A new flow cytometric approach to the analysis of DNA and a second parameter

OBJECTIVE: To describe a new method of analyzing flow cytometry data by continuously variable gating (CVG), which is particularly useful for analysis of cellular DNA plus a second parameter, such Its the proliferation marker Ki-67. STUDY DESIGN: CVG was compared with histogram subtraction (HS) using Overton's cumulative subtraction and with boxed gating (BG). Background suppression (thresholding) was used to remove outliers. The comparisons were carried out on a human bladder cancer cell line (T24) in various phases of cell proliferation and on disaggregated cells of fresh specimens of human prostatic and bladder carcinoma. With similar counts in control and test samples, data did not have to be normalized. RESULTS: In most cases histogram analysis by CVG teas similar to that of BG and HS. However, when true positive values showed only a slight shift in fluorescence intensity, HS generated higher values than CVG. When true positive values exhibited strong fluorescence signals, HS could falsely include minor shifts in fluorescence values, and CVG became move accurate. CVG generated a continuous plot of percent positive events that could be superimposed on the DNA histogram, or a DNA histogram of positive events only could be created. This latter method demonstrated the DNA subpopulations containing most of the positive cells and improved discrimination of small subpopulations. CONCLUSION: The CVG method presents data in unique graphic form from flow cytometry assays of DNA and a second parameter. It is relatively objective and particularly useful in analyzing specimens with multiple DNA populations.