Tracking Entire Units of Transfused Pathogen-Reduced Red Cells in Sickle Cell Patients Using a Novel Acridine Marker

Introduction: Amustaline/glutathione pathogen reduction (PR) of red blood cells (RBCs) is an investigational process designed to reduce the risk of transfusion-transmitted infections and to replace irradiation in preventing transfusion associated-GVHD. Small quantities of acridine, a degradation product of amustaline, can be detected on the surface membranes of circulating PR RBCs by flow cytometry using an acridine-specific monoclonal (2S197-2M1) antibody. We used biotin labelling to validate the use of the acridine marker to track aliquots and entire units of transfused PR-RBCs simultaneously in sickle cell disease (SCD) patients in vivo. Methods: SCD patients (n=6) on monthly simple RBC transfusion protocols received three ~7 mL aliquots of different biotin-dose labeled RBCs: Aliquots drawn from an RBC unit before and after PR treatment (Pre-PR RBCs, PR-RBCs, labeled at 2µg or 6µg biotin), and one from an additional conventional RBC unit (labeled at 18µg biotin). The remaining non-biotinylated PR-RBC and conventional RBC units were also transfused. Flow cytometry for acridine- and biotin-labeled RBCs was performed on samples drawn and frozen at 11 designated time points until 16 weeks post-transfusion during which time the patients also received ~monthly therapeutic conventional RBC transfusions. Acridine antibody was detected with a goat-anti-mouse IgG-phycoerythrin (PE) antibody. Biotin was detected with streptavidin-allophycocyanin (BD Biosciences). Acridine surface density was quantitated using calibrated PE beads (Quantibrite, BD Biosciences). RBC survival for each labelled population at each timepoint was expressed as the proportion of the 1-hour post-transfusion red cell count (RCC) for each biotinylated population and for the non-biotinylated acridine-labeled entire PR-RBC unit. The study was funded by the DHHS Biomedical Advanced Research and Development Authority (HHS010020160009c) and supported in part by a NIH/NHLBI award (K23HL146904 PI: Yee). This study was approved by the Institutional Review Board of Emory University and the Food and Drug Administration for biotinylation of allogeneic RBC transfusion and was registered at clinicaltrials.gov (NCT04426591). Results: Acridine-labeled RBCs from the entire PR RBC unit (peak concentration 7.5-18.7% circulating RBCs) showed near identical survival kinetics over 16 weeks as RBCs from the biotin-labeled PR RBC aliquot (peak concentration 0.6-1.4% of circulating RBCs). The acridine-labeled populations were uniform and discrete but the acridine RBC-surface density declined 83% from a mean of 5,062 PE molecules/RBC at 1-hour to 1,260 PE molecules/RBC by 7 days, decreasing further but remaining detectable at a mean of 110 PE molecules/RBC at 16 weeks. Biotin and acridine labeling revealed non-linear RBC survival curves with interpatient and inter-RBC component variation in the duration and kinetics of survival. Peak circulating concentrations occurred 1-14 days after transfusion for most PR and non-PR components, suggesting initial in vivo sequestration of transfused RBCs, followed by a linear senescence phase of RBC clearance. PR RBCs showed similar survival kinetics to Pre-PR RBCs for the initial ~60 days post-transfusion by biotin labelling, followed by a small relative decline in survival in keeping the relative lifespan documented in normal volunteers with radiolabeling studies (Cancelas et al. Transfusion 2017). Conclusions: Survival of entire PR RBC units up to 16-weeks post transfusion can be tracked in vivo by flow cytometry for RBC surface acridine with similar sensitivity as biotin labeling without extra processing or radiolabeling. SCD patients demonstrated initial sequestration of transfused RBCs. PR RBCs showed survival comparable to RBCs from the same unit prior to PR treatment for the first ~60 days after transfusion. RBC survival with or without PR is highly variable in SCD patients.