Progress in Human Pathology

CUTANEOUS IMMUNOPATHOLOGY The Diagnostic Use of Direct and Indirect Immunofluorescence Techniques in Dermatologic Disease* Terence J. Harrist, M.D., t and Martin C. Mihm, Jr., M.D.+

Abstract Direct and indirect immunofluorescence tests performed on skin biopsy specimens and serum have enriched the diagnostic skills of the practir.ing pathologist. Specific patterns of immunoglobulin and complement deposition have clarified the diagnostic entities within the group of vesiculobullous diseases. The pemphigus group of diseases is characterized by antibodies, usually IgG, directed against the intercellular substance of squamous epithelium. The pemphigoid group of bullous diseases is characterized by antibody, usually IgG, directed against the basement membrane zone. The basement membrane zone deposition of immunoglobulin or complement is linear and localized to the lamina lucida. In dermatitis herpetiformis, granular or speckled IgA deposition in the upper papillary dermis is characteristic; however, other patterns of deposition may occur. Abnormal microfibrillar bundles in the upper papillary dermis have recently been identified in patients with dermatitis herpetiformis. . Immunofluorescence studies of patients with lupus erythematosus are important not only in diagnosis but also in prognosis. The diseased skin of lupus patients contains deposits of immunoglobulin, usually IgG or IgM, at the basement membrane zone in more than 90 per cent of the cases. In discoid lupus erythematosus, clinically normal skin does not contain such deposits. However, in systemic lupus erythematosus, normal sun exposed skin contains *Study supported in part by :\IH grant AI I04\J6-02.

t Clinical Fellow in Dermatopathology, James Homer Wright Laboratories of Pathology, Harvard Medical School and Massachusetts General Hospital, Boston, Massachusetts. *Associate Professor of Pathology and Chief, Dermatopathology Unit, Harvard Medical School. Associate Pathologist, Massachusetts General Hospital. Associate Staff, Peter Bent Brigham Hospital. Consultant in Dermatopathology, Children's Hospital Medical Center, Beth Israel Hospital, and Veterans Administration Medical Center, Boston. Consultant in Dermatopathology, Addison Gilbert Hospital, Gloucester, and Cambridge City Hospital, Cambridge, Massachusetts.

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HUMAN PATHOLOGY- VOLUME 10, NUMBER 6 November 1979

these deposits in approximately 80 per cent of the cases and normal nonsun exposed skin contains them in 50 per cent of the cases. Direct immunofluorescence of normal skin may demonstrate basement membrane zone deposition of immunoglobulin in mixed connective tissue disease and other autoimmune diseases with anti-DNA antibodies. The skin of psoriatic patients may demonstrate in vivo bound IgG within the stratum corneum. Similar circulating antistratum corneum antibodies are found in normal subjects; however, these antibodies do not appear to have access to epidermal binding sites. Patients with lichen planus characteristically have large globular deposits of immunoglobulin and complement in the epidermis and dermis in diseased skin. Granular deposition of IgM and IgG at the basement membrane zone in lichen planus may lead to confusion with lupus erythematosus. Deposition of immunoglobulin and complement is found in and about vessels in early lesions of cutaneous vasculitis. Negative findings in older lesions may be due to immune complex degradation and removal. Further information has accrued concerning the possible role of humoral immunity in graft versus host disease. Granular or linear deposition of immunoglobulin, primarily IgM, has been identified in the skin of patients with acute and chronic graft versus host disease. Identification of infectious agent antigen in tissue using modified direct immunofluorescence techniques promises rapid diagnosis in certain infectious diseases such as Rocky Mountain spotted fever and gonococcemia. The direct and indirect immunofluorescence findings in the foregoing diseases as well as others are discussed in detail along with considerations of pathogenesis. A brief review of methodology is given.

Direct and indirect immunofluorescence techniques are of great value to the cutaneous pathologist both in diagnosis and in research. Since its inception less than two decades ago, immunopathology has substantiated certain classifications of dermatologic disease and has provided clear insight into the pathogenesis of several disease entities. Although of greatest aid in the diagnosis and management of patients with vesiculobullous diseases, these techniques are also useful in evaluating patients with lupus erythematosus and other so-called collagen-vascular diseases. It is the purpose of this review to discuss the immunofluorescence findings in these disorders and, as well, to discuss recent immunofluorescence data concerning epidermal cytoplasmic antigens and psoriasis.

ology laboratory of the Massachusetts General Hospital are outlined. These techniques are similar to those elaborated in Beutner et al.'s comprehensive monograph.' For direct immunofluorescence studies biopsy tissue, usually a 4 mm. punch biopsy specimen, is immediately placed in a towelette saturated with phosphate buffered saline consisting of 0.1M sodium phosphate and 0.15M sodium chloride with a pH 00.3. The wrapped specimen is then placed on ice and transported to the immunopathology laboratory. In some laboratories, tissue is snap frozen and transported in liquid nitrogen. The biopsy specimen is properly oriented in Optimum Cutting Temperature (OCT) Compound (Ames Co., Division of Miles Laborat.ories, Inc., Elkhart, Indiana) and frozen upon a precooled chuck in the cryostat at -25 C. Sect.ions are prepared at a 4 micron thickness and mounted on glass slides. The sections are stored at -70 0 C. overnight. After thawing for 15 minutes, the slides are placed in a stain0

METHODOLOGY

626

The techniques for immunofluorescence studies of skin at the immunopath-

CUTANEOUS IMMUNOPATHOLOGY -

ing tray and immersed in phosphate buffered saline contained in a glass receptacle (Coplin jar or histologic dish) for two 5 minute washes . A magnetic stir bar in the histologic dish and a magnetic stirrer are adjusted for gentle stirring motion. After washing, excess phosphate buffered saline is removed from each slide separately. One drop of appropriately diluted, commercially prepared, fluorescein isothiocyanate (FITC) antisera labeled to IgG, IgM, IgE, IgA. C3, fibrin, and albumin (Hyland. Cappel) is applied individually to separate moist biopsy sections. which are incubated at room temperature for 30 minutes in a moist chamber. Following three 5 minute rinses with phosphate buffered saline, the sections coverslipped with glycerol-phosphate buffered saline-polyvinyl alcohol solution (Elvanol 51-05 ; Gelvatol; Monsanto, St. Louis) and examined using an immunofluorescence micro scope with epi-illumination and high pressure mercury lamp as the light source. For indirect immunofluorescence studies venous blood is obtained under sterile conditions. The serum is separated and stored at _70 0 C. until used. Rhes us or ring tailed monkey esophagus and guinea pig esophagus (Cibco, New York), stored at -70 C., are used as substrate. Dilutions of sera are made with phosphate buffered saline and 4 per cent bovine serum albumin in order to titrate the serum antibody level. Serum dilutions and substrate are incubated at room temperature in a moist chamber for 30 minutes. The slides are then rinsed three times for 10 minutes each. With the application of antisera, processing ensues in the same manner as for direct immunofluorescence. Michel. Milner. and David" first developed a solution containing ammonium sulfate, N-ethyl maleimide and magnesium sulfate in a citrate buffer for transporting biops y specimens for direct immunofluorescence at ambient temperature. They reported comparable dire ct immunofluorescence results for tissue processed in the hold ing solution or when processed in the widel y used frozen tissue technique. Althou gh earlier studies did not support th e reliability of this 0

HARRIST, MIHM

transport medium," Nisengard et al." found a 90 per cent correlation between the immunoreactivity of tissues processed in holding solution and that of those that were snap frozen . Therefore, they advocated use of the holding solution to facilitate and increase the availability of direct immunofluorescence testing .

