Red Blood Cell Storage Research Articles

Continuous-Flow Magnetic Fractionation of Red Blood Cells Based on Hemoglobin Content and Oxygen Saturation—Clinical Blood Supply Implications and Sickle Cell Anemia Treatment

Approximately 36,000 units of red blood cells (RBCs) are used every day in the U.S. and there is a great challenge for hospitals to maintain a reliable supply, given the 42-day expiration period from the blood donation date. For many years, research has been conducted to develop ex vivo storage solutions that limit RBC lysis and maintain a high survival rate of the transfused cells. However, little attention is directed towards potential fractionation methods to remove unwanted cell debris or aged blood cells from stored RBC units prior to transfusion, which could not only expand the ex vivo shelf life of RBC units but also avoid adverse events in transfused patients. Such fractionation methods could also limit the number of transfusions required for treating certain pathologies, such as sickle cell disease (SCD). In this work, magnetic fractionation is studied as a potential technology to fractionate functional and healthy RBCs from aged or sickle cells. It has been reported that during ex vivo RBC storage, RBCs lose hemoglobin (Hb) and lipid content via formation of Hb-containing exosomes. Given the magnetic character of deoxygenated- or met-Hb, in this work, we propose the use of a quadrupole magnetic sorter (QMS) to fractionate RBCs based on their Hb content from both healthy stored blood and SCD blood. In our QMS, a cylindrical microchannel placed inside the center of the quadrupolar magnets is subjected to high magnetic fields and constant field gradients (286 T/m), which causes the deflection of the paramagnetic, Hb-enriched, and functional RBCs from their original path and their collection into a different outlet. Our results demonstrated that although we could obtain a significant difference in the magnetic mobility of the sorted fractions (corresponding to a difference in more than 1 pg of Hb per cell), there exists a tradeoff between throughput and purity. Therefore, this technology when optimized could be used to expand the ex vivo shelf life of RBC units and avoid adverse events in transfused individuals or SCD patients requiring blood exchange therapy.

Questionnaire Survey on Current Red Blood Cell Transport and Storage in Korea for Reducing Wastage.

BackgroundTo ensure safe red blood cell (RBC) transfusion practice, it is important to comply with storage and transport requirements of RBC units. We conducted a comprehensive survey on the practice of RBC transport and storage to explore the awareness of and compliance with the 30-minute rule, the current status of RBC unit transport, and possible utility of temperature indicators (TIs) to reduce RBC wastage.MethodsFrom June to August of 2019, 64 blood bank physicians (14 questions) in 64 secondary- and tertiary-care hospitals and 673 nurses (13 questions) in 42 tertiary-care hospitals replied to a questionnaire survey. The results of the survey were analyzed with descriptive statistics.ResultsAmong the physicians surveyed, 97.0% (N=62) of hospitals had transfusion guidelines in place. The RBC wastage in 2018 ranged from less than five units to more than 200 units. Among the nurses surveyed, 99.4% (N=669) were aware of and complied with the 30-minute rule; 13.5% (N=91) of the nurses had experience of RBC wastage due to violation of the 30-minute rule. Both physicians (67%, N=43) and nurses (83.1%, N=559) responded that TIs would help reduce RBC wastage.ConclusionsThis is the first survey on the practices related to RBC transport and storage in Korea. This study provides fundamental data on current practice for the blood cold chain, insights into RBC wastage, and highlights the utility of TIs.

HPT‐ Axis Activation During Cold Head Out Water Immersion is Not Modified by Moderate Hypoxia

