Instrument-dependent fluorochrome sensitivity in flow cytometric analyses

Flow cytometry has become the preferred technique by which critical clinical evaluations are made such as CD4 counts and aneuploid analyses. Mounting concern has arisen over the numerous techniques, reagents, and different flow cytometric employed to determine these data. Several studies have documented significant differences in results when different flow cytometers are utilized to analyze the same sample. Fluorochrome-dependent instrument sensitivity also has been reported by numerous investigators. As more and more procedures are performed by cytometric analysis, light scatter and fluorescence limitations, which appear to be instrument dependent, demonstrate that not all flow cytometers have the same capabilities. Attempts were made to calculate molecules of equivalent soluble fluorochrome (MESF) values on nine different flow cytometers using fluorescein isothiocyanate (FITC) and R-phycoerythrin (R-PE) labeled microsphere reference standards produced by Flow Cytometry Standards Corporation (FCSC). Dramatic differences were observed in the ability of some cytometers to resolve these microspheres. The diminished resolution appeared to be instrument model and fluorochrome dependent. We propose that diminished fluorescence resolution in certain flow cytometers could be responsible for significant variability in clinical values reported from laboratories utilizing different flow cytometers.