Storage of murine red blood cells enhances alloantibody responses to an erythroid-specific model antigen

Abstract

Background: Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization.

Study design and methods: Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels.

Results: Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage.

Conclusions: These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.

Fig. 1

HOD RBCs stored for 14 days result in higher levels of alloantibodies after transfusion. HOD RBCs were filter leukoreduced and transfused fresh or stored for 14 days and then transfused. Alloantibody response was assessed 14 days after transfusion. (A) Anti-HEL response as determined by ELISA in a representative experiment with five mice per group, transfused with fresh leukoreduced HOD RBCs (–●–) or 14-day-stored leukoreduced HOD RBCs (--▲--); mean and SD are shown with sera dilutions of 1:50–1:500,000. This experiment was repeated five times (five to six mice/group/experiment) with similar results. (B) Anti-HOD response as determined by flow cytometric crossmatch. Representative flow histograms are shown, with serum at a 1:5 dilution from a recipient of fresh HOD RBCs and from a recipient of 14-day-stored HOD RBCs crossmatched with control FVB RBCs (shaded) or HOD RBCs (solid).

Fig. 2

Analysis of HOD RBCs. (A) After filter leukoreduction, WBCs were enumerated using PI and Trucount beads; a 3-log or greater WBC reduction seen. (B) Before transfusion, HEL and Fy³ expression were evaluated by flow cytometry, using polyclonal sera from HEL-immunized mice and the MIMA 29 anti-Fy³ monoclonal antibody, respectively, with control FVB RBCs (shaded), fresh HOD RBCs (solid), and 14-day-stored HOD RBCs (dotted). This experiment was repeated five times, with similar results.

Fig. 3

Posttransfusion HOD RBC survival. To determine 24-hour posttransfusion survival, recipients were bled at 10 minutes, 30 minutes, 2 hours, and 24 hours posttransfusion, and the percentage of transfused HEL-positive RBCs circulating in the recipient was assessed by flow cytometry. (A) HEL expression in control FVB RBCs. (B) Representative plot of HEL-positive RBCs in a recipient after transfusion. (C) Representative plot of posttransfusion HEL-positive RBC survival (mean and SD), with three mice per group. (▲) 14-day-stored leukoreduced HOD; (●) fresh leukoreduced HOD.