[A quantitative measurement of polymorphonuclear leukocyte oxidative metabolism using plasma depleted blood by flow...

We investigated a method measuring the superoxide production in peripheral polymorphonuclear leukocytes (PMN) in whole blood by flow cytometer using DCFH-DA as marker dye. The DCFH-DA which is colorless in its form and is freely permeable into blood cells is converted into DCF, a fluorescent substance, by oxidative reaction of superoxide in PMN. Thus, the superoxide production in PMN is measurable by counting number of fluorescence positive cells and their intensity by flow cytometer. However, when the DCFH-DA is added to the whole blood, a considerable amount of it is trapped not only in PMN but also in red blood cell, thus giving rise an inconsistent incorporation of the DCFH-DA in PMN due to differences in number of red blood cell among samples. Furthermore, there are several anti-oxidative factors in blood plasma. In order to avoid possible effects of plasma oxidative factors and red blood cell-numbers in whole blood on the measurement of PMN-superoxide production, we prepared plasma depleted blood by washing and the number of red blood cells was adjusted to 5 x 10(6) microliters before addition of DCFH-DA. This improved reproducibility and gave within assay coefficient of variance (CV) of 3.62% for a sample with normal value and 7.09% for a low value sample. The value obtained by this method correlated well with those of NBT reduction test (r = -0.887). It is concluded that this method is simple, reliable and useful for the clinical determination of superoxide production instead of NBT test.