Optimizing the diagnosis of chronic granulomatous disease using dihydrorhodamine‐123 assay: Lessons from a large monocentric cohort

The dihydrorhodamine-123 flow cytometry (DHR FC) assay is used to diagnose chronic granulomatous disease (CGD). This study presents the experience of a single center with the DHR FC assay in a large cohort to evaluate its applications and limitations. A total of 179 subjects were included in the study for CGD diagnosis, X-linked CGD (X-CGD) carrier determination, and post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring. We performed the DHR FC assay using a standardized protocol, with additional intracellular myeloperoxidase (MPO) staining for borderline cases. The DHR FC assay demonstrated high diagnostic accuracy. PMA stimulation detected 94% of CGD cases. E. coli stimulation identified all CGD cases, including atypical EROS-deficient (CYBC1) patients (4.3% of the cohort) who exhibited residual reactive oxygen species production (median SI PMA = 40 vs. E. coli = 24). Genetic confirmation revealed that 82% of CGD patients had X-CGD (CYBB), and 18% had autosomal recessive CGD (CYBA/NCF1/NCF2/CYBC1) forms. Borderline results were clarified by MPO staining, which diagnosed three MPO-deficient cases. The assay reliably identified 43 X-CGD carriers out of 45 suspected cases and showed a strong correlation with the real-time quantitative polymerase chain reaction for HSCT chimerism monitoring (r = 0.78, p < 0.0001). The DHR FC assay is a rapid and reliable diagnostic tool for CGD, providing results within hours. Its applications extend to X-CGD carrier detection and post-HSCT chimerism monitoring. Dual-stimulant protocols (PMA + E. coli) and MPO staining enhance diagnostic accuracy. These findings support the use of the DHR FC assay as a versatile and essential tool in CGD management.