VESICULOBULLOUS DISEASES (TABLE 1)

Biopsy Site Selection

The approach to the diagnosis of vesiculobullous diseases includes knowledge of the appropriate site in which biopsy will yield the most reliable and diagnostic results on direct immunofluorescence. In general, biopsy of the border of an early bulla with surrounding skin should be done for optimal results.v " For example . if adequate sampling is performed, direct immunofluorescence demonstrates positive results in virtuall y 100 per cent of the patients with pemphigus, bullous pemphigoid, and herpes gestationis."? In dermatitis herpetiformis, biopsy of skin adjacent to a vesicle is indicated because immunoglobulin deposition is not usually found in the subvesicular dermis.s!" Direct immunofluorescence studies of biopsy specimens of perivesicular skin should be positive in nearly 100 per cent of the cases," It must be emphasized that multiple tissue sections and perhaps multiple biopsy specimens must be processed to ensure detection.': s Biopsy specimens from uninvolved skin from a variety of sites yield positive findings in 63 to 100 per cent of the cases.1O-13 Fry and Seah" demand positive direct immunofluorescence findings in non involved skin in order to make the diagnosis of dermatitis herpetiformis. Pemphigus Vulgaris

Pemphigus is characterized by the presence of autoantibodies directed against the intercellular substance of squamous epithelium. These circulating antibodies, first demonstrated by Beutner and jordon," occur in all forms of pemphigus, which include pemphigus vul-

627

HUMAN PATHOLOGY-VOLUME 10, NUMBER 6 November 1979 TABLE 1.

IMMUNOFLUORESCENCE FINDINGS IN VESICULOBULLOUS DISEASES

% Positive

% Positive

Direct Immunofluorescence

Immunoreactant* Localization

Pemphigus

80-95%

Intracellular substance

IgG, C3

79-90%

Bullous pemphigoid

-100%

BMzt

IgG, C3

70%

Cicatricial pemphigoid

64-83%

BMZ

IgG, C3

Uncommon

100%

BMZ

C3

Rare

-100%

Upper papillary dermis and BMZ

IgA, C3 fibrin

Rare

IgA

Rare

Disorders

Herpes gestationis Dermatitis herpetiforrnis Chronic bullous disease of childhood

BMZ

Principle Immunoreaetants

Indirect Immunofluorescence

*The term "imrnunoreactant" refers to the various immunoglobulins, complement components, and fibrin. tBMZ, epidermal basement membrane zone.

628

garis, pemphigus vegetans, pemphigus foliaceus, contagious Brazilian pemphigus foliaceus (fogo selvagem), and pemphigus erythematosus (Senear-Usher syndrome). Pemphigus antibody is composed of all four IgG subclasses." In certain cases there is evidence that in vivo complement fixation occurs; in vitro complement fixation has only recently been demonstrated. 16 The exact nature of the intercellular antigen is unknown. Intercellular binding substances have been isolated from several mammalian species.!" 18 In the guinea pig one intercellular substance is a protein with a molecular weight of 18,000 containing ribonucleic acid (RNA) and hexose." Immunologically the binding sites of concanavalin A (Con-A) and pemphigus antibody are similar, for the latter can be blocked by Con-A.20 Con-A may bind to a-D-glucopyranosides or a-Nacetyl-D-glucosamides. Because of the blocking phenomenon, Hashimoto et al. have speculated that the pemphigus antibody site may be either a carbohydrate or a glycoprotein moiety containing a-Dglucopyranosides or a-N-acetyl-D-glucosamides. Other studies have shown that the binding sites of Con-A and pemphigus antibody are different. 20a The pemphigus

antigen isolated from human esophagus appears to be a heat labile protein.?" The pemphigus antibody binding substance appears to be more mobile on the membrane of basal and lower spinous cells than on that of the upper spinous and granular cells.21 The more ready aggregation of antigen in the lower epidermal intercellular substance may explain the suprabasilar bulla formation in pemphigus vulgaris. Although intercellular substance antibodies are relatively specific for pemphigus, several conditions may result in false positive pemphigus antibodies or "pemphigus-like antibodies" in serum. These antibodies characteristically exhibit weak intercellular immunofluorescence in low or high titers in such conditions as burns, practolol eruptions, lepromatous leprosy, penicillin induced morbilliform eruptions, lupus erythematosus, myasthenia gravis with either thymoma or thymic hyperplasia, Trichophsum rubrum infections, toxic epidermal necrolysis, strong anti-A and anti-B blood group sera, as well as other cutaneous bullous diseases. 5,8,22-33 Drug induced pemphigus has been associated with penicillamine, phenylbutazone, irgapyrin, and rifampicin.w'"

CUTANEOUS IMl'vIUNOPATHOLOGY-HARRIST,

Drug rashes secondary to penicillin and chlorpromazine have shown deposition of immunoglobulin confined to the basal cell layer of skin without circulating antibodies (probably representing basal cell cytoplasm antibodyj.i" True pemphigus may occur in association with myasthenia

gravis." Tuffanelli's comprehensive review of cutaneous immunopathology describes the causes of false negative reactions in pemphigus, namely, the prozone phenomenon, interference by other antibodies, particularly antinuclear factors, laboratory error, and species or organ specificity of the antibody."

MUIM

Direct study of pemphigus vulgaris by Jordon,40 Nishikawa et al.,"! and Jablonska et al. 42 showed IgG in 80 to 95 per cent of the cases (Fig. 1). IgA and IgM intercellular substance antibodies are found much less frequently. 40, 42.43 Jordon and others reviewed complement activation in pemphigus and foune! CIg in 58 to 66 per cent of the cases, C3 in 100 per cent, and C4 in 66 per cent.:'?..j,j, 45 The complement components were present only in areas of acantholysis. The demonstration of properdin and factor B suggests a possible role of the alternate pathway.v' however, since C3PA is found much more commonly than properdin, activation of

Figure 1. Direct immunofluorescence of skin adjacent to a blister in a patient with pemphigus vulgaris. Granular to confluenr granular IgG is present in the intercellular space. Note absence of deposition at the basemenr membrane zone. (X 'Ill.)

