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.     

Improved storage of erythrocytes by prior leukodepletion: flow cytometric evaluation of stored erythrocytes.

In vivo phagocytosis of senescent red blood cells (RBCs) by macrophages occurs 120 days after their release into the circulation. It depends on two sequential signals that trigger phagocytosis: (1) desialylation of membrane glycoconjugates with the exposure of the penultimate beta-galactosyl residues and (2) exposure of phosphatidylserine in the membrane outer leaflet. Leukodepleted and nonleukodepleted RBCs were compared using flow cytometric procedures to determine whether the in vitro deterioration of RBCs during storage might be attributable to an identical mechanism of desialylation induced by leukocyte neuraminidases, resulting in exposure of beta-galactosyl and subsequently phosphatidylserine residues - signals of senescent RBCs. Without prior leukodepletion, stored RBCs showed an increased population of senescent RBCs (using light scatter measurements), extensive desialylation with the exposure of beta-galactosyl residues (using specific fluorescein isothiocyanate [FITC]-lectins), significant exposure of phosphatidylserine in the outer leaflet of the RBC membrane (using FITC-annexin V), and extensive in vitro phagocytosis (using PKH-26-labeled RBCs). There were minimal changes observed with the leukodepleted RBCs. These results lead to the conclusion that leukocyte enzymes, including neuraminidases, are definitive contributers to the desialylation of RBCs during storage and to the exposure of phosphatidylserine residues. These deleterious effects resulting from highly active leukocyte enzymes are preventable by prior leukodepletion of the stored RBCs. Previously developed flow cytometric procedures to detect in vivo "RBC senescence" have been applied and proved to be reliable criteria to monitor the viability of stored RBCs.     

Phospholipid composition of packed red blood cells and that of extracellular vesicles show a high resemblance and stability during storage

Red blood cells (RBCs) are stored up to 35–42 days at 2–6 °C in blood banks. During storage, the RBC membrane is challenged by energy depletion, decreasing pH, altered cation homeostasis, and oxidative stress, leading to several biochemical and morphological changes in RBCs and to shedding of extracellular vesicles (EVs) into the storage medium. These changes are collectively known as RBC storage lesions. EVs accumulate in stored RBC concentrates and are, thus, transfused into patients. The potency of EVs as bioactive effectors is largely acknowledged, and EVs in RBC concentrates are suspected to mediate some adverse effects of transfusion. Several studies have shown accumulation of lipid raft–associated proteins in RBC EVs during storage, whereas a comprehensive phospholipidomic study on RBCs and corresponding EVs during the clinical storage period is lacking. Our mass spectrometric and chromatographic study shows that RBCs maintain their major phospholipid (PL) content well during storage despite abundant vesiculation. The phospholipidomes were largely similar between RBCs and EVs. No accumulation of raft lipids in EVs was seen, suggesting that the primary mechanism of RBC vesiculation during storage might not be raft -based. Nonetheless, a slight tendency of EV PLs for shorter acyl chains was observed.     

Challenges for red blood cell biomarker discovery through proteomics

Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.     

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.     

Degradation of specific polyunsaturated fatty acids in red blood cells stored at -20 degrees C proceeds faster in patients with schizophrenia when compared with healthy controls

Deficits in red blood cell (RBC) polyunsaturated fatty acids (PUFAs) have been extensively reported in schizophrenia although reports are inconsistent. A possible explanation for this inconsistency is varying storage conditions of blood samples prior to analysis, especially freezer storage temperature. We conducted a prospective investigation of fatty acid degradation rates in RBCs from healthy control subjects when samples from each individual were stored at both -20 degrees C or -70 degrees C. Differences were detected between storage conditions. A second prospective study was conducted to investigate the effect of differential storage conditions on RBC membrane fatty acids from schizophrenic patients. We found that storage at -20 degrees C was associated with reduced levels of PUFAs. Comparison of decay rates suggest that schizophrenics decay approximately twice as rapidly as controls. Furthermore, this phenomenon appears to be specific for the longer chain PUFAs suggesting that an enzymatic process may be responsible, e.g. elevated phospholipase A(2) activity, as opposed to simple chemical oxidation.     

