Diagnostic tests l. Take biopsy specimens of perilesional skin or mucosa with normal and the edge of lesional areas in Michel's transport medium for direct immunofluorescent (IF) tests for in vivo bound pemphigus antibodies. 2. Take blood sample in a red top tube for serum tests by indirect IF (IIF) and ELISA methods for circulating pemphigus antibodies. 3. For added confirmation, take biopsy of a fresh lesion in buffered formalin for light microscopy.

Pemphigus vulgaris and pemphigus foliaceus are autoimmune bullous diseases diagnosed by the presence of in vivo bound and circulating autoantibodies. They usually produce lesions characterized by their clinical and histologic appearance. They can be diagnosed most reliably with three types of studies: 1) Biopsy studies of perilesional skin or mucosa by direct IF afford the most definitive marker (1,2). 2) Two groups of serum tests for pemphigus antibodies afford the most sensitive indicators, IIF tests on two substrates (1,3) and the ELISA method for antibodies to desmoglein 1 and 3 (Dsgl and Dsg3) (4,5). 3) Light microscopy of H&E stained biopsies of fresh lesions can confirm the diagnosis of pemphigus (6). Screening with direct IF and IIF followed by the ELISA methods is indicated for a high sensitivity and specificity in diagnosing pemphigus and other autoimmune bullous diseases. The frequency of positive intercellular IgG in a single biopsy specimen is lower (74% of 106 cases) than in. a single serum study for pemphigus antibodies (>90%), in our experience. Paraneoplastic pemphigus (PNP) has same features of pemphigus vulgaris but it requires special studies (7).

DIRECT IMMUNOFLUORESCENCE: Perilesional biopsies of skin or mucous membranes of pemphigus patients usually reveal IgG, strong IgG4 and frequently also complement component C3 in the intercellular areas. Mucosal biopsies should be taken -0.5 cm. from a lesion because closer sites may be negative. Also, old skin lesions of pemphigus may be negative. Most, but not all, perilesional normal and lesional areas reveal intercellular IgG deposits in active cases because the in vivo reactions of pemphigus antibodies are focal and transient; some cases require repeat direct IF studies. Some experts take two biopsies for direct IF initially, one of perilesional normal and another

at the edge of a lesion. PNP may present with pemphigoid-type deposits. INDICATIONS FOR SERUM STUDIES FOR PEMPHIGUS ANTIBODIES: Indirect IF (IIF) on monkey and guinea pig esophagus for cell surface antibodies and the Dsgl and Dsg3 ELISA* afford key methods both in diagnostic and prognostic studies for all forms of true pemphigus. (See UPDATE on Pemphigus: Confirmatory, prognostic and special studies) (1-4). Indirect IF can detect several disorders. While the sensitivity of the two methods is about the same, the ELISA methods have a greater specificity. PNP requires special tests.

For diagnosis, use both IIF and the ELISA method for maximum sensitivity. IIF also detects pemphigoid and other disorders. ELISA for Dsgl and Dsg3 distinguishes the vulgaris from the foliaceus form of pemphigus

(8-10). Cases that have both skin and mucosal lesions have both Dsg3 and Dsgl antibodies. (Table). In brief, a combination of the IIF tests on two substrates and the two ELISA methods affords the greatest sensitivity and the highest level of confidence in the diagnosis of pemphigus.

Table Dsgl ELISA in 79 cases of Dsg3 psoitive pemphigus vulgaris(8)*
Lesion sites and severity	Mucosal only	Muscosal & minor skin	Muscosal & severe skin	Total cases	Time before treating mean/median
Dsgl postive	0	25	23	48	6 mo	3 mo
Dsgl negative	12	19	0	31	20 mo	12 mo
Total cases	12	44	23	79

* According to one study of 44 active pemphigus vulgaris cases (8), all were Dsg3 positive; only the 20 cases with coexisting skin lesions were also Dsgl positive. All cases listed in the table are Dsg3 positive. In another study, all 23 pemphigus foliaceus cases examined were Dsgl positive and Dsg3 negative (5).

