Antigen retrieval in immunofluorescent testing of bullous pemphigoid

Direct immunofluorescence is an immunopathological technique frequently utilized for diagnosis of vesiculobullous disease such as bullous pemphigoid. Fresh-frozen tissue is required for immunofluorescent testing, making retrospective analysis difficult. In this study, we compared two methods of antigen retrieval in formalin-fixed, paraffin-embedded skin tissue from patients with bullous pemphigoid to determine if archival tissue, after use of an unmasking antigen, can be substituted for fresh-frozen tissue in the immunopathological study of skin. Paraffin-embedded tissue blocks from patients with bullous pemphigoid and patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid were obtained. Sections were mounted on poly-L-lysine-coated slides and the slides were deparaffinized. The methods of antigen retrieval included incubation with trypsin (0.1%) and microwave irradiation in urea (6 M). Antigen retrieval was followed by indirect immunofluorescence. Microwave irradiation was more effective in antigen retrieval than was incubation with trypsin (0.1%). Microwave irradiation in urea (6 M) produced more intense immunofluorescent staining than did trypsinization. Overall, positive basement membrane zone immunofluorescent staining was found in 60% of patients with a diagnosis of classical bullous pemphigoid and in 50% of patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid. Although the frozen-tissue method appeared more effective than the antigen-retrieval method in immunofluorescent testing of skin, the antigen-retrieval method can certainly be considered an option in retrospective studies. Antigen retrieval may be particularly advantageous in patients with eosinophilic spongiotic dermatitis in whom the diagnosis of bullous pemphigoid may not be suspected initially.