Storage Of Erythrocytes Research Articles

Ultrasound-induced molecular delivery to erythrocytes using a microfluidic system.

Preservation of erythrocytes in a desiccated state for storage at ambient temperature could simplify blood transfusions in austere environments, such as rural clinics, far-forward military operations, and during space travel. Currently, storage of erythrocytes is limited by a short shelf-life of 42 days at 4 °C, and long-term preservation requires a complex process that involves the addition and removal of glycerol from erythrocytes before and after storage at -80 °C, respectively. Natural compounds, such as trehalose, can protect cells in a desiccated state if they are present at sufficient levels inside the cell, but mammalian cell membranes lack transporters for this compound. To facilitate compound loading across the plasma membrane via ultrasound and microbubbles (sonoporation), a polydimethylsiloxane-based microfluidic device was developed. Delivery of fluorescein into erythrocytes was tested at various conditions to assess the effects of parameters such as ultrasound pressure, ultrasound pulse interval, microbubble dose, and flow rate. Changes in ultrasound pressure and mean flow rate caused statistically significant increases in fluorescein delivery of up to 73 ± 37% (p < 0.05) and 44 ± 33% (p < 0.01), respectively, compared to control groups, but no statistically significant differences were detected with changes in ultrasound pulse intervals. Following freeze-drying and rehydration, recovery of viable erythrocytes increased by up to 128 ± 32% after ultrasound-mediated loading of trehalose compared to control groups (p < 0.05). These results suggest that ultrasound-mediated molecular delivery in microfluidic channels may be a viable approach to process erythrocytes for long-term storage in a desiccated state at ambient temperatures.

Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery.

Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient. Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42. Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO2 ) levels. Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions.

Enhancement of erythrocyte oxygen transport function after hydrothermic storage under the action of human cord blood low-molecular fraction and the Actovegin drug

We study the effect of a low-molecular fraction (below 10 kDa) derived from human cord blood and Actovegin on the content of various forms of hemoglobin in erythrocytes stored under hypothermia (2-4 ºС) for 21 days. During the long-term storage of erythrocytes, there is known to be a change in the hemoglobin forms ratio toward the decrease of the relative content of oxyhemoglobin and an increase of deoxy- and methemoglobin. As a result, the affinity of hemoglobin for oxygen reduces. The incubation of erythrocytes in a rehabilitation medium with each of the studied low-molecular drugs was found to be crucial to increase the content of oxyhemoglobin and to reduce the amounts of deoxy- and methemoglobin relative to the control. As a result, there was a rise in the oxygenation coefficient, reflecting the oxygen saturation of hemoglobin, up to the level characteristic of freshly isolated erythrocytes. The data obtained show that the human cord blood low-molecular fraction and the Actovegin normalize the ratio of hemoglobin forms in hypothermically stored erythrocytes, but the use of the Actovegin drug is more effective.

Blood Donor Exposome and Impact of Common Drugs on Red Blood Cell Metabolism

Computational models based on recent maps of the red blood cell proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to red blood cell storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic or environmental exposures (“exposome”) may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and post-transfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and off-the-counter drugs in 250 units donated by healthy volunteers from the REDS-III RBC Omics study. Based on high-throughput drug screenings of 1,366 FDA-approved drugs, we report a significant impact of ~65% of the tested drugs on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton-pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR) suggesting that these drugs have a direct, conserved, and significant impact on erythrocyte metabolism. We then focused on ranitidine – a common antiacid - as a representative drug with the potential to improve human erythrocyte storage quality and post-transfusion performances in mice. By combining tracing experiments with 1,2,3-13C3-glucose, proteome integral solubility alteration assays, genetic ablation of S1P synthesis capacity, in silico docking and 1D NMR, we show that ranitidine triggers metabolic mechanisms involving sphingosine 1-phosphate (S1P)-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin.

