Storage Of Erythrocytes Research Articles

Oxygen Transport Rate and Erythrocyte Deformability

To the Editor The implications of the recent article by Frank et al.1 are that erythrocyte storage time is associated with decreased cell membrane deformability and that this change might impede capillary transit of red blood cells and, thereby, tissue oxygenation. Thus, the longer the storage time toward the 6 weeks standard before the blood is discarded, the poorer tissue oxygenation will be. In 2008, we published an article2 demonstrating that the oxygen transport rate from the hemoglobin molecule through the cell membrane to the tissues appears to be unique for each individual tested and that the oxygen transport rate increases during storage (1.72-fold over 8 weeks, 95% confidence interval 1.5–1.95). Thus, the longer the storage time toward the 6 weeks standard before the blood is discarded, the better tissue oxygenation will be. Although on the surface these 2 hypotheses appear to conflict with one another, there are differences between the 2 studies that might explain this conflict. We assessed stored blood outside the context of the capillary, whereas the Frank et al.1 article dealt with red blood cell deformability taking place in the context of the capillary. In other words, the 2 groups addressed completely different variables that contribute to tissue oxygenation capacity of stored blood, the results of which point to all the physical traits of stored blood that must be measured to understand the most important issue of outcomes. Furthermore, though both articles are correct, the contribution of either effect may be outweighed by other characteristics of stored blood, for example, the nitric oxide scavenging effects. The bottom line of these apparently conflicting studies is that more research needs to be done studying blood storage and the selection of red blood cell units for transfusion. Henry Buchwald, MD, PhD Hector J. Menchaca, MD Department of Surgery University of Minnesota Minneapolis, Minnesota [email protected]

Dynamic Simulation and Metabolome Analysis of Long-Term Erythrocyte Storage in Adenine–Guanosine Solution

Although intraerythrocytic ATP and 2,3-bisphophoglycerate (2,3-BPG) are known as direct indicators of the viability of preserved red blood cells and the efficiency of post-transfusion oxygen delivery, no current blood storage method in practical use has succeeded in maintaining both these metabolites at high levels for long periods. In this study, we constructed a mathematical kinetic model of comprehensive metabolism in red blood cells stored in a recently developed blood storage solution containing adenine and guanosine, which can maintain both ATP and 2,3-BPG. The predicted dynamics of metabolic intermediates in glycolysis, the pentose phosphate pathway, and purine salvage pathway were consistent with time-series metabolome data measured with capillary electrophoresis time-of-flight mass spectrometry over 5 weeks of storage. From the analysis of the simulation model, the metabolic roles and fates of the 2 major additives were illustrated: (1) adenine could enlarge the adenylate pool, which maintains constant ATP levels throughout the storage period and leads to production of metabolic waste, including hypoxanthine; (2) adenine also induces the consumption of ribose phosphates, which results in 2,3-BPG reduction, while (3) guanosine is converted to ribose phosphates, which can boost the activity of upper glycolysis and result in the efficient production of ATP and 2,3-BPG. This is the first attempt to clarify the underlying metabolic mechanism for maintaining levels of both ATP and 2,3-BPG in stored red blood cells with in silico analysis, as well as to analyze the trade-off and the interlock phenomena between the benefits and possible side effects of the storage-solution additives.