PurposeThe Bajau, an indigenous population renowned for breath‐hold diving, have comparatively large spleen volumes. This adaptation is purportedly indicative of a greater capacity to store red blood cells, which may aid operations in the aquatic environment. Spleen size in the Bajau is associated with the PDE10A genotype that encodes an enzyme that catalyzes the release of thyroxine (T4) from the thyroid gland. T4 is readily released during cold stress as the terminus to activation of the hypothalamus‐pituitary‐thyroid (HPT) ‐axis to increase metabolic rate. T4, and downstream counterpart triiodothyronine (T3), can also stimulate erythropoiesis by increasing erythropoietin production, which can be further promoted with hypoxia. Thus, exposure to cold water may augment erythropoiesis as hypoxia develops during breath‐hold diving via activation of the HPT‐axis. However, interactions between cold exposure and hypoxia on HPT‐axis activation have not been directly explored. Mountaineering expedition studies indirectly suggest that T4 release is elevated by hypoxia. Thus, this study tested the hypothesis that during cold head out water immersion (HOWI) acute hypoxia augments circulating thyroid hormone concentrations compared to normoxia.MethodsIn a randomized crossover single blind design, 12 healthy adults (27 ± 2 y, 2 women) completed one hour of cold (22.0 ± 0.1⁰C) HOWI breathing either normobaric normoxia (FiO2 = 0.21 ± 00, NORM) or normobaric hypoxia (FiO2 = 0.13 ± 00, HYP). Free (f) and total (t) T3 and T4, and thyroid stimulating hormone (TSH) were measured in venous blood samples obtained before (baseline), during (15‐, 30‐, and 60‐min), and 15 min following immersion (post‐). Arterial oxyhemoglobin saturation (SpO2) and rectal temperature were measured continuously. Data are presented as mean ± SD.ResultsSpO2 was lower in HYP (90 ± 3%) compared to NORM (98 ± 1%, p <.001). Rectal temperature did not differ between HYP and NORM (p > .711) but decreased over time in both trials (HYP ‐ baseline: 37.2 ± 0.4⁰C, post‐: 36.4 ± 0.5⁰C, p<.001, NORM ‐ baseline: 37.2 ± 0.4⁰C, post‐: 36.3 ± 0.5⁰C, p <.001). From baseline to post‐HOWI, fT3 (HYP: +0.3 ± 0.4 pg/mL, NORM: +0.4 ± 0.6 pg/mL, p <.001), fT4 (HYP: +1.6 ± 1.7 pg/mL, NORM: +0.7 ± 1.3 pg/mL, p <.001), tT3 (HYP: +0.3 ± 0.5 ng/mL, NORM: +0.2 ± 0.4 ng/mL, p= .053), and tT4 (HYP: +0.6 ± 0.4 µg%, NORM: +0.4 ± 0.5 µg%, p < .001) increased, but the magnitude did not differ between inspirates (p > .165). Relative to baseline, tT4 at 15 min (HYP: ‐0.2 ± 0.3 µg%, NORM: ‐0.3 ± 0.3 µg%, p = .025), and TSH at 30 min (HYP: ‐0.2 ± 0.2 µIU/mL, NORM: ‐0.2 ± 0.3 µIU/mL, p = .029) and 60 min (HYP: ‐0.2 ± 0.2 µIU/mL, NORM: ‐0.3 ± 0.4 µIU/mL, p = .008) of HOWI were lower, but did not differ between inspirates (p = .921).ConclusionsIncreases in circulating thyroid hormone concentrations during one hour of cold HOWI are not modified by normobaric hypoxia. Thus, moderate hypoxia does not likely affect activation of the HPT‐axis during short duration cold exposure. Further investigation is warranted to determine the possibility of circulating thyroid hormones to augment the erythropoietic response to acute hypoxia during cold exposure.

Storage of red blood cells in alkaline PAGGGM improves metabolism but has no effect on recovery after transfusion

AbstractAdditive solutions are used to limit changes that red blood cells (RBCs) undergo during storage. Several studies have shown better preservation of glucose and redox metabolism using the alkaline additive solution PAGGGM (phosphate-adenine-glucose-guanosine-gluconate-mannitol). In this randomized open-label intervention trial in 20 healthy volunteers, the effect of storage, PAGGGM vs SAGM (saline-adenine-glucose-mannitol), on posttransfusion recovery (PTR) and metabolic restoration after transfusion was assessed. Subjects received an autologous biotinylated RBC concentrate stored for 35 days in SAGM or PAGGGM. As a reference for the PTR, a 2-day stored autologous biotinylated RBC concentrate stored in SAGM was simultaneously transfused. RBC phenotype and PTR were assessed after transfusion. Biotinylated RBCs were isolated from the circulation for metabolomics analysis up to 24 hours after transfusion. The PTR was significantly higher in the 2-day stored RBCs than in 35-day stored RBCs 2 and 7 days after transfusion: 96% (90 to 99) vs 72% (66 to 89) and 96% (90 to 99) vs 72% (66 to 89), respectively. PTR of SAGM- and PAGGGM-stored RBCs did not differ significantly. Glucose and redox metabolism were better preserved in PAGGGM-stored RBCs. The differences measured in the blood bag remained present only until 1 day after transfusion. No differences in RBC phenotype were found besides an increased complement C3 deposition on 35-day RBCs stored in PAGGGM. Our data indicate that despite better metabolic preservation, PAGGGM is not a suitable alternative for SAGM because storage in PAGGGM did not result in an increased PTR. Finally, RBCs recovered from circulation after transfusion showed reversal of the metabolic storage lesion in vivo within a day. This study is registered in the Dutch trial register (NTR6492).

