Marked Inter-Unit Variability and Early Depletion of 2,3-Diphosphoglycerate During Red Blood Cell Storage

Abstract Red blood cell (RBC) transfusion aims to restore tissue oxygen delivery, yet a stored unit’s capacity to unload oxygen depends on 2,3-diphosphoglycerate (2,3-DPG), the principal allosteric modulator of hemoglobin–oxygen affinity. Randomized trials comparing fresher with older units have been consistently null, and all have used storage duration as the exposure variable. We asked whether storage age reflects the metabolic state of individual units. Twenty-four leukodepleted packed RBC units were sampled serially from day 3 to day 42 of standard cold storage. 2,3-DPG was quantified by liquid chromatography–mass spectrometry (LC–MS) with isotopically labelled internal standards, pH was measured in parallel, and trajectories were modelled by linear mixed effects with unit-level random intercepts. At day 3, 2,3-DPG spanned a twenty-fold range across units of identical age (0.8–14.7 µmol/g Hb; median 4.8): some were already near-depleted at the earliest sampled timepoint, while others retained near-physiological concentrations. This heterogeneity collapsed rapidly - median values fell to 27% of day-3 levels by day 7 and by more than 90% by day 14 (β = −0.298 µmol/g Hb per day, p < 0.001) - and were below the limit of detection in most units after day 17. pH declined monotonically to 6.40 ± 0.04 by day 42 (β = −0.0163 units per day, p < 0.001). Units of identical storage age therefore differ markedly in the metabolite that governs oxygen unloading, and virtually all are depleted long before the 42-day expiry, indicating that storage-age dichotomies misclassify the exposure they are intended to represent.