Image- and Fluorescence-Based Test Shows Oxidant-Dependent Damages in Red Blood Cells and Enables Screening of Potential Protective Molecules.

An increase of oxygen saturation within blood bags and metabolic dysregulation occur during storage of red blood cells (RBCs). It leads to the gradual exhaustion of RBC antioxidant protective system and, consequently, to a deleterious state of oxidative stress that plays a major role in the apparition of the so-called storage lesions. The present study describes the use of a test (called TSOX) based on fluorescence and label-free morphology readouts to simply and quickly evaluate the oxidant and antioxidant properties of various compounds in controlled conditions. Here, TSOX was applied to RBCs treated with four antioxidants (ascorbic acid, uric acid, trolox and resveratrol) and three oxidants (AAPH, diamide and H2O2) at different concentrations. Two complementary readouts were chosen: first, where ROS generation was quantified using DCFH-DA fluorescent probe, and second, based on digital holographic microscopy that measures morphology alterations. All oxidants produced an increase of fluorescence, whereas H2O2 did not visibly impact the RBC morphology. Significant protection was observed in three out of four of the added molecules. Of note, resveratrol induced diamond-shape "Tirocytes". The assay design was selected to be flexible, as well as compatible with high-throughput screening. In future experiments, the TSOX will serve to screen chemical libraries and probe molecules that could be added to the additive solution for RBCs storage.

Medical student education in transfusion medicine, part II: Moving forward to building up a "Know How" education program in transfusion medicine for under-graduate medical students.

There is a general trend in changing paradigm in teaching medicine; the emerging concept relies on a competence-based approach. Transfusion is either a discipline or a subsidiary of others depending on the countries and systems; this variability can be explained because transfusion is a medical care that is transdisciplinary. As a collective of professionals in both transfusion medicine practice and education, authors aim to propose a revision of the way education in transfusion medicine is delivered in this era of the 'global competency approach'. They advocate in favor of a Know How on 5 key issues: Diagnosing the patient condition in line with the Patient Blood Management principles; Facing acute blood loss; Addressing compatibility and avoiding immunization; Seeking for maximized benefits and dampening complications; and Inlaying competence within global health care issues, also comprising od economy. The methods used would be those developed for medical education at large, such as assessment tools. The global objective is to deliver the necessary competence to manage patients by an intern/resident. At the end of the curriculum, students should be able to self-evaluate the following items: 1) Do I know why my patient is anemic, thrombocytopenic, bleeding….? 2) Do I know the best approach to treat anemia, thrombocytopenia, bleeding (including the "no treatment" option)? 3) Do I know whether a transfusion approach is appropriate for my patients? 4) Do I know how to evaluate and anticipate benefits from blood transfusion and to avoid side-effects in the patient? 5) Do I know how to avoid unnecessary use of the products?

Detection of Stimulated Erythropoiesis by the RNA-Based 5'-Aminolevulinate Synthase 2 Biomarker in Dried Blood Spot Samples.

BACKGROUND: Despite implementation of the Athlete Biological Passport 10 years ago, blood doping remains difficult to detect. Thus, there is a need for new biomarkers to increase the sensitivity of the adaptive model. Transcriptomic biomarkers originating from immature reticulocytes may be reliable indicators of blood manipulations. Furthermore, the use of dried blood spots (DBSs) for antidoping purposes constitutes a complementary approach to venous blood collection. Here, we developed a method of quantifying the RNA-based 5'-aminolevulinate synthase 2 (ALAS2) biomarker in DBS. MATERIALS: The technical, interindividual, and intraindividual variabilities of the method, and the effects of storage conditions on the production levels of ALAS2 RNA were assessed. The method was used to monitor erythropoiesis stimulated endogenously (blood withdrawal) or exogenously (injection of recombinant human erythropoietin). RESULTS: When measured over a 7-week period, the intra- and interindividual variabilities of ALAS2 expression in DBS were 12.5%-42.4% and 49%, respectively. Following withdrawal of 1 unit of blood, the ALAS2 RNA in DBS increased significantly for up to 15 days. Variations in the expression level of this biomarker in DBS samples were more marked than those of the conventional hematological parameters, reticulocyte percentage and immature reticulocyte fraction. After exogenous stimulation of erythropoiesis via recombinant human erythropoietin injection, ALAS2 expression in DBS increased by a mean 8-fold. CONCLUSIONS: Monitoring of transcriptomic biomarkers in DBS could complement the measurement of hematological parameters in the Athlete Biological Passport and aid the detection of blood manipulations.

