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: MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) 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 G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 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 software-based 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.
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: MCF-7 breast cancer cell lines and heterogeneous cell populations mixed with MCF-7 cells and human peripheral blood lymphocytes (PBL) were fixed with 1% paraformaldehyde and permeabilized with 100% methanol. Cytokeratin and several proliferation-associated cellular antigens (proliferating cell nuclear antigen (PCNA), p53, c-erbB/2 and c-myc) 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 G0G1 peak of MCF-7 cells was 4.3 and the cell cycle phase fractions of G0G1, S and G2M were 44.9, 45.9 and 9.2%, respectively. Fluorescein isothiocyanate, PE and PI fluorescences were detected without interference. The MCF-7 cells expressed cytokeratin, PCNA, p53, c-erbB/2 and c-myc antigen. In the heterogeneous population of MCF-7 cells mixed with PBL, two cellular populations were clearly separated into diploid PBL and aneuploid MCF-7 cells without interference. The CV of G0G1 peak of PBL was 2.3 and the G0G1, S and G2M phase fractions were 85.5, 2.7 and 11.8%, respectively. The DNA index of MCF-7 cells was 1.7, which indicated that the MCF-7 cell line was composed of tumor cells with aneuploid DNA. The CV of the G0G1 peak of the MCF-7 cells was 4.2, and the cell cycle phase fractions were 47.5% for G0G1, 42.3% for S, and 10.2% for G2M. The MCF-7 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 software-based 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.