How malaria parasites reduce the deformability of infected red blood cells

The pathogenesis of malaria is largely due to stiffening of the infected red blood cells (RBCs). Contemporary understanding ascribes the loss of RBC deformability to a 10-fold increase in membrane stiffness caused by extra cross-linking in the spectrin network. Local measurements by micropipette aspiration, however, have reported only an increase of ～3-fold in the shear modulus. We believe the discrepancy stems from the rigid parasite particles inside infected cells, and have carried out numerical simulations to demonstrate this mechanism. The cell membrane is represented by a set of discrete particles connected by linearly elastic springs. The cytosol is modeled as a homogeneous Newtonian fluid, and discretized by particles as in standard smoothed particle hydrodynamics. The malaria parasite is modeled as an aggregate of particles constrained to rigid-body motion. We simulate RBC stretching tests by optical tweezers in three dimensions. The results demonstrate that the presence of a sizeable parasite greatly reduces the ability of RBCs to deform under stretching. With the solid inclusion, the observed loss of deformability can be predicted quantitatively using the local membrane elasticity measured by micropipettes.     

Stored red blood cells: A changing universe waiting for its map(s)

The availability of stored red blood cells (RBCs) for transfusion remains an important aspect of the treatment of polytrauma, acute anemia or major bleedings. RBCs are prepared by blood banks from whole blood donations and stored in the cold in additive solutions for typically six weeks. These far from physiological storage conditions result in the so-called red cell storage lesion that is of importance both to blood bankers and to clinical practitioners. Here we review the current state of knowledge about the red cell storage lesion from a proteomic perspective. In particular, we describe the current models accounting for RBC aging and response to lethal stresses, review the published proteomic studies carried out to uncover the molecular basis of the RBC storage lesion, and conclude by suggesting a few possible proteomic studies that would provide further knowledge of the molecular alterations carried by RBCs stored in the cold for six weeks.     

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia ^1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens ⁴), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs ^{4, 5}. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions³. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers ^{6, 7}, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane ^{8, 9} and mask RBC surface antigens^{10, 11}.     

From stem cells to red blood cells:how far away from the clinical application?

The generation of red blood cells(RBCs)from stem cells provides a solution for deficiencies in blood transfusion.Currently,primary hematopoietic stem cells,embryonic stem cells and induced pluripotent stem cells have shown the potential to produce fully mature RBCs.Here,we discuss the advantages,induction protocols,progress and possible clinical applications of stem cells in RBC production.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol?1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

Heritability of glutathione and related metabolites in stored red blood cells

Red blood cells (RBCs) collected for transfusion deteriorate during storage. This deterioration is termed the “RBC storage lesion.” There is increasing concern over the safety, therapeutic efficacy, and toxicity of transfusing longer-stored units of blood. The severity of the RBC storage lesion is dependent on storage time and varies markedly between individuals. Oxidative damage is considered a significant factor in the development of the RBC storage lesion. In this study, the variability during storage and heritability of antioxidants and metabolites central to RBC integrity and function were investigated. In a classic twin study, we determined the heritability of glutathione (GSH), glutathione disulfide (GSSG), the status of the GSSG,2H⁺/2GSH couple (E_hc), and total glutathione (tGSH) in donated RBCs over 56 days of storage. Intracellular GSH and GSSG concentrations both decrease during storage (median net loss of 0.52±0.63 mM (median ± SD) and 0.032±0.107 mM, respectively, over 42 days). Taking into account the decline in pH, E_hc became more positive (oxidized) during storage (median net increase of 35±16 mV). In our study population heritability estimates for GSH, GSSG, tGSH, and E_hc measured over 56 days of storage are 79, 60, 67, and, 75%, respectively. We conclude that susceptibility of stored RBCs to oxidative injury due to variations in the GSH redox buffer is highly variable among individual donors and strongly heritable. Identifying the genes that regulate the storage-related changes in this redox buffer could lead to the development of new methods to minimize the RBC storage lesion.     

Red blood cell membrane water permeability increases with length of ex vivo storage

Water transport across the red blood cell (RBC) membrane is an essential cell function that needs to be preserved during ex vivo storage. Progressive biochemical depletion during storage can result in significant conformational and compositional changes to the membrane. Characterizing the changes to RBC water permeability can help in evaluating the quality of stored blood products and aid in the development of improved methods for the cryopreservation of red blood cells. This study aimed to characterize the water permeability (L_p), osmotically inactive fraction (b), and Arrhenius activation energy (E_a) at defined storage time-points throughout storage and to correlate the observed results with other in vitro RBC quality parameters. RBCs were collected from age- and sex-matched blood donors. A stopped flow spectrophotometer was used to determine L_p and b by monitoring changes in hemoglobin autofluorescence when RBCs were exposed to anisotonic solutions. Experimental values of L_p were characterized at three different temperatures (4, 20 and 37 °C) to determine the E_a. Results showed that L_p, b, and E_a of stored RBCs significantly increase by day 21 of storage. Degradation of the RBC membrane with length of storage was seen as an increase in hemolysis and supernatant potassium, and a decrease in deformability, mean corpuscular hemoglobin concentration and supernatant sodium. RBC osmotic characteristics were shown to change with storage and correlate with changes in RBC membrane quality metrics. Monitoring water parameters is a predictor of membrane damage and loss of membrane integrity in ex vivo stored RBCs.     

