Atlas of

Tissue
Autoantibodies
Third Edition
THE BINDING SITE LIMITED
P.O. Box 11712

Atlas of Tissue Autoantibodies (3

Birmingham
B14 4ZB
United Kingdom
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R.G. Hughes
M.J. Surmacz
A.R. Karim
A.R. Bradwell
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ISBN: 070442701X
9780704427013
USA $70
 Atlas of

Tissue Autoantibodies
Third Edition

RG Hughes, BSc., DPhil.

MJ Surmacz, BSc(Hons).
AR Karim, BSc(Hons)., PhD.
AR Bradwell, MB ChB, FRCP, FRCPath.
 Atlas of Tissue Autoantibodies

All rights reserved. No part of this publication may be reproduced, stored in a

©A.R. Bradwell 2008

retrieval system or transmitted in any form or by any means - electronic, mechanical,

photocopying, recording or otherwise - without the prior permission of the publisher or
in accordance with the provisions of the Copyright Act 1988

First published in the United Kingdom in 1997

The Binding Site Ltd., P.O. Box 11712, Birmingham, B14 4ZB, UK.
Distributors:

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 Egrève,
France.
The Binding Site, Balmes 243 4o 3a, 08006 Barcelona, Spain.

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

ISBN: 070442701X
9780704427013

ii
 Atlas of Tissue Autoantibodies

Preface to Third Edition

It is high time for a new atlas of autoantibody patterns. It is 9 years since

the last edition so there have been numerous clinical and technical
developments. It is also appropriate to merge the original “Atlas of
Autoantibody Patterns” with the “Advanced Atlas”, into a single volume. This
3rd Edition now encompasses all the tissue patterns within an expanded version
of 230 pages – larger than both previous editions combined. Importantly, we
have kept the same simple format with large photographic images, and
maintained the emphasis on clinical interpretation and clinical relevance.
Image numbers have increased from 76 to 140 and they are of the highest
quality. Many show rare patterns, but that is the intention, so that there is
comprehensive coverage of all relevant clinical issues in a single volume.
There is extensive text on new antigens, now increased to over 120 from the
original 58. There are detailed descriptions of many new brain, liver and skin
antigens together with numerous others. Furthermore, we have increased the
numbers of diagrams and tables from 12 to 20 in order to provide clarity in
those areas that are complex.
We believe this new edition will provide an important update to the field of
autoimmunity and allow those who perform clinical and laboratory work to
better 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 and
complex personality have lost none of their splendour. And fear not the new
multiple antigen arrays; such features were always part of your character. This
new atlas is dedicated to those who are still captivated by your magic.

AR Bradwell July 2008

iii
 Atlas of Tissue Autoantibodies

Can you identify these patterns?

Sera with unusual patterns will be viewed with interest.

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 Atlas of Tissue Autoantibodies

Acknowledgements

We would like to thank the following:-

Mark Drayson, Senior Lecturer, and Tim Plant, Laboratory Manager, from
the 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 have
generously 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 confocal
microscopy. Lakhvir Assi for significant contributions in writing the ANCA and
APS chapters. Graham Mead for challenging conversations, patience and
critical reviews. Simon Hendy from The Binding Site Ltd., who provided the
immunofluorescence tissues and all other materials that made this atlas
possible.

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 Atlas of Tissue Autoantibodies

Contents

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

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

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

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

Choice of Tissue Substrate and Species . . . . . . . . . . . . . . . . . . . . 3

2. Detection of Autoantibodies on Tissues . . . . . . . . . . . . . . . . . . . . . . . 3

Preparation of Tissue Sections . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

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

EIA Protocols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
3. Detection of Autoantibodies Using Enzyme Immunoassays . . . . . . 19

Practical Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Commercial Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Assay Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Quantitation and Assay Calibration . . . . . . . . . . . . . . . . . . . . . . . 24
EIAs and Autoimmune Assessment . . . . . . . . . . . . . . . . . . . . . . . 25

International Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4. Standardisation and Quality Control . . . . . . . . . . . . . . . . . . . . . . . . 27

Quality Control Schemes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Positive Control Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

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

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 Atlas of Tissue Autoantibodies

Smooth Muscle Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

6. Autoimmune Liver Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

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

Gastric Parietal Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 60

7. Gastro-intestinal Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . 59

Intrinsic Factor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Coeliac Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Endomysial/Tissue Transglutaminase Antibodies . . . . . . . . . . . . 65
Gliadin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Reticulin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Chronic Inflammatory Bowel Disease . . . . . . . . . . . . . . . . . . . . . 75

Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
8. Autoimmune Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

Serum Autoantibody Patterns in Different Renal Diseases . . . . . 83

Glomerular Basement Membrane Antibodies . . . . . . . . . . . . . . . 87
Tubular Basement Membrane Antibodies . . . . . . . . . . . . . . . . . . 91
dsDNA Antibodies and Renal Disease . . . . . . . . . . . . . . . . . . . . . 93
Renal Antibodies of Unknown Significance . . . . . . . . . . . . . . . . 94

Autoantibodies Against Steroid Producing Cells . . . . . . . . . . . . . 99

9. Autoimmune Endocrine Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

Diseases Related to Steroidal Cell Antibodies . . . . . . . . . . . . . . . 105

The Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Pancreatic Islet Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 108
The Thyroid Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Thyroglobulin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Thyroid Peroxidase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Thyrotrophin Receptor Antibodies . . . . . . . . . . . . . . . . . . . . . . . . 114
The Pituitary Gland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
Pituitary Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

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 Atlas of Tissue Autoantibodies

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

Indirect Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121

10. Autoimmune Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119

Indirect Immunofluorescence Using Salt-Split Skin . . . . . . . . . . 127

Direct Immunofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
Autoantigens in Skin Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Autoimmune Skin Diseases - Brief Summaries . . . . . . . . . . . . . 134

Clinical and Pathological Significance . . . . . . . . . . . . . . . . . . . . 139

11. Neurological and Muscle Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 139

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

Classification Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176

12. Anti-Phospholipid Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

Lupus Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177

Cardiolipin and β2 Glycoprotein I Antibodies . . . . . . . . . . . . . . 179
Phosphatidylserine Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

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 Atlas of Tissue Autoantibodies

Additional Anti-Phospholipid Antibody Specificities . . . . . . . . . 185

Prothrombin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Annexin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187

Methodology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191
13. Vasculitis and ANCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

Guidelines and Recommendations for Testing . . . . . . . . . . . . . . 192

Proteinase 3 Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Myeloperoxidase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198
Cathepsin G Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Elastase Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Bactericidal Permeability Increasing Factor Antibodies . . . . . . . 201
Lactoferrin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202
Other Atypical ANCA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

Filaggrin Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207

14. Miscellaneous Autoantibody Specificities . . . . . . . . . . . . . . . . . . . . 207

Salivary Duct Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209

Endothelial Cell Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
Sperm Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

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

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 Atlas of Tissue Autoantibodies

Abbreviations
ACA - anti-cardiolipin antibodies
AChR - acetylcholine receptor
ACTH - adrenocorticotrophic hormone
AECA - anti-endothelial cell antibodies
AGNA - anti-glial nuclear antibodies
AIH - autoimmune hepatitis
AIRE - autoimmune regulator
AKA - anti-keratin antibodies
AMA - anti-mitochondrial antibodies
ANA - anti-nuclear antibodies
ANCA - anti-neutrophil cytoplasmic antibodies
ANT - adenine nucleotide translocator
Anx - annexin
ANNA - anti-neuronal nuclear antibodies
APA - anti-phospholipid antibodies
APECED - autoimmune polyendocrinopathy-candidiasis-ectodermal
dystrophy
APF - anti-perinuclear factor antibodies
APS - autoimmune polyglandular syndrome or anti-phospholipid syndrome
ASA - anti-sperm antibodies
ASCA - anti-Saccharomyces cerevisiae antibodies
ASDA - anti-salivary duct antibodies
ASGPR - asialoglycoprotein receptor
β2GPI - β2 glycoprotein I
BMZ - basement membrane zone
BP180 (BPAG2) - bullous pemphigoid antigen 2
BP230 (BPAG1) - bullous pemphigoid antigen 1
BPI - bactericidal permeability increasing factor
C - Celsius
C-ANCA - cytoplasmic ANCA
CCP - cyclic citrullinated peptides
CDC - Centres for Disease Control
cDNA - complementary DNA
CDR - cerebellar degeneration related proteins
C1q - complement component 1, q subcomponent
CNS - central nervous system
CSF - cerebro spinal fluid
CSS - Churg-Strauss syndrome

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 Atlas of Tissue Autoantibodies

DABCO - 1,4-diazobicyclo-(2,2,2)-octane
DNA - deoxyribonucleic acid
DNAH - de novo autoimmune hepatitis
Dsc - desmocollin
dsDNA - double stranded DNA
Dsg - desmoglein
EBA - epidermolysis bullosa acquisita
EIA - enzyme immunoassay
EMA - endomysial antibody
ENA - extractable nuclear antigen
FITC - fluorescein isothiocyanate
FSH - follicle stimulating hormone
GABA - gamma-amino butyric acid
GAD - glutamic acid decarboxylase
GBM - glomerular basement membrane
GH - growth hormone
GI - gastro-intestinal
GPC - gastric parietal cells
GS-ANCA - granulocyte specific ANCA
GST - glutathione S-transferase
HBV - hepatitis B virus
HCV - hepatitis C virus
HEp-2 - human epithelial cell line type 2
HSP - heat shock protein
IA-2 - tyrosine phosphatase
ICA - islet cell antibody
IDCM - idiopathic dilated cardiomyopathy
IDDM - insulin dependent diabetes mellitus
IF - immunofluorescence
IFA - immunofluorescence assay
Ig - immunoglobulin
IIF - indirect immunofluorescence
JDF - Juvenile Diabetes Foundation
kDa - molecular weight in kilo-Daltons
LA - lupus anti-coagulants
LADA - latent autoimmune diabetes in adults
LC - liver cytosol
LEMS - Lambert Eaton myasthenic syndrome
LH - luteinizing hormone
LKM - liver, kidney microsomal

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 Atlas of Tissue Autoantibodies

LKS - liver, kidney and stomach

LM - liver microsomal
LMA - liver membrane antigen
LP - liver pancreas protein
LSP - liver specific membrane lipoprotein
MAG - myelin associated glycoprotein
MG - myasthenia gravis
mGluR - metabotropic glutamate neurotransmitter receptors
MP - microscopic polyangiitis
MPO - myeloperoxidase
mRNA - messenger RNA
MSH - melanocyte stimulating hormone
MuSK - muscle specific receptor tyrosine kinase
NCGN - pauci-immune necrotising crescentic glomerulonephritis
NEQAS - National External Quality Assurance Scheme
NIBSC - National Institute of Biological Standards and Controls
NMO - neuromyelitis optica
P-ANCA - perinuclear ANCA
PBC - primary biliary cirrhosis
PBS - phosphate buffered saline
PDC - pyruvate dehydrogenase complex
PEG - polyethylene glycol
PGSF1a - pituitary gland specific factor
PMNs - polymorphonuclear leukocytes
PN - polyarteritis nodosa
PNP - paraneoplastic pemphigus
PNS - paraneoplastic neurological syndromes and peripheral nervous system
POA - paraneoplastic opsoclonus ataxia
POEMS - polyneuritis, organomegaly, endocrinopathy, monoclonal
gammopathy and skin changes
POMA - paraneoplastic opsoclonus myoclonus ataxia
PR3 - proteinase 3
PRL - prolactin
PSC - primary sclerosing cholangitis
PVA - polyvinyl alcohol
R - reticulin
RA - rheumatoid athritis
RF - rheumatoid factor
RIA - radioimmunoassay
RNA - ribonucleic acid

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 Atlas of Tissue Autoantibodies

RyR - ryanodine receptor

SCLC - small cell lung carcinoma
SDS - sodium dodecyl sulphate
SDS-PAGE - SDS polyacrylamide gel electrophoresis
SLA - soluble liver antigen
SLE - systemic lupus erythematosus
Sm - Smith ENA
SMA - smooth muscle antibody
SNGN - pauci-immune segmental necrotising glomerulonephritis
SPS - stiff person syndrome
ssDNA - single stranded DNA
T3 - triiodothyronine
T4 - thyroxine/tetraiodothyronine
TBM - tubular basement membrane
Tg - thyroglobulin
TPO - thyroid peroxidase
TSH - thyroid stimulating hormone
tTG - tissue transglutaminase
UC - ulcerative colitis
VGCC - voltage gated calcium channels
VGKC - voltage gated potassium channels
WB - western blotting
WG - Wegener’s granulomatosis
WHO - World Health Organisation

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 Introduction

Chapter 1

Introduction

Identification of autoantibodies is an essential part of clinical medicine and

clinical immunology. Autoantibody testing for the assessment of systemic and
organ-specific autoimmune disease has increased progressively since
immunofluorescence (IF) techniques were first used in 1957 to demonstrate
anti-nuclear antibodies. At present, HEp-2 cells and tissue sections allow the
detection of over 100 different autoantibodies and many more have been
reported in specific immunoassays. There are many specific immunoassays for
confirmation and quantitation of these autoantibodies. However, HEp-2 cells
and tissue sections are frequently used to screen for antibodies thus allowing a
more focused choice of specific tests to be used for identification of the
autoantibody specificity. The purpose of this atlas, in conjunction with the Atlas
of HEp-2 patterns, is to facilitate the identification of characterised
autoantibodies and provide a springboard for the reader to identify new
autoantibodies in clinical immunology.
Rodent liver, kidney and stomach tissues are the traditional substrates for
mitochondrial, gastric parietal cell and smooth muscle antibody detection.
HEp-2 cells have largely replaced rodent liver as the routine substrate for ANA
detection; they are a more sensitive substrate which allows identification of
many staining patterns. Complex tissues cannot be replaced by single cell lines
and tissue sections will remain in use. Although monkey tissues have not
replaced the traditional substrates of rodent liver, kidney, and stomach they are
the substrate of choice for detecting many of the organ specific autoantibodies.
Clear examples are the use of monkey oesophagus for the detection of skin and
endomysial antibodies and monkey kidney for the detection of anti-glomerular
basement membrane (GBM) autoantibodies. With respect to GBM, an
additional streptavidin-biotin step increases sensitivity to match that of enzyme
immunoassays (EIA).
The debate over IF assays (IFA) versus antigen specific asssays will
continue, each technique having its own strengths and weaknesses. IF is
simple, inexpensive and allows identification of many patterns. Introduction of
instrumentation allowing automation of IFA may have added ten years or more
to the longevity of these assays. Other techniques such as western blot and EIA

1
 Chapter 1

will be used increasingly because of greater sensitivity and specificity but are
likely to be used predominantly as secondary, or confirmatory tests unless
quantitative 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 rather
than EIA.
In this atlas each well-known autoantibody pattern is described along with
the clinical associations, the autoantigens involved, the original reference for
their identification (where appropriate), plus relevant recent references. These
references may be used as good starting points should the reader want more
detailed and specific information. Brief descriptions are given for the more rare
or clinically irrelevant patterns. Wherever a serum has been available,
photographs have been included to illustrate typical staining patterns.

2
 Detection of Autoantibodies on Tissues

Chapter 2

Detection of Autoantibodies on Tissues

In many ways detection of autoantibodies by IFA has changed very little

Choice of Tissue Substrate and Species

since the first description of anti-nuclear antibodies by Holborow, Weir and

Johnson in 1957. This lack of technical change indicates the power and
simplicity of the method. What has changed over 50 years is the number of
described autoantibodies and the specific requirements for detecting them. This
has occurred through improved quality and consistency of commercial tissue
sections, the availability of more tissue types and the increased specificity and
avidity of the fluorescein-labelled secondary antibodies. For some diseases the
incremental improvements have been of great clinical consequence. For
instance, anti-GBM antibodies, detected on kidney sections, can now be
detected 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 now
endomysial autoantibodies assessed on monkey oesophagus provide diagnostic
specificity and sensitivity of almost 100%.
Rodent tissues are frequently utilised as substrates for IFA but fail to reveal
some autoantibodies due to differences between rodent and human antigens
(Figure 2.1 and 2.2). Furthermore, heterophile antibodies are commonly
observed 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 section
preparation, is rarely available. Of greater importance, however, is the problem
of human immunoglobulins located in the vascular and extravascular
compartments in the tissues. The labelled second antibodies bind to these
molecules in addition to the patients’ autoantibodies. This results in a high
background fluorescence which reduces sensitivity of the assay (Figure 2.2).
Monkey tissues are clearly more similar to human than rodent tissues so are
preferable in terms of antigen and organ structures. They are also readily
available and can be prepared in perfect condition. Unfortunately, monkey and
human immunoglobulins are also very similar, sharing around 98% of their
amino acid sequences. Thus, monkey and human tissues produce similar
background fluorescence when conventional anti-human immunoglobulin
reagents are used (Figure 2.3).

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 Chapter 2

Figure 2.1. Indirect immunofluorescence on rodent tissues. The rodent antigen

(▲) will be similar but not identical to the human antigen and subsequently
occasional autoantibodies may be missed.

Figure 2.2. Indirect immunofluorescence on human tissues. Although the

human autoantigen (●) will be present in human tissue, the staining pattern may
be difficult to identify due to background staining of the endogenous human
immunoglobulin.
The problem of the secondary antibody reacting against monkey
immunoglobulin can be overcome with an antibody that is specific to the 2-3%
of unique epitopes on human immunoglobulins (Figure 2.4). Whilst this is
technically difficult to achieve, production of species-specific anti-human
immunoglobulin antibodies of high affinity is possible. These antibodies
dramatically improve the sensitivity of autoantibody detection on many monkey
tissues and should be used in preference to second antibodies that cross-react
with monkey immunoglobulins. For steroid cell autoantibodies assessed on
monkey adrenal or ovary tissues the sensitivity of IFA using a species-specific
second antibody is 10-20 fold greater than using a conventional second

4
 Detection of Autoantibodies on Tissues

antibody. A similar improvement is observed with bullous pemphigoid

autoantibody positive sera (Figure 2.11).

Figure 2.3. Indirect immunofluorescence on monkey tissues. The significant

homology between monkey and human proteins ensures that the autoantibodies
are 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 protein
homology are maintained and the problem due to background staining of
endogenous immunoglobulins is removed, resulting in improved clarity and a
more sensitive test system.

Reference

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

5
 Chapter 2

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

Preparation of Tissue Sections

of manufacturers. High quality sections may be stored for over a year at 4˚C or
for several years at -40˚C. Whilst it may appear cheaper or more resourceful to
cut one’s own tissues, in practice considerable cost and expertise is required to
produce thin, uniform sections that do not deteriorate with time, particularly
since there are no standard protocols published describing ideal fixatives,
drying procedures or storage conditions. Several commercial products which
fulfil all the quality requirements are available but there are no systematic
comparisons between products from different manufacturers.

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

of which there are many species. The blood group carbohydrate antigens in
monkeys are identical in structure to human antigens and whilst not present on
red cells, they are expressed in many monkey tissues, depending upon secretor
status. Therefore, on occasions, blood group reactions lead to confusing IF
patterns. 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 antibodies
are more rare, although when present they can be of high titre and avidity. The
false positive staining can be removed by addition of AB antigens to the sample
buffer. Occurrence of this false positive AB reaction on monkey oesophagus is
~3%, although as many as 25% of samples will show an improved clarity when
using the AB block (unpublished data). Arguably, any uncertain pattern should
be re-tested with an AB blocking step so that the intensity of genuine staining
can be assessed.

Monkey tissues positive for AB antigens: Endothelium of capillaries and larger

blood 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 striated
muscle, valvular endocardium, proximal renal tubular cells, pulmonary alveolar
lining cells, hepatocytes, liver sinusoidal lining cells, bile ducts and pancreatic
endocrine cells.

The cytoplasm of the Leydig cells of the testis stains positively with antibodies
Mimicking of steroid cell antibodies by AB reactivity

against P450 enzymes. The appearance is similar to capillaries cut in transverse

section and staining positively for AB antigens in capillary endothelial cells.

6
 Detection of Autoantibodies on Tissues

Whilst confusion is unlikely on the ovary, addition of AB antigens may

facilitate interpretation of testis staining (Figure 2.5).

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 the
sample with AB antigens; only the true staining pattern will remain.

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

Heterophile Antibodies

antibodies that give false positive staining patterns on animal tissues and may
mimic clinically relevant IFA patterns. Such heterophile antibodies vary across
different tissues and animal species; rat and to a lesser extent mouse tissues
show heterophile antibody binding (Table 2.1). Most heterophile antibodies do
not stain monkey tissues. When genuine autoantibodies and heterophile
antibodies co-occur the staining pattern may be difficult to assess on rat tissues,
especially for the inexperienced observer. Mouse tissues largely overcome such
problems but may be more expensive. Outlined below are descriptions to be
used 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 tubule
cytoplasmic staining but no brush border staining.
Genuine SMA on mouse tissues: Staining should be seen between the gastric
glands, of the muscularis mucosa and the muscularis externa tissues, and the
blood vessels.
Genuine anti-mitochondrial/striational/myocardial on rat heart: The antibodies
do not show inter-fibre staining.

7
 Chapter 2

References

Hawkins BR, McDonald BL, Dawkins RL. Characterisation of immunofluorescent heterophile

antibodies 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 Immunopathol
1977; 7: 349-363.

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.

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

8
 Detection of Autoantibodies on Tissues

Figure 2.7. Heterophile antibodies on mouse stomach showing staining

between the gastric glands similar to smooth muscle, but blood vessels are
negative. Endomysial antibodies may be confusingly similar but they are IgA
class and are best demonstrated on monkey oesophagus.

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

9
 Chapter 2

Figure 2.9. Heterophile antibodies staining rat liver sinusoids.

Figure 2.10. Heterophile antibodies staining rat heart endomysium.

10
 Detection of Autoantibodies on Tissues

The species of animal used for the fluorescein-conjugated anti-human

Fluorescein-Conjugated Antibodies

immunoglobulin antibody are usually sheep, goat or rabbit. Immunoglobulin

fractions are satisfactory, however affinity purified antibodies are preferable
because they produce lower background staining. Antibodies are labelled at a
fluorescein/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). The
optimal working dilution is established by chessboard titrations. The undiluted
conjugate is stable for at least a year when stored at 4˚C in the dark. In
commercial kits the reagent is supplied pre-diluted and matched for optimal
reactivity with the respective substrate.
When investigating autoimmune disorders, IgG class autoantibodies are
commonly of most clinical relevance. However, this will depend on the
particular autoimmune disorder under investigation as there are certain
instances where IgA or IgM class autoantibodies are the major interest. The
conjugate specificity must therefore be considered in the context of the tissue
substrate and the particular autoantibodies in question. Frequently, laboratories
will use a non-affinity purified fluorescein-conjugated second antibody directed
against IgG, A and M heavy chains. This will detect IgG, A and M antibodies
equally and results are reported without identifying the antibody class, hence all
results are regarded equally and this strategy can lead to over-reporting of
irrelevant antibodies. There is an additional consideration as to whether the
second antibodies should react with the IgG (H+L) or with IgG, A and M heavy
chains. 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 specific
heavy chain of interest. Examples would be the IgA α chain, IgG γ chain or the
IgM µ chain when investigating tissue-transglutaminase, islet cell and myelin
autoantibodies, respectively. In such circumstances one must ensure that the
conjugate recognises all subclasses since autoantibodies can be subclass
restricted. In conclusion, IgM, IgA and IgG class autoantibodies can be
detected separately or in combination, using the appropriate anti-
immunoglobulin antibody conjugates. The argument in favour of detecting
antibodies of a specific class is that there will be no confusion with irrelevant
antibodies of another class.

Ideally, serum samples should be used within a few hours of collection.

Collection and Preparation of Patient Samples

Storage at 4˚C is satisfactory for samples analysed up to a week after collection

whilst for longer periods (months/years) storage at -20˚C is essential. Frequent

11
 Chapter 2

thawing and freezing of samples may lead to reductions in antibody levels; long
term storage may lead to increases in titre due to lyophilisation. It is
recommended to add preservative to samples for any period of storage; this is
achieved by addition of sodium azide (1mg/ml).
The required dilutions of the patient’s sera depend upon the autoantibodies
under investigation. Some are considered to be of significance if present at a
dilution of 1/5 whilst others may only be significant at 1/100 or greater (Table
5.1). The individual chapters provide guidelines on the clinical relevance of
antibodies in relation to their titres. Dilution of samples should be in phosphate
buffered 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 of
serum globulins to the tissues.

Each manufacturer will state a specific immunofluoresence procedure which

Immunofluorescence Assay Procedure

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

1. Slides should be allowed to reach room temperature before opening. This

prevents condensation forming on the substrate which can disrupt the
antigen localisation.
2. Slides should be labelled appropriately, including the date. Labelling
should be with a pencil as ink may subsequently run across the wells and
result in unwanted auto-fluorescence.
3. Place 30-50µl 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 of
PBS especially when agitation is not possible. More important is a high
ratio of wash volume to the number of slides/samples i.e. the use of
coplin jars leads to insufficient washing.
6. The slides should be removed from the wash; excess fluid knocked off
and if necessary, blotted briefly with absorbent paper. The fluorescein-
labelled conjugate (30-50µl) is then added onto each well. Slides are
incubated for a further 30 minutes at room temperature in a light

12
 Detection of Autoantibodies on Tissues

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. Traditional
mounting media are glycerol based (70-90% in PBS pH 8.6), however,
for most substrates a polyvinyl alcohol (PVA) based mounting media will
better maintain staining and improve clarity.*
9. Observe the wells through an epi-fluorescence microscope with filters
optimal for fluorescein detection. Magnification is dependent on the
tissue; when a 50x magnification or above is used an immersion lens is
preferable. Properly prepared fluorescein labelled sections are stable for
several weeks when stored at 4˚C.

Counterstain: The use of counterstain is very much a matter of personal

preference. Some manufacturers provide Evans Blue pre-diluted in the kit
conjugate. Alternatively, a minute amount can be added to the final wash. Care
must be taken as excessive amounts can lead to a loss in sensitivity.

* There are various recipes for mounting medium. We recommend either a

glycerol based mounting medium (90% glycerol in PBS pH 8.6) or a polyvinyl-
alcohol (PVA) based mounting medium. Chemicals can be added to the
mounting medium to reduce fading of fluorescence during illumination. We
recommend DABCO (1,4-diazobicyclo-[2,2,2]-octane, Sigma-Aldrich
Company Ltd.) 2.5g per 100ml of mounting medium.
Johnson GD, Davidson RS, McNamee KC, Russell G, Goodwin D, Holborow EJ. Fading of
immunofluorescence during microscopy: a study of the phenomenon and its remedy. J Immunol
Methods 1982; 55: 231-242.

Sensitivity of IFA is sufficient to detect the majority of clinically significant

Techniques to Increase Sensitivity of IFA

autoantibodies. Indeed, ANA detection using HEp-2 cells is arguably too

sensitive at sample dilutions of 1/40 to 1/80 and should preferably be performed
at 1/160. The argument becomes stronger in the case of samples from the
elderly; the frequency of autoantibodies, especially ANA increases with age.
The quality and proper maintenance of the epi-fluorescence microscope is a
significant factor affecting detection of autoantibodies. Mercury bulbs have a
limited life span and must be regularly replaced as their intensity will
deteriorate with age. Centring of the arc should be assessed weekly to guarantee
a consistent level of sensitivity. Lenses and filters must be checked and cleaned
on a regular basis. Modern epi-fluorescence microscopes will provide a very

13
 Chapter 2

strong level of excitation and in some cases it may be advisable to employ filters
to moderate this intensity. Some autoantibody specificities, such as anti-GBM
are often screened by EIA because it is considered more sensitive but some
simple procedures can increase IFA sensitivity by up to 10 times.
Longer incubation times: The traditional method of increasing sensitivity is to
increase incubation times of the patient’s sera by up to 18 hours. Sensitivity is
increased approximately four-fold but background fluorescence also increases
and the tissues become progressively more fragile with long exposure to liquid
reagents. Typically, pancreatic islet cell antibodies have been detected in this
manner.
Anti-human, species-specific, second antibodies: As mentioned earlier these
reagents enhance sensitivity by reducing background staining (Figure 2.11).
This gain can be further utilised by reducing the sample dilution factor. For
example, adrenal antibodies are normally screened using a 1/5 dilution because
a 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 thus
allowing weaker autoantibodies to be detected. The clinical utility of detecting
weaker autoantibodies would need evaluating in the respective clinical
environment.

Autoantibody staining patterns can also be visualised on tissue sections using

Enzyme Immunohistochemistry Staining on Tissues

enzyme-conjugated second antibodies. The immunoperoxidase technique with

3,3’-diaminobenzidine (DAB) or 3-amino-9-ethyl-carbazole (EAC) as substrate
produces 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 be

considered:
1. Sensitivity is lower, particularly for antigens that are sparsely distributed
such as mitotic spindle antigens in HEp-2 cells.
2. The procedure is more time consuming due to the extra incubation step.

14
 Detection of Autoantibodies on Tissues

Figure 2.11. Monkey oesophagus stained with pemphigoid antibodies and

species-specific (left) and non species-specific (right) secondary antibodies.
Endogenous monkey IgG in the highly vascular lamina propria is not
recognised using the species-specific antibody.

The intensity and patterns of fluorescence should be assessed and recorded.

Interpretation of Results

The intensity can be expressed according to a scale of values compatible with

the guidelines established by the reference centres for established control sera
(e.g. CDC, Atlanta) as negative or + to ++++. A semi-quantitative evaluation
can be obtained by performing serial dilutions of the test serum to endpoint
fluorescence. Such slides should be read from the most dilute sample upwards
and the end point is considered the first sample where a discernable positive
pattern is observed. For confidence in reporting results, the positive and
negative controls must have performed to expectations.

Autoantibodies occur in both physiological and pathological conditions. In

Considerations during interpretation

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

15
 Chapter 2

may be because the antibodies are absent, poor presentation of target antigens
or unsatisfactory assay technique. A further possibility is that the level of
activity of the disease results in adsorption of the autoantibodies by antigens
released into the circulation. In contrast, low titre antibodies may be found in
normal people, relatives of patients with autoimmune conditions and a variety
of diseases such as inflammation and cancer with no autoimmune basis.
Autoantibody levels also increase with age, particularly in women, without
necessarily being harmful. Interpretation must be made with reference to the
individual’s medical history, age and existing conditions. Of equal importance
is awareness of the limitations of the tests and quality control of substrates and
reagents.
The majority of IF patterns are only indicative of autoantibody specificity
and exact specificity must be confirmed by other techniques, such as
immunoblotting or EIA. Where the antigens are known, such specific assays
provide quantitative and definitive results. However, frequently the
autoantigens are not fully characterised and IF is necessary. In many clinical
laboratories EIA and other methods are likely to be secondary, or confirmatory
tests unless quantitative antibody concentrations are clinically essential.
Mixed patterns are a frequent occurrence on tissue sections. These have to
be distinguished by using combinations of tissue such as liver, kidney and
stomach. They also have to be distinguished from heterophile antibodies. For
HEp-2 cells, diluting test sera may help to resolve the titre and specificity of
different autoantibody combinations and hence their clinical relevance.

Identification of immunofluorescent staining is predominantly subjective so

Practical Aspects of Running a Clinical Autoantibody Service

there may be considerable variation in interpretation of results from day to day

and between different observers. Should discrepancies arise, particularly
between past and present specimens, it is important to be able to identify old
slides and compare them with the new slides. For this reason we would
recommend including the following procedures.

1. Only use slides that have numerically identified wells.

Preparation

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 specimen
number.
5. Follow a standardised assay protocol

16
 Detection of Autoantibodies on Tissues

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

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

Reporting

mistakes it is imperative that transcription is carried out by the person who

interpreted the fluorescence. When results have been verified, with particular
reference to the age, sex and diagnosis of the patient, they are authorised for
reporting. The clinical immunologist adds appropriate comments and signs the
result form before dispatch. Autoantibodies are more common in patients over
50 years of age and therefore weaker samples for certain specificities may have
little clinical relevance. This must be aluded to in the report. If a discrepancy
arises between assay runs, the appropriate historical slide may be readily
located by diary date, slide number, and well number and the sample checked.

Identification of autoantibody specificity is generally of clinical interest once

Identification of Antibody Specificity

a patient serum has been shown to contain autoantibodies. In the majority of

cases the staining pattern is no more than suggestive of antibody specificity and
precise specificity must be confirmed by other means.
Immunoblotting: The western blot assay is an essential tool for the
characterisation of many autoantibodies. The following is a brief description
but details should be sought in the relevant publications. Proteins from an
extract of cultured cells are separated by polyacrylamide gel electrophoresis
under denaturing and reducing conditions with sodium dodecyl-sulphate (SDS-
PAGE). Proteins in the gel are then transferred to nitrocellulose paper, which
provides a solid support for the antigens. The nitrocellulose paper is cut into
strips and each is incubated with a serum to be tested. Several methods are in
use for detecting a specific antigen-antibody reaction: enzymes such as
horseradish peroxidase on radiolabelled probes, followed by
chemiluminescence reactions, and autoradiography. Molecular mass markers
and positive and negative controls are run together in each assay. They provide
the necessary comparison bands alongside the patient sera. The main problem
with these assays is that the antigens are not in their native structure but as linear
peptides. This inevitably distorts non-linear epitopes so that results are not

17
 Chapter 2

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

General Reference

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

Karim AR. Immunofluorescence Image Library:

http://www.ii.bham.ac.uk/clinicalimmunology/CISimagelibrary/

18
 Enzyme Immunoassays

Chapter 3
Detection of Autoantibodies Using
Enzyme Immunoassays

Enzyme immunoassays (EIA) are widely used in clinical laboratories for

quantifying thyroid, neutrophil, anti-dsDNA and other autoantibodies. Brief
comments are made on the clinical role of these assays in the relevant chapters,
methods and other technical considerations are discussed here. The concept of
an EIA is similar to that of the immunofluorescent assay, where the conjugated
fluorochrome of the secondary antibody is replaced with an enzyme. There are,
however, marked differences between these two methods which confer certain
practical advantages. The use of specifically selected purified antigens in EIAs
ensures greater assay specificity compared to IFA, where an array of antigens
are presented. EIAs are also more sensitive and give quantifiable results,
although autoantibody concentrations do not necessarily correlate with disease
activity and so this is not always advantageous. EIAs are easily automated,
making them the method of choice for autoantibody monitoring in many
clinical laboratories. This is especially the case when there is a high sample
throughput. This popularity has led to the commercial availability of EIAs for
the detection of most autoantibody specificities where the clinically relevant
antigen has been identified.

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

EIA Protocols

including indirect, sandwich, and competitive assays. Indirect EIAs are the
most commonly used for the detection of autoantibodies and so these are the
focus here. Briefly, autoantigen is adsorbed onto a solid support, most
commonly a 96 well, polystyrene microtitre plate. Any remaining protein
binding sites are blocked to prevent non-specific adsorption of serum
immunoglobulins. The plate is then ready for the determination of autoantibody
levels (Figure 3.1). Alternatively, the plate can be dried and stored in an airtight
container. Here the block will also act as a stabiliser and, when stored
appropriately, the plate should be stable for more than 12 months. It is essential
to ensure that all components are given sufficient time to reach room
temperature prior to commencing the assay.

19
 Chapter 3

Test Protocol

1. Sample dilution: 1:100 is commonly employed as a starting dilution. This is

a greater screening dilution than generally used in IFA, a reflection of the
greater sensitivity of the assay system.

2. Sample addition: 100µl 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 the
recommended period of time (e.g. 30 minutes).

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

4. Conjugate addition: 100µl of diluted enzyme-conjugated anti-

immunoglobulin is added to the wells and the plate is incubated at room
temperature (e.g. 30 minutes). Horse-radish peroxidase (HRP) and alkaline
phosphatase (AP) are the most commonly employed enzymes.

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

6. Substrate: 100µl of substrate is added. The plate is incubated for a further

period to allow the coloured product to form (e.g. 30 minutes). There are many
enzyme substrates available. For HRP 3,3’5,5’Tetramethylbenzidene (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 coloured
end product (p-nitrophenyl).

7. After the last incubation, the reaction is stopped by the addition of 100µl of
stop solution. For HRP the stop solution is a strong acid, e.g. 1M HCl, H2SO4
or H3PO4 and in the case of AP 1M NaOH is used.

8. The wells are then read within 30 minutes (preferably immediately) by

measuring the absorbance of the coloured end product using a
spectrophotometer. The wavelength examined depends on the enzyme substrate
utilised; TMB 450nm and p-Nitrophenyl phosphate 405/650nm.

20
 Enzyme Immunoassays

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

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

Practical Considerations

both individually and as a complete process (some major considerations are

outlined below):

Antigen source: The source and purity of the antigen has a fundamental effect
on the assay performance; several factors should be taken into account.
Evaluation of antigen from different sources is recommended as large variations
in performance may be seen. It is not unknown for the same grade of antigen
from different suppliers to perform differently: phospholipids are a clear
example. To reduce non-specific binding the antigen should be as pure as
possible. The choice between recombinant and native antigen rests on two
considerations. Native antigen of human origin is not always available and so
there remains a question of homology between the human autoantigen and the
antigen from the chosen species. Recombinant antigen is usually derived from
the human sequence and so the amino acid sequence homology will be exact.
However, the recombinant antigen may not be folded correctly nor have had
post translational modifications such as glycosylation. The final choice is often
down to price and availability and is resolved by trial and error.

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

21
 Chapter 3

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

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

Block: Any uncoated regions of the polystyrene surface must be blocked to

prevent serum components from sticking non-specifically to its surface. To
ensure efficient blocking, the volume used must be greater than both the volume
of original antigen coat and that of the diluted sample. This step is generally
carried out at room temperature for a minimum of 30 minutes. Sugars, proteins
and proprietary blocks are all used for this purpose; ultimately the block of
choice is dependent on the antigen being coated. The block may also
incorporate a stabiliser thus allowing longer term storage of the coated plates.

Conjugate specificity: Clinically significant autoantibodies are frequently of

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

Incubation temperature: For a consistent inter-assay performance it is important

to ensure that the running conditions are kept constant. Both antibody binding
kinetics and enzyme activity can be affected by fluctuations in temperature.
Ideally, when running an assay, all the components must be maintained at a set
temperature and the incubations should be performed within a temperature
controlled environment.

Good quality commercial EIAs are available from a number of

Commercial Assays

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 the
running of their EIAs. Colour coding of reagents is a distinct advantage for the
user, as is a common buffer system and identical dilution steps for assays within

22
 Enzyme Immunoassays

Figure 3.2. Photograph showing a commercial kit including colour coded

reagents.
a complementary range. The assays should be optimised, taking many of the
previously mentioned points into consideration. They will also have been
validated for expected performance and have a generous shelf life. As with all
immunodiagnostic assays, to achieve optimum performance, all
recommendations from the manufacturer must be adhered to when running the
assays.

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

performance data. It is therefore necessary to establish performance

characteristics with respect to precision, linearity, sensitivity, stability and the
effect of potentially interfering substances. Such data should be considered for
in-house assays and is generated as a matter of course for commercial assays.

Linearity, precision and stability: Linearity of the assay can be shown by

running a dilution series of several samples followed by calculating the %
recovery. Intra-assay and inter-assay reproducibility can be determined by
testing a number of different samples in multiple repeats within the same assay
run and on separate occasions, respectively. It is also helpful to establish the
performance across separate production batches. Stability is assessed by
repeated testing over a period of time. It is also important to challenge the assay
with extreme temperature conditions and mimic any transient fluctuations of
storage conditions.

