Routine clinical flow cytometric procedures demand rigorous, simple, and reproducible procedures for spectral compensation. The current, often laborious, spectral compensation procedures are the result of variability in instrument settings, instrument performance, and variability in reagents. In particular, the use of tandem dye conjugates necessitates elaborate spectral compensation procedures that need to be applied frequently. Manufacturer, lot number, and handling procedures are considered the key aspects affecting the fluorescence characteristics of tandem dyes. A better understanding of how specific conditions affect the variability in emission spectra of tandem dyes can lead to a considerable increase in reliability of measurements and a potential simplification of setup procedures for routine, clinical flow cytometry. We investigated the effect of light exposure, handling, and storage conditions on the fluorescence characteristics of some common phycoerythrin tandem fluorochromes. In general, PE-Cy5 showed the lowest degradation rates, whereas PE-Cy7 showed the highest. During storage, long-term degradation rates were lowest for reagents packaged using an extra light protective approach. Under these conditions, a degradation rate of 0.9%/month of a PE-Cy7 conjugate decreased to 0.3%/month. As degradation rates were minimized, we studied the effect of slow degradation of a set of tandem dye conjugates on compensation matrix values over several months. Finally, we explored the effect of slow degradation on flow cytometric analysis using the same compensation settings for extended periods for an analysis template with preset regions and gating strategies. ' 2009 International Society for
Advancement of CytometryKey terms degradation; fluorochrome; stability; T-regulatory cells; CD4/CD8; clinical; flow cytometry; compensation THE emission spectra of most fluorochromes are fairly constant. The conjugation of fluorochromes to proteins (e.g., antibodies) may affect their quantum efficiency (i.e., absolute fluorescence intensity) but shifts their entire spectrum by only a few nanometers (1). In contrast, tandem dyes are known to display a considerable degree of variability in fluorescence intensities within different regions of each emission spectrum. A tandem dye is a combination of two fluorochromes (donor and acceptor dye) such that the difference between the excitation and emission maxima (Stokes shift) is significantly increased relative to that of the donor dye. The higher Stokes shift is accomplished by means of fluorescence resonance energy transfer (FRET) between the donor molecule (e.g., phycoerythrin) and acceptor molecule (e.g., cyanine 7) (2-5). The optimum energy transfer level depends on the acceptor-to-donor molar ratio, distance, and orientation and results in significant reduction of donorderived emission, while self-quenching of the acceptor is avoided (3,6,7). The delicate balance between donor and acceptor is the reason that emission spectra of tandem dyes are affected by manufactur...
