Immunohistology in Dermatopathology

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AP119 Immunohistology inDermatopathology

Mark R. Wick, MDUniversity of VirginiaCharlottesville, VA

© College of American Pathologists 2004. Materials are used with the permission of

Mark R. Wick, MD, FCAP.

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AgendaAgendaLecture 1: Immunohistology in non-neoplastic skin diseasesLecture 2: Immunohistology in the evaluationof undifferentiated skin tumors

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Lecture 3: Immunohistology in the analysisof differentiated cutaneous neoplasmsLecture 4: Immunohistologic evaluation ofcutaneous lymphoid infiltrates

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DisclosureDisclosure

The Speaker has no personal, professional, orfinancial interests in any of the productsor techniques that will be covered in the

following lectures.

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Course Objectives

• To understand the general goals of immunohistology in relation to non-neoplastic skin diseases and cutaneous neoplasms

• To be able to relate specific immunofluorescenceabnormalities to particular dermatoses & dermatitides

• To make rational reagent choices in regard to differential diagnosis of skin tumors with immunohistology

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IMMUNOHISTOLOGY INNON-NEOPLASTIC

DERMATOPATHOLOGY

IMMUNOHISTOLOGY INNON-NEOPLASTIC

DERMATOPATHOLOGY

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IMMUNOHISTOLOGY IN NON-NEOPLASTIC

DERMATOPATHOLOGY

IMMUNOHISTOLOGY IN NON-NEOPLASTIC

DERMATOPATHOLOGY• Although immunohistology (IHL) is

conceptualized by pathologists as being a tool for evaluation of tumors,

its principal application in dermatopathology is in non-

neoplastic disease• Paradigms have been in use for 25

years for diagnosis of inflammatory skin diseases by IHL

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IMMUNOHISTOLOGIC METHODS FOR USE IN NON-NEOPLASTIC DERMATOPATHOLOGY

IMMUNOHISTOLOGIC METHODS FOR USE IN NON-NEOPLASTIC DERMATOPATHOLOGY

• Direct Immunofluorescence--One-step procedure using skin biopsy from patient in

question, and fluoresceinated anti-human antibodies (to Ig, C’, fibrin, etc.)

• Indirect Immunofluorescence--Two-step process wherein non-patient substrates (skin; esophagus) are first incubated with patient serum, and then

exposed to tagged anti-human antibodies

• Visualization--Immunofluorescence microscopy with UV excitation is used

with both methods

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SALINE SPLIT-SKIN TECHNIQUE FOR INDIRECT IF MICROSCOPY

SALINE SPLIT-SKIN TECHNIQUE FOR INDIRECT IF MICROSCOPY• Of principal use in distinguishing between bullous pemphigoid and epidermolysis bullosa

acquisita• Biopsies of normal skin are obtained from human

volunteers• Biopsy specimens are incubated in 1M saline at 4oC for

72 hrs.; the epidermis and dermis are subsequently separated from each other manually

• Cleavage occurs between the lamina lucida & the remainder of the epidermal BMZ

• BP antigen is in roof of artificial “blister;” EBA antigen is in the base

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USE OF FORMALIN-FIXED, PARAFFIN-EMBEDDED

SPECIMENS OF SKIN FOR IF

USE OF FORMALIN-FIXED, PARAFFIN-EMBEDDED

SPECIMENS OF SKIN FOR IF• Some accounts in the literature

report success with DIF using formalin-fixed, paraffin-embedded samples of skin, after pretreatment

with 1% NH4OH• Attempts to reproduce this technique

in the speaker’s laboratory have not produced satisfactory results

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SKIN DISEASES THAT OFTEN NECESSITATE IMMUNOHISTOLOGY

FOR DEFINITIVE DIAGNOSIS

SKIN DISEASES THAT OFTEN NECESSITATE IMMUNOHISTOLOGY

FOR DEFINITIVE DIAGNOSIS• Pemphigus variants

• Bullous pemphigoid & congeners• Epidermolysis bullosa acquisita

• Dermatitis herpetiformis• Lupus erythematosus

• Selected cutaneous vasculitides

11Epidermal Basement Membrane Zone Antigens in Bullous Diseases

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PEMPHIGUS VARIANTSClinicopathologic Features