Case Scenario: Hemodynamic Management of Postoperative Acute Kidney Injury

Case Scenario: Hemodynamic Management of Postoperative Acute Kidney Injury

P71: Nitrite and nitric oxide supplementation during erythrocyte storage

Background Several studies have shown that aging red blood cells (RBCs) during storage reduces their ability to improve microvascular perfusion and oxygenation after transfusion. In this study we investigated the role of nitric oxide (NO) and nitrite supplementation during storage of RBCs. In order to study the roles of NO and nitrite in the various biochemical changes during RBC storage, NO-releasing agents, NO releasing nanoparticles (NO-nps) and nitrite were used. Methods Rat cells were stored for 14 days at 4 °C in CDPA-1, during storage NO-releasing agents, NO-nps and nitrite were supplemented at various doses. Biochemical and biomechanical changes were quantified and compared to fresh cells. Additionally, rats were instrumented and exchange transfused with stored cells with and without NO supplementation. Results During the storage duration 2,3-DPG concentration and pH decreased, and increased lactate. NO supplementation with NO-nps slowed down the biochemical changes including the depletion on important antioxidant factors, such as GSH, NADP and NADPH. NO supplementation with NO-nps decreased proteolytic enzymes, including connective tissue-activating peptide III, which participates in tissue remodeling and inflammatory process. Lastly, NO supplementation preserved static and dynamic red cell mechanics, which affect cell-to-cell and cell-to-wall interaction. Transfusion of stored cells without NO supplementation affected small arterioles hemodynamics, including diameter, velocity, blood flow, and oxygen delivery. While, infusion of cells stored under NO supplementation presented superior microvascular hemodynamics and oxygenation. Conclusions Although both NO and its donors and/or their decomposed products may have biological activities, most of the cellular responses to these donors have been postulated to reflect NO-dependent events. NO supplementation during storage preserved cell deformability, preserved antioxidant capacity and preserved microvascular endothelial cell mechanotransduction, and red cell hydrodynamics. Most of the storage changes were prevented or slowed down by NO, NO induced protection seems to be related to decrease oxidative stress in the storage conditions. Therefore, NO supplementation prevents ROS irreversible lesions in the early period of storage, rather then intervening a posteriori through the addition of rejuvenating solutions. Disclosure There is not financial interest. Supported by R01HL052684 P01HL11090.

Spray drying for preservation of erythrocytes: effect of atomization on hemolysis.

Spray drying has the potential to enable storage of erythrocytes at room temperature in the dry state. The spray drying process involves atomization of a liquid into small droplets and drying of the droplets in a gas stream. In this short report, we focus on the atomization process. To decouple atomization from drying, erythrocyte suspensions were sprayed with a two-fluid atomizer nozzle using humid nitrogen as the atomizing gas. The median droplet size was less than 100 μm for all of the spray conditions investigated, indicating that the suspensions were successfully atomized. Hemolysis was significantly affected by the hematocrit of the erythrocyte suspension, the suspension flow rate, and the atomizing gas flow rate (p<0.01 in all cases). Under appropriate conditions, it was possible to achieve less than 2% hemolysis, suggesting that spray drying may be a feasible option for erythrocyte biopreservation.

Decreased Erythrocyte Deformability After Transfusion and the Effects of Erythrocyte Storage Duration

Erythrocyte cell membranes undergo morphologic changes during storage, but it is unclear whether these changes are reversible. We assessed erythrocyte cell membrane deformability in patients before and after transfusion to determine the effects of storage duration and whether changes in deformability are reversible after transfusion. Sixteen patients undergoing posterior spinal fusion surgery were studied. Erythrocyte deformability was compared between those who required moderate transfusion (≥ 5 units erythrocytes) and those who received minimal transfusion (0-4 units erythrocytes). Deformability was measured in samples drawn directly from the blood storage bags before transfusion and in samples drawn from patients before and after transfusion (over 3 postoperative days). In samples taken from the blood storage bags, we compared deformability of erythrocytes stored for a long duration (≥ 21 days), those stored for a shorter duration (<21 days), and cell-salvaged erythrocytes. Deformability was assessed quantitatively using the elongation index (EI) measured by ektacytometry, a method that determines the ability for the cell to elongate when exposed to shear stress. Erythrocyte deformability was significantly decreased from the preoperative baseline in patients after moderate transfusion (EI decreased by 12% ± 4% to 20% ± 6%; P = 0.03) but not after minimal transfusion (EI decreased by 3% ± 1% to 4% ± 1%; P = 0.68). These changes did not reverse over 3 postoperative days. Deformability was significantly less in erythrocytes stored for ≥ 21 days (EI = 0.28 ± 0.02) than in those stored for <21 days (EI = 0.33 ± 0.02; P = 0.001) or those drawn from patients preoperatively (EI = 0.33 ± 0.02; P = 0.001). Cell-salvaged erythrocytes had intermediate deformability (EI = 0.30 ± 0.03) that was greater than that of erythrocytes stored ≥ 21 days (P = 0.047), but less than that of erythrocytes stored <21 days (P = 0.03). The findings demonstrate that increased duration of erythrocyte storage is associated with decreased cell membrane deformability and that these changes are not readily reversible after transfusion.

