

tTests

Add

ison

sdi

seas

eC

ytoc

hrom

eP4

50en

zym

esA

dren

algl

and

1/5

Ova

ry,t

estis

,GPC

Ant

i-pho

spho

lipid

synd

rom

ePh

osph

olip

idbi

ndin

gpr

otei

nsEI

An/

aLu

pus

antic

oagu

lant

s

Aut

oim

mun

ega

strit

isG

astri

cpa

rieta

lcel

ls(G

PC)

Stom

ach

fund

us1/

20Th

yroi

dan

tibod

ies

Aut

oim

mun

ehe

patit

isty

pe1

Smoo

thm

uscl

e(F

-act

in)

Live

r,ki

dney

&st

omac

h1/

40So

lubl

eliv

eran

tigen

(SLA

)

Aut

oim

mun

ehe

patit

isty

pe2

Live

rki

dney

mic

roso

mal

1,2

&3

Live

r&ki

dney

1/20

Live

rcyt

osol

antib

odie

s(L

C1)

Chu

rg-S

traus

ssy

ndro

me

P-A

NC

AN

eutro

phils

1/20

AN

A,C

RP

Coe

liac

dise

ase

Endo

mys

ium

(tTG

)M

onke

yoe

soph

agus

1/10

Glia

din

and

retic

ulin

Cro

hns

dise

ase

ASC

A,

panc

reat

icac

inar

cells

EIA

panc

reas

1/20

P-A

NC

A(a

typi

cal),

CR

P

Der

mat

itis

herp

etifo

rmis

Endo

mys

ium

(tTG

)M

onke

yoe

soph

agus

1/10

Glia

din

and

retic

ulin

Goo

dpas

ture

sSy

ndro

me

Glo

mer

ular

base

men

tmem

bran

e(

-3-c

hain

type

IVco

llage

n)M

onke

yki

dney

1/5

AN

CA

,CR

P

Her

pes

gest

atio

nis

Epid

erm

alba

sem

entm

embr

ane

zone

Mon

key

oeso

phag

us1/

20Sk

inbi

opsy

forI

gGor

C3/

C9

Idio

path

iccr

esce

ntic

glom

erul

onep

hriti

sP-

AN

CA

Neu

troph

ils1/

20A

NA

,CR

P

Idio

path

icdi

late

dca

rdio

myo

path

y(I

DC

M)

-1-

adre

nerg

icre

cept

orEI

An/

aM

yosi

n,tro

pom

yosi

net

c

Insu

linde

pend

ent

diab

etes

mel

litus

Panc

reat

icis

letc

ells

(GA

D65

,IA

2)M

onke

y/hu

man

panc

reas

1/5

-

Isaa

cs

synd

rom

eVo

ltage

gate

dpo

tass

ium

chan

nels

RIA

n/a

-

Tabl

e5.

1.A

utoi

mm

une

dise

ases

and

the

asso

ciat

edau

toan

tibod

ies.

33

Atlas Section

Lam

bert

Eato

nm

yast

heni

csy

ndro

me

Volta

gega

ted

calc

ium

chan

nels

RIA

n/a

-

Lym

phoc

ytic

hypo

phys

itis

Pitu

itary

glan

dau

toan

tigen

sM

onke

ypi

tuita

rygl

and

1/8

-

Mic

rosc

opic

poly

angi

itis

P-A

NC

AN

eutro

phils

1/20

AN

A,C

RP,

mye

lope

roxi

dase

Mor

van

ssy

ndro

me

Volta

gega

ted

pota

ssiu

mch

anne

lsR

IAn/

a-

Mya

sthe

nia

grav

isC

ontra

ctile

elem

ents

ofst

riatio

nalm

uscl

e(ti

tin)

Skel

etal

mus

cle

1/5

Ace

tylc

holin

ere

cept

oran

tibod

ies,

MuS

Kan

tibod

ies,

skel

etal

mus

cle

Myo

card

itis

Myo

sin,

tropo

myo

sin

etc

Mon

key

hear

t1/

5-

Neu

rom

yelit

isop

tica

Aqu

apor

in-4

Rat

/mon

key

cere

bellu

m/m

idbr

ain,

spin

alco

rd1/

60-

Ova

rian

failu

re/in

ferti

lity

Cyt

ochr

ome

P450

enzy

mes

Ova

ry,a

dren

alan

dte

stis

1/5

Thyr

oid

Para

neop

last

icne

urol

ogic

alsy

ndro

me

Hu,

Yo,R

i,M

a,am

phip

hysi

n,C

RM

P-5

Mon

key

cere

bellu

m1/

100

Wes

tern

blot

Para

neop

last

icpe

mph

igus

Epid

erm

alin

ter-c

ellu

larp

rote

ins

Mon

key

oeso

phag

us1/

20M

onke

ybl

adde

r

Pem

phig

oid

(bul

lous

)Ep

ider

mal

base

men

tmem

bran

ezo

neM

onke

yoe

soph

agus

1/20

Skin

biop

syfo

rIgG

orC

3/C

9

Pem

phig

oid

(cic

atric

ial)