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HUMAN PATHOLOGY - VOLUME 10, NUMBER 6

the C3b feedback mechanism is more probable. Indirect immunofluorescence has been variously reported to be positive in 79 to 90 per cent of the cases. 1,13,42 Although antibody titer has been reported to correlate well with disease activity and to be useful in patient management, recent dat.adispute t.heseearlier findings.': 45, 46, 46" It is important to remember that variations of one dilution are within the standard error of indirect immunofluorescence. 5 The exact. role of the antibody in pathogenesis is, as yet, not fully defined. Acantholysis may be the result of complement fixation by pemphigus antibody via the classic pathway." However, acantholysis may be produced in vitro without complement.v-Y The pathogenetic role of pemphigus antibody has been suggested by the demonstration of acantholysis in skin explants and organ cultures and in squamous mucosa after repeated submucosal injections of antibody containing serum. 47-53 Other studies have not been successful in substantiating pemphigus antibody pathogenicity. 54, 55 In two patients with pemphigus, plasmapheresis was associated with a decrease in antibody titer and parallel clinical improvement. 56 Secondary changes with keratinocytes (inhibition of RNA and protein synthesis) may sustain acantholysis." Pemphigus Foliaceus

630

Bystryn and Rodriquez" report nine of 12 patients to have IgG localized mainly in the superficial epidermis intercellular substance on direct immunofluorescence. C3 was identified in 10 of 12 patients within the intercellular substance of the deep epidermis. Intercellular IgM was identified in one patient. No IgA was found. Loss or blocking of lower epidermal intercellular substance antigen was demonstrated in over 70 per cent of the patients. With indirect immunofluorescence, 11 of 12 patients were found to have circulating intercellular substance antibodies, which localized to all layers of the squamous epithelium. One patient had circulating subcorneal antibodies directed against the intercellular substance of the superficial epidermis. Bystryn, Abel, and DeFe0 58 found

November 1979

similar antibodies specific for subcorneal intercellular substance in four of 10 patients with pemphigus foliaceus in an earlier study. The antigen against which the antibody is directed may be an epidermal differentiation antigen and distinct from the antigen of pemphigus vulgaris. Thus, according to the work of Bystryn and coworkers, the subcorneal acantholytic blister of pemphigus foliaceus may be due to either loss of intercellular substance antigens in the lower epidermis or to a specific subcorneal intercellular substance antibody. Either of these two findings may account for the anatomic site of the blister in pemphigus foliaceus. However, in our experience full thickness epidermal intercellular substance deposition of immunoglobulin is by far the most common direct immunofluorescence pattern demonstrated. There appears to be no clear-cut relationship between antibody titer and severity of disease. Pemphigus Vegetans

Two types of pemphigus vegetans are recognized, the Neumann and the Hallopeau types. The Neumann variant biologically acts as pemphigus vulgaris. Confusion reigns concerning the Hallopeau variant, which has a benign course, and pyoderma vegetans, a disorder possibly of infectious etiology. Pyoderma vegetans does not demonstrate intercellular substance antibody on direct or indirect immunoI-luorescence examination. 1,5 In pemphigus vegetans of the Hallopeau type, direct and indirect immunofluorescence studies show intercellular substance deposits of IgG. 59,60 Fogo Selvagem

Immunofluorescence studies reveal results similar to those in pemphigus vulgaris. 35 These findings have been confirmed using immunocytochemical locali-

zation." Pemphigus Erythematosus

This entity, also known as the Senear-Usher syndrome, demonstrates the findings of both pemphigus and

CUTANEOUS IMMUNOPATHOLOGY-HARRIsT, Mr r-rxr

lupus erythematosus on direct immunofluorescence . Basement membrane zone deposition of immunoglobulin and complement is present as well as intercellular substance antibody deposition.P -v' Biopsy specimens should be taken from sun exposed areas, since basement membrane zone deposition of antibody is found in 83 per cent of the cases in sun exposed skin and in only 31 per cent in nonsun exposed skin .!i3. !i4 Antinuclear antibody and pemphigus antibody may be demonstrated on indirect immunofluorescence examination .w- 63. fj4

Bullous Pemphigoid Recent morphologic studies of bullous pemphigoid in various stages of develo pmen t h ave suggested that mast cell m igration and degranulation are the earliest recognized changes .li5 The presence of at least one chemotactic factor with the properties of mast cell-eosinophil chemot actic factor of anaphylaxis m ay explain the sequence o f mast cell deg ra n ulation and subsequen t eosinophil infiltration (eosinophilic papillary microabscesses) that is so characteristic of bullous pemphigoid.5 • 66 The exact role of th e lymphocyte in the pathogenesis of the lesion is not defined. Direct immunofluorescence re veals the deposition of immunoglobulin, most commonly IgG, and complement components , of both classic and alternate pathwa ys, in a linear array at th e basement m embrane zon e. I • 5 Complement deposition is mo re reliab ly demonstrated than immunoglobulin deposition and hence is a more sensiti ve test for bullous pemphigoid (Fig. 2). This is not surprising beca use two IgG molecules and o ne IgM molecule ma y fix numerous C3 molecu tes." C3 was identified in all patients reported by Ahmed et a1. 7 and Wintroub et al.,65 whereas IgG was found in 32 of 36 an d six of nin e patients , respectively. Although the exact nature of the basem ent membrane zone an tigen is unknown , a protein found in epidermal phosp h ate buffered saline so luble p roducts, was able to block pemphigoid a n tibody reac tions with the basement membrane zone.?" The an tige nic determinant may be near th e a-glucosido-,B-

Figure 2. Direct imruu nofluo resceuce of skin at the margin o f a blister in a pa tie n t with b ullo us pe m p h igoid. Linear depositio n of C3 is p resent a t the base ment mem bra ne zone o f a p ila r external root sheath. (X 25.)

galactosido-hydroxylysine componen t of the basement membrane zone.fi8 ;' Id entification o f base m en t me m brane zon e linear IgG has occurred in 65 to 95 per cent of the patients.w- 67. 69.70 IgM, IgA, I gD, and IgE ar e found much less commonly. In vivo bound complement and a n tibody are loc alized to the space bet ween the basal lamina a n d plasma m embranes of the basal cells (Fig. 3A, B).71 , 72 Although pemphigoid antibod y is composed of a ll four IgG subclasses, Sarns'" has shown that most complement fixing pemphigo id an tibody belongs to the IgG 3 subclass. Properdin, fibrin, C4, and Clq are also found at the basement membran e zone."?- 73. H Indirec t immunofluorescence studies for circulating basement membrane zone antib od y (l gG) are positive in ap proximately 70 per cent o f the cases.': 1.1 . 65. 67. 70 Antibody titer, howeve r , does not correlate well with clinical d isease severi ty .75 The path ogenesis o f bullous pemphigoid is unknown. Immunologic injury to

631

HUMAN PATHOLOGY - V O LUME 10, NUM BER 6 N ovember 1979

:

",

•

0

'

TONOFIBRILS PLASMA MEMBRANE , \

LAMINA LUCIDA,

,, \

'., .

BASAL L A MI N,~ \

HJ MI DESMOSOME \ \

,

,-

, /

----ANCHORING FILAMENT

-"

A

... ...

