Two-color multiparametric method for flow cytometric DNA analysis. Standardization of spectral compensation.
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.
Brown
R D
RD
Division of Surgical Pathology, Henry Ford Hospital, Detroit, Michigan 48202.
Zarbo
R J
RJ
Linden
M D
MD
Torres
F X
FX
Nakhleh
R E
RE
Schultz
D
D
Mackowiak
P G
PG
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
D004273:DNA, Neoplasm