A New Method Utilizing Nitroblue Tetrazolium Reduction to Evaluate Superoxide Production of Human Polymorphonuclear Leukocytes at Phagocytosis of Candida albicans

Abstract

A new method utilizing nitroblue tetrazolium (NBT) reduction was to designed to evaluate the superoxide (O^-₂) production of human polymorphonuclear leukocytes (PMN) at phagocytosis of C. albicans. The most ideal experimental condition was set up as follows: 0.2ml each of PMN (2.5×10⁶/ml) and Candida (2.5×10⁷/ml) suspensions with the addition of 0.1ml of 50% autoserum were put into a polyethylene tube for 30 minutes of incubation in a CO₂ incubator followed the measurement of phagocytosis (NBT-phagocytosis) and NBT reduction. The same experimental procedure but without the addition of NBT was performed simultaneously to determine the phagocytosis (PBS-phagocytosis) and candidacidal activity of PMN after 30 and 180 minutes of incubation, respectively. Killing activity was estimated by counting the rate of killed Candida (killing rate) from the culture of the incubated suspension on Sabourud destrose agar. In studies of normal control, positive correlations existed between PBS- and NBT-phagocytic rates (p<0.001) and between NBT reduction and killing rates (p<0.05). The mew method was applied to clinical patients with pulmonary diseases to verify its usefulness, and some were revealed to have various degrees of impaired PMN functions. These experimental results thus confirmed the value of the new method in the analysis of human PMN functions.