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.