Quantification of eumelanin and pheomelanin: stereologic image analysis method.
OBJECTIVE
To develop an ultrastructural stereologic image analysis method allowing quantification of intracellular melanization.


METHODS
First, in the field of image analysis, a newly elaborated segmentation method, SEM2, was compared with a previously described method based on gray level histograms. Only SEM2 allows the segmentation of melanin in micrographs of poorly melanized melanocytes. Second, in the field of stereology, estimation of cell volume remains problematic in the case of mixed cell populations. This problem is approached by the comparison of stereologic alternatives and cytochemistry (L-3, 4-dihydroxyphenylalanine reaction) in epidermal melanocytes and melanoma cells from several in vitro experiments. The cytochemical approach was found to be the best choice.


RESULTS
Concerning the quantification of eumelanin and pheomelanin, the alkali elution method, permitting the specific dissolution of pheomelanin on ultrathin sections, was validated in normal human follicular melanocytes.


CONCLUSIONS
These results allow us to envisage the stereologic quantification of eumelanin and pheomelanin at the ultrastructural level. At present our method is undergoing evaluation by comparison with the present method of reference based on high-performance liquid chromatography.
Donois
E
E
Defense and Cell Regulation Factors Team, University of Bordeaux I, Talence, France.
del Marmol
V
V
Taïeb
A
A
Ghanem
G
G
Surlève-Bazeille
J E
JE
D000293:Adolescent; D000328:Adult; D048429:Cell Size; D002478:Cells, Cultured; D004817:Epidermis; D005260:Female; D006200:Hair Color; D018859:Hair Follicle / Q000737:chemistry / Q000648:ultrastructure; D006801:Humans; D019044:Image Cytometry / Q000379:methods; D007091:Image Processing, Computer-Assisted; D008297:Male; D008543:Melanins / Q000032:analysis*; D008544:Melanocytes / Q000166:cytology / Q000473:pathology / Q000648:ultrastructure; D008545:Melanoma / Q000473:pathology; D014407:Tumor Cells, Cultured
D008543:Melanins; C018362:pheomelanin; C041877:eumelanin