Improved compensation of the fluorochrome AmCyan using cellular controls

Implementation of polychromatic flow cytometry in the clinical laboratory often requires the use of newer fluorochromes, with which experience may be comparatively limited. In the course of implementing polychromatic flow cytometry in our laboratory, we have observed significant differences in compensation values derived for the violet-excited dye, AmCyan, when cells rather than a commercially available set of polystyrene microparticles (BD CompBeads) are used as compensation controls.

Compensation values were calculated for AmCyan and several other fluorochromes using the BD CompBeads Set according to the manufacturer's protocol, and using unstained and singly stained lymphocytes as compensation controls.

When the BD CompBeads Set was used to determine compensation values, spillover from AmCyan into V450 was overcompensated, while spillover from AmCyan into FITC was undercompensated. In contrast, when compensation values were calculated using unstained and singly stained lymphocytes, spillover into V450 and FITC from cells stained brightly with AmCyan-conjugates was compensated appropriately. Although significant differences were observed in the compensation of spillover from AmCyan into V450 and FITC using cells rather than the BD CompBeads Set as compensation controls (P < 0.0001, two-tailed Wilcoxon signed-rank test), such differences were not observed in control experiments using fluorochromes excited by the blue (FITC and PE) or red (APC) lasers.

Improved compensation of the violet-excited dye, AmCyan, is obtained when cells rather than the BD CompBeads Set are used as compensation controls.