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Seymour Brownstein, Kailun Jiang, Kay Lam, Bruce Burns, James Farmer; Immunohistochemical Analysis with a Novel Red Chromagen of Benign and Malignant Melanocytic Lesions of the Conjunctiva. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5894.
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© ARVO (1962-2015); The Authors (2016-present)
In North America, immunohistochemical analysis of heavily pigmented melanocytic lesions of the conjunctiva and uvea rely almost exclusively on the 3, 3 diaminobenzidine (DAB) as a colored substrate. DAB use may require bleaching of samples to remove the confounding melanin pigment, but bleaching can affect cellular antigenicity and may be incomplete. We evaluated an alternative substrate, Vector Red (VR), whose vibrant red color is distinct from the melanin pigment and renders bleaching unnecessary. Using VR, we identified an immunological marker that is sensitive for all melanocytes and another marker that is sensitive and specific for activated/atypical melanocytic lesions.
Retrospective and prospective case series. 8 specimens each of normal conjunctiva and choroid, choroidal melanoma, conjunctival nevus, PAM with mild or no atypia, PAM with moderate or severe atypia, and conjunctival melanoma were obtained from our laboratory from 2005 to 2012. We compared the immunohistochemical profile of these specimens. Each specimen was immunolabeled with dPANMEL, S100, HMB45, Melan A, and Ki67 using the VR substrate. The HMB45 immunolabeled specimens were additionally developed with the DAB substrate with or without overnight 4% H2O2 bleaching. The immunoreactivity data were then analyzed using a 2-tailed Mann-Whitney test (p<0.05 is significant).
The degree of immunoreactivity in specimens developed with VR was similar to those treated with 4% H202 and developed with DAB. Atypical melanocytes were most specifically labeled with HMB45 (93% specific and 88.3% sensitive). The percentage of HMB45 and Ki67 positive cells increased significantly with worsening atypia. dPANMEL and Melan A labeled all benign and malignant melanocytic lesions indiscriminately. 40% of normal conjunctival melanocytes and 58% of PAM specimens stained negative for S100.
We recommend using VR as a standard substrate for the immunohistochemical analysis of melanocytic lesions as the recent VR products with their vivid red colour are relatively inexpensive and do not require bleaching of the samples. We found Melan A and HMB45 to be the 2 minimal markers necessary to fully characterize a given melanocytic lesion. Melan A has superior sensitivity for both benign and malignant melanocytes compared to S100, and HMB45. HMB45 is able to identify atypical melanocytes most specifically.
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