Biomarkers defining the metabolic age of red blood cells during cold storage

Metabolomic investigations of packed red blood cells (RBCs) stored under refrigerated conditions in saline adenine glucose mannitol (SAGM) additives have revealed the presence of 3 distinct metabolic phases, occurring on days 0-10, 10-18, and after day 18 of storage. Here we used receiving operating characteristics curve analysis to identify biomarkers that can differentiate between the 3 metabolic states. We first recruited 24 donors and analyzed 308 samples coming from RBC concentrates stored in SAGM and additive solution 3. We found that 8 extracellular compounds (lactic acid, nicotinamide, 5-oxoproline, xanthine, hypoxanthine, glucose, malic acid, and adenine) form the basis for an accurate classification/regression model and are able to differentiate among the metabolic phases. This model was then validated by analyzing an additional 49 samples obtained by preparing 7 new RBC concentrates in SAGM. Despite the technical variability associated with RBC processing strategies, verification of these markers was independently confirmed in 2 separate laboratories with different analytical setups and different sample sets. The 8 compounds proposed here highly correlate with the metabolic age of packed RBCs, and can be prospectively validated as biomarkers of the RBC metabolic lesion.