Red Blood Cell Storage Research Articles

Measurement of red blood cell deformability during morphological changes using rotating-glass-plate-based scanning optical tweezers.

It is important to measure the deformability of red blood cells (RBCs) before transfusion, which is a key factor in the gas transport ability of RBCs and changes during storage of RBCs in vitro. Moreover, the morphology of RBCs also changes during storage. It is proposed that the change in morphology is related to the change in deformability. However, the efficiency of typical methods that use particles as handles is low, especially in the deformability measurement of echinocyte and spherocytes. Therefore, the deformability of RBCs with different morphologies is hard to be measured and compared in the same experiment. In this study, we developed a cost-effective and efficient rotating-glass-plate-based scanning optical tweezers device for the measurement of deformability of RBCs. The performance of this device was evaluated, and the deformability of three types of RBCs was measured using this device. Our results clearly show that the change of erythrocyte morphology from discocyte to echinocyte and spherocyte during storage in vitro is accompanied by a decrease in deformability.

Red Blood Cell Storage: From Genome to Exposome Towards Personalized Transfusion Medicine

Over the last decade, the introduction of omics technologies—especially high-throughput genomics and metabolomics—has contributed significantly to our understanding of the role of donor genetics and nongenetic determinants of red blood cell storage biology. Here we briefly review the main advances in these areas, to the extent these contributed to the appreciation of the impact of donor sex, age, ethnicity, but also processing strategies and donor environmental, dietary or other exposures - the so-called exposome—to the onset and severity of the storage lesion. We review recent advances on the role of genetically encoded polymorphisms on red cell storage biology, and relate these findings with parameters of storage quality and post-transfusion efficacy, such as hemolysis, post-transfusion intra- and extravascular hemolysis and hemoglobin increments. Finally, we suggest that the combination of these novel technologies have the potential to drive further developments towards personalized (or precision) transfusion medicine approaches.

Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the “first-in-first-out” principle to avoid wastage, whereas most healthcare providers prefer the “last-in-first-out” approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Leukoreduction of red blood cell units decreases dysregulatory micro RNAs during routine storage: An observational study with In-silico analysis.

Red Blood cells (RBCs) bring about harmful consequences during storage. MicroRNA (miRNA) dysregulation in stored RBCs could represent potential biomarkers of storage lesions. Although leukoreduction prevents damage to RBCs, it is uncertain whether leukoreduction of RBCs would impact the dysregulation of miRNAs during storage. This study evaluated the potential role of miRNAs for any alteration of leukoreduced (LR) and non-leukoreduced (NLR) RBCs till 21 days of storage. In this prospective study, thirty male volunteers' blood was equally divided into leukoreduced RBCs (LR) and NLR RBC (NLR) bags and stored till Day 21 at 4-60c. Selected miRNAs were quantified on Days 0 and 21. Further, bioinformatic tools were used to analyze the selected miRNAs and their predicted target genes (mRNAs) and identify the miRNA-mRNA regulatory relationships. A significantly higher fold change values of three miRNAs (miR-96-5p, miR-197-3p, miR-769-3p) were observed in NLR RBCs (p < .05). A significantly higher (p < .05) expression levels of miR-150-5p and miR-197-3p were observed in NLR RBCs till 21 days of storage. Further, the correlation with mRNA quantification confirmed the regulatory role of these miRNAs upon functional pathway enrichment analysis. A higher level of dysregulation of miRNAs was observed in NLR RBCs. Validation from In-Silico analysis suggested the regulatory role of miRNAs in cell apoptosis, senescence, and RBC-related signaling pathways. This indicated that stored LR RBCs would likely have better in vivo survival and function following transfusion. However, an in vivo study of miRNA in RBCs is warranted for conclusive evidence.

ZOOMICS : comparative metabolomics of red blood cells from dogs, cows, horses and donkeys during refrigerated storage for up to 42 days.

