Red Blood Cell Storage Research Articles

Preservation of red blood cell antigenicity in a new storage solution in vitro

Introduction Red blood cell (RBC) storage solution is used for suspending and preserving RBCs for later use in in vitro immunohematology testing. Proper RBC preservation is crucial for obtaining accurate results in RBC phenotyping and pretransfusion antibody screening tests. Haemolysis or RBC antigen degradation during storage can result in inaccurate RBC phenotyping, thereby decreasing the sensitivity of pretransfusion antibody screening and identification assays. The conventional RBC storage solutions usually contain adenosine, adenine, and antibiotics. We designed an RBC storage solution and determined whether it could preserve RBC integrity for 70 days. Materials and Methods The new storage solution has a different formula from that of the conventional solution—in particular, it is strengthened with polyethylene glycol (PEG). The extent of haemolysis and hemagglutination reactivity of the RBC antigen systems, Rh, Duffy, Kidd, Lewis, MNS, P1, and the rare antigen Mia (which has a low prevalence antigen in most parts of the world but a higher prevalence in Taiwan), in the new RBC storage solution was compared with that of the conventionally preserved RBC storage solution. Results The RBCs preserved in the new solution for 70 days retained a similar haemolysis grade as those preserved in the control solution for 28 days. Although both solutions largely preserved RBC antigenicity, the decline in RBC hemagglutination scores in new solution often occurred later than that in the control solution in most antigen phenotyping assays, especially labile antigens such as D, P1, and M. Conclusion The new solution reduces haemolysis more effectively and preserves antigenicity throughout the 70-day storage period. Moreover, Mia antigen is more stable in the experimental group.

Developing an optimized preservation solution for the supercooled storage of red blood cells: A comparison of two extracellular additives

Developing an optimized preservation solution for the supercooled storage of red blood cells: A comparison of two extracellular additives

Intermittent scavenging of storage lesion from stored red blood cells by electrospun nanofibrous sheets enhances their quality and shelf-life

Transfusion of healthy red blood cells (RBCs) is a lifesaving process. However, upon storing RBCs, a wide range of damage-associate molecular patterns (DAMPs), such as cell-free DNA, nucleosomes, free-hemoglobin, and poly-unsaturated-fatty-acids are generated. DAMPs can further damage RBCs; thus, the quality of stored RBCs declines during the storage and limits their shelf-life. Since these DAMPs consist of either positive or negative charged species, we developed taurine and acridine containing electrospun-nanofibrous-sheets (Tau-AcrNFS), featuring anionic, cationic charges and an DNA intercalating group on their surfaces. We show that Tau-AcrNFS are efficient in scavenging DAMPs from stored human and mice RBCs ex vivo. We find that intermittent scavenging of DAMPs by Tau-AcrNFS during the storage reduces the loss of RBC membrane integrity and reduces discocytes-to-spheroechinocytes transformation in stored-old-RBCs. We perform RBC-transfusion studies in mice to reveal that intermittent removal of DAMPs enhances the quality of stored-old-RBCs equivalent to freshly collected RBCs, and increases their shelf-life by ~22%. Such prophylactic technology may lead to the development of novel blood bags or medical device, and may therefore impact healthcare by reducing transfusion-related adverse effects.

Storage of packed red blood cells impairs an inherent coagulation property of erythrocytes.

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.

Premature Macrophage Activation by Stored Red Blood Cell Transfusion Halts Liver Regeneration Post-Partial Hepatectomy in Rats.

