Plasma from mice transfused with stored RBCs enhances bacterial growth in vitro. (A) Plasma (100 μL) was obtained from mice 2 hours after transfusion with 400 μL of fresh RBCs (n = 15), stored RBCs (n = 24), stored RBC-derived supernatant (SN, n = 12), washed stored RBCs (n = 13), or ghosts prepared from stored RBCs (n = 8). Plasma was also obtained from control, untransfused mice (n = 14) or 24 hours after transfusion with stored RBCs (n = 8). Samples were incubated at 37°C with shaking with ∼ 1 × 106 CFU of E coli, as labeled. Bacterial growth was monitored every 30 minutes by absorbance at 600 nm for up to 5 hours. Bacterial growth in plasma obtained from mice 2 hours after transfusion with stored RBCs or washed stored RBCs began diverging from all other groups at 2.5 hours of incubation in vitro, and AUC (in parentheses) for each group was significantly different as indicated. (B) Pooled plasma samples (100 μL) from mice 2 hours after transfusion with 400 μL of fresh RBCs or stored RBCs were supplemented with either ferric citrate (20μM), sodium citrate (20μM), bovine serum albumin (BSA; 80μM), or protoporphyrin IX (20μM), and then incubated at 37°C with shaking with ∼ 1 × 106 CFU of E coli. Bacterial growth was monitored every 30 minutes by absorbance at 600 nm for up to 5 hours in replicates of 5 per group. AUC (in parentheses) for growth in plasma from mice transfused with fresh RBCs, supplemented with or without sodium citrate, BSA, or protoporphyrin IX, differed significantly from the other 3 groups. (C) Pooled plasma (n = 4) were incubated with the iron chelator, DFO (20 μM), or with the iron-chelated form FO (20 μM) and inoculated with E coli as shown for the previous experiment. The AUC (in parentheses) for growth in plasma with DFO significantly differed from all other groups. (D) Pooled plasma (n = 5) was incubated with the iron chelator, 2,2′-dipyridyl (400μM), with or without ferric citrate (133μM) and inoculated with E coli, as shown for the previous experiment. The AUC (in parentheses) for growth in plasma with 2,2′-dipyridyl significantly differed from all other groups; *P < .05. Results are representative of at least 2 experiments and are shown as mean (± SEM). Note that the absence of an error bar is indicative of highly reproducible replicates with pooled plasma.
