Handbook of Direct Immunofluorescence: A Pattern-Based Approach to Skin and Mucosal Biopsies Douglas R Fullen, May P Chan, Aleodor A Andea, David P Arps
Note: Page numbers in italic refer to figures.
Alpha-2-macroglobulin-like-1 protein (A2ML1) 51, 54, 55
A2ML1 see Alpha-2-macroglobulin-like-1 protein (A2ML1)
Anchoring fibrils 81, 87
Annular erythema 39
Antiendothelial antibodies 155
Antineutrophil cytoplasmic antibodies (ANCA) 155
Antinuclear antibodies (ANAs) 39, 40, 45, 119
homogeneous pattern 40, 4142, 42
rim (nuclear membrane) pattern 42, 42
speckled pattern 4041, 42, 43
Anti-p200 pemphigoid 84, 107109
Atrophie blanche see Livedoid vasculopathy (LV)
Basement membrane zone (BMZ) 45, 53, 54, 65, 66, 66, 68, 69, 69, 72, 75, 76, 78, 81, 83, 87, 88, 90, 90, 91, 93, 94, 95, 97, 98, 98, 99, 102, 103, 104, 119, 120, 122, 123, 134, 135, 135, 136, 136, 137, 142, 144, 168
BLE see Bullous lupus erythematosus (BLE)
BMZ see Basement membrane zone (BMZ)
BP see Bullous pemphigoid (BP)
BP180 65, 70, 75, 78, 93, 94, 97, 98, 101, 103, 108
BP230 65, 70, 75, 78, 87, 93, 101, 103
BPAg1 see BP230
BPAg2 see BP180
Brunsting–Perry variant see Mucous membrane pemphigoid
Bullosis diabeticorum 177
Bullous impetigo, 17
Bullous lupus erythematosus (BLE) 68, 84, 8791, 93
Bullous pemphigoid (BP) 7, 10, 15, 51, 6572, 75, 82, 89, 93, 101, 133
urticarial (prebullous) stage 66
Castleman disease 51
Caterpillar bodies 167, 169
CBDC see Chronic bullous dermatosis of childhood (CBDC)
Celiac disease 113
Chronic bullous dermatosis of childhood (CBDC) 93
Chronic ulcerative stomatitis (CUS) 4548
Cicatricial pemphigoid see Mucous membrane pemphigoid (MMP)
Cirrhosis 161
Civatte bodies 134, 134136, 136, 137, 141
Colloid bodies 101, 102, 102, 104, 134, 134136, 136, 137, 141
Connective tissue diseases 3943, 46, 113, 119
Corticosteroids 45, 68, 133, 155
CREST syndrome 39
Crohn disease 81
CUS see Chronic ulcerative stomatitis (CUS)
CUS protein (CUSP) 45
Cytoid bodies 45, 53, 54, 55, 119, 121, 123, 124, 128, 129, 141142 142145
DEJ see Dermoepidermal junction (DEJ)
Deoxyribonucleic acid (DNA) 39, 123, 127
Dermatitis herpetiformis (DH) 113117
Dermatomyositis 39 see also Connective tissue diseases
Dermoepidermal junction (DEJ) 40, 54, 5758, 60, 61, 89, 97, 98, 101, 102, 103, 107, 109, 115, 116, 117, 120, 121, 127, 128, 128, 129, 129, 134, 145, 167, 168, 170, 171, 173, 175
Desmocollin 9, 21, 25, 27, 31, 34
Desmoglein 1 (Dsg1) 3, 9, 15, 21, 31, 34, 51, 57, 58
Desmoglein 3 (Dsg 3) 3, 9, 15, 21, 31, 34, 51, 59
Desmoplakin 51
Desmosomal plaque 3, 9, 15, 57
Desmosome 51
Desquamative gingivitis 75, 101
Discoid lupus erythematosus (DLE) 119 see also Lupus erythematosus (LE)
DLE see Discoid lupus erythematosus (DLE)
DNA see Deoxyribonucleic acid (DNA)
EBA see Epidermolysis bullosa acquisita (EBA)
Eccrine coil 180, 181
EM see Erythema multiforme (EM)
Envoplakin 51, 54, 55
Eosinophilic spongiosis 3, 15, 21, 66
Epidermal transglutaminase (eTG) 113
Epidermolysis bullosa acquisita (EBA) 65, 77, 8184, 89, 93
Epiligrin 75, 78
Epitope spreading 3, 15, 31, 51, 57, 59, 87, 101
Erythema multiforme (EM) 52, 93, 127, 129, 141
Extractable nuclear antigens (ENAs) 39, 41, 42, 43
FDE see Fixed drug eruption (FDE)
Festooning 167, 168, 169, 173, 174
Fibrinoid necrosis 156
Finkelstein's disease 155 see also Hemorrhagic edema of infancy
Fixed drug eruption (FDE) 141