Liver resection is a common treatment for various conditions and often requires blood transfusions to compensate for operative blood loss. As partial hepatectomy (PHx) is frequently performed in patients with a pre-damaged liver, avoiding further injury is of paramount clinical importance. Our aim was to study the impact of red blood cell (RBC) resuscitation on liver regeneration. We assessed the impact of RBC storage time on liver regeneration following 50% PHx in rats and explored possible contributing molecular mechanisms using immunohistochemistry, RNA-Seq, and macrophage depletion. The liver was successfully regenerated after PHx when rats were transfused with fresh RBCs (F-RBCs). However, in rats resuscitated with stored RBCs (S-RBCs), the regeneration process was disrupted, as detected by delayed hepatocyte proliferation and lack of hypertrophy. The delayed regeneration was associated with elevated numbers of hemorrhage-activated liver macrophages (Mhem) secreting HO-1. Depletion of macrophages prior to PHx and transfusion improved the regeneration process. Gene expression profiling revealed alterations in numerous genes belonging to critical pathways, including cell cycle and DNA replication, and genes associated with immune cell activation, such as chemokine signaling and platelet activation and adhesion. Our results implicate activated macrophages in delayed liver regeneration following S-RBC transfusion via HO-1 and PAI-1 overexpression.

Immunomodulatory Properties of Packed Red Blood Cells during Storage

Introduction: Red blood cell (RBC) transfusion may affect the recipient immune system. During RBC storage in an unphysiological environment, RBC quality and function are impaired, the cells bleb extracellular vesicles (EVs), and other bioactive substances accumulate in the storage medium. EVs can carry reactive biomolecules and mediate cell-cell interactions. Thus, EVs could explain RBC transfusion related immunomodulation, particularly after prolonged storage. Methods: We exposed peripheral blood mononuclear cells (PBMCs) to allogeneic RBC supernatant (SN) and EVs from fresh and longer-stored RBC units, diluted plasma, and storage solution SAGM, and studied activation and proliferation of T-cells by flow cytometry, and cytokine secretion of LPS-stimulated PBMCs by enzyme-linked immunosorbent assay (ELISA). Results: Both fresh and longer-stored RBC SN but not EVs induced immunomodulation in recipient cells. RBC SN and diluted plasma augmented the proliferation of particularly CD8⁺ T-cells in a 4-day proliferation assay. T-cell activation by SN was evident already after 5 h as shown by upregulation of CD69. SN suppressed monocyte TNF-α and increased IL-10 secretion while diluted plasma increased secretion of both cytokines. Conclusion: This in vitro study demonstrates that stored RBC SN will have mixed immunomodulatory effects depending on responder cells and conditions, independent of RBC storage age. Fresh RBCs containing relatively few EVs can induce immune responses. Residual plasma in the products may contribute to these effects.

Hypoxic storage of murine red blood cells improves energy metabolism and post-transfusion recoveries.

The Red blood cell (RBC) storage lesion results in decreased circulation and function of transfused RBCs. Elevated oxidant stress and impaired energy metabolism are a hallmark of the storage lesion in both human and murine RBCs. Although human studies don't suffer concerns that findings may not translate, they do suffer from genetic and environmental variability amongst subjects. Murine models can control for genetics, environment, and much interventional experimentation can be carried out in mice that is neither technically feasible nor ethical in humans. However, murine models are only useful to the extent that they have similar biology to humans. Hypoxic storage has been shown to mitigate the storage lesion in human RBCs, but has not been investigated in mice. RBCs from a C57BL6/J mouse strain were stored under normoxic (untreated) or hypoxic conditions (SO2 ~ 26%) for 1h, 7 and 12 days. Samples were tested for metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics and end of storage post transfusion recovery. Hypoxic storage improved post-transfusion recovery and energy metabolism, including increased steady state and 13C3-labeled metabolites from glycolysis, high energy purines (adenosine triphosphate) and 2,3-diphospholgycerate. Hypoxic storage promoted glutaminolysis, increased glutathione pools, and was accompanied by elevation in the levels of free fatty acids and acyl-carnitines. This study isolates hypoxia, as a single independent variable, and shows similar effects as seen in human studies. These findings also demonstrate the translatability of murine models for hypoxic RBC storage and provide a pre-clinical platform for ongoing study.

A deep 96-well plate RBC storage platform for high-throughput screening of novel storage solutions.

