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Abstract:
The volume of oxygen drawn from systemic capillaries down a partial pressure gradient is determined by the oxygen content of red blood cells (RBCs) and their oxygen-unloading kinetics, although the latter is assumed to be rapid and, therefore, not a meaningful factor. Under this paradigm, oxygen transfer to tissues is perfusion-limited. Consequently, clinical treatments to optimize oxygen delivery aim at improving blood flow and arterial oxygen content, rather than RBC oxygen-handling. Whilst the oxygen-carrying capacity of blood is increased with transfusion, previous studies have shown that stored blood undergoes kinetic attrition of oxygen release, which may compromise overall oxygen delivery to tissues, i.e. transport became diffusion-limited. We sought evidence for diffusion-limited oxygen release in viable human kidneys normothermically perfused with stored blood. In a cohort of kidneys that went on to be transplanted, ex-vivo renal respiration correlated inversely with the time-constant of oxygen-unloading from RBCs used for perfusion. Furthermore, the renal respiratory rate did not correlate with arterial O2 delivery unless this factored the rate of oxygen-release from RBCs, as expected from diffusion-limited transport. In kidneys deemed unsuitable for transplantation, perfusion was alternated between stored and rejuvenated RBCs of the same donation to control oxygen-unloading without intervening ischemia and holding all non-RBC parameters constant. Rejuvenated oxygen-unloading kinetics reversibly improved the kidney's oxygen diffusion capacity and increased cortical oxygen partial pressure by 60%. Thus, oxygen delivery to tissues can become diffusion-limited during perfusion with stored blood, which has implications in scenarios such as ex-vivo organ perfusion, major hemorrhage, and pediatric transfusion.

Abstract:
Stored red blood cells (RBCs) incur biochemical and morphological changes, collectively termed the storage lesion. Functionally, the storage lesion manifests as slower oxygen unloading from RBCs, which may compromise the efficacy of transfusions where the clinical imperative is to rapidly boost oxygen delivery to tissues. Recent analysis of large real-world data linked longer storage with increased recipient mortality. Biochemical rejuvenation with a formulation of adenosine, inosine, and pyruvate can restore gas-handling properties, but its implementation is impractical for most clinical scenarios. We tested whether storage under hypoxia, previously shown to slow biochemical degradation, also preserves gas-handling properties of RBCs. A microfluidic chamber, designed to rapidly switch between oxygenated and anoxic superfusates, was used for single-cell oxygen saturation imaging on samples stored for up to 49 days. Aliquots were also analyzed flow cytometrically for side-scatter (a proposed proxy of O2 unloading kinetics), metabolomics, lipidomics, and redox proteomics. For benchmarking, units were biochemically rejuvenated at 4 weeks of standard storage. Hypoxic storage hastened O2 unloading in units stored to 35 days, an effect that correlated with side-scatter but was not linked to posttranslational modifications of hemoglobin. Although hypoxic storage and rejuvenation produced distinct biochemical changes, a subset of metabolites including pyruvate, sedoheptulose 1-phosphate, and 2/3 phospho-d-glycerate, was a common signature that correlated with changes in O2 unloading. Correlations between gas handling and lipidomic changes were modest. Thus, hypoxic storage of RBCs preserves key metabolic pathways and O2 exchange properties, thereby improving the functional quality of blood products and potentially influencing transfusion outcomes.

Keywords:
hypoxia, Hemanext, erythrocyte, hemoglobin, oxidative stress, microfluidics