(P-CB-12) Optimizing ATP and 2,3-DPG of Hypoxically Stored Whole Blood

Hypoxic/hypocapnic processing and subsequent storage of red blood cells (RBCs) using the HemanextONE has been proven to reduce storage lesions and yield RBCs with enhanced qualities¹ by preventing oxidative damage. The deoxygenation process also removes CO₂ resulting in alkalinization of the RBCs, increasing overall glucose utilization and altering the balance between 2,3-DPG and ATP levels. Alkalinization after CO₂ depletion is more pronounced in CPDA1 Whole Blood (WB) than in citrate/phosphate-containing AS3 RBCs, resulting in supraphysiological 2,3-DPG (manifested as high P50) at the expense of depleted ATP. As WB is increasingly being used for hemorrhagic patients, we aim to extend the benefits of hypoxically stored RBCs to WB. We investigated a simple method to restore the DPG/ATP balance in WB processed with HemanextONE by adding varying volumes of CO₂ gas prior to refrigerated storage and monitoring weekly changes in pH, ATP and P50, an important metric demonstrative of the DPG levels used in the absence of validated assays for 2,3-DPG.


Study

Design/Methods:

Four fresh WB units (300mL, CPDA1) were processed to 10±1 SO₂, 32±2mmHg pCO₂ using HemanextONE. Four replicates (n=4) of 4 subunits (unprocessed control, processed WB and those with CO₂ added) were made by placing 40 mL in storage bags made from transparent gas barrier polymer (ClearFoil Z, PAXXUS; polyethylene blood contact surface). Varying volumes of CO₂ (0mL, 6mL or 15mL) were then added before the WB subunits were stored at 4°C. pO₂, pCO₂, pH and SO₂ (ABL90, Radiometer), ATP (DiaSys) and P50 (Loligo Systems) were measured weekly for 3 weeks.


Results/Findings:

The O₂/CO₂ depletion process significantly increased the pH and reduced ATP of processed units to unphysiological levels at days 8 and 14 as opposed to the unprocessed control WB. When CO₂ was added prior to storage, by day 2 pH stabilized. As expected, the lowest pH values were measured for the subunits with the most CO₂ added and the highest pH values were measured for the subunits with the least CO₂ added. Restored ATP levels were correlated with decreased p50 values (Figure A), suggesting that any elevated DPG levels are achieved at the expense of ATP amounts, which aligns with our previous reports on ATP/DPG balance, although in this case we rely on the p50 metric in absence of a validated DPG assay.


Conclusions:

When CPDA1 WB is processed with HemanextONE, O₂ and CO₂ are depleted and in combination with excessive alkalinization result in higher pH and elevated 2-3,DPG (as estimated by P50) at the expense of depleted ATP. As demonstrated in this study, the addition of CO₂ gas after processing restores the ATP/DPG balance, thus potentially providing the benefits that have been previously associated with HemanextONE processed RBCs to WB as well.