Background: Red blood cell (RBC) storage solutions, also known as additive solutions (ASs), first developed in the 1970s, enable extended storage of RBCs. Unfortunately, the advancements in this field have been limited, due to labor intensive and time-consuming serial in vitro and in vivo testing, coupled with very high commercialization hurdles. This study examines the utility of deep 96-well plates for preliminary screenings of novel ASs through comparison of RBC storage with the standard PVC bags in terms of hemolysis and ATP levels, under both normoxic (N) and hypoxic/hypocapnic (H) storage conditions. The necessity for the presence of DEHP, normally provided by PVC bags, is also examined. Materials and methods: A pool of 2 ABO compatible RBC units was split between a bag and a plate. Each plate well contained either 1, 2 or 0 PVC strips cut from standard storage bags to supply DEHP. The H bags and plates were processed in an anaerobic glovebox and stored in O2 barrier bags. Hemolysis and ATP were measured bi-weekly using standard methods. Results: Final ATP and hemolysis values for the plate-stored RBCs were comparable to the typical values observed for 6-week storage of leukoreduced AS-3 RBCs in PVC bags under both N and H conditions. Hemolysis was below FDA and EU benchmarks of 1% and 0.8%, respectively, and excluding DEHP from plates during storage, resulted in an inconsequential increase when compared to bag samples. Discussion: In combination with high-throughput metabolomics workflow, this platform provides a highly efficient preliminary screening platform to accelerate the initial testing and consequent development of novel RBC ASs.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant “enhancers” are used.

Storage with ethanol attenuates the red blood cell storage lesion

BackgroundCurrent management of hemorrhagic shock relies on control of surgical bleeding along with resuscitation with packed red blood cells and plasma in a 1-to-1 ratio. Transfusion, however, is not without consequence as red blood cells develop a series of biochemical and physical changes during storage termed “the red blood cell storage lesion.” Previous data has suggested that ethanol may stabilize the red blood cell membrane, resulting in improved deformability. We hypothesized that storage of packed red blood cells with ethanol would alter the red blood cell storage lesion. MethodsMice underwent donation and storage of red blood cells with standard storage conditions in AS-3 alone or ethanol at concentrations of 0.07%, 0.14%, and 0.28%. The red blood cell storage lesion parameters of microvesicles, Band-3, free hemoglobin, annexin V, and erythrocyte osmotic fragility were measured and compared. In additional experiments, the mice underwent hemorrhage and resuscitation with stored packed red blood cells to further evaluate the in vivo inflammatory impact. ResultsRed blood cells stored with ethanol demonstrated decreased microvesicle accumulation and Band-3 levels. There were no differences in phosphatidylserine or cell-free hemoglobin levels. After hemorrhage and resuscitation with packed red blood cells stored with 0.07% ethanol, mice demonstrated decreased serum levels of interleukin-6, macrophage inflammatory protein-1α, keratinocyte chemokine, and tumor necrosis factor α compared to those mice receiving packed red blood cells stored with additive solution-3. ConclusionStorage of murine red blood cells with low-dose ethanol results in decreased red blood cell storage lesion severity. Resuscitation with packed red blood cells stored with 0.07% ethanol also resulted in a decreased systemic inflammatory response in a murine model of hemorrhage.

Determination of free heme in stored red blood cells with an apo-horseradish peroxidase-based assay.

Transfusion effectiveness of red blood cells (RBCs)has been associated with duration of the storage period. Storage-dependent RBC alterations lead to hemolysis and release of toxic free heme, but the increase of free hemelevels over time is largely unknown. In the current study, an apo-horseradish peroxidase (apoHRP)-based assay was applied to measure levels of free heme atregular intervals or periodically in supernatants of RBCs until a maximum storage period of 42days. Free heme levelsincreased with linear time-dependent kinetics up to day 21 and accelerated disproportionally after day 28 until day 42, as determined with the apoHRP assay. Individual timecourses of free heme in different RBC units exhibited high variability. Notably, levels of free hemoglobin, an established indicator of RBC damage, and those of total hemeincreased with continuous time-dependent linear kinetics over the entire 42day storage period, respectively. Supernatants from RBC units with high levels of free heme ledto inflammatory activation of human neutrophils. In conclusion, determining free heme in stored RBCs with the applied apoHRP assay may become feasible for testing ofRBC storage quality in clinical transfusion medicine.

