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.

Inhibition of HIF‐1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3‐M cells

“…To minimize operator‐ and platform‐dependent differences in settings adjustment, this study adapted methods of establishing fluorescent particle targeted PMT sensitivities and calculation of spectral overlap (22–27) using our recalled settings as a guide. After establishing appropriate compensation for PI (discussed below), Table 1 illustrates the effect of bead‐adjusted versus recalled flow cytometer instrument settings on the day‐to‐day variability of control and BzATP‐stimulated YO‐PRO‐1 fluorescence associated with viable (CD14‐PE pos /PI neg ) monocytes.…”

“…Specifically, when all samples are processed on the day of phlebotomy with our prior method, the coefficient of variance is 0.10 ± 0.07, whereas if any of the samples have delayed processing the day‐to‐day CV in 0.21 ± 0.13 ( P = 0.09). Considering previous work on standardization (20–29), we have revised our method to further homogenize data collection using readily available fluorescent particles and the viability marker propidium iodide to identify responding cells. In addition, the following variables inherent to multicenter clinical assessments were examined: sample age, different instrumentation, and biological variability.…”

Determination of genomic copy number with quantitative microsphere hybridization

“…In fact, probes that were paralogous to other genomic loci produced inconsistent MFI ratios for different individuals with the same genotype (data not shown). Voltage parameters that resulted in either poor separation of spectrally-distinct multiplexed microspheres or broad FL2 peaks were avoided [Bagwell et al, 1989;Brown et al, 1994].…”

The Effect of Number of Histogram Events on Reproducibility and Variation of Flow Cytometric Proliferation Measurement

“…At the present time, quality control criteria for cell cycle analysis of cytokeratin (CK)-gated histograms is largely undefined. 21 This laboratory has previously reported on the advantages of two-color MPA of solid tumors (Fig. 6), which we have used in the analysis of more than 3,000 clinical samples to date.…”