Fig. 3

In vitro analysis oxidative stress and membrane integrity of RBC. A Schematic of storage-induced RBC morphologic change. Healthy discocytes into non-healthy echinocytes and sphero-echinocytes. B-E Oxidative stress markers: ROS, MDA, SOD, and CAT levels. Mean values of ROS B), MDA C), CAT D), and SOD E) in RBC samples at 0, 14, 28, 42, 49, 56, and 63 days. Control (blue) and supercooling (green). Data are shown as the mean ± SD from six biological replicates (n = 6). independent samples t-test, *p < 0.05; ***p < 0.001; ns, nonsignificant. F–H Membrane integrity and morphological changes: MCF, PS exposure, and RBC morphology. F) Mean values of Osmatic fragility test. To characterize RBC membrane integrity, we conducted the osmotic fragility test by resuspending the stored RBCs in a hypotonic solution. Mean cell fragility (MCF) reflects the NaCl concentration at which 50% hemolysis occurs. Data are shown as the mean ± SD from three biological replicates (n = 3). G PS exposure in the supercooled group was significantly lower than that in the control group (statistical result). Data are shown as the mean ± SD from three biological replicates (n = 3). *p < 0.05; ns, nonsignificant. H Morphological changes in the control group were mainly irreversible at the end of the preservation period. The morphological changes in the supercooled group were mainly reversible at the end of the preservation period. Red arrow: irreversible changes (sphero-echinocytes and fragmented RBCs). Scale bar = 10 µm