ORIGINAL ARTICLE Biphasic clearance of incompatible red blood cells through a novel mechanism requiring neither complement nor Fcg receptors in a murine model Justine S. Liepkalns, Eldad A. Hod, Sean R. Stowell, Chantel M. Cadwell, Steven L. Spitalnik, and James C. Zimring BACKGROUND: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruc- tion following antibody binding is typically thought to require complement activation and/or the involvement of Fcg receptors (FcgRs). In the current report, we ana- lyzed mechanisms of HTRs during incompatible transfu- sions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. STUDY DESIGN AND METHODS: C3 and Fcg receptor knockout, splenectomized, Fcg receptor blocking antibody–treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. RESULTS: This HTR model results in both rapid clear- ance and cytokine storm. Neither complement nor FcgRs were required for RBC clearance; in contrast, FcgRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incom- patible RBCs reentered over 24 hours in clodronate- treated mice. CONCLUSION: These data demonstrate a biphasic HTR, the first phase involving sequestration of incom- patible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcgRs. These findings demon- strate novel mechanistic biology of HTRs. C rossmatch-incompatible transfusions consist of infusing donor red blood cells (RBCs) into a recipient who has antibodies against antigens on the donor RBCs. Except for naturally occur- ring blood group antibodies (e.g., the ABO system), alloantibodies to RBC antigens are generated through prior exposure to allogeneic RBCs, typically by transfusion or pregnancy. 1 In general, incompatible transfusions are strictly avoided, as hemolysis of the transfused RBCs can occur with potentially fatal outcomes, known as hemolytic transfusion reactions (HTRs). 2,3 HTRs can occur during amnestic antibody responses or as a result of clerical error and mistransfusion. 4,5 Moreover, in other instances, crossmatch-incompatible RBCs may be purposefully transfused if the immediate risks of hypoxia (e.g., due to anemia) outweigh the potential damage from an induced HTR. Alloantibodies against fetal RBCs can also cause hemolytic disease of the fetus and newborn, resulting in ABBREVIATIONS: DiI = 1,1′-dioctadecyl-3,3,3′3′- tetramethylindocarbocyanine perchlorate; DiO = 3,3′- dihexadecyloxacarbocyanine perchlorate; FcgRs = Fcg receptors; hGPA = human glycophorin A; HTR(s) = hemolytic transfusion reaction(s); MAC = membrane attack complex. From the Center for Transfusion and Cellular Therapies, Depart- ment of Pathology and Laboratory Medicine, Emory University, and the Aflac Cancer Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia; and the Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons–New York Presbyterian Hos- pital, New York, New York. Address reprint requests to: James C. Zimring, MD, PhD, Puget Sound Blood Center, 1551 Eastlake Ave E, Suite 100, Seattle, WA 98102; e-mail: jzimring@psbc.org. Received for publication November 29, 2011; revision received February 8, 2012, and accepted February 15, 2012. doi: 10.1111/j.1537-2995.2012.03647.x TRANSFUSION **;**:**-**. Volume **, ** ** TRANSFUSION 1