Erythrocyte Storage Duration Is Not Associated with Increased Mortality in Noncardiac Surgical Patients

More than 5 million patients receive erythrocyte transfusions in the United States every year. Previous studies linked the storage duration of allogeneic erythrocytes to the risk of severe postoperative complications, especially after cardiac or trauma surgery. Limited data are available for noncardiac surgical patients. We therefore evaluated the association between storage duration of transfused erythrocytes and postoperative all-cause mortality among general surgery patients. Perioperative data corresponding to 63,319 adult, general surgery patients were obtained from our registry and merged with blood product data. Patients receiving solely leukocyte-reduced, allogeneic erythrocyte transfusions were included. Multivariable Cox proportional hazards regression was used to characterize the relationship between median erythrocyte storage duration and postoperative mortality rate, adjusting for characteristics plausibly influencing the storage duration of erythrocytes. Of the 6,994 patients included in the final analysis, 23, 44, 11, 9, and 13% received 1, 2, 3, 4, and ≥5 erythrocyte units, respectively. The authors found no evidence that increasing median storage duration was associated with a difference in the risk of postoperative mortality (hazard ratio, 0.99 [0.94-1.04]; P = 0.64). Analyzing the mean storage duration of erythrocyte units as a function of year of transfusion, the authors demonstrate a relevant decrease in utilization of the oldest blood units, whereas young blood storage duration remains nearly unchanged. The authors' study supports the recent literature in surgical and medical patients and underlines the importance of sufficiently powered randomized trials to finally resolve the erythrocyte storage duration debate.

Understanding the Erythrocyte Storage Lesion

Understanding the Erythrocyte Storage Lesion

Potassium leakage primes stored erythrocytes for phosphatidylserine exposure and shedding of pro‐coagulant vesicles

During storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PS-negative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

Functional consequences of sphingomyelinase-induced changes in erythrocyte membrane structure

Inflammation enhances the secretion of sphingomyelinases (SMases). SMases catalyze the hydrolysis of sphingomyelin into phosphocholine and ceramide. In erythrocytes, ceramide formation leads to exposure of the removal signal phosphatidylserine (PS), creating a potential link between SMase activity and anemia of inflammation. Therefore, we studied the effects of SMase on various pathophysiologically relevant parameters of erythrocyte homeostasis. Time-lapse confocal microscopy revealed a SMase-induced transition from the discoid to a spherical shape, followed by PS exposure, and finally loss of cytoplasmic content. Also, SMase treatment resulted in ceramide-associated alterations in membrane–cytoskeleton interactions and membrane organization, including microdomain formation. Furthermore, we observed increases in membrane fragility, vesiculation and invagination, and large protein clusters. These changes were associated with enhanced erythrocyte retention in a spleen-mimicking model. Erythrocyte storage under blood bank conditions and during physiological aging increased the sensitivity to SMase. A low SMase activity already induced morphological and structural changes, demonstrating the potential of SMase to disturb erythrocyte homeostasis. Our analyses provide a comprehensive picture in which ceramide-induced changes in membrane microdomain organization disrupt the membrane–cytoskeleton interaction and membrane integrity, leading to vesiculation, reduced deformability, and finally loss of erythrocyte content. Understanding these processes is highly relevant for understanding anemia during chronic inflammation, especially in critically ill patients receiving blood transfusions.

