QUANTIFICATION OF EUMELANIN AND PHEOMELANIN - STEREOLOGIC IMAGE-ANALYSIS METHOD

OBJECTIVE: To develop an ultrastructural stereologic image analysis me thod allowing quantification of intra-cellular melanization. STUDY DES IGN: First, in the field of image analysis, a newly elaborated segment ation method. SEM2, was compared with a previously described method ba sed on gray level histograms. Only SEM2 allows the segmentation of mel anin in micrographs of poorly melanized melanocytes. Second, in the fi eld of stereology, estimation of cell volume remains problematic in th e case of mixed cell populations. This problem is approached by the co mparison of stereologic alternatives and cytochemistry (L-3,4-dihydrox yphenylalanine reaction) in epidermal melanocytes and melanoma cells f rom several in vitro experiments. The cytochemical approach was found to be the best choice. RESULTS: Concerning the quantification of eumel anin and pheomelanin, the alkali elution method, permitting the specif ic dissolution of pheomelanin on ultrathin sections, was validated in normal human follicular melanocytes. CONCLUSION: These results allow u s to envisage the stereologic quantification of eumelanin and pheomela nin at the ultrastructural level. At present our method is undergoing evaluation by comparison with the present method of reference base on high-performance liquid chromatography.