" demonstrated positive lupus band tests in 16 of 19 patients with histologically documented, diffuse proliferative or membranous glomerulonephritis, whereas only three of 13 patients had positive lupus band tests in which focal proliferative glomerulonephritis or normal renal findings were documented, These findings have been confirmed by Dantzig et al,144 Also Pennebaker, Gilliam, and Ziffl 45 found that severe renal disease and mortality are greater in patients who have positive lupus band tests with IgG alone or with IgG plus other immunoglobulins. Leif et al.146 describe six patients with glomerulonephritis and positive antinuclear antibody in whom the diagnosis of systemic lupus erythematosus was made on the basis of positive lupus band tests in nonsun exposed, clinically normal skin. To our knowledge, correlation with other single organ disease activity has not been demonstrated. These immunofluorescence findings may diminish or disappear with therapy.P"- 147 Wierzchowiecki et al. 1l9 have demonstrated that the immunoglobulin deposits in clinically normal skin contain antibodies to nuclear and basement membrane zone components. NZB/NZW FI hybrid mice, an experimental model for systemic lupus erythematosus, initially develop focal, granular pattern basement membrane zone immunoglobulin deposition that progresses to a confluent granular pattern.v" Boonpucknavig et al. 149 demonstrated similar findings in patients with systemic lupUS,149 Similar patterns were obtained in mice that were irradiated to form thymine dimers.P" Immunoglobulin and complement were deposited at the basement membrane zone in a pattern similar to that seen in the NZB/NZW Fl hybrid model. The immunoglobulin deposits within the skin correlate with the presence of antibody to native DNA.123

November 1979

If discoid lupus erythematosus is suspected, we recommend that direct immunofluorescence be performed on a skin lesion and that serum antinuclear antibody testing be done. If skin biopsy examination yields a positive lupus band test and the antinuclear antibody is of a high titer, direct immunofluorescence should be performed on sun exposed, clinically normal skin (nontelangiectatic) in order to rule out systemic lupus erythematosus. It should be remembered that 14 to 40 per cent of the patients with discoid lupus may have positive serum antinuclear antibody reactions. 1 When disseminated discoid or systemic lupus is considered, serum antinuclear antibody testing and biopsy of a skin lesion of clinically normal, sun exposed skin should be done. Although a positive lupus band test in nonsun exposed, clinically normal skin is most specific for systemic lupus, there is a high rate of false negative reactions. Although anti-DNA antibodies are highly specific for systemic lupus, they are found in only 60 per cent of the active cases. High titers of antinative DNA correlate with positive lupus band tests in nonsun exposed, clinically normal skin.123, 151 The frequency of positive antinuclear antibody tests does not correlate with cutaneous immunofluorescence. us Scleroderma Direct immunofluorescence studies in scleroderma have been uniformly negative in many series. I, 5,13 The positivity of direct immunofluorescence in rare cases of scleroderma may be attributable to biopsy of telangiectatic skin, which may yield a basement membrane zone band of low intensity staining." ]ablonska et al. 152 report immunoglobulin deposition at t.he basement membrane zone in telangiectatic lesions of the face, but direct immunofluorescence studies of specimens from indurated nontelangiectatic lesions were negative. It is important to remember that immunoglobulin deposition may occur nonspecifically in t.elangiectatic or chronically inflamed or sun damaged skin. With fluorescein labeled antirenal glomerular conjugate, foci of intercollagenous staining for connective tissue an-

CUTANEOUS IMMUNOPATHOLOGY-HARRIST, MIHM

t~gens were identified in scleroderma pa-

tients and were absent in lupus erythematosus and normal skin biopsy

specimens.w Mixed Connective Tissue Disease Sharp et al.I54.155 described a group of patients with a benign connective tissue disease with features of systemic lupus erythematosus with scleroderma or polymyositis or both. In contrast with typical systemic lupus, there is high titer antibody directed against the ribonuclease sensitive antigenic component of extractable nuclear antigen . In Gilliam and Prystowsky's study of 15 patients in whom there was epidermal speckled nuclear staining on direct immunofluorescence of normal sun exposed skin and high titer antibody to ribonuclease sensitive extractable nuclear antigen, five showed basement membrane zone immunoglobulin deposits.P" Winkelmann, Carapeto, and Jordon 157 found immu noglobulin deposition at the basement membrane zone in seven of nine patients with mixed connective tissue disease in sun exposed skin lesions. IgM was found singly in six cases, IgG and IgM in one case, and C3 in one case. The remaining two cases demonstrated vascular C3 in two and IgM in one case. Patterns were not otherwise specified. Up to one-third of the patients with mixed connective tissue disease may demonstrate basement membrane zone deposition of immunoglobulins in nonsun exposed, clinically normal skin. 140. 141

Other Disorders In rheumatoid arthritis, perivascular immunoglobulin and complement were found in biops y specimens of clinicall y normal skin in 20 of 32 cases. Basement membrane zone deposition was absent in all 30. Rarely rheumatoid arthritis has produced deposition of immunoglobulins at the epide rmal basement membrane zone.P" Another study reported negative basement membrane zone findings in 20 patients. 13 In dermatom yositis, immunoglobulin deposited along the basement membrane zone has been rarely demonstrated in telangiectatic lesions. 128, 151) Biopsy of normal-

ly exposed skin for direct immunofluorescence should readily distinguish systemic lupus erythematosus from dermatomyositis in most cases. CUTANEOUS VASCULITIS

The disease entities com posing the spectrum of necrotizing vasculitis are clinicopathologic syndromes .t w Knowledge of both clinical and histopathologic findings is necessary before a specific diagnosis can be rendered. Abundant evidence implicates immune mechanisms, primarily immune complex mediation, in the pathogenesis of necrotizing vasculi-

tides."! Direct immunofluorescence studies ha ve supported the hypothesis of immune complex mediation of most necrotizing vasculitides.I62.104 Use of this technique in diagnostic pathology is limited but helpful in light of the following con siderations. First, early clinical lesions should be subjected to biopsy examination. Cream et al.,165 using the guinea pig, demonstrated complete removal of immune complexes within 18 hours after injection of antigen in an Arthus reaction . Cochrane et al.,166 in an Arthus model, found complete removal of antigen-antibody complexes within 24 hours. If no early clinical lesion can be identified, cutaneous injection of histamine may be employed to induce immune complex deposition by increasing vessel wall permeability.I67-169 According to Gower et al.,169 deposition of immunoglobulin an d C3 in vessel walls was most prominent in biopsies carried out one hour and four hours after histamine injection. Therefore, the deposition of immunoglobulins, complement, and fibrin in vessels is markedly dependent on the time course of the individual vasculitic lesion . Secondly, deposition of immunoglobulins, complement. and fibrin in and about dermal vessels may represent a secondary change, such as that seen in various inflammatory dermatoses . However, the presence of immunoglobulin, complement, and fibrin in and abou t vessel walls without other changes on direct immunofluo~·escence eX~I?ination is highly suggest.!ve of vasculitis.we

641

HUMAN PATHOLOGY -

VOLU~[E

10. NUMBER 6 seru~

Figure 8. Direct immunofluorescence of a lesion in a patient with cutaneous vasculitis. Homogeneous vascular wall deposition and granular perivascular deposition of 19G are evident. (X 25.)

