Flow cytometry and the stability of phycoerythrin‐tandem dye conjugates

Abstract

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...

Practical guidelines for the high‐sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry

“…It also has a very low detection frequency of PNH phenotypes in normal samples (approximately four cells with Type III PNH phenotype per million). This method also has an advantage for labs performing infrequent testing in that FITC and PE conjugates have a longer shelf life than conjugates made with tandem dyes as they are not prone to breakdown of the tandem dye components (33).…”

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“…The supernatant was discarded and cells resuspended into 0.5 ml of PBS. All reagents were kept cold and protected from light and exposure to the air; all stained samples were run promptly after processing to minimize any effect of degradation of tandem-dyes after fixation (42).…”

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“…If this is not possible (e.g., lack of availability from an antibody manufacturer), each new lot of antibody should be appropriately titrated to ensure that the flow cytometry data will be comparable to that obtained using the previous lot. The central lab and the site processing lab must take great care to ship and store the antibody cocktail at 4 °C and avoid light exposure to minimize degradation that can occur, especially with tandem dyes [8][9] .…”