Although refrigerated storage slows the metabolism of volunteer donor RBCs, which is essential in transfusion medicine, cellular aging still occurs throughout this in vitro process. Storage-induced microerythrocytes (SMEs) are morphologically altered senescent RBCs that accumulate during storage and are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhi) and morphologically normal RBCs (CFSElo), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhi RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhi RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhi RBC membranes. CFSEhi RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen perfusion ex vivo. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElo RBCs resembled those of short-stored RBCs. CFSEhi RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in these storage-induced senescent RBCs targeted for clearance.

European guidelines require packed red blood cells (pRBC) to be stored at 2–6 °C. However, negative temperature shifts can occur especially in prehospital transfusion. We investigated the impact of sub-zero temperature exposure on pRBC storage quality. At day 6 post donation (D6), three cohorts (14 pRBC) were put on a supercooled table for 10 h at either − 1 °C, -5 °C, and − 11 °C and compared to a control cohort. Hemolysis, pH and plasma biochemistry were evaluated weekly until D49. Storage-induced micro-erythrocytes (SMEs) were quantified as a surrogate marker for transfusion recovery. The primary endpoint was compliance with European storage standards at D42. The three sub-zero-exposed cohorts met standards at D42. Differences in hemolysis, pH, plasma biochemistry, or SMEs between exposed and control cohorts were non-statistically and/or non-clinically significant. Ten hours exposure to sub-zero temperatures down to -11 °C by conduction maintains storage quality of pRBCs, enabling a wiser risk assessment for potential transfusion use.

Red blood cells (RBC) ability to circulate is closely related to their surface area-to-volume ratio. A decrease in this ratio induces a decrease in RBC deformability that can lead to their retention and elimination in the spleen. We recently showed that a subpopulation of small RBC with reduced projected surface area accumulated upon storage in blood bank concentrates, but data on the volume of these altered RBC are lacking. So far, single cell measurement of RBC volume has remained a challenging task achieved by a few sophisticated methods some being subject to potential artifacts. We aimed to develop a reproducible and ergonomic method to assess simultaneously RBC volume and morphology at the single cell level. We adapted the fluorescence exclusion measurement of volume in nucleated cells to the measurement of RBC volume. This method requires no pre-treatment of the cell and can be performed in physiological or experimental buffer. In addition to RBC volume assessment, brightfield images enabling a precise definition of the morphology and the measurement of projected surface area can be generated simultaneously. We first verified that fluorescence exclusion is precise, reproducible and can quantify volume modifications following morphological changes induced by heating or incubation in non-physiological medium. We then used the method to characterize RBC stored for 42 days in SAG-M in blood bank conditions. Simultaneous determination of the volume, projected surface area and morphology allowed to evaluate the surface area-to-volume ratio of individual RBC upon storage. We observed a similar surface area-to-volume ratio in discocytes (D) and echinocytes I (EI), which decreased in EII (7%) and EIII (24%), sphero-echinocytes (SE; 41%) and spherocytes (S; 47%). If RBC dimensions determine indeed the ability of RBC to cross the spleen, these modifications are expected to induce the rapid splenic entrapment of the most morphologically altered RBC (EIII, SE, and S) and further support the hypothesis of a rapid clearance of the small RBC subpopulation by the spleen following transfusion.