title: A comparison of melanin bleaching and azure blue counterstaining in the immunohistochemical diagnosis of malignant melanoma.
abstract: Distinguishing heavily pigmented melanocytes from melanophages on routine hematoxylin and eosin slides can be difficult. Melanin bleaching with potassium permanganate solution is a traditional means of removing melanin from tissues and can be used before immunohistochemical staining to remove any pigment that might be confused with the brown chromogen diaminobenzidine. Azure B stains melanin granules green-blue, easily contrasts with diaminobenzidine, and may be used as a counterstain on unbleached sections after immunohistochemical staining. To our knowledge, studies comparing melanin bleaching with azure B counterstaining in the immunohistochemical evaluation of malignant melanomas have not been performed. Paraffin sections from 33 heavily pigmented malignant melanomas were bleached with a 3.0-g/L potassium permanganate solution, immunohistochemically stained for S-100 and HMB-45, and counterstained with hematoxylin. Unbleached sections were similarly stained for S-100 and HMB-45 and counterstained with azure B. To establish optimal permanganate concentrations, a variable number of sections were bleached with lower permanganate concentrations ranging from 0.125 to 2.5 g/L. S-100 antigenicity was preserved at all permanganate concentrations, whereas HMB-45 antigenicity was abolished at concentrations of 0.5 g/L and greater. At permanganate concentrations from 0.125 to 0.5 g/L, both antigenicities were preserved; however, melanin was incompletely removed. Complications of bleaching included tissue damage and loss of cytologic detail. Positive immunohistochemical staining was observed in azure B counterstained sections. Azure B stained melanin greenblue and was easily distinguished from the brown diaminobenzidine chromogen, regardless of the antibody tested. Neither tissue damage nor loss of cytologic detail was observed. We conclude that the use of azure B counterstaining is superior to permanganate bleaching in the histologic evaluation of heavily pigmented cutaneous malignant melanomas.
authors.affiliation: Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Kligora
authors.forename: K P
authors.initials: KP
authors.lastname: Fair
authors.forename: M S
authors.initials: MS
authors.lastname: Clem
authors.forename: J W
authors.initials: JW
authors.lastname: Patterson
mesh_terms: D000951:Antigens, Neoplasm; D001399:Azure Stains; D006801:Humans; D007124:Immunoenzyme Techniques; D008543:Melanins / Q000378:metabolism*; D008544:Melanocytes / Q000378:metabolism / Q000473:pathology*; D008545:Melanoma / Q000378:metabolism / Q000473:pathology*; D058950:Melanoma-Specific Antigens; D008547:Melanophores / Q000378:metabolism / Q000473:pathology*; D009363:Neoplasm Proteins / Q000378:metabolism; D010858:Pigmentation / Q000187:drug effects; D011196:Potassium Permanganate / Q000494:pharmacology; D009418:S100 Proteins / Q000378:metabolism; D012878:Skin Neoplasms / Q000378:metabolism / Q000473:pathology*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: D000951:Antigens, Neoplasm; D001399:Azure Stains; D008543:Melanins; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D009418:S100 Proteins; D011196:Potassium Permanganate; C025819:Azure B