642

The characteristic findings in early lesions of vasculitis include the deposition of immunoglobulin within vessel walls and in perivascular spaces (Fig. 8).162164, 167-t70 Complement and fibrin may be deposited in a granular, homogeneous, or fibrillar array similar to the patterns of immunoglobulin deposition. IgG and IgM are found most often, while IgA is identified less commonly. IgD has also been observed.F" The size of the vessel in which immunoglobulins, complement, and fibrin are deposited varies with the type of vasculitis, i.e., small and medium sized arteries in the polyarteritis nodosa group and in the postcapillary venules in "hypersensitivity" vasculitis." Both normal and diseased vessels may show deposition; t67 however, the deposits are confined to the vessel walls (not perivascular) in the histologically normal vessels. Immune electron microscopic localization has shown immunoglobulin and complement between endothelial cells, pericytes, and layers of the basal lamina.t'"- 168 Late findings include fibrin deposition and neutrophil accumulation.l'" Granular deposition of immunoglobulin, complement, and fibrin may also occur at the basement membrane zone in lesions of vasculitis.w' This deposition may pose a problem in differential diagnosis, which can be resolved by following the approach to collagen-vascular disease already outlined. Immunoglobulin, complement, and fibrin deposition has been described in Wegener's granulomatosis, systemic lupus erythematosus, rheumatoid vasculitis, and

November /979

. sicknes~.t73-18t The spectrum of hepatitis associated vasculitis, including polyarteritis nodosa, urticaria, and hypersensitivity vasculitis, is characterized by the presence of immunoglobulin, hepatitis antigen, and complement in vessel walls. 182-l84 Certain forms of cryoglobulinemia may be associated with vasculitis.l'" Deposition of immune complexes, usually composed of IgM-rheumatoid factor directed against IgG, identical to those found in the circulation, occurs.186-188 Recently hepatitis B antigen has been identified in cryoglobulins.r" In Henoch-Schoenlein purpura, a "hypersensitivity" vasculitis with a recurrent course and spontaneous resolution, IgA and complement are found within dermal vessels described as capillaries (probably venules) of both involved and uninvolved skin. 161. t90. isi Other immunoglobulins are less often demonstrated and stain less iutensely.l'" Interestingly enough, normal skin from patients with Berger's nephritis, possibly related to Henoch-Schoenlein purpura, may show small vessel deposition of IgA and C3. 192 This observation awaits confirmation. Perhaps the major mechanism of tissue damage in these two diseases is fixation of complement via the alternate pathway. The evidence for this type of activation inclucles the usual absence of early complement components and the fact that Henoch-Schoenlein purpura may occur in patients in the absence of C2. 192. rsa Granuloma faciale, thought by some to be a localized form of vasculitis, presents as a violaceous plaque occurring about the head and neck. It has been shown to contain IgA, IgM, IgG, and C3 about vessels and at the basement membrane zone. 162 Necrotizing vasculitis, in the view of Fauci.r" is thought to be initiated by circulating immune complexes in antigen excess that are deposited in vessel walls. Immune complexes of intermediate size appear to be the most important.ws- 195 Deposition occurs locally as a result of increased vascular permeability secondary to platelet derived and basophil derived amines. Complement is subsequently fixed, releasing polymorphonuclear leukocyte chemotactic factors. As neutrophils infiltrate the wall, release of lysosomal enzymes results in necrosis of the vessel wall and fibrin deposition.

CUTANEOUS IMMUNOPATHOLOGY -HARRIST, Mn-IM

OTHER DERMATOSES Psoriasis A new avenue of research in psoriasis has opened within the last 10 years. Krogh and Tonder.t'" using a n:ixed agglutination technique, f?und ~m~un~­ globulin (lgG and IgM) 111 ps~natlC epIdermis. C3 and rheumatoid factor activity were identified as well. Sever.al authors identified in vivo bound IgG Il1 the intercellular areas of the stratum corneum in psoriatic lesions.197-I99 T~ese antibodies were more widespread 111 early and mature lesions than in resolving ones. Comparable titers of circulating antistratum corneum antibody were present in psoriasis patients and controls.v" However, the antibody in normal subjects usually does not bind to the stratum corneum of the epidermis.l'" The accessibility of the antibody to the stratum corneum in psoriatic patients may be due at least partially to discontinuity i~ the ~asal lamina of blood vessels and epidermis as described by Braverman et a1. 200 Cormane et aPOl described antinuclear antibodies to parakeratotic nuclei of psoriatic skin. These immunoglobulins were present on neutrophils and lymphocytes of the infiltrate in psoriatic skin and Munro microabscesses. They were not demonstrated in serum. The foregoing findings at the present time are of little diagnostic value, but have spurred hypotheses concerning the pathogenesis of psoriasis. Significant reduction of T cell lymphocyte numbers was demonstrated in 60 psoriasis patients. 202 The reduction paralleled increased severity of disease. A possible T cell subpopulation deficiency is implied by the disparate responses to phytohemagglutinin and Con-A. These findings may be the result of the interaction of genetic and immunologic factors with possibly infectious agents (viruses).

Lichen Planus Lichen planus is an idiopathic eruption characterized by pruritic, polygonal, violaceous papules of the extremities or by reticulate white papules in the oral mucosa. Lichen planopilaris, i.e., lichen planus with a predisposition for fol-

licular involvement, may present as cicatricial alopecia. The characteristic direct immunofluorescence finding is the presence of numerous colloid deposits (globular or ovoid bodies) containing immunoglobulin and complement (Fig. 9).13 Although the majority of colloid bodies are identified within the papillary dermis, occasional colloid bodies are found within the epidermis as well as in the reticular dermis.203.204 Although colloid bodies are not pathognomonic for lichen planus, they are highly suggestive of this disorder if found in large quantity, in clusters, or in the deep papillary and upper reticular dermisY,203-206 In small numbers they may even be found in clinically normal skin. 203,207 In sever.al series colloid bodies have been noted Il1 approximately 85 to 100 per cent of the biopsy specimens of skin lesions. 1. 13, 203-205 Although IgM has been identified in virtually all colloid bodies, IgG, IgA, C3, fibrin, and albumin are found less frequently. Baart de la Faille-Kuyper and Baart de la Faille 204 reported granular IgM deposition at the basement membrane zone in all 40 patients with lichen planus studied. This finding was focal and was associated with granules of IgM dispersed among adjacent inflammatory and epidermal cells. These authors also identified focal linear IgG and granular as well as linearly deposited complement components at the basement membrane zone. Using the immunoperoxidase technique, Shousha and Svirbel y206 found similar basement membrane ZOne deposits of IgM and IgG in seven of 11 and in four of 13 cases, respectively. Also, intraepithclial IgG and IgM were identified. Basement membrane zone immunoglobulin deposits were demonstrate? ~n two of .i~ patients by Abell et aJ.203 Fibrin deposition occurred as a homogeneous band at. t.he epidermal basement membrane zone with irregular strands extending into the dermis.!" The dep?sition .of fibrin increased with the quantIty of Illflammatory infiltrate and the age of the lesion.s?' Fibrin was also present in vascu1ar 1ocation. . 204 . a penvascu . lar walls an d m Epidermal cell transformation is the probable mechanism of colloid body formation.F" Ultrastructurally, colloid bodies 643