Procesiranje i skladištenje krvnih komponenti u uslovima COVID-19 pandemije

The spread of the COVID-19 virus has a strong influence on blood collection, maintaining a stable supply of all blood components and the safety of the transfusion itself. SARS-CoV-2 has a long incubation period (1-14 days, on average 5-6 days, longest reported 24 days) and causes asymptomatic infection in a large number of patients, which is a great challenge in a recruitment of blood donors and achieving a safe transfusion. Precise recommendations and precautions have been adopted regarding the criteria for temporary refusal of blood donors during the COVID-19 pandemic, organization of mobile teams and collection sites, disposal of medical waste, examination of potential donors and mandatory body temperature measurement. Although transmission of COVID-19 via blood and blood components has not been demonstrated, some countries have also introduced mandatory NAT testing for SARS-CoV-2 as a part of blood screening testing. Also, proactive measures have been taken, such as temporary storage of blood in quarantine for 14 days after collection, while special attention is paid to efficient management of blood component stocks and development of a collection plan, in order to avoid shortage of certain blood components or their expiration. The first step in this regard is to revise the measures which have the aim for improving the usability of blood components, ie reducing waste of stocks, which primarily refers to the temporary extension of the shelf life of blood components. Extending the shelf life of erythrocytes (longer than 35 to 49 days, which is defined at the national level) should be considered as early as possible, because once a shortage of erythrocytes occurs, they will be issued long before the expiration date. Previous studies have not shown significant side effects of erythrocyte transfusion with extended shelf life, so it is possible to consider the flexibility of blood processing and erythrocyte storage conditions with mandatory internal process validation and component quality control. The shelf life of platelet concentrate should be extended from 5 days to 7 or even 8 days, with mandatory bacteriological testing or pathogen inactivation. Another option to increase the platelet supply for prophylactic purposes is to reduce the platelet dose by dividing the existing components. Frozen fresh plasma has the longest shelf life (up to 3 years), so maintaining stable reserves is much safer than for cellular components. Liquid plasma (never previously frozen) has a shelf life of 7-40 days, and can be used in conditions of reduced freezer capacity, shortage of staff working on blood processing or for the production of convalescent plasma. Pathogen inactivation of plasma and platelets allows 3-6 log reduction of SARS-CoV-2 and MERS-CoV. The decision to introduce some of the methods of pathogen inactivation should be made taking into account the costs and resources required for implementation. For countries that do not have pathogenic inactivation already in routine practice, its rapid introduction is a big task. For now, the risk of SARS-CoV-2 transmission through the blood appears to be very low, although our understanding of the virus and behavior during a pandemic will improve over time. In this regard, pathogen inactivation of convalescent plasma should also be considered.

A Study of Some Biochemical Properties of Ecto-ATPase (Nucleotidase) in Erythrocytes of the Black Sea Thornback Ray Raja clavata L.

Biochemical properties and some kinetic characteristics of whole-erythrocyte ecto-ATPase (EC 3.6.1.5) were studied in the Black Sea thornback ray Raja clavata L. It was shown that enzyme activity varied from 1.5 to 3.5 nmol Pi/min/μL of packed cells and peaked in the presence of 3.0-6.0 mM Mg2+. Ecto-ATPase exhibited broad substrate specificity towards splitting nucleoside triphosphates. The enzyme was tolerant to changes in ambient pH and exhibited maximum activity in the pH range from 5.5 to 7.7. The enzyme also showed high substrate affinity, with the Michaelis constant (KM) found to be 12.0 ± 1.0 μM and Vmax being 2.35 0.2 nmol Pi/ min/μL of packed cells. Erythrocyte ecto-ATPase in R. clavata was sustainable to erythrocyte storage at 4°C for four days and exhibited a tolerance to high concentrations of urea.

Homogeneous Deformation of Native Erythrocytes During Long-Term Storage

Purpose of the study — to evaluate biomechanical regularities of deep deformation of native erythrocytes’ membranes during long-term (up to 32 days) storage of erythrocyte suspension.Materials and methods. The method for addressing the said problem was atomic-force spectroscopy. The measured value was hHz comprizing the depth to which the probe immersion process was described by interaction with a homogeneous medium. Empirical and theoretical dependence of the interaction force F (nN) on the probe immersion depth h (nm) — F (h) were obtained. Bar charts of relative frequency density of Young’s modulus E were built.Results. Modulus E changed from 9.3±3.2 kPa — for 3 days of storage, to 22.7±8.7 kPa — for 32 days. Coefficients of skewness were 0.52±0.04 (for day 3) and 0.82±0.09 (for day 32 d), hHz value remaining constant.Conclusion. Progressively as erythrocyte suspension was stored, erythrocyte membranes to the depth of 700 nm deflected homogeneously in spite of 2.4-fold increase of Young’s modulus.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

Hypoxic storage of erythrocytes slows down storage lesions and prolongs shelf-life.