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage.

Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality.

Single Cell RNA-Seq Analysis of Human Red Cells.

Human red blood cells (RBCs), or erythrocytes, are the most abundant blood cells responsible for gas exchange. RBC diseases affect hundreds of millions of people and impose enormous financial and personal burdens. One well-recognized, but poorly understood feature of RBC populations within the same individual are their phenotypic heterogeneity. The granular characterization of phenotypic RBC variation in normative and disease states may allow us to identify the genetic determinants of red cell diseases and reveal novel therapeutic approaches for their treatment. Previously, we discovered diverse RNA transcripts in RBCs that has allowed us to dissect the phenotypic heterogeneity and malaria resistance of sickle red cells. However, these analyses failed to capture the heterogeneity found in RBC sub-populations. To overcome this limitation, we have performed single cell RNA-Seq to analyze the transcriptional heterogeneity of RBCs from three adult healthy donors which have been stored in the blood bank conditions and assayed at day 1 and day 15. The expression pattern clearly separated RBCs into seven distinct clusters that include one RBC cluster that expresses HBG2 and a small population of RBCs that express fetal hemoglobin (HbF) that we annotated as F cells. Almost all HBG2-expessing cells also express HBB, suggesting bi-allelic expression in single RBC from the HBG2/HBB loci, and we annotated another cluster as reticulocytes based on canonical gene expression. Additional RBC clusters were also annotated based on the enriched expression of NIX, ACVR2B and HEMGN, previously shown to be involved in erythropoiesis. Finally, we found the storage of RBC was associated with an increase in the ACVR2B and F-cell clusters. Collectively, these data indicate the power of single RBC RNA-Seq to capture and discover known and unexpected heterogeneity of RBC population.

Automatic Classification of Red Blood Cell Morphology Based on Quantitative Phase Imaging

Classification of the morphology of red blood cells (RBCs) plays an extremely important role in evaluating the quality of long-term stored blood, as RBC storage lesions such as transformation of discocytes to echinocytes and then to spherocytes may cause adverse clinical effects. Most RBC segmentation and classification methods, limited by interference of staining procedures and poor details, are based on traditional bright field microscopy. In the present study, quantitative phase imaging (QPI) technology was combined with deep learning for automatic classification of RBC morphology. QPI can be used to observe unstained RBCs with high spatial resolution and phase information. In deep learning based on phase information, boundary curvature is used to reduce inadequate learning for preliminary screening of the three shapes of unstained RBCs. The model accuracy was 97.3% for the stacked sparse autoencoder plus Softmax classifier. Compared with the traditional convolutional neural network, the developed method showed a lower misclassification rate and less processing time, especially for RBCs with more discocytes. This method has potential applications in automatically evaluating the quality of long-term stored blood and real-time diagnosis of RBC-related diseases.

