Three parameter flow cytometric analysis for simultaneous detection of cytokeratin, proliferation associated antigens and DNA content.

The analysis of the cell cycle distributions by univariate flow cytometric DNA measurement has been widely applied in the clinic to determine kinetic parameters of human malignancies. A common problem with measurements of cell cycle phase distributions in tumor biopsy material is the presence of non-malignant diploid cells. Furthermore, such a static measurement might not be accurate enough to describe the dynamic process of cell proliferation. For this purpose alternative methods have been developed to include BrdUrd incorporation or the presence of intrinsic proliferation associated markers such as PCNA or Ki67-Ag into the analysis. However, the presence of nonmalignant diploid cells will influence also these bivariate analyses, especially in case of DNA-diploidy of the tumor cells. Here we present a three parameter flow cytometric assay based on the simultaneous detection of cytokeratin, DNA and a proliferation associated marker, such as BrdUrd, PCNA or Ki67-Ag. Based on the presence of cytokeratin, epithelial cells can be selected for a detailed cell cycle analysis. This method can be applied to frozen tissue, which makes this assay useful for multicentre clinical studies.