Fogo selvagem (FS) 15
FS see Fogo selvagem (FS)
Gammopathy 25, 31, 51
Gluten-sensitive enteropathy (GSE) 113
GSE see Gluten-sensitive enteropathy (GSE)
Halogenoderma 9
Hemidesmosome 51, 65
Hemorrhagic edema of infancy 155
Henoch–Schönlein purpura (HSP) 150151, 155158
Hepatitis C 133, 167
Herpes gestationis see Pemphigoid gestationis (PG)
Histone 39
HSP see Henoch-Schönlein purpura (HSP)
Human leukocyte antigen (HLA) 3, 15, 75, 81, 97, 113, 133, 155
Hydroxycloroquine 45
Hypersensitivity vasculitis see Leukocytoclastic vasculitis (LCV)
IEN see Intraepidermal neutrophilic (IEN) variant
IgA paraneoplastic pemphigus 25
IgA pemphigus see Immunoglobulin A (IgA) pemphigus
IgA vasculitis 155
IgG4 15, 18, 21, 65, 66, 75, 108
IgG/IgA pemphigus 3134
Immunoglobulin A (IgA) pemphigus 2526
Inflammatory bowel disease 9, 31, 81
Integrin 75, 78
Interface dermatitis/mucositis 141145
Interface tissue reaction 141
Intraepidermal neutrophilic (IEN) variant 25, 27, 28
La (SSB) 42, 43
LABD see Linear IgA bullous dermatosis (LABD)
Lamina densa 65, 67, 71, 87, 89
Lamina lucida 6567, 71, 89, 93
Laminin 332 (laminin 5) 75, 78
Laminin γ-1 107, 109
LCV see Leukocytoclastic vasculitis (LCV)
LE see Lupus erythematosus (LE)
Leukocytoclasis 149, 150, 156
Leukocytoclastic vasculitis (LCV) 149152
Lichen planopilaris 133
Lichen planus (LP) 45, 52, 65, 101, 133137
Lichen planus pemphigoides (LPP) 101104
Linear IgA bullous dermatosis (LABD) 9395
Livedoid vasculopathy (LV) 177, 179
LP see Lichen planus (LP)
LPP see Lichen planus pemphigoides (LPP)
Lupus band 57, 58, 60, 88, 122, 123, 128, 136
Lupus band test (LBT) 120123, 127
Lupus erythematosus (LE) 39, 57, 58, 60, 119124, 127, 128, 136, 150, 178 see also Connective tissue diseases
Lymphoma 25, 51, 113
Max-Joseph space 101
Milia 75, 81, 107, 167, 173
Mixed connective tissue disease 39
Monkey esophagus 5, 9, 18, 21, 26, 33, 59, 69, 116
Mucous membrane pemphigoid (MMP) 45, 65, 7578, 8284, 93, 108
autoimmune disease 75
Brunsting–Perry variant 75
Myeloma 25
NC16A 65, 70, 98, 101, 103
Neutrophilic karyorrhexis 87, 89
Neutrophilic spongiosis 21, 25, 26
Nikolsky sign 3, 15, 57
Nonbullous neutrophilic lupus erythematosus 8788
n-serrated pattern 68, 77, 78, 82, 95, 107, 109
Paraneoplastic autoimmune multiorgan syndrome (PAMS) 51 see also Paraneoplastic pemphigus (PNP)
Paraneoplastic pemphigus (PNP) 5155, 101
PCT see Porphyria cutanea tarda (PCT)
PE see Pemphigus erythematosus (PE)
Pemphigoid gestationis (PG) 9799
Pemphigus erythematosus (PE) 5761
Pemphigus foliaceus (PF) 3, 1518, 21, 31, 57
Pemphigus herpetiformis (PH) 2123, 31
Pemphigus vegetans 913
Hallopeau type (HT) 9
Neumann type (NT) 9
Pemphigus vulgaris (PV) 38, 15, 21, 25, 31
Penicillamin 57, 65, 87
Periplakin 51, 54, 55
PF see Pemphigus foliaceus (PF)
PG see Pemphigoid gestationis (PG)
Pigmented purpuric dermatoses (PPD) 161, 163
Plectin 51
PNP see Paraneoplastic pemphigus (PNP)
Porphyria cutanea tarda (PCT) 167171, 173
Porphyrin 169, 173
Pruritic urticarial papules and plaques of pregnancy (PUPPP) 97
Pseudoporphyria 173175
PUVA-induced blisters 127, 128
PV see Pemphigus vulgaris (PV)
Pyodermatitis-pyostomatitis vegetans 9
Rat bladder 54, 103
Ribonucleoprotein (RNP) 41
Ro (SSA) 41, 42, 43
‘Row of tombstones’ 3, 5
Salt-split skin (SSS) 67, 71, 76, 89, 93, 98, 108
Sandwich double antibody immunofluorescence method 66
