Abstract
Objective:
To investigate whether multiparameter flow cytometric analysis of solid tumor specimens, including gynecologic tumors, which were stained triply with phycoerythrin (PE), fluorescein isothiocyanate (FITC) and propidium iodide (PI), can be performed simultaneously without interference from normal diploid cell populations and spectral overlap on a standard flow cytometer.
Methods:
MCFโ7 breast cancer cell lines and heterogeneous cell populations mixed with MCFโ7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferationโassociated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, cโerbB/2 and cโmyc) were labeled with PE and FITC, which was followed by DNA staining using PI. These labeled cells were measured on a standard FACScan flow cytometer equipped with a 488 nm single laser.
Results:
The coefficient of variation (CV) of the G~0~G~1~ peak of MCFโ7 cells was 4.3 and the cell cycle phase fractions of G~0~G~1~, S and G~2~M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCFโ7 cells expressed cytokeratin, PCNA, p53, cโerbB/2 and cโmyc antigen. In the heterogeneous population of MCFโ7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCFโ7 cells without interference. The CV of G~0~G~1~ peak of PBL was 2.3 and the G~0~G~1~, S and G~2~M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCFโ7 cells was 1.7, which indicated that the MCFโ7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G~0~G~1~ peak of the MCFโ7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G~0~G~1~, 42.3% for S, and 10.2% for G~2~M. The MCFโ7 cells expressed cytokeratin, but the PBL did not.
Conclusions:
Multiparameter flow cytometer analysis was useful to determine DNA ploidy status, phase fraction of the cell cycle and expression of cellular antigens and selective cytokeratin expression allowed epithelial originated tumor cells to be differentiated from normal stromal cells. This analysis could be performed without interference of spectral overlaps of fluorochromes using softwareโbased algorithmic compensation of spectral overlaps. Thus, this method offers new possibilities for multiparameter flow cytometric analysis and its use should be extended to future studies of the diagnosis, treatment and prediction of prognosis of the neoplasm.