Generation of procoagulant collagen- and thrombin-activated platelets in platelet concentrates derived from buffy coat: the role of processing, pathogen inactivation, and storage.

BACKGROUND: Collagen- and thrombin-activated (COAT) platelets (PLTs), generated by dual-agonist stimulation with collagen and thrombin (THR), enhance THR generation at the site of vessel wall injury. There is evidence that higher amounts of procoagulant COAT PLTs are associated with stroke, while a decreased ability to generate them is associated with bleeding diathesis. Our aim was to study PLT functions, particularly the ability to generate COAT PLTs, in PLT concentrates (PCs) from buffy coat. Thus, we investigated the effect of processing, pathogen inactivation treatment (amotosalen-UVA), and PC storage. STUDY DESIGN AND METHODS: Two PCs from five donors each were pooled and split in two bags; one of them was pathogen inactivated and the other one was left untreated (n = 5). Flow cytometric analyses were performed immediately after PC preparation (Day 1) and thereafter on Days 2, 5, 7, and 9 in treated and untreated PCs to measure the reactivity of PLTs (CD62P and PAC-1), the content and secretion of dense granule after stimulation with different agonists, and the percentage of COAT PLTs after dual stimulation with convulxin (agonist of the collagen receptor GPVI) and THR. RESULTS: Preparation of PCs resulted in a significant decrease of COAT PLTs and in an impaired response to adenosine 5'-diphosphate sodium (ADP). Storage further decreased ADP response. Minor differences were observed between untreated or amotosalen-UVA-treated PCs. CONCLUSION: Preparation of PCs from buffy coats decreased the ability to generate COAT PLTs and impaired PLT response to ADP.

Medical student education in transfusion medicine: Proposal from the "European and Mediterranean initiative in transfusion medicine".

A large body of observations indicate that there is an inconsistent knowledge of Transfusion Medicine among health care professionals as well as inconsistent knowledge in all aspects of the transfusion process, from blood donation to transfusion on the ward. It is obvious to consider that appropriate education in Transfusion Medicine should be achieved in the education of specialists who will prescribe transfusion on a regular basis (hematologists, critical care specialists, anaethesiologists and others.) However,we also believe that education in Transfusion Medicine should also be delivered to almost all other medical specialists who may prescribe blood components. The variability in education of undergraduates in medical schools is universal most likely due to an absence of a predefined common platform. This paper, therefore, focuses on education at the undergraduate level and advocates coverage of the essential physiology and pathophysiology of blood as applied to blood transfusion as well as the medical and societal aspects of issues related to blood donation. It proposes incremental levels of training in Transfusion Medicine, with what is being therefore referred to as 'A', 'B', 'C' etc. curricula in ascending order of complexity; for example, 'A' and 'B' levels would involve medical, midwifery and nursing students, covering a broad base of the subject: they will be detailed in the present essay; ongoing further curricula will focus on physicians and other professionals working within the area or with responsibility for different aspects of the transfusion chain. It is intended that these courses include aspects of donor care, patient care and the appropriate use, safety and effectiveness of blood products. Next, it is advocated that curricula are addressed not only for high-income countries but also for middle- and low-income ones.

Low-Frequency Blood Group Antigens in Switzerland.

BACKGROUND: High-frequency blood group antigens (HFA) are present in >90% of the human population, according to some reports even in >99% of individuals. Therefore, patients lacking HFA may become challenging for transfusion support because compatible blood is hardly found, and if the patient carries alloantibodies, the cross-match will be positive with virtual every red cell unit tested. METHODS: In this study, we applied high-throughput blood group SNP genotyping on >37,000 Swiss blood donors, intending to identify homozygous carriers of low-frequency blood group antigens (LFA). RESULTS: 326 such individuals were identified and made available to transfusion specialists for future support of patients in need of rare blood products. CONCLUSION: Thorough comparison of minor allele frequencies using population genetics revealed heterogeneity of allele distributions among Swiss blood donors which may be explained by the topographical and cultural peculiarities of Switzerland. Moreover, geographically localized donor subpopulations are described which contain above-average numbers of individuals carrying rare blood group genotypes.

Redox Proteomics and Platelet Activation: Understanding the Redox Proteome to Improve Platelet Quality for Transfusion.