23
 Chapter 3

Sensitivity and specificity – The diagnostic sensitivity of an assay can be

determined by studying a suitable number of patients with known disease states.
Within the test group it is important to include both the disease group in
question and a significant number of samples from patients with disease states
for which the differential diagnosis is carried out. It is also advisable to use
normal blood donors as another control group, this may highlight unexpected
observations for the disease state controls. The positive and negative predictive
values of a clinical assay are dependent on the sample population under
investigation; there will be a marked difference in calculated performance
between screening the general population and when testing a group of patients
which have been selected on grounds of clinical symptoms.

Effect of interfering substances – There are a number of substances that are

recognised to interfere with certain immunodiagnostic assays. It is therefore
necessary to establish whether there is interference for any assay and, if
identified, to be aware of the degree of such interference. Substances that
should be taken into consideration include: rheumatoid factor, elevated
immunoglobulin levels, lipids, haemolysed samples and bilirubin. Other
potential interfering substances may be relevant when considering particular
assays.

In order to allow quantitative and comparable reporting of results, EIAs

Quantitation and Assay Calibration

should be calibrated against international standards, and units quoted in either

international units or micrograms per millilitre. This is predominantly the case
where recognised standards are available; however, this is more often the
exception than the rule. Where no standard is available, one of two options is
implemented: a cut-off control is used or, alternatively, a calibration curve is
generated against an internal reference. One must consider that the returned
value is not a direct measure of concentration; it is rather a reflection of the
combination of autoantibody avidity and titre. Autoantibodies are often of
lower avidity, but this will differ from sample to sample. There is also the
possibility that a sample has mixed populations of autoantibodies covering a
range of avidities.

Cut-off – If a simple indication of negative, low, medium and high values is

adequate, then results may be reported as a multiple of an established cut-off
control. This is a crude form of calibration and does not take into account the
sigmoidal nature of the true calibration curve. An advantage of this simple
calibration is that this takes up only one test well on the microtitre plate and so

24
 Enzyme Immunoassays

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

Calibration curve – Where there is benefit in returning a quantitative result, a

precise calibration curve needs to be established. An EIA calibration curve
covering the whole range of autoantibody concentrations is expected to be
sigmoidal in shape. In order to establish an equation to describe such a curve a
4 parameter logit fit is performed; this requires a minimum of 5 points,
preferably 7, across the whole curve. To ensure reproducibility and prevent
calibration drift across multiple batches it is necessary to create an internal
reference against which all calibration material is standardised. Where
international standards are available, the internal reference should be calibrated
against this, otherwise a suitable serum should be chosen; due to the variable
nature of autoantibodies, a pool of sera is preferable. When a calibration curve
is set, special emphasis should be given to the region where a differential
diagnosis could be the outcome, this is usually at the lower end of the curve.
Accurate quantification of high autoantibody values is generally of less
importance, although reproducibility remains essential.

EIAs are available for the determination of most autoantibody specificities

EIAs and Autoimmune Assessment

where the autoantigen has been clearly described. However, the choice to
perform an EIA as opposed to another immunodiagnostic technique will depend
on several factors including cost and throughput. A quantitative result does not
always provide additional value when identifying autoantibodies. In many
cases, the distinction between presence or absence of autoantibody is helpful
and in other cases an indication of low-, medium- and high-titres can be
sufficient. In such circumstances, the decision to use an EIA for autoantibody
determination will be for purposes other than a clinical requirement, e.g. there
may be a cost advantage due to a high sample throughput. Reporting the
numerical result of an EIA does not demand the same skill and experience
which is necessary to interpret IFA staining patterns.
Interestingly, a number of laboratories choose to use EIAs as an initial screen
for ANA determination. This may appear counter-intuitive as HEp-2 IFA tests
are generally cheaper. Apart from the benefits of automation, the reasoning in
favour of this approach is that an ANA EIA has specifically selected
autoantigens, those of most clinical relevance. As the antigens on the plate are
known, one can further identify the specificity of positive samples by using
single-specificity EIAs. This approach will detect the majority of ANAs but a
proportion of ANA samples will be missed. Any unidentified autoantibodies

25
 Chapter 3

will probably be of limited clinical importance: specificities will be for minor

antigens of little known significance.
Selective screening for autoantibodies is most efficiently performed using a
system where the relevant autoantigens are all present, such as cerebellum when
looking for paraneoplastic antibodies, monkey oesophagus when testing for
autoimmune skin diseases or HEp-2 cells when investigating a connective
tissue disease query. Once positive samples are identified then more specific
assays can be employed to confirm the autoantibody specificity inferred from
the immunofluorescent staining pattern. However, when a clinician has a strong
suspicion of a particular condition and there are known autoantibody profiles,
with both high sensitivity and specificity, a direct request may be made for a
particular marker, which will be determined by EIA. Tissue transglutaminase
and the M2 mitochondrial antigen are clear examples for coeliac disease and
primary biliary cirrhosis, respectively, as are anti-CCP antibodies with respect
to rheumatoid arthritis. Occasionally, there are conditions which are defined by
the autoantibody profile, such as anti-phospholipid syndrome (Chapter 12).
Other than the lupus anticoagulant test, there is no alternative to using EIAs for
determination of these antibodies.

26
 Standardisation and Quality Control

Chapter 4
Standardisation and Quality Control

Recognising IF patterns is relatively straightforward but assessing IF

intensity and its clinical significance requires considerable experience.
Definitive decisions on sensitivity can only be made from practice and by
regularly viewing batches of normal, or allegedly normal, sera in order to obtain
the picture of a ‘negative’ background. When there is doubt, a clinician should
be involved and the final judgement made in the context of the patient’s clinical
symptoms.
Organ-specific autoantibodies are relatively easy to identify and interpret.
After only a short training period most members of staff are able to recognise
different patterns of the more common anti-nuclear antibody types and to
distinguish positive staining of β cells of the islets of Langerhans, zona
glomerulosa of adrenal, theca cell layer of developing follicles etc. However,
in composite blocks of liver, kidney and stomach, autoantibody patterns are less
clear because the observer is searching for patterns in three tissues, frequently
against a background of other staining. There are also a huge variety of
staining patterns which may be ‘new’ even to someone with many years
experience. The problems are enhanced when employing rodent tissues due to
heterophile 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 some
patterns that do not relate to the patient’s diagnosis may be of interest. The
clinicians can choose to ignore the results or one may indicate that they are of
no known significance. Such results may be reviewed from time-to-time to
assess their importance.

27
 Chapter 4

There are few human autoantibody standards relative to the number of

International Standards

known specificities for immunofluorescence assays, apart from anti-nuclear

antibodies. It is not apparent why there has been such little progress. The
following is a list of the materials that have been prepared in stable form for
general use:

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

1. Human primary biliary cirrhosis serum: 67/183

MRC Research Standard A. 100 units per ampoule.

2. Human anti-thyroid microsome serum: 66/387

No official status. 1,000 units per ampoule.

3. Human anti-thyroglobulin serum: 65/93

1st International Reference Preparation 1978. 1,000 International Units
per vial.

4. Human rheumatoid arthritis serum: 64/2 (WHO 1066)

1st British Standard, 100 International Units per ampoule.

5. Human anti-islet cell serum: 97/550

1st WHO Reference Reagent 1999, 20 units per ampoule (also 100 units
of anti-GAD65 and anti-IA-2).

6. Native (ds)DNA antibody: WHO Wo80

1st WHO Reference Reagent 1985. 100 International Units per ampoule

7. Human anti-smooth muscle (anti-actin) serum: W1062

WHO International Reference Reagent.

- Sapporo monoclonal antibodies (HCAL for IgG and EY2C9 for IgM).
Cardiolipin Antibody Standards

Available from: Centres for Disease Control and Prevention (CDC), 1600
Clifton Road, Atlanta, GA 30333.

28
 Standardisation and Quality Control

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

- Louisville (‘Harris’) calibrators for the measurement of anti-cardiolipin IgG

and IgM antibodies. Available from: Louisville APL Diagnostics, Inc., 2622
NASA Pwky STE G2 Seabrook, TX 77586 USA.

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

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 and
comprise normal or pathological human sera. Results are reported as U/ml or
titre and for qualitative responses are positive or negative.

- General Autoimmune Serology (rheumatoid factor, thyroid peroxidase

antibody, mitochondrial antibody, liver-kidney microsome antibody, smooth
muscle antibody and gastric parietal cell antibody).
- Antibodies to Nuclear and Related Antigens (anti-nuclear antibody, dsDNA
antibody, ENA antibodies, HEp-2 cell nuclear substrate and centromere
antibodies).
- Phospholipid Antibodies (cardiolipin antibody, β2 glycoprotein I antibody
and phosphatidylserine antibody).
- ANCA and GBM Antibodies (neutrophil cytoplasmic antibody, proteinase 3
antibody, myeloperoxidase antibody and glomerular basement membrane
antibody).
- Acetylcholine Receptor Antibodies (AChR antibody).
- Bullous Dermatosis and Coeliac Disease Antibodies (skin basement
membrane antibody, desmosome antibody, gliadin antibody, endomysial
antibody and tissue transglutaminase antibody).
- Andrology (sperm antibodies).

2. The College of American Pathologists provides a scheme for autoimmune

serology which is available from The College of American Pathologists, 325
Waukgan Road, Northfield, Illinois 60093-2750, USA. Samples are distributed
from one to three times per year.
- Special Immunology (S2) including: mitochondrial, smooth muscle and
glomerular basement membrane antibodies.
- Coeliac Disease (CES) including IgA endomysium antibodies.

29
 Chapter 4

- Gastrointestinal (GIH) including mitochondrial and gastric parietal cell

antibodies.

3. INSTAND e.v., Ubierstr 20, Üsseldorf, D-40223, Germany.

- Special Autoimmune including mitochondrial, smooth muscle, liver-kidney
microsome, gastric parietal cell, endomysium and Purkinje antibodies.

Several commercial companies provide quality control schemes for

autoimmune testing including The Binding Site Ltd., P.O. Box 11712,
Birmingham, B14 4ZB, U.K.

Many are available from commercial sources including The Binding Site
Positive Control Sera

Ltd., which has antibodies against the following antigens:

Adrenal/ovary/testis steroidal cell Liver/kidney microsomal (LKM)
ANA Mitochondrial
C-ANCA (proteinase 3) Myelin associated glycoprotein (MAG)
P-ANCA (myeloperoxidase) Pancreatic islet cells
dsDNA Pemphigoid (bullous)
ENA Pemphigus vulgaris
Endomysium (IgA) Paraneoplastic pemphigus (PNP)
Gastric parietal cells Reticulin 1 (IgA)
Gliadin Skeletal muscle fibres (striational)
Glomerular basement membrane Smooth muscle (SMA)
(GBM) Yo (PCA-1)
Glutamic acid decarboxylase (GAD)
Hu (ANNA-1)

Reference

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

30
 Atlas Section

Chapter 5

Atlas Section Introduction

The remaining chapters in this book describe the immunofluorescence

staining patterns of autoantibodies. Each chapter is self contained and based
upon autoimmune disease groups, rather than tissue substrate usage. A
summary table is to be found at the beginning of each chapter, this highlights
the major staining patterns, common specificities and related disease diagnosis.
The animal species of the tissue substrates are indicated for each image. On
monkey tissues the second antibody is sheep anti-human species-specifc IgG
FITC unless otherwise stated.
The figures showing immunofluorescent patterns are taken at various
magnifications to optimally identify the autoantibodies and demonstrate
confusing patterns. Photographs were taken over a period of time and several
image capture devices were used. A variety of objectives were employed, with
a x50 water immersion lens for the higher magnifications. Subsequent
photographic enlargement was usually employed in order to optimally highlight
the relevant features.
The summary table overleaf is designed to allow rapid identification of
antibody patterns, associated diseases and vice versa. Also, the lowest
significant titre is highlighted for each specificity. Awkward telephone
conversations can be turned into authoritative accounts of target autoantigens,
disease associations and other test requirements with a quick scan of this table
and those at the beginning of each respective chapter.

31
32
Table 5.1. Autoimmune diseases and the associated autoantibodies.

Lowest
Chapter 5

Clinical Diagnosis Autoantigens Preferred Tissue Significant Other Relevant Tests

Titre
Addison’s disease Cytochrome P450 enzymes Adrenal gland 1/5 Ovary, testis, GPC
Anti-phospholipid syndrome Phospholipid binding proteins EIA n/a Lupus anticoagulants
Autoimmune gastritis Gastric parietal cells (GPC) Stomach fundus 1/20 Thyroid antibodies
Autoimmune hepatitis type 1 Smooth muscle (F-actin) Liver, kidney & stomach 1/40 Soluble liver antigen (SLA)
Autoimmune hepatitis type 2 Liver kidney microsomal 1, 2 & 3 Liver & kidney 1/20 Liver cytosol antibodies (LC1)
Churg-Strauss syndrome P-ANCA Neutrophils 1/20 ANA, CRP
Coeliac disease Endomysium (tTG) Monkey oesophagus 1/10 Gliadin and reticulin
ASCA, EIA
Crohn’s disease 1/20 P-ANCA (atypical), CRP
pancreatic acinar cells pancreas
Dermatitis herpetiformis Endomysium (tTG) Monkey oesophagus 1/10 Gliadin and reticulin
Glomerular basement membrane
Goodpasture’s Syndrome Monkey kidney 1/5 ANCA, CRP
(α-3-chain type IV collagen)
Herpes gestationis Epidermal basement membrane zone Monkey oesophagus 1/20 Skin biopsy for IgG or C3/C9
Idiopathic crescentic
P-ANCA Neutrophils 1/20 ANA, CRP
glomerulonephritis
Idiopathic dilated
β-1-adrenergic receptor EIA n/a Myosin, tropomyosin etc
cardiomyopathy (IDCM)
Insulin dependent Monkey/human
Pancreatic islet cells (GAD65, IA2) 1/5 -
diabetes mellitus pancreas
Isaac’s syndrome Voltage gated potassium channels RIA n/a -
 Lambert Eaton myasthenic
Voltage gated calcium channels RIA n/a -
syndrome
Lymphocytic hypophysitis Pituitary gland autoantigens Monkey pituitary gland 1/8 -
Microscopic polyangiitis P-ANCA Neutrophils 1/20 ANA, CRP, myeloperoxidase
Morvan’s syndrome Voltage gated potassium channels RIA n/a -
Contractile elements of Acetylcholine receptor antibodies,
Myasthenia gravis Skeletal muscle 1/5
striational muscle (titin) MuSK antibodies, skeletal muscle
Myocarditis Myosin, tropomyosin etc Monkey heart 1/5 -
Rat/monkey
Neuro myelitis optica Aquaporin-4 cerebellum/mid brain, 1/60 -
spinal cord
Ovarian failure/infertility Cytochrome P450 enzymes Ovary, adrenal and testis 1/5 Thyroid
Paraneoplastic neurological Hu, Yo, Ri, Ma,
Monkey cerebellum 1/100 Western blot
syndrome amphiphysin, CRMP-5
Paraneoplastic pemphigus Epidermal inter-cellular proteins Monkey oesophagus 1/20 Monkey bladder
Pemphigoid (bullous) Epidermal basement membrane zone Monkey oesophagus 1/20 Skin biopsy for IgG or C3/C9
Pemphigoid (cicatricial) Epidermal basement membrane zone Monkey oesophagus 1/20 Skin biopsy for IgG or C3/C9
Pemphigus Epidermal inter-cellular proteins Monkey oesophagus 1/20 Direct IF on patient’s skin
Intrinsic factor antibodies,
Pernicious anaemia Gastric parietal cells (GPC) Stomach fundus 1/20
thyroid antibodies
Polyarteritis nodosa C-ANCA Neutrophils 1/20 ANA

Primary biliary cirrhosis Mitochondrial M2 antigen Liver, kidney, stomach 1/20 CRP, ANA (gp210, sp100), SMA

Primary sclerosing cholangitis P-ANCA Neutrophils 1/20 CRP

Atlas Section

33
34
Lowest
Clinical Diagnosis Autoantigens Preferred Tissue Significant Other Relevant Tests
Titre
Chapter 5

Cyclic citrullinated peptide (CCP), EIA, Rheumatoid factor (RF),

Rheumatoid arthritis 1/20
granulocyte specific ANA neutrophils ANA
Glutamic acid decarboxylase 67 Western blot, pancreatic islet cell
Stiff- person syndrome Monkey cerebellum 1/50
(GAD67) antibodies
Thymoma (myasthenia Contractile elements of
Skeletal muscle 1/5 Acetylcholine receptor antibodies
gravis with thymoma) striational muscle (titin)
GPC, pancreatic islet cell
Thyroglobulin (Tg),
Thyroid diseases - all types Monkey thyroid 1/20 antibodies, steroidal cell Abs,
thyroid peroxidase (TPO)
EIAs (Tg, TPO & TSH-R)
Ulcerative colitis P-ANCA (atypical) Neutrophils 1/20 CRP, ASCA
P-ANCA
Wegener’s granulomatosis Neutrophils 1/20 ANA
C-ANCA

Table 5.1. Autoimmune diseases and the associated autoantibodies.

 Liver Diseases

Chapter 6

Autoimmune Liver Diseases

More than 20 autoantigens have been described in the literature to be

associated with autoimmune liver diseases, although very few are tissue
specific. The more commonly reported associations are anti-M2 antibodies
reported in over 96% of patients with primary biliary cirrhosis, anti-smooth
muscle antibodies (more specifically anti-actin) associated with autoimmune
hepatitis type I, and liver kidney microsomal antibodies associated with
autoimmune hepatitis type II.
These antibodies are usually detected by indirect immunofluorescence on
rodent tissues. Autoantibodies may occasionally be missed due to the
differences between rodent and human antigens, however, rodent is generally
satisfactory. Heterophile reactions frequently occur on rat tissues making
interpretation difficult. Monkey tissues are preferable, particularly when
combined with secondary antibodies that are anti-human, species-specific, as
described in Chapter 2. Staining patterns are similar on monkey and rodent
tissues with the exception of liver/kidney microsomal antibodies. However,
monkey liver demonstrates bile caniculi antibodies and liver cytosolic
antibodies, which are not visible on rat liver. Background staining is lower with
monkey tissues and there is much less heterophile staining.

35
 Chapter 6

Autoantigen Clinical Associations

Smooth Muscle (SMA) AIH, PBC, viral hepatitis
Mitochondria (AMA) PBC

AIH, hepatitis C-virus infection,

Liver Kidney Microsomal
hepatitis D-virus infection, APS
(LKM1, 2 and 3)
type 1 and drug induced hepatitis

AIH, hepatitis C-virus infection,

Liver Cytosol (LC1)
autoimmune cholangitis
Soluble Liver Antigen (SLA)/ AIH, PBC, autoimmune
Liver Pancreas Protein (LP) hepatitis overlap syndrome
Glutathione-S-Transferase (GST) A1-1 AIH, PBC
Table 6.1. Summary of the major liver autoantibodies and associated diseases
detected on rat or mouse liver/kidney/stomach. PBC = primary biliary cirrhosis;
AIH = autoimmune hepatitis; APS = autoimmune polyglandular syndrome.

Kidney Cortex Kidney Medulla

Liver
Stomach

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

36
 Liver Diseases

Smooth Muscle Antibodies

Antigen: Anti-smooth muscle antibodies have several target antigens found in

the filaments of smooth and striated muscle (Table 6.2).

Muscle Filament Antigens

6nm Microfilament (thin filament) Actin, myosin, tropomyosin and troponin
10nm Intermediate Filament Vimentin, desmin and cytokeratin
25nm Filament (microtubules) Tubulin
Table 6.2. Antigens of smooth muscle antibodies.

Anti-actin antibodies are the most clinically relevant, at high titres they are very
specific for autoimmune hepatitis type 1. In eukaryotic cells actin functions as
an important structural molecule of the cytoskeleton as well as being involved
in contraction and relaxation of muscle. Filamentous actin (F-actin) is the
polymerised form of globular subunits (G-actin) which have a molecular weight
of 42kDa.

Clinical associations: Anti-smooth muscle antibodies are associated with

autoimmune hepatitis type 1, primary biliary cirrhosis, chronic viral hepatitis B
and C, non-alcoholic fatty liver disease, alcoholic cirrhosis, other autoimmune
inflammatory diseases and can also be found in apparently healthy individuals.
Anti-actin smooth muscle antibodies are frequently detected at high titres in
autoimmune hepatitis type 1 (50-80%) and primary biliary cirrhosis (20%). At
a titre of 1/40 or greater, they are considered to be a specific marker for
autoimmune hepatitis type 1. Anti-SMA associated with other diseases often
react with a variety of other cytoskeletal antigens and are usually present at
lower titres.

Detection: IIF using rodent LKS sections (Figure 6.1) and anti-actin smooth
muscle antibodies, usually of the IgG subclass, reveals a classic homogeneous
staining of the interglandular actin fibres and muscularis mucosae, mesangial
cells of the glomeruli (SMA-G), and intracellular fibrils of the renal tubule
(SMA-T) and peritubular areas (Figure 6.2). Anti-SMA antibodies with
reactivity to additional antigens may also be observed to stain sub-membranous
actin around hepatocytes (producing a polygonal pattern) and the muscle layer
around blood vessels (SMA-V). Anti-actin antibodies can be detected on HEp-
2 cells although LKS sections are the preferred substrate. Anti-nuclear

37
 Chapter 6

antibodies are frequently found in combination with anti-SMA.

References

Johnson GD, Holborow EJ, Glynn LE. Antibody to smooth muscle in patients with liver disease.
Lancet 1965; 2: 878-879.

Bottazzo G-F, Florin-Christensen A, Fairfax A, Swana G, Doniach D, Groeschel-Stewart U.

Classification of smooth muscle autoantibodies detected by immunofluorescence. J Clin Pathol
1976; 29: 403-410.

Granito A, Muratori L, Muratori P, Pappas G, Guidi M, Cassani F et al. Antibodies to filamentous

actin (F-actin) in type 1 autoimmune hepatitis. J Clin Pathol 2006; 59: 280-284.

Villalta D, Bizzaro N, Da Re M, Tozzoli R, Komorowski L, Tonutti E. Diagnostic accuracy of four

different immunological methods for the detection of anti-F-actin autoantibodies in type 1
autoimmune hepatitis and other liver-related disorders. Autoimmunity 2008; 41: 105-110.

Figure 6.2. SMA on rat stomach (left), liver (centre) and kidney (right). Note
staining of interglandular actin fibres and muscularis mucosae in the stomach,
muscle layer around blood vessels in the liver, mesangial cells of the glomeruli
and intracellular fibrils of the renal tubule and peritubular areas in the kidney.

38
 Liver Diseases

Figure 6.3. Monkey stomach (left), liver (centre) and kidney (right) stained with
SMA.

Figure 6.4. SMA with a specificity for actin staining monkey kidney at high
magnification.

39
 Chapter 6

Mitochondrial Antibodies

Antigen: There are several mitochondrial autoantigens (Table 6.3.), although

anti-M2 antibodies are the most frequently detected and of greatest clinical
utility. Anti-mitochondrial M2 antibodies react with a variety of subunits of
pyruvate 2-oxo-acid dehydrogenase complex (Table 6.4.). This enzyme is
located on the inner mitochondrial membrane and is involved in energy
metabolism. PDC-E2 is the dominant subunit and the related antibodies are
virtually indicative of primary biliary cirrhosis.

Associated Disease (Antibody

Antibody Antigen
Frequency)
Syphilis (96%), SLE and other
M1 Cardiolipin
autoimmune diseases
PBC (96%) and occasionally in
2-oxo-acid dehydrogenase
M2 other chronic liver diseases and
complex
systemic sclerosis
Pseudolupus erythematosus
M3 Unknown
syndrome
PBC with M2 (up to 55%), mixed
M4 Sulphite oxidase form of PBC and chronic
aggressive hepatitis
Undefined collagen diseases,
visceral lupus erythematosus,
M5 Possibly cardiolipin complex
autoimmune haemolytic anaemia,
anti-phospholipid syndrome
Mono-amino oxidases,
M6 Iproniazid-induced hepatitis
Iproniazid

Table 6.3. Mitochondrial autoantigens.

It should be noted that there are two numbering systems for mitochondrial
antibody classification in use, one based on Storch, 1981 (AMA 1-10) and
another based on Berg and Klein, 1992 (AMA 1-9). Types 1-6 are identical in
both systems; however types 7, 8 and 9 of the Berg classification are different
to those in the Storch classification as they are not detected by
immunofluorescence.

40
 Liver Diseases

Antigen MW (kDa) Incidence in PBC

E2-subunit of pyruvate dehydrogenase
70-74 >95%
complex (PDC-E2)
E2-subunit of branched chain 2-oxo-acid
52 52-55%
dehydrogenase complex (BCOADC-E2)
E2-subunit of 2-oxo-glutarate dehydrogenase
48 39-88%
(OGDC-E2)
E1a-subunit of pyruvate dehydrogenase
41 41-66%
complex (PDC- E1a)
E3 dihydrolipoamide dehydrogenase binding
50-55 90-95%
protein (E3-BP/Protein X)
E1§ subunit of pyruvate dehydrogenase
36 2-7%
(PDC-E1§)
Table 6.4. Antigens of M2 antibodies. There is a recombinant protein (pML-
MIT3) which co-expresses the immunodominant epitopes of PDC-E2,
BCOADC-E2 and OGDC-E2 (Moteki et al. 1996). This protein is utilised in
EIA and immunoblotting assays.

Clinical associations: Clinically, anti-M2 antibodies are important as they are

strongly associated with PBC. Anti-mitochondrial antibodies are also detected
in asymptomatic patients who later go on to develop PBC. M4 and M8
antibodies, when detected, are typically found alongside M2 antibodies and are
associated with a more aggressive disease and rapid onset of terminal cirrhosis.
Naturally occurring anti-mitochondrial antibodies (NOMAs) are detected in
people who are in close contact with patients with PBC or their sera, although
rarely in patients with PBC. These antibodies are directed against different
antigenic epitopes to AMA and their clinical significance is uncertain.

Detection: IIF using rat or mouse LKS composite blocks is considered to be the
gold standard for detection of AMA in PBC with a specificity of almost 100%.
AMA at titres of 1/40 or greater are recognised as being specific for PBC; they
are also one of the three criteria used for diagnosis. Anti-M2 antibodies show
cytoplasmic granular fluorescence in the liver hepatocytes, proximal tubules of
the kidney (stronger in P1 and P2 than P3) and parietal and chief cells of the
stomach (Figure 6.5 and 6.6). Other types of AMA can sometimes be identified
by differences in the intensity and areas of staining produced (Table 6.6). IIF
using HEp-2 cells can detect AMA, although LKS composite blocks are
considered to be more sensitive and reliable. On HEp-2 cells, AMA will
produce course granular speckles in the cytoplasm (Figure 6.7). There are many
EIAs for the detection of M2 antibodies, however all EIA negative samples

41
 Chapter 6

Figure 6.5. Anti-M2 antibodies showing differential cytoplasmic granular

fluorescence of the proximal tubules (P1, P2 and P3) in rat kidney.

Figure 6.6. Cytoplasmic granular fluorescence in the parietal and chief cells of
rat stomach (left) and in rat liver hepatocytes (right) by anti-mitochondrial M2
antibodies.

42
 Liver Diseases

should be confirmed by IFA in clinically suspicious cases. AMA may mask

other staining patterns such as anti-GPC antibodies.

Nuclear Antibodies

ANA which specifically recognise sp100 and gp210 are also found in the sera
of PBC patients (see Atlas of HEp-2 patterns, Third Edition). On HEp-2 cells
anti-sp100 and anti-gp210 antibodies will produce a multiple nuclear dot and
nuclear membrane pore immunofluorescent patterns respectively. In a study by
Bogdanos et al. (2003), the presence of anti-sp100 antibodies and AMA
correlated with recurrent urinary tract infection in women with no evidence of
liver disease. These findings add support to the idea that E.coli infection has a
role in the development of PBC.

Figure 6.7. Anti-mitochondrial M2 antibodies on HEp-2 cells showing granular

filamentous staining of mitochondria in the cell cytoplasm.

43
 Chapter 6

Figure 6.8. Comparison of anti-mitochondrial M2 antibodies on rat (left) and

monkey stomach (right).

References

Walker JG, Doniach D, Roitt IM, Sherlock S. Serological tests in diagnosis of primary biliary
cirrhosis. Lancet 1965; 1: 827-831.

Berg PA, Klein R. Antimitochondrial antibodies in primary biliary cirrhosis and other disorders:
definition and clinical relevance. Dig Dis 1992; 10: 85-101.

Moteki S, Leung PS, Coppel RL, Dickson ER, Kaplan MM, Munoz S et al. Use of a designer triple
expression hybrid clone for three different lipoyl domains for the detection of antimitochondrial
autoantibodies. Hepatology 1996; 24: 97-103.

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

Bogdanos DP, Baum H, Butler P, Rigopoulou EI, Davies ET, Ma Y et al. Association between the
primary biliary cirrhosis specific anti-sp100 antibodies and recurrent urinary tract infection. Dig
Liver Dis 2003; 35: 801-805.

Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005; 353: 1261-1273.

Andrejevic S, Bonaci-Nikolic B, Sefik-Bukilica M, Petrovic R. Clinical and serological follow-up

of 71 patients with anti-mitochondrial type 5 antibodies. Lupus 2007; 16: 788-793.

44
 Liver Diseases

Liver/Kidney Microsomal Antibodies

Antigen: The target antigens of liver/kidney microsomal antibodies are P450

cytochromes (Table 6.5); a large and diverse family of enzymes characterised
by the presence of a haem pigment.

Antibody Antigen Molecular Weight

LKM1 P4502D6/CYP2D6 50kDa
LKM2 P4502C9/CYP2C9 50kDa
Uridine diphosphate (UDP)
LKM3 55kDa
glucuronosyl transferase
Table 6.5. LKM autoantibody antigens.
CYP2D6 and CYP2C9 are involved in phase one metabolism to detoxify
xenobiotics such as drugs, metals, industrial and naturally occurring chemicals.
UDP-glucuronosyl transferase catalyses glucuronidation in phase two
detoxification to produce non-toxic products for excretion. Liver/kidney
microsomal antibodies may also recognise other P450 cytochrome antigens;
these require characterisation with specific assays.

Clinical associations: Anti-LKM1 antibodies are detected in autoimmune

hepatitis type 2 (80-95%), hepatitis C (~7%) and also hepatitis associated with
autoimmune polyglandular disease type 1. Anti-LKM2 antibodies are less
frequently detected and are usually associated with drug (e.g. tienilic acid)
induced hepatitis. Anti-LKM3 antibodies, also rarely detected, are associated
with AIH type 2 (8-19%), hepatitis C (~6%) and hepatitis D (6-13%).

Detection: IIF using a screening dilution of 1/20 on LKS composite blocks

enables detection of anti-LKM antibodies (Figure 6.9). The origin of the tissue
should be considered carefully as differences in staining pattern can occur. Anti-
LKM1 antibodies on rat tissue will show homogeneous staining of the
hepatocytes and tubules of the inner cortex of the kidney, i.e. the proximal renal
tubules, however the distal tubules (some close to the glomeruli) are negative or
only weakly positive. This allows anti-LKM1 antibodies to be easily
distinguished from anti-mitochondrial antibodies which stain both the proximal
and distal renal tubules (Table 6.6). Variations in LKM-1 antibody staining have
been shown in AIH and hepatitis C infections, suggesting additional
specificities. Comparison of LKM-1 positive sera on rat and monkey tissues
further supports this possibility. In an in-house study (unpublished data) 21
samples showed typical staining on rat liver and kidney. Of these 8 were similar

45
 Chapter 6

on monkey tissues, 8 showed minimal kidney staining and 4 showed selective

proximal tubular staining. Examples are shown in Figures 6.10 and 6.11. One
sample showed typical staining on monkey tissues but on rodent tissue stained
only the liver.
Anti-LKM2 antibodies on mouse tissue show strong positive staining of
centrolobular hepatocytes and in contrast to LKM1 there is weaker staining of
periportal hepatocytes. The renal tubules in the outer cortex of the kidney are
positive, i.e., around the glomeruli but the inner cortex tubules are only weakly
positive.
Anti-LKM3 antibodies stain rat and primate tissue similarly to LKM1.
However, anti-LKM3 antibodies will also bind microsomes in primate thyroid,
adrenal and pancreas. Specificity of LKM antibodies can be confirmed using
EIA, western blot and radioligand assays.

References

Rizzetto M, Swana G, Doniach D. Microsomal antibodies in active chronic hepatitis and other
disorders. Clin Exp Immunol 1973; 15: 331-344.

Crivelli O, Lavarini C, Chiaberge E, Amoroso A, Farci P, Negro F et al. Microsomal autoantibodies

in chronic infection with the HBsAg associated delta (δ) agent. Clin Exp Immunol 1983; 54: 232-
238.

Homberg JC, Andre C, Abuaf N. A new anti-liver-kidney microsome antibody (anti-LKM2) in

tienilic acid-induced hepatitis. Clin Exp Immunol 1984; 55: 561-570.

Homberg JC, Abuaf N, Bernard O, Islam S, Alvarez F, Khalil SH et al. Chronic active hepatitis
associated with antiliver/kidney microsome antibody type 1: a second type of "autoimmune"
hepatitis. Hepatology 1987; 7: 1333-1339.

Bradwell AR, Elias E, Milkaewicz P, Haigh T, Peycke S, Drayson M. Detection of autoantibodies

in liver diseases using monkey tissues. Clin Chem 1998; 44: S6:44.

Strassburg CP, Manns MP. Liver cytosol antigen type 1 autoantibodies, liver kidney microsomal
autoantibodies and liver microsomal autoantibodies. In: Autoantibodies. Shoenfeld Y, Gershwin
ME, Meroni PL, editors. 2nd Edition. Elsevier Science; 2007.

46
 Liver Diseases

Figure 6.9. LKM1 antibodies showing homogeneous fluorescence of the

hepatocytes and proximal renal tubules in rat liver and kidney.

Figure 6.10. LKM1 staining monkey liver (right), but kidney is negative (left).

47
 Chapter 6

Figure 6.11. LKM1 staining monkey kidney (left) and liver (right).

Liver Cytosol Antibodies

Antigen: The target antigen of anti-liver cytosol antibodies is

formiminotransferase cyclodeaminase. This is a 62kDa intracellular enzyme
found in the cytosol of hepatocytes and is involved in the metabolism of folate
(B12), an essential water-soluble vitamin.

Clinical associations: Anti-liver cytosol 1 antibodies are associated with

autoimmune hepatitis type 2 (20-30%) and hepatitis C-virus infection (2-10%).
Antibody titres correlate with disease activity in autoimmune hepatitis type 2.
Anti-LC1 antibodies may be easily missed as anti-LKM1 antibodies are
frequently found to coexist.

Detection: IIF using rodent or monkey liver and anti-LC1 antibodies produces
homogeneous cytoplasmic staining of the liver hepatocytes (screening dilution
1/20). With rodent liver the hepatocyte layer around the central vein is spared,

48
 Liver Diseases

however with monkey liver there is more uniform staining of the whole hepatic
lobule (Figure 6.12). Co-occurrence of anti-LKM antibodies will mask the
central-lobular sparing observed in rodent tissues. In comparison to anti-LKM
antibodies, anti-LC1 will not stain the proximal tubules of the kidney. Anti-LC1
antibodies can also be detected using western blotting, immunoprecipitation
and counter-immunoelectrophoresis.

References

Martini E, Abuaf N, Cavalli F, Durand V, Johanet C, Homberg JC. Antibody to liver cytosol (anti-
LC1) in patients with autoimmune chronic active hepatitis type 2. Hepatology 1988; 8: 1662-1666.

Muratori L, Cataleta M, Muratori P, Lenzi M, Bianchi FB. Liver/kidney microsomal antibody type
1 and liver cytosol antibody type 1 concentrations in type 2 autoimmune hepatitis. Gut 1998; 42:
721-726.

Beland K, Lapierre P, Marceau G, Alvarez F. Anti-LC1 autoantibodies in patients with chronic

hepatitis C-virus infection. J Autoimmun 2004; 22: 159-166.

Figure 6.12. Rat (left) and monkey (right) liver showing homogeneous
cytoplasmic fluorescence due to LC1 antibodies.

49
 Chapter 6

Kidney Liver Stomach

Proximal and distal Homogeneous diffuse Parietal and chief
M1
tubules diffuse + + cells diffuse +
Granular distal tubules
Parietal cells +++,
M2 +++, proximal tubule Granular ++
chief cells +
++ (P1/P2>P3)
Granular proximal and
M5(rare) distal tubules Granular ++ Villous tip cells ++
(P1/P2>P3)
Proximal tubules Enteroendocrine
M6(rare) (P1/P2>P3) granular Granular ++ cells (APUD cells)
++ ++
Proximal tubules
diffuse homogeneous
LKM1 Homogeneous +++ -
+++, distal tubules
negative
Centrolobular
Proximal tubules hepatocytes +++,
LKM2 -
positive (P1/P2>P3) periportal hepatocytes
weakly positive
LKM3 As LKM1 As LKM1 -
Homogeneous
LC1 - -
cytoplasmic +++
Table 6.6. Summary of the major liver autoantibody staining patterns on rodent
LKS. For illustration of nephron structure see Figure 8.2 (including proximal
P1, P2, P3 and distal tubule location).

Soluble Liver Antigen Antibodies (Liver Pancreas Protein)

Antigen: Anti-SLA and anti-LP antibodies were reported to be identical by

Wies et al. (2000). Although the specificity of these antibodies continues to be
debated, the evidence strongly suggests that one of the target antigens is a
50kDa cytosolic transfer ribonucleoprotein complex (tRNP (ser) sec) which is
involved in the incorporation of selenocysteine into polypeptide chains. Other
proposed antigenic targets include α-enolase and catalase. It is likely that anti-
SLA antibodies are heterogeneous in their target antigens.

Clinical associations: Anti-SLA/LP antibodies are associated with

autoimmune hepatitis (10-30%), cryptogenic hepatitis (25%) and PBC/AIH

50
 Liver Diseases

overlap syndrome. Patients diagnosed with cryptogenic hepatitis often have

AIH; this is supported by their positive response to immunosuppressive therapy.
Anti-SLA/LP antibodies were originally used as markers for AIH type 3,
however these patients are now generally regarded as having AIH type 1 due to
their similar clinical and biological features. Titres of anti-SLA/LP antibodies
are thought to correlate with disease activity and also the presence of these
autoantibodies correlates with an increased relapse rate following corticosteroid
withdrawal.

Detection: Anti-SLA/LP antibodies cannot be detected using IIF, possibly

because the fixation procedure does not preserve the antigen. They can be
detected using EIA and RIA which use cytosolic liver extracts or recombinant
antigens.

References

Manns M, Gerken G, Kyriatsoulis A, Staritz M, Meyer zum Buschenfelde KH. Characterisation of

a new subgroup of autoimmune chronic active hepatitis by autoantibodies against a soluble liver
antigen. Lancet 1987; 1: 292-294.

Wies I, Brunner S, Henninger J, Herkel J, Kanzler S, Meyer zum Buschenfelde KH et al.

Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis. Lancet 2000;
355: 1510-1515.

Torres-Collado AX, Czaja AJ, Gelpí C. Anti-tRNP(ser)sec/SLA/LP autoantibodies. Comparative

study using in-house ELISA with a recombinant 48.8 kDa protein, immunoblot, and analysis of
immunoprecipitated RNAs. Liver Int 2005; 25: 410-419.

Asialoglycoprotein Receptor Antibodies

Antigen: The transmembrane asialoglycoprotein receptor is expressed at high

density on the surface of periportal liver cells. It has a role in the internalisation
of asialoglycoproteins by binding a terminal galactose residue in coated pits.

Clinical associations: Anti-ASGPR antibodies are most frequently detected in

active AIH types 1 and 2 (70-90%), where antibody titre correlates with disease
activity. The antibodies are not specific and have been found in patients with
PBC, primary sclerosing cholangitis (PSC), chronic hepatitis B and alcoholic
liver disease.

Detection: IIF cannot be used to detect anti-ASGPR antibodies. They can be

detected using EIA and radioimmunofiltration assay (RIFA), however these

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 Chapter 6

assays are difficult to establish as they require chemically purified ASGPR

which is not widely available.

References

McFarlane IG, McFarlane BM, Major GN, Tolley P, Williams R. Identification of the hepatic asialo-
glycoprotein receptor (hepatic lectin) as a component of liver specific membrane lipoprotein (LSP).
Clin Exp Immunol 1984; 55: 347-354.

Czaja AJ, Pfeifer KD, Decker RH, Vallari AS. Frequency and significance of antibodies to
asialoglycoprotein receptor in type 1 autoimmune hepatitis. Dig Dis Sci 1996; 41: 1733-1740.

Schreiter T, Liu C, Gerken G, Treichel U. Detection of circulating autoantibodies directed against

the asialoglycoprotein receptor using recombinant receptor subunit H1. J Immunol Methods 2005;
301: 1-10.

Glutathione S-Transferase A1-1 Antibodies

Antigen: Glutathione S-transferase A1-1 is a phase II multi-drug metabolising

enzyme.

Clinical associations: A study by Kato et al. (2004), reported anti-GST A1-1

antibodies in autoimmune hepatitis (~16%) and primary biliary cirrhosis
(~10%). The antibodies are frequently found alongside ANA, SMA and AMA.
Patients positive for anti-GST A1-1 antibodies have been reported to have
severe clinical features and a poor prognosis.

Detection: IIF using rat LKS composite blocks will allow detection of anti-
GST A1-1 antibodies. Kato et al. (2004) described the pattern on liver as
staining of the cytoplasm of perivenous hepatocytes resulting in a zonation
image, with stronger staining adjacent to the vessels and decreased towards the
periphery. On the kidney, the antibodies were observed to stain the proximal
tubules and different segments of distal tubules. Immunoblotting using
recombinant GST A1-1 can also be used to detect anti-GST A1-1 antibodies.

References

Wesierska-Gadek J, Grimm R, Hitchman E, Penner E. Members of the glutathione S-transferase

gene family are antigens in autoimmune hepatitis. Gastroenterology 1998; 114: 329-335.

Kato T, Miyakawa H, Ishibashi M. Frequency and significance of anti-glutathione S-transferase

autoantibody (anti-GST A1-1) in autoimmune hepatitis. J Autoimmun 2004; 22: 211-216.

52
 Liver Diseases

Other Liver Autoantigens

There are many other antibodies associated with liver diseases, however, most
of them cannot be detected by IIF and are of limited clinical significance. A
selection of these are described below.

Liver Microsomal (LM): These antibodies react with hepatic cytochrome

P450 CYP1A2. They can be detected using IIF, immunoblotting and
radioligand assay (RLA). The IIF pattern shows liver microsomal staining
which predominates in perivenous hepatocytes and is absent in the kidney. Co-
existing LKM antibodies can mask the liver microsomal antibody staining
pattern. Liver microsomal antibodies may also recognise other cytochrome
P450 antigens which cannot be characterised using IIF. The antibodies are
detected in the sera of patients with dihydralazine-induced hepatitis. Also, a
small proportion of patients with autoimmune polyglandular syndrome type 1-
APS1 (see Chapter 9, Endocrine Diseases) will have anti-LM antibodies. In
these circumstances they are a specific marker for an additional disease
component (AIH type 1).

References

Nataf J, Bernuau J, Larrey D, Guillin MC, Rueff B, Benhamou JP. A new anti-liver microsome
antibody: a specific marker of dihydralazine-induced hepatitis. Gastroenterology 1986; 90: 1751.

Obermayer-Straub P, Perheentupa J, Braun S, Kayser A, Barut A, Loges S et al. Hepatic

autoantigens in patients with autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy.
Gastroenterology 2001; 121: 668-677.

Dihydrolipoamide Dehydrogenase (E3): This is the shared E3 subunit of PDC

(pyruvate dehydrogenase complex), OGDC (2-oxoglutarate dehydrogenase
complex) and BCOADC (branched-chain 2-oxoacid dehydrogenase complex).
Anti-dihydrolipoamide dehydrogenase (E3) antibodies are detected via EIA and
immunoblotting in HCV (~53%), PBC (~27%) and HBV (~2%). In HCV
infection, anti-dihydrolipoamide dehydrogenase antibodies are associated with
increased risk of developing abnormal liver function, liver cirrhosis, arthritis
and elevated α-fetoprotein levels.

References

Maeda T, Loveland BE, Rowley MJ, Mackay IR. Autoantibody against dihydrolipoamide
dehydrogenase, the E3 subunit of the 2-oxoacid dehydrogenase complexes: significance for primary
biliary cirrhosis. Hepatology 1991; 14: 994-999.

53
 Chapter 6

Wu YY, Hsu TC, Chen TY, Liu TC, Liu GY, Lee YJ et al. Proteinase 3 and dihydrolipoamide
dehydrogenase (E3) are major autoantigens in hepatitis C virus (HCV) infection. Clin Exp Immunol
2002; 128: 347-352.

Carbonic Anhydrase II: Is a zinc metal enzyme found in the cholangiocytes

of the liver, pancreatic duct cells, gastric parietal cells, distal renal tubular cells
and acinar cells of the salivary glands. Anti-carbonic anhydrase II antibodies are
detected via ELISA and immunoblotting in AMA negative PBC (~50%), AMA
positive PBC (~43%), AIH (~50%), HCV (~25%) and HBV (20-40%).
However, they are not specific to liver disease and have also been found in SLE,
scleroderma, Sjögren’s syndrome, polymyositis, dermatomyositis,
endometriosis and autoimmune pancreatitis.

References

Gordon SC, Quattrociocchi-Longe TM, Khan BA, Kodali VP, Chen J, Silverman AL et al.
Antibodies to carbonic anhydrase in patients with immune cholangiopathies. Gastroenterology
1995; 108: 1802-1809.

Hosoda H, Okawa-Takatsuji M, Tanaka A, Uwatoko S, Aotsuka S, Hasimoto N et al. Detection of

autoantibody against carbonic anhydrase II in various liver diseases by enzyme-linked
immunosorbent assay using appropriate conditions. Clin Chim Acta 2004; 342: 71-81.

Liver Membrane Antigen (LMA): Liver membrane antibodies bind to an

uncharacterised antigen found on the hepatocyte plasma membrane (Figure
6.13). Many potential antigens have been suggested, however it is probable that
the target antigen is heterogeneous. IIF reveals characteristic staining of the
hepatocyte plasma membrane. LM antibodies have been detected in a variety of
liver diseases including AIH, PBC, viral hepatitis and also in SLE. The
antibodies can be detected using isolated hepatocytes and IF, EIA, RIA or
immunoblotting.

References

Hopf U, Meyer zum Buschenfelde KH, Arnold W. Detection of a liver-membrane autoantibody in

HBsAg-negative chronic active hepatitis. N Engl J Med 1976; 294: 578-582.

Matsuo I, Ikuno N, Omagari K, Kinoshita H, Oka M, Yamaguchi H et al. Autoimmune reactivity of

sera to hepatocyte plasma membrane in type 1 autoimmune hepatitis. J Gastroenterol 2000; 35:
226-234.

Liver Specific Membrane Lipoprotein (LSP): This was the first liver
membrane antibody to be described. LSP is a heterogeneous liver preparation
containing more than 20 proteins, with molecular weights of 5-220kDa. Only

54
 Liver Diseases

two of the antigens have been characterised; these are the asialoglycoprotein
receptor (ASGPR) and alcohol dehydrogenase (ADH). Antibodies to LSP are
not specific for liver diseases and therefore, are rarely measured.

References

Meyer zum Buschenfelde KH, Miescher PA. Liver specific antigens. Purification and
characterization. Clin Exp Immunol 1972; 10: 89-102.

McFarlane IG, Wojcicka BM, Zucker GM, Eddleston AL, Williams R. Purification and
characterization of human liver-specific membrane lipoprotein (LSP). Clin Exp Immunol 1977; 27:
381-390.

Ma Y, Gaken J, McFarlane BM, Foss Y, Farzaneh F, McFarlane IG et al. Alcohol dehydrogenase: a

target of humoral autoimmune response in liver disease. Gastroenterology 1997; 112: 483-492.

Figure 6.13. Comparison of liver membrane antibodies on rat (left) and monkey
(right) liver.

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 Chapter 6

Bile Duct: These are found in patients with chronic liver diseases but are of no
known clinical significance (Figure 6.14).

Figure 6.14. Autoantibodies staining bile duct and hepatocytes.

Bile Canalicular: In 1966, Johnson et al., described the first bile canaliculi
antibodies (Figure 6.15 and 6.16). They are associated with AIH, however, their
clinical relevance remains uncertain.

Figure 6.15. Comparison of antibodies to bile canaliculi on rat liver, also

demonstrating heterophile staining (left) and a much clearer pattern on monkey
liver (right).

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 Liver Diseases

Figure 6.16. Bile canaliculi antibodies staining monkey liver/kidney (left) and
liver at high magnification (right).

References

Johnson GD, Holborow EJ, Glynn LE. Antibody to liver in lupoid hepatitis. Lancet 1966; 2: 416-
418.

Diederichsen H. Hetero-antibody against bile canaliculi in patients with chronic, clinically active
hepatitis. Acta Med Scand 1969; 186: 299-302.

Biliary Epithelial Cell: These antibodies recognise a number of unidentified

antigens on the hepatocyte cell surface. Anti-biliary epithelial cell antibodies
have been found in PSC (~63%), PBC (~37%), AIH (~16%) and normal
controls (~8%).

References

Xu B, Broome U, Ericzon BG, Sumitran-Holgersson S. High frequency of autoantibodies in patients

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 Chapter 6

with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44
and production of interleukin 6. Gut 2002; 51: 120-127.

Karrar A, Broome U, Sodergren T, Jaksch M, Bergquist A, Bjornstedt M et al. Biliary epithelial cell
antibodies link adaptive and innate immune responses in primary sclerosing cholangitis.
Gastroenterology 2007; 132:1504-1514.

Liver Transplant and de novo Autoimmune Hepatitis

The occurrence of de novo AIH (DNAH) is a rare phenomenon affecting ~2-5%

of patients 0.7-9.5 years after undergoing liver transplantation. Patients affected
with DNAH have detectable autoantibodies including ANA, SMA, LKM, P and
C-ANCA. DNAH is reported to improve after standard treatment for AIH
(steroids and azathioprine). Monitoring of patients having undergone liver
transplant for AIH related antibodies may prove to be invaluable in identifying
patients at risk of developing DNAH and subsequent graft loss.

References
Kerkar N, Hadzic N, Davies ET, Portmann B, Donaldson PT, Rela M et al. De-novo autoimmune
hepatitis after liver transplantation. Lancet 1998; 351: 409-413.

Riva S, Sonzogni A, Bravi M, Bertani A, Alessio MG, Candusso M et al. Late graft dysfunction and
autoantibodies after liver transplantation in children: preliminary results of an Italian experience.
Liver Transpl 2006; 12: 573-577.

General References

Alvarez F, Berg PA, Bianchi FB, Bianchi L, Burroughs AK, Cancado EL et al. International
Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J
Hepatol 1999; 3: 929-938.

Czaja AJ, Norman GL. Autoantibodies in the diagnosis and management of liver disease. J Clin
Gastroenterol 2003; 37: 315-329.

Vergani D, Alvarez F, Bianchi FB, Cancado EL, Mackay IR, Manns MP et al. Liver autoimmune
serology: a consensus statement from the committee for autoimmune serology of the International
Autoimmune Hepatitis Group. J Hepatol 2004; 41: 677-683.

Zachou K, Rigopoulou E, Dalekos GN. Autoantibodies and autoantigens in autoimmune hepatitis:

important tools in clinical practice and to study pathogenesis of the disease. J Autoimmune Dis
2004; 1: 2.

Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005; 353: 1261-1273.

Czaja AJ. Autoimmune liver disease. Curr Opin Gastroenterol 2006; 22: 234-240.

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 Gastro-intestinal Diseases

Chapter 7
Gastro-intestinal Autoimmune Diseases

The autoimmune aspects of gastro-intestinal diseases have been studied in

great detail, however, evidence of an abnormal immune response having a role
in disease pathogenesis differs between the individual disorders. The
association of anti-gastric parietal cell (GPC) antibodies with pernicious
anaemia, especially in conjunction with anti-intrinsic factor antibodies, has
been recognised for over 30 years and autoantibody determination has an
established role as part of the differential diagnosis. In coeliac disease,
knowledge and understanding of autoantibody association has evolved steadily.
Anti-reticulin (R1) antibodies were identified first, these were superseded by
the more sensitive anti-gliadin antibodies which themselves were, in turn,
superseded by the superior diagnostic performance of endomysial antibody
(EMA) detection. Other autoantibodies have been reported to be associated
with coeliac disease, although possibly of most interest are recent reports of an
improved specificity when using modified gliadin peptides.
Although, studied for an equal period of time, the association and role of
autoantibody assessment in diagnosis of inflammatory bowel disease (IBD) and
patient management remains uncertain and controversial. Ulcerative colitis
(UC) and Crohn’s disease (CD) present with very similar symptoms and a
physical examination will rarely differentiate between the two. Examination by
endoscopy and biopsy are almost always required, even here a number of cases
remain un-differentially diagnosed. Treatment and disease management can
differ so available tests to differentiate the two would be beneficial. Many
autoantibody specificities have been investigated, some have even shown
significant specificity, however, none have been shown to have the sensitivity
required to become established as routine assays. The most encouraging data
comes from combining autoantibody detection, for example atypical ANCAs
and antibody levels against a mannan antigen of the cell wall from
Saccharomyces cerevisiae (ASCA). All three of the aforementioned gastro-
intestinal conditions are discussed within this chapter.

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 Chapter 7

Autoantigen/ Associated Autoimmune

Screening Substrate
Autoantibody Diseases
Pernicious anaemia,
GPC Rodent stomach
autoimmune gastritis
Intrinsic factor EIA Pernicious anaemia

Endomysial (tTG) Monkey oesophagus Coeliac disease

Reticulin (R1) Rodent LKS Coeliac disease
Gliadin EIA Coeliac disease
Atypical ANCA Ethanol-fixed neutrophils Ulcerative colitis
ASCA EIA Crohn’s disease
Table 7.1. Summary of gastro-intestinal autoantigens, screening substrates and
associated autoimmune diseases.

Gastric Parietal Cell Antibodies

Antigen: Anti-GPC antibodies target the α (92kDa) and β (60-90 kDa) subunits
of the gastric H+/K+ ATPase, a proton pump responsible for acidification of the
gastric juice. It is located in the intracellular and apical membranes of the
parietal cells.

Clinical associations: This autoantibody specificity is associated with

autoimmune gastritis type A (chronic atrophic gastritis) and pernicious anaemia
(90%), the antibody class is predominantly IgG. Whenever there is a suspicion
of pernicious anaemia, the serum should also be tested for intrinsic factor
antibodies; when these two antibodies are found together there is a very strong
correlation with pernicious anaemia. The antibodies are also found in patients
with associated organ specific diseases such as insulin-dependent diabetes
mellitus and endocrine diseases such as hypothyroidism and Addison’s disease.
Antibodies are also reported in the healthy population although generally of a
low titre; the incidence of anti-GPC antibodies is between 2% and 15% in the
general population, primarily dependent on age.

Detection: The antibodies are not species-specific and can be detected on

stomach sections from a multitude of species, although the usual substrate for
gastric parietal cell autoantibodies is rat or mouse stomach (Figure 7.1).
Screening is usually carried out at 1/20. The staining pattern shows a distinct

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 Gastro-intestinal Diseases

fine granular fluorescence which is exclusive to the parietal cells. One should
take care when using rat stomach as potentially confusing heterophile staining
is common in this tissue (Figure 7.3 and 7.4). Genuine GPC staining on rat
tissues does not show any heterophile kidney or liver staining. However,
genuine anti-GPC antibodies can co-occur with heterophile antibodies: here the
genuine anti-GPC staining will be dominant and a dilution series should resolve
interpretation. Heterophile antibodies on mouse stomach may show staining of
the gastric gland cell but also show strong inter-gland staining which obscures
interpretation. Anti-mitochondrial antibodies give similar staining to anti-GPC
antibodies on stomach sections, but unlike anti-GPC antibodies they also bind
the mitochondrial antigens in the kidney and liver. It is therefore useful to run
liver and kidney sections in combination with stomach sections.

Figure 7.1. GPC antibodies on rat (left) and monkey (right) stomach.

Intrinsic Factor Antibodies

Antigen: Intrinsic factor (~70kDa) is a glycoprotein secreted by the parietal

cells of the stomach mucosa. It binds vitamin B12 and has a role in its
absorption in the ileum. The malabsorption of vitamin B12 can be investigated
using the ‘Schilling test’.

Clinical associations: Between 50 and 70 % of patients with pernicious

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 Chapter 7

anaemia will have anti-intrinsic factor antibodies. Vitamin B12 deficiency can
also be due to a deficiency of intrinsic factor or malabsorption, as apposed to
solely being caused by anti-intrinsic factor antibodies.

Detection: Anti-intrinsic factor antibodies are generally detected by radio-

immunoassays although EIAs are available. The EIAs will detect both type I
antibodies, those that block formation of the vitamin B12 – intrinsic factor
complex, and type II antibodies which bind the complex as well as intrinsic
factor alone thus inhibiting absorption of vitamin B12.

References

Taylor KB, Roitt IM, Doniach D, Couchman KG, Shapland C. Autoimmune phenomena in
pernicious anaemia: gastric antibodies. Br Med J 1962; 2: 1347-1352.

de Aizpurua HJ, Toh BH, Ungar B. Parietal cell surface reactive autoantibody in pernicious
anaemia demonstrated by indirect membrane immunofluorescence. Clin Exp Immunol 1983; 52:
341-349.

Gleeson PA, Toh BH. Molecular targets in pernicious anaemia. Immunol Today 1991; 12: 233-238.

Toh BH, Alderuccio F. Parietal cell and intrinsic factor autoantibodies. In: Autoantibodies. 2nd
Edition. Eds. Shoenfeld Y, Gershwin ME, Meroni PL. Elsevier Science; 2007.

Figure 7.2. Schematic of stomach mucosal glands.

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Figure 7.3. Heterophile antibodies on rat stomach (left) and mouse stomach
(right).

Figure 7.4. Heterophile antibodies staining rat liver, kidney and stomach
composite block.

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 Chapter 7

Coeliac Disease

Coeliac disease is an immune-mediated disorder of the intestine with an

estimated incidence in the general population of 0.1 – 0.5% and is due to
intolerance of gluten proteins consumed in a diet containing wheat, rye or
barley. Symptoms include the malabsorption of nutrients, diarrhoea, weight
loss, anaemia and bone disease. These symptoms can often be subtle so good
diagnostic assays are essential.
Predisposition to coeliac disease has a genetic element; 99% of patients
express the HLA-DQ2 and/or HLA-DQ8 alleles although only a minority of
individuals expressing these alleles will have coeliac disease. Coeliac disease
also has a higher incidence in relatives of affected individuals (x5-20), patients
with thyroid disease (x4), insulin-dependent diabetes mellitus (x5-7) and
selective IgA deficiency (x10-16). Gluten intolerance can also present itself as
dermatitis herpetiformis, patients can have abnormal gut biopsies but limited or
absent gastrointestinal symptoms.
Patients with coeliac disease are at increased risk of malignancies, further
emphasising the importance of diagnosis and management of patients.
Diagnosis of coeliac disease has changed considerably as disease specific
antibodies have been identified, including EMA, anti-tissue transglutaminase
(tTG) and anti-gliadin antibodies. Anti-reticulin antibodies are also associated
with coeliac disease, although their use has been mostly superseded due to the
increased sensitivity of the former specificities (Table 7.2). It must be
emphasised that currently, the definitive diagnosis is identification of villous
atrophy by examination of a small intestinal biopsy followed by a clear clinical
improvement once the patient is on a gluten free diet.
Antibodies Sensitivity (%) Specificity (%)
Endomysial - IgA ~95 >98
Endomysial - IgG ~40 >98
Tissue transglutaminase - IgA >95 >98
Tissue transglutaminase - IgG ~40 >98
Gliadin - IgA ~80 80-90
Gliadin - IgG ~80 >80
Reticulin RI - IgA 60-95 95-100
Table 7.2. Sensitivity and specificity for antibodies in coeliac disease.
Anti-actin antibodies, a cause of interference when interpreting EMA
staining, are reported by some to be associated with the level of villous atrophy;
this has yet to be substantiated by larger studies. Any establishment of anti-

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actin determination in coeliac disease assessment is hindered by lack of

sensitivity, more importantly, specificity because anti-actin antibodies are found
in a number of autoimmune conditions.

Endomysial/Tissue Transglutaminase Antibodies

Antigen: The endomysium is the supporting structure that surrounds the

smooth and striated muscle fibres in the lower two thirds of the oesophagus. It
contains collagen and reticulin together with the endomysial target antigen,
which has been shown to be the enzyme tissue transglutaminase (tTG). This
enzyme (76kDa) catalyses the cross-linking of glutamine residues and primary
amines, these residues can be cross-linked to tTG itself. It has a function in
wound healing as well as stabilising the extra-cellular matrix. Tissue
transglutaminase is neither species-specific nor organ-specific, being found in
the oesophagus of human, monkey, rat and many other species and in many
tissues such as stomach, jejunum, liver and umbilical cord.

Clinical associations: IgA antibodies to tTG are highly specific for patients
with coeliac disease and dermatitis herpetiformis. In some studies, up to 100%
of patients with coeliac disease have been detected. In certain patient groups
the assay performance is a little weaker, for example false negative results can
occur in children with mild disease, so additional markers may be useful for
these patients. Also, coeliac disease is 5-20 times more common in IgA
deficient patients, when compared to the general population. In such cases, IgG
class antibodies should be considered, despite their lower sensitivity (~40%).

Detection: Monkey oesophagus, jejunum, or human umbilical cord can all be

used for endomysial antibody detection by IFA. Monkey oesophagus is the
tissue of choice using a screening dilution of 1/10. The lower two thirds of the
oesophagus contains squamous epithelium and the muscularis mucosa
(endomysial positive tissue) so the same section can be used for detection of
EMA and skin antibodies. EMA patterns show as a honeycomb/chicken wire-
like pattern surrounding the smooth muscle fibres (Figure 7.5). In human
umbilical cord, staining for EMA is in the blood vessels but the intensity is
weaker and the pattern is more difficult to interpret. Care should be taken when
identifying EMA on the oesophagus, the muscularis mucosa should not only
stain positive but the clear honeycomb structure must also be observed.
Difficulties may arise when SMA co-exist with the EMA staining (Figure 7.8
and 7.9), resulting in a general positive staining of the muscularis mucosa
region. It is recommended to use a species-specific conjugate to improve the
clarity of staining and several dilutions of the sample may also be advantageous.

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 Chapter 7

Monkey oesophagus and jejunum make a useful tissue combination (Figure

7.6) as it is easier to distinguish smooth muscle antibodies from endomysial
antibodies on jejunum by studying its interglandular smooth muscle areas
(Figure 7.7). EIAs have been available since 1997, when the endomysial
antigen was first identified. The first examples used a crude extract from guinea
pig liver, however, the subsequent availability of a recombinant human tTG

Figure 7.5. Monkey oesophagus at low magnification showing endomysial

staining of the muscularis mucosa, seen as a network of fibres surrounding the
smooth muscle cells.

Figure 7.6. Oesophagus (left) and jejunum (right) at low magnification.

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 Gastro-intestinal Diseases

antigen improved the specificity of this assay considerably. The overall

expected performance of EIAs for IgA is >95% sensitivity, >98% specificity
and IgG 40% sensitivity and >98% specificity.

Figure 7.7. Monkey jejunum showing endomysial staining of the central part of
the villi, seen as a network of fibres surrounding the muscle cells.

Figure 7.8. Smooth muscle and endomysial antibodies staining monkey

oesophagus.

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 Chapter 7

Figure 7.9. Monkey oesophagus stained with smooth muscle and anti-nuclear
antibodies.

Gliadin Antibodies

Antigen: The protein mass of wheat flour is gluten, and gliadin, the ethanol-
soluble fraction of gluten, is the toxic factor in coeliac disease. It consists of
polypeptides with molecular weights of 16-40kDa, rich in glutamine (37%) and
proline (17%) residues. The α, β, γ and ω gliadin peptides all have toxic effects
and are differentiated by their mobility.
The enzyme tTG can modify gliadin residues causing deamidation of
glutamine to form glutamic acid residues. The resulting increased negative
charge of the modified gliadin peptides enhances their binding to HLA class II
membrane proteins DQ2 and DQ8, thus having the potential to activate specific
T-cells against the modified peptides. Theoretically, these cells could provide T-
cell help, eventually leading to antibody production against the modified gliadin
peptides. HLA markers DQ2 and DQ8 predispose an individual to development
of coeliac disease.

Clinical associations: Anti-gliadin antibodies are associated with coeliac

disease and dermatitis herpetiformis and were the traditional investigation
before the development of EMA IFA and tTG EIA. In some series, IgG
antibodies are found in 90-100% of patients with coeliac disease whilst IgA
antibodies are found in ~90%. A combination of the IgG and IgA tests produces

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a similar diagnostic sensitivity to EMA. However, gliadin tests are less specific
with false positive rates of around 20% for IgG and IgA antibodies. Substantial
work by Schwertz et al. (2004), investigating a plethora of modified gliadin
peptides culminated in identification of certain peptides with much greater
specificity for coeliac disease, when assessed by EIA. It is of interest that IgA-
deficient patients have a ten-fold increase in coeliac disease compared with
controls, an argument for measuring both IgG and IgA antibodies in these
patients.

Detection: Anti-gliadin antibodies are usually detected by EIA where the α

gliadin fraction is used. The expected performance for gliadin IgA assays is
80% sensitivity, 80-90% specificity, and for IgG the sensitivity is 80% and
specificity is >80%. The use of specific deamidated gliadin peptides gives a
much greater specificity for coeliac disease, without compromising sensitivity.
It is too early to assume that these modified assays will be used routinely
alongside anti-tTG determination. However, they may provide additional value
when assessing younger children; EMA/tTG antibodies can be absent in
younger children (<5 years of age) with mild disease. Also, the assays may be
advantageous when assessing IgA deficient individuals; IgG class anti-gliadin
antibodies are more sensitive for coeliac disease than IgG class EMA/tTG
antibodies.
IgG and IgA gliadin antibodies can be detected by IFA if gliadin is added to
a section of monkey kidney, prior to sample incubation, where it binds to the
reticulin antigen. This technique is not as sensitive as EIA and not quantitative
(Figure 7.10).

Figure 7.10. IgA gliadin antibodies on gliadin coated monkey kidney (left) and
non-coated kidney (right).

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 Chapter 7

Reticulin Antibodies

Antigen: There are five reticulin antibodies defined by their reactivity with rat
LKS sections (Table 7.3). Reticulin (R1) antigens comprise collagen/fibrous
structural components that are between the hepatocytes and the endothelial cells
of the sinusoids in the liver and elsewhere. R1 is the only type which has
clinical associations but diagnostically it has largely been replaced by
endomysial and tTG testing. The importance of the others is unclear and the
antigens have not been characterised although they are related to heterophile
reactions (Chapter 2).
Staining Patterns R1 R2 Rs R3 R4
++ +
Liver sinusoids +/- + +
Fig 7.16 (Kupffer)
+
Kidney peritubular - - - -
Fig 7.11
+
Kidney periglomerular - - - -
Fig 7.11
+ +
Stomach submucosa - - -
Fig 7.13 Fig 7.15
+ +
Stomach intragastric glands - - -
Fig 7.13 Fig 7.15
+ +
Blood vessels - - -
Fig 7.12 Fig 7.14
+
Perivascular - - - -
Fig 7.14
Table 7.3. Summary of reticulin antibody staining patterns in the relevant
tissues.
Clinical associations: Serum IgG and IgA antibodies to reticulin R1 are found
in coeliac disease. IgG antibodies are present in approximately 70% of children
with untreated coeliac disease whereas IgA is found in 90-95% of coeliac
disease patients. Approximately 20% of patients with Crohn’s disease and other
gastro-intestinal disorders also have these antibodies. The titre falls following
treatment with a gluten free diet in both children and adults. The relationship
between these antibodies and the pathogenesis of coeliac disease is unknown.
IgA class reticulin R1 antibodies are strongly associated with endomysial
antibodies. Testing for endomysial antibodies on monkey oesophagus has
largely replaced the diagnostic use of R1 antibodies by IFA because of its
greater diagnostic specificity for coeliac disease. R1 antibodies are found in 5%
of normal individuals and, non-diagnostically, in patients with rheumatic

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 Gastro-intestinal Diseases

diseases. IFA staining patterns of IgG and IgA reticulin R1 are identical so IgA
specific conjugated antibodies should be used, the usual screening dilution is
1/20.

Detection:
R1 – these antibodies can be distinguished from R2 by their staining in the
kidney. The latter stains only the blood vessels whilst R1 stains peritubular and
periglomerular reticulin fibres. Furthermore only R1 stains human and monkey
tissues suggesting R2 may be a heterophile phenomenon.
R2 – antibodies can be distinguished from R1 by their staining in the kidney
(above).
Rs – is the reticulin type most frequently encountered in routine IFA testing on
rodent tissues. Extensive staining of all liver sinusoids is seen and the brush
border of the kidney tubules is sometimes strongly positive suggesting a
relationship with heterophile antibodies (Figure 7.17).

References
Alp MH, Wright R. Autoantibodies to reticulin in patients with idiopathic steatorrhoea, coeliac
disease, and Crohn's disease, and their relation to immunoglobulins and dietary antibodies. Lancet
1971; 2: 682-685.

Unsworth DJ, Manuel PD, Walker-Smith JA, Campbell CA, Johnson GD, Holborow EJ. New
immunofluorescent blood test for gluten sensitivity. Arch Dis Child 1981; 56: 864-868.

Dieterich W, Ehnis T, Bauer M, Donner P, Volta U, Riecken EO et al. Identification of tissue

transglutaminase as the autoantigen of celiac disease. Nat Med 1997; 3: 797-801.

Clemente MG, Musu MP, Frau F, Brusco G, Sole G, Corazza GR et al. Immune reaction against the
cytoskeleton in coeliac disease. Gut 2000; 47: 520-526.

Mäki M, Mustalahti K, Kokkonen J, Kulmala P, Haapalahti M, Karttunen T et al. Prevalence of

Celiac disease among children in Finland. N Engl J Med 2003; 348: 2517-2524.

Lenhardt A, Plebani A, Marchetti F, Gerarduzzi T, Not T, Meini A et al. Role of human-tissue

transglutaminase IgG and anti-gliadin IgG antibodies in the diagnosis of coeliac disease in patients
with selective immunoglobulin A deficiency. Dig Liver Dis 2004; 36: 730-734.

Rostom A, Dubé C, Cranney A, Saloojee N, Sy R, Garritty C et al. Celiac disease summary,

evidence report/technology assessment no. 104. AHRQ Publication No. 04.E029-1. Rockville, MD:
Agency for Healthcare Research and Quality. June 2004.

Schwertz E, Kahlenberg F, Sack U, Richter T, Stern M, Conrad K et al. Serologic assay based on
gliadin-related nonapeptides as a highly sensitive and specific diagnostic aid in celiac disease. Clin
Chem 2004; 50: 2370-2375.

Alaedini A, Green PH. Autoantibodies in celiac disease. Autoimmunity 2008; 41: 19-26.

71
 Chapter 7

Figure 7.11. Rat kidney showing R1 staining on the peritubular and

periglomerular tissues.

Figure 7.12. Rat liver including a blood vessel. R1 staining is on the blood
vessel wall connective tissue and on reticulin fibres throughout the liver
parenchyma as hair-like fibres (only seen with R1). The sinusoids may show
varying degrees of staining.

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 Gastro-intestinal Diseases

Figure 7.13. Rat stomach showing R1 staining around the gastric parietal cells
and in the muscularis mucosa.

Figure 7.14. Rat kidney showing R2 as a fibrillary network in perivascular

tissues.

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Figure 7.15. Rat stomach showing R2 staining between the mucous glands and
in the muscle layers as a fine mesh of fibres.

Figure 7.16. Rat liver showing Rs staining of liver sinusoids with the
hepatocytes unstained.

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Figure 7.17. Rat kidney showing Rs staining of the tubule brush border with no
reticulin staining.

Chronic Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic idiopathic inflammatory

condition of the intestines, not related to infection. The condition generally
refers to one of two diseases: ulcerative colitis (UC) or Crohn’s disease (CD).
They have distinctly different pathologies, however, presenting symptoms can
be very similar including abdominal pain and intermittent diarrhoea, which on
occasions can be bloody. Disease differentiation is therefore established from
a combination of patient history, physical examination, endoscopy and biopsy.
All the same, a number of cases remain undifferentiated. In such circumstances
a serology test to differentiate between the two conditions would be very useful.
Many autoantibodies have been reported to be associated with these conditions;
some of which are summarised here. In spite of this, evidence for an
autoimmune element in IBD is mostly circumstantial and remains to be firmly
established.
Ulcerative colitis: This is a chronic disease of unknown aetiology with a
prevalence of up to 1 per 1000. It presents as inflammation of the mucosa and
submucosa resulting in mucosal ulcerations affecting the rectum, colon and
large intestine. Treatment includes the use of immunosuppressant therapy and
in severe cases, colectomy. Autoantibodies against intestinal mucosal cells in

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UC were first described in 1959. However, they are of no diagnostic use since
many normal subjects have the same autoantibodies as do patients with
cirrhosis and urinary tract infections.
Crohn’s disease: This is a non-specific granulomatous inflammatory condition,
with a prevalence of around 1 per 25,000, predominantly affecting the lower
end of the small intestine. Affected areas are discontinuous, more variable than
UC, and any part of the gastro-intestinal tract can be involved. Treatment is
largely restricted to anti-inflammatory drugs, and some success has been shown
with monoclonal antibodies against tumour necrosis factor α (TNF-α).

Serological markers in IBD

Pancreatic antibodies: Pancreatic antibodies are said to be a specific marker in

~20% of patients with CD and their presence may be associated with pancreatic
insufficiency. Pancreatic antibody levels do not correlate with disease activity
and rarely occur in other family members with CD. Two types of antibodies can
be identified by their pattern of staining. Type 1 antibodies (Figure 7.18) are
characterised by a drop-like fluorescence in the pancreatic acini whilst type 2
antibodies (Figure 7.19) give a fine speckled pattern in the acinar cells. Any
possible ABO antibodies should be removed by adsorption as they may produce
similar patterns (Figure 7.20).

ANCA: Antibodies against neutrophil cytoplasmic antigens (ANCA) are a vital

tool for the investigation of primary, systemic and small vessel vasculitides.
The respective autoantigens have been identified and are well characterised (see
Chapter 13). Specific antibodies against neutrophil antigens have been
reported in up to 70% of patients with ulcerative colitis. Here, the staining
pattern is atypical ANCA comprising a combination of cytoplasmic and
perinuclear fluorescence (Figure 13.9). Several autoantigens have been
proposed including elastase, α-enolase, histones and high mobility group
proteins (HMG1 & HMG2), however no studies have proved definitive.
Atypical ANCAs do not fluctuate with disease activity and are not specific for
UC; they are present in up to 20% of CD patients, found in primary sclerosing
cholangitis and frequently reported in autoimmune hepatitis. Initial screening
is performed on ethanol-fixed neutrophils, and samples should be considered
positive when an atypical or P-ANCA staining pattern is observed that is shown
to be unrelated to anti-MPO or ANA activity.

ASCA: Antibodies against mannan from Saccharomyces cerevisiae (ASCA)

occur more frequently and at a higher titre in patients with CD (60-80%), when

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compared to UC (up to 15%). IgA and IgG class antibodies are useful, when
found in combination, the specificity for CD increases significantly but
sensitivity is low. The antibodies are determined by EIA. In isolation,
determination of ASCA levels is of limited value. Combined determination of
ASCA by EIA and ANCA by IF is reported to improve differentiation between
ulcerative colitis and Crohn’s disease. One report suggests sensitivity,
specificity and positive predictive value for pANCA+ve/ASCA-ve for Crohn’s
disease of 56%, 92% and 95% respectively and for pANCA-ve/ASCA+ve in
UC, the same values were 44%, 98% and 88%. The sensitivity of this
combination is of limited utility and the gastroenterologist may well prefer to
rely on biopsy, thus negating the requirement for the test.

OmpC: Antibodies against Escherichia coli outer membrane porin C (anti-

OmpC) have been reported in up to 55% of patients with CD, although other
reports suggest less than half this frequency. These antibodies are also found in
UC, albeit at a much lower frequency. The utility of this and other recent
markers is yet to be established; evidence suggests that when found in
combination with other markers there is an increased risk of more severe
disease.

References

Broberger O, Perlmann P. Autoantibodies in human ulcerative colitis. J Exp Med 1959 ; 110: 657-
674.

Seibold F, Mörk H, Tanza S, Müller A, Holzhüter C, Weber P et al. Pancreatic autoantibodies in

Crohn's disease: a family study. Gut 1997; 40: 481-484.

Quinton JF, Sendid B, Reumaux D, Duthilleul P, Cortot A, Grandbastien B et al. Anti-

Saccharomyces cerevisiae mannan antibodies combined with antineutrophil cytoplasmic
autoantibodies in inflammatory bowel disease: prevalence and diagnostic role. Gut 1998; 42: 788-
791.

Conrad K, Schmechta H, Klafki A, Lobeck G, Uhlig HH, Gerdi S et al. Serological differentiation
of inflammatory bowel diseases. Eur J Gastroenterol Hepatol 2002; 14: 129-135.

Bossuyt X. Serologic markers in inflammatory bowel disease. Clin Chem 2006; 52: 171-181.

Jaskowski TD, Litwin CM, Hill HR. Analysis of serum antibodies in patients suspected of having
inflammatory bowel disease. Clin Vaccine Immunol 2006; 13: 655-660.

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Figure 7.18. Type 1 pancreatic antibodies from a patient with Crohn’s disease
showing drop-like staining in the acinar cells.

Figure 7.19. Type 2 pancreatic antibodies from a patient with Crohn’s disease
showing fine speckles in the acinar cells.