The use of omics technologies in human transfusion medicine has improved our understanding of the red blood cell (RBC) storage lesion(s). Despite significant progress towards understanding the storage lesion(s) of human RBCs, a comparison of basal and post-storage RBC metabolism across multiple species using omics technologies has not yet been reported, and is the focus of this study. Blood was collected in a standard bag system (CPD-SAG-Mannitol) from dogs (n=8), horses, bovines, and donkeys (n=6). All bags were stored at 4°C for up to 42 days (i.e., the end of the shelf life in Italian veterinary clinics) and sampled weekly for metabolomics analyses. In addition, data comparisons to our ongoing Zoomics project are included to compare this study's results with those of non-human primates and humans. Significant interspecies differences in RBC metabolism were observed at baseline, at the time of donation, with bovine showing significantly higher levels of metabolites in the tryptophan/kynurenine pathway; dogs showing elevated levels of high-energy compounds (especially adenosine triphosphate and S-adenosyl-methionine) and equine (donkey and horse) RBCs showing almost overlapping phenotypes, with the highest levels of free branched chain amino acids, glycolytic metabolites (including 2,3-diphosphoglycerate), higher total glutathione pools, and elevated metabolites of the folate pathway compared to the other species. Strikingly, previously described metabolic markers of the storage lesion(s) in humans followed similar trends across all species, though the rate of accumulation/depletion of metabolites in energy and redox metabolism varied by species, with equine blood showing the lowest degree of storage lesion(s). These results interrogate RBC metabolism across a range of mammalian species and improve our understanding of both human and veterinary blood storage and transfusion.

Variability of extracellular vesicle release during storage of red blood cell concentrates is associated with differential membrane alterations, including loss of cholesterol-enriched domains.

Transfusion of red blood cell concentrates is the most common medical procedure to treat anaemia. However, their storage is associated with development of storage lesions, including the release of extracellular vesicles. These vesicles affect in vivo viability and functionality of transfused red blood cells and appear responsible for adverse post-transfusional complications. However, the biogenesis and release mechanisms are not fully understood. We here addressed this issue by comparing the kinetics and extents of extracellular vesicle release as well as red blood cell metabolic, oxidative and membrane alterations upon storage in 38 concentrates. We showed that extracellular vesicle abundance increased exponentially during storage. The 38 concentrates contained on average 7 × 1012 extracellular vesicles at 6weeks (w) but displayed a ∼40-fold variability. These concentrates were subsequently classified into 3 cohorts based on their vesiculation rate. The variability in extracellular vesicle release was not associated with a differential red blood cell ATP content or with increased oxidative stress (in the form of reactive oxygen species, methaemoglobin and band3 integrity) but rather with red blood cell membrane modifications, i.e., cytoskeleton membrane occupancy, lateral heterogeneity in lipid domains and transversal asymmetry. Indeed, no changes were noticed in the low vesiculation group until 6w while the medium and the high vesiculation groups exhibited a decrease in spectrin membrane occupancy between 3 and 6w and an increase of sphingomyelin-enriched domain abundance from 5w and of phosphatidylserine surface exposure from 8w. Moreover, each vesiculation group showed a decrease of cholesterol-enriched domains associated with a cholesterol content increase in extracellular vesicles but at different storage time points. This observation suggested that cholesterol-enriched domains could represent a starting point for vesiculation. Altogether, our data reveal for the first time that the differential extent of extracellular vesicle release in red blood cell concentrates did not simply result from preparation method, storage conditions or technical issues but was linked to membrane alterations.

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 0-165mM of trehalose or 0-165mM 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 110mM. 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.