Conventional storage conditions of erythrocytes cause storage lesions. We propose that hypoxic storage conditions, involving removal of oxygen and replacement with helium, the changes in stored erythrocytes under hypoxic condition were observed and assessed. Erythrocytes were divided into two equal parts, then stored in conventional and hypoxic conditions, separately. Blood gas analysis, hemorheology, and hemolysis were performed once a week. Energy metabolism and membrane damage were monitored by enzyme-linked immunosorbent assay. Phosphatidylserine exposure was measured by flow cytometry. P50 was measured and the oxygen dissociation curve(ODC) plotted accordingly. Erythrocyte morphology was observed microscopically. In the 9th week of storage, the hemolysis of the hypoxia group was 0.7%; lower (p < .05) than that of the control group and still below the threshold of quality requirements. The dissolved oxygen and pO2 were only 1/4 of that in the control group (p < .01); the adenosine triphosphate, glucose, and lactic acid levels were decreased (p < .05), while the 2,3-diphosphoglycerate levels were increased relative to that in the control group (p < .01). There were no statistically significant differences in membrane damage, deformability, and aggregation between the two groups. In addition, the ODC of the two groups was shifted to the left but this difference was not statistically different. Basically similar to the effect of completely anaerobic conditions. Erythrocytes stored under hypoxic conditions could maintain a relatively stable state with a significant decrease in hemolysis, reduction of storage lesions, and an increase in shelf-life.

Rapid and continuous on-chip loading of trehalose into erythrocytes.

Freeze-drying is a promising approach for the long-term storage of erythrocytes at room temperature. Studies have shown that trehalose loaded into erythrocytes plays an important role in protecting erythrocytes against freeze-drying damage. Due to the impermeability of the erythrocyte membrane to trehalose, many methods have been developed to load trehalose into erythrocytes. However, these methods usually require multistep manual manipulation and long processing time; the adopted protocols are also diverse and not standardized. Thus, we develop an osmotically-based trehalose-loading microdevice (TLM) to rapidly, continuously, and automatically produce erythrocytes with loaded trehalose. In the TLM, trehalose is loaded through the erythrocyte membrane pores induced by hypotonic shock; then, the trehalose-loaded erythrocytes are rinsed to remove hemoglobin molecules and cell fragments, and the extracellular solution is restored to the isotonic state by integrating a rinsing-recovering design. First, the mixing function and the rinsing-recovering function were confirmed using a fluorescent solution. Then, the performance of the TLM was evaluated under various operating conditions with respect to the loading efficiency of trehalose, the hemolysis rate of erythrocytes (ϕ), the recovery rate of hemoglobin in erythrocytes (φ), and the separation efficiency of the TLM. Finally, the preliminary study of the freeze-drying of erythrocytes with loaded trehalose was accomplished using the TLM. The results showed that under the designated operating conditions, the loading efficiency for human erythrocytes reached ~21mM in ~2min with a ϕ value of ~17% and a φ value of ~74%. This study provides insights into the design of the on-chip loading of trehalose into erythrocytes and promotes the automation of life science studies on biochips.