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 Gastro-intestinal Diseases

Figure 7.20. Heterophile/ABO antibodies from a normal person on monkey

pancreas.

Figure 7.21. Autoantibodies staining colonic goblet cells in ulcerative colitis.

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Figure 7.22. Villous tip antibody of unknown significance from a patient with
acute gastroenteritis (courtesy of F.X. Huchet, Institute Pasteur, Paris).

General References

Keren DF. Autoimmune disease of the gastrointestinal tract. In: Clinical and Laboratory Evaluation
of Human Autoimmune Diseases. Eds. Bylund DJ, Keren DF, Nakamura RM. American Society of
Clincal Pathology; 2002.

Goeken JA. Immunologic testing for celiac disease and inflammatory bowel disease. In: Manual of
Molecular and Clinical Laboratory Immunology. 7th Edition. Eds. Rose NR, Hamilton RG, Detrick
B. ASM Press, Washington DC, USA; 2006.

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 Renal Diseases

Chapter 8
Autoimmune Renal Diseases

Immunological mechanisms are responsible for most cases of glomerular

damage and occasionally for damage to the tubules and interstitial tissues of the
kidney. Antibodies alone can cause damage in at least four ways. Direct
glomerular damage in Goodpasture's syndrome where circulating antibodies
against renal antigens in the glomeruli can be detected by IIF on human or
monkey tissues. Glomerular damage via immune complexes and complement
deposition in glomerular basement membranes; immunoglobulins and
complement deposition can be detected in renal biopsies immunochemically.
Induction of vasculitis and clotting in glomerular capillaries and other vessels
as shown by fibrin deposits. Finally, renal failure by deposition of
immunoglobulin light chains in renal tubules in multiple myeloma.

Disease/Pathology Proteins Frequently Identified

Mixed granular GBM deposits of IgG,
SLE
A, M, C# and F*
Membranous glomerulonephritis Granular GBM IgG and C#
Post streptococcal Irregular, granular GBM IgG,A,M and
glomerulonephritis C#
Vasculitis, ANCA mediated Largely fibrinogen* and some IgM in
vasculitis capillaries
GBM disease Linear GBM deposits of IgG and C#
IgA nephropathy + Henoch- Mesangial deposits of IgA, IgG, C3 and
Schönlein purpura fibrinogen
Immunoglobulins and light chains in the
Myeloma, amyloid, SLE etc
tubules
#Complement proteins of the classical pathway are deposited alongside IgG.
C1q, C3 and C4 are found but C9 is well detected due to heavy chain
deposition and minimal background staining. *Fibrinogen deposits occur in
capillary loops if there is a vasculitic component and clotting.

Table 8.1. Renal diseases and biopsy patterns.

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Methodology

Routine diagnosis of renal diseases is by assessment of renal tissues usually

in the form of needle biopsies. The detection of immune complexes,
complement and fibrin deposits in renal biopsies, by direct
immunofluorescence or more commonly immunoenzyme techniques, plays a
central role in renal disease diagnosis. Specimens are examined histologically
and then immunohistochemically using fluorochrome or enzyme labelled
antibodies for deposits of immunoglobulins, complement proteins, and
fibrinogen (Figures 8.3 - 8.8). A useful antibody screening panel comprises
anti-G, A, M and anti-complement. Any immune deposits should be assessed in
the context of the clinical and the histological appearance.
IIF is used to detect pathogenic circulating autoantibodies against renal
tissues in Goodpasture's syndrome and tubular basement membrane (TBM)
disease. Antibodies that react with the kidney but have no apparent clinical
importance include antibodies against Bowman's capsule, different parts of the
tubules and collecting ducts (Figures 8.16 - 8.19). It is important that when
kidney tissues are prepared for IIF they must be fresh, since autolysis is rapid.

Figure 8.1. Illustration of kidney structure indicating the position of the cortex,
medulla and nephrons.

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 Renal Diseases

Figure 8.2. Diagrammatic representation of a nephron and collecting duct

system. Of particular note are the different sections of the convoluted tubule
P1, P2 and P3.

Serum Autoantibody Patterns in Different Renal Diseases

Initial laboratory assessment of autoantibodies should include tests for

ANCA and ANA since GBM disease, systemic vasculitis and SLE may produce
similar clinical features.
Autoimmune Disease Anti-dsDNA ANCA Anti-GBM

GBM disease - +/- +++

SLE +++ +/- -
Wegener's granulomatosis - +++ -
Microvascular polyarteritis +/- +++ +/-
IgA nephropathy - - -
Table 8.2. Summary of renal diseases and related autoantibodies.

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Figure 8.3.

Figure 8.4.
Renal biopsy showing IgG granular basement membrane staining in a patient
with membranous glomerulonephritis (Figure 8.3) and complement C9
granular staining of a sclerosed glomerulus in a patient with post-infectious
glomerulonephritis (Figure 8.4).

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 Renal Diseases

Figure 8.5.

Figure 8.6.
Renal biopsy showing fibrinogen deposits in capillary loops in a patient with
microvasculitis of the kidney (Figure 8.5) and IgG linear basement membrane
staining in a patient with Goodpasture’s syndrome (Figure 8.6).

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Figure 8.7. Renal biopsy showing mesangial IgA deposition in a patient with
IgA nephropathy.

Figure 8.8. Renal biopsy stained with free kappa (fluorescein) and free lambda
(rhodamine) in a patient with primary amyloid disease, IgG kappa paraprotein
in the serum and free monoclonal kappa in the urine. Free kappa is deposited
in the blood vessels and both kappa and lambda (yellow) light chains are found
in the tubular epithelial cells.

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 Renal Diseases

Glomerular Basement Membrane Antibodies

Antigen: Glomerular basement membrane (GBM) antibodies target type IV

collagen, which is found in the basement membranes in the kidney, lung, lens,
cochlear, brain and testis. Type IV collagen contains three α chains and a
globular domain at the C-terminal end (NC1) (Figure 8.9). The NC1 portion of
the α-3 chain in type IV collagen contains the main Goodpasture's antigen and
it is these antibodies which are responsible for the linear immunofluorescence
pattern seen in the kidneys. However, patients with other forms of
glomerulonephritis as well as GBM disease may have antibodies to other
basement membrane components but the precise antigens remain unknown.

Figure 8.9. Schematic interpretation of the α-3 type IV NCl terminus, the
glomerular basement membrane target antigen.

Clinical associations: Anti-GBM antibodies cause rapidly progressive

glomerulonephritis with or without lung haemorrhage (Goodpasture's
syndrome). Goodpasture's syndrome has an incidence rate of 0.5-1
cases/million per year with two disease peaks in the third and seventh decades
of life. Antibody titres are known to correlate with disease activity and therefore
are used to monitor disease progress. A lower concentration of anti-GBM
antibodies is also associated with better renal survival. Approximately 32% of
all GBM positive patients will also be positive for ANCA, the majority of which
will be MPO positive. There are conflicting reports as to whether these patients
respond better to therapy and have better renal survival as the relationship
between the two antibodies remains unclear. Anti-GBM antibodies are also
associated with transplant glomerulopathy and graft loss in transplants.

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Detection: Anti-GBM antibodies were previously detected via IIF on blood

group O human kidney; monkey tissues are now used due to reduced
background staining and improved preservation of tissue (Figure 8.10), where
the usual screening dilution is 1/5. The GBM antigen is relatively inaccessible
due to protein folding, pre-treatment with 6M urea makes the antigen more
accessible and therefore enhances sensitivity (Figure 8.11). However, general
recommendations are that all positive samples should still be confirmed by a
secondary test such as EIA.

References

Scheer RL, Grossman MA. Immune aspects of the glomerulonephritis associated with pulmonary
hemmorhage. Ann Int Med 1964; 60: 1009-1021.

Hellmark T, Johansson C, Wieslander J. Characterisation of anti-GBM antibodies involved in

Goodpasture's syndrome. Kidney Int 1994; 46: 823-829.

Borza DB, Bondar O, Todd P, Sundaramoorthy M, Sado Y, Ninomiya Y, et al. Quaternary

organization of the goodpasture autoantigen, the alpha 3(IV) collagen chain. Sequestration of two
cryptic autoepitopes by intrapromoter interactions with the alpha4 and alpha5 NC1 domains. J Biol
Chem 2002; 277: 40075-40083.

Levy JB, Hammad J, Coulthart A, Dougan T, Pusey CD. Clinical features and outcome of patients
with both ANCA and anti-GBM antibodies. Kidney Int 2004; 66: 1535-1540.

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Figure 8.10. High magnification of monkey kidney with GBM staining.

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Figure 8.11. Glomerular basement membrane staining on monkey kidney

without urea pre-treatment (left). The tissue was pre-treated with urea in order
to reduce protein folding making the antigen more accessible and therefore
increasing autoantibody binding (right).

Figure 8.12. Poorly prepared tissue with shrinkage of the glomerular tuft from
the sides of the Bowman’s capsule.

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 Renal Diseases

Tubular Basement Membrane Antibodies

Antigen: Tubular basement membrane antibodies target an unknown antigen,

suggested to be a 58kDa protein, in the tubular basement membrane of the
kidney.

Clinical associations: The clinical significance of anti-TBM antibodies is

unclear and they may just be an incidental finding. Anti-TBM antibodies are
detected in a small proportion of patients with tubulointerstitial nephritis in
association with lupus nephritis, allograft rejection and methicillin sensitisation.
Tubulointerstitial nephritis affects renal tubules and interstitial tissue, with
infiltration by plasma cells and mononuclear cells. Anti-TBM antibodies can
also be detected in serum from patients with glomerulonephritis and GBM
disease.

Detection: IIF on monkey kidney reveals staining of the tubular basement

membrane (Figure 8.13). This staining can be enhanced by using a urea buffer
and streptavidin-FITC as previously described for the GBM antibody (Figure
8.14). In renal biopsies TBM antibodies are observed to activate complement.

References

Steblay R, Rudofsky U. Renal tubular disease and autoantibodies against tubular basement
membrane induced in guinea pigs. J Immunol 1971; 107: 589-594.

Brentjens JR, Matsuo S, Fukatsu A, Min I, Kohli R, Anthone R et al. Immunologic studies in two
patients with anti-tubular basement membrane nephritis. Am J Med 1989; 86: 603-608.

Lindqvist B, Lundberg L, Wieslander J. The prevalence of circulating anti-tubular basement

membrane-antibody in renal diseases, and clinical observations. Clin Neph 1994; 41: 199-204.

Audard V, Hellmark T, El Karoui K, Noël LH, Pardon A, Desvaux D et al. A 59-kD renal antigen
as a new target for rapidly progressive glomerulonephritis. Am J Kidney Dis 2007; 49: 710-716.

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Figure 8.13. Monkey kidney pre-treated with urea showing tubular basement
membrane staining.

Figure 8.14. Monkey tissues with urea pre-treatment showing GBM and
tubular basement membrane staining.

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dsDNA Antibodies and Renal Disease

Antigen: The target antigen of anti-dsDNA antibodies is the phosphate

backbone of the dsDNA helix; in comparison, the less frequently detected anti-
ssDNA antibodies react with the exposed nucleotides of the ssDNA. In lupus
nephritis it is has been suggested that the anti-dsDNA antibodies also react with
α-actinin (100kDa) as well as dsDNA in circulation or in the glomeruli.

Clinical associations: Anti-dsDNA antibodies are detected more frequently

and at higher titres in systemic lupus erythematosus (SLE) patients with lupus
nephritis. The presence of anti-dsDNA antibodies or an increase in titre
correlates with an increased risk of lupus nephritis flare. Therefore it is useful
to monitor anti-dsDNA antibody levels and subsequently respond with the
appropriate therapy when titres increase. Other antibodies have also been
associated with lupus nephritis and these include anti-ribosomal P, anti-histone,
anti-C1q, anti-Sm, anti-nucleosome and anti-α-actinin.

Detection: There are a number of immunoassays which can be used to detect

anti-dsDNA antibodies and these differ in the sensitivity, affinity, and class of
antibodies that they detect. The EIA assay is the most sensitive detecting both

Figure 8.15. Immunofluorescence of Crithidia luciliae shows a characteristic

double spot pattern in the presence of anti-dsDNA whilst only the nucleus is
fluorescent with non-dsDNA nuclear antibodies.

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high and lower affinity antibodies usually of the IgG class. In comparison, the
Farr radioimmunoassay (RIA) detects all antibody classes, however mainly
those of a higher affinity. The less frequently utilised PEG RIA detects both
higher and lower affinity antibody populations. IIF using Crithidia luciliae will
also detect anti-dsDNA antibodies of the IgG class (Figure 8.15).

References

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

Jaekel H-P, Trabandt A, Grobe N, Werle E. Anti-dsDNA antibody subtypes and anti-C1q antibodies:
toward a more reliable diagnosis and monitoring of systemic lupus erythematosus and lupus
nephritis. Lupus 2006; 15: 335-345.

Ng KP, Manson JJ, Rahman A, Isenberg DA. Association of antinucleosome antibodies with disease
flare in serologically active clinically quiescent patients with systemic lupus erythematosus.
Arthritis Rheum 2006; 55: 900-904.

Renaudineau Y, Deocharan B, Jousse S, Renaudineau E, Putterman C, Youinou P. Anti-alpha-actinin

antibodies: A new marker of lupus nephritis. Autoimmun Rev 2007; 6: 464-468.

Renal Antibodies of Unknown Significance

Figure 8.16. Mesangial staining on rat (left) and to a lesser extent, on monkey
kidney (right).

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Figure 8.17. Autoantibody staining of Bowman’s capsule on rat (left) but

monkey kidney (right) is negative suggesting a heterophile reaction.

Figure 8.18. Heterophile staining of proximal tubule brush border on rat (left)
and monkey kidney (right).

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Figure 8.19. Antibody against collecting ducts on rat (left) and monkey kidney
(right).

References

Ford PM. A naturally occurring human antibody to loops of Henle. Clin Exp Immunol 1973; 14:
569-572.

Gaarder PI, Heier HE. A human autoantibody to renal collecting duct cells associated with thyroid
and gastric autoimmunity and possibly renal tubular acidosis. Clin Exp Immunol 1983; 51: 29-37.

Konishi K, Hayashi M, Saruta T. Renal tubular acidosis with autoantibody directed to renal
collecting-duct cells. N Engl J Med 1994; 331: 1593-1594.

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Chapter 9

Autoimmune Endocrine Diseases

Fifty years ago Hashimoto's thyroiditis became the first organ-specific

autoimmune disease to be identified by demonstration of autoantibodies from
these patients binding human thyroid tissues. The target antigens of thyroid
autoantibodies from patients with a variety of thyroid disorders including
Hashimoto's thyroiditis have now been defined and include thyroglobulin,
thyroid peroxidase and the thyrotrophin receptor. Target antigens in other
autoimmune endocrine diseases such as autoimmune polyglandular disease,
Addison's disease and insulin dependent diabetes mellitus (IDDM) are less well
characterised. Consequently IIF using pituitary gland, pancreas and steroid
producing cells remains the technique of choice especially for initial screening.
Monkey endocrine glands are possibly the best substrate as they are similar to
human tissues, unlike rat and rabbit glands which show clear differences in
steroidal cell enzymes.

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Screening
Autoantibody/
Substrate Associated Autoimmune Diseases
autoantigen
(Dilution)

IDDM, LADA (latent autoimmune diabetes

Pancreatic islet cell Pancreas
in adults), autoimmune gestational diabetes
(ICA) antibodies (1/5)
and healthy relatives of affected patients

Pancreas IDDM and autoimmune poly-

Insulin
(1/5) endocrinopathies

GAD65 (Glutamic Pancreas IDDM, LADA and healthy relatives of

acid decarboxylase) (1/5) affected patients

Adrenal gland (1/5)

Ovary (1/5) Addison’s disease, premature ovarian
Steroidal cell
failure and autoimmune polyglandular
antibodies Testis (1/5) disease type 1, 2 and 3
Placenta (1/5)

Hashimoto’s disease (autoimmune

Thyroid gland hypothyroidism), autoimmune
Thyroglobulin (Tg)
(1/20) hyperthyroidism, thyroid adenomas and
carcinomas

Thyroid peroxidase Thyroid gland Autoimmune hypothyroidism, postpartum

(TPO) (1/20) thyroiditis and Grave's hyperthyroidism

Lymphocytic hypophysitis,
hyperprolactinaemia, thyroid disorders,
Pituitary gland Pituitary gland
growth hormone deficiency,
antibodies (1/5)
hypopituitarism, Addison's disease and
inactive pituitary disorders

Table 9.1. Summary of endocrine autoantibodies, associated diseases and

recommended screening titres. IDDM = insulin dependent diabetes mellitus,
also referred to as type 1 autoimmune diabetes mellitus.

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Autoantibodies Against Steroid Producing Cells

Antigens: Autoantibodies against steroid producing cells react with

cytochrome P450 enzymes. These targets are steroid dehydrogenases (Figure
9.1) which convert cholesterol to aldosterone, testosterone, progesterone and
α-hydroxylase (P450c17) is found in the adrenal gland, testis and
cortisol. 17α
the ovary and converts pregnenolone to 17-OH-pregnenolone and dehydro-
epiandrosterone. 21ß-hydroxylase (P450c21) is only found in the adrenal
gland (zona glomerulosa, fasciculata and reticularis) and converts 17-OH
progesterone to 11-deoxycortisol and progesterone into 11-deoxy-
corticosterone. Cholesterol desmolase (P450scc) is the first rate-limiting
enzyme in the steroid hormone biosynthesis pathway and converts cholesterol
to pregnenolone. P450scc is found in the adrenal gland, testis, ovary and
placenta.

Figure 9.1. Schematic illustrating steroid hormone biosynthesis from

cholesterol. Arrows indicate the pathways and the red lines the blockade that
results from specific enzyme deficiencies.

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 Chapter 9

Adrenal Gland Testis

Enzyme
Ovary Placenta
(MW) Fasciculata/
Glomerulosa Leydig Cells
Reticularis
P450 c17
- + + + -
(~57kDa)
P450c21
+ + - - -
(~56kDa)
P450scc
+ + + + +
(~60kDa)

Table 9.2. Summary of steroidal cell antibody staining patterns.

Clinical associations: Autoantibodies against steroid producing cells are

associated with the following diseases: Addison's disease (~50-70%), premature
ovarian failure (~30-70%) and autoimmune polyglandular disease type 1 (~60-
80%), 2 (~40-80%) and 3 (~11%). Anti-steroidal cell antibodies are less
frequently detected in Grave's disease, insulin dependent diabetes mellitus,
Cushing's syndrome and healthy patients.

Detection: IIF using monkey adrenal gland (Figure 9.3), ovary (Figure 9.4),
testis (Figure 9.6) and placenta (Figure 9.8) will reveal staining of steroid
dehydrogenases by the respective autoantibodies (Table 9.2). The adrenal gland
contains P450c17, P450c21 and P450scc; autoantibodies will stain the cell
cytoplasm of the granulosa and fasciculate layers. The testis contains P450c17
and P450scc; here autoantibodies will stain the interstitial tissues between the
seminiferous tubules. All the stroma cells of the ovary have the potential to
produce P450c17 and P450scc and therefore may be stained by autoantibodies.
However, staining is most frequently observed within the theca cells (for
orientation on ovary section see Figure 9.7). The placenta contains P450scc and
autoantibodies will stain the syncytiotrophoblast cells. In the sample population
studied by Boe et al. (2004) the autoantibodies against P450c21 and P450scc
were predominantly of the IgG1 subclass. Positive samples should also be
tested on monkey thyroid, pancreas and LKS cryosections as many patients will
have autoantibodies to other antigens which may add difficulty to interpretation.
For example AMA shows a speckled staining pattern which may hide a weaker
and less uniform steroidal cell antibody pattern on adrenal gland. Anti-steroid
dehydrogenase antibody specificity can be confirmed using EIA, western
blotting, immunoprecipitation assays and radiobinding assays.

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Cortex

Medulla

Figure 9.2. Transverse section of the adrenal gland showing the cortex and
medulla. The steroid producing cells are in the cortex of which the outer
glomerulosa layer has the most intense staining. The adrenal gland is stained
with a primary antibody from a patient with Addison’s disease and a peroxidase
labelled second antibody.

Figure 9.3. Positive staining of monkey adrenal gland using a sample from a
patient with Addison’s disease.

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Figure 9.4. Monkey ovary (top) with positive staining of the theca cells
surrounding the follicles and sporadic staining of the stromal cells. False
positive staining (bottom) showing antibody binding to blood vessels.

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 Endocrine Diseases

Figure 9.5. Transverse section of monkey testis showing the seminiferous

tubules and the interstitial tissues containing Leydig cells. The section was
stained with a peroxidase labelled second antibody and a primary antibody
from a patient with Addison’s disease.

Figure 9.6. Monkey testis showing positive staining (green) of the Leydig cells.
This section is counterstained with ethidium bromide to highlight the tissue
structure.

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Figure 9.7. Illustration of follicle development in the ovary. All stroma cells
have the potential to produce steroid hormones; however autoantibody staining
is usually against the theca cells which surround the developing follicles,
scattered lipid-rich luteinising stromal cells, and enzymatically active stromal
cells. Key for follicle development; 1) primordial follicle, 2) unilaminar
primary follicle, 3) multilaminar primary follicle, 4) secondary follicle, 5)
graafian follicle (mature follicle), 6) corpus luteum and 7) corpus albicans.

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Diseases Related to Steroidal Cell Antibodies

Addison's Disease: Is a rare autoimmune disease also known as primary

adrenocorticol deficiency. Gradual destruction of the adrenal gland leads to
adrenocorticol insufficiency and clinical symptoms which may include
weakness, anorexia, nausea and vomiting, weight loss, cutaneous and mucosal
pigmentation, hypotension and hypoglycaemia.

Autoimmune Polyglandular Syndrome (APS) Type 1, 2 and 3

There are three main types of autoimmune polyglandular disease which are
characterised by the endocrinopathies found within them. It should be noted that
this disease is not to be confused with the anti-phospholipid syndrome (APS).

APS Type 1: Also known as candida endocrinopathy and autoimmune

polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). APS type 1
is a rare disorder which usually presents in the first decade of life and is caused
by mutations in the autoimmune regulator (AIRE) gene. The disease is
characterised by the presence of two out of three major components;
mucocutaneous candidiasis (which usually appears first) with chronic
hypoparathyroidism and/or autoimmune Addison’s disease. Complete disease
characteristics may not develop until 20 years of age, supported by the finding
that only 50% of patients are found to present with all three components of the
disease. Many other endocrine and non-endocrine diseases may also be present.
These include gonadal failure in females, autoimmune hepatitis, diabetes
mellitus, enamel dysplasia, keratopathy and malabsorption. Identification of
specific disease manifestations may be aided by analysis of the autoantibodies
present. For example antibodies against aromatic L-amino acid decarboxylase,
P450IA2/CYP1A2 and tryptophan hydroxylase are considered to be markers of
autoimmune hepatitis within APS.

APS Type 2: Also known as Schmidt’s syndrome, it is a rare syndrome (1.4 - 2

cases per 100,000) usually affecting middle aged women and very rarely
presents in childhood. APS type 2 is associated with HLA-DR3 and HLA-DR4
alleles. It is characterised by the presence of autoimmune Addison’s disease
(100%) with autoimmune thyroid diseases (69-82%) and/or IDDM (30-52%).

APS Type 3: Is defined by the presence of an autoimmune thyroid disease and

other autoimmune diseases, albeit with the exclusion of Addison’s disease.

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Figure 9.8. Antibodies from a patient with Addison’s disease staining the
syncytiotrophoblasts of monkey placenta.

References

Anderson JR, Goudie RB, Gray K, Stuart-Smith DA. Immunological features of idiopathic
Addison's disease: an antibody to cells producing steroid hormones. Clin Exp Immunol 1968; 3:
107-117.

Sotsiou F, Bottazzo GF, Doniach D. Immunofluorescence studies on autoantibodies to steroid-

producing cells, and to germline cells in endocrine disease and infertility. Clin Exp Immunol 1980;
39: 97-111.

Uibo R, Aavik E, Peterson P, Perheentupa J, Aranko S, Pelkonen R et al. Autoantibodies to

cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease
types I and II and in isolated Addison's disease. J Clin Endocrinol Metab 1994; 78: 323-328.

Boe AS, Bredholt G, Knappskog PM, Hjelmervik TO, Mellgren G, Winqvist O et al. Autoantibodies
against 21-hydroxylase and side-chain cleavage enzyme in autoimmune Addison's disease are
mainly immunoglobulin G1. Eur J Endocrinol 2004; 150: 49-56.

Soderbergh A, Myhre AG, Ekwall O, Gebre-Medhin G, Hedstrand H, Landgren E et al. Prevalence

and clinical associations of 10 defined autoantibodies in autoimmune polyendocrine syndrome type
I. J Clin Endocrinol Metab 2004; 89: 557-562.

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Coco G, Dal Pra C, Presotto F, Albergoni MP, Canova C, Pedini B et al. Estimated risk for
developing autoimmune Addison's disease in patients with adrenal cortex autoantibodies. J Clin
Endocrinol Metab 2006; 91: 1637-1645.

Betterle C, Zanchetta R, Chen S, Furmaniak J. Antibodies to adrenal, gonadal tissues and

steroidogenic enzymes. In: Autoantibodies. 2nd Edition. Eds. Shoenfeld Y, Gershwin ME, Meroni
PL. Elsevier Science; 2007.

The Pancreas

The endocrine portion of the pancreas is comprised mainly of islets of

Langerhans cells. These are the main hormone producing cells of the pancreas
and can be further subdivided into 5 cell types, based on the hormones they
secrete (Table 9.3). The exocrine portion is composed of acinar cells,
centroacinar cells and intercalated ducts. The acinar cells secrete digestive
enzymes into an alkaline buffer produced by the duct cells, which is then
utilised in the small intestine.
Proportion of Islet of
Islet of Langerhans Cell Type
Langerhans Cells
Insulin and amylin secreting cells (β-cells) ~ 70%
Glucagon secreting cells (α-cells) ~ 20%
Somatostatin secreting cells (δ-cells) ~ 5-10%
Pancreatic polypeptide secreting cells (PP-cells) ~ 1-2%
Vasoactive-intestinal peptide secreting cells and
<1%
mixed secretion cells
Table 9.3. Islet of Langerhans cell types.

The distribution of α and β-cells varies within the islets of human and monkey
pancreas. In human pancreas the β-cells tend to be in the centre and the α-cells
tend to be in the periphery. In monkey pancreas the reverse is true with the β
and α-cells tending to be present in the periphery and centre respectively.

Reference

Gartner LP, Hiatt JL editors. Color Atlas of Histology. 3rd Edition. Lippincott Williams and
Wilkins; 2000.

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Pancreatic Islet Cell Antibodies

Antigens: Pancreatic islet cell antibodies (ICA) react with a number of antigens
in the cytoplasm and on the membranes of the islet of Langerhans cells,
including antigens in the glucagon producing cells (α-cells), insulin producing
cells (β-cells) and somatostatin producing cells (δ-cells).

Insulin: A small (~6kDa) essential hormone secreted in response to high blood

sugar levels by the β-cells of the pancreatic islets. It is a flexible protein
comprised of an A chain (21 amino acids) and a B chain (30 amino acids) linked
via disulfide bonds to form 3 α helices. Insulin promotes carbohydrate, protein
and fat metabolism by binding to specific receptors located within the plasma
membranes of muscle, fat and liver cells.

GAD: Glutamic acid decarboxylase (GAD) is found in the central and

peripheral nervous systems, pancreatic islet cells, testis, ovaries, thymus and
stomach. It is responsible for catalysing the α-decarboxylation of L-glutamic
acid into gamma-amino butyric acid (GABA). GABA functions as an
inhibitory neurotransmitter in the brain and is involved in the control and
release of insulin from secretory granules. GAD exists as two isoforms which
share 68% sequence homology and have molecular weights of 65 and 67kDa.
GAD65 is a membrane anchored protein responsible for vesicular GABA
production whereas GAD67 is a cytoplasmic protein responsible for
cytoplasmic GABA production. Diabetic patients, in comparison to patients
with stiff person syndrome (see Chapter 11 page 154), will have antibodies
which react with conformational epitopes of GAD65 and not GAD67 unless
there is additional neurological involvement.

IA-2: Tyrosine phosphatase is a transmembrane protein of 106kDa expressed in

the brain and pancreas. It catalyses the removal of phosphate groups from
phosphorylated tyrosine residues.

Clinical associations: Anti-pancreatic islet cell antibodies are detected in

IDDM (~70-80%), latent autoimmune diabetes in adults - LADA (~50-75%),
close relatives of affected patients (~5-10%) and in autoimmune gestational
diabetes. Antibody titres are linked to β cell destruction and titres will decrease
within the first year of the disease. Detection of antibodies in unaffected
relatives is associated with progression to diabetes in the future.

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 Endocrine Diseases

Figure 9.9. Monkey pancreas showing non-restricted staining of the α and β

islet cells (left) and restricted staining of the β islet cells (right).

Figure 9.10. Comparison of incubation times for ICA , 30 minutes (left) and 18
hours (right).

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Anti-insulin antibodies are reported in IDDM (40-80%), autoimmune

polyendocrinopathies and also in patients receiving thiol containing drugs.

Anti-GAD65 antibodies are reported in IDDM (65-85%), LADA (70-95%),

healthy relatives of diabetic patients (~80%) and also in a small percentage of
type 2 (non-insulin dependent) diabetic patients. Healthy relatives of diabetic
patients may be anti-GAD65 antibody positive for up to 8 years before disease
onset. Anti-GAD65 antibodies are associated with an earlier requirement for
insulin and increased frequency of autoimmune thyroid disease.

Anti-IA-2 antibodies have been detected in IDDM (~50-75%), LADA (~11%)

and in healthy relatives of affected patients. Healthy relatives positive for anti-
IA-2 antibodies are at an increased risk of progression to diabetes in the future.

Detection: IIF using human (blood group O) or monkey pancreas and an ICA
positive sample will show staining of all islet cells (non-restricted ICA) or
staining limited to the β-cells (restricted ICA) (Figure 9.9). Anti-insulin and/or
anti-GAD65 antibodies will show staining of the β-cells in the pancreatic islets.
Incubation of sera overnight can increase sensitivity approximately eight fold
(Figure 9.10), although this should be done with care as the tissue will become
delicate. Difficulty in pattern interpretation may be caused by ABO blood group
antibodies binding acinar cells. These antibodies can be blocked by diluting
samples in a buffer containing AB blood group antigens rather than the usual
PBS. Positive samples should also be tested on LKS sections to identify any
additional antibodies, for example ANA or mitochondrial antibodies which may
add confusing patterns. Antibodies to recombinant GAD65 and insulin can also
be detected using RIA and EIAs. Anti-IA-2 antibodies are usually detected
using RIA and the appropriate recombinant antigen. EIAs are available,
however RIA are often the preferred choice because of their superior sensitivity
and specificity.

Standardisation between ICA assays was improved by the introduction of an

international serum (1988), where neat, 1/2, 1/4, 1/8 and 1/16 serum dilutions
corresponded to 80, 40, 20, 10 and 5 Juvenile Diabetes Foundation (JDF) units
respectively. This serum has subsequently (1999) been aliquoted, prepared to
WHO guidelines and is available from the National Institute of Biological
Standards and Control (NIBSC, 97/550). Each ampoule contains 20
international units of islet cell antibodies and it is also a recognised standard for
GAD65 and IA-2 antibodies. Sera containing less than 10 international units

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 Endocrine Diseases

are considered to be low titre and have no increased risk of developing IDDM.
Sera containing 20 international units or more are described as strongly positive
and sera above 40 international units are calculated to have a positive predictive
value of 85% for the development of IDDM

References

Botazzo GF, Florin-Christensen A, Doniach D. Islet-cell antibodies in diabetes mellitus with

autoimmune polyendocrine deficiencies. Lancet 1974; 2: 1279-1282.

Rabin DU, Pleasic SM, Shapiro JA, Yoo-Warren H, Oles J, Hicks JM et al. Islet cell antigen 512 is
a diabetes-specific islet autoantigen related to protein tyrosine phosphatases. J Immunol 1994; 152:
3183-3188.

Hallberg A, Juhlin C, Berne C, Kampe O, Karlsson FA. Islet cell antibodies: variable
immunostaining of pancreatic islet cells and carcinoid tissue. J Intern Med 1995; 238: 207-213.

Daw K, Ujihara N, Atkinson M, Powers AC. Glutamic acid decarboxylase autoantibodies in stiff-
man syndrome and insulin-dependent diabetes mellitus exhibit similarities and differences in
epitope recognition. J Immunol 1996; 156: 818-825.

Lan MS, Wasserfall C, Maclaren NK, Notkins AL. IA-2, a transmembrane protein of the protein
tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus. Proc
Natl Acad Sci U S A 1996; 93: 6367-6370.

Verge CF, Gianani R, Kawasaki E, Yu L, Pietropaolo M, Jackson RA et al. Prediction of type I

diabetes in first-degree relatives using a combination of insulin, GAD, and ICA512bdc/IA-2
autoantibodies. Diabetes 1996; 45: 926-933.

Lohmann T, Hawa M, Leslie RD, Lane R, Picard J, Londei M. Immune reactivity to glutamic acid
decarboxylase 65 in stiff-man syndrome and type 1 diabetes mellitus. Lancet 2000; 356: 31-35.

Bingley PJ, Bonifacio E, Mueller PW. Diabetes Antibody Standardization Program: first assay
proficiency evaluation. Diabetes 2003; 52: 1128-1136.

Mayr A, Schlosser M, Grober N, Kenk H, Ziegler AG, Bonifacio E et al. GAD autoantibody affinity
and epitope specificity identify distinct immunization profiles in children at risk for type 1 diabetes.
Diabetes 2007; 56: 1527-1533.

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The Thyroid Gland

The thyroid gland weighs approximately 15-20g and consists of two lobes
located either side of the upper trachea. The main function of the thyroid gland
is to synthesise and secrete the hormones thyroxine (T4) and calcitonin.
Thyroxine, a derivative of thyroglobulin, acts to regulate basal metabolic rate
via stimulation of mitochondrial respiration and oxidative phosphorylation.
Calcitonin aids calcium homeostasis causing a reduction of the concentration of
calcium in the blood. The main autoimmune thyroid disorders are:

Autoimmune hypothyroidism: Also known as Hashimoto's disease and

autoimmune thyroiditis. Underactivity of the thyroid gland is demonstrated by
low levels of thyroxine (T4) and triiodothyronine (T3), and high levels of
thyroid stimulating hormone (TSH). Clinical presentation may include fatigue,
weakness, mental impairment, cold intolerance, weight gain and dry, thick,
yellow skin.

Autoimmune hyperthyroidism: Overactivity of the thyroid gland in autoimmune

hyperthyroidism results in increased levels of thyroid hormones. Clinical
presentation may include hyperactivity, fatigue, weight loss, heat intolerance
and excessive sweating, rapid heart rate and irregular menstrual flow.

Grave’s disease: A hyperthyroid disorder similar to autoimmune

hyperthyroidism caused by autoantibodies to the TSH receptor. Disrupted TSH
receptor function will be demonstrated by increased thyroxine (T4) levels and
reduced TSH levels. Clinical presentation will be similar to autoimmune
hyperthyroidism and may also include ophthalmopathy.

Autoimmune thyrotoxicosis: A hyperthyroid condition with a clinical

presentation similar to other hyperthyroid disorders. It is caused by an excess
amount of thyroxine hormone which may be the result of over production,
increased uptake by the thyroid gland, or due to a leakage in the gland because
of damage to the storage function.

Postpartum thyroiditis: A disorder due to inflammation of the thyroid gland.

Initially, excess thyroid hormone results in hyperthyroidism, followed by
hypothyroidism once hormone levels have become depleted. Normal thyroid
function will return after 3-6 months when the thyroid gland has fully
recovered.

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Thyroglobulin Antibodies

Antigen: Thyroglobulin is a 660kDa dimer which is water soluble and found

within the thyroid cells and follicles. In response to thyroid stimulating
hormone, thyroglobulin is synthesised in the rough endoplasmic reticulum,
iodised and then stored in the colloid. Simultaneously, previously synthesised
thyroglobulin exits the colloid and is split via hydrolysis and proteolysis into
two iodinated amino acids, tetraiodothyronine (T4) and triiodothyronine (T3).

Clinical associations: Anti-thyroglobulin antibodies usually appear before

anti-TPO antibodies and therefore may be the first presenting antibody in
thyroid disorders. These antibodies are more frequently detected in females
than males and are associated with geographical areas of iodine deficiency.
Anti-thyroglobulin antibodies have been associated with autoimmune
hypothyroidism (~75%), autoimmune hyperthyroidism (~30%), thyroid
adenomas and carcinomas (10-50%).

Detection: IIF using monkey thyroid and anti-thyroglobulin antibodies will

show staining of the thyroid follicles (Figure 9.11). EIAs, radioimmunoassays
and haemagglutination can also be used to detect antibodies specific for
thyroglobulin.

Thyroid Peroxidase Antibodies

Antigen: Thyroid peroxidase (TPO - formerly known as thyroid microsome) is

a glycosylated membrane-bound enzyme of approximately 107kDa found in the
apical membrane of thyroid follicle cells. Thyroid peroxidase oxidises iodide to
an activated intermediate, which then combines with tyrosine residues of
thyroglobulin in the lumen of the colloid follicle.

Clinical associations: Anti-thyroid peroxidase antibodies appear after initial

thyroid tissue damage and are more frequently detected in females and in
patients from iodine deficient geographical areas. Anti-TPO antibodies are
reported in autoimmune hypothyroidism (~100%), postpartum thyroiditis
(~66%), Grave’s hyperthyroidism (~75%), primary Sjögren's syndrome
(~11%), secondary Sjögren's syndrome (~17%), rheumatoid arthritis (~7%) and
healthy blood donors (~10%). Positivity for anti-TPO antibodies is also
associated with an increased risk of miscarriages and infertility in males.

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Detection: Using IIF, anti-TPO antibodies will stain the thyroid epithelial cells
of monkey thyroid (Figure 9.12). EIA, radioimmunoassays and
haemagglutination are also used to detect autoantibodies to TPO.

Thyrotrophin Receptor Antibodies

(Thyrotrophin stimulating hormone receptor antibodies)

Antigen: The thyrotrophin receptor is a transmembrane protein of

approximately 100kDa, which is found attached to the surface of the thyrocyte
plasma membrane. Thyroid stimulating hormone binds to the receptor and
subsequently stimulates thyroglobulin synthesis and release. Orbital tissues are
also reported to contain thyrotrophin receptors.

Clinical associations: Anti-thyrotrophin receptor antibodies have been

associated with autoimmune thyrotoxicosis, Grave's disease, primary
hypothyroidism, autoimmune hypothyroidism and also in non-thyroid diseases
such as SLE and systemic sclerosis. Antibodies have different effects
depending on the region of the receptor which is targeted. The antibodies can
either stimulate, inhibit or have no identifiable effect on the thyrotrophin
receptor. Discriminating between these antibodies can prove useful for disease
diagnosis and subsequent treatment. Titres of anti-thyrotrophin receptor
antibodies may correlate with disease activity in Grave's disease and therefore
may be a useful predictor of potential relapse.

Detection: Anti-thyrotrophin receptor antibodies cannot be detected using IIF;

however EIAs, radioimmunoassays and haemagglutination assays are used.