Blood banks use pathogen inactivation (PI) technologies to increase the safety of platelet concentrates (PCs). The characteristics of PI-treated PCs slightly differ from those of untreated PCs, but the underlying reasons are not well understood. One possible cause is the generation of oxidative stress during the PI process. This is of great interest since reactive oxygen species (ROS) act as second messengers in platelet functions. Furthermore, there are links between protein oxidation and phosphorylation, another mechanism that is critical for cell regulation. Current research efforts focus on understanding the underlying mechanisms and identifying new target proteins. Proteomics technologies represent powerful tools for investigating signaling pathways involving ROS and post-translational modifications such as phosphorylation, while quantitative techniques enable the comparison of the platelet resting state versus the stimulated state. In particular, redox cysteine is a key player in platelet activation upon stimulation by different agonists. This review highlights the experiments that have provided insights into the roles of ROS in platelet function and the implications for platelet transfusion, and potentially in diseases such as inflammation and platelet hyperactivity. The review also describes the implication of redox mechanism in platelet storage considerations.

Antioxidant power as a quality control marker for completeness of amotosalen and ultraviolet A photochemical treatments in platelet concentrates and plasma units.

BACKGROUND: Pathogen inactivation treatments such as INTERCEPT aim to make sure blood and blood-derived products are free of pathogens before using them for transfusion purposes. At present, there is no established quality control assay that assesses the completeness of the treatment. As INTERCEPT is a photochemical treatment known to generate reactive oxygen species we sought to use the antioxidant power (AOP) of the blood product as a marker of treatment execution. In this perspective, we evaluated an electrochemically based miniaturized system, the EDEL technology, for measuring the AOP in both platelet concentrates (PCs) and plasma. STUDY DESIGN AND METHODS: Aliquots were withdrawn from PCs or plasma units before and after INTERCEPT treatment and a few microliters were directly deposited into the EDEL sensor for the AOP measurement. The result is expressed in EDEL, an arbitrary unit (micromolar equivalent of ascorbic acid). RESULTS: The INTERCEPT treatment resulted in a significant decrease of the AOP. An AOP threshold of 66.5, 89.0, 59.8, and 131.5 EDEL was determined for apheresis PCs collected from female and male donors, buffy coat PCs, and plasma units, respectively. Below the threshold value, INTERCEPT treatment is considered to be executed. Additionally, we showed that the presence of the photosensitizer in combination with the ultraviolet A illumination is required to observe the AOP decrease. CONCLUSION: The measurement of the AOP of PCs and plasma units can be used to document the completeness of the INTERCEPT treatment.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT.

In vitro study of platelet function confirms the contribution of the ultraviolet B (UVB) radiation in the lesions observed in riboflavin/UVB-treated platelet concentrates.

BACKGROUND: Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ultraviolet (UV)A- and riboflavin/UVB-induced platelet (PLT) lesions in vitro. Particular attention is given to the effect of UVB alone on PLTs. STUDY DESIGN AND METHODS: Buffy coat-derived PLT concentrates (PCs) were treated with amotosalen/UVA, riboflavin/UVB, or UVB alone and compared to untreated PCs throughout storage. In vitro PLT function was assessed by blood gas and metabolite analyses, flow cytometry-based assays (CD62P, JC-1, annexin V, PAC-1), hypotonic shock response, and static adhesion to fibrinogen-coated wells. RESULTS: In our experimental conditions, riboflavin/UVB-treated PCs showed the most pronounced differences compared to untreated and amotosalen/UVA-treated PCs. The riboflavin/UVB treatment led to a significant increase of anaerobic glycolysis rate despite functional mitochondria, a significant increase of CD62P on Day 2, and a decrease of JC-1 aggregates and increase of annexin V on Day 7. The expression of active GPIIbIIIa (PAC-1) and the adhesion to fibrinogen was significantly increased from Day 2 of storage in riboflavin/UVB-treated PCs. Importantly, we showed that these lesions were caused by the UVB radiation alone, independently of the presence of riboflavin. CONCLUSION: The amotosalen/UVA-treated PCs confirmed previously published results with a slight increase of PSLs compared to untreated PCs. Riboflavin/UVB-treated PCs present significant in vitro PSLs compared to untreated PCs. These lesions are caused by the UVB radiation alone and probably involve the generation of reactive oxygen species. The impact of these observations on clinical use must be investigated.

In vitro assays and clinical trials in red blood cell aging: Lost in translation.