Scarring 65, 75, 77, 81, 82, 107, 113, 119, 120, 133, 167, 173
Scl-70 41, 42, 43
Scleroderma 39, 41 see also Systemic sclerosis
Senear–Usher syndrome see Pemphigus erythematosus (PE)
Seroreversion 55
Sjögren syndrome 39 see also Connective tissue diseases
annular erythema in 39
Sm antigen 41
Solar elastosis 127, 128
SPD variant see Subcorneal pustular dermatosis (SPD) variant
Stasis dermatitis 177
Stevens–Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN) 141
‘String of beads’ 93
Subacute cutaneous lupus erythematosus (SCLE) 119 see also Lupus erythematosus (LE)
Subcorneal pustular dermatosis (SPD) variant 25, 26, 28
Sweat gland 128, 178
Systemic lupus erythematosus (SLE) 87, 119
Systemic sclerosis 39, 41 see also Connective tissue diseases
Tissue transglutaminase (tTG) 113
Type IV collagen 68, 71, 87
Type VII collagen 75, 78, 81, 84, 87, 89, 90, 93, 108, 109, 123
Tzanck smear 3
Ulcerative colitis 11, 81
Ultraviolet light 57, 127, 167, 173
Uroporphyrinogen decarboxylase 167, 169
Urticarial vasculitis 149
u-serrated pattern 77, 82, 83, 83
Vacuolar interface dermatitis 39, 40, 57, 141, 142, 144
Vancomycin 93
Waldenstrom macroglobulinemia 51
Wickham's striae 101, 133
Chapter Notes

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Handbook of Direct Immunofluorescence A Pattern-Based Approach to Skin and Mucosal Biopsies
Handbook of Direct Immunofluorescence A Pattern-Based Approach to Skin and Mucosal Biopsies
Douglas R Fullen MD Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA May P Chan MD Associate Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA Aleodor A Andea MD Professor of Pathology and Dermatology, University of Michigan, Ann Arbor, MI, USA David P Arps MD Dermatopathologist, Consolidated Pathology Consultants, Libertyville, IL, USA
© 2018 JP Medical Ltd.
Published by JP Medical Ltd,
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The rights of Douglas R Fullen, May P Chan, Aleodor A Andea and David P Arps to be identified as authors of this work have been asserted by them in accordance with the Copyright, Designs and Patents Act 1988.
All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission in writing of the publishers. Permissions may be sought directly from JP Medical Ltd at the address printed above.
All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book.
Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the authors assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book.
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British Library Cataloguing in Publication Data
A catalogue record for this book is available from the British Library
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A catalog record for this book is available from the Library of Congress
Richard Furn
Development Editor:
Gavin Smith
Editorial Assistant:
Katie Pattullo
Designers Collective Ltd
Handbook of Direct Immunofluorescence: A Pattern-Based Approach to Skin and Mucosal Biopsies is a guide for practicing general pathologists, dermatopathologists, and oral and ophthalmic pathologists who interpret biopsies from patients with inflammatory and blistering disorders requiring direct immunofluorescence examination. The book will also serve as a resource for clinicians who care for patients with these disorders and trainees who are studying in these fields.
