Chromaticity analysis of immunostained tumor specimens.

In order to evaluate the correlation between immunohistochemical and morphometric data on the same histological sections, we have developed a flexible color image analyzer (Microcomputer-Assisted Picture Processing System type II, MAPPS-II), and established an effective method to analyze the immunostained colorectal neoplasms based on the color recognition theory of human visual system. Colorectal adenomas and adenocarcinomas were stained with a monoclonal antibody C 12, which recognizes abnormal H antigen, using Avidin-Biotin method and diaminobenzidine (DAB, brown dye). Nuclei were stained with Hematoxylin (blue dye). Density and colorimetric analyses revealed two results: (A) Separation of immunostained brown area from blue nuclei was best performed by plotting the representative sample areas on a standard chromaticity diagram, which displays the hue and saturation of colors simulating color of human visual system. (B) After separation of immunostained areas, usual density analysis was useful for the assay of nuclear morphometric information. Using these programs, normal mucosa was negative for C 12, and showed low nuclear/cytoplasmic ratio (NCR). Adenoma was occasionally focally positive for C 12, and showed medium NCR. Carcinomas were C 12 positive, and showed high NCR. Our method permits nuclear counterstaining by hematoxylin instead of low contrast methyl green, which will widen the field of combined immunohistochemical and morphometric study.