Cerus Corporation, Concord, CA, USA, California, United States
Background/Case Studies: A simple clinical method to assess clearance of transfused RBCs would help predict the physiologic activity of antibodies or drugs implicated in hemolytic reactions. Pathogen reduction of RBCs (PR-RBCs) with amustaline/glutathione is an investigational process to reduce transfusion-transmitted infections. We describe a novel tool to track PR-RBC survival in vivo by flow cytometry (FACS) using a monoclonal antibody specific for membrane-bound acridine, a byproduct of the PR process. Data were derived from Phase 2 RBC survival studies in a healthy volunteer and in sickle cell anemia (SCA) patients.
Study
Design/Methods: A healthy volunteer received a 5 mL aliquot of autologous 35-day-stored 51Cr and biotin-labelled PR-RBCs during a RBC recovery and survival study (Clinicaltrials.gov NCT03384407). In a separate study of SCA using biotin-labelled RBCs, two patients received three ~7 mL aliquots of different biotin dose levels of labeled RBCs: Two aliquots from an RBC unit before and after PR treatment (Pre-PR RBCs, 2µg biotin; PR-RBCs, 6 µg biotin), and one from a conventional RBC unit (Control, 18µg biotin). The remaining PR-RBC and Control RBC units were transfused concurrently. The proportion and antigen density of circulating acridine-labeled cells in recipient blood was determined by FACS using an acridine antibody (2S197-2MI).
Results/Findings: The healthy volunteer study demonstrated that after transfusion of 5 ml of PR-RBCs the acridine label could be detected by FACS at 24 hours (0.33% circulating RBCs) and up to 112 days post transfusion (0.06% circulating RBCs). In the biotin study of SCA patients, the acridine assay detected 10.0-12.3% circulating PR-RBCs 24 hours after transfusion of the entire PR-RBC units. The density of acridine RBC surface antigen (Table) was equivalent to the 18µg biotin label 1-hour after transfusion and declined homogeneously ~50% at 24 hours post-transfusion and by >80% on day 7 but remained stable near the 2 µg biotin label level through the 98-110-day observation period. Biotin and acridine labeling resulted in similar linear RBC survival curves. Conclusions: Circulating PR-RBC can be detected in vivo following transfusion of small RBC aliquots or entire RBC units using FACS for RBC surface acridine, with similar sensitivity as biotin labels and increased sensitivity compared to 51Cr labels. Transfusion of PR RBCs provides a potential method to rapidly assess the physiologic activity of antibodies and drugs, including therapeutic monoclonal antibodies, that may impact RBC clearance in vivo, without additional processing or use of radiolabels. A standardized method and criteria for defining rapid clearance are in development.
Importance of research: We describe a novel method for tracking pathogen reduced RBC survival in vivo without radiolabeling or further manipulation.
