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Optimizing the diagnosis of chronic granulomatous disease using dihydrorhodamine-123 assay: Lessons from a large
Lodoeva Oiuna1, Varlamova Tatiana2, Yukhacheva Daria3
1Laboratory of Transplantation Immunology and Immunotherapy, Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology, Moscow, Russia.
Insights
The dihydrorhodamine-123 flow cytometry (DHR FC) assay is a reliable tool for diagnosing chronic granulomatous disease (CGD). This assay also effectively identifies X-linked CGD carriers and monitors stem cell transplant success.
Area of Science:
- Immunology
- Hematology
- Clinical Diagnostics
Background:
- Chronic granulomatous disease (CGD) is a primary immunodeficiency affecting phagocyte function.
- Accurate diagnosis of CGD and carrier status is crucial for patient management and genetic counseling.
- Monitoring post-hematopoietic stem cell transplantation (HSCT) chimerism is essential for assessing transplant efficacy.
Purpose of the Study:
- To evaluate the diagnostic accuracy and versatility of the dihydrorhodamine-123 flow cytometry (DHR FC) assay.
- To assess the DHR FC assay's utility in identifying X-linked CGD carriers.
- To determine the DHR FC assay's effectiveness in monitoring HSCT chimerism.
Main Methods:
- A cohort of 179 subjects underwent DHR FC assay for CGD diagnosis, carrier determination, and HSCT chimerism monitoring.
- Standardized DHR FC protocol with additional intracellular myeloperoxidase (MPO) staining for borderline cases.
- Dual stimulation with phorbol myristate acetate (PMA) and E. coli was employed.
Main Results:
- The DHR FC assay showed high diagnostic accuracy, detecting 94% of CGD cases with PMA and all cases with E. coli stimulation.
- Atypical EROS-deficient (CYBC1) patients were identified, accounting for 4.3% of the cohort.
- The assay reliably identified 43 out of 45 suspected X-CGD carriers and correlated strongly with quantitative PCR for HSCT chimerism (r=0.78).
Conclusions:
- The DHR FC assay is a rapid, reliable, and versatile tool for diagnosing CGD, detecting carriers, and monitoring HSCT chimerism.
- Dual-stimulant protocols and MPO staining enhance diagnostic precision.
- The DHR FC assay is an essential component of comprehensive CGD management.
Abstract:
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.
