Blood Bank Storage Research Articles

Measurement and analysis of ionic leakage profiles in refrigerated human red blood cells using dielectrophoresis and inductively coupled mass spectroscopy.

Human red blood cells (RBCs) undergo ionic leakage through passive diffusion during refrigerated storage, affecting their quality and health. We investigated the dynamics of ionic leakage in human RBCs over a 20-day refrigerated storage period using extracellular ion quantification and dielectrophoresis (DEP). Four type O- human blood donors were examined to assess the relationship between extracellular ion concentrations (Na+, K+, Mg2+, Ca2+, and Fe2+), RBC cytoplasm conductivity, and membrane conductance. A consistent negative correlation between RBC cytoplasm conductivity and membrane conductance, termed the "ionic leakage profile" (ILP), was observed across the 20-day storage period. Specifically, we noted a gradual decline in DEP-measured RBC cytoplasm conductivity alongside an increase in membrane conductance. Further examination of the electrical origins of this ILP using inductively coupled plasma mass spectrometry revealed a relative decrease in extracellular Na+ concentration and an increase in K+ concentration over the storage period. Correlation of these extracellular ion concentrations with DEP-measured RBC electrical properties demonstrated a direct link between changes in the cytoplasmic and membrane domains and the leakage and transport of K+ and Na+ ions across the cell membrane. Our analysis suggests that the inverse correlation between RBC cytoplasm and membrane conductance is primarily driven by the passive diffusion of K+ from the cytoplasm and the concurrent diffusion of Na+ from the extracellular buffer into the membrane, resulting in a conductive reduction in the cytoplasmic domain and a subsequent increase in the membrane. The ILP's consistent negative trend across all donors suggests that it could serve as a metric for quantifying blood bank storage age, predicting the quality and health of refrigerated RBCs.

Biological and genetic determinants of glycolysis: Phosphofructokinase isoforms boost energy status of stored red blood cells and transfusion outcomes

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging invivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis invitro and invivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes.

Identifying transcriptomic profiles of iron–quercetin complex treated peripheral blood mononuclear cells from healthy volunteers and diabetic patients

Peripheral blood is an alternative source of stem/progenitor cells for regenerative medicine owing to its ease of retrieval and blood bank storage. Previous in vitro studies indicated that the conditioned medium derived from peripheral blood mononuclear cells (PBMCs) treated with the iron–quercetin complex (IronQ) contains potent angiogenesis and wound-healing properties. This study aims to unveil the intricate regulatory mechanisms governing the effects of IronQ on the transcriptome profiles of human PBMCs from healthy volunteers and those with diabetes mellitus (DM) using RNA sequencing analysis. Our findings revealed 3741 and 2204 differentially expressed genes (DEGs) when treating healthy and DM PBMCs with IronQ, respectively. Functional enrichment analyses underscored the biological processes shared by the DEGs in both conditions, including inflammatory responses, cell migration, cellular stress responses, and angiogenesis. A comprehensive exploration of these molecular alterations exposed a network of 20 hub genes essential in response to stimuli, cell migration, immune processes, and the mitogen-activated protein kinase (MAPK) pathway. The activation of these pathways enabled PBMCs to potentiate angiogenesis and tissue repair. Corroborating this, quantitative real-time polymerase chain reaction (qRT-PCR) and cell phenotyping confirmed the upregulation of candidate genes associated with anti-inflammatory, pro-angiogenesis, and tissue repair processes in IronQ-treated PBMCs. In summary, combining IronQ and PBMCs brings about substantial shifts in gene expression profiles and activates pathways that are crucial for tissue repair and immune response, which is promising for the enhancement of the therapeutic potential of PBMCs, especially in diabetic wound healing.

Hematological Changes in Whole Blood Stored in Blood Bags Containing CPDA-1 Over a Period of 28 Days