LIGHT MICROSCOPY: Histologic studies reveal intraepidermal or intraepithelial, acantholytic bullae. Particularly in cases that are negative by direct IF, histology may yield key findings. Since acantholysis may also appear in other bullous diseases including Hailey Hailey disease, Darier's disease and transient acantholytic dermatosis, serum studies for pemphigus antibodies are indicated for confirmation.

FORMS: OF TRUE PEMPHIGUS: Pemphigus vulgaris may also present clinically as the vegitans form of pemphigus. Similarly, pemphigus foliaceus may also present clinically as pemphigus erythematosus or herpetiform pemphigus. other forms can be distinguished by diagnostic laboratory studies. IgA pemphigus cases have intercellular deposits of IgA; some of these ("90%) also have IgA class pemphigus antibodies (9).
PNP shares some clinical, histologic, direct IF and serologic features of pemphigus vulgaris and has some distinct features (7). In PNP, direct. IF frequently reveals basement membrane zone IgG and/or C3, as in pemphigoid, and weak or negative intercellular deposits. The ELISA method reveals Dsg3 antibodies (10). PNP is characterized by the presence of plakin and other bands in immunoprecipitation (7) and immunoblot (11) tests. In our experience, complement IIF tests reveal distinctive cell surface antibodies in fatal PNP cases. Also, while IIF gives typical intercellular patterns, they may be primarily of the IgG1 rather than the IgG4 subclass in PNP.

References
1. Beutner EH, Lever WF, Witebsky E, Jordon RE, Chertock B. Autoantibodies in pemphigus vulgaris. Responses to an intercellular substance of epidermis. J Am Med Assoc 192:6828,1965. and Beutner EH, Jordon RE, Chorzelski TP. The immunopathology of pemphigus and bullous pemphigoid. J Invest Dermatol 51:63-80,1968.
2. Beutner EH, Chorzelski TP, Jablonska S. Clinical significance of immunofluorescence tests of sera and skin in bullous diseases. "Immunopathology of the Skin" Edited EH Beutrer et al. J Wiley NYC, 3rd ed: 177-206, 1987.
3. Sabolinski ML, Beutner EH, Krasny S et al. Substrate Specificity of Anti -Epithelial Antibodies of Pemphigus Vulgaris and Pemphigus Foliaceus Sera in Immunofluorescence Tests on Monkey and Guinea Pig Esophagus Sections. J Invest Dermatol 88: 545-549, 1987.
4. Amagai M, Hashimoto T, Green KJ, et al. Antigen specific immunoadsorption of pathogenic autoantibodies in pemphigus foliaceus. J Invest. Dermatol 104:895-901,1995.
5. Amagai M, Tsunoda K, Zillikens D, et al. Clinical phenotype is defined by the anti-des-moglien autoantibody profile. J Am Acad Dermatol 40:167-70,1999.
6. Lever WF. Pemphigus and pemphigoid. Charles Thomas, Springfield, IL 1965;1-266.
7. Anhalt GJ, Kim S, Stanley JR, Korman NJ, Jabs DA, Kory M, Izumi H, Ratrie H, Mutasim D, Ariss-Abdo L, Labib RS. An Autoimmune Mucocutaneous Disease Associated with Neoplasia. N. Engl J Med 323:1729-35,1990.
8. Harman KE, Gratian MJ, Black MM et al. The clinical significance of autoantibodies to desmoglein 1 in 78 cases of pemphigus vulgaris. J Invest Dermatol 112, 569, 1999 (Abstr). 9. Chorzelski TP, Beutner EH, Kowalewski C et al. IgA pemphigus foliaceus with a clinical presentation of pemphigus herpetiformis. J Am Acd Dermatol 24: 839-44, 1991.
10. Amagai M, Nishikawa T, Nousari HC, Hashimoto T. Antibodies against Desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice. J Clin Invest 102:775-82,a998. 11. Hashimoto T, Amagai M, Wantanabe K, Chrorzelski TP, Bhogal BS, Black MM, Stevens 3P, Boorsma DM, Korman NJ, Gamou S, Shimizu N, Nishikawa T. Characterizationi of Paraneoplastic Pemphigus Autoantigens by Immunoblot Analysis. J Invest Dermatol 104:829-34,1995.

Copyright © 2001 Beutner Labs, Inc.
Published with permission.