The age of erythrocyte concentrates (EC) in transfusion medicine and the adverse outcomes when transfusing long-term-stored EC are highly controversial issues. Whereas the definition of a short-term-stored EC or a long-term-stored EC is unclear in clinical trials, data based on in vitro storage assays can help defining a limit in addition of the expiration date. The present review merges together these data in order to highlight an EC age cut-off and points out potential misleading consideration. The analysis of in vitro data highlights the presence of reversible and irreversible storage lesions and demonstrates that red blood cells (RBC) exhibit two limits during storage: one around 2 weeks and another one around 4 weeks of storage. Of particular importance, the first lesions to appear, i.e. the reversible ones, are per se reversible once transfused, whereas the irreversible lesions are not. In clinical trials, the EC age cut-off for short-term storage is in general fewer than 14 days (11 ± 4 days) and more disperse for long-term-stored EC (17 ± 13 days), regardless the clinical outcomes. Taking together, EC age cut-off in clinical trials does not totally fall into line of in vitro aging data, whereas it is the key criteria in clinical studies. Long-term-stored EC considered in clinical trials are not probably old enough to answer the question: "Does transfusion of long-term-stored EC (older than 4 weeks) result in worse clinical outcomes?" Depending on ethical concerns and clinical practices, older EC than currently assayed in clinical trials should have to be considered. These two worlds trying to understand the aging of erythrocytes and the impact on patients do not seem to speak the same language.

Differences between calcium-stimulated and storage-induced erythrocyte-derived microvesicles.

Microvesicles (MVs), or microparticles, are a complex, dynamic and functional part of cells. Red blood cell (RBC)-derived MVs are naturally produced in vivo (during normal aging processes or in several diseases) as well as ex vivo during cold storage of RBCs, or in vitro by ATP depletion or treatment with Ca(2+) and calcium ionophore. All these MVs are equivalently classified according to their size and/or surface markers. Nevertheless, their content in proteins can differ and a few differences in terms of lipid raft proteins, notably stomatin and flotillin-2, have been reported. Based on two-dimensional gel electrophoreses, the present study highlights the differences between MVs induced during storage of RBCs (storage-MVs) and MVs stimulated by Ca(2+) entry (Ca-MVs). Upon treatment, Ca-MVs are formed following a clear recruitment of Ca(2+)-binding proteins (sorcin, grancalcin, PDCD6) and particularly annexins (4 and 5). Therefore, it emerges that different molecular pathways are available to produce similar MVs by disturbing the membrane/cytoskeleton interactions. Interestingly, these differences provide non-negligible pieces of information on the parent cells, and the mechanisms and modes of actions involved in the formation of MVs. In addition to biophysical characterization, protein analysis is important to classify these cellular corpuscles and evaluate their potential impacts in diseases or transfusion medicine.

Molecular RHD screening of RhD negative donors can replace standard serological testing for RhD negative donors.

This work aims to assess the value of a generalized molecular RHD screening strategy which could replace routine serological screening of weak D by indirect antiglobulin test. Three independent studies were performed at the two Blood Transfusion Services Berne and Zurich. Donors investigated were 652 RhD negative, but RhC and/or RhE positive, 17,391 mainly Rhccee, and 8200 with normal RhCcEe phenotype distribution. In study I single samples, in studies II and III minipools of 24 and 20 donor samples were tested, respectively. Among 26,243 phenotypically RhD negative blood donors, 65 carriers of RHD alleles were identified. Thirty-one of them were redefined as RhD positive.

Physiology of iron metabolism.

A revolution occurred during the last decade in the comprehension of the physiology as well as in the physiopathology of iron metabolism. The purpose of this review is to summarize the recent knowledge that has accumulated, allowing a better comprehension of the mechanisms implicated in iron homeostasis. Iron metabolism is very fine tuned. The free molecule is very toxic; therefore, complex regulatory mechanisms have been developed in mammalian to insure adequate intestinal absorption, transportation, utilization, and elimination. 'Ironomics' certainly will be the future of the understanding of genes as well as of the protein-protein interactions involved in iron metabolism.

Phosphoproteomics and morphology of stored human red blood cells treated by protein tyrosine phosphatases inhibitor.