Age-Related Modulations in Erythrocytes under Blood Bank Conditions

Background: During storage of erythrocytes, storage lesions are formed that reduce the safety and efficacy of the stored blood. Thus, there is a need to understand the changes that occur during storage. Most studies have focused on storage of a mixed population of erythrocytes. The aim of this study is to analyze the changes in young and old erythrocytes over the course if storage. Materials and Methods: Blood was collected from the blood bank at the Kempegowda Institute of Medical Sciences (KIMS) Hospital (Bengaluru, India) and stored for 35 days in CPDA-1 at 4°C. Every 5 days, erythrocytes were separated based on the blood’s age using a Percoll-BSA gradient. Young and old erythrocytes obtained were used for analysis of the following oxidative stress (OS) markers: hemoglobin (Hb), hemolysis, mechanical fragility, antioxidant enzymes (superoxide dismutase and catalase [CAT]), superoxides, sialic acid, glutamic oxaloacetate transaminase (GOT), glucose, plasma membrane redox system (PMRS), total antioxidant capacity-cupric ion reducing antioxidant capacity assay (TAC_CUPRAC), lactate dehydrogenase (LDH), lipid peroxidation products (malondialdehyde [MDA] and conjugate dienes), and protein oxidation products (advanced oxidation protein products and protein sulfhydryls). Result: Young cells had higher amounts of Hb, sialic acid, GOT, LDH, TAC_CUPRAC, CAT, and superoxides compared to old cells. Old cells, however, had higher PMRS and MDA levels with respect to young cells. Discussion: Young cells could endure OS during storage more efficiently than old cells. In conclusion, the efficacy of stored blood depends on the ratio of young cells in the population. This study highlights the prospects of storing young erythrocytes for a prolonged shelf life of blood.

Extreme blood-boosting capacity of an Antarctic fish represents an adaptation to life in a sub-zero environment.

Blood doping, the practice of boosting the oxygen carrying capacity of blood, is an illegal strategy used by human athletes to enhance aerobic capacity and athletic performance. Interestingly, the practice of boosting blood oxygen carrying capacity is also naturally prevalent in the animal kingdom via the splenic release of stored erythrocytes. Here, we demonstrate that an Antarctic notothenioid fish, the bald notothen (Pagothenia borchgrevinki), is a master of this practice. Because of the sub-zero environment these fish inhabit, they sequester a large proportion of erythrocytes in the spleen during times of inactivity to reduce the energetic and physiological costs associated with continuously pumping highly viscous blood around the body. However, in response to metabolically demanding situations (i.e. exercise and feeding), these fish contract the spleen to eject stored erythrocytes into circulation, which boosts blood oxygen carrying capacity by up to 207% (cf. exercise-induced increases of ∼40-60% in a range of other vertebrates and ∼5-25% in blood-doping athletes). By evaluating cardiorespiratory differences between splenectomized (unable to release erythrocytes from the spleen) and sham-operated individuals, we demonstrate the metabolic benefits (i.e. aerobic scope increase of 103%) and the cardiovascular trade-offs (i.e. ventral aortic blood pressure and cardiac workload increase of 12% and 30%, respectively) associated with the splenic blood-boosting strategy. In conclusion, this strategy provides bald notothens with an extraordinary facultative aerobic scope that enables an active lifestyle in the extreme Antarctic marine environment, while minimizing the energetic and physiological costs of transporting highly viscous blood during times of reduced energetic demand.

Upotreba autolognih hemoprodukata u mirnodopskim uslovima, vanrednim situacijama i ratu

