Determination of neutrophil function by measuring superoxide production with whole blood flow cytometry

The function of neutrophil can be evaluated by measuring oxidative metabolism using chemiluminescence, tetrazolium dye reduction or the others. Those results are not always satisfactory which would be caused by subtle difference in each preparation of the reagents and the lack of reproducibility. Recently, flow cytometric procedures for semi-quantitating superoxide production in neutrophils have been developed to evaluate their function. This procedure, which requires only small amount of whole blood, can easily and rapidly yield reproducible and reliable data. In this study, we optimized analytical conditions and then determined reference interval to evaluate neutrophil function of patients with various disorders. Optimal concentrations and incubation times of DCFH-DA and PMA were 5 mumol/l for 15 minutes and 25 micrograms/ml for 20 minutes, respectively. Production of superoxide in neutrophil was represented by relative fluorescence intensity(RFI) with assay coefficient of variance(CV) of 4.0-11.1%. Neutrophils had to be examined within 2 hours after venipuncture to obtain reliable data. Reference interval was determined as 170.4 +/- 58.7(mean +/- SD) RFI. Neutrophil function of patients with neutropenia, paroxysmal nocturnal hemoglobinuria(PNH), renal failure, systemic lupus erythematosus(SLE), myeloperoxidase deficiency, myelodysplastic syndrome(MDS), and diabetes mellitus were within the reference interval as evaluated by this method. Only neutrophils of chronic granulomatous disease, which is known to give clearly low superoxide production, showed actually decreased value. These results indicate that this procedure would be clinically useful for diagnosis of patient with impaired neutrophil function.