Acker, J. P. | bioRxiv Science

bioRxiv2026

Enhancing Supercooled Red Blood Cell Storage: The Membrane-Stabilizing Effect of Ethanol

Hypothermic storage is constrained by the progressive depletion of energy reserves, curtailing the shelf-life of organs, cell therapies, and blood products. High sub-zero supercooling helps preserve energy homeostasis by slowing catabolic processes; however, the resulting injury in this setting is not primarily driven by energy depletion. Here, we investigated whether low-dose ethanol could prevent forms of injury that arise independently of disrupted energy homeostasis and remain unaddressed in supercooled storage. Human red blood cells treated with 4% (v/v) ethanol were stored 4 {degrees}C, -4 {degrees}C, or -8 {degrees}C and subject to a series of functional assessments and integrated metabolomic/lipidomic profiling after 21 and 42 days of storage. Metabolomics data showed that energy homeostasis was better preserved at lower temperatures, yet these supercooled conditions simultaneously intensified hemolysis and caused a marked depletion of lysophospholipid species that did not occur at 4 {degrees}C. Ethanol blunted these effects, cutting hemolysis by [~]50 % at -4 {degrees}C, by [~]85 % at -8 {degrees}C, and attenuating lysophospholipid depletion. These results uncover a previously unrecognized, lipid-centric injury that arises during supercooled storage and establish low-dose ethanol as a simple, readily deployable countermeasure that could help extend storage intervals of diverse biological systems. SUMMARYCellular preservation has traditionally focused on maintaining energy metabolism during hypothermic storage, but whether this is sufficient at high sub-zero temperatures remains unclear. Human red blood cells were stored for up to 42 days at 4{degrees}C, -4{degrees}C, or -8{degrees}C with or without 4% ethanol and evaluated using functional assays and integrated metabolomic and lipidomic profiling. Although supercooling preserved energy homeostasis, it increased hemolysis and caused pronounced lysophospholipid depletion. Ethanol reduced hemolysis by approximately 50% at -4{degrees}C and 85% at -8{degrees}C, attenuated lysophospholipid loss, and produced comparatively modest changes in cellular metabolism. These findings identify membrane integrity as a critical determinant of preservation outcome and support strategies that protect membrane stability alongside metabolic homeostasis.

cell biology↗

bioRxiv2018

Label-free assessment of red blood cell storage lesions by deep learning

Blood transfusion is a life-saving clinical procedure. With millions of units needed globally each year, it is a growing concern to improve product quality and recipient outcomes.\n\nStored red blood cells (RBCs) undergo continuous degradation, leading to structural and biochemical changes. To analyze RBC storage lesions, complex biochemical and biophysical assays are often employed.\n\nWe demonstrate that label-free imaging flow cytometry and deep learning can characterize RBC morphologies during 42-day storage, replacing the current practice of manually quantifying a blood smear from stored blood units. Based only on bright field and dark field images, our model achieved 90% accuracy in classifying six different RBC morphologies associated with storage lesions versus human-curated manual examination. A model fitted to the deep learning-extracted features revealed a pattern of morphological changes within the aging blood unit that allowed predicting the expiration date of stored blood using solely morphological assessment.\n\nDeep learning and label-free imaging flow cytometry could therefore be applied to reduce complex laboratory procedures and facilitate robust and objective characterization of blood samples.

bioinformatics↗