Review: In vitro generation of red blood cells for transfusion medicine: Progress,prospects and challenges

In vitro generation of red blood cells (RBCs) has the potential to circumvent the shortfalls in global demand for blood for transfusion applications. The conventional approach for RBC generation has been from differentiation of hematopoietic stem cells (HSCs) derived from cord blood, adult bone marrow or peripheral blood. More recently, RBCs have been generated from human induced pluripotent stem cells (hiPSCs) as well as from immortalized adult erythroid progenitors. In this review, we highlight the recent advances to RBC generation from these different approaches and discuss the challenges and new strategies that can potentially make large-scale in vitro generation of RBCs a feasible approach.     

The immune-stimulation capacity of liposome-treated red blood cells

Our in vivo studies on a rat model established safety of transfusing liposome-treated red blood cells (RBCs) but identified the potential for immune modulation as related to transfusion efficacy of liposome-treated RBCs. The aim of this study was at assessing the impact of liposome-induced membrane changes on the immune profile of liposome-treated RBCs by (a) evaluating their interaction with endothelial cells and monocytes; and (b) the resulting immune response derived from this interaction, in the form of cytokine release, adhesion molecules expression and phagocytosis. Unilamellar liposomes were synthesized to contain unsaturated phospholipids (1,2-dioleoyl-sn-glycero-3-phosphocholine [DOPC]:CHOL, 7:3?mol%). The human RBCs immune profile was assessed by incubating control and DOPC-treated RBCs with human umbilical vein endothelial cells (HUVECs) and monocytes. Cytokine release measured by Luminex technology, vascular cell adhesion molecule (VCAM)-1 and E-selectin on HUVECs measured by flow cytometry, and the erythrophagocytic activity of monocytes by monocyte monolayer assay (MMA) were determined. Fibroblast growth factor [FGF]-2 was the only cytokine released by HUVECs that remained increased after incubation with DOPC-treated RBCs compared to control throughout storage. The expression of both VCAM-1 (15.3?±?5.6% versus 6.3?±?0.9%, p?=?0.008) and E-selectin (18.0?±?6.3% versus 6.6?±?0.7%, p?=?0.004) by HUVECs were significantly increased after incubation with DOPC-treated RBCs at day 2 of storage. The MMA resulted in phagocytic indexes of zero for both control and DOPC-treated RBCs at day 2 and 42 of storage. The liposome treatment did not result in significant changes to the immune profile of stored DOPC-treated RBCs. These findings combined with previous in vivo results, make liposome treatment a potential candidate for application in RBC preservation and open the possibility for clinical use with other cell types.     

Effects of erythrocyte flexibility on microvascular perfusion and oxygenation during acute anemia

Responses to exchange transfusion using red blood cells (RBCs) with normal and reduced flexibility were studied in the hamster window chamber model during acute moderate isovolemic hemodilution to determine the role of RBC membrane stiffness in microvascular perfusion and tissue oxygenation. Erythrocyte stiffness was increased by 30-min incubation in 0.02% glutaraldehyde solution, and unreacted glutaraldehyde was completely removed. Filtration pressure through 5-microm pore size filters was used to quantify stiffness of the RBCs. Anemic conditions were induced by two isovolemic hemodilution steps using 6% 70-kDa dextran to a hematocrit (Hct) of 18% (moderate hemodilution). The protocol continued with an exchange transfusion to reduce native RBCs to 75% of baseline (11% Hct) with either fresh RBCs (RBC group) or reduced-flexibility RBCs (GRBC group) suspended in 5% albumin at 18% Hct; a plasma expander (6% 70-kDa dextran; Dex70 group) was used as control. Systemic parameters, microvascular perfusion, capillary perfusion [functional capillary density (FCD)], and oxygen levels across the microvascular network were measured by noninvasive methods. RBC deformability for GRBCs was significantly decreased compared with RBCs and moderate hemodilution conditions. The GRBC group had a greater mean arterial blood pressure (MAP) than the RBC and Dex70 groups. FCD was substantially higher for RBC (0.81 +/- 0.07 of baseline) vs. GRBC (0.32 +/- 0.10 of baseline) and Dex70 (0.38 +/- 0.10 of baseline) groups. Microvascular tissue Po(2) was significantly lower for Dex70 and GRBC vs. RBC groups and the moderate hemodilution condition. Results were attributed to decreased oxygen uploading in the lungs and obstruction of tissue capillaries by rigidified RBCs, indicating that the effects impairing RBC flexibility are magnified at the microvascular level, where perfusion and oxygenation may define transfusion outcome.     