Introduction: Blood storage is needed to ensure a readily available and safe blood supply. During blood storage in blood bank, biochemical and physical properties of RBCs are altered because of storage conditions. In normal conditions in the body circulation, these do not occur as optimum pH, temperature, nutrient concentration and waste products removal are maintained. The aim of this study was to observe the changes in whole blood stored in blood bags containing CPDA-1 over a period of 28 days.
 Materials and Methods: This was a hospital based observational study conducted in Blood Bank, National Medical College and Teaching Hospital (NMCTH), Birgunj. A total of 450 ml blood was drawn from 50 healthy volunteer donors into blood bags containing CPDA–1 anticoagulant and placed on the quarantine shelf of the blood bank refrigerator maintained at 2-8o centigrade. The blood bags were screened for HCV, HBsAg, syphillis and HIV. Ten milliliters blood was withdrawn from the blood bags on 1st, 7th, 14th, 21st, and 28th day and tested for hematological parameters using Yumizen H550 (HORIBA) fully automated hematology analyzer.
 Results: At the end of 28 days, whole blood storage in CPDA-1 revealed significant decrease in WBC (T = 4.79, p < 0.01) and platelets count (T=180.09, p < 0.01). Hematocrit increased significantly after 21 days of storage (T = 40.86, p < 0.01). MCHC and MCH also demonstrated significant increase during the storage period (T = 37.32, p < 0.01) and (T = 29.38, p < 0.01), respectively.
 Conclusion: WBC and platelets are significantly altered (decreased) when stored in blood bags containing CPDA- 1 after one week period. The study recommends whole blood transfusion before seventh day of donation in order to avoid the adverse outcome of hematological changes in stored blood. In case it has to be used beyond one week, then it should be leukodepleted before storage.

Dielectrophoretic detection of electrical property changes of stored human red blood cells.

The ability to transport and store a large human blood inventory for transfusions is an essential requirement for medical institutions. Thus, there is an important need for rapid and low-cost characterization tools for analyzing the properties of human red blood cells (RBCs) while in storage. In this study, we investigate the ability to use dielectrophoresis (DEP) for measuring the storage-induced changes in RBC electrical properties. Fresh human blood was collected, suspended in K2-EDTA anticoagulant, and stored in a blood bank refrigerator for a period of 20 days. Cells were removed from storage at 5-day intervals and subjected to a glutaraldehyde crosslinking reaction to "freeze" cells at their ionic equilibrium at that point in time and prevent ion leakage during DEP analysis. The DEP behavior of RBCs was analyzed in a high permittivity DEP buffer using a three-dimensional DEP chip (3DEP) and also compared to measurements taken with a 2D quadrupole electrode array. The DEP analysis confirms that RBC electrical property changes occur during storage and are only discernable with the use of the cell crosslinking reaction above a glutaraldehyde fixation concentration of 1.0w/v%. In particular, cytoplasm conductivity was observed to decrease by more than 75% while the RBC membrane conductance was observed to increase by more than 1000% over a period of 20 days. These results show that the presented combination of chemical crosslinking and DEP can be used as rapid characterization tool for monitoring electrical properties changes of human RBCs while subjected to refrigeration in blood bank storage.

Blood bank storage of red blood cells increases RBC cytoplasmic membrane order and bending rigidity.

Blood banks around the world store blood components for several weeks ensuring its availability for transfusion medicine. Red blood cells (RBCs) are known to undergo compositional changes during storage, which may impact the cells’ function and eventually the recipients’ health. We extracted the RBC’s cytoplasmic membrane (RBCcm) to study the effect of storage on the membranes’ molecular structure and bending rigidity by a combination of X-ray diffraction (XRD), X-ray diffuse scattering (XDS) and coarse grained Molecular Dynamics (MD) simulations. Blood was stored in commercial blood bags for 2 and 5 weeks, respectively and compared to freshly drawn blood. Using mass spectrometry, we measured an increase of fatty acids together with a slight shift towards shorter tail lengths. We observe an increased fraction (6%) of liquid ordered (lo) domains in the RBCcms with storage time, and an increased lipid packing in these domains, leading to an increased membrane thickness and membrane order. The size of both, lo and liquid disordered (ld) lipid domains was found to decrease with increased storage time by up to 25%. XDS experiments reveal a storage dependent increase in the RBCcm’s bending modulus κ by a factor of 2.8, from 1.9 kBT to 5.3 kBT. MD simulations were conducted in the absence of proteins. The results show that the membrane composition has a small contribution to the increased bending rigidity and suggests additional protein-driven mechanisms.