Introduction In industrialized countries, autologous transfusion (AT) of blood and/or autologous blood products is often used. Objective To perform an analysis on the possibilities of using autologous blood products (ABP) in peace and in war. Methods In peacetime conditions, all AT strategies can be performed: 1) planned preoperative autologous blood and/or blood components collection (PPAB-BCC); 2) acute normovolemic hemodilution (ANH); 3) intraoperative blood salvage of autologous blood; 4) postoperative blood salvage; 5) administration of autologous platelets; 6) the use of autologous fibrin glue and autologous tissue adhesives; 7) the collection and storage of autologous erythrocytes and autologous fresh plasma. In emergency situations and war, the most applicable strategies are: a) ANH; b) intraoperative blood salvage; c) postoperative blood salvage; d) application of autologous fibrin glue and autologous tissue adhesives. Results In peace. PPAB-BCC was used for 314 patients, who were planned for elective surgery. AB was collected using intraoperative blood salvage in the amount of 520 ml for 281 patients. ANH was performed for 30 patients. For 20 of those, 780ml of blood was collected and they were operated on for colon cancer with rectum amputation and one nephrectomy was performed on a patient with single kidney neoplasm. ANH was performed on 9 orthopedic patients. Intraoperative autologous blood salvage in peaceful conditions was performed for 52 adult patients who underwent cardiac surgery procedures. The amount of autologous blood collected and reinfused ranged from 1,500 to 3,500 ml per patient. With the help of a Cell-Saver, autologous blood salvage was performed on 63 patients during pediatric heart surgery. The amount of intraoperative salvaged autologous blood per operation ranged from 210 ml to 620 ml. During the 1990s, doctors at the Military Medical Academy (MMA) used plateletrich autologous plasma on 38 patients who had diseased blood vessels replaced with dacron grafts. In war. During the civil war on the territory of former Yugoslavia, intraoperative salvage of autologous blood was performed on 25 wounded patients who suffered from closed pneumothorax due to blast injury, in which cased 500 ml to 1500 ml of autologous blood was collected. With the help of the 'Pleur-Evac autotransfusion system', immediately after collection, reinfused to the wounded patient during the helicopter transport to the MMA. Autologous fibrin glue was used to treat 10 wounded patients during reconstructive surgery procedures. Conclusion In peacetime conditions, the AT program should be organized in every hospital that has adequate conditions for its implementation. The implementation of AT is very important because it can serve as an alternative to homologous transfusion. Our experiences from the war on the territory of the former Yugoslavia tell us that certain strategies of the AT program can successfully be implemented even in emergency situations and during war.

Equine blood reticulocytes: reference intervals, physiological and pathological changes

High-sensitivity haematology analysers detect equine blood reticulocytes, but their analysis and quantitation is largely unstudied. We assessed whether equine reticulocytes are affected by pathological or physiological factors, determined reference intervals, and examined reticulocyte and related, erythron parameters using 6-years’ data (Advia 2120) from our hospital archives. Data was categorised according to horse age (foals < 12 months), breed (hot-, warm-, cold-blooded), presence of anaemia and various diseases. In adults, reticulocytes (× 109/L) were 20% lower in cold-blooded horses compared to others (4.14 ± 2.3, 25 vs 5.15 ± 3.3, 157; data shown as mean ± SD, n; p < 0.05.) There was no gender effect. In thoroughbreds, foals had 50% higher reticulocytes than adults (7.5 ± 4.5, 18 vs 5.0 ± 3.5, 56). We determined the first reference interval for healthy adult horses as 5.0 ± 3.2,182, 1.16–13.1. Reticulocyte counts were 120% higher with marked anaemia (11.0 ± 9.4, 6) but 36% with other diseases (6.9 ± 4.2, 74), versus reference mean. Erythrocyte size (MCV)/reticulocyte haemoglobin content (CHr) were 21/14% lower in foals versus adults and 12/37% higher with marked anaemia. Immature reticulocytes (IRF-H) were 60% lower in foals versus adult thoroughbreds, and 87% with marked anaemia. In conclusion, equine reticulocytes are only mildly increased by physiological parameters (breed, age, but not gender), various non-blood diseases (colic, dysproteinaemia) and marked anaemia. Reticulocyte counts are substantially lower in horses versus other species; their clinical use is limited to marked anaemias. This is consistent with the greater erythrocyte storage and buffering capacity of the spleen of the horse. Finally, this study supports the theory of a reticulocyte maturation role for equine spleen.

Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes.

The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.

Estimation of Achievable Oxygen Consumption Following Transfusion With Rejuvenated Red Blood Cells

