Alternative Bleaching Techniques in the Immunohistochemical Evaluation of Melanocytic Lesions of the Conjucntiva and Uvea

Abstract

Abstract: : Purpose: During the past two years we have been studying the immunohistochemical profile of melanocytic lesions of the conjunctiva and uvea, for which melanin pigment must be bleached out for adequate formulation of diagnoses. We found that bleaching with potassium permanganate and oxalic acid before the application of the antibodies (the current method of bleaching) diminished and altered the cellular antigenicity in the melanocytic lesions (except for S–100 and MIB–1). Our objective was to investigate a method of bleaching based on hydrogen peroxide (H₂O₂) and disodium hydrogen phosphate, and compare this to the currently used method for the assessment of these lesions. Methods: Retrospective and prospective case series. Eighteen specimens of melanocytic lesions were obtained through the ocular pathology registry at the University of Ottawa Eye Institute from May 2001 to March 2004. The specimens consisted of: 4 conjunctival nevi, 3 PAM without atypia, 4 PAM with atypia, and 7 uveal melanomas. Sections from the specimens received the following histochemical stains: hematoxylin–eosin, and periodic acid–Schiff (PAS), and immunohistochemical stains: S–100 protein, HMB45 (anti–gp100), and pan melanoma cocktail (anti–melan–A, anti–tyrosinase, HMB45). Two separate bleaching solutions were used which contained 3% and 4% H₂0₂, respectively, and 10 g/L disodium hydrogen phosphate. The quality of bleaching and retention of the reaction product were evaluated for all of the specimens. Results: Immunohistochemical staining using S100, HMB45, and pan melanoma cocktail followed by bleaching with 3% H₂0₂ showed effective melanin removal with retention of the reaction product in 100% of the uveal melanoma specimens, 75% of compound nevi ,and only 57% of PAM. Bleaching with 4% H₂0₂ yielded effective removal of melanin in 100% of all lesions studied, with retention of a slightly less vivid reaction product. Conclusions: The immunohistochemical staining for melanocytic markers followed by the H₂0₂ method of bleaching is superior to currently used techniques in evaluating heavily pigmented melanocytic lesions. Since the initial part of our study, we have utilized only the 4% H₂0₂ bleach in light of the above results, with some recent instances of incomplete bleaching which still allows for accurate diagnoses. Our findings are being further updated and will be included at the meeting.

Keywords: melanoma • immunohistochemistry • pathology: human