HUMAN PATHOLOGY- VOLUi\IE 10, NUMBER 6 November 1979

are composed of tightly packed aggregates of fine filaments. ~U!'f-2111 Immunoglobulin and complement (as well as other plasma factors) adhere to the colloid bodies. 2 06 Whether the antibody initiates the formation of the colloid body or is a phenomenon secondary to altered epidermal cell antigens is unknown. It is im-

644

portant to remember that various other dermatoses, such as eczema, erythema multiforme, systemic lupus erythematosus and dermatomyositis, may show colloid bodies on direct immunofluorescence.203 In fact, some patients may demonstrate lesions that on clinical, histopathologic, and immunopathologic

Figure 9. Direct immunofluorescence in lichen planus and lichen planopilaris. A, Skin lesion in a patient with lichen planus. Globular deposition (colloid bodies) of IgM as well as other immunoglobulins, complement, and fibrin were present in large numbers, in clusters, and in the reticular dermis. 13. Skin lesion in a patient. with lichen planopilaris. Globular deposits of IgM. other immunoglobulins, and complement were present about the external root sheath. (A, X 25, [3, X 16,)

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MIHM

grounds lichen planus cannot be distinguished from lupus erythematosus.vv'[" Abell and Ramnarain-t! demonstrated loss of basement membrane zone antigen in the lesions of lichen planus. The loss of basement membrane zone antigen may be due either to destruction by the inflammatory infi ltrate or to decreased basement membrane synthesis by epidermal basal cells. Intercellular substance antigen. however, was present except in a few rete. These findings may explain the formation of sub-basal clefts and bullae and the lack of acantholysis in the lesions of lichen planus. The pathogenesis of lichen planus is unknown. As already cited, there is much evidence implicating humoral immunity in the evolution of the lesion. Recent studies using E rosette techniques and identification of surface markers have confirmed earlier observations that the predominant infiltrating inflammatory cell is the T Iymphocyte.P'v 216 Thus, cell mediated immune phenomena may play a role as well. The primary epidermal event is damage to the basal cell of unknown etiology.s" Whether this injury is immunologically mediated or whether the altered cell evokes a secondary immune response is not yet known.

walls of the superficial vascular plexUS.13.219·221 Deposition of other immunoglobulins is rare. Vascular staining. however, may occur deeper within the dermis.P' Basement membrane zone immunoglobulin deposition in a homogeneous, granular, or fibrillar array is usually less intense than th e vascular stain in g. Epstein, Tuffanelli, and Epstein 221 found positive vascular immunofluorescence in 22 of 22 biopsy specimens and basement membrane zone immunofluorescence in 21 of 22 biopsy specimens of skin lesions in patients with active porphyria cutanea tarda. Positive findings were present in biopsy specimens from clinically normal, medial arm skin in 15 of 22 patients. In patients with inactive porphyria cutanea tarda onl y six of 10 showed positive immunofluorescence in specimens from the dorsum of the hand and one of 10 from clinically normal. medial arm skin. In all five patients with erythrohepatic protoporphyria there was positive immunofluorescence in noninvolved skin. In general, the intensity of immunofluorescence was greater in erythrohepatic protoporphyria than in porphyria cutanea tarda,

Porphyria

I nformation is rapidly accruing concerning direct and indirect immunofluorescence findings in various disorders. Deposits of IgM and C3 have been observed in dermal vessels and along the basement membrane zone in fresh lesions of pityriasis lichenoides.222.223 Similar findings have been documented in er ythema multiforme.F" In one patient with erythema gyratum repens, basement membrane zone granular deposition of IgG and C3 was identified in the skin lesion and in clinically normal skin, both in the active and the resolving phases of the disease.v" Immunoglobulin and complement have been demonstrated at the basement membrane zone and in vessels in both normal skin and cutaneous lesions of patients with atopic and contact de rmatitis.225a, 225b Direct immunofluorescence of typical lesions in lichen niditus was negative for immunoglobin and complement in six patients studied.i'" However, a band of fibrin-

Porphyria is a metabolic disease, either inherited or acquired, characterized by abnormal porphyrin metabolism. Because porphyrins are potent photosensitizers, most forms of porphyria are characterized by cutaneous photosensitivity eruptions. Cutaneous blisters may occur in congenital erythropoietic porphyria, erythrohepatic protoporphyria, variegate porphyria, and porphyria cutanea tarda.P" Microscopically, the lesions contain PAS positive, diastase resistant hyalin about blood vessels at the basement membrane zone and in the dermis. The amount of hyalin and its location vary with the type of porphyria. Although the deposition of immunoglobulins is secondary to primary vessel damage, the pattern of deposition is characteristic. IgG and much less commonly IgM are deposited in an intense homogeneous array about and in vessel

Other Disorders

645

HUMAN PATHOLOGY-VOLUME 10, "eMBER

ogen was demonstrated at the basement membrane zone in one patient. In one patient with typical lichen rriditus, one papule that was clinically atypical for the disorder demonstrated angular deposits of IgG in the upper derrnis.s'" The principal direct immunofluorescence finding in granuloma annulare was found to be the deposition of fibrin in zones ofso-called necrobiosis.P" IgM and C3 have also been observed in blood vessels and at the basement membrane zone. In a study of eight patients with cutaneous sarcoidosis, immunoglobulin deposition was identified in the skin lesions of five. 228 IgM was found in vessel walls in five patients and at the basement membrane zone in two patients. Fibrin was identified within granulomas in all specimens. However, two immunohistochemical studies, totaling 14 patients, demonstrated fibrin localized in the granulomas within cutaneous lesions and IgG, IgM, and complement localized only in the basement membrane zone. 22 9 , 2~O Said and Mannukselav" studied 20 positive Kveim reaction biopsy specimens and found IgM in vessels within the granulomas in 19 and IgG in one. C3 was present in vessel walls within the granulomas in nine of 12 Kveim reaction sites. Other studies have found no immunoglobulins or complement deposition in Kveim reactions.F" Lichen myxedematosus (papular mucinosis) is a progressive disorder of unknown etiology in which mucin is deposited in the upper dermis. An unusual basic serum gamma globulin has been identified in these patients.w'- 2~4 Deposits of IgG have been identified in zones of mucinosis in some patients but not in others.2M-237 In

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November 1979

pretibial myxedema, another cutaneous mucinosis, dermal deposition of an IgG, so-called long acting thyroid stimulator, has been dernonstrated.F" Although Stringa et al.239 did not demonstrate immunoglobulins within amyloid deposits in six patients with lichen amyloidosus, MacDonald, Black, and Ramnarainv? identified immunoglobulins and complement, particularly IgM and C3, in all biopsy specimens from 47 patients with the same disease.

