[Evaluation of leukocyte oxidative metabolism using whole blood samples by chemiluminescence and flow cytometric assays].
To examine granulocyte oxidative metabolism (GOM) quantitatively, we evaluated two methods, one for assaying luminol-dependent chemiluminescence (CL) and the other for changes in fluorescence by dichlorofluorescin (DCF). The CL assay was carried out using a lumiphotometer (TD-4000, Laboscience) after stimulating granulocytes in whole blood by n-formyl-methonyl-leucyl-phenylalanine (FMLP, 100 nmol/l) or by opsonized zymosan (OZ, 2 mg/ml). To reduce hemoglobin (Hb) interference, each sample was diluted with autologous plasma to a Hb concentration of 3 g/dl. The DCF assay was performed on whole blood samples (100 microliters) labeled with DCF (5 mumol/l) following stimulation by phorbol myristate acetate (PMA, 100 ng/ml). Changes in fluorescence were determined using a flow cytometer that gave the delta mean channel fluorescence intensity (DMCF) as an indicator of GOM. The coefficients of variance (CV) in within-run assays for the CL and the DCF were 10% and 5%, respectively. These CV-values were almost the same even when separated granulocytes as a test sample were used instead of whole blood. CL determined by FMLP stimulation in 27 renal failure patients on hemodialysis (HD), was significantly lower than that in 10 normal controls, but no difference was found in that determined by OZ stimulation. In HD patients, the DMCF values tended to increase in those at 15 min and the end of HD sessions compared to the value prior to HD. This suggested that the HD membrane and/or extracorporeal circulation stimulates GOM.(ABSTRACT TRUNCATED AT 250 WORDS)
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D000328:Adult; D005434:Flow Cytometry; D005609:Free Radicals; D006098:Granulocytes / Q000378:metabolism*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007676:Kidney Failure, Chronic / Q000378:metabolism / Q000503:physiopathology; D008163:Luminescent Measurements; D008165:Luminol; D010100:Oxygen / Q000378:metabolism*; D010587:Phagocytosis; D006435:Renal Dialysis
D005609:Free Radicals; D008165:Luminol; D006861:Hydrogen Peroxide; D010100:Oxygen