This book consists of an introductory section that describes immunofluorescence techniques and protocols, followed by 29 chapters covering the range of conditions which the pathologist or clinician is likely to encounter using direct immunofluorescence examination. Each chapter succinctly describes the clinical presentation, pathogenesis, histopathology, direct immunofluorescence and, when appropriate, indirect immunofluorescence and enzyme-linked immunosorbent assays for the specific disorders. Multiple images show the histopathologic and direct immunofluorescence findings of the entities discussed in each chapter. An additional strength of this book is the inclusion of nonspecific direct immunofluorescence findings that are commonly observed in practice. A limited number of essential references are included in each chapter.
We believe this book will aid in understanding the technical aspects and problems that affect specimen procurement, transportation and processing for direct immunofluorescence examination. The pattern-based approach to direct immunofluorescence will serve as a logical framework for learning about these disorders, for reaching diagnoses, for understanding when additional serologic studies may be helpful, and in recognizing nonspecific findings on biopsy specimens submitted for this purpose.
Douglas R Fullen
May P Chan
Aleodor A Andea
David P Arps
January 2018
To Angela, Peter, Aileen and my mentors, Dr J T Headington,
Dr J A Reed and Dr N S McNutt, for their support and encouragement
To Alice, Danny and all of my mentors
To my son, Andrew
To Joyce, Jennifer, Nicholas, Monisha, Nikolai, David, Gideon and Ladd
Introduction: immunofluorescence techniques and protocols  
Immunofluorescence is a laboratory technique used to document the presence of auto-antibodies either in tissues or circulating in blood. It provides useful information for the diagnosis and treatment of certain skin disorders. All immunofluorescence tests are based on the detection of fluorescence-linked antibodies against immunoglobulins, complement or proteins such as fibrinogen.
Fluorescent techniques use fluorochromes: when stimulated with light of a specific wavelength, these substances emit light with a longer wavelength. The most commonly used fluorochrome in dermatologic immunofluorescence is fluorescein isothiocyanate (FITC). Less commonly, tetramethylrhodamine isothiocyanate (TRITC) is employed. The fluorochrome is linked to the antibody via a thiocarbamide linkage which does not destroy its immunoreactivity.1
Biopsy and tissue transport
Choosing an appropriate skin biopsy site is an important component for optimal results using direct immunofluorescence (DIF) and the selection depends on the suspected clinical diagnosis. In the absence of a favored clinical diagnosis perilesional skin should be sampled.2 However, depending on the clinical differential diagnosis the biopsy protocol can be modified as follows:
For autoimmune blistering diseases, the optimal biopsy is from the perilesional area.2,3 In connective tissue diseases, the biopsy should be taken from active lesions in evolution if chronic cutaneous lupus is suspected, or from both lesional and nonlesional sun-protected skin if systemic lupus erythematosus is considered (see Chapter 20). In cases where vasculitis is suspected, the biopsy should be collected from recent lesions, preferably under 24 hours of evolution.3 Since often a lesional biopsy for histologic diagnosis is collected at the same time with the DIF biopsy, one can consider performing a biopsy at the edge of the lesion and bisecting the tissue. This practice is controversial, with some authors opposing it due to the risk of inadvertently detaching the epidermis; however, one study seems to suggest that bisection of the tissue does not result in a higher rate of inadequate specimens.4
For lesions of the oral mucosa, it is recommended that two biopsies be taken, one from lesional tissue and one from adjacent normal mucosa. A punch biopsy is usually performed; however, incisional biopsy is the technique of choice in case of hard palate and gingival lesions because punch at these sites often provides unsatisfactory tissue for DIF.57
If the test is not performed immediately and the specimen needs to be transported to another location (which is more often the case), the biopsies should be placed in Michel (or Zeus) transport medium which contains ammonium sulfate, N-ethyl maleimide and magnesium sulfate in citrate buffer8 and allows the specimens to maintain immunoreactivity for about 2 weeks at room temperature (up to 6 months in some cases).7,9 Alternatively, the specimen can be placed on saline-soaked gauze if it is submitted directly to the laboratory to be processed the same day or snap-frozen immediately if the test is performed on site.
