The dihydrorhodamine flow cytometry based assay is the more commonly used diagnostic screening test for CGD in reference laboratories and larger medical centers.
This test is based on the principle that nonfluorescent DHR (dihydrorhodamine) 123 when phagocytosed by normal activated neutrophils (after stimulation with PMA – phorbol myristate acetate) can be oxidized by hydrogen peroxide, produced during the activated neutrophil respiratory oxidative burst, to rhodamine 123, a green fluorescent compound, which can be detected by flow cytometry.
Therefore, the detected fluorescence is an indirect measure of neutrophil function and oxidative burst. The top left-hand panel demonstrates normal neutrophil activation resulting in a robust oxidative burst and a complete shift of the peak (containing the cells) to the right indicating DHR oxidation to rhodamine. This shift can potentially be reported as a stimulation index (SI), which is the ratio of the mean fluorescent intensity of stimulated cells over unstimulated cells. In other words, the SI is the ratio of cells with rhodamine to the cells with DHR.