PEMPHIGUS VARIANTSClinicopathologic Features

• Subclassified as pemphigus vulgaris, p. foliaceus, p. vegetans, & p. erythematosus based on level of

acantholysis in the epidermis & selected other clinical features

• Fogo selvagem (“endemic Brazilian pemphigus”) is probably an infectious disease

• Immunological (antibody-mediated) attack on plakoglobin, desmoglein, and other components of desmosomal complexes results in acantholysis in

pemphigus• IgG1 and C’3 are observed in a mosaic pattern,

outlining individual keratinocytes• P. erythematosus also demonstrates the presence

of a “lupus band” of Ig/C3 at the BMZ

13Pemphigus

14Indirect Immunofluorescence for IgG in Pemphigus Vulgaris

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SUBEPIDERMAL IMMUNOLOGICAL BULLOUS DISEASES

SUBEPIDERMAL IMMUNOLOGICAL BULLOUS DISEASES

• Bullous pemphigoid• Epidermolysis bullosa acquisita

• Herpes gestationis• Dermatitis herpetiformis

• Linear IgA disease• Bullous disease of childhood

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DERMATITIS HERPETIFORMIS:Clinicopathologic Features

DERMATITIS HERPETIFORMIS:Clinicopathologic Features

• Onset usually in adulthood• Intensely itchy, pustular and bullous lesions that

tend to cluster on the dorsal extremities• Association with other immunologic or quasi-immune diseases such as lymphocytic thyroiditis,

sprue, & pernicious anemia• Responds to dapsone and gluten-free diet

• Biopsy shows aggregates of neutrophils in the papillary dermis, becoming confluent and

detaching the overlying epidermis

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SIMILARITIES & DIFFERENCES IN THE IgA-RELATED BULLOUS DERMATOSESSIMILARITIES & DIFFERENCES IN THE

IgA-RELATED BULLOUS DERMATOSES

• DH, LID, & BDC all show an association with the HLA allele B8

• Pattern of IgA deposition differs; granular and discontinuous at BMZ in DH; linear at

BMZ in LID/BDC• Only patients with DH show circulating

antibodies to smooth muscle (anti-endomysial Aby) & have intestinal disease

clinically• The 3 diseases are considered to

represent a “family” of conditions

18Dermatitis Herpetiformis

19Direct Immunofluorescence for IgA in Dermatitis Herpetiformis

20Direct Immunofluorescence for IgA in Linear IgA Dermatosis

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BULLOUS PEMPHIGOIDClinicopathologic FeaturesBULLOUS PEMPHIGOIDClinicopathologic Features

• Bimodal; seen in childhood and again in elderly patients

• Tense bullae that break spontaneously• Trunk, extremities, and mucosal surfaces

affected relatively equally• In the eyes, the “cicatricial” variant is

important• Subepidermal cleft microscopically;

mixed, usually scant inflammatory infiltrate• Linear IgG and/or C3 at the BMZ by DIF

22Bullous Pemphigoid

23Direct Immunofluorescence for IgG in Bullous Pemphigoid

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EPIDERMOLYSIS BULLOSA ACQUISITA

Clinicopathological Features

EPIDERMOLYSIS BULLOSA ACQUISITA

Clinicopathological Features• Generally more restricted in lesional

distribution than BP• Rare cases of disseminated EBA do resemble

pemphigoid clinically• Potential association with visceral malignancy,

unlike BP• May be histologically-identical to BP

• Definable as different from pemphigoid only by detailed IHL

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Epidermolysis Bullosa Acquisita

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HERPES GESTATIONISHERPES GESTATIONIS

• Basically a form of bullouspemphigoid that occurs during or immediately following pregnancy• Differs from classic BP only in showing a restriction to C3 (no

Ig) at the BMZ by DIF

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A PRACTICAL ALTERNATIVE TO SALINE SPLIT SKIN PREPSA PRACTICAL ALTERNATIVE

TO SALINE SPLIT SKIN PREPS• DIF on patient biopsies; those with linear

staining for IgG at BMZ and subepidermalblisters are then studied further to

distinguish BP from EBA• Paraffin sections are stained for collagen

type IV; BP antigen is superficial to CIV (which is in lamina densa), and EBA

antigen is deep to it. CIV is therefore in roof of EBA blisters & base of

BP blisters

28Immunohistology for Collagen Type IV in Lesional Skin of Bullous Pemphigoid

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CUTANEOUS LUPUS ERYTHEMATOSUS

