title: Two-color multiparametric method for flow cytometric DNA analysis. Standardization of spectral compensation.
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 lowered the coefficient of variation percentage in 95% of cases, the greatest differences (maximum, 3.4%; mean, 1.81%) in tumors of highest intensity CK-FITC. The authors concluded that use of cell-based compensation standards (HIGFS) with intense green signals that mimic clinical tumor samples will avoid spurious aneuploidy and maximize resolution of near-diploid abnormalities.
authors.affiliation: Division of Surgical Pathology, Henry Ford Hospital, Detroit, Michigan 48202.
authors.forename: R D
authors.initials: RD
authors.lastname: Brown
authors.forename: R J
authors.initials: RJ
authors.lastname: Zarbo
authors.forename: M D
authors.initials: MD
authors.lastname: Linden
authors.forename: F X
authors.initials: FX
authors.lastname: Torres
authors.forename: R E
authors.initials: RE
authors.lastname: Nakhleh
authors.forename: D
authors.initials: D
authors.lastname: Schultz
authors.forename: P G
authors.initials: PG
authors.lastname: Mackowiak
mesh_terms: D000704:Analysis of Variance; D003116:Color; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009369:Neoplasms / Q000473:pathology*; D011003:Ploidies; D012015:Reference Standards; D012044:Regression Analysis; D013194:Staining and Labeling
chemical_list: D004273:DNA, Neoplasm