Useful Immunohistochemical Technique in Heavily Pigmented Malignant Melanoma
Michio Shimizu, Yusuke Hosonuma, Hiroshi Yamaguchi, Koji Nagata, Masanori Yasuda. Saitama Medical University International Medical Center, Hidaka, Saitama, Japan
Background: Immunohistochemistry is often used in the diagnosis of malignant melanoma. However, evaluation can be challenging in heavily pigmented cases because immunohistochemical staining for HMB-45 and Melan-A antibodies using diaminobenzidine (DAB) as the chromogen reveals a brown product, which cannot distinguish melanocytes from melanophages. In such cases, alkaline phosphatase-fast red detection method may be conducted in some institutions. Here, we specifically paid attention to the process of counterstaining in immunohistochemistry.
Design: Fifteen examples of heavily pigmented malignant melanoma were immunohistochemically evaluated by using HMB-45, Melan-A, and microphthalmia transcription factor (MiTF). Immunohistochemistry was performed using four different stains, Giemsa stain, methylene blue stain, toluidine blue stain and cresyl violet stain, as counterstains, and then these four counterstains were compared with one another in terms of their staining quality, handling time, facility and cost.
Results: Thirty minutes was the appropriate time to obtain adequate metachromasia in melanin pigment. All four counterstains showed melanin and melanophages as green blue staining, which can easily contrast with DAB, regardless of the antibody tested. On the other hand, positive melanoma cells showed as brown in most cases. Among the four counter stains described above, methylene blue stain was the most useful because of its better staining quality, shorter handling time and easier handling. Regarding the immersion time, methylene blue stain was only three minutes and was shorter than the other stains (10 to 30 minutes). In areas that were difficult to evaluate immunohistochemically, the addition of immunohistochemical staining using MIB-1 and CD68 was found to be useful.
Conclusions: Modified immunohistochemical technique by counterstain with methylene blue is the most useful diagnostic tool in evaluating heavily pigmented malignant melanoma. It should be performed when encountering heavily pigmented melanocytic lesions, not only melanoma but also blue nevi, pigmented epithelioidmelanocytoma, and pigmented spindle cell variant of Spitz nevi.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 293, Wednesday Morning