Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans’ assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.
Background: Characteristics of red blood cells (RBCs) are influenced by donor variability. This study assessed quality and metabolomic variables of RBC subpopulations of varied biologic age in red blood cell concentrates (RCCs) from male and female donors to evaluate their contribution to the storage lesion. Study Design and Methods: Red blood cell concentrates from healthy male (n = 6) and female (n = 4) donors were Percoll separated into less dense ("young", Y-RCCs) and dense ("old", O-RCCs) subpopulations, which were assessed weekly for 28 days for changes in hemolysis, mean cell volume (MCV), hemoglobin concentration (MCHC), hemoglobin autofluorescence (HGB), morphology index (MI), oxygen affinity (p50), rigidity, intracellular reactive oxygen species (ROS), calcium ([Ca 2+ ]), and mass spectrometry-based metabolomics. Results: Young RCCs having disc-to-discoid morphology showed higher MCV and MI, but lower MCHC, HGB, and rigidity than O-RCCs, having discoid-tospheroid shape. By Day 14, Y-RCCs retained lower hemolysis and rigidity and higher p50 compared to O-RCCs. Donor sex analyses indicated that females had higher MCV, HGB, ROS, and [Ca 2+ ] and lower hemolysis than male RBCs, in addition to having a decreased rate of change in hemolysis by Day 28. Metabolic profiling indicated a significant sex-related signature across all groups with increased markers of high membrane lipid remodeling and antioxidant capacity in Y-RCCs, whereas O-RCCs had increased markers of oxidative stress and decreased coping capability. Conclusion: The structural, functional, and metabolic dissimilarities of Y-RCCs and O-RCCs from female and male donors demonstrate RCC heterogeneity, where RBCs from females contribute less to the storage lesion and age slower than males.
Background Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). Methods Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. Results Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). Conclusions Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.
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