References

Campbell PN, Doniach D, Hudson RV, Roitt IM. Auto-antibodies in Hashimoto's disease
(lymphadenoid goitre). Lancet 1956; 271: 820-821.

Burek, CL. Autoimmune diseases of the thyroid and adrenal glands . In: Clinical and Laboratory
Evaluation of Human Autoimmune Diseases. Editors Nakamura RM, Keren DF, Bylund DJ. ASCP
Press; 2002.

Davies TF, Ando T, Lin RY, Tomer Y, Latif R. Thyrotropin receptor-associated diseases: from
adenomata to Graves disease. J Clin Invest 2005; 115: 1972-1983.

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Figure 9.11. Monkey thyroid showing staining with anti-thyroglobulin

antibodies in the thyroid follicles.

Figure 9.12. Anti-thyroid peroxidase (TPO) staining in the thyroid epithelial

cells on monkey tissue.

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The Pituitary Gland

The pituitary gland is located beneath the brain, weighs approximately 0.4-
0.9g, is bean shaped and consists of two parts, the anterior and posterior
pituitary. In response to hormone stimulation from the hypothalamus, pituitary
gland cells will secrete a number of hormones (Table 9.4). Subsequently these
will stimulate other endocrine organs to secrete more specific hormones.
Anterior Pituitary Cells Hormones Secreted
Somatotrophs Growth hormone (GH)
Lactotrophs Prolactin (PRL)
Adrenocorticotrophic hormone (ACTH), α-
Corticotrophs melanocyte stimulating hormone (α-MSH)
and β-endorphin

Thyrotrophs Thyroid stimulating hormone (TSH)

Follicle stimulating hormone (FSH) and
Gonadotrophs
luteinizing hormone (LH)

Posterior Pituitary Cells Oxytocin and anti-diuretic hormone

Table 9.4. Pituitary cells and related hormones.

Pituitary Diseases

Autoimmune diseases of the pituitary gland are rare but the predominant
disorder is lymphocytic hypophysitis.

Lymphocytic hypophysitis: Also known as autoimmune hypophysitis. In this

disorder the pituitary gland becomes enlarged due to infiltration of lymphocytes
and hormonal secretion will be dysfunctional. Clinical symptoms may include
headache and changes in visual field, hypopituitarism and hyperprolactinaemia
may also develop.

Hypopituitarism: The underactive pituitary gland is demonstrated by decreased

levels of pituitary hormones.

Hyperprolactinaemia: Characterised by increased levels of the hormone

prolactin, responsible for preparing breasts for milk production.

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Pituitary Gland Antibodies

Antigen: A number of pituitary gland autoantigens have been reported

including prolactin (~23kDa), growth hormone (~23kDa), pituitary gland
specific factor 1a (PGSF1a) (~16kDa), PGSF2 (~27kDa), α-enolase (~49kDa)
and some uncharacterised cytoplasmic antigens.

Clinical associations: Anti-pituitary antibodies are rarely detected and are

associated with a wide range of autoimmune diseases including lymphocytic
hypophysitis, hyperprolactinaemia, thyroid disorders, growth hormone
deficiency, hypopituitarism, Addison's disease, inactive pituitary disorder and
other autoimmune disorders.

Detection: IIF using monkey pituitary gland and anti-pituitary gland antibodies
will show granular cytoplasmic staining of the pituitary cells (Figure 9.13). It
is advisable to use a conjugate which contains anti-human IgG, IgA and IgM
antibodies to help increase sensitivity for anti-pituitary antibodies. Only anti-
pituitary antibodies at a high titre (>1/8) should be considered as autoimmune
markers of pituitary impairment. Anti-pituitary antibodies at low titre (<1/8)
may be found in patients with pituitary adenomas or patients who are otherwise
healthy. Antigen specificity of the pituitary gland antibodies can be determined
using immunoblotting, radioligand assays and EIA.

Figure 9.13. Antibodies against prolactin in monkey pituitary gland.

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References

Bottazzo GF, Pouplard A, Florin-Christensen A, Doniach D. Autoantibodies to prolactin-secreting

cells of human pituitary. Lancet 1975; 2: 97-101.

Tanaka S, Tatsumi KI, Kimura M, Takano T, Murakami Y, Takao T et al. Detection of autoantibodies
against the pituitary-specific proteins in patients with lymphocytic hypophysitis. Eur J Endocrinol
2002; 147: 767-775.

De Bellis A, Bizzarro A, Bellastella A. Pituitary antibodies and lymphocytic hypophysitis. Best

Pract Res Clin Endocrinol Metab 2005; 19: 67-84.

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Chapter 10

Autoimmune Skin Diseases

Autoimmune skin diseases comprise a number of separate conditions

including bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus and
paraneoplastic pemphigus. From a clinical viewpoint most of these diseases are
readily distinguished, although this differentiation depends on three separate
features. The first is clinical appearance, however, clinical symptoms can be
very similar and in some cases treatment is very different. The other two
features, based on immunofluorescence testing for autoantibodies, are therefore
essential. These are the identification of immunoglobulin and complement
deposition in the skin of affected patients and detection of the relevant
autoantibody in serum.
The autoantibodies can be demonstrated by IIF using patients’ serum,
monkey oesophagus is the tissue substrate of choice. The IIF patterns are
described in Table 10.2. A species-specific anti-human IgG second antibody is
recommended and ensures minimal background and interference. Skin, lip,
tongue, anal canal and oesophagus from different species have been used;
guinea pig, rat and mouse tissues are less specific and do not detect a
significant number of patients. Alternatively the autoantibodies can be
demonstrated, in association with complement deposition, on perilesional skin
by direct IF. The direct IF patterns are described in Table 10.4. Since direct IF
on biopsy material is more sensitive, but more traumatic and expensive, serum
tests are frequently used as an initial screening procedure. If negative, a biopsy
assessed by direct IF may be required.
As with most IFAs, if the specificity needs to be confirmed then a specific
immunoassay must be used. There is growing interest in identifying the antigen
targets so that pathogenesis can be understood and this has led to the use of
western blot or EIA tests in research laboratories. Another procedure is IIF on
normal human salt-split skin. Salt treatment results in a separation of the tissue
within the basement membrane zone (BMZ) thus allowing further
differentiation of BMZ staining autoantibodies (Table 10.3). Also included
within this chapter are brief descriptions of the more common skin autoantigens
and related diseases: Table 10.1 summarises these associations.

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Pemphigus vulgaris

Linear IgA bullous

Herpes gestationis

bullosa acquisita
IgA pemphigus
Paraneoplastic

Epidermolysis

herpetiformis
pemphigoid

pemphigoid
Pemphigus

Pemphigus
pemphigus

dermatosis
Cicatricial
foliaceus

Bullous
Desmoglein ● ● ● ●
Desmocollin ● ● ● ●
BP230 ● ● ● ●
BP180 ● ● ● ●
Periplakin ● ●
Envoplakin ● ●
Plectin ● ●
Collagen VII ● ●
Desmoplakin ●
●
170kDa

●
Antigen

Uncein

Laminin 5 ●
●
190kDa

●
Antigen
II-2

LAD-1 ●
Table 10.1. Autoimmune skin diseases and associated autoantigens.

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Indirect Immunofluorescence

Methodology

Indirect immunofluorescence is used to detect circulating autoantibodies in

the sera of patients with autoimmune skin diseases. Patient sera samples should
be diluted 1/20 and serial dilutions used to assess the titre of these antibodies.
Monkey oesophagus is the substrate of choice, although other substrates such as
guinea pig oesophagus are suggested to be more sensitive for detecting certain
antibodies, for example those to the BMZ. For interpretation of tissue
orientation see Figures 10.4, 10.5 and 10.14. Monkey bladder should be used
as a substrate if paraneoplastic pemphigus is suspected as this will reveal
staining of the transitional epithelium. Autoantibodies to the epidermal basal
cell layer (Figure 10.10) are occasionally found; these may recognise several
different target antigens. They are of little clinical significance and are reported
in 1.5% of normal subjects and at a higher frequency in patients with drug-
induced skin diseases and chronic hepatitis B virus infections. Most laboratory
monkeys are blood group AB secretors and these antigens are expressed in
many tissues including squamous epithelium and gastric mucosa. Consequently,
blood group O patients with high titre IgG class AB antibodies will produce
atypical pemphigus patterns. Whilst the staining usually spares intercellular
spaces around the cells of the basal layer and tends to be variable, weak
pemphigus antibodies may be obscured. It is, therefore, important to adsorb sera
that produce confusing patterns and retest. This is achieved by adding a
concentrate of blood group antigens into the sample dilution buffer (Figure
10.8). Fluorescein-labelled, species-specific, conjugate against the appropriate
human immunoglobulin should be applied additionally to the substrate.
Antibody patterns are visualised using a fluorescence microscope (for pattern
descriptions and examples see Table 10.2, Figures 10.2, 10.3, 10.6 and 10.7).

References

Jordon RE, Beutner EH, Witebsky E, Blumental G, Hale WL, Lever WF. Basement zone antibodies
in bullous pemphigoid. JAMA 1967; 200: 751-756.

Kanitakis J. Indirect immunofluorescence microscopy for the serological diagnosis of autoimmune

blistering skin diseases: a review. Clin Dermatol 2001; 19: 614-621.

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Disease Indirect Immunofluorescence Pattern

IgG autoantibodies to epidermal cell surface
Pemphigus vulgaris antigens of stratified squamous epithelia.
Characteristic ‘chicken wire pattern’
IgG (usually IgG4) autoantibodies to
Pemphigus foliaceus intercellular spaces of stratified squamous
epithelia
IgG autoantibodies to epithelium cell surfaces
Paraneoplastic pemphigus and BMZ. NB: IgG autoantibodies to
transitional epithelium in bladder
IgA pemphigus IgA autoantibodies to subcorneal or
intercellular spaces of squamous epithelia
Bullous pemphigoid IgG autoantibodies to the BMZ (80%)
Herpes gestationis IgG autoantibodies to the BMZ (20%)
Cicatricial pemphigoid IgG autoantibodies to the BMZ (30%)
IgG (less frequently IgM, IgA and
Epidermolysis bullosa
complement) autoantibodies to the BMZ
acquisita
(50%)
Linear IgA bullosa dermatosis IgG and IgA cell-surface autoantibodies
Epidermal basal cell layer Staining of basal cell layer of stratified
antibodies squamous epithelium
Table 10.2. Indirect immunofluorescence skin disease patterns.

Figure 10.1. Patient with bullous skin disease.

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Figure 10.2. Monkey oesophagus showing binding of pemphigoid IgG

autoantibodies along the BMZ.

Figure 10.3. IgG pemphigus autoantibodies labelling epidermal cell surface

antigens of stratified squamous epithelia producing the characteristic ‘chicken
wire’ pattern on monkey oesophagus.

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Figure 10.4. Schematic of a skin section, indicating the epidermis, dermis and
basement membrane zone (BMZ). The area within the box is enlarged in Figure
10.5.

Figure 10.5. (Enlargement of the boxed area in Figure 10.4). Diagrammatic

representation of the structural organisation of BMZ, basal cells, desmosomes
and hemidesmosomes at the dermal/epidermal junction within the skin. The
main desmosomal antigens are desmoglein, desmocollin, periplakin,
envoplakin and desmoplakin. The main hemidesmosomal antigens are plectin,
BP230 and BP180.

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Figure 10.6. Paraneoplastic pemphigus autoantibodies showing cytoplasmic

staining of basal epidermal cells on monkey oesophagus. When paraneoplastic
pemphigus is suspected, samples should be tested on monkey bladder sections
(Figure 10.7).

Figure 10.7. Monkey bladder showing staining of the transitional epithelium

surrounding the lumen by paraneoplastic pemphigus autoantibodies.

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Figure 10.8. Typical pemphigus (left), atypical pemphigus before (centre) and
after (right) the addition of blood group antigens, on monkey oesophagus.

Figure 10.9. Monkey oesophagus showing squamous epithelium and the

underlying lamina propria. The tissue has been stained with a serum from a
patient with pemphigus and a peroxidase labelled second antibody.

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Figure 10.10. Antibodies staining cells in the basal cell layer of the stratified
squamous epithelia of monkey oesophagus.

Indirect Immunofluorescence Using Salt-Split Skin

Methodology

Sodium chloride split skin can be used to detect autoantibodies to skin

components (example shown in Figure 10.11). A large proportion of
autoimmune skin diseases have similar clinical features and consequently may
be difficult to distinguish from one another. The use of salt-split skin may help
overcome this problem as BMZ antigens are separated: for pattern description
see Table 10.3. Detailed methodology of how to carry out the procedure can be
found elsewhere (Gammon et al. 1984). Briefly, the addition of 1.0 M NaCl to
normal skin separates the basement membrane zone at the lamina lucida leaving
the antigens BP230, BP180 and LAD97 on the epidermal side and laminin 5,
collagen VII and components of the lamina densa on the dermal side. Patient
sera should be diluted 1/5 and added to the split-skin, followed by addition of
the appropriate conjugate.

Reference
Gammon WR, Briggaman RA, Inman AO 3rd, Queen LL, Wheeler CE. Differentiating anti-lamina
lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M
sodium chloride-separated skin. J Invest Dermatol 1984; 82:139-144.

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Disease IIF Pattern on Salt-Split Skin

Usually epidermal side, although some sera
Bullous pemphigoid
may bind both sides
Usually epidermal side, although some sera
Cicatricial pemphigoid
may bind both sides
Epidermolysis bullosa
Usually dermal side
acquisita
Herpes gestationis Epidermal side
Linear IgA bullous Usually epidermal side, although a minority
dermatosis may bind to dermal or both sides
Pemphigus erythematosus Faint binding to epidermal side
Table 10.3. Patterns of different skin diseases on salt-split skin.

Figure 10.11. Bullous pemphigoid antibodies on salt-split skin showing staining

of the epidermal side of the BMZ.

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Direct Immunofluorescence

Methodology

Direct immunofluorescence is used to detect autoantibodies or complement

components which have been deposited in the patient's skin (Figures 10.12 and
10.13). The skin sample used should contain an early lesion and perilesional
skin, thus avoiding any potentially misleading results due to changes to the
epithelium by pathogenic autoantibodies. Anti-human fluorescein-labelled
conjugates against the relevant specificity i.e. IgG, IgA, IgM or complement
components-C3, C9, C1q and fibrinogen should be applied to the skin samples.
Antibody patterns can then be visualised through a fluorescence microscope.
Table 10.4 describes the fluorescent patterns produced by the autoantibodies
and complement which are deposited within the skin layers. Light microscopy
with peroxidase or alkaline phosphatase labelled antibodies may be more
informative with respect to the structure of the biopsy specimen.

Figure 10.12. Human skin biopsy showing C3 deposited along the BMZ.

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Disease Direct Immunofluorescence Pattern

Linear/granular intercellular IgG (less
Pemphigus vulgaris
frequently IgA and IgM) and/or C3
Pemphigus foliaceus Linear/granular intercellular IgG and/or C3
Intercellular IgG and/or C3 in the epidermis
Paraneoplastic pemphigus
and linear/granular staining to the BMZ
IgA pemphigus
- subcorneal pustular dermatosis - IgA deposition to the cell surfaces of the
(SPD) Type 1 uppermost epidermis
- intra-epidermal neutrophilic - IgA deposition to the cell surfaces of the
IgA dermatosis (IEN) Type 2 entire epidermis
Linear IgG (less frequently IgA and IgM)
Bullous pemphigoid
and/or C3 in the BMZ
Linear C3 with or without IgG along the
Herpes gestationis
BMZ
Linear IgG (less frequently IgA and IgM)
Cicatricial pemphigoid
and/or C3 in the BMZ
Epidermolysis bullosa acquisita Broad linear IgG/C3 in the BMZ
Linear IgA bullosa dermatosis Linear IgA and/or C3 in the BMZ
Cell surface IgG, IgA and/or C3 deposits
Pemphigus herpetiformis
along the dermal papillae

Table 10.4. Patterns observed for autoimmune skin diseases by direct

immunofluorescence.

References

Jordon RE, Triftshauser CT, Schroeter AL. Direct immunofluorescent studies of pemphigus and
bullous pemphigoid. Arch Dermatol 1971; 103: 486-491.

Morrison LH. Direct immunofluorescence microscopy in the diagnosis of autoimmune bullous

dermatoses. Clin Dermatol 2001; 19: 607-613.

Mutasim DF, Adams BB. Immunofluorescence in dermatology. J Am Acad Dermatol 2001; 45: 803-
822.

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Figure 10.13. IgG deposited intercellularly within the epidermis and along the
BMZ in a human skin biopsy.

Autoantigens in Skin Diseases

The type of autoimmune skin diseases can be inferred from the observed
immunofluorescent pattern. However, the specific skin antigens recognised by
the autoantibodies producing these patterns can only be determined using more
specific assays such as EIA and immunoblotting. What follows is a brief
description of reported skin autoantigens; the more commonly associated skin
disorders are also mentioned.

Desmoglein (Dsg): Is a member of the cadherin family. It is a desmosomal

glycoprotein (Dsg1 = 160kDa, Dsg3 = 130kDa) consisting of an extracellular
domain containing six calcium binding sites, a transmembrane domain and an
intercellular domain which links desmoglein via plakoglobulin to the
cytoskeleton. Desmoglein functions as an adhesion molecule and has an
important role in the formation of tissue integrity. Anti-Dsg1 and anti-Dsg3
antibodies have been detected in the following diseases: paraneoplastic
pemphigus, pemphigus foliaceus, endemic pemphigus foliaceus, pemphigus
vulgaris - mucocutaneous type, IgA pemphigus and pemphigus herpetiformis.
In addition, anti-Dsg3 antibodies alone have also been associated with
pemphigus vulgaris - the mucosal dominant type.

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Desmocollin (Dsc): Is a glycoprotein found in desmosomes (Dsc1 = 130kDa,

Dsc2 = 115kDa) that functions as an adhesion molecule. Desmocollin
antibodies have been detected in the following diseases: IgA pemphigus,
paraneoplastic pemphigus, pemphigus herpetiformis and endemic pemphigus
foliaceus.

BP230 (also known as BPAG1): BP230 is a 230kDa cytoplasmic protein of the

plakin family which is found in hemidesmosomes. It is involved in linking the
keratin cytoskeleton to hemidesmosomes. Anti-BP230 antibodies were
originally detected in bullous pemphigoid; however, they have also been
reported in paraneoplastic pemphigus, herpes gestationis and linear IgA bullous
dermatosis.

BP180 (also known as BPAG2 and collagen XVII): Is a 180kDa transmembrane

collagenous protein found in hemidesmosomes, which functions as a cell-
surface receptor contributing to the maintenance of dermo-epidermal cohesion.
Anti-BP180 antibodies were originally detected in bullous pemphigoid and
have also been reported in cicatricial pemphigoid, herpes gestationis and linear
IgA bullous dermatosis. The majority of autoantibodies from bullous
pemphigoid and herpes gestationis sera recognise epitopes within the NC16A
subdomain, the immunodominant region of BP180.

LAD-1: Is a 97kDa protein found in the extracellular domain of BP180. Anti-

LAD-1 antibodies have been detected in linear IgA bullous dermatosis.

Periplakin: Is a 190kDa protein found in the desmosomes and epidermal

cornified envelope. Anti-periplakin antibodies have been detected in
paraneoplastic pemphigus and endemic pemphigus foliaceus.

Envoplakin: Is a 210kDa protein which is thought to be involved in linking the

cornified envelope to desmosomes and keratin filaments. Anti-envoplakin
antibodies have been detected in paraneoplastic pemphigus and endemic
pemphigus foliaceus.

Plectin: A member of the plakin family, plectin is a cytoplasmic

hemidesmosomal protein of approximately 500kDa which is involved in the
attachment of keratin filaments. Anti-plectin antibodies have been detected in
paraneoplastic pemphigus and, rarely, in bullous pemphigoid.

Collagen VII: Is a 290kDa protein which is a major component of anchoring

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fibrils. Anti-collagen VII antibodies are detected in epidermolysis bullosa

acquisita and linear IgA bullous dermatosis. The majority of epidermolysis
acquisita autoantibodies are directed against epitopes within the non-
collagenous domain (NC1) of collagen VII.

Desmoplakin: Desmoplakin I, II and III are desmosomal proteins with

estimated molecular weights of 250, 210, and 81kDa respectively.
Desmoplakin I forms an interlinked dimer between its parallel rod-like domain;
desmoplakin II and III are not thought to dimerise, possibly due to absence of
required regions of the rod-like domain. Anti-desmoplakin I and II antibodies
have been reported in paraneoplastic pemphigus.

Figure 10.14. Schematic interpretation of the desmosome structure (Garrod

1996). The target antigens in the desmosomes comprise specific intercellular
adhesion molecules of 100-500nm that mediate intercellular adhesion and link
cells to the intermediate filament cytoskeleton that spans the cytoplasm.

170kDa Antigen: A 170kDa uncharacterised keratinocyte polypeptide. Anti-

170kDa antibodies have been infrequently detected in paraneoplastic
pemphigus.

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200kDa Antigen: A 200kDa uncharacterised antigen located in the lamina

lucida. The associated antibodies have been detected in anti-p200 pemphigoid.

Uncein: Is a component of anchoring fibrils. Anti-uncein antibodies have been

occasionally detected in epidermolysis bullosa acquisita.

α Subunit of Laminin 5: Laminin 5 is essential for epidermal cell attachment.

Its main functions include linking epidermal basal cells to the papillary dermis,
initiating hemidesmosome formation, accelerating basement membrane
formation and enhancing the recovery of damaged skin. Anti-laminin 5
antibodies have been detected in cicatricial pemphigoid.

Melanocytes: These are cells found in the skin and eye that synthesise the
enzyme tyrosinase, which catalyses the oxidation of tyrosine to produce
melanin. Melanin-related antibodies and those that specifically target the
enzyme tyrosinase have been reported in vitiligo, alopecia areata and alopecia
totalis.

Hair Follicle Structures: Antibodies to an unknown hair follicle structure have

been reported in alopecia areata.

190kDa Antigen: Anti-190kDa antibodies to an as yet unidentified antigen have

been reported in paraneoplastic pemphigus.

Autoimmune Skin Diseases - Brief Summaries

There are many autoimmune skin diseases; a brief description along with the
most frequently associated autoantigens (in brackets) is given below.

Pemphigus Vulgaris (desmoglein): This disease usually occurs between the 3rd
and 6th decade of life, affecting both genders equally. The disease is split into
two subgroups based upon the bodily areas the disease affects. The mucosal
dominant type exhibits mucosal lesions with minimal skin involvement. In
comparison, the mucocutaneous type exhibits extensive skin blisters as well as
erosions with mucosal involvement.

Pemphigus Foliaceus (desmoglein 1): Affected patients are observed to have

thin-roofed skin blisters which easily rupture, producing painful crusting
lesions. The disease is epidermal specific and cell-cell detachment occurs
within the subcorneal layer of the skin. Fogo selvagem is a clinically identical

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form of pemphigus foliaceus which occurs in regions of Brazil and Columbia.

This is an endemic disease and an environmental trigger is strongly suspected
to be involved in disease development.

Paraneoplastic Pemphigus (desmoglein 1 and 3, periplakin, BP230,

envoplakin, desmoplakin 1 and 2, plectin): Is an uncommon disease
characterized by severe oral mucositis and sometimes blisters or erosions of
other mucous membranes. Patients usually have malignant neoplasm, for
example malignant lymphoma, chronic lymphocytic leukaemia, thymoma,
bronchogenic squamous cell carcinoma and sarcoma, but occasionally benign
neoplasm may be present. Pulmonary injury is a common complication and may
ultimately lead to respiratory failure.

IgA Pemphigus/Intercellular IgA Dermatosis (desmocollin, desmoglein): Is a

rare vesiculobullous skin disease, within which there are two major subtypes.
(1) Subcorneal pustular dermatosis (SPD) type. Presents as superficial pustular
skin lesions over the entire body (particularly where skin surfaces touch) and
also subcorneal pustules in the dermis and epidermis.
(2) Intra-epidermal neutrophilic IgA dermatosis (IEN) type. Presents as
atypical skin lesions over the whole body comprising numerous pustules and
crusts.

Bullous Pemphigoid (BP230, plectin, BP180): Is a common sub-epidermal

blistering disease with an incidence of approximately 10 cases per million
population. Incidence is higher in men than women and usually affects people
60-80 years of age, although there are rare reports of childhood bullous
pemphigoid. Bullous pemphigoid typically presents as tense round or oval
bullae in symmetric arrangement located on flexor surfaces of the arms, legs,
groin, axilla, and lower abdomen (Figure 10.1). Mucous membranes may be
involved but is rarely the presenting feature.

Herpes Gestationis/Gestational Pemphigoid (BP230, BP180): Is a sub-

epidermal bullous disease which affects approximately 1 in 50,000 pregnant
women in the 2nd and 3rd trimester or immediately post-partum. The disease
may recur with subsequent pregnancies, menstruation and use of oral
contraceptive. Herpes gestationis presents as extremely itchy lesions on the
abdomen which may spread to the extremities. Newborns may have some
lesions, be low weight and there is a risk of premature delivery.

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Cicatricial Pemphigoid/Benign Mucous Membrane Pemphigoid (BP180,

laminin 5): Is a sub-epidermal bullous disease, which primarily involves the
oral and ocular mucous membranes although other mucosal sites may also be
involved. Cicatricial pemphigoid affects women twice as often as men and
usually presents in 6th to 8th decades of life, although early adult onset can
occur. Erosions and ulcerations heal with scarring, and complications include
blindness and airway obstruction. Cicatricial pemphigoid has also been
associated with malignancies in some patients.

Linear IgA Bullous Dermatosis (BP230, BP180, collagen VII, LAD-1): Is a

subepidermal skin disease with an incidence of less than 0.5 per million in
Western Europe with an average onset age of 60 years. Linear IgA bullous
dermatosis can be induced by drugs e.g. antibiotics. Lesions can consist of tense
arciform bullae similar to bullous pemphigoid or as groups of papulo vesicles
similar to dermatitis herpetiformis. The lesions will be distributed
symmetrically on trunk, limbs, face, scalp, hands and feet. Mucosal
involvement can occur in 60-80% of cases affecting eyes, nose, throat, mouth
and oesophagus. Chronic bullous disease of childhood is clinically and
immunopathologically similar to linear IgA bullous dermatosis. There is also an
increased risk of developing various malignancies, particularly
lymphoproliferative type reported in up to 5% of cases.

Vitiligo (melanocyte antigens): The detection of anti-melanocyte autoantibodies

has provided strong evidence that autoimmune mechanisms are involved in the
development of vitiligo. This disorder is characterised by the presence of milk-
white lesions on the skin which may change in size and shape. Damage to
melanocytes leads to increased UV sensitivity and therefore increased risk of
developing melanoma. The condition has a world wide incidence of
approximately 1-2% and usually develops before 20 years of age.

Alopecia Areata (melanocyte antigens, hair follicle structures): This disorder

has a lifetime risk of 1-2% for the general population and is characterised by
non-scarring hair loss. Alopecia areata can affect either gender at any age. Hair
loss may reverse completely or progress to alopecia totalis (loss of all scalp
hair) or to alopecia universalis (loss of all body hair). The aetiology of this
disorder is not clear although immune mediated mechanisms are implicated.

Epidermolysis Bullosa Acquisita (NC1 domain of collagen VII, uncein): Is a

rare skin disease which usually affects adults in the 4th to 5th decades of life. It
is characterised by vesicles and bullae on skin surfaces which are susceptible to

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frictional trauma e.g. knees, elbows, fingers and toes. Vesicles and bullae may
be preceded by an intense itch. There may also be variable mucous membrane
involvement which if the eyes are involved can lead to blindness. Vesicles and
bullae may heal with scarring and pigmentation alteration.

Anti-p200 pemphigoid (p200): Is a recently reported autoimmune subepidermal

blistering disorder which may be mistaken for epidermolysis bullosa acquisita
because direct and indirect immunofluorescence patterns are reported to be
identical. However, clinical presentation may mimic bullous pemphigoid,
linear IgA dermatosis or dermatitis herpetiformis. The as yet unidentified
200kDa antigen is a component of dermal/epidermal junctions, distinct from
those already identified.

Pemphigus Herpetiformis (desmoglein 1 and 3): This disorder exhibits similar

clinical features to dermatitis herpetiformis: erythematous, urticarial plaques
and vesicles in a herpetiform arrangement. However, the detection of anti-
desmoglein 1 autoantibodies is more characteristic of pemphigus foliaceus.

References

Waldorf DS, Smith CW, Strauss AJ. Immunofluorescent studies in pemphigus vulgaris.
Confirmatory observations and evaluation of technical considerations. Arch Dermatol 1966; 93: 28-
33.

Anhalt GJ, Kim SC, Stanley JR, Korman NJ, Jabs DA, Kory M et al. Paraneoplastic pemphigus. An
autoimmune mucocutaneous disease associated with neoplasia. N Engl J Med 1990; 323: 1729-
1735.

Garrod DR. Epithelial development and differentiation: the role of desmosomes. The Watson Smith
Lecture 1996. J R Coll Physicians Lond 1996; 30: 366-373.

Fishman P, Merimski O, Baharav E, Shoenfeld Y. Autoantibodies to tyrosinase: the bridge between

melanoma and vitiligo. Cancer 1997; 79: 1461-1464.

Ishii K, Amagai M, Komai A, Ebihara T, Chorzelski TP, Jablonska S et al. Desmoglein 1 and
desmoglein 3 are the target autoantigens in herpetiform pemphigus. Arch Dermatol 1999; 135: 943-
947.

Hashimoto T. Immunopathology of IgA pemphigus. Clin Dermatol 2001; 19: 683-689.

Lin MS, Arteaga LA, Diaz LA. Herpes gestationis. Clin Dermatol 2001; 19: 697-702.

Hacker MK, Janson M, Fairley JA, Lin MS. Isotypes and antigenic profiles of pemphigus foliaceus
and pemphigus vulgaris autoantibodies. Clin Immunol 2002; 105: 64-74.

Allen J, Wojnarowska F. Linear IgA disease: the IgA and IgG response to the epidermal antigens

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demonstrates that intermolecular epitope spreading is associated with IgA rather than IgG
antibodies, and is more common in adults. Br J Dermatol 2003; 149: 977-985.

Allen J, Wojnarowska F. Linear IgA disease: the IgA and IgG response to dermal antigens
demonstrates a chiefly IgA response to LAD285 and a dermal 180-kDa protein. Br J Dermatol
2003; 149:1055-1058.

Kozlowska A, Hashimoto T, Jarzabek-Chorzelska M, Amagai A, Nagata Y, Strasz Z et al.

Pemphigus herpetiformis with IgA and IgG antibodies to desmoglein 1 and IgG antibodies to
desmocollin 3. J Am Acad Dermatol 2003; 48: 117-122.

Tobin DJ. Characterization of hair follicle antigens targeted by the anti-hair follicle immune
response. J Investig Dermatol Symp Proc 2003; 8: 176-181.

Thoma-Uszynski S, Uter W, Schwietzke S, Hofmann SC, Hunziker T, Bernard P et al. BP230- and
BP180-specific auto-antibodies in bullous pemphigoid. J Invest Dermatol 2004; 122: 1413-1422.

Bekou V, Thoma-Uszynski S, Wendler O, Uter W, Schwietzke S, Hunziker T et al. Detection of

laminin 5-specific auto-antibodies in mucous membrane and bullous pemphigoid sera by ELISA. J
Invest Dermatol 2005; 124: 732-740.

Salato VK, Hacker-Foegen MK, Lazarova Z, Fairley JA, Lin MS. Role of intramolecular epitope
spreading in pemphigus vulgaris. Clin Immunol 2005; 116: 54-64.

Woodley DT, Ram R, Doostan A, Bandyopadhyay P, Huang Y, Remington J et al. Induction of

epidermolysis bullosa acquisita in mice by passive transfer of autoantibodies from patients. J Invest
Dermatol 2006; 126: 1323-1330.

Dilling A, Rose C, Hashimoto T, Zillikens D, Shimanovich I. Anti-p200 pemphigoid: a novel

autoimmune subepidermal blistering disease. J Dermatol 2007; 34: 1-8.

Di Zenzo G, Calabresi V, Grosso F, Caproni M, Ruffelli M, Zambruno G. The intracellular and

extracellular domains of BP180 antigen comprise novel epitopes targeted by pemphigoid
gestationis autoantibodies. J Invest Dermatol 2007; 127: 864-873.

Kemp EH, Gavalas NG, Gawkrodger DJ, Weetman AP. Autoantibody responses to melanocytes in
the depigmenting skin disease vitiligo. Autoimmun Rev 2007; 6: 138-142.

Zhu X, Zhang B. Paraneoplastic pemphigus. J Dermatol 2007; 34: 503-511.

General References
Kolanko E, Bickle K, Keehn C, Glass LF. Subepidermal blistering disorders: a clinical and
histopathologic review. Semin Cutan Med Surg 2004; 23: 10-18.

Eming R, Hertl M. Autoimmune Diagnostics Working Group. Autoimmune bullous disorders. Clin
Chem Lab Med 2006; 44: 144-149.

Nousari CH, Anhalt GJ. Skin Diseases. In: Manual of Molecular and Clinical Laboratory
Immunology. 7th Edition. Editors Rose NR, Hamilton RG, Detrick B. ASM Press, Washington DC,
USA; 2006.

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Chapter 11

Neurological and Muscle Diseases

Autoantibodies to specific antigens of Purkinje cells and other neurones are

found in a variety of neurological and muscle diseases. Many are associated
with paraneoplastic syndromes (Table 11.1). However, they are also found in
neurological diseases of unknown aetiology and on occasion in normal
individuals. Paraneoplastic neurological syndromes (PNS) are relatively
common in patients with ovarian tumours (15%) and small cell lung carcinomas
(SCLC 12%) but are found at lower frequency in patients with other tumours,
for example those of the stomach, prostate and breast. Only a few of these
patients have autoantibodies and whilst of great interest, they are relatively
uncommon. A recent audit of neuro-immunology test results showed that of
1025 samples sent from referring neurologists only 47 were positive for PNS
autoantibodies, the most common being Hu. There are many PNS
autoantibodies described in the literature; here we present the most common in
detail as well as mentioning a number of the more rare specificities.
The PNS autoantibodies are usually named after the initials of the original
patients but in several cases a histological classification has also been used. For
clarity, both are used here.

Clinical and Pathological Significance

Some autoantibodies associated with PNS are helpful in diagnosis and may
allow more informed treatment. For example, it was clearly shown in a multi-
centre trial that the presence of Hu antibodies in patients with small cell lung
tumours, but no neurological features, was an indicator of relatively limited
disease, good response to therapy and longer survival. Thus, the immune system
and anti-Hu antibodies in particular, may inhibit the growth of the tumour.
Unfortunately, the PNS autoantibodies are not always diagnostic of the
respective diseases and they have been found in other diseases and occasionally
in normal controls. Doubts about the clinical importance of the antibodies will
also remain whilst there is no clear pathogenic link and the functions of the
antigens are not understood. In the majority of cases, pathogenesis is more
likely to be due to a cellular immune response than a direct action of the
antibodies.

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Autoantibody/
Detection Substrate Clinical Associations
Autoantigen
Cerebellum and rodent
Hu (ANNA-1) Small cell lung carcinoma
LKS
Yo (PCA-1) Cerebellum Breast and lung carcinoma
Breast and small cell lung
Ri (ANNA-2) Cerebellum
carcinoma
Tr (PCA-Tr) Cerebellum Hodgkin’s disease
Small cell lung carcinoma and
Amphiphysin Cerebellum
breast tumours
Small cell lung carcinoma and
CV2 (CRMP5) Cerebellum
thymomas
PCA2 Cerebellum Lung malignancies
Lung, testis, parotid, breast and
Ma1, 2(Ta) and 3 Cerebellum and testis
colon tumours
Benign monoclonal
MAG Peripheral nerve
paraproteins
Rat/monkey
Aquaporin-4 cerbellum, midbrain, Neuromyelitis optica
spinal cord
Stiff person syndrome, breast
GAD67 Cerebellum and pancreas and colon tumours, small cell
lung cancer and diabetes
Small cell lung carcinoma and
ANNA3 Cerebellum
adenocarcinoma
mGluR1 Cerebellum Hodgkin’s disease
Striational/ Thymoma, myasthenia gravis
Skeletal muscle
titin and lung carcinoma
Zic4 Cerebellum Small cell lung carcinoma
Lambert Eaton myasthenic
AGNA Rat cerebellum
syndrome
Myocardial Cardiac muscle Myocarditis

Table 11.1. Neurological and muscle disease antibodies and associated

diseases. Except where stated otherwise, monkey tissue should be used.

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Posner provided useful guidelines for assessing the clinical relevance of anti-
neuronal antibodies:
1. A particular antibody must be present in more than one patient with a
similar neurological disorder and corresponding tumour, and the
occurrence of both false-negative and false-positive tests for auto-
antibodies should be rare.
2. The concentration of the antibody in the serum should be relatively high.
3. A higher titre in CSF than in serum would suggest intrathecal synthesis
and would provide evidence for a neurologically relevant antibody.
4. The antibodies should react with a symptomatic part of the nervous
system and the nature of the antigen must be identified by both
immunohistochemistry and western blots on neuronal proteins.

Methods
Screening of sera for PNS autoantibodies is commonly performed by indirect
immunofluorescence and immunoperoxidase staining on cerebellum
cryosections (for interpretation of tissue orientation see Figures 11.1-11.3).
Rodent cerebellum sections are frequently used, however monkey tissues are
preferable for reasons of antigenic similarity. It has been claimed that some
antigens may require careful preparation of the neuronal tissues: intracardiac
perfusion with 4% paraformaldehyde has been recommended when the tissue is
used for the detection of anti-GAD, anti-amphiphysin and particularly CV2
antibodies. However, the more common antigens, Yo, Hu, Ri and Tr seem
stable, as do GAD and amphiphysin, so the requirement for specialised
preservation techniques is debatable. Only IgG antibodies are considered to be
clinically relevant. Sera are screened at 1/50 and 1/500, high titres (>1/100) are
normally of clinical significance. CSF titres of the antibodies may be
proportionately higher than the corresponding sera (after correction for protein
dilution) which supports a pathogenic role of the antibodies and is associated
with the increased likelihood of a tumour.
Any samples which show specific staining patterns must be tested on rodent
liver/kidney/stomach sections to assess the occurrence of anti-mitochondrial,
ANA and other antibodies which can make interpretation more difficult. The
co-occurrence of ANA with Hu antibodies has been reported to be
approximately 29% and with mitochondrial antibodies 15%. On rare occasions,
the anti-mitochondrial antibodies can mask Hu antibodies on cerebellum
sections.
Once the presence of a neuronal-specific antibody has been identified, the
specificity is then confirmed by western blot analysis. The western blot is
performed on primate cerebellum extract, also the inclusion of recombinant

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proteins is recommended. On occasions where the specificity cannot be

determined, the results should be reported as positive but atypical. It is possible
that the conformational epitopes on the proteins are lost due to the lineariation
of the proteins when used in western blot analysis. Alternatively, the specificity
identified by the immunofluorescence may be due to co-localisation of other
neuronal-specific antigens.

a)

b)

Figure 11.1. Schematic diagrams illustrating the location a) and gross structure
b) of the cerebellum. Figure 11.1a) used with permission from Wolters Kluwer
(Clinically Oriented Anatomy, 1999).

