dihydrorhodamine test

In normal neutrophils and monocytes, phagocytosis activates NADPH oxidase, which generates reactive oxygen species (superoxide, hydrogen peroxide) as part of the oxidative (respiratory) burst, critical for killing catalase-positive organisms. The DHR test exploits this: dihydrorhodamine 123, a nonfluorescent dye, is loaded into neutrophils and the cells are stimulated with phorbol myristate acetate (PMA) to trigger oxidative burst. If NADPH oxidase is functional, reactive oxygen species oxidize DHR to rhodamine 123, a fluorescent compound detectable by flow cytometry. In patients with CGD, due to mutations in genes encoding NADPH oxidase components (most commonly X-linked CYBB, but also autosomal recessive forms like NCF1, NCF2, CYBA), there is little to no fluorescence shift, indicating failure of the oxidative burst.

Clinically, DHR testing has largely replaced the older nitroblue tetrazolium (NBT) test due to its higher sensitivity, quantitative nature, and ability to distinguish carrier states (X-linked carriers show a bimodal population of fluorescent and non-fluorescent cells due to random X-inactivation, Lyonization).

CGD classically presents with recurrent infections by catalase-positive organisms (e.g., Staphylococcus aureus, Burkholderia cepacia, Serratia marcescens, Nocardia, Aspergillus), recurrent pneumonias, lymphadenitis, hepatic/perianal abscesses, and granuloma formation (due to persistent, poorly degraded antigenic material driving chronic inflammation). Diagnosis is confirmed by low/absent DHR fluorescence, and genetic testing can identify the specific mutation. Treatment includes prophylactic antibiotics (e.g., trimethoprim-sulfamethoxazole) and antifungals (e.g., itraconazole), interferon-gamma therapy, and potentially hematopoietic stem cell transplantation for severe cases.