ABSTRACT: The process of protein phosphorylation is involved in numerous cell functions. In particular, phosphotyrosine (pY) has been reported to play a role in red blood cell (RBC) functions, including the cytoskeleton organization. During their storage before transfusion, RBCs suffer from storage lesions that affect their energy metabolism and morphology. This study investigated the relationship between pY and the storage lesions. To do so, RBCs were treated (in the absence of calcium) with a protein tyrosine phosphatase inhibitor (orthovanadate [OV]) to stimulate phosphorylation and with 3 selective kinase inhibitors (KIs). Erythrocyte membrane proteins were studied by western blot analyses and phosphoproteomics (data are available via ProteomeXchange with identifier PXD039914) and cell morphology by digital holographic microscopy. The increase of pY triggered by OV treatment (inducing a global downregulation of pS and pT) disappeared during the storage. Phosphoproteomic analysis identified 609 phosphoproteins containing 1752 phosphosites, of which 41 pY were upregulated and 2 downregulated by OV. After these phosphorylation processes, the shape of RBCs shifted from discocytes to spherocytes, and the addition of KIs partially inhibited this transition. The KIs modulated either pY or pS and pT via diverse mechanisms related to cell shape, thereby affecting RBC morphology. The capacity of RBCs to maintain their function is central in transfusion medicine, and the presented results contribute to a better understanding of RBC biology.

Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

BACKGROUND: Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. STUDY DESIGN AND METHODS: This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. RESULTS: We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. CONCLUSIONS: Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly.

The Effect of the Donor's and Recipient's Sex on Red Blood Cells Evaluated Using Transfusion Simulations.

The hypothesis of the potential impact of the sex of red blood cell (RBC) concentrate (RCC) donors, as well as the sex of the recipients, on the clinical outcome, is still under evaluation. Here, we have evaluated the sex impact on RBC properties using in vitro transfusion models. Using a "flask model", RBCs from RCCs (representing the donor)-at different storage lengths-were incubated in a sex-matched and sex-mismatched manner with fresh frozen plasma pools (representing the recipient) at 37 °C, with 5% of CO2 up to 48 h. Standard blood parameters, hemolysis, intracellular ATP, extracellular glucose and lactate were quantified during incubation. Additionally, a "plate model", coupling hemolysis analysis and morphological study, was carried out in similar conditions in 96-well plates. In both models, RBCs from both sexes hemolyzed significantly less in female-derived plasma. No metabolic or morphological differences were observed between sex-matched and -mismatched conditions, even though ATP was higher in female-derived RBCs during incubations. Female plasma reduced hemolysis of female- as well as male-derived RBCs, which may be related to a sex-dependent plasma composition and/or sex-related intrinsic RBC properties.

In vitro-transfusional model for red-blood-cell study: the advantage of lowering hematocrit.

BACKGROUND: The quality of red blood cells (RBCs) stored in red cell concentrates (RCCs) is influenced by processing, storage and donor characteristics, and can have a clinical impact on transfused patients. To evaluate RBC properties and their potential impact in a transfusion setting, a simple in vitro-transfusional model has been developed. MATERIALS AND METHODS: Transfusion was simulated by mixing a washed RBC pool from two male-derived RCCs stored at 4°C with a pool of 15 male-derived fresh frozen plasma (FFP) units, representing the recipient, at a hematocrit (HCT) of 30% ("control" setting) or 5% (alternative model). The mixtures were incubated at 37°C, 5% of CO2 up to 48 h. Different metabolites, hemolysis and microvesicles (MVs) were quantified at several incubation times and RBC-morphology changes and deformability after incubation. For each model, biological triplicates have been investigated with RCCs at storage days 2 and 43. RESULTS: The 5%-HCT model restored the 2,3-DPG level and maintained the ATP level. Furthermore, glucose consumption and corresponding lactate production were increased in the 5%- vs the 30%-HCT condition. Lower hemolysis was observed with 5%-HCT, but only at day 2. However, morphological analysis by digital holographic microscopy (DHM) revealed a decreased fraction of discocytes at 5% rather than at 30% of HCT at storage day 2 but at day 43, the trend was inverted. Concordantly, RBCs incubated at 5% of HCT were more deformable than at 30% at day 43 (p<0.0001). DISCUSSION: Higher metabolic activity of RBCs in the 5%-HCT condition was promoted by a higher glucose availability and limited cell-waste accumulation. The conditions of the new proposed model thus enabled rejuvenation of RBCs and maintained them in a physiological-close state in contrast to the 30%-HCT model. It may be used as a first approach to evaluate e.g., the impact of donor and recipient characteristics on RBC properties.