Chronic Granulomatous Disease (CGD) is typically caused by mutations in one of the six genes coding for the various components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, which is responsible for the production of reactive oxygen species (ROS)(Leiding and Holland, 2020; Sacco et al., 2020; Yu et al., 2021) from molecular oxygen after electron transfer by NADPH, leading to the formation of metabolites causing the breakdown of bacteria (Bartosz, 2009). Mutations in the NADPH oxidase genes lead to 1) impaired NADPH activity in phagocytes, 2) a decrease or complete lack of ROS generation with a concomitant increase is granuloma formation in various organs, and 3) an increase in infections with bacteria and fungi, particularly catalase-positive organisms, including Nocardia, Listeria, Aspergillus, Candida, Serratia, Klebsiella, Escherichia coli, Staphylococcus, Burkholderia cepacia, and Helicobacter pylori species (Bortoletto et al., 2015; Yu et al., 2021). The incidence of CGD is approximately 1/200,000 births in the US and occurs most commonly as an X-linked disease affecting the gp91phox protein product. Supplemental Table 1 shows the remaining cases of CGD which occur via an autosomal recessive mode of inheritance, affecting one of the other five autosomal gene products, p22phox, p40phox, p47phox, p67phox, RAC, or the NADPH complex chaperone protein EROS (Donko et al., 2022; Kim et al., 2013).

The Dihydrorhodamine-123 (DHR-123) flow cytometry-based assay is typically part of diagnostic testing for CGD patients. DHR-123 is a small uncharged and nonfluorescent molecule which has been utilized for the detection of reactive oxygen species generation (O'Gorman and Corrochano, 1995; Walrand et al., 2003). Upon neutrophil activation by a variety of stimuli, intracellular non-fluorescent DHR-123 is oxidized to brightly fluorescent rhodamine by the reactive oxidative species in concert with myeloperoxidase. Intracellular rhodamine emits bright green fluorescence (peak intensity at 525 nm), when excited by a blue laser at 488 nm. In the flow cytometry-based read of this assay, neutrophils of healthy individuals will emit a homogeneous bright green signal, while CGD patient neutrophils demonstrate no or very low fluorescence. In the DHR flow based assay, the neutrophils of carriers with the X-linked CGD mutation will exhibit two populations of neutrophils, one with very high fluorescence, consistent with those cells expressing the wild type X chromosome gp91phox protein and a second population with no or very low fluorescence consistent with those cells expressing the abnormal (CGD mutated) variant of the gp91phox protein on the X chromosome (Cornaby and O'Gorman, 2024; O'Gorman and Corrochano, 1995).

While patients with CGD have been effectively treated with antimicrobial prophylaxis, the only available curative therapy is hematopoietic stem cell transplantation (HSCT) or less frequently cure via the participation in novel gene therapy based clinical trials (www.clinicaltrials.gov). CGD patients that proceed to transplant are monitored post-transplant to assess engraftment status. By regular monitoring, potential complications can be identified early allowing for appropriate interventions (Lowsky and Messner, 2015; Sureda et al., 2024). Engraftment of donor neutrophils in the patient is monitored by measuring the percentage donor chimerism which is currently assessed using a variety of molecular methods including short tandem repeats (STR), RT-PCR, and more recently, next-generation sequencing (NGS) assays(Blouin et al., 2024; Brow et al., 2024; Liacini et al., 2023). Molecular chimerism assay turnaround times are highly variable ranging from several hours to days using molecular based methods. In addition, during post-transplant engraftment monitoring it is desirable to measure engraftment (percent donor chimerism) within specific myeloid and lymphocyte subsets. For all molecular based chimerism assays, assessment of donor engraftment within specific leukocyte subsets requires sufficient sample volume as well as time-consuming pre-processing steps to enrich for the cells of interest. With the flow cytometry-based assay, donor chimerism can be assessed within the neutrophil population without any preanalytical specimen manipulation. The flow-based assay requires minimal sample volume, (less than 1 mL of whole blood), can be performed rapidly (less than 2 h), and is relatively inexpensive making it a potentially attractive method to measure donor chimerism more frequently (Table 1). Additionally, the method is statistically very robust due to its ability to measure tens of thousands of individual cells (Givan, 2011; Givan, 2001; O'Neill et al., 2013) which in turn allows for the accurate and sensitive detection of very small populations of cells, (Borowitz et al., 2022).

The fluorescence generated in the flow cytometry assay is derived from byproducts of a pre-loaded dye which becomes oxidized via an in-vitro stimulated oxidative burst within single neutrophils. This assay provides a rapid functional evaluation of normal oxidative burst (donor engraftment) vs abnormal oxidative burst (residual CGD patient neutrophils) following transplant with the percent normal being equivalent to the percent donor chimerism. For these reasons, we hypothesize that the assay may provide a rapid and accurate assessment of donor neutrophil engraftment (percent donor chimerism) in the CGD patient population post-transplant.

Other than one reported case study (N = 1), there have been no studies to evaluate the potential and/or performance characteristics of the DHR-123 flow cytometry-based assay to be used as a test to monitor donor chimerism in CGD patients' post-transplant. In this report, we investigate the performance characteristics of the DHR-123 flow cytometry-based assay as a measure of donor chimerism, compare the result of the flow cytometry assay to donor chimerism results obtained via molecular methods, and provide a patient case series illustrating the potential utility of the flow cytometry-based assay.