Chronic Granulomatous Disease (CGD) identification and carrier state detection by flow cytometric analysis of granulocyte respiratory burst

Chronic granulomatous disease includes a group of genetically inhereted diseases in which there is a reduced capacity of phagocytes to release union superoxide (O₂ ^-) upon infection of cells with microrganisms or chemotactic factors. Diagnosis depend upon demonstration of reduced production of O₂ ^- by neutrophils activated with phorbol-myrestate-acetate (PMA). Nitro blue tetrazolium (NBT) reduction test is the commonly used tecniquefor the identification of CGD patients. However, this method does not allow the identification of patients with variant-CGD that display a residual production of reactive oxygen. Recently, novel tests for the diagnosis of CGD (based on flow-cytometry analysis) have been introduced. We investigated anion superoxide production in PMA-activated neutrophils of 28 healthy adult donors, 30 children with the clinical suspect of CGD, and 5 of the X-linked form of CGD (X-CGD) by flow cytometric with dihydrorhodamine 123, NBT salts precipitation and dismutase-inhibitable cytochrome-c reduction. Each of these assays allowed correct identification of 5 CGD patients among 30 studied, although the NBT test failed to recognize a low production of O₂ ^- in a patient then found affected from a variant form of the disease. These three diagnostic assays were then used to study anion superoxide release in neutrophils derived from 7 mothers or sisters of X-CGD patients. Interestingly, in one of the CGD carriers we found that about 6% of DHR-loaded granulocytes failed to respond to PMA stimulation, thus indicating that this subject was an X-CGD carrier as confirmed by mutational analysis of CYBB gene. In contrast, the both cytochrome- c reduction test and the NBT test failed to detect the carrier state in this subject. On the basis of our results we conclude that flow cytometric analysis of anion superoxide production by PMN, is an effective, rapid and accurate tool for diagnosis of Chronic Granulomatous Disease and allows precise identification of X-CGD-carrier.