Effect of red blood cell storage time in pediatric cardiac surgery patients: A subgroup analysis of a randomized controlled trial

ObjectiveThis study aimed to determine whether or not transfusion of fresh red blood cells (RBCs) reduced the incidence of new or progressive multiple organ dysfunction syndrome compared with standard-issue RBCs in pediatric patients undergoing cardiac surgery. MethodsPreplanned secondary analysis of the Age of Blood in Children in Pediatric Intensive Care Unit study, an international randomized controlled trial. This study included children enrolled in the Age of Blood in Children in Pediatric Intensive Care Unit trial and admitted to a pediatric intensive care unit after cardiac surgery with cardiopulmonary bypass. Patients were randomized to receive either fresh (stored ≤7 days) or standard-issue RBCs. The primary outcome measure was new or progressive multiple organ dysfunction syndrome, measured up to 28 days postrandomization or at pediatric intensive care unit discharge, or death. ResultsOne hundred seventy-eight patients (median age, 0.6 years; interquartile range, 0.3-2.6 years) were included with 89 patients randomized to the fresh RBCs group (median length of storage, 5 days; interquartile range, 4-6 days) and 89 to the standard-issue RBCs group (median length of storage, 18 days; interquartile range, 13-22 days). There were no statistically significant differences in new or progressive multiple organ dysfunction syndrome between fresh (43 out of 89 [48.3%]) and standard-issue RBCs groups (38 out of 88 [43.2%]), with a relative risk of 1.12 (95% CI, 0.81 to 1.54; P = .49) and an unadjusted absolute risk difference of 5.1% (95% CI, −9.5% to 19.8%; P = .49). ConclusionsIn neonates and children undergoing cardiac surgery with cardiopulmonary bypass, the use of fresh RBCs did not reduce the incidence of new or progressive multiple organ dysfunction syndrome compared with the standard-issue RBCs. A larger trial is needed to confirm these results.

Sphingosine 1-phosphate has a negative effect on RBC storage quality.

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n= 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 μM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.

5612976 FIRST ADMINISTRATION OF HYPOXIC BLOOD IN A TRANSFUSION-DEPENDENT PATIENT

Background: Many patients with inherited and acquired anemias such as sickle cell disease, thalassemia and myelodysplastic syndromes (MDS) are transfusion-dependent, requiring regularly scheduled red blood cell (RBC) transfusions.1 Chronic transfusions are associated with a range of complications, such as transfusion-related adverse events (AEs) and iron overload.1 Frequent transfusions may also have a negative impact on patient quality of life (QoL)1 and access is often difficult. To reduce transfusion burden in dependent patients, Hemanext Inc. (Lexington, MA, United States) has developed a CE mark certified device to process and store red blood cells hypoxically - CPD/PAGGSM leukocytes-reduced (LR), O2/CO2 reduced.2 By reducing oxidative stress, hypoxic blood storage has been shown to improve the quality and function of RBCs,3 demonstrating that lower volumes of hypoxic RBCs are required to resuscitate from hemorrhagic shock and maintain hemodynamics than conventional RBCs in animal models.4 This may have several benefits in transfusion-dependent patients, including increasing the interval between transfusions and hence potential to reduce iron overload. Aims/Methods: A pilot clinical study in Norway is investigating the safety of a single administration of hypoxic RBCs in patients with hematologic malignancies and patients with burns. The primary outcome of the study is the occurrence of AEs at Day 7±1 post-transfusion. Secondary outcomes include assessment of AEs from enrollment to subsequent RBC transfusion (or Day 28) and the evolution of hemoglobin (Hb) levels pre- and post-transfusion. For the subset of patients with hematologic malignancies, the transfusion trigger is set to Hb <9.0 g/dL, and two units of hypoxic RBCs are administered. Results: In August 2022, following his informed consent, the first patient was transfused with hypoxic RBCs; this patient was also the first to receive hypoxic blood within Hemanext’s overall clinical program. The patient was an 80-year-old man who initially presented with anemia and diagnostic workup revealed bone marrow smear with morphological features of erythroid dysplasia with ring sideroblasts in 30% of cells and mutation in the SF3B1 gene. After initial response to treatment, he became transfusion-dependent, and has received 2 RBC units every 2 weeks for a year. The patient had a pre-transfusion Hb level of 8.1 g/dL and received 2 units of O+ hypoxic RBCs over 2 hours without any complications. No adverse events were recorded on Day 1 and the patient experienced no adverse events by Day 7. Further outcomes will be reported. Conclusion: Hypoxic RBCs processed with the CPD/PAGGSM LR, O2/CO2 reduced system were well tolerated in this patient; Hb level was within the target range at follow-up, supporting that hypoxically stored RBCs function appropriately. The study is ongoing and will continue to assess hypoxic RBCs in patients with hematologic malignancies and patients with burns. Enrolment within the wider clinical program – including studies investigating hypoxic RBC administration in patients with thalassemia in Italy and sickle cell disease in the US – is planned.

