A NEW FLOW CYTOMETRIC APPROACH TO THE ANALYSIS OF DNA AND A 2ND PARAMETER

OBJECTIVE: To describe a new method of analyzing flow cytometry data b y continuously variable gating (CVG), which is particularly useful for analysis of cellular DNA plus a second parameter, such Its the prolif eration marker Ki-67. STUDY DESIGN: CVG was compared with histogram su btraction (HS) using Overton's cumulative subtraction and with boxed g ating (BG). Background suppression (thresholding) was used to remove o utliers. The comparisons were carried out on a human bladder cancer ce ll line (T24) in various phases of cell proliferation and on disaggreg ated cells of fresh specimens of human prostatic and bladder carcinoma . With similar counts in control and test samples, data did not have t o be normalized. RESULTS: In most cases histogram analysis by CVG teas similar to that of BG and HS. However, when true positive values show ed only a slight shift in fluorescence intensity, HS generated higher values than CVG. When true positive values exhibited strong fluorescen ce signals, HS could falsely include minor shifts in fluorescence valu es, and CVG became move accurate. CVG generated a continuous plot of p ercent positive events that could be superimposed on the DNA histogram , or a DNA histogram of positive events only could be created. This la tter method demonstrated the DNA subpopulations containing most of the positive cells and improved discrimination of small subpopulations. C ONCLUSION: The CVG method presents data in unique graphic form from fl ow cytometry assays of DNA and a second parameter. It is relatively ob jective and particularly useful in analyzing specimens with multiple D NA populations.