PMID: 7513943May 1, 1994Paper

Two-color multiparametric method for flow cytometric DNA analysis. Standardization of spectral compensation

American Journal of Clinical Pathology
R D BrownP Mackowiak

Abstract

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 two-color propidium iodide (PI)/fluorescein isothiocyanate (FITC) system may be observed if compensation is adjusted using an inappropriate standard, or if changes to fluorescence detector high-voltage settings are made without corresponding readjustment of fluorescence compensation. 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 standards: a PI/CK-FITC-stained T24 cell line calibrator overstained to achieve a high-intensity green fluorescence standard (HIGFS) with manually set compensation and automated compensation settings derived from commercial phycoerythrin/low intensity FITC beads (LIGF). Both compensation 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 lo...Continue Reading

Citations

Mar 17, 2006·Human Mutation·Heather L NewkirkJoan H M Knoll
Apr 24, 2008·Cytometry. Part B, Clinical Cytometry·N L Korpi-SteinerL C Denlinger

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