Estimated median density identifies donor age and sex differences in red blood cell biological age.

BACKGROUND: Donors possess heterogeneous red cell concentrates (RCCs) in terms of the biological age of their red blood cells (RBCs) as a direct result of various donor-dependent factors influencing rates of erythropoiesis. This study aimed to estimate the median biological age of RBCs in RCCs based on donor age and sex to investigate inherent differences in blood products' biological ages over hypothermic storage using estimated median densities (EMDs). STUDY DESIGN: Sixty RCCs were collected from four donor groups; male and female teenagers (17-19 years old) and seniors (75+ years old). A Percoll density-based separation approach was used to quantify the EMDs indicative of biological age. EMD and mean corpuscular hemoglobin (MCHC) were compared by correlation analyses. RESULTS: Differences in the median biological age of RCC units were observed with male donors having significantly higher EMDs compared to females (p < .001). Teen male donors possessed the highest EMDs with significantly elevated levels of biologically aged RBCs compared to both female donor groups, regardless of storage duration (p < .05). Throughout most of the 42-day storage period, senior donors, particularly senior females, demonstrated the strongest correlation between EMD and MCHC (R2 > 0.5). CONCLUSIONS: This study provides further evidence that there are inherent differences between the biological age profiles of RBCs between blood donors of different sex and age. Our findings further highlight that biological age may contribute to RBC quality during storage and that donor characteristics need to be considered when evaluating transfusion safety and efficacy.

Red cell concentrates from teen male donors contain poor-quality biologically older cells.

BACKGROUND AND OBJECTIVES: Donor factors influence the quality characteristics of red cell concentrates (RCCs) and the lesions that develop in these heterogeneous blood products during hypothermic storage. Teen male donors' RCCs contain elevated levels of biologically old red blood cells (RBCs). The aim of this study was to interrogate the quality of units of different donor ages and sexes to unravel the complex interplay between donor characteristics, long-term cold storage and, for the first time, RBC biological age. MATERIALS AND METHODS: RCCs from teen males, teen females, senior males and senior females were density-separated into less-dense/young (Y-RBCs) and dense/old RBCs (O-RBCs) throughout hypothermic storage for testing. The unseparated and density-separated cells were tested for haematological parameters, stress (oxidative and osmotic) haemolysis and oxygen affinity (p50). RESULTS: The O-RBCs obtained from teen donor samples, particularly males, had smaller mean corpuscular volumes and higher mean corpuscular haemoglobin concentrations. While biological age did not significantly affect oxygen affinity, biologically aged O-RBCs from stored RCCs exhibited increased oxidative haemolysis and decreased osmotic fragility, with teenage male RCCs exhibiting the highest propensity to haemolyse. CONCLUSION: Previously, donor age and sex were shown to have an impact on the biological age distribution of RBCs within RCCs. Herein, we demonstrated that RBC biological age, particularly O-RBCs, which are found more prevalently in male teens, to be a driving factor of several aspects of poor blood product quality. This study emphasizes that donor factors should continue to be considered for their potential impacts on transfusion outcomes.

Comparing two extracellular additives to facilitate extended storage of red blood cells in a supercooled state.

Background: Adenosine triphosphate (ATP) levels guide many aspects of the red blood cell (RBC) hypothermic storage lesions. As a result, efforts to improve the quality of hypothermic-stored red cell concentrates (RCCs) have largely centered around designing storage solutions to promote ATP retention. Considering reduced temperatures alone would diminish metabolism, and thereby enhance ATP retention, we evaluated: (a) whether the quality of stored blood is improved at -4°C relative to conventional 4°C storage, and (b) whether the addition of trehalose and PEG400 can enhance these improvements. Study Design and Methods: Ten CPD/SAGM leukoreduced RCCs were pooled, split, and resuspended in a next-generation storage solution (i.e., PAG3M) supplemented with 0-165 mM of trehalose or 0-165 mM of PEG400. In a separate subset of samples, mannitol was removed at equimolar concentrations to achieve a fixed osmolarity between the additive and non-additive groups. All samples were stored at both 4°C and -4°C under a layer of paraffin oil to prevent ice formation. Results: PEG400 reduced hemolysis and increased deformability in -4°C-stored samples when used at a concentration of 110 mM. Reduced temperatures did indeed enhance ATP retention; however, in the absence of an additive, the characteristic storage-dependent decline in deformability and increase in hemolysis was exacerbated. The addition of trehalose enhanced this decline in deformability and hemolysis at -4°C; although, this was marginally alleviated by the osmolarity-adjustments. In contrast, outcomes with PEG400 were worsened by these osmolarity adjustments, but at no concentration, in the absence of these adjustments, was damage greater than the control. Discussion: Supercooled temperatures can allow for improved ATP retention; however, this does not translate into improved storage success. Additional work is necessary to further elucidate the mechanism of injury that progresses at these temperatures such that storage solutions can be designed which allow RBCs to benefit from this diminished rate of metabolic deterioration. The present study suggests that PEG400 could be an ideal component in these solutions.

