A rapid, specific assay for superoxide release from phagocytes in small volumes of whole blood

A rapid, specific assay was developed to measure the release of superoxide anion from stimulated phagocytic cells in small volumes of whole blood. The assay is based on the chemiluminescence that results when lucigenin (bis-N-methyl acridinium nitrate) is reduced by superoxide. Heparinized whole blood, at volumes from 0.2 mL to a single drop from a 20-gauge needle, was mixed with lucigenin and either a soluble or particulate stimulus (phorbol myristate acetate or opsonized zymosan particles, respectively) in a standard volume of 1 mL. The chemiluminescence was measured at 3-minute intervals for a 30-minute period in a luminometer capable of automated operation. Characteristic plots of chemiluminescence versus time were obtained. This assay is rapid and simple, obviates the need to isolate leukocytes from whole blood, is specific for superoxide, can be performed with both soluble and particulate stimuli, and is sensitive enough to detect characteristic responses with as little as a single drop of blood. Patients with chronic granulomatous disease were readily identified using this assay. The ability to use very small blood volumes makes the assay suitable for use in prenatal diagnosis. The main disadvantage of this assay compared to the often-employed slide nitroblue tetrazolium test, with respect to testing for chronic granulomatous disease, is that carriers of x-linked chronic granulomatous disease are not readily detected with this chemiluminescence assay, as they are with the nitroblue tetrazolium test.