Alloimmunization in dogs after transfusion: A serial cross-match study.

BackgroundCross‐matching is performed to determine the serological compatibility of donor and recipient blood. Current guidelines recommend that cross‐matching should be performed in dogs when an initial transfusion was performed more than 4 days ago or when the transfusion history is unknown.HypothesisDetermination at what time point alloantibodies are detected in dogs after transfusion. The hypothesis was that dogs would form alloantibodies within 4 days after a transfusion.AnimalsTwenty‐one anemic dogs were transfused and monitored for at least 4 subsequent days. Exclusion criteria were persistent red blood cell (RBC) agglutination and a previous transfusion.MethodsProspective observational study. Cross‐matching was performed before the initial DEA 1‐compatible transfusion and on days 1, 2, 3, and 4 and if possible, between day 5 and 28, using the tube method without enhancement (major cross‐match, recipient controls); recipients were monitored for transfusion reactions.ResultsIn 12/21 dogs a positive cross‐match (microscopic degree of agglutination [AD] 1+ to 2+) was observed within 4 days after the transfusion. In a nonlinear regression model, no significant association was detected between type of anemia (P = .41), RBC storage time (P = .44), immunosuppressive treatment (P = .75) nor transfusion volume (P = .70) and the occurrence of positive cross‐matches within 4 days after transfusion. Another 4 dogs developed a positive cross‐match (microscopic AD 1+ to 2+) after 6 to 13 days.Conclusions and Clinical ImportanceBecause production of alloantibodies was detected as early as 1 day after transfusion, cross‐matching should be performed before every subsequent transfusion.

Evaluation of selected hematological, biochemical and oxidative stress parameters in stored canine CPDA-1 whole blood

Blood transfusions are mainly given to intensive care patients; therefore, additional complications that could arise from storage lesions in preserved blood should be avoided. It has been shown that human stored red blood cells are subject to changes that are considered to be a number of interdependent processes involving metabolic disarrangement and oxidative stress. The aim of our study was to determine alterations in selected hematological and biochemical parameters and to assess whether and when oxidative stress is a significant phenomenon in stored dog CPDA-1 whole blood. Ten ½ unit bags of whole blood donated from dogs and preserved with CPDA-1 (anticoagulant containing citrate, phosphate, dextrose and adenine) were stored for 5 weeks. Each week, a 9 ml sample was drawn aseptically to measure hematological parameters, selected metabolites, free hemoglobin content, osmotic fragility, antioxidant enzyme activity, total antioxidant capacity, malondialdehyde concentration and protein carbonyl content.The results revealed an MCV decrease in the first week of storage and then a gradual increase; osmotic fragility decreased at that time and remained low throughout the study period. Leukodepletion became significant in the fourth week of storage. The free hemoglobin concentration continuously increased, with the greatest changes observed in the last two weeks of storage. The total antioxidant capacity changed in a reverse manner. Superoxide dismutase and glutathione peroxidase activities decreased from week 0 to week 3, and catalase activity tended to decrease over time. The highest malondialdehyde concentrations in blood supernatant were measured in the first week of storage, and the carbonyl concentration increased after 35 days.Hematological changes and oxidative stress are already present in the first week of storage, resulting in depletion of the antioxidant system and subsequent accumulation of oxidation products as well as erythrocyte hemolysis, which are most pronounced at the end of the storage period.

Metabolic reprogramming under hypoxic storage preserves faster oxygen unloading from stored red blood cells

