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Chapter 11.18 : Flow Cytometric Test for Chronic Granulomatous Disease
Normal neutrophil function is of great importance in the host defense against bacterial and fungal infections. As neutrophils recognize, adhere to, and phagocytose an invading organism, they generate an oxidative burst which results in the reduction of molecular oxygen to superoxide. The superoxide produced is rapidly converted to hydrogen peroxide (H2O2, which kills the pathogen). In chronic granulomatous disease (CGD), microbial killing is defective because neutrophils from patients with CGD lack a respiratory burst. The assay described here uses dihydrorhodamine-123 (DHR-123) and phorbol 12 myristate 13 acetate (PMA) to measure oxidative burst activity, which is easily converted to hydrogen peroxide. DHR, a nonfluorescent compound, reacts with hydrogen peroxide and is oxidized to rhodamine-123, a green fluorescent compound. Cellular fluorescence is measured using flow cytometry.