Photomultiplier voltage setting: possible important source of variability in molecular equivalents of soluble fluorochrome (MESF) calculation?

We evaluated the effect of flow cytometer photomultiplier (PMT) voltage setting on molecular equivalents of soluble fluorochrome (MESF) calculation by using two different quantitative microbead calibrating kits as calibrating standards and a third one as an unknown testing sample of stable intensity of fluorescence. Based on the analysis of residual values derived from linear regression and on the determinations of the resolution index, we demonstrated that, on a FACScan instrument, the window of photomultiplier setting for fluorescein isothiocyanate (FITC) fluorescence determinations is 590-670 V. However, the best region was in the range of 610-650 V. Within the 590-670 PMT voltage region, the evaluation of the same testing sample at different PMT voltages, at 590 V, gave MESF percentage differences of 15% and 13% on the two lower unknown standards of intensity compared to the values obtained at the nearby 630 V setting. In the analyzed PMT region, logarithmic amplifier response was globally maintained, whereas evaluation of the same unknown testing sample in both linear (Lin) and logarithmic (Log) amplification demonstrated (at the higher intensities of fluorescence) Log-Lin minimal differences within the 610-650 PMT voltage range. According to our data, it can be stated that PMT voltages between 610 and 650 provide the best instrument performances and that PMT setting may be proposed as a source of variability in MESF calculation that deserves a careful evaluation in quality-control trials.