AbstractStored red blood cells (RBCs) incur biochemical and morphological changes, collectively termed the storage lesion. Functionally, the storage lesion manifests as slower oxygen unloading from RBCs, which may compromise the efficacy of transfusions where the clinical imperative is to rapidly boost oxygen delivery to tissues. Recent analysis of large real-world data linked longer storage with increased recipient mortality. Biochemical rejuvenation with a formulation of adenosine, inosine, and pyruvate can restore gas-handling properties, but its implementation is impractical for most clinical scenarios. We tested whether storage under hypoxia, previously shown to slow biochemical degradation, also preserves gas-handling properties of RBCs. A microfluidic chamber, designed to rapidly switch between oxygenated and anoxic superfusates, was used for single-cell oxygen saturation imaging on samples stored for up to 49 days. Aliquots were also analyzed flow cytometrically for side-scatter (a proposed proxy of O2 unloading kinetics), metabolomics, lipidomics, and redox proteomics. For benchmarking, units were biochemically rejuvenated at 4 weeks of standard storage. Hypoxic storage hastened O2 unloading in units stored to 35 days, an effect that correlated with side-scatter but was not linked to posttranslational modifications of hemoglobin. Although hypoxic storage and rejuvenation produced distinct biochemical changes, a subset of metabolites including pyruvate, sedoheptulose 1-phosphate, and 2/3 phospho-d-glycerate, was a common signature that correlated with changes in O2 unloading. Correlations between gas handling and lipidomic changes were modest. Thus, hypoxic storage of RBCs preserves key metabolic pathways and O2 exchange properties, thereby improving the functional quality of blood products and potentially influencing transfusion outcomes.

Effect of Hematocrit Injury on the Survival Rate of Advanced Malignant Tumors and Its Clinical Significance.

Objective To investigate the effect of logistic multivariate analysis on the survival rate of advanced malignant tumors and to evaluate the effect of erythrocyte storage injury on the survival rate of advanced malignant tumors and its clinical significance. Methods A retrospective analysis was performed on 120 advanced cancer patients who received blood transfusion in Shaanxi Cancer Hospital from March 2018 to June 2019, and the risk factors for death were analyzed. A total of 72 advanced cancer patients admitted to hospital from March 2019 to June 2021 were included in the study. The patients with red blood cell transfusion storage time ≤ 14 d were the study group (n = 36), and the patients with red blood cell transfusion storage time > 14 d were the control group (n = 36). Compare the total efficiency of blood transfusion. The levels of Hb, erythrocyte count, hematocrit (HCT), blood oxygen saturation (SPO2), creatinine (Cr), erythrocyte deformability index, whole blood, erythrocyte, and hemoglobin before and after blood transfusion were compared, and the adverse reactions of blood transfusion were recorded. Results Dyspnea and delirium were significantly associated with patient survival time (P < 0.05). Red blood cell storage time ≤ 14 days, Lym% < 12%, lactate dehydrogenase (LDH) > 500 U/L, and ALB < 30 g/L were significantly correlated with survival time. Karnofsky performance status (KPS) < 30, delirium, LDH > 500 U/L, and albumin (ALB) < 30 g/L were independent influencing factors of survival (P < 0.05). The overall effective rate of the research group was higher (P < 0.05). The incidence of adverse reactions in the study group was lower (P < 0.05). The levels of Hb, red blood cell count, and HCT in the study group were higher (P < 0.05). Compared with the control group, the SPO2 level and the red blood cell deformability index were higher in the study group (P < 0.05). After blood transfusion, the level of (diphosphoglycerate) DPG in the study group was higher than that in the control group (P < 0.05). The length of hospital stay in the study group was significantly shortened (P < 0.05). The nosocomial infection rate and case fatality rate in the study group were significantly reduced (P < 0.05). Conclusion Red blood cell storage time ≤ 14 d, LYM% < 12%, LDH > 500 U/L, and ALB < 30 g/L are all significantly correlated with survival time. KPS < 30, delirium, LDH > 500 U/L, and ALB < 30 g/L were independent factors for survival (P < 0.05). Transfusion of red blood cells stored for ≤14 days in patients with advanced malignant tumors can significantly increase the effective infusion rate, improve anemia status, shorten hospital stay, and reduce mortality and risk of nosocomial infection and is worthy of clinical promotion.