Red blood cells contain enzymatically active GPx4 whose abundance anticorrelates with hemolysis during blood bank storage

The antioxidant function of the phospholipid hydroperoxide glutathione peroxidase (GPx4) is vital for the homeostasis of many cell types, from neoplastic cells to normal erythroid precursors. However, some functional proteins in erythroid precursors are lost during the development of red blood cells (RBCs); whether GPx4 is maintained as an active enzyme in mature RBCs has remained unclear. Our meta-analyses of existing RBC proteomics and metabolomics studies revealed the abundance of GPx4 to be correlated with lipid-anchored proteins. In addition, GPx4 anti-correlated with lyso-phospholipids and complement system proteins, further supporting the presence of active GPx4 in mature RBCs. To test the potential biological relevance of GPx4 in mature RBCs, we correlated the rate of hemolysis of human RBCs during storage with the abundance of GPx4 and other heritable RBC proteins. Of the molecules that anti-correlated with the rate of hemolysis of RBCs, proteins that mediate the cellular response to hydroperoxides, including GPx4, have the greatest enrichment. Western blotting further confirmed the presence of GPx4 antigenic protein in RBCs. Using an assay optimized to measure the activity of GPx4 in RBCs, we found GPx4 to be an active enzyme in mature RBCs, suggesting that GPx4 protects RBCs from hemolysis during blood bank storage.

DEHT is a suitable plasticizer option for phthalate-free storage of irradiated red blood cells.

Due to increasing concerns about possible endocrine-disrupting properties, the use of the plasticizer di(2-ethylhexyl) phthalate (DEHP) will be banned in future blood storage. Di(2-ethylhexyl) terephthalate (DEHT) provides sufficient red blood cell (RBC) quality during conventional blood bank storage. It is important that a new plasticizer also maintains acceptable quality during exposure to high cell stress, such as irradiation, which is commonly used to prevent graft-versus-host disease. A total of 59 RBC units were collected and processed in polyvinyl chloride (PVC)-DEHT or PVC-DEHP blood bags combined with either saline-adenine-glucose-mannitol (SAGM) or phosphate-adenine-glucose-guanosine-saline-mannitol (PAGGSM) additive solution. All units were X-ray irradiated on day 2 post-collection. Sampling for assessment of parameters of storage lesion was performed on day 2 pre-irradiation and day 14 and 28 post-irradiation. Though irradiation increased cell stress, DEHT/PAGGSM and current common European preference DEHP/SAGM were equally affected up to 14 days post-irradiation for all measured parameters. At day 28, haemolysis and microvesicle count were slightly increased in DEHT, whereas extracellular potassium ions, glucose, lactate, pH, mean corpuscular volume and microvesicle phosphatidylserine remained unaffected by plasticizer choice throughout storage. No individual unit exceeded 0.8% haemolysis, not even in DEHT/SAGM, the combination overall most affected by irradiation. Of the four combinations, membrane stability was least impacted in DEHP/PAGGSM. We demonstrate that DEHT is a suitable plasticizer for storage of RBCs after X-ray irradiation cell stress. This strengthens the option of DEHT as a viable non-phthalate substitute for DEHP.

An Insight into the Stages of Ion Leakage during Red Blood Cell Storage.

Packed red blood cells (pRBCs), the most commonly transfused blood product, are exposed to environmental disruptions during storage in blood banks. In this study, temporal sequence of changes in the ion exchange in pRBCs was analyzed. Standard techniques commonly used in electrolyte measurements were implemented. The relationship between ion exchange and red blood cells (RBCs) morphology was assessed with use of atomic force microscopy with reference to morphological parameters. Variations observed in the Na+, K+, Cl−, H+, HCO3−, and lactate ions concentration show a complete picture of singly-charged ion changes in pRBCs during storage. Correlation between the rate of ion changes and blood group type, regarding the limitations of our research, suggested, that group 0 is the most sensitive to the time-dependent ionic changes. Additionally, the impact of irreversible changes in ion exchange on the RBCs membrane was observed in nanoscale. Results demonstrate that the level of ion leakage that leads to destructive alterations in biochemical and morphological properties of pRBCs depend on the storage timepoint.