Clinicopathologic Features

CUTANEOUS LUPUS ERYTHEMATOSUS

Clinicopathologic Features

• Scaly, erythematous lesions, particularly in sun-exposed skin areas

• Discoid form is limited to the skin• Histology is generally that of a lichenoid &

interface dermatitis, making IHL desirable for further Dx

• DIF shows granular deposition of Igand/or C3 at the epidermal BMZ

• “Lupus band” is present in systemic form

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BULLOUS LUPUS ERYTHEMATOSUS

31Cutaneous Lupus Erythematosus– Direct Immunofluorescence for IgG

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IHL IN OTHER NON-BULLOUS DISEASES OF THE SKIN

IHL IN OTHER NON-BULLOUS DISEASES OF THE SKIN

• Lichen planus/planopilaris-- deposition of IgM, IgG, IgA, and/or C3 on “colloid bodies”

(damaged keratinocytes) at or above the BMZ; usually not necessary for definitive diagnosis

• Vasculitides-- deposition of immune complexes in arterioles or venules of the dermis; Ig class

varies with type of vasculitis• Drug-induced dermatitides show no consistent

pattern of immunological abnormality

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Lichen planus– Hematoxylin& Eosin Stain and Direct

ImmunofluorescenceFor IgM

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Cinical & HistologicImages of Cutaneous

Small-VesselVasculitis

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Direct Immunofluorescence for IgAIn Henoch-Schonlein Vasculitis of Skin

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REASONS FOR DESIRABILITY OF IMMUNOSTAINS FOR

INFECTIOUS AGENTS

REASONS FOR DESIRABILITY OF IMMUNOSTAINS FOR

INFECTIOUS AGENTS1. In-situ demonstration of infectious organisms in the

context of tissue responses in histologic sections

2. Rapid, specific identification of organisms that may be difficult to

culture or extremely slowly-growing

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COMMERCIAL IMMUNOSTAINS FOR VIRAL AGENTS IN

CUTANEOUS HUMAN DISEASE

COMMERCIAL IMMUNOSTAINS FOR VIRAL AGENTS IN

CUTANEOUS HUMAN DISEASE

• Herpes simplex I & II• Zoster-Varicella virus

• Cytomegalovirus• Human papillomavirus (pan-capsid

protein)• Epstein Barr virus (indirect detection

via EBV latent membrane protein)

Herpes simplex Dermatitis: Clinical ImageHerpes simplex Dermatitis: Clinical Image

Herpetic Dermatitis: Histologic ImagesHerpetic Dermatitis: Histologic Images

Herpes Simplex: Positive ImmunostainHerpes Simplex: Positive Immunostain

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IMMUNOSTAINS FOR FUNGAL AGENTS IN HUMAN DISEASE

IMMUNOSTAINS FOR FUNGAL AGENTS IN HUMAN DISEASE

• Specific “private” antibodies against Trichosporon, Candida, Aspergillus, & Histoplasma (also possibly available

through the CDC)• Monoclonal antibody to Pneumocystis

carinii (Dako; clone 3F6)• Cross-reactivity between commercial

polyclonal antibodies to mycobacteria and Sporothrix, Coccidioides, & Histoplasma

Fungal Pathogens: Positive ImmunostainsFungal Pathogens: Positive Immunostains

C. immitisC. immitis S. schenckiiS. schenckii

Cutaneous PneumocystosisCutaneous Pneumocystosis

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IMMUNOSTAINS FOR MYCOBACTERIA

IMMUNOSTAINS FOR MYCOBACTERIA

• Polyclonal antibodies available (Dako) against M. paratuberculosis, M. duvali, &

Bacillus Calmette-Guerin• Cross-reactivity is seen between those

agents and M. tuberculosis, M. leprae, and “atypical” mycobacteria

• Principal advantage over conventional histochemical stains is increased

sensitivity

M. Marinum: Positive ImmunostainM. Marinum: Positive Immunostain

Immunohistology in Dermatopathology

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