Standardizing blood dose using body surface area and analyze the effect of blood storage on hemoglobin increment within pediatric patients

Introduction: Pediatric patients exhibit a wide variation in weight which results in diverse transfusion practices. This study aims to standardize red blood cell (RBC) doses according to body surface area (BSA) and to analyze the role of RBC storage in post-transfusion hemoglobin levels. Material and methods: In this original prospective cohort study on hospitalized pediatric patients aged up to 14, we classified patients into transfusion-dependent (n = 31) and non-transfusion-dependent (n = 158). The non-transfusiondependent group was further classified into ≤10 kg (n = 72) or &gt;10 kg (n = 86) according to body weight (bw). We derived a regression equation between BSA and blood dose in non-transfusion-dependent subjects, and modified the equation by fixing blood dose to 15 mL/kg bw for only BSA based blood dose. We measured pre-transfusion and post-transfusion hemoglobin (Hb) levels, and ascertained effects of blood storage ≤15 days (n = 15) and &gt;15 days (n = 16) on posttransfusion Hb. Results: Pediatric patients ≤10 kg and &gt;10 kg bw (n = 158); mean ± standard deviation of weight and BSA were 4.5 ± 3.1 kg; 22.9 ± 10.4 kg; 0.26 ± 0.14 m 2 ; 0.9 ± 0.28) m 2 respectively. The regression equation ≤10 kg and &gt;10 kg bw when adjusted. Blood dose fixed at 15 mL/kg bw adjusted blood dose ≤10 kg; 15 mL/kg bw = −19.12 + 329.69x BSA m 2 . The regression equation &gt;10 kg bw: adjusted blood dose &gt;10 kg bw; 15 mL/kg bw = −158.8 + 563.3x BSA (m 2 ). The adjusted blood dose with BSA did not exceed 20 mL/kg bw. No significant differences were observed in pre and post-transfusion Hb in transfusion-dependent (n = 31) versus non-transfusion-dependent patients (n = 158) due to the RBC storage duration. Conclusions: RBC blood dose can be standardized by regression equation between standardized RBC dosage and BSA. Post-transfusion Hb is not dependent on days of RBC storage at the blood bank.

Early and Late-Phase 24h Responses of Stored Red Blood Cells to Recipient-Mimicking Conditions.

The 24-hour (24 h) post-transfusion survival of donor red blood cells (RBCs) is an important marker of transfusion efficacy. Nonetheless, within that period, donated RBCs may encounter challenges able to evoke rapid stress-responses. The aim of the present study was to assess the effect of exposure to plasma and body temperature upon stored RBCs under recipient-mimicking conditions in vitro from the first hours “post-transfusion” up to 24 h. For this purpose, packed RBCs from seven leukoreduced CPD/SAGM units were reconstituted with plasma of twenty-seven healthy individuals and incubated for 24 h at 37oC. Three units were additionally used to examine stress-responses in 3-hour intervals post mixing with plasma (n = 5) until 24 h. All experiments were performed in shortly-, medium-, and long-stored RBCs. Hemolysis, redox, morphology, membrane protein binding and vesiculation parameters were assessed. Even though spontaneous hemolysis was minimal post-reconstitution, it presented a time-dependent increase. A similar time-course profile was evident for the concentration of procoagulant extracellular vesicles and the osmotic fragility (shortly-stored RBCs). On the contrary, mechanical fragility and reactive oxygen species accumulation were characterized by increases in medium-stored RBCs, evident even from the first hours in the recipient-mimicking environment. Finally, exposure to plasma resulted in rapid improvement of morphology, especially in medium-stored RBCs. Overall, some RBC properties vary significantly during the first 24 h post-mixing, at levels different from both the storage ones and the standard end-of-24 h. Such findings may be useful for understanding the performance of RBCs and their possible clinical effects −especially on susceptible recipients− during the first hours post-transfusion.