To Determine the Morphological Changes in Red Blood Cells During Storage in Blood Banks

Background: During storage of blood, the red blood cells undergo shape changes which cause fragility and endothelial interaction leading to deterioration the quality of blood in blood banks.Objectives: The aim of this study is to determine the morphological changes in red blood cells during storage in blood banks. Material and Methods: In this experimental study, a total 20 healthy volunteers between 17 to 40 years blood donors-Blood bags were taken, ten from each center i.e. MMCTH blood bank Mardan and KTH blood bank Peshawar. The specimen analysis was done at IBMS (Institute of Basic Medical Sciences) of KMU (Khyber Medical University) Peshawar. The exclusion criteria were People with anemia, hepatitis B &C, HIV and syphilis. The duration of this study was six months. The inform consent was taken from each donor. The total blood 250 ml from vein in cubital fossa from each blood donor was collected in 250ml pediatric blood bag with CPDA-1 solution. Blood bags were put up in the blood bank at +2 to +6 °C and stored till 20 days. Blood specimen of about 5cc were collected in 5cc syringe from each blood bag on 0, 5th,10th ,15th and 20th day for following parameters and thin film red blood cell was prepared for examination by light microscope. Morphological changes in RBCs examined via light microscope as well as grading the RBCs status in the peripheral blood film, the occurrence of distorted RBC simply in random fields; such as +1(scored 1 to 5 altered RBC present in each field), +2 (an average of 6 to 15 altered RBC in each field), +3(16 to 25 altered RBC in each field) and +4(more than 25 altered RBC present in each field). The multi head light microscope NIKON eclipse 50 was used for examination of peripheral blood slide and we took images of randomly selected field. The image J software was used for slide examination.Results: The morphological analysis of red blood cells, count of 200 cells in each blood slide in randomly selected fields are: On day 0 the majority of cells were normally shaped (97.95±1.297 (mean±SD).With increasing storage time, the percentage of morphologically abnormal red cells rose sharply. Mean percentage of abnormal cells on day 5, 10, 15 and 20 was 28.80±10.00, 51.73±12.47, 64.78±14.66 and 68.10±7.92 respectively. This increase in percentage of abnormally shaped cells was significant as determined by one way ANOVA (p =0.001). There was a big difference of percentage of abnormal RBCs on day 0 and in = 5 to= 10 days and in = 15 to = 20 days of blood storage. The mean values of day 0 of abnormal cells was 2.05±1.297 (Mean ± Std. Deviation), abnormal cells in= 5 to= 10 days was 40.26± 16.101 (Mean ± Std. Deviation) and on day = 15 and in = 20 day was 66.44± 11.75. The mean difference from day 0 to day 20 was 63.93±10.45 (Mean ± Std. Deviation).The one way ANOVA was significant, P= 0.001.Conclusion: This study confirms the hematological and morphological changes, when blood stored at 2 °C to 6 °C for up to 21 days. The significant morphological changes were observed on 5th day of blood storage. These findings suggested that approximately a week old stored blood is as good as the fresh blood; however, significant morphological and biochemical changes begin to appear after the first week of storage and these changes aggravate with time. Hence in order to achieve best possible transfusion outcomes, stored blood up to one week can be utilized.

To Determine the Morphological Changes in Red Blood Cells During Storage in Blood Banks

Background: During storage of blood, the red blood cells undergo shape changes which cause fragility and endothelial interaction leading to deterioration the quality of blood in blood banks.Objectives: The aim of this study is to determine the morphological changes in red blood cells during storage in blood banks. Material and Methods: In this experimental study, a total 20 healthy volunteers between 17 to 40 years blood donors-Blood bags were taken, ten from each center i.e. MMCTH blood bank Mardan and KTH blood bank Peshawar. The specimen analysis was done at IBMS (Institute of Basic Medical Sciences) of KMU (Khyber Medical University) Peshawar. The exclusion criteria were People with anemia, hepatitis B &C, HIV and syphilis. The duration of this study was six months. The inform consent was taken from each donor. The total blood 250 ml from vein in cubital fossa from each blood donor was collected in 250ml pediatric blood bag with CPDA-1 solution. Blood bags were put up in the blood bank at +2 to +6 °C and stored till 20 days. Blood specimen of about 5cc were collected in 5cc syringe from each blood bag on 0, 5th,10th ,15th and 20th day for following parameters and thin film red blood cell was prepared for examination by light microscope. Morphological changes in RBCs examined via light microscope as well as grading the RBCs status in the peripheral blood film, the occurrence of distorted RBC simply in random fields; such as +1(scored 1 to 5 altered RBC present in each field), +2 (an average of 6 to 15 altered RBC in each field), +3(16 to 25 altered RBC in each field) and +4(more than 25 altered RBC present in each field). The multi head light microscope NIKON eclipse 50 was used for examination of peripheral blood slide and we took images of randomly selected field. The image J software was used for slide examination.Results: The morphological analysis of red blood cells, count of 200 cells in each blood slide in randomly selected fields are: On day 0 the majority of cells were normally shaped (97.95±1.297 (mean±SD).With increasing storage time, the percentage of morphologically abnormal red cells rose sharply. Mean percentage of abnormal cells on day 5, 10, 15 and 20 was 28.80±10.00, 51.73±12.47, 64.78±14.66 and 68.10±7.92 respectively. This increase in percentage of abnormally shaped cells was significant as determined by one way ANOVA (p =0.001). There was a big difference of percentage of abnormal RBCs on day 0 and in = 5 to= 10 days and in = 15 to = 20 days of blood storage. The mean values of day 0 of abnormal cells was 2.05±1.297 (Mean ± Std. Deviation), abnormal cells in= 5 to= 10 days was 40.26± 16.101 (Mean ± Std. Deviation) and on day = 15 and in = 20 day was 66.44± 11.75. The mean difference from day 0 to day 20 was 63.93±10.45 (Mean ± Std. Deviation).The one way ANOVA was significant, P= 0.001.Conclusion: This study confirms the hematological and morphological changes, when blood stored at 2 °C to 6 °C for up to 21 days. The significant morphological changes were observed on 5th day of blood storage. These findings suggested that approximately a week old stored blood is as good as the fresh blood; however, significant morphological and biochemical changes begin to appear after the first week of storage and these changes aggravate with time. Hence in order to achieve best possible transfusion outcomes, stored blood up to one week can be utilized.