Effect of stress actions on some hematological and biochemical parameters of rat blood and on energetic intermolecular interactions in lipid extract under effect of light radiation

Comparative study has been carried of effect of the three-day long starvation, running, and their combination on morphological parameters of rat blood, lipid metabolism, and activity of blood Na,K-ATPase. Different effect has been shown of these stress factors on the blood erythrocyte composition. Starvation is accompanied by the most pronounced release of stored erythrocyte into blood, which results in a significant decrease both of the total amount of reticulocytes and the complete absence of reticulocytes of the I stage of maturity (the youngest). The running on treadmill led to a significant increase of the total amount of blood reticulocytes and to multiple increase of immature reticulocytes (RC-I and RC-II), which can indicate some stress of the bone marrow erythroid stem line. The curve of acid resistance of blood reticulocytes has shown the animal to experience the greatest stress at a combination of starvation and running. Starvation and running produced different effects on blood lipid characteristics. The content of triacylglycerides (TAG) in blood rose by 40% at starvation and decreased by 30% at running, a similar tendency being found for index of atherogeneity. The fatty acid composition of blood phospholipids at running and its combination with starvation practically did not differ from control. A change of Na,K-ATPase, which is so characteristic of reaction to various kinds of stress, sharply fell at starvation (by 22%), but increased at running (by 13%) and decreased markedly at combination of these actions. Absorption spectra of lipid extracts of the whole blood of the rats submitted to various stress actions showed that extracted from blood (at different amount depending on the kind of action) is an organic substance with coupled bonds, which absorbs light in the diapason of 360-620 nm. The absorption of light in the diapason of 400-410 nm has been found to belong to the Soret band of ferroheme and ferriheme. The shift of the Soret band indicates electron transitions in the iron cation. By the change and disappearance of the Soret band, it is possible to judge about the processes occurring in the lipid extract. The disappearance of the Soret band in the lipid extract indicates formation in it of steady radicals as a result of the ferriheme disintegration due to accumulation of energy in porphyrin, which does not seem to occur in the blood cell membranes. The iron atom in the ferriheme molecule is known to accept electron and yields a part of energy probably to porphyrin. Then ferriheme yields electron and becomes ferriheme with excess of energy in porphyrin. Hence, at admission of the next electron to the iron atom the porphyrin molecule is to get rid of the energy obtained earlier to prevent its disintegration. The heme is possible to be an accumulator and distributor of energy in tissue.

Erythrocyte storage increases rates of NO and nitrite scavenging: implications for transfusion-related toxicity

Storage of erythrocytes in blood banks is associated with biochemical and morphological changes to RBCs (red blood cells). It has been suggested that these changes have potential negative clinical effects characterized by inflammation and microcirculatory dysfunction which add to other transfusion-related toxicities. However, the mechanisms linking RBC storage and toxicity remain unclear. In the present study we tested the hypothesis that storage of leucodepleted RBCs results in cells that inhibit NO (nitric oxide) signalling more so than younger cells. Using competition kinetic analyses and protocols that minimized contributions from haemolysis or microparticles, our data indicate that the consumption rates of NO increased ~40-fold and NO-dependent vasodilation was inhibited 2-4-fold comparing 42-day-old with 0-day-old RBCs. These results are probably due to the formation of smaller RBCs with increased surface area: volume as a consequence of membrane loss during storage. The potential for older RBCs to affect NO formation via deoxygenated RBC-mediated nitrite reduction was also tested. RBC storage did not affect deoxygenated RBC-dependent stimulation of nitrite-induced vasodilation. However, stored RBCs did increase the rates of nitrite oxidation to nitrate in vitro. Significant loss of whole-blood nitrite was also observed in stable trauma patients after transfusion with 1 RBC unit, with the decrease in nitrite occurring after transfusion with RBCs stored for >25 days, but not with younger RBCs. Collectively, these data suggest that increased rates of reactions between intact RBCs and NO and nitrite may contribute to mechanisms that lead to storage-lesion-related transfusion risk.

Storage-Induced Changes in Erythrocyte Membrane Proteins Promote Recognition by Autoantibodies

Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.

Alterations of red blood cell metabolome during cold liquid storage of erythrocyte concentrates in CPD–SAGM

Erythrocyte concentrates for transfusion purposes represent a life-saving therapeutics of primary relevance in the clinical setting. However, efforts have been continuously proposed to improve safety and efficacy of long-term stored red blood cells. By means of liquid chromatography coupled with Q-TOF mass spectrometry, we were able to perform an untargeted metabolomics analysis in order to highlight metabolic species (i.e. low molecular biochemicals including sugars, lipids, nucleotides, aminoacids, etc.), both in red blood cells and supernatants, which showed fluctuations against day 0 controls over storage duration on a weekly basis. We could confirm and expand existing literature about the rapid fall of glycolytic rate and accumulation of glycolysis end products. A shift was observed towards the oxidative phase of pentose phosphate pathway, in response to an exacerbation of oxidative stress (altered glutathione homeostasis and accumulation of peroxidation/inflammatory products in the supernatant). The present study provides the first evidence that over storage duration metabolic fluxes in red blood cells proceed from pentose phosphate pathway towards purine salvage pathway, instead of massively re-entering glycolysis via the nonoxidative phase. This article is part of a Special Issue entitled: Integrated omics.