GRAFT VERSUS HOST DISEASE Graft versus host disease occurs most commonly in patients who undergo bone marrow transplantation.v" However, it may occur after infusion of unirradiated blood products.w' The acute form of the disease usually occurs one to three weeks after transplantation and is characterized by a red maculopapular rash that begins about the head and neck and spreads to the extremities and trunk. 243 On occasion, lichen planus-like eruptions may be present.244.245 Chronic graft versus host disease occurs months after transplantation and mayor may not be preceded by the acute form. The skin manifestations are those of hyperpigmentation and generalized scaling, leading to atrophy or scleroderma-like changes. 243.240 The histopathologic features have been described.v" Ullman et a1.248 described the direct. immunofluorescence findings in two cases of graft versus host disease (Fig. 10). A skin biopsy specimen from one patient with the acute form disclosed granular IgG, IgM, and C3 in vessel walls and C3 deposition at

Figure 10. Direct inunuuofluorescence of a skin lesion in a patient with chronic graft versus host disease, Granular to confluent gt'anular basement membrane zone deposition of IgiH. as depicted. is characteristic of but not pathognomonic of gran VCI'SUS host disease, (:3 was demousumed as well. (X 40.)

646

CUTANEOUS IMMUNOPATHOLOGY - HARRIST, MlHM

the epidermal basement membrane zone. A biopsy specimen from a patient with the chronic form of the disease contained granular IgM, IgA, and C3 in vessel walls as well as IgM deposits at the epidermal basement membrane zone. Spielvogel, Goltz, and Kersey-" reported granular deposits of IgM in the basement membrane zone in a patient with chronic graft versus host disease and scleroderma-like changes. Tsoi et aP49 reported a series of 88 patients with allogeneic (HLA identical and mixed leukocyte culture nonreactive) bone marrow grafts with and without graft versus host disease; 32 had the acute form and 12, the chronic form. Basement membrane zone granular or linear IgM deposition was present in 86 per cent of the patients with the chronic form and in 39 per cent of the patients with the acute form. No IgM deposits were found in four patients with syngeneic grafts, and only weak IgM deposits were present in 11 per cent of 58 normal subjects who underwent biopsy examination. The intensity and incidence of the IgM deposits increased as graft versus host disease developed. The intensity of immunofluorescence was also greater in the chronic form when compared to the acute form. No statistical differences were found between the direct immunofluorescence findings in normal subjects and those of short and long term allogeneic recipients without graft versus host disease when compared to the direct immunofluorescence findings in allogeneic recipients with resolved acute disease. The observations listed and the presence in two patients of a circulating IgG directed against the basal cells of the epidermis suggest a possible role for humoral immunity in the pathogenesis of graft versus host disease.>"

INFECTIOUS DISEASES In recent years direct or modified direct immunofluorescence techniques have been of value in the diagnosis of certain infectious diseases. By use of FITC labeled antibody or FITC labeled antigens, demonstration of the corresponding infectious agent antigen or infectious agent specific antibody has been accomplished. In other diseases immunofluorescence find-

ings have been important contributions to search for the pathogenetic mechanIsms. Soltani, Choy, and Lorincz/" labeled fragmented T. pallidum organisms with FITC. The FITC labeled antigen was layered upon 12 biopsy specimens of lesions from 11 patients with proven secondary syphilis. Nine of the 12 specimens revealed gran ular dermal fluorescence in areas containing many plasma cells. Control slides from biopsy specimens of normal skin and various dermatoses were negative. Secondary syphilitic lesions in 12 rabbits revealed similar findings in 11 of 12 animals studied.v' This technique may be a useful diagnostic adjunct in lesions with plasma cell proliferations or in patients with papulosquamous eruptions. Woodward et a1. 252 reported two patients in whom the diagnosis of Rocky Mountain spotted fever was made using indirect immunofluorescence techniques with pink macules on day 4 and with purpuric lesions on day 8 of the illness. By using a FITC labeled rabbit antibody directed against guinea pig antigen and guinea pig antibody to R. rickettsii, coccal and bacillary forms were identified in rnacrophages. Control skin specimens were negative. Similar immunofluorescence findings have been demonstrated in experimentally infected Rhesus monkeys.v" Use of this technique may allow firm diagnosis within four hours after skin biopsy in this potentially life threatening disease. However, further work in this area is needed. Prompt laboratory confirmation of measles (rubeola) is usually not available. Fulton and Middleton>" described an indirect immunofluorescence test that may alleviate the problem. Nasopharyngeal cells from 24 patients in the prodromal phase or with clinically diagnosed measles were obtained by suction and placed in culture medium. Sequentially, according to established procedures, standardized measles virus reference antigen and FITC labeled antiglobulin were placed on the nasopharyngeal cells. Eighteen of the 24 were positive; however, acid buffer elution was needed to remove masking globulins in 10 of the 18. In patients with lepromatous leprosy, a granular band of IgM at the basement ~he

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NU~JBER

membrane zone has been demonstrated in both clinically involved and uninvolved skin in 35 per cent of the cases. 255 However, Quismorio et a1. 26 found IgM deposition in uninvolved skin from 10 of 13 patients with lepromatous leprosy. IgM may also be demonstrated along collagen and elastic fibers of the dermis." Rarely IgG may be deposited at the basement membrane zone. The significance of the antibodies is unknown.P" A reactional state of leprosy, Lucio's phenomenon, is histologically a necrotizing vasculitis.P" It usually occurs in patients from Central America with diffuse lepromatous Ieprosy.P" Direct immunofluorescence reveals deposits ofIgM, IgG, C3, and Clg in dermal blood vessels and in perivascular tissues. The concomitant demonstration of circulating mixed cryoglobulins, rheumatoid factor activity, and immune complexes (Raji cell assay) suggests immune complex mediation.P" In another reactional state, erythema nodosum leprosum, deposition of immunoglobulin, complement, and mycobacterial antigens similar to that seen in an Arthus reaction has been described.P? Gonococcal sepsis is characterized by intermittent fever, arthritis, and a hemorrhagic vesiculopustular eruption. The patients commonly do not have clinical evidence of venereal disease.F" Although the histopathologic picture can be highly characteristic.i"? certitude in diagnosis is often lacking owing to the rarity of positive culture of N. gonorrhoeae and lack of demonstration of organisms using tissue Gram staining techniques.w! Blood cultures are positive in only 20 per cent of the cases.?" Barr and Danielsson.t" however, were able, by using FITC labeled antigono coccus globulin, to demonstrate gonococcal antigens in 14 of 16 smears of skin lesions from patients with gonococcal sepsis. Using the same antibody, Kahn and Danielssonr" found "distorted cocci" and aggregated antigenic material in a perivascular location in skin lesions from two patients. Braun-Falco and Scherer.v" using direct immunofluorescence, found C3 deposition in and about vessels of the dermis as well as at the basement membrane zone in skin lesions in one patient with gonococcal sepsis. No IgG, IgM, IgA, or C4 was observed. These observations suggest possible alternate path-

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way activation by bacterial endotoxin lipopolysaccharide derived from disintegrating organisms.v" Antisera to fungal antigens may also be helpful in the diagnosis of dermatophytoses. 265

