(503) Immunologic Effects of the Hemolytic Components in Stored Aging Red Blood Cells

Introduction: FDA standard allows red blood cell (RBC) storage to 42 days. Over time, cellular degradation occurs accumulating degradation products. Drabkin’s assay is used to quantify cell free hemoglobin (CFH) but other derivatives of hemolysis are not measured. Blood at 42 days may not be as effective or safe as younger RBCs due to hemolysis. We sought to quantify hemolytic products and evaluate the impact upon innate and adaptive immune function.

Methods: Whole blood was obtained commercially and RBCs stored in AS-3 solution. Samples were evaluated on day 1 and weekly for 6 weeks. Quantification of bound and unbound heme (novel heme assay), CFH (Drabkin’s Assay and ELISA), iron, haptoglobin and hemopexin. Immune cell function evaluated by ELISpot using donor specific PBMCs stimulated with concentrated RBCs, storage supernatant, and both. Statistics using ANVOA, p < 0.05 for statistical significance.

Results: 10 samples from 9 healthy donors. Analyses compared day 1 to day 42. RBC count, hgb and RDW remained constant. Hgb (g/dL) in supernatant increased from 0.01 to 0.8 (p < 0.005) indicating hemolysis. Supernatant pH decreased from 6.7 to 6.4 (p < 0.005). CFH (uM) via Drabkin’s increased from 0.01 to 0.06 (p < 0.001). Bound heme (uM) increased from 49.8 to 615.8 (p < 0.0001) and unbound or ‘free’ heme (uM) increased from 0.5 to 32 (p < 0.0001). Haptoglobin (219.2 to 211.5 (p= 0.4) ng/mL) and hemopexin (4 to 2 (p= 0.96) ng/mL) did not significantly change. Immune function is reported as a function of treated sample vs. positive control. Concentrated RBCs increased LPS-induced TNF-α production on day 1, which was attenuated by day 42 (1.6x vs 0.9x (p= 0.09)), and decreased CD3/CD28-induced IFN-ɣ production on day 1 and trended lower on day 42 (0.8x vs 0.3x (p= 0.54)). Supernatant increased LPS-induced TNF-α production on day 1 and continued to trend upwards by day 42 (1.4x to 2x (p= 0.3)), and supernatant decreased CD3/CD28-induced IFN-ɣ production on day 1 and day 42 (0.45x to 0.74x (p= 0.14)).

Conclusions: These findings show that although CFH levels meet FDA standard, there are appreciable changes in heme moieties that may be clinically relevant. Future exploration is warranted to best understand the clinical context of increase in bound and unbound heme and resultant alterations in immune function.