Standardization of a Flow Cytometric Assay for Phagocyte Respiratory Burst Activity

The authors optimized the flow cytometric dichlorofluorescin (DCFH)-oxidation assay for buffy coat neutrophil and monocyte respiratory burst activity. Sample handlings were minimized, monocytes identified with a CD14 antibody, and viability evaluated with propidium iodide. Sodium citrate was a better anticoagulant than heparin, with a more intense Yersinia enterocolitica (YER)-induced dichlorofluorescein (DCF)-fluorescence intensity and a higher proportion of DCF-positive cells. EDTA was unsuitable as an anticoagulant with reduced cell viability and poor DCF response. Exposure of cells to YER, phorbol myristate acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) elicited two neutrophil subpopulations, one with low and the other with high forward light scattering properties. FMLP induced only a marginal DCF response, but after YER or PMA, virtually all neutrophils responded with an increased DCF production. During optimal conditions, the resulting DCF- fluorescence histogram was two-peaked, and the subset of cells with increased forward light scattering properties corresponded to the cells with intense DCF-fluorescence. A similar heterogeneity was frequently but not always observed amongst monocytes. The results indicate that in the peripheral blood there are at least two neutrophil and monocyte populations. One is an effective responder to stimuli, the other exhibiting a moderate response only. Properly optimized, the DCFH-oxidation assay may be used for evaluating neutrophil and monocyte subsets in a clinical setting.