Chronic granulomatous disease (CGD) is a group of congenital immunodeficiencies characterized by defective intracellular oxidative killing in phagocytic leukocytes, caused by gene defects in NADPH oxidase enzyme complex. Mutations in at least four gene products, gp91-phox, p22-phox, p47-phox, and p67-phox, have been found to cause the phenotype of CGD. While gp91-phox gene defects are inherited as X-linked recessive traits, the other three are autosomal recessive (AR) genes. The diagnosis of CGD is made by measuring oxidase activity after polymorphonuclear leukocyte (PMN) activation by appropriate stimuli. Flow cytometric analysis has largely replaced other technically demanding methods in diagnosing CGD and detecting X-linked CGD gene carriers. This method, however, has not been successfully used in the detection of AR-CGD carriers. In order to apply the quantitative power of flow cytometry to the diagnosis of CGD, we modified methods for flow cytometric analysis of granulocyte respiratory burst. We were able to distinguish CGD patients, AR-CGD gene carriers, and unaffected relatives by this optimized flow cytometric analysis.

Materials and Methods: Peripheral blood leukocytes were isolated from members of an AR-CGD family and from normal controls. Isolated leukocytes were incubated with H2DCFDA and then stimulated with a submaximal concentration of PMA for 15 minutes. Catalase was then added to stop the reaction. PMN were gated based on cell size and granularity and the fluorescence intensities measured. Stimulation indexes (SI) were the ratios of the mean fluorescence intensities of stimulated PMN and those of unstimulated PMN.

Results: PMN from the CGD patient showed almost no response to PMN stimulation (SI=1.27) while PMN from normal subjects showed significant respiratory burst (SI ranged from 30.1 to 82.9, n = 4). Both parents showed subnormal responses (SI=3.35 and 5.94). Some other asymptomatic family members also showed subnormal responses (SI ranged from 2.71 to 4.84, n=4, but other asymptomatic family members showed normal responses (SI ranged from 47.8 to 60.1, n=3. We conclude that this modified method will facilitate detection of AR-CGD carriers and is useful in genetic counseling, prenatal diagnosis and screening for new CGD genes in the general population.