Influence of Red Blood Cell Storage Time on Clinical Course and Cytokine Profile in Septic Shock Patients

Background: Previous investigations have suggested beneficial effects of fresh versus stored red blood cell transfusion in critically ill patients. The present study investigates the effects of red blood cell storage time on the clinical course and hemodynamic and laboratory parameters in patients with septic shock. Patients and Methods: 18 patients with septic shock received 2 erythrocyte units stored for ≤ 7 days (n = 8) or > 7 days (n = 10). The sequential organ failure assessment (SOFA) score was calculated for 7 days. Hemodynamic parameters (cardiac index, extravascular lung water) were determined using transpulmonary thermodilution. Laboratory parameters (lactate, base excess, C-reactive protein, procalcitonin, IL-1β, IL-6, TNF-α, sVCAM-1, sICAM-1) were monitored before and 1, 3, 6, 12, 24, and 48 h after transfusion. The Mann-Whitney-U test and Neumann test were used for group comparison and trend assessment, respectively. Results: We failed to observe significant differences with respect to SOFA scores between patients receiving fresh or stored erythrocytes. However, a significant trend towards an improvement in the SOFA score was found in the group receiving fresh erythrocytes (p < 0.01). No significant differences in hemodynamic or laboratory parameters were found between both groups. Conclusion: While the present findings do not provide clear-cut evidence supporting beneficial effects of fresh red blood cells in septic shock, they warrant larger randomized studies to confirm or refute such effects.

Storage-Induced Changes Promote Erythrocyte Recognition by Autoantibodies,

Abstract 3371Physiological erythrocyte removal is associated with a selective increase in the expression of senescent cell antigens related to band 3, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. In order to understand the molecular basis for this phenomenon we investigated whether erythrocyte storage leads to increased expression of non-physiological neoantigens.Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using plasma containing erythrocyte autoantibodies from selected patients with autoimmune hemolytic anemia (N=6). Patient plasma autoantibody binding to erythrocytes increased with erythrocyte storage time, while the exact opposite was observed for plasma from healthy volunteers, showing that erythrocyte antigenicity changes during storage. SDS polyacrylamide gel analysis combined with proteomics revealed band 3 to be a dominant target of the autoantibodies. From vesicles that are formed during erythrocyte storage, the autoantibodies precipitated protein complexes distinct from those recognized on erythrocytes, indicating that they have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the control immunoprecipitates.These findings support the hypothesis that disturbed erythrocyte aging during storage of erythrocyte units contributes to transfusion-induced alloantibody and autoantibody formation. Disclosures:No relevant conflicts of interest to declare.

Reducing noninfectious risks of blood transfusion.

As screening for transfusion-associated infections has improved, noninfectious complications of transfusion now cause the majority of morbidity and mortality associated with transfusion in the United States. For example, transfusion-related acute lung injury, transfusion-associated circulatory overload, and hemolytic transfusion-reactions are the first, second, and third leading causes of death from transfusion, respectively. These complications and others are reviewed, and several controversial methods for prevention of noninfectious complications of transfusion are discussed, including universal leukoreduction of erythrocyte units, use of male-only plasma, and restriction of erythrocyte storage age.