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Figure 11.2. Diagram of the cerebellar folia grey matter indicating the tissue
layers and cell types.

ML

PC PC

GL
ML
WM

Figure 11.3. Monkey cerebellum, showing Purkinje cells stained with an

autoantibody from a patient with breast carcinoma and a peroxidase-labelled
second antibody. The molecular layer (ML) is folded around the Purkinje cell
(PC) layer whilst the more dense granular layer (GL) merges with the white
matter (WM).

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Yo Antibodies (PCA-1)

Antigen: Immuno-electron microscopy has shown that anti-Yo antibodies bind

to clusters of ribosomes, the granular endoplasmic reticulum and to the Golgi
complex vesicles of Purkinje cells. Western blot analysis usually shows
antibodies against three proteins of 34, 52 and 62kDa. These are designated
cerebellar degeneration related proteins, CDR 34, CDR62 (CDR1) and CDR62
(CDR2). CDR2 is the major autoantigen and is expressed in the cerebellum,
brainstem and intestinal mucosa.

Clinical associations: These antibodies are associated with subacute cerebellar

ataxia in patients with carcinomas of the ovary and breast (90% of cases) and
rarely, tumours of the uterus, fallopian tube and lung. 99% of cases are female.
Clinical features are not confined to cerebellar dysfunction and may include
peripheral neuropathy. Patients with suggestive clinical features should be
screened for the presence of anti-Purkinje cell antibodies on cerebellar tissues.
Apart from rare exceptions, anti-Yo antibodies occur with gynaecological
cancers and their presence warrants a focused search for an occult tumour.
Presently, the major role for the identification of anti-Yo antibody is for early
tumour diagnosis that might allow early treatment, although neurological
improvement is unusual.

Detection: An IFA screen on cerebellar cryosections will identify Yo

antibodies. Antibodies stain the cytoplasm of Purkinje cells in a granular
fashion and spare the nucleus (Figure 11.4 and 11.5). Autoantibody titres above
1/500 by IIF are regarded as positive, yielding specificities approaching 100%.
It is important to differentiate between anti-Yo antibodies and non-Yo Purkinje
cell antibodies that can be associated with Hodgkin’s disease (Tr/PCA-Tr) and
PCA-2 antibodies. Specificity should be confirmed by western blot analysis.
References

Greenlee JE, Brashear HR. Antibodies to cerebellar Purkinje cells in patients with paraneoplastic
cerebellar degeneration and ovarian cancer. Ann Neurol 1983; 14: 609-613.

Shams'ili S, Grefkens J, deLeeuw B, van den Bent M, Hooijkaas H, van der Holt B et al.
Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50
patients. Brain 2003; 126: 1409-1418.

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Figure 11.4. Monkey cerebellum showing granular staining of Purkinje cell

cytoplasm by anti-Yo antibodies.

Figure 11.5. High magnification of anti-Yo antibody staining on monkey

cerebellum.

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Tr Antibodies (PCA-Tr)

Antigen: The Tr antigen is presumed to be a protein due to sensitivity to pepsin

digestion, however it has not been detected on immunoblots. This may be due
to the antibody being directed against conformational epitopes.

Clinical associations: There is a strong link between the presence of the anti-
Tr antibodies and paraneoplastic cerebellar degeneration in patients with
Hodgkin’s disease (80%), originally indicated by Trotter et al., 1976. The anti-
Tr antibody titre tends to drop after treatment of Hodgkin's disease. The
cerebellar degeneration is usually irreversible, although one study showed
remission of the cerebellar degeneration in 14% of patients; this was most
striking in the younger patients.

Detection: An IFA screen on cerebellar cryosections will identify anti-Tr

antibodies. On occasions (~7%), the antibody is only identifiable in the CSF.
The antibodies will stain the cytoplasm of the Purkinje cells, similar to anti-Yo.
They also stain the dendritic spines of the Purkinje cells, which show as
characteristic multiple dots in the molecular layer (Figure 11.6). As no common
bands have been identified for the Tr antigen on western blots the identification
of anti-Tr antibodies must be strictly based on the immunofluorescent criteria.

Figure 11.6. Tr antibody staining the cytoplasm and dendrites of Purkinje cells
on monkey cerebellum (courtesy of A. Vincent, John Radcliffe Hospital,
Oxford).

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 Neurological and Muscle Diseases

References

Trotter JL, Hendin BA, Osterland CK. Cerebellar degeneration with Hodgkin’s disease. An
immunological study. Arch Neurol 1976; 33: 660-661.

Graus F, Dalmau J, Valldeoriola F, Ferrer I, Rene R, Marin C et al. Immunological characterization

of a neuronal antibody (anti-Tr) associated with paraneoplastic degeneration and Hodgkin's disease.
J Neuroimmunol 1997; 74: 55-61.

Bernal F, Shams'ili S, Rojas I, Sanchez-Valle R, Saiz A, Dalmau J et al. Anti-Tr antibodies as

markers of paraneoplastic cerebellar degeneration and Hodgkin's disease. Neurology 2003; 60:
230-234.

PCA-2 Antibodies

Antigen: It is suggested that anti-PCA-2 antibodies are directed against a

280kDa protein in cerebellar cortical extracts, although at least one serum with
a similar immunofluorescent staining pattern has been shown not to identify
such a band on western blot analysis.

Clinical associations: These more rare antibodies (single case series reported
in the literature) have been associated with lung malignancies, ~50% of cases
were small cell lung carcinomas (SCLC). They are associated with progressive
multifocal neurological syndromes in most cases. The report showed 50% of
samples had other co-existing anti-neuronal antibodies with anti-CRMP5/CV2
being the most common.

Detection: These antibodies were characterised on mouse cerebellum

cryosections, which were fixed in 10% phosphate-buffered formalin. They
were reported to stain the Purkinje cell cytoplasm and the dendritic processes.
The cytoplasm of cells in the granular layer showed a faint reticular pattern.
The antibodies also stained neurons of the peripheral nervous system such as
the myenteric neurones of the stomach. They must be distinguished from anti-
Yo and anti-Tr antibodies which are associated with ovary and breast
carcinomas, and Hodgkin's disease, respectively.

Reference

Vernino S, Lennon VA. New Purkinje Cell Antibody (PCA-2): Marker of Lung Cancer-Related
Neurological Autoimmunity. Ann Neurol 2000; 47: 297-305.

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Hu Antibodies (ANNA-1)

Antigen: The Hu antigens comprise a family of similar proteins (HuD,

HuC/ple21, Hel-N1, Hel-N2) which differ by alternative splicing of their
mRNAs. The antigens are thought to have a crucial role in neuronal
development and maintenance, although the function of the antigens within the
associated tumour is unknown.

Clinical associations: These antibodies are associated with paraneoplastic

encephalomyelitis, sensory neuropathy and rarely, autonomic neuropathies with
gastrointestinal dysmotility. Small cell lung carcinomas are found in 80% of
cases, other reported tumours include neuroblastomas, prostate tumours,
rhabdosarcomas, seminomas and adenocarcinomas of the gall bladder. Patients
with acute or subacute encephalomyelitis or sensory neuropathy should be
examined for the presence of anti-Hu. If this antibody is found, a search for an
underlying tumour should be primarily directed towards detection of small cell
lung carcinoma. If the initial search is negative, the tumour may become
clinically apparent many months after the onset of neurological symptoms.

Detection: An IFA screen on cerebellar cryosections will identify Hu

antibodies. The antibodies stain neuronal nuclei with nucleolar sparing, of both
the central nervous system (CNS) and the peripheral nervous system (PNS)
(Figure 11.7). The cytoplasm of the neurons stains less intensely. Staining of
the nuclei of the myenteric plexus in the stomach will be seen when the sample
is run on liver, kidney and stomach composite blocks (Figure 11.8). Co-
occurrence of Hu antibodies with either ANA or mitochondrial antibodies is
reported to be 29% and 15%, respectively. These specificities are therefore
taken into consideration when interpreting the immunofluorescent pattern on
the cerebellum sections. Also, on occasions, the Hu staining pattern on the
cerebellum can be masked by the presence of mitochondrial antibodies.
Specificity is confirmed by western blot analysis.

References

Wilkinson PC, Zeromski J. Immunofluorescence detection of antibodies against neurones in

sensory carcinomatous neuropathy. Brain 1965; 88: 529-538.

Graus F, Elkon KB, Cordon-Cardo C, Posner JB. Sensory neuronopathy and small cell lung cancer.
Antineuronal antibody that also reacts with the tumor. Am J Med 1986; 80: 45-52.

Graus F, Keime-Guibert F, Rene R, Benyahia B, Ribalta T, Ascaso C et al. Anti-Hu-associated

paraneoplastic encephalomyelitis: analysis of 200 patients. Brain 2001; 124: 1138-1148.

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 Neurological and Muscle Diseases

Figure 11.7. Anti-Hu antibodies staining neuronal nuclei in Purkinje and other
cells of monkey cerebellum.

Figure 11.8. Anti-Hu antibodies on mouse stomach staining several myenteric

plexus neuronal nuclei.

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Ri Antibodies (ANNA-2)

Antigen: Ri antibodies recognise two proteins of 50kDa and 80kDa which are
encoded by the Nova-1 and Nova-2 genes. The antigens are highly conserved
neuronal-specific RNA binding proteins which appear to have a role in the post-
migratory maturation of neurons.

Clinical associations: These rare antibodies are found with the clinical features
of predominant axial ataxia and ocular movement disorders
(opsoclonus/myoclonus). Paraneoplastic opsoclonus ataxia (POA) and
paraneoplastic opsoclonus myoclonus ataxia (POMA) are both terms used to
describe the symptoms. The relative incidence of the antibodies in females and
males is 2:1. Breast or small cell lung carcinomas are found in 75% of cases,
carcinomas of the ovaries, fallopian tubes, bladder and cervix are present less
frequently. Presence of the antibodies has been reported in cases of ovarian
carcinomas without the presence of paraneoplastic neurological syndromes.
The detection of anti-Ri antibodies should prompt a careful search for an
underlying tumour, especially breast cancer and small cell lung carcinoma.
Occasionally no tumour can be found, although the presence of an occult
tumour cannot be ruled out and this makes close follow-up advisable.

Detection: Anti-Ri antibodies can be identified on a cerebellar IFA screen. The

staining pattern is similar to anti-Hu antibodies showing positive neuronal
nuclei with nucleolar sparing and less intense staining of the neuronal
cytoplasm. However, unlike Hu antibodies, they are specific for neuronal
nuclei of the central nervous system. Hence, no staining is observed in the
neuronal nuclei of the myenteric plexus of the stomach. This differentiation can
be difficult and the IF pattern is only an indication of specificity so reliable
identification should be based on results from western blotting of cerebellar
extracts and recombinant proteins.

References

Luque FA, Furneaux HM, Ferziger R, Rosenblum MK, Wray SH, Schold SC et al. Anti-Ri: an
antibody associated with paraneoplastic opsoclonus and breast cancer. Ann Neurol 1991; 29: 241-
251.

Pittock SJ, Lucchinetti CF, Lennon VA. Anti-Neuronal Nuclear Autoantibody Type 2:
Paraneoplastic Accompaniments. Ann Neurol 2003; 53: 580-587.

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Anti-Neuronal Nuclear Antibodies Type 3

Antigen: Anti-neuronal nuclear antibodies (type 3) recognise a 170kDa brain

protein. Little is known about the function of the antigen, although it has been
suggested that it has a role in cell cycle regulation due to only being identified
in terminally differentiated cells (neurons and podocytes). Interestingly, ~30%
of samples have co-existing anti-neuronal antibodies, CV2/CRMP-5 (20%) and
ANNA-1 (10%).

Clinical associations: These are amongst the most rare paraneoplastic

neurological antibodies. They have been associated with small cell lung
carcinomas and adenocarcinomas. The reported neurological symptoms are
diverse and usually multifocal, amongst these are cerebellar ataxia,
sensorimotor neuropathies, myelopathy, brain stem and limbic encephalopathy.

Detection: ANNA-3 antibodies can be identified on a cerebellar IFA screen.

One major difference to Hu and Ri is that there is no staining observed in the
cytoplasm of neurons. They do not stain the neurons of the myenteric plexus
nor do they stain the neurons of the granular layer. The most intense staining is
seen in the Purkinje and Golgi cell nuclei. Of note, the majority of sera also
stain the podocytes in the glomeruli of the kidney. Specificity can be confirmed
on western blots of cerebellar extract where a 170kDa band is observed.

References

Chan KH, Vernino S, Lennon VA. ANNA-3 Anti-Neuronal Nuclear Antibody: Marker of Lung
Cancer-Related Autoimmunity. Ann Neurol 2001; 50: 301-311.

Pittock SJ, Kryzer TJ, Lennon V.A. Paraneoplastic Antibodies Coexist and Predict Cancer, Not
Neurological Syndrome. Ann Neurol 2004; 56: 715-719.

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CV-2/CRMP-5 Antibodies

Antigen: The antibodies bind to the collapsin response-mediator brain protein-

5 (CRMP-5), a member of a family of five cytosolic phosphoproteins. The
molecular weight of CRMP-5 is reported as 62-66kDa and it is expressed in a
subpopulation of oligodendrocytes, a subset of sensory peripheral neurones and
Schwann cells.

Clinical associations: This autoantibody specificity was first reported in 1996

as anti-CV2 antibodies and was described as a novel antibody reacting with
oligodendrocytes in patients with PNS. The antibody is found in patients with
PNS (cerebellar degeneration, encephalomyelitis or limbic encephalitis) in
association with lung carcinomas (~75%), most of which are SCLC, thymomas
(6%) and other tumours. Further reported associations are with optic neuritis,
chorea and cranial neuropathy. The autoantibody is as frequent as the anti-Yo
PNS autoantibodies.

Detection: The antibodies can be detected by IFA on cerebellum (Figure 11.9),

brainstem and spinal cord. Staining is observed in the cytoplasm of a
subpopulation of oligodendrocytes within the white matter, Purkinje somata are
spared. The antibodies also bind the axons of both myelinated and
unmyelinated fibres in peripheral nerve. On western blot of soluble brain
proteins the antibodies bind a 62-66kDa protein.

References

Honnorat J, Antoine JC, Derrington E, Aguera M, Belin MF. Antibodies to a subpopulation of glial
cells and a 66 kDa developmental protein in patients with paraneoplastic neurological syndromes.
J Neurol Neurosurg Psychiatry 1996; 61: 270-278.

Yu Z, Kryzer TJ, Griesmann GE, Kim K, Benarroch EE, Lennon VA. CRMP-5 neuronal
autoantibody: marker of lung cancer and thymoma-related autoimmunity. Ann Neurol 2001; 49:
146-154.

Antoine JC, Honnorat J, Camdessanche JP, Magistris M, Absi L, Mosnier JF et al. Paraneoplastic
anti-CV2 antibodies react with peripheral nerve and are associated with a mixed axonal and
demyelinating peripheral neuropathy. Ann Neurol 2001; 49: 214-221.

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Figure 11.9. Monkey cerebellum showing staining of oligodendrocytes by CV2

antibodies (courtesy of A Church, University College London Hospitals).

Figure 11.10. High magnification of CV2 antibody staining on monkey

cerebellum.

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Glutamic Acid Decarboxylase Antibodies

Antigen: Glutamic acid decarboxylase has two isoforms, GAD65 and GAD67,
which share 68% sequence homology. The enzyme catalyses the conversion of
glutamic acid to gamma aminobutyric acid (GABA), an inhibitory
neurotransmitter. Both enzymes are expressed in the central nervous system,
pancreatic islet cells, testis, oviduct and ovary.

Clinical associations: Antibodies to GAD65 are associated with insulin

dependent diabetes mellitus (see Chapter 9 page 108) while anti-GAD67
antibodies are found in up to 60% of patients with stiff person syndrome (SPS).
SPS is a rare but severe neurological disease, characterised by progressive
skeletal muscle stiffness with painful spasms. Underlying tumours are
occasionally found in patients with the anti-GAD67 antibodies: breast tumours,
colonic tumours and small cell lung carcinoma. Notably, type 1 autoimmune
diabetes mellitus is observed in up to 60% of cases.

Detection: Anti-GAD antibodies are detected by IIF on both primate

cerebellum and pancreas. In the pancreas, GAD65 is found in the β cells and
GAD67 is found in the α cells. Immunocytochemical staining in the cerebellum
shows binding to the granular layer, the neuronal nuclei of the granular cells are
spared (Figure 11.11) and immunoreactivity is confined to peripheral GABA-
ergic terminals of the cerebellar glomeruli. Mitochondrial antibodies can give a
similar pattern (Figure 11.12), but can be reliably detected on rodent liver,
kidney stomach sections.

References

Solimena M, Folli F, Denis-Donini S, Comi GC, Pozza G, De Camilli P et al. Autoantibodies to

glutamic acid decarboxylase in a patient with stiff-man syndrome, epilepsy, and type I diabetes
mellitus. N Engl J Med 1988; 318: 1012-1020.

Rakocevic G, Raju R, Dalakas MC. Anti-Glutamic acid decarboxylase antibodies in the serum and
cerebrospinal fluid of patients with stiff person syndrome. Arch Neurol 2004; 61: 902 - 904.

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Figure 11.11. Monkey cerebellum showing staining of the granular layer by

GAD antibodies.

Figure 11.12. Antibodies against mitochondria on monkey cerebellum.

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Amphiphysin Antibodies

Antigen: Amphiphysin (128kDa) has two isoforms and exists as a dimer. It is

involved in clathrin-mediated endocytosis and is found in synaptic vesicles
where it is required for recycling of the vesicles. As well as expression in
neurons it is also found in certain endocrine cells, the retina and spermatocytes.

Clinical associations: Paraneoplastic SPS, subacute sensory neuropathy and

sensorimotor peripheral neuropathy have all been associated with anti-
amphiphysin antibodies. SCLC and breast tumours are the most common
neoplasms.

Detection: The antibodies can be detected by IIF on cerebellar cryosections

where staining of the neuropil in the molecular layer and intense granular
staining of the perikarya in the granular layer is observed (Figure 11.13). Other
paraneoplastic anti-neuronal antibodies are found in 74% of cases.
Confirmation of specificity must be determined by western blot analysis of
cerebellar extracts.

References

De Camilli P, Thomas A, Cofiell R, Folli F, Lichte B, Piccolo G, et al. The synaptic vesicle-
associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast
cancer. J Exp Med 1993; 178: 2219-2223.

Pittock SJ, Lucchinetti CF, Parisi JE, Benarroch EE, Mokri B, Stephan CL et al. Amphiphysin
autoimmunity: paraneoplastic accompaniments. Ann Neurol 2005; 58: 96-107.

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Figure 11.13. Amphiphysin antibodies staining monkey cerebellum.

Figure 11.14. High magnification of amphiphysin antibodies staining monkey

cerebellum.

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Ma Antibodies

Antigen: There are three reported Ma antigens (Ma1/Ma, Ma2/Ta and Ma3), all
were identified from cDNA expression libraries. They are expressed in the
nucleoli of neuronal cells. All three show significant homology, however, only
the Ma2 antigen has been recognised by all sera positive for anti-Ma antibodies.

Clinical associations: Approximately 78% of patients with anti-Ma2

antibodies have associated tumours of the testis and up to 40% of patients with
cerebellar ataxia and lung tumours have this antibody. Patients tend to be
young, between 22 and 45 years of age. Anti-Ma1 and Ma3 antibodies are more
common in older patients who tend to develop a wider range of cerebellar
symptoms and most have tumours other than germ cell neoplasms, including
lung (large cell), parotid, breast and colon.

Detection: These antibodies are recognised by IIF on fixed cerebellar

cryosections and will show binding to the neuronal nucleoli (Figure 11.15).
Although specificity should be determined by western blots, the antibodies do
show differential binding to non-neuronal tissue. Anti-Ma1 antibodies will bind
to testicular germ cells, anti-Ma2 are specific to the central nervous system and
Ma3 is expressed in several systemic tissues, including testis, trachea and
kidney.

References

Ahern GL, O'Connor M, Dalmau J, Coleman A, Posner JB, Schomer DL et al. Paraneoplastic
temporal lobe epilepsy with testicular neoplasm and atypical amnesia. Neurology 1994; 44: 1270-
1274.

Dalmau J, Gultekin SH, Voltz R, Hoard R, DesChamps T, Balmaceda C et al. Ma1, a novel neuron-
and testis-specific protein, is recognized by the serum of patients with paraneoplastic neurological
disorders. Brain 1999; 122: 27-39.

Rosenfeld MR, Eichen JG, Wade DF, Posner JB, Dalmau J. Molecular and clinical diversity in
paraneoplastic immunity to Ma proteins. Ann Neurol 2001; 50: 339-348.

Leyhe T, Schüle R, Schwärzler F, Gasser T, Haarmeier T. Second primary tumor in anti-Ma1/2-

positive paraneoplastic limbic encephalitis. J Neurooncol 2006; 78: 49-51.

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Figure 11.15. Monkey cerebellum showing binding of neuronal nuclei by Ma

antibodies (left) and Ma1 antibodies staining monkey testis (right).

Metabotropic Glutamate Neurotransmitter Receptor Antibodies

Antigen: The metabotropic glutamate neurotransmitter receptors (mGluR1-5)

are understood to modulate excitatory synaptic transmission in the central
nervous system and to be involved in neural plasticity and cerebellar motor
learning. The antigen target is mGluR1 (MW ~140kDa) and binding of the
antibodies to the receptors can block their function.

Clinical associations: Anti-mGluR1 antibodies have been reported in patients

with Hodgkin’s disease and cerebellar ataxia. The symptoms of cerebellar
ataxia often occur during remission and so can follow the discovery of the
lymphoma by months or years. This specificity of antibody is very rare,
however, in two reported cases the neurological outcome appeared to be better
than in patients with other paraneoplastic neurological antibody specificities.

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Detection: The antibodies (IgG) are currently considered partially characterised

and are reported to bind Purkinje cell bodies and their dendritic spines which
immunocytochemically will be observed as punctuate staining on the molecular
layer of the cerebellum.

References

Sillevis Smitt P, Kinoshita A, De Leeuw B, Moll W, Coesmans M, Jaarsma D et al. Paraneoplastic

cerebellar ataxia due to autoantibodies against a glutamate receptor. N Engl J Med 2000; 342: 21-
27.

Shams'ili S, Grefkens J, de Leeuw B, van den Bent M, Hooijkaas H, van der Holt B et al.
Paraneoplastic cerebellar degeneration associated with antineuronal antibodies: analysis of 50
patients. Brain 2003; 126: 1409-1418.

Zic4 Antibodies

Antigen: Zic4 is one of the five identified zinc finger proteins of the cerebellum
(Zic1-5). The proteins are considered to work in co-operation with one another
during cerebellar development. The target antigen, Zic4, has a reported
molecular weight of 37kDa and is considered to be partially characterised as a
paraneoplastic neurological antigen.

Clinical associations: Eighty percent of reported patients with anti-Zic4

antibodies have paraneoplastic neurological degeneration. In over 90% of cases
the associated tumour is small cell lung carcinoma (SCLC). In the majority of
cases (80%) anti-Zic4 antibodies co-exist with other paraneoplastic
neurological antibodies. When the antibodies are found in isolation they are
predominantly associated with cerebellar syndromes. Up to 16% of patients
with SCLC may have anti-Zic4 antibodies.

Detection: The antibodies can be found in both the serum and cerebrospinal
fluid of patients and immunocytochemically they are reported to bind the
neuronal nuclei of the cerebellum. The co-existing paraneoplastic neurological
antibodies are most frequently Hu and CV2 and their presence may add
difficulties to the identification of Zic4 antibodies.

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Reference

Bataller L, Wade DF, Graus F, Stacey HD, Rosenfeld MR, Dalmau J. Antibodies to Zic4 in
paraneoplastic neurologic disorders and small-cell lung cancer. Neurology 2004; 62: 778-782.

Anti-Glial Nuclear Antibodies

Antigen: Although an immunocytochemical staining pattern has been

described for these antibodies no antigen has been isolated or characterised.

Clinical associations: This is a recently described specificity. In one report of

24 cases, it was shown to be associated with Lambert Eaton myasthenic
syndrome (LEMS) and the related tumour was SCLC.

Detection: To date, staining has only been described in the rat cerebellum
where staining of the Bergmann glia in the Purkinje cell layer was observed.
Staining of isolated glia in the white matter was also reported.

Reference

Graus F, Vincent A, Pozo-Rosich P, Sabater L, Saiz A, Lang B et al. Anti-glial nuclear antibody:
marker of lung cancer-related paraneoplastic neurological syndromes. J Neuroimmunol 2005; 165:
166-171.

Myelin Associated Glycoprotein Antibodies

Antigen: The antigenic target of myelin associated glycoprotein (MAG)

antibodies is reported to be against the oligosaccharide epitope on the
glycoprotein. Two glycolipids also have the oligosaccharide epitope: sulphated
3-glucuronyl lactosaminyl paragloboside and sulphated 3-glucuronyl
paragloboside. MAG is involved in neurofilament spacing and is expressed in
the peripheral nerve myelin at the periaxonal and paranodal regions as well as
the Schmidt-Lantermann incisures. Expression of MAG in the central nervous
system myelin is considered to be lower.

Clinical associations: Patients with anti-MAG antibodies (IgM) can present

with a slow but progressive sensory neuropathy due to demyelination of the
peripheral nerves. This demyelination is likely to be directly mediated by the

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antibodies. Benign monoclonal paraproteins (and occasionally paraproteins

associated with other diseases including Waldenstrom’s macroglobulinaemia)
may be associated on rare occasions with polyneuropathy and anti-MAG
antibodies.
Approximately 5% of patients with multiple myeloma have associated
axonal degeneration of peripheral nerves, particularly in association with
osteosclerotic bone lesions (3% of cases). In these cases, the paraprotein is
usually of IgG lambda or IgA lambda type and there is no specificity for the
MAG antigen. The acronym POEMS is used for the full syndrome of
polyneuritis, organomegaly, endocrinopathy, monoclonal gammopathy and skin
changes.

Detection: The antibodies are usually IgM kappa, can be readily identified on
cryosections of peripheral nerve and are frequently of high titre. Typically the
antibodies stain the periaxonal and outer myelin membranes (Figure 11.16),
also the Schmidt-Lanterman incisures are frequently observed. Further features
can include staining of the compact myelin in addition to the typical staining of
non-compact myelin. The antibodies can also be detected by EIAs and western
blots.

References

Latov N, Braun PE, Gross RB, Sherman WH, Penn AS, Chess L. Plasma cell dyscrasia and
peripheral neuropathy: identification of the myelin antigens that react with human paraproteins.
Proc Natl Acad Sci USA 1981; 78: 7139-7142.

Miralles GD, O'Fallon JR, Talley NJ. Plasma-cell dyscrasia with polyneuropathy. The spectrum of
POEMS syndrome. N Engl J Med 1992; 327: 1919-1923.

Lopate G, Kornberg AJ, Yue J, Choksi R, Pestronk A. Anti-myelin associated glycoprotein

antibodies: variability in patterns of IgM binding to peripheral nerve. J Neurol Sci 2001; 188: 67-
72.

Renaud S, Steck A, Latov N. Neuropathies associated with monoclonal gammopathy. In: Clinical
Neuroimmunology. Second Edition. Editors Antel J, Birnbaum G, Hartung HP, Vincent A. Oxford
University Press Inc., New York; 2006.

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Figure 11.16. Anti-MAG antibodies binding monkey sciatic nerve at low

power.

Figure 11.17. Anti-MAG antibodies staining monkey optic nerve at high power
showing the characteristic inner and outer myelin staining of axons.

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Aquaporin-4 Antibodies (NMO-IgG)

Antigen: Aquaporin-4 is the dominant water channel protein in the CNS. This
transmembrane protein is concentrated in astrocytic foot processes at the blood-
brain barrier, and partly co-localises with lamin. The protein is also found in
the distal collecting tubules of the kidney medulla and parietal cells of the deep
gastric mucosa.

Clinical associations: Antibodies against aquaporin-4 are specific serum

markers for neuromyelitis optica (NMO); their detection forms part of the
diagnostic criteria for this condition. NMO, also known as Devic’s syndrome,
is a relapsing demyelinating condition and its distinction from multiple sclerosis
(MS) can be challenging. It has a female preponderance and its prevalence is
reported to be higher amongst Black, Asian and Indian populations (>10% of
demyelinating conditions). NMO has a worse prognosis than MS, death is
commonly due to respiratory failure and antibody presence indicates a poor
visual prognosis. Treatment includes immunosuppression and plasma
exchange, this indicates a pathogenic role of the antibodies as does the
relationship of disease severity and antibody titre.

Detection: Determination of antibodies against aquaporin-4 allows early

discrimination between NMO and MS. The antibodies (IgG) can be detected by
IIF on cerebellar, midbrain and spinal cord tissue. Alternatively, transfected
human embryonic kidney (HEK) cells have been used. Mouse aquaporin-4 has
only 95% amino-acid homology with the human protein and so monkey tissue
may be preferable. The IF pattern shows staining of the pia, subpia and
Virchow-Robin spaces, particularly distinctive around microvessels of the CNS
(Figure 11.18 and 11.19). Sensitivity for NMO is reported to be between 73 and
91%; this will depend both on species choice of the tissue substrate and assay
type.

References

Lennon VA, Wingerchuk DM, Kryzer TJ, Pittock SJ, Lucchinetti CF, Fujihara K et al. A serum
autoantibody marker of neuromyelitis optica: distinction from multiple sclerosis. Lancet 2004; 364:
2106-2112.

Lennon VA, Kryzer TJ, Pittock SJ, Verkman AS, Hinson SR. IgG marker of optic-spinal multiple
sclerosis binds to the aquaporin-4 water channel. J Exp Med 2005; 202: 473-477.

Lana-Peixoto MA. Devic's neuromyelitis optica: a critical review. Arq Neuropsiquiatr 2008; 66:
120-138.

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Figure 11.18. Anti-aquaporin-4 antibodies showing linear staining of the pia

and subpia in the molecular layer of monkey cerebellum. Distinct staining of
the granular layer can also be observed (courtesy of T Vincent, Centre
Hospitalier Universitaire, Montpellier).

Figure 11.19. A higher magnification of Figure 11.18 illustrating distinct linear

staining of the pial membranes between two folds of the molecular layer.

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Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular

junction which presents as fatigue and muscle weakness due to impaired
neuromuscular transmission. Occurrence of mysasthenia gravis is estimated at
8.3 cases per million. It can affect all age groups with a female to male ratio of
3:2. Incidence in women peaks in the 20’s and 30’s and for men in their 50’s
and 60’s. Between 10 and 13% of patients with MG have an associated
thymoma. Autoantibody targets in MG include the acetyl choline receptor
(AChR), striated muscle, titin, the ryanodine receptor (RyR), a muscle specific
kinase (MuSK) and two cytokines IFN-α and IL12.

Figure 11.20. Diagram illustrating the molecular structure of skeletal muscle.

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Acetyl Choline Receptor Antibodies

Antigen: The antigen is the nicotinic acetylcholine receptor (AChR), a

pentameric membrane protein of ~280kDa in size.

Clinical associations: Autoantibodies against the AChR are found in up to 85%

of patients with MG (sero-positive myasthenia gravis). The antibodies are not
detected in healthy individuals or in patients with other neurological disorders,
specificity is therefore very high. The concentration of antibodies is not related
to the severity of symptoms, possibly due to variations in target epitopes and
strength of binding.

Detection: Anti-AChR antibodies are traditionally detected by

immunoprecipitation although several EIA assays are available.

References

Lindstrom JM, Seybold ME, Lennon VA, Whittingham S, Duane DD. Antibody to acetylcholine
receptor in myasthenia gravis. Prevalence, clinical correlates, and diagnostic value. Neurology
1976; 26: 1054-1059.

Vincent A. Antibody-mediated disorders of the neuromuscular junction. In: Clinical

Neuroimmunology. Second Edition. Editors Antel J, Birnbaum G, Hartung HP, Vincent A. Oxford
University Press Inc., New York; 2006.

Striational Muscle Antibodies

Antigen: The antibodies (IgG) react against contractile elements of the

striational muscle: recognised antigens include actin, myosin and titin. Anti-
titin antibodies are the most extensively investigated; titin is a giant structural
cytoplasmic protein of 3000kDa. It extends the length of the sarcomere, from
the M line to the Z line (Figure 11.20). A smaller fragment, myasthenia gravis
titin-30 (MGT-30) contains the major immunogenic region. The ryanodine
receptor (RyR), a calcium release channel found in the sarcoplasmic reticulum,
is another recognised striational muscle autoantigen.

Clinical associations: Striational muscle antibodies are diagnostically

important antibodies found in association with myasthenia gravis. Traditionally
these have been reported to have a strong association with MG patients who
have a thymoma. Subsequent investigations showed this correlation to be only
significant if late onset MG is excluded (Table 11.2) and their absence virtually

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excludes a thymoma in a young person. The association with thymoma is

possibly due to thymic abnormalities inducing antibody formation against local
striated muscle fibres which then bind to major locomotor muscles. A recent
investigation has shown that at presentation in patients with thymoma and MG,
over 70% have antibodies against IFNα and over 50% have antibodies against
IL-12.
Occurence
MG with thymoma 90 %
MG without thymoma >60 yrs 55%
MG without thymoma <40 yrs 6%
MG without thymoma <20 yrs Rare

Table 11.2. Occurence of MG with and without thymoma.

Detection: Anti-striational muscle antibodies are best detected by IIF using

cryosections of rodent, human or monkey skeletal muscle (Figure 11.21). The
antibodies can also be observed on cardiac muscle. EIA and western blot
techniques on muscle extracts can also be used.

Figure 11.21. Monkey skeletal muscle showing anti-striational muscle

antibody staining.

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References

Buckley C, Newsom-Davis J, Willcox N, Vincent A. Do titin and cytokine antibodies in MG

patients predict thymoma or thymoma recurrence? Neurology 2001; 57: 1579-1582.

Romi F, Skeie GO, Gilhus NE, Aarli JA. Striational antibodies in myasthenia gravis: reactivity and
possible clinical significance. Arch Neurol 2005; 62: 442-446.

Muscle Specific Receptor Tyrosine Kinase Antibodies

Antigen: The antibody recognises extracellular determinants of muscle-

specific receptor tyrosine kinase (MuSK), which mediates agrin-induced
clustering of the AChRs during synapse formation. Agrin is a protein produced
by nervous tissue and is essential for neuromuscular synapse formation.

Clinical associations: Up to 70% of MG “seronegative” patients (MG without

anti-AChR antibodies) are reported to have IgG antibodies against the MuSK.
The male to female ratio for anti-MuSK antibodies is 1:3. 10-20% of MG
patients are “AChR antibody seronegative” although children with MG are
more frequently “AChR antibody seronegative”. The clinical picture can be
further complicated on the rare occasions when anti-AChR antibodies are found
in conjunction with anti-MuSK antibodies as the antibodies may have opposing
effects.

Detection: The antibodies can be detected by several methods including EIA

techniques.

References

Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A. Auto-antibodies to the

receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor
antibodies. Nat Med 2001; 7: 365-368.

Diaz-Manera J, Rojas-Garcia R, Gallardo E, Juarez C, Martinez-Domeno A, Martinez-Ramirez S et

al. Antibodies to AChR, MuSK and VGKC in a patient with myasthenia gravis and Morvan's
syndrome. Nat Clin Pract Neurol 2007; 3: 405-410.

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Voltage Gated Calcium Channel Antibodies

Antigen: Voltage gated calcium channels (VGCC) consist of multiple

transmembrane protein subunits; the α subunit contains the Ca2+ channel. The
channels are found at the motor nerve terminal and control the release of ACh.
There are several types of VGCCs, P/Q, N and L types, which are characterised
by their binding of neurotoxins. The most commonly targeted is the P/Q type
VGCC (>85%), although 30-50% of patients with Lambert Eaton myasthenic
syndrome (LEMS) have antibodies which recognise the N type VGCCs.
Antibodies against the L type VGCC are rare in LEMS patients.

Clinical associations: LEMS is a disorder of neuromuscular transmission due

to a decrease in the presynaptic release of ACh. The majority of LEMS patients
have antibodies against VGCCs. These antibodies have been shown to impair
the function of the presynaptic nerve terminals and they are implicated in the
pathology of the syndrome; patients have been shown to respond to
plasmaphoresis. About 60% of LEMS patients with anti-VGCC antibodies
have SCLCs, these tumours are much less common in LEMS patients without
the anti-VGCC antibodies. Other symptoms such as cerebellar dysfunction
may mask those of LEMS and detection of the antibodies may therefore be
useful in the identification of the syndrome.

Detection: Screening for antibodies against the P/Q type VGCCs is performed
by radioimmunoassays.

References

Lennon VA, Kryzer TJ, Griesmann GE, O'Suilleabhain PE, Windebank AJ, Woppmann A et al.
Calcium-channel antibodies in the Lambert-Eaton syndrome and other paraneoplastic syndromes.
N Engl J Med 1995; 332: 1467-1474.

Lang B, Vincent A. Autoimmunity to ion-channels and other proteins in paraneoplastic disorders.

Curr Opin Immunol 1996; 8: 865-871.

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Voltage Gated Potassium Channel Antibodies

Antigen: The antigen, voltage gated potassium channel (VGKC), consists of

four transmembrane α subunits that interact with the intracellular β subunits.
The VGKCs are located in paranodal and terminal regions of myelinated axons
where their role is to stabilise the membrane potential and hence regulate nerve
excitability.

Clinical associations: IgG antibodies against VGKCs have been reported in a

number of neurological conditions. They were originally associated with Isaac’s
syndrome; patients with Isaac’s syndrome have neuromyotonia which presents
as hyperexcitable motor neurones. Other symptoms include muscle stiffness,
muscle twitches and a general weakness of the muscles. The antibodies are also
associated with Morvans’s syndrome, which like Isaac’s syndrome, presents
with neuromyotonia but includes an involvement with the central nervous
system. It is proposed that anti-VGKC antibodies inhibit ion channel activity
and this reduction of available VGKCs reduces nerve repolarisation, resulting
in prolonged neurotransmitter release. The antibody levels do not show a
correlation with the severity of symptoms, possibly due to variations of
antibody specificities for particular epitopes. Thymomas have been associated
in a number of cases as have SCLCs, especially where there is CNS
involvement.

Detection: Antibody screening of patients with neuromuscular

hyperexcitability should include VGKC antibodies as well as those against the
AChR and skeletal muscle. Immunohistochemistry does on occasions allow the
detection of the antibodies by staining hippocampal neurons, however, the more
common procedure is a radioimmunoprecipitation assay.

References

Sinha S, Newsom-Davis J, Mills K, Byrne N, Lang B, Vincent A. Autoimmune aetiology for

acquired neuromyotonia (Isaacs' syndrome). Lancet 1991; 338: 75-77.

Vernino S, Lennon VA. Ion channel and striational antibodies define a continuum of autoimmune
neuromuscular hyperexcitability. Muscle Nerve 2002; 26: 702-707.

Pozo-Rosich P, Clover L, Saiz A, Vincent A, Graus F. Voltage-gated potassium channel antibodies

in limbic encephalitis. Ann Neurol 2003; 54: 530-533.

