Leukoreduction Decreases Alloimmunogenicity of Transfused Murine HOD RBCs.

Abstract Abstract 640 Introduction: Alloimmunization against transfused red blood cell (RBC) antigens can lead to hemolytic transfusion reactions as well as difficulties in locating compatible units of blood for future transfusions. Despite the clinical significance, factors influencing rates of RBC alloimmunization are not clearly defined. Leukoreduction decreases rates of HLA alloimmunization, because HLA antigens are carried on white blood cells (WBCs). However, most human RBC antigens are not carried on WBCs, and the effect of leukoreduction on RBC alloimmunization is not known. Many potential variables, including the large number of RBC antigenic differences between donor and recipient, make this question difficult to answer in the setting of human transfusion medicine. We hypothesized that WBCs in transfused RBC units would increase alloimmunization to an RBC specific antigen, and used a reductionist murine model to investigate this issue. Materials and Methods: Transgenic HOD donors, with RBC specific expression of the model antigen hen egg lysozyme (HEL) linked to a multi-pass human Duffy antigen (Fyb), were bled into CPDA-1 to give a final storage medium concentration of 14%. Leukoreduction was performed over a Pall neonatal leukoreduction filter, with residual WBCs quantified by propridium iodide and Trucount beads. Expression of HEL and Fyb on HOD RBCs was assessed by flow cytometry prior to transfusion. Leukoreduced or non-leukoreduced pRBCs (75 μL) were transfused via lateral tail vein, and post-transfusion survival of HOD RBCs was determined by analysis of HEL and Fybexpression at 10 and 30 minutes, 2 and 24 hours following transfusion. Alloimmunization was assessed 2 weeks later by HEL-specific ELISA and flow cytometric crossmatching (using HOD or control FVB RBCs). Statistical significance (p Results: Alloantibodies against HOD RBCs were detectable by HEL-specific ELISA in recipients following a single transfusion of non-leukoreduced or leukoreduced RBCs. In 4 of 5 experiments (n=50 mice total), non-leukoreduced HOD RBCs were statistically significantly more immunogenic than leukoreduced HOD RBCs (sera diluted 1:50). Alloantibodies against HOD RBCs were detected in a minority of mice by flow cytometric crossmatching, which is less sensitive than ELISA. By histogram overlay of sera crossmatched with HOD RBCs compared to control FVB RBCs, 6/25 recipients of non-leukoreduced RBCs and 2/25 recipients of leukoreduced RBCs had a detectable anti-HOD response. In each experiment, there was a >3 log10 decrease in leukocytes after leukoreduction. HEL and Fyb expression was comparable as determined by flow cytometry in samples pre- and post-leukoreduction. Mean 24 hour post-transfusion survival was 98.7% (95% CI 97-100%) and 97.9% (95% CI 95.4-100%) in recipients of non-leukoreduced and leukoreduced RBCs, respectively. Conclusions: Leukoreduction of murine HOD RBCs decreases alloimmunogenicity of the RBC specific HEL antigen. This decrease is subtle and is better detected by HEL-specific ELISA than by less sensitive flow cytometric crossmatching. Although one explanation for these findings is that the WBCs themselves are contributing to the increased immunogenicity by providing a danger signal, it is also possible that non-specific alloreactivity of transfused WBCs or other cells is playing a role. Furthermore, it cannot be ruled out that changes in platelets or RBCs induced by the leukoreduction filter are involved. However, the observed changes in immunogenicity cannot be explained by a decrease in HEL expression post-leukoreduction or by altered clearance of leukroreduced RBCs after transfusion, as HEL and Fyb expression, as well as 24 hour post-transfusion survival of HOD RBCs, were similar in both groups. If the immunogenicity of other RBC antigens is also decreased after leukoreduction, then patients at highest risk of RBC alloimmunization may benefit from receiving solely leukoreduced blood products. Furthermore, the development of higher efficiency and/or modified leukoreduction filters may be warranted. Disclosures: No relevant conflicts of interest to declare.