Effect of Anti-Oxidative of Ethyl Pyruvate and Taurine on the Red Blood Cell Storage at 4 ℃

To investigate the anti-oxidative effect of ethyl pyruvate (EP) and taurine (TAU) on the quality of red blood cells stored at 4±2 ℃, hemolysis, energy metabolism and lipid peroxidation of the red blood cells in the preservation solution were studied at different intervals. At 4±2 ℃, the deleukocyte red blood cells were stored in the citrate-phosphate-dextrosesaline-adenine-1 (CPDA-1) preservation (control group), preservation solution with EP (EP-AS), and TAU (TAU-AS) for long-term preservation. The enzyme-linked immunoassay and automatic blood cell analyzer were used to detect hemolysis and erythrocyte parameters. Adenine nucleoside triphosphate (ATP), glycerol 2,3-diphosphate (2,3-DPG) and malondialdehyde (MDA) kits were used to test the ATP, 2,3-DPG and MDA concentration. During the preservation, the rate of red blood cell hemolysis in EP-AS and TAU-AS groups were significantly lower than that in CPDA-1 group (P<0.01). The MCV of EP-AS group was increased with the preservation time (r=0.71), while the MCV of the TAU-AS group was significantly lower than that in the other two groups (P<0.05). The concentration of ATP and MDA in EP-AS and TAU-AS groups were significantly higher than that in CPDA-1 group at the 14th day (P<0.01). The concentrations of 2,3-DPG in the EP-AS and TAU-AS groups were significantly higher than that in the CPDA-1 group from the 7th day (P<0.01). EP and TAU can significantly reduce the red blood cell hemolysis rate, inhibit the lipid peroxidation level of red blood cells, and improve the energy metabolism of red blood cells during storage. The mechanism of EP and TAU may be related to their antioxidation and membrane protection effect, so as to improve the red blood cell quality and extend the preservation time.

Extracellular Vesicles From Stored Red Blood Cells Convey Heme and Induce Spic Expression on Human Monocytes.

During cold storage, red blood cells (RBC) undergo irreversible morphological changes, hemolysis, and extracellular vesicles (EVs) release. It is known that reinfused stored RBC triggers immune reactions within T-lymphocytes and increases pro-oxidative and immunomodulatory metabolites in the plasma of healthy individuals. Monocytes/macrophages are the primary cells in charge of both uptake of excess heme and recognition of such EVs; however, the effect of reinfusion of stored RBC has not been investigated upon monocytes. Hence, the purpose of this study was to characterize how EVs released by cold-stored RBC impact monocytes. For this, we adopted an ex vivo protocol to mimic autologous blood transfusion. Colorimetric and metabolomic analyses confirmed that cold-stored RBC released increased heme levels. Transcriptomic analysis demonstrated that cold-stored RBC increased the transcript levels of heme-oxygenase 1 and Spic in monocytes, along with an induction of heme-oxygenase-1, CX3CR1, and CD206- known markers of macrophages M2-phenotype. Finally, we have demonstrated that the EVs released by cold-stored RBC were enriched in heme and conveyed to monocytes, triggering Spic expression and an acute production of reactive oxygen species. Finally, our study demonstrates that hemolysis resulting from autologous blood transfusion modifies gene expression patterns in monocytes towards an M2-like phenotype. Furthermore, we highlight the role of EVs released by cold-stored RBC in this scenario, which may act like a damage-associated molecular pattern inducing Spic in monocytes.

NonFreezable Preservation of Human Red Blood Cells at -8 °C.

Red blood cell (RBC) preservation is very important in human health. The RBCs are usually preserved at 4 ± 2 °C without freezing or at a very low temperature (-80 °C or liquid nitrogen) with deep freezing. Herein, non freezable preservation of RBCs at a subzero temperature is reported to prolong the preservation time compared with that at 4 ± 2 °C. By adding glycerol and poly(ethylene glycol) (PEG) (average number molecular weight 400, PEG-400) into the preservation solution, the freezing point is decreased and the hemolysis is kept low. The cell metabolism of stored RBCs at -8 °C is reduced, and the shelf life of RBCs extends up to at least 70 days. At the end of preservation, the pH decreases a little bit to demonstrate the low metabolic rate of RBCs stored at subzero temperatures. After quick washing, the RBC survival rate is ca. 95%. The adenosine triphosphate, 2,3-diphosphoglycerate, and cell deformation ability of the washed RBCs are maintained at a high level, while the malondialdehyde is relatively low, which verifies the high quality of RBCs stored at this condition.