Effects of Transfusion of Stored Red Blood Cells on Renal Ischemia-Reperfusion–Induced Hepatic Injury in Rats

Renal ischemia-reperfusion (IR) injures the liver as well as the kidneys. Transfusion of stored red blood cells (RBCs) triggers inflammatory responses, oxidative stress, and activation of innate immunity. In the present study, we investigated the effect of transfusion of stored RBCs on renal IR-induced hepatic injury. Sprague-Dawley rats were randomly divided into 3 groups based on the following treatments: rats subjected to sham operation (sham group), rats subjected to the induction of renal IR only (RIR group), and rats transfused with stored RBCs 1 hour after the start of reperfusion (RIR-TF group). Renal ischemia was induced for 1 hour, and reperfusion was allowed for 24 hours. After reperfusion, blood and liver tissue samples were obtained. Serum levels of aspartate and alanine aminotransferase were increased in the RIR-TF group compared with those in the RIR and sham groups. The hepatic mRNA expression levels of heme oxygenase-1 and neutrophil gelatinase-associated lipocalin were increased in the RIR-TF group compared with those in the RIR and sham groups. The mRNA expression level of high mobility group box-1 was also increased in the RIR-TF group compared with that in the RIR group. The transfusion of stored RBCs exacerbates renal IR-induced liver damage. Oxidative stress may be responsible for hepatic injury.

The time-course linkage between hemolysis, redox, and metabolic parameters during red blood cell storage with or without uric acid and ascorbic acid supplementation.

Oxidative phenomena are considered to lie at the root of the accelerated senescence observed in red blood cells (RBCs) stored under standard blood bank conditions. It was recently shown that the addition of uric (UA) and/or ascorbic acid (AA) to the preservative medium beneficially impacts the storability features of RBCs related to the handling of pro-oxidant triggers. This study constitutes the next step, aiming to examine the links between hemolysis, redox, and metabolic parameters in control and supplemented RBC units of different storage times. For this purpose, a paired correlation analysis of physiological and metabolism parameters was performed between early, middle, and late storage in each subgroup. Strong and repeated correlations were observed throughout storage in most hemolysis parameters, as well as in reactive oxygen species (ROS) and lipid peroxidation, suggesting that these features constitute donor-signatures, unaffected by the diverse storage solutions. Moreover, during storage, a general "dialogue" was observed between parameters of the same category (e.g., cell fragilities and hemolysis or lipid peroxidation and ROS), highlighting their interdependence. In all groups, extracellular antioxidant capacity, proteasomal activity, and glutathione precursors of preceding time points anticorrelated with oxidative stress lesions of upcoming ones. In the case of supplemented units, factors responsible for glutathione synthesis varied proportionally to the levels of glutathione itself. The current findings support that UA and AA addition reroutes the metabolism to induce glutathione production, and additionally provide mechanistic insight and footing to examine novel storage optimization strategies.

Understanding the Potential Role of Nanotechnology in Liver Fibrosis: A Paradigm in Therapeutics.