The acidosis-induced right shift of the HbO2 dissociation curve is maintained during erythrocyte storage

Background and objectives. In fresh blood, tissue hypoxia increases microcirculatory acidosis, which enhances erythrocyte O2 unloading and increases the amount of available O2. Storage of erythrocytes increases the HbO2 affinity and reduces O2 unloading. We examined the development of the affinity change during a period of 5 weeks of storage by present blood bank standards, and investigated to what extent acidosis offsets the affinity change. Materials and methods. Blood from volunteer donors was processed and stored as erythrocyte concentrates (EC). At 2–5 day intervals, EC were drawn from the bags and suspended in plasma and crystalloids to an Hb ≈ 10 g/dL. The suspensions were adjusted to give a pH of 7.40, 7.10, 6.80 or 6.30 and equilibrated with different gas mixtures to SO2 0, 25, 50, 75 and 100%. Measurements of the PO2/SO2 pairs at each pH were used to calculate the position of the HbO2 curve and its P50 value. Results. A significant leftward shift in the HbO2 curve was established after 1 week of storage; after 2.5 weeks only minor further changes were observed. Acidification right-shifted the HbO2 curve, after 2.5 weeks of storage the curve at pH 7.10 was similar to that for fresh blood at pH 7.40. Calculations of extractable O2 showed that the left-shifted HbO2 curve of stored EC could be advantageous at a low arterial PO2. Conclusions. The rightward shift of the HbO2 curve due to acidosis is well maintained in stored erythrocytes, a moderate pH decrease offsets the storage-induced increased HbO2 affinity.

Oxidative stress-dependent oligomeric status of erythrocyte peroxiredoxin II (PrxII) during storage under standard blood banking conditions

Although biochemical properties of 2-Cys peroxiredoxins have been extensively studied in various cell lines and organisms, redox-induced structural transitions of peroxiredoxin II (PrxII) in human erythrocytes certainly warrant further investigation. In this work, cytosol and membrane ghosts of both fresh erythrocytes (cells obtained just after blood collection) and 28-day stored erythrocytes were analyzed by proteomics tools. We demonstrated that in fresh red blood cells PrxII exhibits four different oligomeric states in cytosol, whereas no PrxII complexes are in the membrane. The highest molecular weight PrxII protein complex (440 kDa) was proven to derive from the association between tetrameric catalase (CAT, 232 kDa) and decameric PrxII, whereas oligomers at 140, 100 and 67 kDa resulted to be homo-polymeric complexes composed of variable copies of PrxII monomeric subunits. Interestingly, the 440 kDa complex contained both reduced and oxidized (disulphide-linked dimers) PrxII decamers. Upon oxidative stress (28-day storage), the PrxII oligomers at 100 kDa in the cytosol disappeared and the CAT–PrxII hetero-oligomeric complex at 440 kDa is converted to a higher molecular weight structure (480 kDa) due to the presence therein of cross-linked species of PrxII and hemoglobin. More interestingly, oxidized red cell membranes contained the CAT–PrxII complex detected in 0-day cytosol as a consequence of protein recruitments induced by oxidative stress, however it showed a greater percentage of PrxII dimers. Finally, since the adoption of distinct PrxII structures is known to be closely related to different functions, peroxidase activity assays were performed demonstrating a positive reaction for oligomers at 440 kDa (both in cytosol and membrane compartment) and at 140 kDa. Our results contribute to clarify structural and functional switching of peroxiredoxin II in erythrocytes, thus possibly opening new scenarios in the biological roles played by this protein in defense mechanisms against oxidative stress, especially with the reference to red cell storage lesions.

Analysis and clinical relevance of microparticles from red blood cells

The mechanisms involved in the formation of red blood cell (RBC) microparticles in vivo as well as during erythrocyte storage are reviewed, and the potential role of microparticles in transfusion medicine is described. Microparticles release is an integral part of the erythrocyte ageing process, preventing early removal of RBCs. Proteomics analyses have outlined the key role of band 3-ankyrin anchoring complex and the occurrence of selective RBC membrane remodelling mechanisms in microparticles formation. The presence of several RBC antigens, expressed on microparticles, has been demonstrated. The potential deleterious effects of RBC microparticles in transfused recipients, including hypercoagulability, microcirculation impairment and immunosuppression, are discussed. Formation and role of RBC microparticles are far from being completely understood. Combining various approaches to elucidate these mechanisms could improve blood product quality and transfusion safety. Implementation of RBC microparticles as biomarkers in the laboratory routine needs to overcome technical barriers involved in their analysis.