Tan KM, Lennon VA, Klein CJ, Boeve BF, Pittock SJ. Clinical spectrum of voltage-gated potassium
channel autoimmunity. Neurology 2008; 70: 1883-1890.

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Myocardial Antibodies

Antigen: A multitude of autoantibody specificities have been described in sera

from patients with idiopathic dilated cardiomyopathy (IDCM) and other types
of carditis. These include antibodies against structural antigens (myosin, actin,
laminin, tropomyosin, vimentin, desmin and tubulin). Of these, the α chain of
cardiac myosin is the most extensively studied. Proposed non-structural
antigens include heat shock protein 60 (HSP60) and to a greater extent, the
adenine nucleotide translocator (ANT) found in the inner mitochondrial
membrane. However, the most recent and compelling evidence is for antibodies
against a region of the second extracellular loop of the β-1-adrenergic receptor,
a transmembrane receptor found almost exclusively in cardiac myocytes.

Clinical associations: The clinical utility of many of these autoantibodies has

been questioned as they have been reported in normal subjects as well as other
disease groups. This is particularly the case for many of the non-organ specific
antibody specificities. However, there is significant data indicating an
association of the more organ-specific specificities with IDCM. IDCM presents
as left ventricular enlargement which can result in congestive heart failure,
systemic pulmonary embolisms, arrhythmias and cardiomegaly. The anti-β-1-
adrenergic receptor antibodies have also been reported in asymptomatic patients
and may be useful to identify patients who have an autoimmune association
with their disease. Anti-myocardial antibodies of high titre (>1/160) have also
been reported in postpartum cardiomyopathy and adriamycin-induced
cardiomyopathy. Lower titre antibodies may be found in hypertrophic
cardiomyopathy and alcoholic cardiomyopathy.

Detection: Human or monkey tissue is commonly used for the demonstration

of anti-cardiac muscle antibodies by IIF, a titre of >1/40 is considered
significant (Figure 11.22). Monkey tissue may be preferred due to reduced
background staining and care should particularly be taken when using rat
myocardium as heterophile antibody staining can occur. Positive samples can
be tested on skeletal muscle which could identify cross-reacting striational
antibodies, although the relationship between the two is unclear. The two
predominantly reported patterns are described below:
Anti-fibrillary antibodies bind to cytoplasmic contractile antigens, for example,
this will show the A-bands in the case of anti-myosin antibodies.
Anti-sarcolemma antibodies bind to the myocyte sarcolemma sheath and show
as an outer membrane fluorescence on transverse sections (Figure 11.23).
More specific assays, such as EIAs and western blots, can be used to confirm

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suspected specificities. EIAs are more sensitive and so may pick up more
positive samples, in the case of anti-ANT antibodies, up to six times as many
samples may be identified. A very specific EIA used to detect anti-β-1-
adrenergic receptor antibodies utilises a synthetic peptide that corresponds to
the second extracellular loop of this receptor.

Figure 11.22. Monkey cardiac muscle showing anti-fibrillary antibody pattern.

Figure 11.23. Anti-sarcolemma antibody binding monkey cardiac muscle.

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References

Maisch B, Deeg P, Liebau G, Kochsiek K. Diagnostic relevance of humoral and cytotoxic immune
reactions in primary and secondary dilated cardiomyopathy. Am J Cardiol 1983; 52:1072-1078.

Limas CJ, Goldenberg IF, Limas C. Autoantibodies against beta-adrenoceptors in human idiopathic
dilated cardiomyopathy. Circ Res 1989; 64: 97-103.

Caforio AL, Mahon NJ, Tona F, McKenna WJ. Circulating cardiac autoantibodies in dilated
cardiomyopathy and myocarditis: pathogenetic and clinical significance. Eur J Heart Fail 2002; 4:
411-417.

Jahns R, Boivin V, Hein L, Triebel S, Angermann CE, Ertl G et al. Direct evidence for a beta 1-
adrenergic receptor-directed autoimmune attack as a cause of idiopathic dilated cardiomyopathy. J
Clin Invest 2004; 113: 1419-1429.

Caforio AL, Tona F, Bottaro S, Vinci A, Dequal G, Daliento L et al. Clinical implications of anti-
heart autoantibodies in myocarditis and dilated cardiomyopathy. Autoimmunity 2008; 41 :35-45.

General References

Brain WR, Daniel PM, Greenfield JG. Subacute cortical cerebellar degeneration and its relation to
carcinoma. J Neurol Neurosurg Psychiatry 1951; 14: 59-75.

Graus F, Cordon-Cardo C, Posner JB. Neuronal antinuclear antibody in sensory neuronopathy from
lung cancer. Neurology 1985; 35: 538-543.

Moll JW, Antoine JC, Brashear HR, Delattre J, Drlicek M, Dropcho EJ et al. Guidelines on the
detection of paraneoplastic anti-neuronal-specific antibodies: report from the Workshop to the
Fourth Meeting of the International Society of Neuro-Immunology on paraneoplastic neurological
disease, held October 22-23, 1994, in Rotterdam, The Netherlands. Neurology 1995; 45: 1937-1941.

Moore KL, Dalley AF, editors. Clinically Oriented Anatomy. 4th Edition. Lippincott Williams and
Wilkins; 1999.

Graus F, Delattre JY, Antoine JC, Dalmau J, Giometto B, Grisold W et al. Recommended diagnostic
criteria for paraneoplastic neurological syndromes. J Neurol Neurosurg Psychiatry 2004; 75: 1135-
1140.

Karim AR, Hughes RG, Winer JB, Williams AC, Bradwell AR. Paraneoplastic neurological
antibodies: a laboratory experience. Ann N Y Acad Sci 2005; 1050: 274-285.

Antel J, Birnbaum G, Hartung HP, Vincent A, editors. Clinical Neuroimmunology. 2nd Edition.
Oxford University Press; 2006.

Karim AR, Hughes RG, El Lahawi M, Bradwell AR. Paraneoplastic neurological antibodies;
Chapters 77, 78 and 79. In: Autoantibodies. 2nd Edition. Editors Shoenfeld Y, Gershwin ME,
Meroni PL. Elsevier Science; 2007.

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 Anti-Phospholipid Syndrome

Chapter 12

Anti-Phospholipid Syndrome

In 1983 Harris et al. in the laboratory of Hughes identified a group of patients

with SLE in whom positivity for anti-cardiolipin antibodies was associated with
an increased risk of thrombosis (Hughes syndrome). Re-named as anti-
phospholipid syndrome (APS) and now defined as an autoimmune disorder
characterised by recurrent vascular thrombosis or pregnancy loss. APS is often
divided into two types; primary APS where the disease occurs alone or
secondary APS where it is found alongside other autoimmune diseases,
frequently SLE. However, the clinical and laboratory features of primary and
secondary APS do not differ. A minority of patients may be diagnosed as having
catastrophic APS, an acute and devastating syndrome characterised by
simultaneous vascular occlusions in at least three organ systems over a period
of days or weeks; this syndrome has a mortality rate of approximately 50%.

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Classification Criteria

A patient cannot be classified as suffering from APS by the clinical

symptoms alone, it is also dependent on laboratory criteria; at least one clinical
and one laboratory criterion must be satisfied. These are the Sapporo criteria,
described in 1999 following the Eighth International Symposium of
Antiphospholipid Antibodies. The Sapporo criteria were reviewed and updated
in 2005, resulting in the international consensus statement on the classification
criteria for definite anti-phospholipid syndrome (Miyakis et al. 2006). These
guidelines continue to be debated and may be subject to further change.

1. Vascular Thrombosis
Clinical Criteria

− One or more clinical episodes of arterial, venous, or small vessel

thrombosis occurring within any tissue or organ.
2. Complications of Pregnancy
− One or more unexplained deaths of morphologically normal foetuses at
or beyond the 10th week of gestation or
− One or more premature births of morphologically normal neonates at or
before the 34th week of gestation or
– Three or more unexplained, consecutive, spontaneous abortions before
the 10th week of gestation.

1. Lupus Anticoagulant
Laboratory Criteria

– Lupus anticoagulant present in the plasma on two or more occasions at

least twelve weeks apart, detected according to the guidelines of the
International Society on Thrombosis and Haemostasis.
2. Anti-cardiolipin antibodies
– Anti-cardiolipin IgG or IgM antibodies in serum or plasma, present on
two or more occasions at a moderate or high titre (>40GPL or MPL or
>99th percentile) at least twelve weeks apart, measured by a standardised
EIA.
3. Anti-β2 Glycoprotein I Antibodies
– Anti-β2-glycoprotein I IgG or IgM antibodies in serum or plasma,
present on two or more occasions at least twelve weeks apart, measured
by a standardised EIA (>99th percentile).

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References

Harris EN, Gharavi AE, Patel SP, Hughes GV. Evaluation of the anti-cardiolipin test: Report of a
standardized workshop held 4 April 1986. Clin Exp Immunol 1987; 68: 215-222.

Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: An
update. Thromb Haemost 1995; 74: 1185-1190.

Wilson WA, Gharavi AE, Koike T, Lockshin MD, Branch DW, Piette JC et al. International
consensus statement on preliminary classification criteria for definite antiphospholipid syndrome.
Arthritis Rheum 1999; 42: 1309-1311.

Levine JS, Branch DW, Rauch J. The Antiphospholipid Syndrome. N Engl J Med 2002; 346: 752-
763.

Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R et al. International consensus
statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).
J Thromb Haemost 2006; 4: 295-306.

Galli M, Reber G, de Moerloose P, de Groot PG. Invitation to a debate on the serological criteria that
define the antiphospholipid syndrome. J Thromb Haemost 2008; 6: 399-401.

Lupus Anticoagulants

Lupus anticoagulants (LAs) are immunoglobulins which prolong

phospholipid-dependent coagulation tests (Table 12.1). The term ‘lupus
anticoagulant’ itself is contradictory as well as a misnomer; LAs are frequently
related to thrombotic events in vivo and at least 50% of patients with LAs do
not have SLE.
The presence of LAs is strongly predictive of clinical thromboembolic
events and they have a 95% sensitivity and ~100% specificity for APS. The
identification of LAs requires the evaluation of different components of the
coagulation system (Figure 12.1) and so more than one screening test must be
carried out.
Intrinsic Pathway Extrinsic Pathway Final Common Pathway

Activated partial Dilute prothrombin time Dilute Russell viper venom

thromboplastin time (APTT) (dPT) time (dRVVT)

Kaolin clotting time (KCT) Textarin time

Colloidal silica clotting time Taipan snake venom time

Table 12.1. Screening assays for lupus anticoagulants, details of such assays can
be found elsewhere (e.g. Brandt et al. 1995).

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Figure 12.1. The Clotting Pathway (yellow stars indicate the phospholipid
dependent steps).

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 Anti-Phospholipid Syndrome

The recommended guidelines for the detection of LA were established by the

subcommittee on lupus anticoagulants/phospholipid-dependent antibodies of
the scientific and standardisation committee of the International Society of
Thrombosis and Haemostasis (Brandt et al. 1995). In order to establish that
there is no other coagulation deficiency present, a sample should show each of
the following:
1. Prolongation of at least one phospholipid-dependent clotting test.
2. Evidence of inhibitory activity shown by the effect of patient plasma on
pooled normal plasma.
3. Evidence that the inhibitory activity is dependent on phospholipid. This
can be inferred if clotting time is shortened after the addition of
exogenous phospholipids. Also, LAs must be carefully distinguished
from other coagulopathies that may give similar laboratory results or may
occur concurrently with LAs.

Reference

Brandt JT, Triplett DA, Alving B, Scharrer I. Criteria for the diagnosis of lupus anticoagulants: An
update on behalf of the subcommittee on lupus anticoagulants/antiphospholipid antibody of the
scientific and standardisation committee of the ISTH. Thromb Haemost 1995; 74: 1185-1190.

Cardiolipin and β2-Glycoprotein I Antibodies

Anti-phospholipid antibodies (APA) were originally identified due to a high

incidence of false positive results in the Wassermann reaction, a serological test
for syphilis. This assay, developed more than 100 years ago, utilises a tissue
extract containing cardiolipin and phosphatidylcholine. The false positive
results correlated with the development of SLE. In 1983, Harris et al. (working
in the laboratory of Professor Graham Hughes) developed a radioimmunoassay
for the detection of anti-cardiolipin antibodies. They found a strong correlation
between high titre anti-cardiolipin antibodies and the occurrence of thrombosis
in SLE. Subsequently, in 1985 such patients were classified as having anti-
cardiolipin syndrome and in 1987 the condition was renamed more
appropriately anti-phospholipid syndrome (APS). APA have also been detected
in relatives of APS/SLE patients, suggesting that there is some genetic
predisposition to developing the syndrome. McNeil et al. (1990) identified that
the binding of APA to cardiolipin requires the presence of a protein co-factor,
β2-glycoprotein I (β2GPI). The cofactor is not a requirement for anti-
cardiolipin antibodies produced in response to infection (e.g. syphilis) which
bind directly to cardiolipin. Other anionic phospholipids in the presence of

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β2GPI can also be used to detect APA and several other autoantibody protein
targets have been associated with APS, including prothrombin and annexin V.

Cardiolipin Antibodies

Antigen: Anti-cardiolipin antibodies (ACA) associated with APS will only be

detected when the cofactor β2GPI is present in the assay. This is because the
antibodies are in fact directed against epitopes on the cofactor. However,
binding of antibodies to these epitopes is partially dependent on the cofactor
being presented on the phospholipid layer. Cardiolipin (diphosphatidylglycerol)
is an abundant phospholipid found in bacterial and inner mitochondrial
membranes and accounts for 10% of the phospholipids in bovine heart muscle,
which is an ideal source of antigen for immunoassays.

Figure 12.2a) Schematic representation of the structure of cardiolipin (R =

aliphatic moiety of the fatty acid). b) Schematic representation of
phospholipids (phosphatidylserine Y = serine, phosphatidylinositol Y =
inositol, phosphatidic acid Y = hydrogen, phosphatidylglycerol Y = glycerol,
phosphatidylethanolamine Y = ethanolamine and phosphatidylcholine Y =
choline).
Clinical associations: ACA are detected in APS (80-90%), SLE (12-30%), first
degree relatives of SLE/APS patients, infectious diseases (e.g. hepatitis and
malaria), young healthy controls (1-5%) and other autoimmune diseases. In
infectious disease ACA are usually transient and at low titre. Patients positive

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for APA, although not diagnosed with APS, especially those with SLE, should
be monitored quarterly or yearly as 50-70% may develop APS within 20 years.
IgG and IgM ACA at high titre (>40 GPL or MPL) is reported to be associated
with thrombosis and foetal loss. IgG antibodies are more prevalent than IgM
antibodies, although occasionally isolated IgM or IgA antibodies may be
detected. ACA titre is reported to decrease before a thrombotic episode,
possibly due to the presence and subsequent binding to their respective
antigens. Care should be taken with the interpretation of IgM results, this may
in part be due to a risk of interference from rheumatoid factor. The significance
of IgA ACA is debated; consequently they are not included in the diagnostic
criteria for APS. However, they are reported to be the dominant isotype for APS
in certain ethnic populations such as Afro-Caribbeans and Afro-Americans.
Running the β2GPI assay in conjunction can help to compensate for the lower
specificity of the ACA assay.

Detection: ACA are routinely detected by EIA. Special attention should be

given to the type of plate, source of β2GPI, source and purity of cardiolipin and
buffers used as these can all affect the sensitivity of the assay. To ensure
consistency and interlaboratory agreement antibody titre is reported in GPL
(IgG)/MPL (IgM) units, where one unit is equivalent to 1µg/ml of affinity
purified anti-cardiolipin antibody. IgG and/or IgM ACA at titres of >40 GPL or
MPL when present on at least two occasions at least 12 weeks apart are
recognised as part of the classification criteria for APS. Recognised standards
include the Louisville APA (the reference preparation from which MPL and
GPL are defined), UK reference preparation (97/656) and Sapporo monoclonal
antibodies (HCAL for IgG and EY2C9 for IgM). Despite great efforts in
standardisation significant variation still exists between these assays. For
example, in the October 2006 UK NEQAS scheme a sample moderate to strong
positive for anti-cardiolipin IgG antibodies was incorrectly identified by 4% of
participants as negative and by 16% of participants as weakly positive.

β2-Glycoprotein I Antibodies

Antigen: β2GPI (apolipoprotein H) is 326 amino acids in length, with a

predicted molecular weight of 36.3kDa and is glycosylated at four sites which
result in a final MW of ~50kDa. It is considered to be a natural inhibitor of
coagulation. The protein consists of five domains of around 60 amino acids
each and is described as orientated in a rigid ‘fish hook’ shape. Domain V has
a concentrated region of positively charged amino acids which interact with
anionic phospholipids; within this region is a hydrophobic group which attaches

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Figure 12.3. Schematic representation of β2GPI antigen structure, see text for
details.

the protein to the phospholipid bilayer (Figure 12.3). Domains III and IV are
considered linking domains and have three and one glycosylation sites
respectively. Domains I and II are presented furthest from the lipid bi-layer and
the majority of autoantibodies against β2GPI are reported to be targeted to
specific regions of domain I. The epitopes presented by amino acids (40-43) are
cited as especially dominant.

Clinical associations: Anti-β2GPI antibodies are more sensitive than ACA for
diagnosis of APS, being detected in 98% of patients, 3-10% of patients sera are
only anti-β2GPI antibody positive. The antibodies are also detected in SLE (11-
39%) and infectious diseases. It is recommended that SLE patients positive for
APA be monitored quarterly or annually as 50-70% may develop APS within 20
years. Antibody titre has been reported to correlate with symptoms of APS. As
with ACA assay care should be taken with the interpretation of IgM results as
false positive results are known to occur. The significance of IgA anti-β2GPI
antibodies is debated; consequently they are not included in the diagnostic
criteria for APS. However, they are reported to be, as for ACA, the dominant

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isotype for APS in certain ethnic populations such as Afro-Caribbeans and Afro-
Americans.

Detection: Anti-β2GPI antibodies are routinely detected by EIA. Special care

should be given to the selection of plates, buffers and source of β2GPI to be
used. Some APA will only bind to β2GPI after it has bound phospholipid and
therefore undergone a conformational change, whereas some APA will bind to
β2GPI in isolation in its native conformation. Therefore it is essential to test for
APA using both anti-cardiolipin (where β2GPI is the essential cofactor) and
anti-β2GPI (in isolation) assays. IgG antibodies are more prevalent than IgM
antibodies; occasionally isolated IgM or IgA antibodies may be found. Other
assays which combine β2GPI with other phospholipids e.g. phosphatidylserine
are also available. Currently there are no international standards for anti-β2GPI
assays although the European Forum on Antiphospholipid Antibodies
recommend the use of the monoclonal preparations of HCAL and EY2C9. Anti-
β2GPI antibodies have also been detected on endothelial cells (Figure 12.4),
however, this is a not routine procedure due to lack of specificity.

Figure 12.4. Endothelial cell staining of an umbilical vein by antibodies against

β2GPI.

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References

Harris EN, Gharavi AE, Boey ML, Patel BM, Mackworth-Young CG, Loizou S et al.
Anticardiolipin antibodies: detection by radioimmunoassay and association with thrombosis in
systemic lupus erythematosus. Lancet 1983; 2: 1211-1214.

McNeil HP, Simpson RJ, Chesterman CN, Krilis SA. Anti-phospholipid antibodies are directed
against a complex antigen that includes a lipid-binding inhibitor of coagulation: beta 2-glycoprotein
I (apolipoprotein H). Proc Natl Acad Sci USA 1990; 87: 4120-4124.

de Groot PG, Bouma B, Lutters BCH, Simmelink MJA, Derksen RHWM, Gros P. Structure-
function studies on β2-glycoprotein 1. J Autoimmun 2000; 15: 87-89.

Tincani A, Allegri F, Sanmarco M, Cinquini M, Taglietti M, Balestrieri G et al. Anticardiolipin

antibody assay: a methodological analysis for a better consensus in routine determinations-a
cooperative project of the European Antiphospholipid Forum. Thromb Haemost 2001; 86: 575-583.

Sanmarco M, Roll P, Gayet S, Oksman F, Johanet C, Escande A et al. Combined search for anti-β2-
glycoprotein I and anticardiolipin antibodies in antiphospholipid syndrome: contribution to
diagnosis. J Lab Clin Med 2004; 144: 141-147.

de Laat B, Derksen RH, Urbanus RT, de Groot PG. IgG antibodies that recognize epitope Gly40-
Arg43 in domain I of beta 2-glycoprotein I cause LAC, and their presence correlates strongly with
thrombosis. Blood 2005; 105: 1540-1545.

Ioannou Y, Pericleous C, Giles I, Latchman DS, Isenberg DA, Rahman A. Binding of

antiphospholipid antibodies to discontinuous epitopes on domain I of human beta(2)-glycoprotein
I: mutation studies including residues R39 to R43. Arthritis Rheum 2007; 56: 280-290.

Phosphatidylserine Antibodies

Antigen: Phosphatidylserine is found in animals, plants and micro-organisms,

making up ~10% of the total phospholipid. It is primarily located in the inner
lipid bilayer of the plasma membrane and the greatest concentration is found
within the myelin of brain tissue. Anti-phosphatidylserine antibodies recognise
the cofactors β2GPI or prothrombin bound to phosphatidylserine.

Clinical associations: Anti-phosphatidylserine antibodies are detected in

patients with APS, SLE, healthy relatives of these patients and in a small
percentage of other healthy controls. Although not currently included in the
Sapporo criteria the presence of anti-phosphatidylserine antibodies is closely
correlated to that of anti-cardiolipin and anti-β2GPI antibodies. The antibodies
are reported to be associated with the clinical features of APS; however this
relationship is not confirmed by all studies.

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Detection: Anti-phosphatidylserine antibodies recognise the cofactors β2GPI

or prothrombin; recognition is enhanced by the cofactors binding to
phosphatidylserine on the EIA plates. The antibodies are usually of IgG or IgM
class and rarely IgA. Anti-phosphatidylserine EIA may have some utility in
identifying a minority of patients with APS who are positive for anti-
phosphatidylserine antibodies and negative for anti-β2GPI and anti-cardiolipin
antibodies.

References

Radway-Bright EL, Ravirajan CT, Isenberg DA. The prevalence of antibodies to anionic
phospholipids in patients with the primary antiphospholipid syndrome, systemic lupus
erythematosus and their relatives and spouses. Rheumatology 2000; 39: 427-431.

von Landenberg P, Schölmerich J, von Kempis J, Lackner KJ. The combination of different
antiphospholipid antibody subgroups in the sera of patients with autoimmune diseases is a strong
predictor of thrombosis. Immunobiology 2003; 207: 65-71.

Additional Anti-Phospholipid Antibody Specificities

The internationally recognised criteria for APS preclude the reporting of

anti-phospholipid specificities other than anti-cardiolipin/β2GPI. Nevertheless,
research into APA has generated data on autoantibodies to other anionic
phospholipid specificities: phosphatidylinositol, phosphatidic acid and
phosphatidylglycerol. Investigations have also included the determination of
zwitterionic phospholipid specificities: phosphatidylcholine and phosphatidyl-
ethanolamine. As for anti-cardiolipin antibodies in APS the antibodies are not
likely to be targeted against the phospholipid itself. The more probable targets
are phospholipid binding proteins presented by the phospholipids; such proteins
are not restricted to β2GPI alone.
Studies have shown sera samples can have APA in the absence of anti-
cardiolipin/β2GPI autoantibodies, albeit a rare event. However, when one
considers the increased costs of implementing further assays, the lack of
conclusive data, and that the potential improvements in sensitivity are limited it
is reasonable to conclude that these assays would not add significant benefit to
the diagnosis of APS. Alternatively, one can consider this an area where the
availability of additional assays is required to allow for the generation of more
conclusive data.
Perhaps of more interest are investigations into a correlation of the presence
of APA and reproductive failure. In the normal population 10-15% of
pregnancies have an unfavourable outcome and between 2 and 3% of the
population have APA. Whereas, ~15% of women with a history of recurrent

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miscarriage have APA, and in the absence of treatment up to 90% of

pregnancies in this group of patients can be unsuccessful. McIntyre and
Wagenknecht (2000) reported an association of anti-phosphatidyl ethanolamine
antibodies with thrombosis, recurrent pregnancy loss, and many other
symptoms associated with APS. Ulcova-Gallova et al. (2005) reported that
patients with two or more IVF failures or three or more spontaneous
miscarriages were more frequently reported to have APA against
phosphatidylinositol and phosphatidylserine than against cardiolipin or β2GPI
alone. These observations have not been confirmed by all studies and so the
significance of these antibodies continues to be debated.
Further complicating this area of research is the lack of standardisation of
methodology in the determination of these specificities. Some have proposed
the use of an EIA coated with a mix of phospholipids. However, this would not
specifically identify patients with APS according to guidelines, and so further
assays would still be required to be carried out on all patient samples. The
availability of individually optimised EIAs coated with separate phospholipids
allows the user to either focus on cardiolipin and β2GPI or determine the
prevalence of other APA in their patient population.

References

Mcintyre JA, Wagenknecht DR. Anti-phosphatidylethanolamine (aPE) antibodies: a survey. J

Autoimmun 2000; 15: 185-193.

Hornstein MD, Davis OK, Massey JB, Paulson RJ, Collins JA. Antiphospholipid antibodies and in
vitro fertilization success: a meta-analysis. Fertil Steril 2000; 73: 330-333.

Ulcova-Gallova Z, Krauz V, Novakova P, Milichovska L, Micanova Z, Bibkova K et al. Anti-

phospholipid antibodies against phosphatidylinositol, and phosphatidylserine are more significant
in reproductive failure than antibodies against cardiolipin only. Am J Reprod Immunol 2005; 54:
112-117.

Prothrombin Antibodies

Antigen: Prothrombin (Factor II) was first identified as a cofactor of lupus

anticoagulants in 1959 by Loeliger. It is a 72kDa vitamin K-dependent
glycoprotein which has a high affinity for anionic phospholipids. In vivo,
prothrombin is converted to thrombin which catalyses the conversion of
fibrinogen to fibrin.

Clinical associations: Anti-prothrombin antibodies have been reported to be

detected in 50-90% of APS patients, depending on the method of detection. The

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association of these antibodies with thrombosis and the clinical manifestations

of APS is debated. There is no current recommendation for the routine
measurement of these antibodies and therefore they should only be measured as
part of clinical research studies.

Detection: Double immunodiffusion and two-dimensional immuno-

electrophoresis have both been utilised in the detection of anti-prothrombin
antibodies; however EIA is now the usual method. Detection of these antibodies
is dependent on antigen presentation which is affected by the presence of
phospholipids and the type of EIA plates used. For example, when plain
polystyrene, gamma irradiated or phosphatidylserine coated plates are used 0,
~55 and ~90% of sera from APS patients were shown to be positive. As with
anti-β2GPI antibodies, it is thought that antibodies bind to neo-epitopes
produced when prothrombin binds to phosphatidylserine. The antibodies are
predominantly IgG, less frequently IgM and rarely IgA class. Although both
bovine and human sources of prothrombin have been used, human prothrombin
is the preferred antigen.

References

Loeliger A. Prothrombin as co-factor of the circulating anticoagulant in systemic lupus

erythematosus? Thromb Diath Haemorrh 1959; 3: 237-256.

Amengual O, Atsumi T, Koike T. Specificities, properties and clinical significance of

antiprothrombin antibodies. Arthritis Rheum 2003; 48: 886-895.

Galli M, Borrelli G, Jacobsen EM, Marfisi RM, Finazzi G, Marchioli R et al. Clinical significance
of different antiphospholipid antibodies in the WAPS (warfarin in the antiphospholipid syndrome)
study. Blood 2007; 110: 1178-1183.

Horita T, Ichikawa K, Kataoka H, Yasuda S, Atsumi T, Koike T. Human monoclonal antibodies

against the complex of phosphatidylserine and prothrombin from patients with the antiphospholipid
antibodies. Lupus 2007; 16: 509-516.

Annexin Antibodies

Antigens: Annexins (Anx) are a family of structurally related proteins which

bind to phospholipids in a calcium dependent manner. Sera from APS patients
predominantly recognise annexin II and V. Annexin II is an endothelial cell
surface receptor which functions as a cofactor for plasmin generation and to
localise fibrinolytic activity to the cell surface. It is found on the surface
membrane of endothelial cells and monocytes, and on the brush border

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membrane of placental syncytiotrophoblasts. Annexin V is a potent

anticoagulant which is highly expressed by placental trophoblasts and found
abundantly on the apical surface of syncytiotrophoblasts.

Clinical associations: Anti-annexin antibodies are associated with APS, they

are also found, albeit less frequently, in other autoimmune diseases such as RA
and systemic sclerosis. Patients with anti-annexin antibodies are often positive
for anti-prothrombin and anti-β2GPI antibodies as well. Anti-annexin II and V
antibodies have been proposed to be associated with the clinical features of APS
including thrombosis, recurrent miscarriages and IVF-embryo transfer failure.
However, this relationship is not confirmed by all studies.

Detection: Anti-annexin antibodies usually of the IgG or IgM class are detected
using EIA and recombinant annexin as an antigen. The type of EIA plate used
is important as this can affect the presentation of the antigen.

References
Lakos G, Kiss E, Regeczy N, Tarjan P, Soltesz P, Zeher M et al. Antiprothrombin and antiannexin
V antibodies imply risk of thrombosis in patients with systemic autoimmune diseases. J Rheumatol
2000; 27: 924-929.

Rand JH, Wu XX. The Annexins: A target of antiphospholipid antibodies. In: The Antiphospholipid
Syndrome II: Autoimmune Thrombosis. Asherson RA, Cervera R, Piette J-C, Shoenfeld Y Editors.
Elsevier Science B.V; 2002.

Cesarman-Maus G, Ríos-Luna NP, Deora AB, Huang B, Villa R, del Carman Cravioto M et al.
Autoantibodies against the fibrinolytic receptor annexin 2, in antiphospholipid syndrome. Blood
2006; 107: 4375-4382.

General References
Asherson RA, Cervera R, Piette J-C, Shoenfeld Y editors. The Antiphospholipid Syndrome II:
Autoimmune Thrombosis. Elsevier Science B.V; 2002.

Galli M, Luciani D, Bertolini G, Barbui T. Anti-beta 2-glycoprotein I, antiprothrombin antibodies,

and the risk of thrombosis in the antiphospholipid syndrome. Blood 2003; 102: 2717-2723.

Salmon JE, Girardi G, Lockshin MD. The antiphospholipid syndrome as a disorder initiated by
inflammation: implications for the therapy of pregnant patients. Nat Clin Pract Rheumatol 2007; 3:
140-147.

de Laat B, Mertens K, de Groot PG. Mechanisms of disease: antiphospholipid antibodies-from

clinical association to pathologic mechanism. Nat Clin Pract Rheumatol 2008; 4: 192-199.

Shoenfeld Y, Meroni PL, Cervera R. Antiphospholipid syndrome dilemmas still to be solved: 2008
status. Ann Rheum Dis 2008; 67: 438-442.

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 Vasculitis and ANCA

Chapter 13

Vasculitis and ANCA

Neutrophils (polymorphonuclear leukocytes, PMNs) are by far the most

abundant white cells in the blood, representing approximately 45-60% of
circulating leukocytes. These cells function as professional phagocytes, by
rapidly identifying and engulfing invading pathogens. They have a
characteristic multi-lobed nucleus (Figure 13.1) and a densely packed
cytoplasm, filled predominantly with granules. There are four major families of
granules (primary, secondary, tertiary and secretory), each containing an array
of proteolytic enzymes, adhesion molecules, receptors and anti-microbial
proteins, all necessary for neutrophil function. Some of these proteins have been
identified as target antigens for anti-neutrophil cytoplasmic antibodies (ANCA)
(Table 13.1).

ANCA have been used since 1982 as diagnostic markers for primary,
systemic and small-vessel vasculitides. There are also a wide range of other
diseases associated with the presence of ANCA including inflammatory bowel
diseases, liver diseases and connective tissue diseases. Immunofluorescence
assays utilising human peripheral blood neutrophils are the best method for
ANCA detection, combined with EIA to confirm specificity. Anti-
myeloperoxidase and anti-proteinase 3 are the main antibodies detected. On
ethanol-fixed neutrophils, a perinuclear with nuclear extension (P-ANCA) and
granular cytoplasmic (C-ANCA) immunofluorescent staining pattern are
produced respectively. A few samples may only be positive by IFA or EIA, as
antigen presentation may differ between the two methods. However, the
International Consensus Statement on Testing and Reporting ANCA strongly
recommends that all samples positive by immunofluorescence should have
specificity confirmed by EIA.

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Antigen Pattern Clinical Associations

WG (~90%), MP (~50%), CSS (~30%),
pauci-immune necrotising crescentic
Proteinase-3 C-ANCA glomerulonephritis (minority), systemic
(PR3) sclerosis (~20%), acute reactive arthritis
(~4%), chronic reactive arthritis (~2%),
ulcerative colitis (~17%), RA (~17%)
WG (15%), MP (~50%), CSS (~40%),
renal limited rapidly progressive
Myeloperoxidase glomerulonephritis (~50%), systemic
P-ANCA
(MPO) sclerosis (18%), PN (~62%), chronic reactive
arthritis (8%), ulcerative colitis (~10%), RA
(~47%), idiopathic pauci-immune necrotising
crescentic glomerulonephritis (~80%)
Atypical Ulcerative colitis (~40%), Crohn's disease
Cathepsin G
P-ANCA (~28%), systemic sclerosis, SLE, RA,
autoimmune liver diseases
Ulcerative colitis, sclerosing cholangitis, WG,
Elastase P-ANCA
MP, renal insufficiency, cocaine-induced
midline destructive lesions (~68%)
Bactericidal Systemic vasculitis, systemic sclerosis, RA,
P-ANCA
permeability SLE, cystic fibrosis, primary sclerosing
or Atypical
increasing factor cholangitis, autoimmune hepatitis, Crohn's
(BPI) disease, ulcerative colitis
Chronic reactive arthritis (~16%), ulcerative
colitis (~13%), RA (~35%),
polymyositis/dermatomyositis (~27%),
Lactoferrin P-ANCA
primary biliary cirrhosis (~36%),
autoimmune hepatitis (~29%), autoimmune
cholangitis (~100%), primary sclerosing
cholangitis (~22%)
Table 13.1. Summary of the main ANCA antigens, their staining patterns on
ethanol-fixed neutrophils and clinical associations (WG - Wegener's
granulomatosis, MP - microscopic polyangiitis, CSS - Churg-Strauss syndrome,
PN - polyarteritis nodosa, RA - rheumatoid arthritis, SLE - systemic lupus
erythematosus).

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Methodology

Neutrophil isolation and fixation

Human neutrophil slides of a high quality are widely available from
commercial sources, however some laboratories prefer to prepare their own.
Human neutrophils are isolated from whole blood, dispensed onto glass slides
then dried and fixed in ethanol, formalin or methanol. Care should be taken
throughout the procedure to prevent neutrophil activation as this may result in
confusing or abnormal staining patterns. Standardisation between laboratories
can be achieved by participation in quality control schemes.

Figure 13.1. Highly purified neutrophils isolated from peripheral blood.

Neutrophils are stained using the Giemsa (Ramonowski) staining method. The
image was taken at 40X magnification.
Fixation of neutrophils with ethanol allows the positively-charged
cytoplasmic granule proteins to migrate towards the negatively-charged nuclear
membrane. Antibodies with specificity for the positively-charged protein
therefore give a perinuclear (P-ANCA) fluorescent staining pattern. In
comparison, negatively-charged cytoplasmic granule proteins will remain in the
cytoplasm and their respective antibodies will give the cytoplasmic (C-ANCA)
fluorescent staining pattern. Fixation with formalin results in cross-linking of

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the antigens to other cytoplasmic proteins, consequently migration is prevented.

Therefore true P-ANCA samples will bind the antigens in their native location,
thus giving a granular cytoplasmic pattern on formalin-fixed neutrophils. The
majority of nuclear antigens are sensitive to formalin fixation. Consequently,
anti-nuclear antibody (ANA) samples or samples containing ANA as well as
ANCA will have weaker staining on formalin fixed-neutrophils. Granulocyte
specific (GS)-ANA samples which produce a perinuclear or nuclear staining
pattern on ethanol-fixed neutrophils are negative on formalin-fixed neutrophils
and HEp-2 cells. Anti-GS-ANA have been reported in up to ~68% of
rheumatoid arthritis patients. There are also a number of antibodies which will
produce atypical P and C-ANCA patterns on ethanol-fixed neutrophils, some of
these will remain positive on formalin-fixed neutrophils (Table 13.2). Atypical
P-ANCA samples with more sensitive antigens will be negative on formalin-
fixed neutrophils. As an alternative to formalin, methanol can also be used for
neutrophil fixation. Atypical P-ANCA negative on formalin-fixed neutrophils,
may be positive on methanol-fixed neutrophils. These methanol-positive,
atypical P-ANCA are often associated with ulcerative colitis. In comparison,
only approximately 20% of vasculitis P-ANCA are positive on methanol-fixed
neutrophils.

Ethanol-Fixed Formalin-Fixed
Antibody Type HEp-2
Neutrophils Neutrophils
C-ANCA Cytoplasmic Granular/cytoplasmic Negative
P-ANCA Nuclear/perinuclear Granular/cytoplasmic Negative
GS-ANA Nuclear/perinuclear Reduced/none Negative
Atypical ANCA Cytoplasmic/perinuclear Cytoplasmic/none Negative
P-ANCA+ANA Nuclear/perinuclear Cytoplasmic/reduced Nuclear
ANA Nuclear/perinuclear Reduced/none Nuclear
Table 13.2. ANCA types and their IFA patterns using different fixatives.

Guidelines and Recommendations for Testing

Sample dilutions
Patient serum samples should be diluted at 1/20 although a dilution of 1/40
may help to reduce 'false positives'. Titration may be useful for samples
containing ANA or other cytoplasmic antibodies in addition to ANCA, as one
pattern may disappear before the other. When monitoring disease, it is useful
to include a previous serum sample in order to show changes in antibody levels.

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Evans blue counterstain should only be used in moderation as significant

amounts of weak positive fluorescence may be masked.

Conjugates
ANCA are usually of the IgG class therefore fluorescein labelled anti-human
IgG is usually used in screening since anti-IgM and anti-IgA may produce false
positive results. However, there have been reports of IgA antibodies in ~82% of
patients with Henoch-Schönlein purpura and IgM antibodies in severe
pulmonary haemorrhage. Therefore, if these conditions are suspected the
appropriate conjugate should be used. IgG1 and IgG4 subclasses are the most
common ANCA found in vasculitic diseases, whereas IgG3 and IgG2 ANCA
are associated with more active disease in systemic vasculitis and renal
involvement in Wegener's granulomatosis.

Important clinical indications for ANCA testing

· Glomerulonephritis, especially rapidly progressive glomerulonephritis
· Pulmonary haemorrhage especially pulmonary-renal syndrome
· Cutaneous vasculitis with systemic features
· Multiple lung nodules
· Chronic destructive disease of the upper airways
· Long-standing sinusitis or otitis
· Subglottic tracheal stenosis
· Mononeuritis multiplex or other peripheral neuropathy
· Retro-orbital mass
· Undiagnosed inflammatory disease

When these clinical features are present, the demonstration of ANCA is

probably 95% sensitive and 90% specific for Wegener's granulomatosis or
microscopic polyangiitis. IFA alone detects 90-95% of all ANCA-positive sera
in patients with Wegener's granulomatosis, microscopic polyangiitis and pauci-
immune segmental/crescentic necrotising glomerulonephritis (NCGN/SNGN),
whilst EIA for PR3 and MPO-ANCA are about 90% sensitive.