Biopsies for DIF should not be placed in formalin. Tissues accidentally immersed in formalin for <10 min may still yield positive results for pemphigoid and dermatitis herpetiformis (but not for pemphigus). Longer exposure to formalin compromises the DIF diagnosis for all entities.10
For indirect immunofluorescence (IIF), blood is collected without anticoagulant. Serum is separated from the clotted blood. If it is not tested immediately it should be stored at –20°C.
Direct immunofluorescence
Direct immunofluorescence is used to identify the presence of autoantibodies, complement and fibrinogen bound to tissue antigens. It is a one-step procedure in which fluorescent-labelled antibodies against immunoglobulins, complement and fibrinogen are incubated with the patient's skin biopsy. The usual protocol consists of the following steps:
Positive controls for IgG, IgA, IgM, and C3 are run with each DIF batch. The laboratory needs to identify recent tissue samples positive for each antibody that can be used as positive controls. Slides are cut and stored at –80°C. Control slides are stained in an identical fashion to the patients' slides. They are replaced as needed and as good control tissue becomes available. One slide per patient will also be run with the primary antibody step omitted (only reaction buffer) to serve as a negative reagent control.
Controls are routinely reviewed by a laboratory director and any problems are discussed with the technical staff. Staining is repeated for any isolated failure; antibody incubation times may need to be readjusted if problem persists.
Indirect immunofluorescence
Indirect immunofluorescence (IIF) is used to detect the presence of circulating autoantibodies. IIF is a two-step procedure.
In the first step the patient's serum is incubated with frozen section slides from an appropriate substrate (such as human epidermis, monkey esophagus or rat bladder). If autoantibodies are present in the serum they will bind to the substrate.
In the second step a FITC-labelled antibody of defined type (usually anti-IgG or anti-IgA) is incubated with the tissue. The usual protocol consists of the following steps:
The initial serum dilution is usually 1:10 or 1:80. If it is positive, higher dilutions are used to determine the serum titer.2 Control sera from known positive and negative patients are run along with the samples.
Salt-split skin
This technique is used in autoimmune subepidermal blistering diseases to map the location of the antigen with respect to the lamina lucida. It can be employed for both DIF and IIF; however, it is usually used for the latter. The protocol entails incubation of the patient's perilesional skin (for DIF) or normal human skin (for IIF) with 1M NaCl solution for 48–72 hours. This step induces an artificial split through lamina lucida. The regular protocols for DIF or IIF are employed next and the biopsy is examined under a fluorescent microscope.
  1. Mohan KH, Pai S, Rao R, Sripathi H, Prabhu S. Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol 2008;74:415–419.
  1. Huilgol SC, Bhogal BS, Black MM. Immunofluorescence of the immunobullous disorders part one: Methodology. Indian J Dermatol Venereol Leprol 1995;61:187–195.
  1. Aoki V, Sousa JX, Jr., Fukumori LM, et al. Direct and indirect immunofluorescence. An Bras Dermatol 2010;85:490–500.
  1. Loh E, Armstrong AW, Fung MA. Pre-bisection of a single skin biopsy does not produce technically inadequate specimens for direct immunofluorescence: a review of 3450 specimens. J Cutan Pathol 2014;41:890–892.
  1. Sano SM, Quarracino MC, Aguas SC, et al. Sensitivity of direct immunofluorescence in oral diseases. Study of 125 cases. Med Oral Patol Oral Cir Bucal 2008;13:E287–291.
  1. Siegel MA. Intraoral biopsy technique for direct immunofluorescence studies. Oral Surg Oral Med Oral Pathol 1991;72:681–684.
  1. Tapia JL, Neiders ME, Suresh L. Indications and procedures for direct immunofluorescence biopsies of the oral mucosa. Quintessence Int 2015;46:247–253.
  1. Michel B, Milner Y, David K. Preservation of tissue-fixed immunoglobulins in skin biopsies of patients with lupus erythematosus and bullous diseases – preliminary report. J Invest Dermatol 1972;59:449–452.
  1. Vaughan Jones SA, Salas J, McGrath JA, et al. A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel's medium. Dermatology 1994;189:131–132.
  1. Arbesman J, Grover R, Helm TN, Beutner EH. Can direct immunofluorescence testing still be accurate if performed on biopsy specimens after brief inadvertent immersion in formalin? J Am Acad Dermatol 2011;65:106–111.