(443) Hemolytic Components in Aging Blood: "Are Things Really Better With Age?"

Introduction: Currently, the FDA allows red blood cells to be stored for up to 42 days, and requires that at least 75% of the cells remain in circulation after 24 hours post-infusion, and that hemolysis not exceed 1% at the end of the storage period. To determine extent of hemolysis, the FDA relies on Drabkin’s assay to report hemolysis as increases in cell free hemoglobin, but it does not account for other potential hemolytic modifiers such as heme and iron moieties. Although blood at 42 days meets the “gold standard” requirement, it may not be as effective or safe compared to “younger” blood due to these other hemolytic modifiers, so we evaluated these modifiers over standard storage time to quantify them and the potential influence on immunologic parameters.

Methods: Donor collected whole blood was collected and placed into red blood cell concentrates in AS-5 storage solution. These concentrates were evaluated on days 1, 7, 14, 21, 28, 35, 42, and 49 (±1 day) for bound and unbound heme, cell-free hemoglobin (Drabkin's Assay), and total heme. Red cells, storage supernatant, and combination of red cell/supernatants were additionally co-incubated with donor specific PBMCs to evaluate innate and adaptive immune function using ELISpot.

Results: Preliminary data for four separate donors for weeks 1-3 are presented. Between weeks 1-3, the mean change (normalized to baseline) for donors 1 and 2 for bound heme was -1083 to -149% and -101 to -121.9%, respectively. The mean quantity of total (bound and unbound heme in µM) between weeks 1-3 for all four donors was 337.25 ± 42 (n=4) whereas cell free hemoglobin for the same donors was 0.2 ± 0.5 µM. This demonstrates a significantly higher amount of heme bound to proteins other than hemoglobin that also substantially increases over RBC storage duration. The immunologic findings are currently pending.

Conclusions: These preliminary findings show that although cell-free hemoglobin levels remain less than 1% as per the FDA standard, appreciable changes in heme moieties may be clinically relevant in transfusion of certain populations. Future studies should explore if changes in other hemolytic molecules should be evaluated to develop new storage guidelines that may influence ‘storage lesion’ and could influence immunologic function.