Trouble shooting
Background fluorescence can be reduced by including 1% bovine serum
albumin in the serum diluent and wash solution.

It is important that activation of the cells is prevented otherwise partial or

variable degranulation occurs with ragged cell margins and bright granules in
the background. This will produce the following features when assessing
autoantibodies:

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1. Reduced intensity of staining for some patterns.

2. C-ANCA may appear as multiple dots rather than a speckled/homogeneous
pattern, particularly for autoantibodies directed against low concentration
antigens.
3. Neutrophils that are partially activated show loss of granules in the cell
periphery so that P-ANCA and C-ANCA may be difficult to distinguish and
unusual patterns may be seen.

Guidelines for ANCA Testing

Immunofluorescence of ethanol-fixed neutrophils should be the first choice
of ANCA test for screening new vasculitis patients, since 10% of ANCA-
positive sera in patients with Wegener's granulomatosis or microscopic
polyangiitis are demonstrated only by IFA (i.e. negative by EIA). Formalin-
fixed neutrophils should be used with caution to confirm P-ANCA with MPO
specificity as some anti-MPO samples may appear negative. EIA should be used
to confirm any IFA positives, to provide numerical results for serial studies and
to detect occasional IFA negative antibodies in clinically suspicious situations.
Occasionally secondary ANCA specificity can develop in patients, particularly
in the case of Wegener's granulomatosis or microscopic polyangiitis, so EIA
testing may be useful.

References

Luqmani RA, Bacon PA, Moots RJ, Janssen BA, Pall A, Emery P et al. Birmingham Vasculitis
Activity Score (BVAS) in systemic necrotizing vasculitis. Q J Med 1994; 87: 671-678.

Savige J, Gillis D, Benson E, Davies D, Esnault V, Falk RJ et al. International Consensus Statement
on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA). Am J Clin Pathol
1999; 111: 507-513.

Savige J, Dimech W, Fritzler M, Goeken J, Hagen EC, Jennette JC et al. Addendum to the
International Consensus Statement on testing and reporting of antineutrophil cytoplasmic
antibodies. Quality control guidelines, comments, and recommendations for testing in other
autoimmune diseases. Am J Clin Pathol 2003; 120: 312-318.

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Figure 13.2. A flow diagram describing the assays and report-back systems used
for the determination of ANCA associated vasculitis according to the
International Consensus Statement.

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Proteinase 3 Antibodies

Antigen: Proteinase 3 (PR3) is a 29kDa serine protease found in the azurophilic

granules of neutrophils and in peroxidase-positive lysosomes of monocytes.
PR3 has a non-proteolytic antimicrobial activity against bacteria and fungi.
When released in inflammatory conditions, PR3 can degrade collagens,
proteoglycans, elastin, other connective tissue components and all four IgG
subclasses.

Clinical associations: Anti-PR3 antibodies are detected in patients with the

following diseases: Wegener's granulomatosis (~90%), microscopic
polyangiitis (~50%), Churg-Strauss syndrome (~30%), a minority of pauci-
immune necrotising crescentic glomerulonephritis (NCGN), systemic sclerosis
(~20%), acute reactive arthritis (~4%), chronic reactive arthritis (~2%),
ulcerative colitis (~17%) and rheumatoid arthritis (~17%). Wegener's
granulomatosis (WG)-positive PR3 patients are more prone to relapse when
anti-PR3 antibody titres increase and may have more rapidly progressive renal
failure.

Detection: Ethanol-fixed neutrophils reveal a classical C-ANCA pattern of

cytoplasmic granular fluorescence with central interlobular accentuation
(Figure 13.3). With formalin-fixed neutrophils, the fluorescence is still
cytoplasmic and granular but with less interlobular accentuation (Figure 13.4).
All positive samples should be confirmed by EIA. EIAs utilising PR3 capture,
rather than direct adsorption of PR3 to the polystyrene, have been reported to
increase sensitivity for Wegener's granulomatosis. This is likely to depend on
which EIAs are compared.

References

Goldschmeding R, van der Schoot CE, ten Bokkel Huinink D, Hack CE, van den Ende ME,
Kallenberg CG et al. Wegener's granulomatosis autoantibodies identify a novel
diisopropylfluorophosphate-binding protein in the lysosomes of normal human neutrophils. J Clin
Invest 1989; 84: 1577-1587.

Jennette JC, Hoidal JR, Falk RJ. Specificity of anti-neutrophil cytoplasmic autoantibodies for
proteinase 3. Blood 1990; 75: 2263-2264.

Wiik A. What you should know about PR3-ANCA. An Introduction. Arthritis Res 2000; 2: 252-254.

Kallenberg CG. Pathogenesis of PR3-ANCA associated vasculitis. J Autoimmun 2008; 30: 29-36.

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Figure 13.3. Ethanol-fixed neutrophils stained with anti-PR3 antibodies,

producing the characteristic C-ANCA pattern of cytoplasmic granular
fluorescence with central interlobular accentuation.

Figure 13.4. Anti-PR3 antibodies staining formalin-fixed neutrophils.

Fluorescence is cytoplasmic and granular with less interlobular accentuation.

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Myeloperoxidase Antibodies

Antigen: The target antigen of anti-MPO antibodies is myeloperoxidase a

140kDa covalently-linked dimer found in the positively charged azurophilic
granules of neutrophils and lysosomes of monocytes. Myeloperoxidase in
combination with H2O2 catalyses oxidation of chloride ions to hydrochlorous
acid. Hydrochlorous acid can kill phagocytised bacteria and viruses and in
combination with metabolites, inactivates protease inhibitors such as α-anti-
trypsin in blood and tissues.

Clinical associations: Anti-MPO antibodies are detected in patients with the

following diseases: Wegener's granulomatosis (~15%), microscopic
polyangiitis (~50%), Churg-Strauss syndrome (~40%), renal limited rapidly
progressive glomerulonephritis (~50 %), systemic sclerosis (~18%),
polyarteritis nodosa (~62 %), chronic reactive arthritis (~8%), ulcerative colitis
(~10%), rheumatoid arthritis (~47%), idiopathic pauci-immune necrotising
crescentic glomerulonephritis (~80%) and also, less frequently, in anti-GBM
disease, SLE, IgA nephropathy and drug-induced glomerulonephritis. Anti-
MPO positive patients are usually older in comparison to anti-PR3 positive
patients and titres of anti-MPO antibodies frequently persist during disease
remission. Anti-MPO positive patients are less likely to relapse and may have
a less progressive form of glomerulonephritis.

Detection: Immunofluorescence on ethanol-fixed neutrophils reveals a

classical P-ANCA pattern of perinuclear fluorescence with nuclear extension
(Figure 13.5). However, samples containing anti-MPO, anti-MPO and ANA or
ANA may look similar on ethanol-fixed neutrophils. It is possible to
discriminate between true anti-MPO and ANA samples using formalin-fixed
neutrophils and HEp-2 cells. Formalin-fixation denatures the majority of
nuclear antigens, therefore ANA become weaker or negative. Anti-MPO
antibodies on formalin-fixed neutrophils will be seen as a cytoplasmic granular
fluorescent pattern (Figure 13.6). All positive samples should be confirmed by
EIA. The use of an MPO capture EIA may increase sensitivity, although
possibly to a limited extent, for microscopic polyangiitis.

References
Franssen CFM, Stegeman CA, Kallenberg CGM, Gans ROB, De Jong PE, Hoorntje SJ et al. Anti-
proteinase 3 and anti-myeloperoxidase associated vasculitis. Kidney Int 2000; 57: 2195-2206.

Schmitt WH. Newer insights into the aetiology and pathogenesis of myeloperoxidase associated
autoimmunity. Jpn J Infect Dis 2004; 57: S7-8.

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 Vasculitis and ANCA

Figure 13.5. Ethanol-fixed neutrophils stained with anti-MPO antibodies,

producing the characteristic P-ANCA pattern of perinuclear fluorescence with
nuclear extension.

Figure 13.6. Anti-MPO antibodies showing conversion to a cytoplasmic,

granular staining pattern on formalin-fixed neutrophils.

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Cathepsin G Antibodies

Antigen: Cathepsin G is a 26kDa protease found in the azurophilic granules of

neutrophils. It acts as a monocyte chemoattractant at sites of inflammation and
is involved in platelet function.

Clinical associations: Anti-cathepsin G antibodies are often detected in

inflammatory bowel diseases, for example in 41% of active ulcerative colitis
(UC), 13% of inactive UC and in 29% of Crohn's disease patients. Presence of
anti-cathepsin G antibodies is associated with a more severe colitis and
consequently antibody measurement may have a utility in monitoring disease
activity. Anti-cathepsin G antibodies have been reported in patients with a
number of connective tissue diseases including systemic sclerosis, SLE and
rheumatoid arthritis. They have also been detected in patients with several liver
diseases including autoimmune hepatitis, primary biliary cirrhosis and primary
sclerosing cholangitis. This illustrates their lack of utility as a specific
diagnostic marker.

Detection: Immunofluorescence on ethanol-fixed neutrophils produces an

atypical P-ANCA staining pattern. Confirmation of specificity can be
confirmed by either EIA or western blot.

Reference

Kuwana T, Sato Y, Saka M, Kondo Y, Miyata M, Obara K et al. Anti-cathepsin G antibodies in the
sera of patients with ulcerative colitis. J Gastroenterol 2000; 35: 682-689.

Elastase Antibodies

Antigen: Human neutrophil elastase is a member of the chymotrypsin family of

serine proteases and is closely related to PR3 in both function and homology.
When released at inflammatory sites, elastase digests and degrades elastin and
type IV collagen, it is inhibited by α-1 anti-trypsin.

Clinical associations: Anti-elastase antibodies are rarely detected in

autoimmune diseases although they have been found occasionally in patients
with the following diseases: ulcerative colitis, sclerosing cholangitis, Wegener's
granulomatosis and microscopic polyangiitis. Wiesner et al. (2004) reported the
detection of anti-elastase antibodies in ~68% of patients with cocaine-induced
midline destructive lesions.

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Detection: Immunofluorescence on ethanol-fixed neutrophils will produce a P-

ANCA staining pattern. Specificity to elastase can be confirmed using direct or
capture EIAs with purified, native human neutrophil elastase as an antigen.
Anti-elastase antibodies have also been detected using a transformed, ethanol-
fixed human mast cell line.

Reference

Wiesner O, Russell KA, Lee AS, Jenne DE, Trimarchi M. Gregorini G et al. Anti-neutrophil
cytoplasmic antibodies reacting with human neutrophil elastase as a diagnostic marker for cocaine-
induced midline destructive lesions but not autoimmune vasculitis. Arthritis Rheum 2004; 50: 2954-
2965.

Bactericidal Permeability Increasing Factor Antibodies

Antigen: Bactericidal permeability increasing factor (BPI) is a 55kDa

membrane-associated protein found in the azurophilic granules of neutrophils
which exhibits bacteriostatic and bactericidal effects against a wide range of
gram-negative bacteria.

Clinical associations: The significance and utility of measuring anti-BPI

antibodies remains unknown although they have been detected in a wide range
of autoimmune related diseases including systemic vasculitis, systemic
sclerosis, rheumatoid arthritis, SLE, cystic fibrosis, primary sclerosing
cholangitis, autoimmune hepatitis, Crohn's disease and ulcerative colitis.

Detection: Immunofluorescence of ethanol-fixed neutrophils reveals a P-

ANCA or atypical staining pattern. Specificity to BPI can be confirmed using
either EIA or western blot.

References

Zhao MH, Jones SJ, Lockwood CM. Bactericidal/permeability-increasing protein (BPI) is an

important antigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) in vasculitis. Clin Exp
Immunol 1995; 99: 49-56.

Khanna D, Aggarwal A, Bhakuni DS, Dayal R, Misra R. Bactericidal/Permeability-Increasing

Protein and Cathepsin G are the major antigenic targets of Anti-neutrophil Cytoplasmic
Autoantibodies in Systemic Sclerosis. J Rheumatol 2003; 30: 1248-1252.

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Lactoferrin Antibodies

Antigen: Lactoferrin is a 78kDa iron-binding protein found in milk, tears,

mucosal secretions and specific granules of granulocytes. It exhibits
bacteriostatic and bactericidal properties and when released stimulates
phagocytic activity in neutrophils.

Clinical associations: Anti-lactoferrin antibodies have been reported in a wide

range of diseases but usually at a low incidence. Included amongst these
diseases are chronic reactive arthritis (~16%), ulcerative colitis (~13%),
rheumatoid arthritis (~35%), polymyositis/dermatomyositis (~27%), primary
biliary cirrhosis (~36%), autoimmune hepatitis (~29%), autoimmune
cholangitis (~100%) and primary sclerosing cholangitis (~22%).

Detection: A P-ANCA pattern is observed on ethanol-fixed neutrophils. Anti-

lactoferrin antibodies can also be detected by EIA and western blotting.

Reference

Ohana M, Okazaki K, Hajiro K, Uchida K. Antilactoferrin antibodies in autoimmune liver diseases.

Am J Gastroenterol 1998; 93: 1334-1339.

Other Atypical ANCA

In addition to the previously mentioned ANCA antigens Table 13.3 lists some
of the more uncommon antigens, which at present have limited clinical
significance. ANCA directed against these antigens are more successfully
detected using more sensitive techniques such as EIA and western blot, as IIF
will often be negative.

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Antigen Pattern Clinical Associations References

Beta glucuronidase Inflammatory bowel Roozendaal et al.

P-ANCA
(75kDa) disease (IBD) 1998

Lysozyme Systemic sclerosis,

P-ANCA Khanna et al. 2003
( 16.5kDa) IBD
h-lamp-2 (human
lysosomal- Necrotising and
associated C-ANCA crescentic Kain et al. 1995
membrane protein- glomerulonephritis
2), (120kDa)
Primary sclerosing
Alpha enolase P and C- cholangitis, ulcerative Roozendaal et al.
(47kDa) ANCA colitis and Crohn’s 1998
disease
Primary sclerosing
Catalase (240kDa, cholangitis, ulcerative Roozendaal et al.
P-ANCA
tetrameric) colitis, Crohn’s 1998
disease
Systemic vasculitis,
Azurocidin P and C- cystic fibrosis,
Zhao et al. 1996
(27kDa) ANCA chronic active
hepatitis
HMG1 and 2 (high Autoimmune
mobility group, hepatitis, primary
non-histone biliary cirrhosis,
chromosomal P-ANCA rheumatoid arthritis, Uesugi et al. 1998
protein), (29kDa SLE, Sjögren’s
and 28kDa syndrome, ulcerative
respectively) colitis
Smooth
cytoplasmic,
Autoimmune
unlike
Actin (43kDa) hepatitis Orth et al. 1997
granular C-
ANCA
staining
Table 13.3. Atypical ANCA staining patterns and their clinical associations.

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Figure 13.7. Ethanol-fixed neutrophils stained with a mixed P and C-ANCA

sample.

Figure 13.8. Smooth C-ANCA on ethanol-fixed neutrophils.

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Figure 13.9. Atypical ANCA binding ethanol-fixed neutrophils.

References

Kain R, Matsui K, Exner M, Binder S, Schaffner G, Sommer EM et al. A novel class of autoantigens
of anti-neutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the
lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane
protein in glomerular endothelial cells. J Exp Med 1995; 181: 585-597.

Zhao MH, Lockwood CM. Azurocidin is a novel antigen for anti-neutrophil cytoplasmic
autoantibodies (ANCA) in systemic vasculitis. Clin Exp Immunol 1996; 103: 397-402.

Orth T, Gerken G, Kellner R, Meyer zum Büschenfelde KH, Mayet WJ. Actin is a target antigen of
anti-neutrophil cytoplasmic antibodies (ANCA) in autoimmune hepatitis type-1. J Hepatol 1997;
26: 37-47.

Uesugi H, Ozaki S, Sobajima J, Osakada F, Shirakawa H, Yoshida M et al. Prevalence and

characterization of novel pANCA, antibodies to the high mobility group non-histone chromosomal
proteins HMG1 and HMG2, in systemic rheumatic diseases. J Rheumatol 1998; 25: 703-709.

Roozendaal C, Zhao MH, Horst G, Lockwood CM, Kleibeuker JH, Limburg PC et al. Catalase and
α-enolase: two novel granulocyte autoantigens in inflammatory bowel disease (IBD). Clin Exp
Immunol 1998; 112: 10-16.

Khanna D, Aggarwal A, Bhakuni DS, Dayal R, Misra R. Bactericidal/permeability-increasing

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protein and cathepsin G are the major antigenic targets of antineutrophil cytoplasmic autoantibodies
in systemic sclerosis. J Rheumatol 2003; 30: 1248-1252.

General References

Davies DJ, Moran JE, Niall JF, Ryan GB. Segmental necrotising glomerulonephritis with
antineutrophil antibody: possible arbovirus aetiology? Br Med J. 1982; 285: 606.

Savige J, Pollock W, Trevisin M. What do antineutrophil cytoplasmic antibodies (ANCA) tell us?
Best Pract Res Clin Rheumatol 2005; 19: 263-276.

Sinico RA, Di Toma L, Maggiore U, Bottero P, Radice A, Tosoni C et al. Prevalence and clinical
significance of antineutrophil cytoplasmic antibodies in Churg-Strauss syndrome. Arthritis Rheum
2005; 52: 2926-2935.

Kallenberg CG, Heeringa P, Stegeman CA. Mechanisms of Disease: pathogenesis and treatment of
ANCA-associated vasculitides. Nat Clin Pract Rheumatol 2006; 2: 661-670.

Bosch X, Guilabert A, Font J. Antineutrophil cytoplasmic antibodies. Lancet 2006; 368: 404-418.

Kallenberg CG. Antineutrophil cytoplasmic autoantibody-associated small-vessel vasculitis. Curr

Opin Rheumatol 2007; 19: 17-24.

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 Miscellaneous Autoantibody Specificities

Chapter 14

Miscellaneous Autoantibody Specificities

Numerous autoantibody specificities are now used as reliable diagnostic

markers in well characterised autoimmune diseases. However, there are many
which have a more ambiguous clinical utility and a small selection of these is
discussed below.

Filaggrin Antibodies

Antigen: Young et al. (1979) detected antibodies in rheumatoid arthritis (RA)

patients which showed reactivity to the epithelial cells of the stratum corneum
in rat oesophagus. These were initially termed anti-keratin antibodies (AKA);
however, subsequent work revealed that they actually recognise filaggrin
(filament aggregating protein). Anti-perinuclear factor antibodies (APF) have
also been described, these recognise profilaggrin within the keratohyalin
granules of buccal mucosal epithelia cells. The two antibodies react with almost
identical antigens and are now generally referred to as anti-filaggrin antibodies
(AFA). Filaggrin has a post-translational modification by peptidyl-arginine
deiminase (PAD) which converts the arginine residues to citrulline, a non-
standard amino acid. It aggregates with keratin intermediate filaments and
facilitates in their alignment. The citrullinated residues on filaggrin are the
principle epitopes recognised by AFA. The cyclic citrullinated peptide (CCP)
EIA employed peptides based on these epitopes. The second generation assay
(CCP2) utilises further modified synthetic peptides and is used as a specific
assay for RA.

Clinical associations: AFA are detected in patients with RA (both positive and
negative for rheumatoid factor) and occasionally in other systemic rheumatic
diseases and in up to 3% of healthy subjects. The presence of AFA precedes
clinical symptoms of RA in otherwise healthy subjects and their titres correlate
with clinical parameters of the disease.

Detection: Samples are only considered to be positive for AFA when the
characteristic staining pattern of linear, laminated fluorescence of the stratum
corneum in the middle third of rat oesophagus is observed (Figure 14.1).

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Homogeneous fluorescence affecting the stratum corneum, fluorescence of the

stratum basale and stratum spinosum should be excluded, as these patterns are
not specific for RA. Pre-treatment of sections with PBS plus 0.5% w/v Triton
X-100 increases sensitivity for AFA (Storch 2000). EIA using second generation
synthetic citrullinated cyclic peptides (CCP2) are very specific (>95%) and
sensitive (60-77%) for RA. Anti-CCP2 antibodies can aid early diagnosis of
RA, especially in patients with undifferentiated arthritis, and are also linked to
a more aggressive disease progression. AFA and anti-CCP2 assays are
considered to be more sensitive than RF for RA, it probably only remains a
matter of time before anti-CCP2 antibodies are included alongside RF as a
recommended disease marker for the classification of RA.

Figure 14.1. Filaggrin antibodies showing linear, laminated staining on rat

oesophagus.

References

Young BJ, Mallya RK, Leslie RD, Clark CJ, Hamblin TJ. Anti-keratin antibodies in rheumatoid
arthritis. Br Med J 1979; 2: 97-99.

Vincent C, Serre G, Lapeyre F, Fournié B, Ayrolles C, Fournié A et al. High diagnostic value in
rheumatoid arthritis of antibodies to the stratum corneum of rat oesophagus epithelium, so-called
'antikeratin antibodies'. Ann Rheum Dis 1989; 48: 712-722.

Sebbag M, Simon M, Vincent C, Masson-Bessière C, Girbal E, Durieux JJ et al. The antiperinuclear

208
 Miscellaneous Autoantibody Specificities

factor and the so-called antikeratin antibodies are the same rheumatoid arthritis-specific
autoantibodies. J Clin Invest 1995; 95: 2672-2679.

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

Dubucquoi S, Solau-Gervais E, Lefranc D, Marguerie L, Sibilia J, Goetz J et al. Evaluation of anti-

citrullinated filaggrin antibodies as hallmarks for the diagnosis of rheumatic diseases. Ann Rheum
Dis 2004; 63: 415-419.

Salivary Duct Antibodies

Antigen: It has been proposed that anti-salivary duct antibodies (ASDA) react
with a currently unidentified antigen found within the cytoplasm of epithelial
cells lining the excretory ducts of the salivary gland. In the majority of cases
this has shown to be false-positive reactions against ABO blood group antigens.

Clinical associations: In the 1960s ASDA were initially suggested to be

detected in Sjögren’s syndrome (10-58%) and rheumatoid arthritis (22-45%).
However, true ASDA are now believed to be much rarer and the majority of
ASDA staining observed is due to blood group antibodies binding to AB
antigens in the salivary tissue.

Figure 14.2. Monkey parotid gland showing staining of the secretory ducts
which is removed by adsorption of the serum with ABO blood group antigens.

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Detection: IIF using monkey salivary gland and ASDA will show staining of
the intracytoplasmic granules in the primary ductules and collecting ducts
(Figure 14.2). The old world macaques species of monkey, often used as a
substrate source are known to express human type AB blood group antigens on
the striated ducts of salivary glands. Adsorption of positive samples with AB
antigens will remove any false-positive staining. Anti-ABO blood group
antibodies also produce false-positive staining in other tissues such as the
pemphigus-like chicken-wire pattern on monkey oesophagus. Since the
observation of this false-positive staining (Goldblatt et al. 2000) we have been
unable to find any true positive samples.

References

Bertram U, Halberg P. A specific antibody against the epithelium of the salivary ducts in the sera
from patients with Sjögren’s syndrome. Acta Allergol 1964; 19: 458-466.

Goldblatt F, Beroukas D, Gillis D, Cavill D, Bradwell A, Rischmueller M et al. Antibodies to AB

blood group antigens mimic anti-salivary duct autoantibodies in patients with limited sicca
symptoms. J Rheumatol 2000; 27: 2382-2388.

Endothelial Cell Antibodies

Antigen: Anti-endothelial cell antibodies (AECA) react with many different

antigens found within the endothelial cells of large arteries such as the aorta,
large veins such as the umbilical cord vein and also those found in the
microvasculature. Endothelial cells from different tissues are known to express
different antigens. Consequently, the specific antigens targeted by AECA
largely remain unknown, however identified antigens include β2GPI, heat
shock protein 60, α-enolase and phospholipids.

Clinical associations: Due to the diversity of recognised antigens, AECA are

associated with a wide range of diseases other than APS (refer to Chapter 12).
Because of this, they lack utility as specific disease markers, although in some
patients titres have been shown to correlate with disease activity.

Detection: Due to the lack of specificity there is no substrate in routine use for
the detection of AECA by IIF. However, human umbilical cord (Figure 12.4),
neonatal rat lung tissue or endothelial cells from other tissues can be used.
Western blots, RIA and EIA can also be used to detect AECA. Anti-
phospholipid and anti-β2 glycoprotein I antibodies are discussed in more detail
in Chapter 12.

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 Miscellaneous Autoantibody Specificities

References

Meroni PL, Youinou MD. Endothelial cell autoantibodies. In: Autoantibodies. Eds., Peter JB,
Shoenfeld Y, Elsevier Science B.V; 1996.

Wusirika R, Ferri C, Marin M, Knight DA, Waldman WJ, Ross P Jr et al. The assessment of anti-
endothelial cell antibodies in scleroderma-associated pulmonary fibrosis. A study of indirect
immunofluorescent and western blot analysis in 49 patients with scleroderma. Am J Clin Pathol
2003; 120: 596-606.

Youinou P. New target antigens for anti-endothelial cell antibodies. Immunobiology 2005; 210: 789-
797.

Yu F, Zhao MH, Zhang YK, Zhang Y, Wang HY. Anti-endothelial cell antibodies (AECA) in
patients with propylthiouracil (PTU)-induced ANCA positive vasculitis are associated with disease
activity. Clin Exp Immunol 2005; 139: 569-574.

Sperm Antibodies

Antigen: The target antigens of anti-sperm antibodies (ASA) remain unknown.

However, several potential antigens have been proposed: prolactin-inducible
protein, clusterin, alcohol dehydrogenase, annexin I, annexin III, BRCA1-
associated ring domain protein 1, heat shock 27kDa protein, isocitrate
dehydrogenase, lactoylglutathione lyase, NG-dimethylarginine, syntenin 1,
dimethylaminohydrolase 1 and peroxiredoxin 2. The most recent proposal
antigen is SPRASA (sperm protein reactive with anti-sperm antibodies), which
is found on the inner membrane of the acrosome.

Clinical associations: ASA have been associated with infertile couples and are
also found in fertile men; consequently not all ASA cause infertility. Other
associations include ulcerative colitis and otherwise healthy patients after
vasectomy. The effects ASA have on fertility are dependent upon the location
of the antigens recognised. ASA against the head may interfere with sperm
penetration and hence fertilisation. It is suggested that alterations to the integrity
of the sperm plasma membrane, caused by ASA, may adversely affect its
function in the fertilisation process. ASA against the tail-tip alone are probably
not pathogenic.

Detection: IIF using monkey testis or fresh spermatozoa will allow detection of
ASA. Fluorescence may be observed in the acrosome, equatorial region, post
acrosomal region, tail (Figure 14.3) and nucleus. ASA are predominantly IgG.

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IgM and IgA are rarely found and when identified, usually co-exist with IgG.
Agglutination assays using normal sperm are often used to diagnose
immunological infertility. EIAs are also used to detect ASA to specific antigens.

References

Tung KSK. Human sperm antigens and antisperm antibodies. Clin Exp Immunol 1975; 20: 93-104.

Rossato M, Galeazzi C, Ferigo M, Foresta C. Antisperm antibodies modify plasma membrane

functional integrity and inhibit osmosensitive calcium influx in human sperm. Hum Reprod 2004;
19: 1816-1820.

Figure 14.3. Antibodies against sperm tails shown in a mature monkey testis
(courtesy of F.X. Huchet, Institute Pasteur, Paris).

212
 Index

Index

A.

ABO blood group reactions 6, 121, 210

Acetyl choline receptor (AChR) antibodies 166 - 167
Actin antibodies 32, 35, 37 - 39, 64 - 65
Addison’s disease 32, 60, 98, 100 - 106
Adrenal gland 99-101
Alopecia areata 136
AMA (see mitochondrial antibodies)
Amphiphysin antibodies 33, 140-141, 156 - 157
ANCA (see anti-neutrophil cytoplasmic antibodies)
Annexin antibodies 187
Anti-neuronal nuclear antibodies type -3 (ANNA-3) 140, 151
Anti-neutrophil cytoplasmic antibodies (ANCA) 32 - 34, 59 - 60, 76,

Atypical ANCA 76, 192, 200 - 205

189 - 206

C-ANCA 189 - 196

P-ANCA 32 - 34, 189 - 199
Anti-nuclear antibodies (ANA) 43
Anti-phospholipid syndrome (APS) 32, 175 - 188
Anti-Saccharomyces cerevisiae antibodies (ASCA) 32, 59 - 60, 76 - 77
Aquaporin-4 antibodies (NMO-IgG) 33, 140, 164 - 165
ASCA (see anti-Saccharomyces cerevisiae antibodies)
Asialoglycoprotein receptor antibodies (ASGPR) 51 - 52
Autoimmune gastritis 32, 60
Autoimmune hepatitis (AIH) type 1 and 2 35 - 39, 45 - 57
Autoimmune hypophysitis (see lymphocytic hypophysitis)
Autoimmune polyglandular syndrome (APS) Type 1, 2 and 3 97 - 106
Autoimmune skin disease 119 - 138
Autoimmune thyrotoxicosis 112, 114

B.

Bactericidal permeability increasing factor (BPI) antibodies 201

Basal cell antibodies 127
Bile canalicular antibodies 56 - 57
Bile duct antibodies 56
Biliary epithelial cell antibodies 57

213
 Index

BP180 & BP230 120, 124, 127, 132

Bullous pemphigoid 122, 128, 130, 135
β2-Glycoprotein I (β2GPI) antibodies 176, 179 - 183

C.

Carbonic anhydrase II 54
Cardiolipin antibodies 176, 179 - 182
Cathepsin G antibodies 190, 200
Cholangitis 36, 76
Churg-Strauss syndrome 32, 196 - 198
Cicatrical pemphigoid 128, 130, 136
Coeliac disease 32, 59 - 60, 64 - 71
College of American Pathologists 29 - 30

Crohn’s disease (CD) 32, 59 - 60, 75 - 78

Crithidia luciliae 93

CV-2/CRMP5 antibodies 140 - 141, 152 - 153

Cyclic citrullinated peptide (CCP) 34, 207
Cytochrome P450 enzymes 32, 33, 45, 53, 99 - 100

D.

DABCO 13
de novo autoimmune hepatitis 58
Dermatitis herpetiformis 32, 64 - 65
Desmocollin 120, 124, 132
Desmoglein 120, 124, 131
Desmoplakin 120, 124, 133
Diabetes (see insulin dependent diabetes mellitus)
Dihydrolipoamide dehydrogenase (E3) 53
dsDNA antibodies 93

E.

Elastase antibodies 190, 200 - 201

Endocrine disease 97 - 118
Endomysial antibodies (EMA) (also see tissue transglutaminase) 59, 60,

Endothelial cell antibodies 210

64 - 69

Envoplakin 120, 124, 132

214
 Index

Enzyme Immunoassay (EIA) 19 - 26

Calibration 24 - 25
Commercial assays 22 - 23
Protocol 19 - 21
Validation 23 - 24
Enzyme immunohistochemistry 14
Epidermolysis bullosa acquisita 122, 128, 130, 136 - 137
Ethanol-fixed neutrophils 192, 197 - 199
Evans blue 13

F.

F-actin 37
Filaggrin antibodies 207 - 208
Fluorescein conjugated antibodies 11
Formalin-fixed neutrophils 192, 197 - 199

G.

G-actin 37
Gastric parietal cell (GPC) antibodies 32, 33, 59 - 61
Gastro-intestinal autoimmune diseases 59 - 80
Gliadin antibodies 59 - 60, 64, 68 - 69
Glial nuclear antibodies (AGNA) 140, 161
Glomerular basement membrane (GBM) antibodies 81 - 92
Glutamic acid decarboxylase (GAD65 & GAD67) 32, 34, 98, 108 - 111,

Glutathione-S-transferase (GST) 36, 52

140 - 141, 154 - 155

Goodpasture’s antigen (see glomerular basement membrane)

Goodpasture’s syndrome 32, 81 - 92
Gp210 43
Grave’s disease 98, 100, 112, 114

H.

Hashimoto’s thyroiditis 97 - 98, 112

Hepatitis (see autoimmune hepatitis)
HEp-2 cells 43
Herpes gestationis 32, 128, 130, 135
Heterophile antibodies 7 - 10, 61, 63, 70, 79

215
 Index

Hodgkin’s disease 146 - 159

Hu antibodies (ANNA-1) 33, 139 - 141, 148 - 149
Hyperthyroidism 112
Hypothyroidism 60, 98, 112 - 114

I.

IA-2 108 - 111

Idiopathic crescentic glomerulonephritis 32, 198
Idiopathic dilated cardiomyopathy (IDCM) 32, 172
IgA pemphigus 122, 130, 135
Immunoblotting 17 - 18
Immunofluoresence assay (IFA)
Interpretation 15 - 16
Procedure 12 - 13
Reporting 17
Inflammatory bowel disease (IBD) 59, 75 - 80
INSTAND 30
Insulin dependent diabetes mellitus (IDDM) 32, 60, 64, 98, 100, 107 - 111
International standards 28 - 29
Intrinsic factor antibodies 59, 61 - 62
Islet cell antibodies (ICA) 98, 108 - 111

J.

Jejenum 65 - 67
Juvenile Diabetes Foundation (JDF) units 110

L.

Lactoferrin antibodies 202

LAD-1 132
LAD97 127
Lambert Eaton myasthenic syndrome (LEMS) 33, 170
Latent autoimmune diabetes in adults (LADA) 108 - 110
Linear IgA bullous dermatosis 128, 136
Liver cytosol antigen (LC1) 36, 48 - 50
Liver disease (see autoimmune liver disease)
Liver kidney microsomal (LKM) type 1, 2 & 3 32, 35 - 36, 45 - 53
Liver membrane antigen 54

216
 Index

Liver microsomal (LM) antibodies 53

Liver specific membrane lipoprotein (LSP) 54
Louisville calibrators 29, 181
Lupus anticoagulant 176 - 177
Lupus nephritis 93
Lymphocytic hypophysitis 33, 98, 116

M.

Ma (Ma1, 2 &3) antibodies 33, 140, 158

Metabotropic glutamate neurotransmitter receptor antibodies 140, 159
Methanol-fixed neutrophils 192
Microscope maintenance 13 - 14
Microscopic polyangiitis 33, 193 - 198
Mitochondrial antibodies (AMA) 36, 40 - 44, 50
M2 antibodies 33, 35, 40 - 44, 50
Modified gliadin peptide antibodies 69
Monkey bladder 121, 125
Morvan’s syndrome 33, 171
Mounting medium 13
Muscle disease 166-174
Muscle specific receptor tyrosine kinase (MUSK) antibodies 169
Myasthenia gravis 33, 166 - 168
Myelin associated glycoprotein (MAG) antibodies 140, 161 - 163
Myeloperoxidase (MPO) antibodies 190, 198 - 199
Myocardial antibodies 172 - 173
Myocarditis 33, 172

N.

Naturally occurring anti-mitochondrial antibodies (NOMAs) 41

NEQAS 29
Neuromyelitis optica (NMO) 33, 164 - 165
Neutrophil antibodies (see anti-neutrophil cytoplasmic antibodies)
NIBSC 28

O.

OmpC antibodies 77
Ovarian antibodies 99-105

217
 Index

P.

Pancreatic antibodies 76 - 78
Pancreatic islet cell antibodies (see Islet cell antibodies)
Paraneoplastic neurological syndrome (PNS) 33, 139 - 161
Paraneoplastic pemphigus 33, 122, 125, 130, 135
Pemphigoid (also see autoimmune skin diseases) 33, 123
Pemphigus (also see autoimmune skin diseases) 33, 123, 126
Pemphigus erythematosus 128
Pemphigus foliaceus 122, 130, 134
Pemphigus herpetiformis 130, 137
Pemphigus vulgaris 122, 130, 134
Periplakin 120, 124, 132
Pernicious anaemia 33, 59 - 62
Phosphatidic acid antibodies 185
Phosphatidylcholine antibodies 185
Phosphatidylethanolamine antibodies 185
Phosphatidylinositol antibodies 185
Phosphatidylserine antibodies 184
Pituitary gland antibodies 98, 117
Placenta antibodies 98-100, 106
Plectin 132
Polyarteritis nodosa 33
Postpartum thyroiditis 112 - 113
Primary biliary cirrhosis (PBC) 28, 33, 35, 36 - 57
Primary hypothyroidism 114
Primary sclerosing cholangitis (PSC) 33, 51, 57, 76
Proteinase 3 (PR3) antibodies 190, 196 - 197
Prothrombin antibodies 186
Purkinje cell 143-147
Purkinje cytoplasmic antibodies type 2 (PCA-2) 140, 147
Pyruvate dehydrogenase complex 40-41

Q.

Quality control schemes 29 - 30

R.

Rat oesophagus 208

218
 Index

Renal disease 81-94

Reticulin antibodies (R1) 59 - 60, 64, 70 - 73
Reticulin antibodies (R2) 71 - 74
Reticulin antibodies (Rs) 71, 74, 75
Rheumatoid arthritis 34, 207 - 208
Ri antibodies (ANNA-2) 33, 141, 150
Ryanodine receptor (RyR) antibodies 167

S.

Salivary duct antibodies 209

Salt-split skin 119, 127 - 128
Sapporo monoclonal antibodies 28, 181 - 183
Skeletal muscle 166-168
Skin disease 119-138
Small cell lung carcinoma (SCLC) 140, 147-148, 150, 152, 156, 160-161,

Smooth muscle antibodies (SMA) 35 - 39, 65

170-171

Soluble liver antigen (SLA) 36, 50 - 51

Sp100 43
Species-specific conjugates 3 - 5, 119
Sperm antibodies 211
Steroidal cell antibodies 105
Stiff-person syndrome (SPS) 34, 154 - 156
Striational muscle antibodies 33, 167 - 168
Systemic lupus erythematosus (SLE) 93, 175, 179 - 182

T.

Testis antibodies 98-100, 103, 158-159

Theca cells 100, 102, 104
Thymoma 167-169
Thyroglobulin (Tg) antibodies 34, 97 - 98, 113 - 115
Thyroid autoimmune disease 112-115
Thyroid peroxidase (TPO) antibodies 34, 97 - 98, 113 - 115
Thyrotrophin receptor antibodies 114
Tissue-transglutaminase (tTG) antibodies 32, 64 - 69
Titin antibodies (see striational muscle antibodies)
Tr antibodies (PCA-Tr) 140 - 141, 146
Tubular basement membrane antibodies 91 - 92

219
 Index

Type 1 autoimmune diabetes mellitus (see insulin dependent diabetes mellitus)

U.

Ulcerative colitis (UC) 34, 59 - 60, 75 - 79

V.

Vasculitis 189-206
Vitiligo 136
Voltage gated calcium channel (VGCC) antibodies 33, 170
Voltage gated potassium channel (VGKC) antibodies 33, 171

W.

Wegener’s granulomatosis 34, 190, 193-198

Y.

Yo antibodies (PCA-1) 33, 140, 144 - 145

Z.

Zic4 antibodies 160

220
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