Transfusion of stored RBCs. Transfusions of stored RBCs lead to increased RBC clearance, tissue iron delivery, and circulating NTBI levels, compared with transfusions of fresh RBCs, stored RBC-derived supernatant, or ghosts prepared from stored RBCs. All transfusion recipients were male C57BL/6 mice (8-12 weeks of age). The results are presented as mean (± SEM) except where specified. (A) Leukoreduced fresh FVB/NJ mouse RBCs (< 24-hour storage; n = 3; □) and stored RBCs (2-week storage; n = 5; ■) were transfused (400 μL at 17.0-17.5 g/dL of hemoglobin), and survival of transfused RBCs was calculated by dual-label flow cytometric tracking at 10 minutes, 30 minutes, 1 hour, 2 hours (only for stored RBCs), and 24 hours after transfusion. The results are from 1 representative experiment and are presented as mean (± SD). (B) A representative image of spleens obtained from mice 2 hours after transfusion with fresh RBCs or stored RBCs. (C) Mean spleen weight of mice transfused with fresh RBCs (n = 13) and stored RBCs (n = 13). (D) Aliquots (400 μL) of fresh RBCs (n = 13), stored RBCs (n = 13), washed stored RBCs (n = 13), stored RBC-derived supernatant (SN; n = 12), and ghosts prepared from stored RBCs (n = 8) were transfused. Total iron was measured in organs obtained at necropsy 2 hours after transfusion; the increases in iron are shown compared with levels measured in control, untransfused mice (n = 12). The results are combined from 3 separate experiments. (E) Mice were transfused as labeled (n = 5 per group) and plasma NTBI levels were measured 2 hours after transfusion. Note that the absence of an error bar indicates undetectable NTBI levels. The results are representative of 2 separate experiments; *P < .05; **P < .01; ***P < .001 compared with fresh RBC transfusions.