EPIDERMAL ANTIGENS Each major anatomic compartment of the epidermis contains a distinct set of antigens against which antibodies may be directed. Much of the preceding discussion has been devoted to these specifically directed antibodies and antigens. For example, pemphigus antibody is directed against intercellular substance antigen, pemphigoid antibody is directed against basement membrane zone antigens, and stratum corneum antibody is directed against stratum corneum antigen. The cytoplasm and nuclei of epidermal cells may also act as antigens that elicit antibody production. Three types of anticytoplasmic antibodies are recognized: antibodies to antigens present in all keratinocytes (G-CYT), antibodies to antigens present only in keratinocytes of the upper epidermis (U-CYT), and antibodies to antigens present only in basal cells (BCL).266 Bystryn'"" found circulating BCL antibodies in 38 (1.5 per cent of 2500) sera. In 13 patients with titers greater than 1:80, 10 had cutaneous bullous diseases. The BCL antibody is not species specific, is probably directed against more than one cytoplasmic antigen, and may only be an isoantibody. BCL antibody has been identified in seven of 42 patients with drug reactions, in burn patients, in patients with drug induced toxic epidermal necro1ysis, and in two patients with fixed drug eruption secondary to phenophthalein. 38. 2(lH-270 U-CYT antibodies were found in the sera of 21 per cent of 32 normal individuals and in sera of 64 per cent of the patients with various medical and derrnatologic diseases. m. m G-CYT antibodies were identified in the sera of 8 per cent of 32 normal persons and in the sera of 20 per cent of 53 patients with malignant melanorna.v" Recently Bystryn, Nash, and Robins 266 demonstrated the absence of U-CYT and BCL antigens in four basal cell carcinomas

CUTANEOUS IMMUNOPATHOLOGY-HARRIST,

and three squamous cell carcinomas. G-CYT antigens were present in all seven neoplasms. U-CYT and BCL antigens are retained in keratoacanthoma. Antinuclear antibodies h ave recently been discussed and classified by Burnham.F" CONCLUSION Immunofluorescence techniques have become a valuable diagnostic aid available to the pathologist. Their diagnostic value is most clear-cut in the evaluation of the patient with a vesiculobullous disorder or connective tissue diseas e. Although man y questions ar e still unanswered, the information ga thered via applica tion of these techniques in other cutaneous disea ses has led to gre ate r insight in to, and understanding of, many pathogen etic processes and mechanisms. Continuing use of direct and indirect immunofluorescence in cutaneous disease is warranted. REFERENCES I. Beumer, E. H .• et a l. (Ed itors): Immunop ath ology of the Skin : Labelled Ant ibod y Studies. Stroudsbu rg . Pennsy lvania, Dowden. Hutchinson. an d Ross Co.• 1973. 2 . Michel. B.• et al.: J. In vest. Dermatol.,59:449, 1973. 3. Skeete. M. V. H ., et al.: CHn. Exp . Dermatol., 2:49. 1977. 4. Nisengard, R. J. . et al.: Arch. Derrnatol., 114:1329,1978. 5. Cooperative study. Arch. Dermatol., 111:371, 1975. 6 . Bean , S. F.: J. Derrnatol, Surg., 2 : 148, 1976. 7. Ahmed , A. R., et al. : Arc h. Derm atol. ,113 :1043, 1977 . 8. J ordon, R. E.: Prog . Derrn atol., 9: I , 1975. 9. Katz, S. 1., et al. : J. Clin . In vest., 5 7:1434, 1976 . 10. Cho r zelski, T . P., et al.: J. In vest. Der matol., 56 :373. 197 1. 11 . Van del' Meer, ]. n.: Br. J. Dermatol., 81 :493 , 1969 . 12. Fry, L. , et al.: Br .J . Derrnat ol., 90: 137,1974. 13. T uffanelli, D. L.: J. Invest. Der matol., 65 :143, 1975. 14. Beumer, E. H ., et al.: Proc. Soc. Exp . Biol. Med ., 111 :505 , 1964. 15 . Sams, W. M., et al. : ] . Lab. Clin , Med., 82:249, 1973. 16 . Hash imoto, T ., et al.: Arch . Derma tol., 114:11 9 1, 1978. 17 . Shu, S.-Y.• et al.: J . Invest. Derrnatol., 61 :270, 1973. 18. Ahlin. R.J ., et al.: J. H yg. Epidemiol. Microbiol, Immunol. (Praha), 13 :321,1969.

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19. Miyagawa, S., et al .: Acta De rrn. Ven ereol. (Stockh .). 57 :7. 197 7. 20. Hashimoto . K.. et al.: J. In vest. De rm a tol ., 52 :423, 1974. 211a. Nish ikawa, T. , et al.: Acta Derm. Vene re ol , (Stockh .), 55:3 09, 1975. 20b. Shu , S., et al.: J. Invest. Derrnatol., 61:270, 1973. 21. Takigawa, M.. et aI.: J. In ves t. Dermatol. , 71: 182, 1978. 22. Thivolet .j ., et al.: Bull. Soc. Fr. De rmatol. Syphiligs., 74 :300, 1967. 23. Ablin, R.]., et aI.: Vox Sang.• 16 :73, 1969. 24. Quismorio, F. P., et aI.: Clin, Exp. Imm\.lI1OI., 8 :70 1, 197L 25. Am os, H. E., et al.: Br. Med . J .• 1 :598, 1975. 26. Quisrnorio, F. P., et al.: Arch. De rrnatol., 111 :331.1975. 27. Fellner, M. r, et aI.: Br. J. De rrn atol. , 89 :115 , 1973. 28. Wh itt ingham . 5., et al.: Br. J. De rm atoI., 84 : I , 1971. 29 . Peck, S. M. , et al.: J. In vest, De r rnato l., 58 :133, 1972 . 30 . Grab, P. j. , et al.: ] . Invest. Dermatol., 49 :285, 1967. 3 1. Olsen, K.• et al.: Acta Derm. Vener eo l, (Stockh.), 52 :389, 1972. 32. Cram. D. K., et al.: Arc h. Derrnatol. , 109 :235 , 1974. 33 . Ikai, K., et al.: Derrnatologica, 152 :304 , 1976 . 34, Tan, S. G. , et al.: Br . J. Dermatol., 95 :99 , 1976. 35. Sparrow, G. P. : Br. J . Derrnatol., 98 :10 3, 1978. 36 . Beume r, E . H., et al.: Autosensitization in Pemphigus and Pemph igo id. Sp ringfi eld, Illinois, Charles C Thomas (Publishe r s), 1970. 37. Gang e, R. W.• et al.: Br . J. Dermatol., 95:445, 1976. 38. Stein. K. M.• et al.: Br. J. Dermatol. , 86:246, 1972. 39. Maize. J. C., et al.: Arch. Derrn atol., 111: 1334, 1975. 40. Jordon. R. E.: J. Invest. Derrnatol., 65 :162, 1975. 4 1. Nishikawa , T ., et al.: Clin . Exp . Derrnatol., 3 :57 , 1968. 42. J ablonska, S.: Int. J. Dermatol. , 14 :83, 1975 . 43. Be um er , E. H., et al.:J . l n vest. De r matol .•51:63 , 1968. 44. Jordo n, R. E., et aI.:J . Inv est. De rrna tol. , 63 :256, 1974. 45. Jord on , R. E.: J. Invest. Derrnatol., 67:366 , 1976. 46 . Weissman, V., et aI.: J. In vest. Derrnatol., 71 :107, 1978. 46