Epid

erm

alba

sem

entm

embr

ane

zone

Mon

key

oeso

phag

us1/

20Sk

inbi

opsy

forI

gGor

C3/

C9

Pem

phig

usEp

ider

mal

inte

r-cel

lula

rpro

tein

sM

onke

yoe

soph

agus

1/20

Dire

ctIF

onpa

tient

ssk

in

Pern

icio

usan

aem

iaG

astri

cpa

rieta

lcel

ls(G

PC)

Stom

ach

fund

us1/

20In

trins

icfa

ctor

antib

odie

s,th

yroi

dan

tibod

ies

Poly

arte

ritis

nodo

saC

-AN

CA

Neu

troph

ils1/

20A

NA

Prim

ary

bilia

ryci

rrho

sis

Mito

chon

dria

lM2

antig

enLi

ver,

kidn

ey,s

tom

ach

1/20

CR

P,A

NA

(gp2

10,s

p100

),SM

A

Prim

ary

scle

rosi

ngch

olan

gitis

P-A

NC

AN

eutro

phils

1/20

CR

P

Chapter 5

34

Clin

ical

Diagn

osis

Autoantigens

PreferredTissue

Low

est

Sign

ificant

Titre

Other

Relevan

tTests

Rhe

umat

oid

arth

ritis

Cyc

licci

trulli

nate

dpe

ptid

e(C

CP)

,gr

anul

ocyt

esp

ecifi

cA

NA

EIA

,ne

utro

phils

1/20

Rhe

umat

oid

fact

or(R

F),

AN

A

Stiff

-per

son

synd

rom

eG

luta

mic

acid

deca

rbox

ylas

e67

(GA

D67

)M

onke

yce

rebe

llum

1/50

Wes

tern

blot

,pan

crea

ticis

letc

ell

antib

odie

sTh

ymom

a(m

yast

heni

agr

avis

with

thym

oma)

Con

tract

ileel

emen

tsof

stria

tiona

lmus

cle

(titin

)Sk

elet

alm

uscl

e1/

5A

cety

lcho

line

rece

ptor

antib

odie

s

Thyr

oid

dise

ases

-all

type

sTh

yrog

lobu

lin(T

g),

thyr

oid

pero

xida

se(T

PO)

Mon

key

thyr

oid

1/20

GPC

,pan

crea

ticis

letc

ell

antib

odie

s,st

eroi

dalc

ellA

bs,

EIA

s(T

g,TP

O&

TSH

-R)

Ulc

erat

ive

colit

isP-

AN

CA

(aty

pica

l)N

eutro

phils

1/20

CR

P,A

SCA

Weg

ener

sgr

anul

omat

osis

P-A

NC

AC

-AN

CA

Neu

troph

ils1/

20A

NA

Tabl

e5.

1.A

utoi

mm

une

dise

ases

and

the

asso

ciat

edau

toan

tibod

ies.

More than 20 autoantigens have been described in the literature to beassociated with autoimmune liver diseases, although very few are tissuespecific. The more commonly reported associations are anti-M2 antibodiesreported in over 96% of patients with primary biliary cirrhosis, anti-smoothmuscle antibodies (more specifically anti-actin) associated with autoimmunehepatitis type I, and liver kidney microsomal antibodies associated withautoimmune hepatitis type II.

These antibodies are usually detected by indirect immunofluorescence onrodent tissues. Autoantibodies may occasionally be missed due to thedifferences between rodent and human antigens, however, rodent is generallysatisfactory. Heterophile reactions frequently occur on rat tissues makinginterpretation difficult. Monkey tissues are preferable, particularly whencombined with secondary antibodies that are anti-human, species-specific, asdescribed in Chapter 2. Staining patterns are similar on monkey and rodenttissues with the exception of liver/kidney microsomal antibodies. However,monkey liver demonstrates bile caniculi antibodies and liver cytosolicantibodies, which are not visible on rat liver. Background staining is lower withmonkey tissues and there is much less heterophile staining.

35

Liver Diseases

Autoimmune Liver Diseases

Chapter 6

Chapter 6

36

Table 6.1. Summary of the major liver autoantibodies and associated diseasesdetected on rat or mouse liver/kidney/stomach. PBC = primary biliary cirrhosis;AIH = autoimmune hepatitis; APS = autoimmune polyglandular syndrome.

Figure 6.1. A cryosection from composite block of mouse liver, kidney andstomach tissue stained with anti-mitochondrial and anti-smooth muscleantibodies, labelled with peroxidase conjugate.

Autoantigen Clinical AssociationsSmooth Muscle (SMA) AIH, PBC, viral hepatitisMitochondria (AMA) PBC

Liver Kidney Microsomal(LKM1, 2 and 3)

AIH, hepatitis C-virus infection,hepatitis D-virus infection, APS

type 1 and drug induced hepatitis

Liver Cytosol (LC1) AIH, hepatitis C-virus infection,autoimmune cholangitisSoluble Liver Antigen (SLA)/Liver Pancreas Protei

Autoantibodies

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