The liver is a vital organ that plays a crucial role in the physiological operation of the human body. The liver controls the body's detoxification processes as well as the storage and breakdown of red blood cells, plasma protein and hormone production, and red blood cell destruction; therefore, it is vulnerable to their harmful effects, making it more prone to illness. The most frequent complications of chronic liver conditions include cirrhosis, fatty liver, liver fibrosis, hepatitis, and illnesses brought on by alcohol and drugs. Hepatic fibrosis involves the activation of hepatic stellate cells to cause persistent liver damage through the accumulation of cytosolic matrix proteins. The purpose of this review is to educate a concise discussion of the epidemiology of chronic liver disease, the pathogenesis and pathophysiology of liver fibrosis, the symptoms of liver fibrosis progression and regression, the clinical evaluation of liver fibrosis and the research into nanotechnology-based synthetic and herbal treatments for the liver fibrosis is summarized in this article. The herbal remedies summarized in this review article include epigallocathechin-3-gallate, silymarin, oxymatrine, curcumin, tetrandrine, glycyrrhetinic acid, salvianolic acid, plumbagin, Scutellaria baicalnsis Georgi, astragalosides, hawthorn extract, and andrographolides.

Long-term storage protocol of reagent red blood cells treated with 0.01M dithiothreitol (DTT) for pre-transfusion testing of patients receiving anti-CD38 therapy, daratumumab

ABSTRACTObjective:Use red blood cell stabilizer to store the antibody screening and antibody identification reagent red blood cells (RBCs) treated with 0.01 mol/L DTT and investigate its value in the pre-transfusion examinations of patients treated with daratumumab.Method:Determined the optimal incubation time for the 0.01 mol/L DTT-treated RBCs method by evaluating the effect of treatment at different time points. Added ID-CellStab to store DTT-treated RBCs, determined the maximum shelf life of reagent RBCs by monitoring the hemolysis index, and assessed changes in the antigenicity of blood group antigens on the surface of RBCs during storage with antibody reagents.Result:A protocol for long-term storage of reagent red blood cells treated with the 0.01 mol/L DTT method was established. The optimal incubation time was 40-50 min. RBCs could be stored stably for 18 days after adding ID-CellStab. The protocol was able to eliminate pan-agglutination caused by daratumumab, with no significant changes in the antigens of most blood group systems, except for some attenuation of K antigen and Duffy blood group system antigens during the storage period.Conclusion:The storage protocol of reagent RBCs based on the 0.01 mol/L DTT method does not affect the detection of most blood group antibodies and retains a certain degree of detection ability for anti-K antibodies, allowing patients treated with daratumumab to quickly perform pre-transfusion examinations, making up for the shortcomings of currently commercial reagent RBCs.

Deep ensemble learning enables highly accurate classification of stored red blood cell morphology

Changes in red blood cell (RBC) morphology distribution have emerged as a quantitative biomarker for the degradation of RBC functional properties during hypothermic storage. Previously published automated methods for classifying the morphology of stored RBCs often had insufficient accuracy and relied on proprietary code and datasets, making them difficult to use in many research and clinical applications. Here we describe the development and validation of a highly accurate open-source RBC morphology classification pipeline based on ensemble deep learning (DL). The DL-enabled pipeline utilized adaptive thresholding or semantic segmentation for RBC identification, a deep ensemble of four convolutional neural networks (CNNs) to classify RBC morphology, and Kalman filtering with Hungarian assignment for tracking changes in the morphology of individual RBCs over time. The ensembled CNNs were trained and evaluated on thousands of individual RBCs from two open-access datasets previously collected to quantify the morphological heterogeneity and washing-induced shape recovery of stored RBCs. Confusion matrices and reliability diagrams demonstrated under-confidence of the constituent models and an accuracy of about 98% for the deep ensemble. Such a high accuracy allowed the CNN ensemble to uncover new insights over our previously published studies. Re-analysis of the datasets yielded much more accurate distributions of the effective diameters of stored RBCs at each stage of morphological degradation (discocyte: 7.821 ± 0.429 µm, echinocyte 1: 7.800 ± 0.581 µm, echinocyte 2: 7.304 ± 0.567 µm, echinocyte 3: 6.433 ± 0.490 µm, sphero-echinocyte: 5.963 ± 0.348 µm, spherocyte: 5.904 ± 0.292 µm, stomatocyte: 7.080 ± 0.522 µm). The effective diameter distributions were significantly different across all morphologies, with considerable effect sizes for non-neighboring classes. A combination of morphology classification with cell tracking enabled the discovery of a relatively rare and previously overlooked shape recovery of some sphero-echinocytes to early-stage echinocytes after washing with 1% human serum albumin solution. Finally, the datasets and code have been made freely available online to enable replication, further improvement, and adaptation of our work for other applications.