Cold storage of platelets in platelet additive solution maintains mitochondrial integrity by limiting initiation of apoptosis-mediated pathways.

Cold storage of platelets in plasma maintains hemostatic function and is an attractive alternative to room temperature platelets (RTPs). We have recently shown that functional differences between cold-stored platelets (CSPs) and RTPs after 5-day storage are associated with mitochondrial respiration and that CSPs in platelet (PLT) additive solution (PAS) can maintain hemostatic function for at least 15 days. This study tested the hypothesis that cold storage in PAS preserves mitochondrial integrity by reducing PLT apoptosis. CSPs and RTPs in plasma or PAS were stored and assayed for up to 15 days for mitochondrial function and integrity, mitochondrial-associated mRNA transcript expression, apoptotic proteins, and apoptotic flow cytometry metrics. CSP preserved mitochondria-associated mRNA comparable to baseline levels, improved mitochondrial respiration, and minimized depolarization to Day 15. Additionally, CSPs had minimal induction of caspases, preservation of plasma membrane integrity, and low expression of pro-apoptotic Bax. Storage in PAS appeared to be protective for RTPs in some parameters and enhanced the effects of CSPs. Mitochondrial function and molecular analyses defined CSP priming as distinctly different from the well-documented RTP storage lesion. While current blood bank storage at room temperature is limited to 5 to 7 days, refrigeration and storage in PAS for up to 15 days may represent an opportunity to enhance inventories and access to PLT hemostatic support for bleeding patients.

Time dependent changes in red blood cells during storage in the local blood banks of Khyber Pakhtunkhwa.

This project aimed at determining time-dependent ultrastructural and haematological changes taking place in blood stored in local blood banks of Khyber Pakhtunkhwa. It was a longitudinal study with repeated measures design. Twenty healthy blood donors participated in this study. An amount of 250ml blood was collected from each donor and stored in Citrate Phosphate Dextrose Adenine-1 (CPDA-1)- containing blood bags. Within first four hours, baseline samples were taken while subsequent samples were obtained at 5 days interval till day 20th. Structural changes in RBCs were observed under light and scanning electron microscope (SEM) at different intervals. Furthermore, haematological parameters and osmotic fragility were also determined. Remarkable alterations were seen in RBCs morphology. From 5th day onwards, multiple visible spicules were observed on the RBC's outer membrane and more than 2/3rd cells were abnormal at day 20. There was a significant reduction in RBCs count and haemoglobin concentration while the remaining parameters remained unchanged. Osmotic fragility increased significantly over time, with <1% haemolysis noted in baseline samples as compared to 2.4% haemolysis on day 20th (p≤0.0001). Prolonged storage of blood results in distorted RBCs morphology and increased fragility. Transfusion of such cells would potentially result in rapid lysis in patients with hepatosplenomegaly and conditions requiring multiple blood transfusions.

Computational Identification and Characterization of New microRNAs in Human Platelets Stored in a Blood Bank.

