Molecular Determinants of Human Red Blood Cell Survival in Murine Circulation

Abstract

Introduction: Transfusion of human red blood cells (RBCs) is an irreplaceable life-saving medical intervention. However, as the expression of surface proteins on donor RBCs can regulate their circulatory lifespan, these proteins can affect the efficiency of transfusion. A murine preclinical transfusion model was used to better understand the impact of these proteins on human RBC survival. Methods: Human peripheral blood RBCs were obtained from qualified blood donors as aliquots of leukoreduced, packed CP2D/AS1 RBCs, or separately collected from healthy research donors. RBCs were biotinylated at densities ≤18 mg/ml following established protocols. Unlabeled or biotinylated human RBCs were transfused into immunodeficient NCG mice treated with chlodronate and cobra venom factor before and after transfusion. Human RBCs in murine circulation were identified with FITC-conjugated anti-human CD235a antibodies and/or PE-conjugated streptavidin. Aliquots of transfused RBCs were analyzed by flow cytometry for expression levels of the surface molecules CD35, CD36, CD38, CD44, CD47, CD55, CD59, CD147, CD233 and CD235a before transfusion, and also in peripheral blood samples collected from mice after transfusion. In some studies, human RBCs were treated with antibodies against selected surface molecules before transfusion. Results: Samples collected from transfused mice at serial timepoints showed significantly decreased expressions over time of several human RBC surface molecules: CD47, CD55, CD59, CD147, CD233 and CD235a. In contrast, there were no significant changes in RBC expression of CD35, CD36, CD38 and CD44 in the samples. Among the surface molecules showing lower levels over time, there was a significant positive correlation between CD47 or CD59 expression levels on human RBCs and circulatory survival of those RBCs in the mice. When mixtures of RBCs from two donors were transfused simultaneously, those RBCs with higher CD47 or CD59 expression demonstrated significantly longer survival in murine peripheral blood in comparison with the co-transfused RBCs. Furthermore, pre-incubation of human RBCs with antibodies against CD47 or CD59 prior to transfusion markedly reduced their survival in murine circulation. In contrast, antibodies against CD55, CD147, CD233 or CD235a had no effect on human RBC circulation. Conclusions: Our data show that CD47 and CD59 expression on human RBCs increases their survival after transfusion, and suggest that pre-transfusion profiling of CD47 and CD59 molecules could help predict the efficiency of human RBC transfusion.