Quality assessment of red blood cell concentrates from blood donors at the extremes of the age spectrum: The BEST collaborative study.

BACKGROUND: Blood donors at the extremes of the age spectrum (16-19 years vs. ≥75 years) are characterized by increased risks of iron deficiency and anemia, and are often underrepresented in studies evaluating the effects of donor characteristics on red blood cells (RBC) transfusion effectiveness. The aim of this study was to conduct quality assessments of RBC concentrates from these unique age groups. STUDY DESIGN: We characterized 150 leukocyte-reduced (LR)-RBCs units from 75 teenage donors, who were matched by sex, and ethnicity with 75 older donors. LR-RBC units were manufactured at three large blood collection centers in the USA and Canada. Quality assessments included storage hemolysis, osmotic hemolysis, oxidative hemolysis, osmotic gradient ektacytometry, hematological indices, and RBC bioactivity. RESULTS: RBC concentrates from teenage donors had smaller (9%) mean corpuscular volume and higher (5%) RBC concentration compared with older donors counterparts. Stored RBCs from teenage donors exhibited increased susceptibility to oxidative hemolysis (>2-fold) compared with RBCs from older donors. This was observed at all testing centers independent of sex, storage duration, or the type of additive solution. RBCs from teenage male donors had increased cytoplasmatic viscosity and lower hydration compared with older donor RBCs. Evaluations of RBC supernatant bioactivity suggested that donor age was not associated with altered expression of inflammatory markers (CD31, CD54, and IL-6) on endothelial cells. CONCLUSIONS: The reported findings are likely intrinsic to RBCs and reflect age-specific changes in RBC antioxidant capacity and physical characteristics that may impact RBC survival during cold storage and after transfusion.

Hypothermic storage of leukoreduced red blood cells for greater than 21 days is a safe alternative to irradiation.

BACKGROUND: Irradiation of red blood cells (RBCs) inactivates residual donor T lymphocytes to prevent transfusion-associated graft-vs-host disease (TA-GVHD) but can have adverse effects on recipients and inventory management. Reported incidence of TA-GVHD is lower when leukoreduced RBCs and older blood products are transfused; therefore, the impact of leukoreduction and storage was evaluated as an alternative prevention strategy. STUDY DESIGN AND METHODS: Effectiveness of leukoreduction filters on white blood cell (WBC) proliferation was evaluated by filtering buffy coat (BC) products and isolating residual WBCs. Additionally, leukoreduced RBCs were spiked with 5 × 106 WBCs on Day 21 of hypothermic storage, then stored and processed on Days 7, 14, and 21 to obtain residual WBCs to investigate the impact of hypothermic storage on their viability and proliferative ability. Viability of residual WBCs was assessed by staining with annexin V and an antibody cocktail for flow cytometry analysis. Proliferative ability was assessed by placing carboxyfluorescein diacetate succinimidyl ester-labeled residual WBCs into culture for 6 days with phytohemagglutinin before flow cytometry assessment. RESULTS: Filtration of BC units depleted WBCs, particularly T lymphocytes, to 0.001% ± 0.003% cells/unit, although proliferative activity remained consistent with prefiltration levels of WBCs. WBCs in stored RBCs remained viable even on Day 21 of storage; however, the proliferative activity decreased to 0.24% ± 0.41%. CONCLUSIONS: Hypothermic storage of RBCs for 21 days or more is sufficient to inactivate T lymphocytes, which may help prevent TA-GVHD when irradiated RBCs are not available.

Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells.

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. Video abstract.

Assessment of extracellular vesicles using IFC for application in transfusion medicine.

Extracellular vesicles (EVs) have been shown to be involved in various physiological and pathophysiological processes. With respect to Transfusion Medicine, the accumulation of EVs in blood products during hypothermic storage is an indicator of the storage lesion and reportedly correlates with adverse effects after transfusion, including but not limited to immunomodulation, activation of coagulation, endothelial activation, and others. To optimally reduce such an impact on blood product quality degradation and improve post-transfusion outcomes, better methods for detection, enumeration, characterisation by size and phenotype, and functional involvement of EVs in different pathophysiological and physiological processes are required. Currently, Imaging Flow Cytometry (IFC) technology provides the most comprehensive assessment of EV subsets in different body fluids. The unique ability of IFC to detect EVs of 20 nm size by registration of a single pixel of fluorescence signal makes this approach highly promising for comprehensive studies of EVs. In this review, we will focus on the recent breakthrough and advantages of using the ImageStreamX MKII IFC platform for the detection and characterisation of EVs and its future prospects for routine application of IFC in Transfusion Medicine.

Donor-dependent aging of young and old red blood cell subpopulations: Metabolic and functional heterogeneity.

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 ([Ca2+ ]), 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-to-spheroid 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 [Ca2+ ] 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.

Objective assessment of stored blood quality by deep learning.

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.