Effect of low concentration of melatonin on the quality of stored red blood cells <em>in vitro</em>

Introduction. Oxidative stress is one of the important causes of red blood cells (RBCs) storage lesion. As a hormone, melatonin (MT) is also an effective antioxidant, however the pro- and antioxidative properties of MT depend on the cell type, redox state, as well as experimental conditions.Aim of this study — to investigate the protective effects of low concentration of MT on the stored RBCs in vitro.Materials and methods. Leukofi ltered RBCs were incubated in MAP RBC additive solution with or without 150 pg/mL of MT for 42 days under blood bank conditions. The morphology, aggregation index, methemoglobin (MetHb), m alondialdehyde (MDA), glucose, lactic acid and ATP of RBCs were detected on days 0, 7, 14, 21, 28, 35 and 42 to observe the protective effects of MT during the storage of RBCs.Results. During RBCs s torage, the number of deformed RBCs, relative hemolysis rate, aggregation index, MDA and MetHb were signifi cantly affected by both storage time (p < 0.0001) and melatonin (p < 0.01), and they had interaction only on the number of deformed RBCs (p < 0.0001). The concentration of glucose, lactic acid and ATP were affected by storage time (p < 0.0001), but not by MT concentration (p > 0.05). The number of deformed RBCs, relative hemolysis rate, MDA and MetHb in MT group were signifi cantly lower than that in control group at the end of storage stage (p < 0.05).Conclusion. Our study showed low hypnotic drug concentration of MT is speculated to have protective effects on the quality of stored RBCs through antioxidative mechanism.

Effects of Freshly Irradiated vs Irradiated and Stored Red Blood Cell Transfusion on Cerebral Oxygenation in Preterm Infants

Gamma irradiation of leukoreduced red blood cells (RBCs) prevents transfusion-associated graft-vs-host disease but also exacerbates storage lesion formation in RBCs. It is unknown whether freshly irradiated RBCs are more efficacious than irradiated and stored RBCs in preterm infants with high transfusion requirements. To examine whether transfusion of freshly irradiated vs irradiated and stored RBC components improves cerebral oxygen delivery in preterm infants with anemia. This single-center, double-blinded, proof-of-concept randomized clinical trial was conducted at the neonatal intensive care unit of Wellington Regional Hospital in Wellington, New Zealand, between December 1, 2017, and November 30, 2018. Participants were preterm infants (<34 weeks' gestation at birth) who were at least 14 days of age and had anemia. Participants underwent nonurgent transfusions, and these episodes were randomized to the intervention group (in which the infants received a transfusion of RBCs that were freshly irradiated on the day of transfusion) or control group (in which the infants received a transfusion of RBCs that were irradiated and stored for up to 14 days). Data were analyzed using the evaluable population approach. Transfusion of freshly irradiated RBCs. The prespecified primary outcome was the change in cerebral regional oxygen saturation (crSO2) from baseline (immediately before) to immediately after the transfusion. The prespecified secondary outcomes were the change in cerebral fractional tissue oxygen extraction (cFTOE) at different time points (immediately after, 24 hours after, and 120 hours or 5 days after transfusion). Outcomes were measured by blinded clinicians using near-infrared spectroscopy. A covariate-adjusted linear mixed model was used to quantify mean treatment effects and account for multiple transfusions in some infants. A total of 42 infants (mean [SD] gestational age, 26 [10] weeks and 3 days; 29 [69%] boys) were enrolled in the trial and underwent 64 transfusion episodes, which were randomized to the intervention (n = 31) or control (n = 33) group. Compared with infants in the control group, those in the intervention group showed a covariate-adjusted mean increase in crSO2 (2.0 percentage points; 95% CI, 1.2-2.8 percentage points) and a mean decrease in cFTOE (0.02; 95% CI, 0.01-0.04) immediately after transfusion. These differences were sustained up to 120 hours or 5 days after transfusion. There were negligible mean changes in crSO2 or cFTOE in infants in the control group at any of the follow-up time points. Results of this trial showed that transfusion of freshly irradiated RBCs conferred a small advantage in cerebral oxygenation for at least 5 days after transfusion compared with transfusion of irradiated and stored RBC components. On-demand irradiation of RBC components may be considered to optimize oxygen delivery in the recipient, but this physiological finding requires further research. ANZCTR Identifier: ACTRN12617001581358.

High-Throughput Metabolomics Platform for the Rapid Data-Driven Development of Novel Additive Solutions for Blood Storage.