Storage differentially impacts alloimmunization to distinct red cell antigens following transfusion in mice.

The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens. RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion. Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation. These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization.

A study on the correlation between hemoglobin concentration and the storage quality of suspended red blood cells prepared from the whole blood of Tibetan male residents.

Previous studies reported that the blood of Tibetans living at different altitudes may vary slightly. There is evidence that the harsh environmental conditions at high altitudes, such as low pressure and hypoxia, may affect the morphology and hemorheology of red blood cells (RBCs). Hypoxia would increase the levels of hemoglobin ([Hb]) and hematocrit (Hct), potentially increasing blood hyperviscosity and compromising blood collection and transfusions. Therefore, it is critical to investigate the in vitro storage quality of Tibetan RBCs. In this study, the in vitro quality of suspended RBCs (SRBCs) prepared from whole blood (WB) of Tibetan residents with varying Hb concentrations ([Hb]) was measured during storage, and the relationship between the major factors in RBC storage and [Hb] was studied. The WB of Tibetan men was divided into three groups based on [Hb] levels (group A: 120 < Hb ≤ 185 g/L; group B: 185 < Hb ≤ 210 g/L; group C: Hb > 210 g/L). The SRBCs prepared from WB were examined aseptically on days 1, 14, 21, and 35 after storage. [Hb] was not correlated with mean corpuscular volume (MCV), adenosine triphosphate (ATP), pH, P50, and hemolysis. There was a moderate or strong negative association between platelets (PLT) and [Hb] from days 1 to 35, and the PLT number of group C was lower than group A during storage. Group C had the highest change rates of electrolytes, glucose, and lactate, and there were moderate or strong positive correlations between lactate and [Hb] (r = 0.3772, p = 0.0045), glucose and [Hb] (r = 0.5845, p < 0.0001), Na+ and [Hb] (r = 0.3966, p = 0.0027), and K+ and [Hb] (r = 0.4885, p = 0.0002). Group B had the highest change rates of 2,3-DPG on day 35, and there was a negative correlation between 2,3-DPG and [Hb] (r = -0.4933, p = 0.0001). These new data on the [Hb] could have implications for researchers wishing to study the storage quality of Tibetan SRBCs, particularly in the context of erythrocyte metabolism, and we propose finding a new, suitable alternative solution for plateau SRBCs, particularly the blood with [Hb] greater than 185 g/L. Our results could have important implications for researchers wishing to study the potential framework of high-altitude-induced SRBC storage lesions.

UPLC-MS/MS method for quantitative determination of the advanced glycation endproducts Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine.

During blood storage, red blood cells (RBCs) undergo physical, chemical, and metabolic changes that may contribute to post-transfusion complications. Due to the hyperglycemic environment of typical solutions used for RBC storage, the formation of advanced glycation endproducts (AGEs) on the stored RBCs has been implicated as a detrimental chemical change during storage. Unfortunately, there are limited studies involving quantitative determination and differentiation of carboxymethyl-lysine (CML) and carboxyethyl-lysine (CEL), two commonly formed AGEs, and no reported studies comparing these AGEs in experimental storage solutions. In this study, CML and CEL were identified and quantified on freshly drawn blood samples in two types of storage solutions, standard additive solution 1 (AS-1) and a normoglycemic version of AS-1 (AS-1N). To facilitate detection of the AGEs, a novel method was developed to reliably extract AGEs from RBCs, provide Food and Drug Administration (FDA) bioanalytical guidance criteria, and enable acceptable selectivity for these analytes. Ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS) was utilized to identify and quantify the AGEs. Results show this method is accurate, precise, has minimal interferences or matrix effects, and overcomes the issue of detecting AGE byproducts. Importantly, AGEs can be detected and quantified in both types of blood storage solutions (AS-1 and AS-1N), thereby enabling long-term (6 weeks) blood storage related studies.

