A Novel Method for DNA Cytometry of Exfoliated Cells

Understanding proliferative status of samples from precancerous lesions can be difficult when proliferating cells are mixed among majority of normal cells. Here, we report a novel flow cytometry method to enable detection of proliferative cell populations. Using flow cytometry, we found a high correlation between microsphere diameter to the width of the forward scattered signal (FSC_W) and the fluorescent microsphere diameter to the width of the side fluorescent signal (RFL_W). In addition, the amount of fluorescent intensity of fluorochrome-stained DNA highly correlated to that of the calculated intensities of side fluorescent signals (RFL_A). Next, human buccal cells and human umbilical vein endothelial cells (HUVECs) were measured by flow cytometry to calculate the ratios of nuclear and cell diameters (N/C ratio) of individual cells using the FSC_W and RFL_W values, as these values correspond to the lengths of the cell and nucleus, respectively. By drawing scattergrams of cell lengths and N/C ratios using measurement values of buccal cells and HUVECs, respectively, these cells could be discriminated as low-N/C ratio group (buccal cells) and high-N/C ratio group (HUVECs). In addition, cell cycle analyses of HUVECs, which exhibited high-N/C ratio were performed to obtain the cell proliferation index (CPIx), an original index that represents the proliferative characteristics of cell populations. CPIx values of HUVECs well represented the proliferative conditions of each cell culture. Finally, when low-proliferative HUVECs and high-proliferative Hela cells were mixed and measured in this system, CPIx values of mixed samples were well correlated with the proportion of Hela cells, suggesting that CPIx quantitatively represents proliferative status of samples. Therefore, this method is expected to substantially contribute to improvements in cancer testing such as cervical cancer screening.