Red blood cell transfusion is a life-saving intervention, and storage is a logistic necessity to make ~110 million units available for transfusion every year worldwide. However, storage in the blood bank is associated with a progressive metabolic decline, which correlates with the accumulation of morphological lesions, increased intra- and extra-vascular hemolysis upon transfusion, and altered oxygen binding/off-loading kinetics. Prior to storage, red blood cells are suspended in nutrient formulations known as additive solutions to prolong cellular viability. Despite a thorough expansion of knowledge regarding red blood cell biology over the past few decades, only a single new additive solution has been approved by the Food and Drug Administration this century, owing in part to the limited capacity for development of novel formulations. As a proof of principle, we leveraged a novel high-throughput metabolomics technology as a platform for rapid data-driven development and screening of novel additive solutions for blood storage under both normoxic and hypoxic conditions. To this end, we obtained leukocyte-filtered red blood cells (RBCs) and stored them under normoxic or hypoxic conditions in 96 well plates (containing polyvinylchloride plasticized with diethylhexylphthalate to concentrations comparable to full size storage units) in the presence of an additive solution supplemented with six different compounds. To inform this data-driven strategy, we relied on previously identified metabolic markers of the RBC storage lesion that associates with measures of hemolysis and post-transfusion recovery, which are the FDA gold standards to predict stored blood quality, as well as and metabolic predictors of oxygen binding/off-loading parameters. Direct quantitation of these predictors of RBC storage quality were used here—along with detailed pathway analysis of central energy and redox metabolism—as a decision-making tool to screen novel additive formulations in a multiplexed fashion. Candidate supplements are shown here that boost-specific pathways. These metabolic effects are only in part dependent on the SO2 storage conditions. Through this platform, we anticipate testing thousands of novel additives and combinations thereof in the upcoming months.

Tangential flow filtration facilitated washing of human red blood cells: A proof-of-concept study.

Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Effect of Hypoxic Blood Infusion on Pulmonary Physiology.

The ability to store red blood cells (RBCs) and other components for extended periods of time has expanded the availability and use of transfusion as a life-saving therapy. However, conventional RBC storage has a limited window of effective preservation and is accompanied by the progressive accumulation of a series of biochemical and morphological modifications, collectively referred to as “storage lesions.” These lesions have been associated with negative clinical outcomes (i.e., postoperative complications as well as reduced short-term and long-term survival) in patients transfused with conventionally stored blood with older and deteriorated transfused red cells. Hence, there is an increased unmet need for improved RBC storage. Hypoxic storage of blood entails the removal of large amounts of oxygen to low levels prior to refrigeration and maintenance of hypoxic levels through the entirety of storage. As opposed to conventionally stored blood, hypoxic storage can lead to a reduction of oxidative damage to slow storage lesion development and create a storage condition expected to result in enhanced efficacy of stored RBCs without an effect on oxygen exchange in the lung. Hypoxic blood transfusions appear to offer minimal safety concerns, even in patients with hypoxemia. This review describes the physiology of hypoxically stored blood, how it differs from conventionally stored blood, and its use in potential clinical application, such as massively transfused and critically ill patients with oxygenation/ventilation impairments.

Proof of concept: hypoxanthine from stored red blood cells induces neutrophil activation.

Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO2. Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex® assay. In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

Deep Learning-Based Phenotypic Assessment of Red Cell Storage Lesions for Safe Transfusions.

This study presents a novel approach to automatically perform instant phenotypic assessment of red blood cell (RBC) storage lesion in phase images obtained by digital holographic microscopy. The proposed model combines a generative adversarial network (GAN) with marker-controlled watershed segmentation scheme. The GAN model performed RBC segmentations and classifications to develop ageing markers, and the watershed segmentation was used to completely separate overlapping RBCs. Our approach achieved good segmentation and classification accuracy with a Dice's coefficient of 0.94 at a high throughput rate of about 152 cells per second. These results were compared with other deep neural network architectures. Moreover, our image-based deep learning models recognized the morphological changes that occur in RBCs during storage. Our deep learning-based classification results were in good agreement with previous findings on the changes in RBC markers (dominant shapes) affected by storage duration. We believe that our image-based deep learning models can be useful for automated assessment of RBC quality, storage lesions for safe transfusions, and diagnosis of RBC-related diseases.