Erythrocyte storage induces a nonphysiological increase in hemoglobin-oxygen affinity (quantified by low p50, the oxygen tension at 50% hemoglobin saturation), which can be restored through biochemical rejuvenation. The objective was to mathematically model the impact of transfusing up to 3 standard allogeneic units or rejuvenated units on oxygen delivery (DO2) and oxygen consumption (VO2). Oxygen dissociation curves were generated from additive solution-1 red blood cell (RBC) leukoreduced units (n = 7) before and after rejuvenation following manufacturer's instructions. Two of these units were used to prepare standard or rejuvenated donor RBC and added to samples of fresh whole blood. These admixtures were used to construct an in vitro transfusion model of postoperative anemia and determine a linear equation for calculating the sample p50, which was subsequently used to calculate DO2 and VO2 after simulated transfusions. Whole blood-packed red blood cell unit admixture p50s could be predicted from a linear model including the p50 of its components, the mass fraction of the transfused component, and interaction terms (R2 = .99, P < 0.001). Transfusion with standard units slightly, but significantly, increased projected DO2 compared with rejuvenated units (P = 0.03), but rejuvenated units markedly increased projected VO2 (P = 0.03). Standard units did not significantly change VO2 relative to pre-transfusion levels (P > 0.1). Using high-p50, rejuvenated RBC in simulated transfusions greatly improved projected VO2, indicating the potential for increased end-organ oxygen availability compared with standard transfusion. Patient capacity to increase cardiac output after cardiac surgery may be limited. Transfusing high-p50 RBC in this setting may improve the perioperative care of these patients.

Nano alterations of membrane structure on both γ-irradiated and stored human erythrocytes

Purpose: Storage and ionizing radiation of human red blood cells (RBC) produce alterations on RBC membranes and modify their normal shape and functionality. We investigated early morphological and biochemical changes in RBC due to those stressing agents at the nanoscale level and their impact on blood quality.Materials and methods: Whole blood samples from healthy donors were γ-irradiated with 15, 25, 35, and 50 Gy. Non-irradiated and non-stored RBC were used as control samples. Irradiated blood samples were stored separately at 4 °C and analyzed immediately and after 5 and 13 d. Atomic force microscopy (AFM), osmotic fragility and Raman spectroscopy were used to detect morphological and biochemical changes.Results: RBC function is challenged by both irradiation and storage. The storage procedure caused nanometric variations over the surface of RBC membrane for both irradiated and non-irradiated cells. The membrane of RBC became more fragile, while the biochemical fingerprint of hemoglobin (Hb) remained unaltered.Conclusions: Our work shows that the irradiation procedure leads to an increase in the number and size of nanovesicles along with the dose. The functionality of RBC can be affected from changes in the roughness, becoming more fragile and susceptible to breakage.

Vitamin C as a modulator of oxidative stress in erythrocytes of stored blood

AimTo determine the effects of Vitamin C (VC-ascorbic acid) as an additive on erythrocytes of stored blood. BackgroundOxidative stress (OS) plays a major role in the formation of storage lesion of erythrocytes. Antioxidants, such as VC could be beneficial in combating oxidative damage during storage. Materials and methodsBlood obtained from male Wistar rats was stored at 4°C in anticoagulant solution citrate-phosphate-dextrose-adenine solution. Blood samples were divided into 3 groups – (i) Controls, (ii) VC 10 (VC at a concentration of 10mM), (iii) VC 30 (VC at a concentration of 30mM). Markers of OS in erythrocytes such as – hemoglobin, superoxides, antioxidant enzymes (superoxide dismutase; SOD, catalase and glutathione peroxidase), hemolysis, lipid peroxidation products (conjugate dienes and malondialdehyde), protein oxidation products and ascorbic acid were determined on days 0, 10 and 15 of storage. ResultsAddition of ascorbic acid to the storage solution contributed to the protection of erythrocytes from oxidative damage. Ascorbic acid at a concentration of 30mM decreased SOD levels and increased protein sulfhydryls (P-SH) levels on day 15 showing that higher concentration supplemented the inherent antioxidant defense system of erythrocytes during blood storage. ConclusionVC proved to be effective in combating OS during blood storage. However, further exploration of antioxidants as additives and the erythrocyte storage lesion would result in better management of blood storage.

Oxidative stress and antioxidant defenses during blood processing and storage of erythrocyte concentrates

Oxidative lesions start accumulating in cells when the oxidant attacks overwhelm the antioxidant defenses. This review will briefly describe red blood cell storage lesions with emphasis on the consequences of oxidation and the cellular defense mechanisms, as well as the methods that can be used to monitor them. The sources of variability in red blood cell storage capacity depend on the donor characteristics, the product processing and the storage conditions. Suggestions to improve the product quality in order to ensure the best efficacy and safety for the transfused patient are also discussed.

Contribution of plasma membrane lipid domains to red blood cell (re)shaping

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.