Diagnosis and carrier detection of chronic granulomatous disease in five families by flow cytometry

Background: The application of flow cytometric assays, for determination of phagocyte respiratory burst (ROB) activity, to the investigation of chronic granulomatous disease (CGD) may lead to improved laboratory detection of patients and carriers and indicate the nature of the molecular defect. To evaluate the diagnostic capability of flow cytometry an investigation of 5 CGD families was undertaken. Methods: Phorbol myristate acetate (PMA)-induced neutrophil ROB was determined using dihydrorhodamine 123 (DHR) and flow cytometric analysis in 26 members of 5 CGD families (2:X-CGD;3:autosomal recessive CGD). Results: Neutrophils from W-CGD patients displayed absence of reactivity. Female carriers demonstrated dual fluorescence peaks of high and low intensity indicative of normal and abnormal populations, respectively. Normal ROB activity was observed in a boy whose X-CGD was successfully treated by bone marrow transplantation. Reduced ROB activity was observed in 3 patients with autosomal-recessive CGD compared with their parents and siblings, The patterns of flow cytometric reactivity correlated with the different molecular defects identified. Absence of the p22(phox) membrane component of the NADPH oxidase complex resulted in a significantly reduced level of respiratory burst activity which was comparable to that observed in X-CGD. whereas reduced but detectable levels of respiratory burst activity were observed in a patient with diminished levels of p22(phox) and in a patient with deficiency of the cytosolic p47(phox) component. Conclusions: The DHR flow cytometric assay offers a sensitive diagnostic screening test for CGD and furthermore may provide an indication of the likely underlying molecular defect.