Fig. 1.

Storage of HOD RBCs profoundly enhances transfusion-induced alloimmunization. WT C57BL/6 mice were transfused i.v. with stored or freshly collected HOD RBCs. a) Flow cytometric cross-match of anti-HOD RBC antibodies measured in sera of naïve untransfused mice and mice transfused with RBCs stored for 7 or 14 days, or freshly collected. Anti-RBC antibodies were calculated by subtracting the MFI of a serum sample incubated with RBCs lacking the HOD antigen from the MFI of a paired sample incubated with HOD + RBCs. n = 3–8 mice/group; results are representative of 3 independent experiments. b) Monocyte chemoattractant protein-1 (MCP-1) in serum 2–4 h following transfusion of RBCs stored for indicated times prior to transfusion measured by cytometric bead array. c) Inflammatory cytokines in serum as in (b) from mice transfused with RBCs stored for 14 days (+ RBCs) or not transfused (− RBCs); Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), MCP-1, and keratinocyte-derived chemokine (KC). n = 2–7 mice/group; results are representative of 4 independent experiments. d) Anti-HEL antibodies, quantified by ELISA, in sera diluted 1:50 from mice transfused with RBCs stored for 14 days and either washed with PBS or unwashed prior to transfusion. Unwashed samples contain storage supernatant. n = 5 mice/group; results are representative of 2 independent experiments. NS, not significant. e) Serum anti-HEL antibodies in mice that received stored (14 day-old), freshly collected (Fresh), or Fresh RBCs 4 h following i.p. administration of 100 μg poly(I:C) (Fresh + PIC). Error bars indicate standard deviation. n = 5 mice/group; results are representative of 2–3 independent experiments. **p < 0.01; ***p < 0.001; ****p < 0.0001.