Platelet concentrate (PC) transfusions are widely used to save the lives of patients who experience acute blood loss. MicroRNAs (miRNAs) comprise a class of molecules with a biological role which is relevant to the understanding of storage lesions in blood banks. We used a new approach to identify miRNAs in normal human platelet sRNA-Seq data from the GSE61856 repository. We identified a comprehensive miRNA expression profile, where we detected 20 of these transcripts potentially expressed in PCs stored for seven days, which had their expression levels analyzed with simulations of computational biology. Our results identified a new collection of miRNAs (miR-486-5p, miR-92a-3p, miR-103a-3p, miR-151a-3p, miR-181a-5p, and miR-221-3p) that showed a sensitivity expression pattern due to biological platelet changes during storage, confirmed by additional quantitative real-time polymerase chain reaction (qPCR) validation on 100 PC units from 500 healthy donors. We also identified that these miRNAs could transfer regulatory information on platelets, such as members of the let-7 family, by regulating the YOD1 gene, which is a deubiquitinating enzyme highly expressed in platelet hyperactivity. Our results also showed that the target genes of these miRNAs play important roles in signaling pathways, cell cycle, stress response, platelet activation and cancer. In summary, the miRNAs described in this study, have a promising application in transfusion medicine as potential biomarkers to also measure the quality and viability of the PC during storage in blood banks.

The impact of circulation in a heart-lung machine on function and survival characteristics of red blood cells.

Extracorporeal circulation is accompanied by changes in red blood cell morphology and structural integrity that affect cell function and survival, and thereby may contribute to the various side effects of heart–lung machine‐assisted surgery. Our main objectives were to determine the effect of circulation of red blood cells in a stand‐alone extracorporeal circuit on several parameters that are known to be affected by, as well as contribute to red blood cell aging. As a source of RBCs, we employed blood bank storage units of different ages. In order to assess the relevance of our in vitro observations for the characterization of extracorporal circulation technology, we compared these changes in those of patients undergoing extracorporeal circulation‐assisted cardiac surgery. Our results show that circulation in a heart–lung machine is accompanied by changes in red blood cell volume, an increase in osmotic fragility, changes in deformability and aggregation behavior, and alterations in the exposure of phosphatidylserine and in microvesicle generation. RBCs from 1‐week‐old concentrates showed the highest similarities with the in vivo situation. These changes in key characteristics of the red blood cell aging process likely increase the susceptibility of red blood cells to the various mechanical, osmotic, and immunological stress conditions encountered during and after surgery in the patient’s circulation, and thereby contribute to the side effects of surgery. Thus, aging‐related parameters in red blood cell structure and function provide a foundation for the validation and improvement of extracorporeal circulation technology.

Vesiculation of Red Blood Cells in the Blood Bank: A Multi-Omics Approach towards Identification of Causes and Consequences.

Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown. In this study, we combined morphological, immunochemical, proteomic, lipidomic, and metabolomic analyses to obtain an integrated understanding of the mechanisms underlying microvesicle generation during the storage of red blood cell concentrates. Our data indicate that changes in membrane organization, triggered by altered protein conformation, constitute the main mechanism of vesiculation, and precede changes in lipid organization. The resulting selective accumulation of membrane components in microvesicles is accompanied by the recruitment of plasma proteins involved in inflammation and coagulation. Our data may serve as a basis for further dissection of the fundamental mechanisms of red blood cell aging and vesiculation, for identifying the cause-effect relationship between blood bank storage and transfusion complications, and for assessing the role of microvesicles in pathologies affecting red blood cells.

Linking Stored Red Blood Cell Metabolism to Transfusion Recipient Iron Homeostasis Pathophysiology in Critically-Ill Children

