2-COLOR MULTIPARAMETRIC METHOD FOR FLOW CYTOMETRIC DNA ANALYSIS

Spectral overlap of green fluorescence signals into the red detector ( red-minus-green compensation) is one potential source of variation in two-color flow cytometric DNA analysis. Suboptimal compensation in a t wo-color propidium iodide (PI)/fluorescein isothiocyanate (FITC) syste m may be observed if compensation is adjusted using an inappropriate s tandard, or if changes to fluorescence detector high-voltage settings are made without corresponding readjustment of fluorescence compensati on. To quantitate the influence of red-minus-green compensation on the quality of DNA histograms, data from 60 dual PI/cytokeratin (CK)-FITC stained carcinomas were acquired in parallel using two compensation s tandards: a PI/CK-FITC-stained T24 cell line calibrator overstained to achieve a high-intensity green fluorescence standard (HIGFS) with man ually set compensation and automated compensation settings derived fro m commercial phycoerythrin/low intensity FITC beads (LIGF). Both compe nsation standards gave similar DNA hyperdiploidy results (DNA index, 1 .1-2.8). However, LIGF standard yielded two falsely hypodiploid peaks (DNA index, .7 and .9). Eight left-skewed peaks became DNA diploid and symmetric, respectively, with the HIGFS. Use of HIGFS lowered the coe fficient of variation percentage in 95% of cases, the greatest differe nces (maximum, 3.4%; mean, 1.81%) in tumors of highest intensity CK-FI TC. The authors concluded that use of cell-based compensation standard s (HIGFS) with intense green signals that mimic clinical tumor samples will avoid spurious aneuploidy and maximize resolution of near-diploi d abnormalities.