Upregulation of miR-20a-5p as the Potential MicroRNA Marker in Red Blood Cell Storage Lesion.

Packed red blood cells (PRBCs) can be preserved for 42 days, and stored PRBCs have slow, dangerous changes over time during storage. miRNA is approximately 22 nucleotides long, a small single-stranded noncoding RNA molecule. miRNA guides by pairing bases with their downstream target mRNA to regulate negative expression. They are essential in many life processes, including cell differentiation, proliferation, and apoptosis. Therefore, miRNA alterations may represent possible biomarkers of PRBC storage lesions. This study is aimed at validating the miR-20a-5p in PRBC storage. Study Design and Methods. A total of 20 PRBC samples were divided into day 1 and day 20 storage groups. Total miRNA was extracted and quantified by probe-based RT-qPCR assays to explore the potential role of miRNAs in PRBC storage lesions. Upregulated miR-20a-5p in PRBC storage on day 20 compared to day 1. MiR-20a-5p promoted cell survival, which may affect the downstream regulation and decrease PRBC viability in prolonged storage. On this basis, this detection may help to assess the quality of stored PRBCs.

PROLONGED STORAGE OF THAWED RED BLOOD CELLS

Introduction. In modern transfusion practice, both in peacetime and in military conditions, red blood cells (RBCs) are widely used as the main component of donor blood. Cryopreserved red blood cells are considered the most safe and high-quality RBC-containing environment. However, the storage period of thawed RBCs after cryopreservation is limited to 24 hours, and significantly complicates their use. Therefore, extending the storage period of thawed RBCs is relevant for the blood service. Research objective: study the RBCs morphological state and functional completeness that were cryopreserved at -40ºС and stored for 7 days at a temperature of +2ºС - +4ºС after thawing.&#x0D; Materials and methods. The object of the study were RBCs that were cryopreserved at -40ºС and stored for 7 days at a temperature of +2ºС - +4ºС after thawing. Deglycerolization of the thawed red blood cells, cryopreserved at -40ºС, required three time washing by using reverse cytoagglomeration. Thawed RBCs were re-suspended in lactate-sucrose-phosphate solution. After RBC thawing and storage for 7 days (186 doses) in the suspension the following indicators were studied: free hemoglobin, extracellular potassium, adenosine triphosphoric acid (ATP), 2,3-diphosphoglycerate (2,3-DPG), hematocrit, degree of hemoglobin affinity to oxygen (P50,), viscosity coefficient, osmotic stability, electrophoretic mobility of erythrocytes. as well as the total number of cells lost and recovered.&#x0D; Results. After storage for 7 days of suspension of thawed RBCs at a temperature of +2ºС - +4ºС indicators of free hemoglobin (0,62±0,02 g/l), extracellular potassium (2,7±0,3 mmol/l), hematocrit (0,4±0,02 l/l) were within normal limits. Osmotic resistance (0,46±0,02%), electrophoretic mobility (0,94±0,04 µm·cm·V-1·s-1) of RBCs, suspension viscosity factor (5,5±0,20mPa·С) did not exhibit changes in comparison with normal values. High levels of ATP indicators (3,0±0,2 µmol/gHb) and 2,3-DPG (10,5±1,3 µmol/gHb) were established. Indicator Р50 (24,1±1,3 hPa) corresponded to low hemoglobine affinity for oxygen. After 7-day storage at +2ºС - +4ºС total cell loss was insignificant and amounted to 5,6±0,4%. High percentage of viable thawed RBCs 94,4±0,5% was shown.&#x0D; Conclusions. Deglycerolization of thawed red blood cells, cryopreserved at -40ºС, by reverse cytoagglomeration, as well as use of lactate-sucrose-phosphate solution for washed RBCs resuspending promote prolongation of thawed RBCs storage period up to 7 days at +2ºС - +4ºС in viable condition.