Identification of a complement receptor 1 peptide for inhibition of immune hemolysis

Complement sensitization of red blood cells (RBCs) can cause life-threatening hemolytic anemias. We have previously shown that complement receptor 1 (CR1) derivatives specifically the N-terminal region with decay accelerating activity (DAA) for inactivation of a key enzyme in the complement cascade can reduce complement-mediated RBC destruction in vitro and in an in vivo mouse model of hemolytic transfusion reaction. In the present study, we have modeled the N-terminal CR1 molecule based on the X-ray crystal structure of decay accelerating factor and the NMR structure of a homologous CR1 domain. Based on the homology model, we identified a 34-mer peptide encompassing the putative DAA which in vitro reduced hemolysis, C3a release and surface C3 deposition. More importantly, this peptide at 0.6 mM was effective in prolonging survival of transfused incompatible RBCs in vivo. Our results indicate that CR1-based structure-function studies may provide insights for developing structure-derived transfusion therapeutics in the future.     

Liposomes composed of unsaturated lipids for membrane modification of human erythrocytes

Abstract

Previous studies have shown that certain saturated lipids protect red blood cells (RBCs) during hypothermic storage but provide little protection during freezing or freeze-drying, whereas various unsaturated lipids destabilize RBCs during hypothermic storage but protect during freezing and freeze-drying. The protective effect of liposomes has been attributed to membrane modifications. We have previously shown that cholesterol exchange and lipid transfer between liposomes composed of saturated lipids and RBCs critically depends on the length of the lipid acyl chains. In this study the effect of unsaturated lipids with differences in their number of unsaturated bonds (18:0/18:1, 18:1/18:1, 18:2/18:2) on RBC membrane properties has been studied. RBCs were incubated in the presence of liposomes and both the liposomal and RBC fraction were analyzed by Fourier transform infrared spectroscopy (FTIR) after incubation. The liposomes caused an increase in RBC membrane conformational disorder at suprazero temperatures. The fluidizing effect of the liposomes on the RBC membranes, however, was found to be similar for the different lipids irrespective of their unsaturation level. The gel to liquid crystalline phase transition temperature of the liposomes increased after incubation with RBCs. RBC membrane fluidity increased linearly during the first 8 hours of incubation in the presence of liposomes. The increase in RBC membrane fluidity was found to be temperature dependent and displayed Arrhenius behaviour between 20 and 40°C, with an activation energy of 88 kJ mol^-1. Taken together, liposomes composed of unsaturated lipids increase RBC membrane conformational disorder, which could explain their cryoprotective action.     

Survival of the fittest?--survival of stored red blood cells after transfusion.

During the last 90 years many developments have taken place in the world of blood transfusion. Several anticoagulants and storage solutions have been developed. Also the blood processing has undergone many changes. At the moment, in The Netherlands, red blood cell (RBC) concentrates (prepared from a whole blood donation and leukocyte-depleted by filtration) are stored for a maximum of 35 days at 4 degrees C in saline adenine glucose mannitol (SAGM). Most relevant studies show that approximately 20% of the RBCs is lost in the first 24 hr after transfusion. Even more remarkable is that the average life span is 94 days after a storage period of 42-49 days. Such observations create the need for a parameter to measure the biological age of RBCs as a possible predictor of the fate of RBCs after transfusion. The binding of IgG to RBCs can lead to recognition and subsequent phagocytosis by macrophages. This occurs during the final stages of the RBC life span in vivo. We determined the quantity of cell-bound IgG during storage, and found considerable variation between RBCs, but no significant storage-related change in the quantity of cell-bound IgG. The significance of this finding for predicting the survival of transfused RBCs in vivo remains to be established. Hereto we developed a flow cytometric determination with a sensitivity of 0.1% for the measurement of survival in vivo based on antigenic differences. This technique has various advantages compared with the 'classical' 51Cr survival method.     

Self-rotation of red blood cells in optical tweezers: prospects for high throughput malaria diagnosis

A simple and sensitive approach for detection of malarial parasite in blood samples is demonstrated. The approach exploits our finding that, in hypertonic buffer, a normal red blood cell (RBC) rotates by itself when trapped by an optical tweezers. The rotational speed increases linearly at lower trap-beam powers and more rapidly at higher powers. In contrast, under the same experimental conditions, RBC having a malarial parasite does not rotate. The rotational speeds of other RBCs from malaria-infected sample are of an order of magnitude less than that for normal RBC and also increase much more slowly with an increase in trap beam power than that for normal RBC. The difference in rotational speeds could be exploited for the diagnosis of malaria.