Corpuscular Fragility and Metabolic Aspects of Freshly Drawn Beta-Thalassemia Minor RBCs Impact Their Physiology and Performance Post Transfusion: A Triangular Correlation Analysis In Vitro and In Vivo.

The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal+) donors (n = 18 each). It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules (e.g., phosphocreatine) and purine metabolism factors (IMP, hypoxanthine) were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.

Survey of blood centre readiness regarding removal of DEHP from blood bag sets: The BEST Collaborative Study.

Di(2-ethylhexyl) phthalate (DEHP) must be removed from blood bag sets in Europe by 27 May 2025. DEHP is known to interact with the red blood cell (RBC) membrane, resulting in reduced haemolysis and thus prolonging shelf-life. Current non-DEHP alternatives result in increased haemolysis requiring reconsideration of the RBC shelf-life. Although the immediate impact of eliminating DEHP is to the European community, the non-DEHP movement could affect blood bag set availability globally. The purpose of this survey is to understand blood centre readiness regarding the transition to non-DEHP blood collection and storage systems. A 24-question on-line survey was completed by members of the Biomedical Excellence for Safer Transfusion Collaborative research network. Responses were obtained from 16 blood collection or processing institutions. A majority of respondents (12/16) indicated that both shelf-life and haemolysis were equally important in selecting non-DEHP blood bag sets. Six respondents would accept a lower RBC product shelf-life compared to current practice. Respondents were not clear on the best non-DEHP vinyl material or RBC storage solution. Three European blood centres indicated they have developed non-DEHP transition plans. One challenge identified regarding the transition to non-DEHP is the extensive validation testing that will be required. Blood centres in Europe are concerned with meeting the sunset date for DEHP, considering that limited non-DEHP blood bag and RBC storage solutions are currently available. Banning DEHP in Europe, which may have global ramifications, represents a major challenge not yet fully understood by the transfusion medicine community.

The relationship between glycosylated hemoglobin level and red blood cell storage lesion in blood donors.

Glycosylated hemoglobin (HbA1c), not routinely screened in blood donors, is associated with morphological, biochemical, and functional abnormalities of red blood cells (RBCs) and with enhanced oxidative stress. We aimed to explore HbA1c levels in blood donors and their effect on RBC storage. An analytical cross-sectional study was conducted on 875 eligible blood donors aged 18-60 years from May 1, 2021, to August 30, 2021. Two selected groups of donors (HbA1c <6.5%, n=10; HbA1c ≥ 6.5%, n=10) exhibiting as similar as possible baseline values (such as age, sex, and living habits, etc.) were recruited for blood donation in leukoreduced CPDA-1units. RBC morphological, biochemical, structural, and oxidative stress states were measured during 5-35 days of storage. Elevated HbA1c prevalence was 37%, including 31.7% (277/875) in the prediabetes range (HbA1c 5.7%-6.4%) and 5.4% (47/875) in the diabetes range (HbA1c ≥ 6.5%). Age, body mass index (BMI), smoking, and alcohol consumption were the main factors influencing the HbA1c levels. During storage, high-HbA1c group had abnormal RBC morphology, impaired membrane function, and ion imbalance (higher mean corpuscular volume, distribution width, hemolysis rate, potassium ion efflux, and phosphatidylserine exposure) as compared with low HbA1c group. Additionally, RBC oxidative stress was significantly increased in donors with high HbA1c levels during 21-35 days. Blood donors proportion with abnormal HbA1c levels was relatively high, and donor HbA1c levels may be associated with stored RBCs capacity. Our study provides new insights into the different effects of donor HbA1c levels on RBC storage lesions.

Storage conditions determine the characteristics of red blood cell derived extracellular vesicles