Background: Transfusion of packed red blood cells (pRBCs) is a common, life-saving therapy for many critically-ill children. However, routine blood bank storage results in the progressive accumulation of a series of RBC modifications, termed the "storage lesion," that reduce RBC recovery in vivo, may affect transfusion recipient physiology, and may be implicated in transfusion-related adverse events. In particular, circulating non-transferrin-bound iron (NTBI) in recipients can be increased after pRBC transfusion. NTBI promotes bacterial growth, induces oxidative injury, and inversely correlates with post-transfusion recovery, all of which may increase the risk of sepsis, multiorgan failure, and death. We aimed to determine whether the metabolic state of donor pRBCs at the time of transfusion correlates with recipient NTBI levels, which serves as a marker of hemolysis and iron homeostasis in critically-ill pediatric patients. Study Design/Methods: Critically-ill pediatric patients, aged &lt; 21-years-old with a minimum weight of 5kg in the pediatric intensive care unit at a single-site tertiary care hospital, who required pRBC transfusion, were prospectively enrolled after parental consent (n = 49 patients). Patient blood samples were obtained pre-transfusion and 4 hours following the patient's first qualifying pRBC transfusion, and analyzed for markers of iron and heme homeostasis, hemolysis, liver function, and inflammation utilizing standard laboratory methods. pRBC transfusate (1 mL) was frozen at -80°C immediately at the end of transfusion for each transfused patient. Hydrophilic and lipophilic compounds were extracted separately using methanol:acetonitrile:water or methanol, respectively, before analysis using high-throughput LC-MS based metabolomics and lipidomics platforms to determine the relative abundance of small molecule compounds. Metabolite levels in the pRBC transfusates were correlated with the differences in pre- and post-transfusion clinical laboratory values and selected patient parameters (e.g. blood type, patient age, and storage age of the pRBC unit at the time of transfusion), using Pearson correlation coefficients and associated p-values. Results: The relative levels of 221 unique metabolites and lipids in the pRBC transfusates were correlated with the corresponding transfusion recipient's clinical laboratory values and selected patient parameters. Analysis of Pearson's correlations between the levels of these compounds in the donor pRBC transfusates and the pre- and post-transfusion differences in the recipients' NTBI levels (ΔNTBI) demonstrated statistically significant correlations for 24 metabolites (R = 0.28-0.43, p = 0.0022-0.0476, see Table). Analysis of recipient clinical laboratory biomarkers associated with recipient ΔNTBI revealed a positive correlation with increases in serum iron (R = 0.41, p = 0.0034) but not with other changes in recipient clinical laboratory biomarkers. The age of the stored pRBCs at the time of transfusion did not correlate with ΔNTBI levels (R = 0.24, p = 0.0912). Conclusions: Statistically significant correlations of donor pRBC transfusate metabolites with transfusion recipient ΔNTBI are primarily seen with hypoxanthine, lactate, metabolites of glutathione homeostasis, amino acids, unsaturated free fatty acids, acyl-carnitines, and metabolites of indole and tryptophan metabolism. These data suggest that higher levels of oxidative stress, proteolysis, membrane remodeling in stored donor pRBCs, and, possibly, microbiome metabolism in the pRBC donor, are associated with significant increases in recipient plasma NTBI and serum iron levels, likely from hemolysis of storage-damaged RBCs. The levels of these metabolic biomarkers of the RBC storage lesion correlate better with ΔNTBI than the storage age of the donor unit, supporting the notion that pRBC metabolic age is not equivalent to pRBC chronologic age, and that the former may be more relevant to transfusion recipient pathophysiology in critically-ill children. Table. Disclosures Nemkov: Omix Technologies, Inc.: Equity Ownership, Other: Founder. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder.

Storage Primes Erythrocytes for Necroptosis and Clearance.

Like nucleated cells, erythrocytes (red blood cells, RBCs) are capable of executing programmed cell death pathways. RBCs undergo necroptosis in response to CD59-specific pore-forming toxins (PFTs). The relationship between blood bank storage and RBC necroptosis was explored in this study. Human RBCs were stored in standard blood bank additive solutions (AS-1, AS-3, or AS-5) for 1 week and hemolysis was evaluated in the context of necroptosis inhibitors and reactive oxygen species (ROS) scavengers. Activation of key factors including RIP1, RIP3, and MLKL was determined using immunoprecipitations and western blot. RBC vesiculation and formation of echinocytes was determined using phase-contrast microscopy. The effect of necroptosis and storage on RBC clearance was determined using a murine transfusion model. Necroptosis is associated with increased RBC clearance post-transfusion. Moreover, storage in AS-1, AS-3, or AS-5 sensitizes RBCs for necroptosis. Importantly, storage-sensitized RBCs undergo necroptosis in response to multiple PFTs, regardless of specificity for CD59. Storage-sensitized RBCs undergo necroptosis via NADPH oxidase-generated ROS. RBC storage led to RIP1 phosphorylation and necrosome formation in an NADPH oxidase-dependent manner suggesting the basis for this sensitization. In addition, storage led to increased RBC clearance post-transfusion. Clearance of these RBCs was due to Syk-dependent echinocyte formation. Storage-induced sensitization to RBC necroptosis and clearance is important as it may be relevant to hemolytic transfusion reactions.

