A quantitative assay of oxidative metabolism by neutrophils in whole blood using flow cytometry

A large number of purified and washed PMNLs are required to monitor generation of active oxygen derivatives in most in vitro studies and this can preclude investigations in small children. The present method has enabled us to measure the oxidative burst (generation of hydrogen peroxide) of PMNLs in a small amount of whole blood using 2′,7′-dichlorofluorescin diacetate, phorbol myristate acetate and flow cytometry. Optimal conditions for this determination were evaluated and the reaction was found to be independent of the absolute numbers of PMNLs and other types of cell in whole blood. The present method will be of value in investigations of the leukocyte metabolism of patients not only with chronic granulomatous disease (CGD) but also with various infectious diseases.