Extracellular vesicles (EVs) are released during the storage of red blood cell (RBC) concentrates and might play adverse or beneficial roles throughout the utilization of blood products (transfusion). Knowledge of EV release associated factors and mechanism amends blood product management. In the present work the impact of storage time and medium (blood preserving additive vs isotonic phosphate buffer) on the composition, size, and concentration of EVs was studied using attenuated total reflection infrared (ATR-IR) spectroscopy, microfluidic resistive pulse sensing (MRPS) and freeze-fraction combined transmission electron micrography (FF-TEM). The spectroscopic protein-to-lipid ratio based on amide and the C–H stretching band intensity ratio indicated the formation of various vesicle subpopulations depending on storage conditions. After short storage, nanoparticles with high relative protein content were detected. Spectral analysis also suggested differences in lipid and protein composition, too. The fingerprint region (from 1300 to 1000 cm−1) of the IR spectra furnishes additional information about the biomolecular composition of RBC-derived EVs (REVs) such as adenosine triphosphate (ATP), lactose, glucose, and oxidized hemoglobin. The difference between the vesicle subpopulations reveals the complexity of the REV formation mechanism. IR spectroscopy, as a quick, cost-effective, and label-free technique provides valuable novel biochemical insight and might be used complementary to traditional omics approaches on EVs.

A Dynamic Constraint-based Modelling (DCBM) Approach With Alternative Metabolic Objective Functions Predicts The impact of Oxidative Stress on Stored Red Blood Cells (RBCs)

Mathematical metabolic modelling is a systematic endeavour to allow identifying the main causes of an observed metabolic change and to estimate the consequences of an imposed metabolic perturbation regarding a biosystem. Dynamic Constraint-based modelling (DCBM) has delivered promising results in metabolic engineering and in bioprocess design by providing mechanistically relevant systems-level knowledge of a network of bioreactions. Here, we seek to establish a DCBM approach that leverages convex optimization and nonlinear regression mathematical toolkit to estimate dynamic intracellular metabolic flux distributions in stored Red Blood Cells (RBCs) for transfusion purposes. First, we developed an ad-hoc metabolic network including 77 reactions and 74 metabolites, second, we adapted Flux Variability Analysis (FVA) technique to quantify the connection between exometabolomic dynamics and the dynamics of feasible intracellular reaction flux ranges. We have obtained fine-grained flux range dynamics of the intracellular reactions for the benchmark data published in (Bordbar et al., 2016). Then, we defined four objective functions regarding the accumulation of oxidative stress in stored RBCs for performing a dynamic Flux Balance Analysis (DFBA). In all four cases, time-resolved flux predictions were obtained respecting the imposed equality and inequality constraints. Last, we adapted a quadratic programming (QP) approach to calculate the Euclidean distance between the dynamic optimum flux vectors. The DCBM approach we have developed herein along with the developed metabolic network showed being suitable for the computational analysis of RBCs metabolic behaviour, and it is thought to be useful for other biosystems.

A study to assess the relationship between donor uric acid levels and supernatant hemolysis in stored packed red blood cell units.

Most of the red blood cell (RBC) storage lesions can be attributed to oxidative stress encountered by the RBCs throughout the duration of their storage. Various donor variables at the time of donation may be responsible for the total antioxidant capacity of the supernatant and thus, the "storability" and the magnitude of development of these RBC storage lesions. It is known that uric acid (UA) is responsible for more than 60% of the TAC of the blood. This study aims to explore the relationship between donor UA levels and the difference in percentage hemolysis, an important RBC storage lesion, on day 1 and day 21, in stored packed RBCs (PRBCs) units. The serum UA of 100 healthy voluntary male blood donors was estimated at the time of blood donation. The percentage hemolysis in the supernatant of the leukoreduced citrate phosphate dextrose/saline-adenine-glucose-mannitol RBC units (n = 100) prepared from these donors was calculated on day 1 and day 21. The difference in percentage hemolysis between donors with high normal serum UA levels (>7 mg/dL) was compared to that of the donors with low normal serum UA levels (<5 mg/dL) to observe the effect of donor UA levels on the difference in percentage hemolysis. The mean of the differences in percentage hemolysis in the supernatant in low UA group (<5 mg/dL) was higher than the mean of the differences in percentage hemolysis in the supernatant in high UA group (>7 mg/dL) and this was statistically significant (P < 0.001). The donor serum UA level and difference in percentage hemolysis on day 21 and day 1 were found to be negatively co-related. Higher levels of serum UA of blood donors seem to have a protective effect on the stored PRBC units as shown in this study. Hence, the potential of UA as one of the constituents of RBC additive solutions might lead to the enhancement of the quality of stored PRBC units by decreasing the RBC storage lesions.