Increased Methylation of Deamidated Asparagines and Aspartates in Stored Red Blood Cells from Glucose 6-Phosphate Dehydrogenase-Deficient Blood Donors

Blood transfusion is a life-saving intervention for millions of recipients worldwide every year. However, refrigerated storage of red blood cells (RBCs) for up to 42 days promotes impairments in energy and redox homeostasis, which impact RBC hemolytic propensity and post-transfusion performance of the storage-damaged RBC. Since mature RBCs are devoid of de novo protein synthesis - owing to the lack of organelles and nuclei - they evolved metabolic mechanisms to cope with oxidative stress. Two of these involve (i) activation of the pentose phosphate pathway (PPP), which generates reducing equivalents (NADPH) to preserve glutathione homeostasis and recharge NADPH-dependent antioxidant enzymes; and (ii) recycling of oxidatively damaged proteins via methylation of dehydrated and deamidated aspartate and asparagine residues, a process that consumes methionine as the main methyl group donor. Thus, we hypothesize that the latter mechanism is relevant to routine blood bank storage, especially in glucose-6-phosphate dehydrogenase (G6PD)-deficient donors. In this routinely accepted donor population (~10% of donors of African descent), mutations of G6PD, the rate-limiting enzyme of the PPP, result in instability and decreased enzymatic activity (e.g., <1% residual activity in the Mediterranean variant).Within the framework of the REDS-III RBC-Omics study, packed RBC units (n=599) were stored under blood bank conditions for 10, 23 and 42 days and profiled for oxidative hemolysis (following AAPH insult) and time-dependent metabolic dysregulation of the trans-sulfuration pathway. The rate of methionine consumption positively correlated with storage age and oxidative hemolysis. In a separate cohort, RBCs from G6PD-deficient and G6PD-normal donors were also collected and metabolically profiled during storage. The G6PD-deficient donors had higher pre-storage methionine levels as compared against G6PD-sufficient donors, although one week of storage was sufficient to significantly flip this ratio (p<0.01). To ensure that the phenomenon was driven by storage-induced oxidative insults, RBCs were also stored under normoxic, hyperoxic (SO2 >95%), or hypoxic conditions (SO2 = 20%, 10%, 5% of < 3%) for up to 42 days. Of note, hypoxia decreased methionine consumption rates and increased S-adenosylmethionine/S-adenosylhomocysteine ratios in comparison to normoxic control RBCs, while opposite trends were observed in response to oxidative stress. In parallel, RBCs were incubated under normoxic or hypoxic conditions for 16h, in presence or absence of a generic methyltransferase inhibitor, adenosine dialdehyde, or hydrogen peroxide (0.1 or 0.5%). 13C,15N-Methionine uptake and consumption rates were faster in response to pro-oxidant insults and were prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and the formation of methyl-Asp on key structural proteins and enzymes, including band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of GAPDH) and/or residues interacting with the N-terminal cytosolic domain of band 3. Hypoxic incubation with hydrogen peroxide phenocopied normoxic conditions in the presence of pro-oxidant insults, suggesting that the beneficial role of hypoxia in preventing methionine consumption and protein methylation is attributable to decreased oxidative stress. G6PD-deficient donor RBCs had significantly (p<0.0001) higher levels of methylated-deamidated Asn residues in hemoglobin, band 3, and GAPDH, as compared to stored G6PD-normal donor RBCs.Although methylation of basic amino acid residues is an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated Asn residues is a non-epigenetic post-translational sensor of oxidative stress and refrigerated storage in anucleate human RBCs. This damage repair mechanism is exacerbated in RBCs from G6PD-deficient donors, as a compensatory mechanism for the lack of normal PPP function. DisclosuresD'Alessandro:Hemanext inc: Membership on an entity's Board of Directors or advisory committees; Omix Technologies inc: Equity Ownership. Nemkov:Omix Technologies inc: Equity Ownership. Yoshida:Hemanext inc: Employment. Dunham:Hemanext inc: Employment. DuMont:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Hansen:Omix Technologies inc: Equity Ownership. Spitalnik:Hemanext inc: Membership on an entity's Board of Directors or advisory committees. Zimring:Rubius Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells

Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank. Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway. In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3. While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.