Red Blood Cell Storage Lesion Research Articles

Established and theoretical factors to consider in assessing the red cell storage lesion

AbstractThe collection and storage of red blood cells (RBCs) is a logistical necessity to provide sufficient blood products. However, RBC storage is an unnatural state, resulting in complicated biological changes, referred to collectively as the “storage lesion.” Specifics of the storage lesion have been studied for decades, including alterations to cellular properties, morphology, molecular biology of carbohydrates, proteins and lipids, and basic metabolism. Recently, mass spectrometry–based “omics” technology has been applied to the RBC storage lesion, resulting in many new observations, the initial effects of which are more information than understanding. Meanwhile, clinical research on RBC transfusion is considering both the efficacy and also the potential untoward effects of transfusing stored RBCs of different ages and storage conditions. The myriad biological changes that have now been observed during the storage lesion have been extensively reviewed elsewhere. This article focuses rather on an analysis of our current understanding of the biological effects of different elements of the storage lesion, in the context of evolving new clinical understanding. A synopsis is presented of both established and theoretical considerations of the RBC storage lesion and ongoing efforts to create a safer and more efficacious product.

Red blood cell storage affects the stability of cytosolic native protein complexes.

Refrigerated storage of red blood cell (RBC) units promotes the progressive accumulation of the so-called storage lesions, a widespread series of alterations to morphology, metabolism, and proteome integrity of stored RBCs. However, while storage lesions targeting the RBC membrane fraction have been widely documented, the cytosolic fraction is as yet an underinvestigated cause of the technical inconveniences related to the high abundance of hemoglobin. By exploiting a recently ideated preparative two-dimensional clear native electrophoresis, followed by mass spectrometry analysis, we could monitor the changes of soluble multiprotein complexes (MPCs) in RBCs after 0, 21, and 35 days of storage under standard blood banking conditions. Data indicate a substantial storage-dependent alteration of RBC MPCs, particularly of those involved in energy and redox metabolism, confirming previous evidence about the progressive dysregulation of these pathways in long-stored units. The use of native gel-based proteomics to investigate MPCs present in the RBC cytosolic fraction proved to be a powerful tool. Results collected represent a preliminary advance in the knowledge of the key role of native cytosolic MPCs in context of RBC storage lesion. Multiprotein organization and interacting partners of some key enzymes have been found to change during storage duration, suggesting that future studies will be needed to assess whether such alterations could influence their activity and efficiency.

The effects of an overnight holding of whole blood at room temperature on haemoglobin modification and in vitro markers of red blood cell aging.

Some effects of the red blood cell (RBC) storage lesion are well documented whereas others are not. Whether a period of room temperature hold (RTH) during RBC production enhances the RBC storage lesion has remained controversial. In this study, we compared whole blood (WB)-derived RBCs produced after 24-h RTH with rapidly cooled (RC) RBCs and tested them for classical metabolic markers and signs of oxidative damage. SAGM-RBCs were prepared from mixed and split pairs (n=12) of WB units. RBCs prepared after a 24-h period of RTH on day+1 after collection (RTH-RBCs) were compared with RC-RBCs. All RBCs were stored at 4°C for 42days with assay of in vitro variables on days+1, +15, +22, +29 and +42. The study examined standard quality parameters, glutathione, catalase and superoxide dismutase (SOD) activities, and indicative markers of oxidative cell damage including post-translational haemoglobin modification, malondialdehyde (MDA), and phosphatidylserine expression. RTH-RBCs exhibited decreased levels of potassium (1·98±0·26 vs. 5·23±0·65mmol/l) and of 2,3-diphosphoglycerate (2,3-DPG) on day+1 compared with RC-RBCs. Haemolysis rate on day+42 was higher in RTH-RBCs than in RC-RBCs (0·52±0·13 vs. 0·37±0·12%). The phosphatidylserine expression amounted to 0·25±0·20% in RTH-RBCs and 0·07±0·12% in RC-RBCs. Haemoglobin modification was not different between both RBC groups. RTH-RBCs showed slightly higher MDA concentration on days +29 and +42. RC-RBCs and RTH-RBCs show only small differences of classical in vitro parameters and no relevant differences in antioxidative metabolism and oxidative haemoglobin modification. These findings do not explain the loss observed in in vivo survival studies with RBCs.

Microparticles from red blood cell transfusion products induce a strong inflammatory host response

Red blood cell (RBC) transfusion is associated with increased morbidity and mortality in the critically ill. Adverse effects of transfusion may be mediated by red blood cell storage lesion. In this study, we hypothesized that MPs from stored RBC bags would induce a more pronounced host response than MPs from fresh RBC bags and that this response is dose dependent.

Storage of RBCs results in an increased susceptibility for complement‐mediated degradation

The objective of this study was to elucidate whether complement activation occurs during the storage of RBCs in newly formulated PAGGS-M storage medium. The reason for red blood cell (RBC) storage lesions is not yet fully understood. The contribution of complement to RBC storage lesion has not been extensively characterised. We investigated the surface expression of CD35, CD55, CD59 and CD47, as well as deposition of C3d, using flow cytometry over a storage period of up to 42 days on a weekly basis. C3d and the immunoglobulins IgG, IgM and IgA were additionally investigated via the direct antiglobulin test (DAT). The effect of contact with homologous serum for 30 min at 37 °C was also performed for C3d and CD35 and is subsequently termed as a 'transfusion simulation (TS)'. A weak but significant increase of C3d was observed prior to TS (anova P = 0.0103), whereas a stronger increase from 74.0 ± 12.4 to 101.2 ± 9.7 was observed post-TS (anova; P < 0.0001). These findings were confirmed by the DAT. CD35, CD55 and CD47 demonstrated a decrease in their expression over storage time (anova; P < 0.0001 each). The majority of changes occurred following 14 days. There was neither a decrease of CD59 observed nor an increase of IgG, IgM and IgA. RBCs are becoming increasingly susceptible to spontaneous complement deposition following TS, which might be associated with the decrease of C35 and CD55 by proteolytic cleavage and vesiculation during storage. As the impact of storage lesions is rather controversial, institutions involved in blood collection and administration of blood products should focus on carrying out research on the prevention of storage lesions.

Red blood cell storage lesion.

To summarize current understanding of the mechanisms responsible for changes occurring during red blood cell (RBC) storage, collectively known as the storage lesion, and to review the biological and clinical consequences of increasing storage time of RBCs. Human and veterinary clinical studies, experimental animal model studies, and reviews of the RBC storage lesion with no date restrictions. Experimental studies have characterized the evolution of human RBC and supernatant changes that occur during storage and form the basis for concern about the potential for harm from long-term storage of RBCs. Although 4 randomized controlled trials of varying sizes failed to find an association between RBC storage time and negative clinical outcomes, a recent meta-analysis and numerous observational clinical studies have demonstrated that transfusion of old versus fresh stored RBCs is associated with an increased risk of morbidity and mortality, particularly among trauma victims and cardiac surgery patients. Potential clinical consequences of RBC transfusion following development of the storage lesion include risk of organ dysfunction, organ failure, infections, and death. Experimental animal models have contributed to the evidence supporting adverse consequences of the RBC storage lesion. Studies on relevant RBC storage issues such as the effect of different preservative solutions and leukoreduction have been completed. Transfusion with RBCs stored for 42 days increases mortality in dogs with experimental sepsis. Storage of RBCs induces progressive biochemical, biomechanical, and immunologic changes that affect red cell viability, deformability, oxygen carrying capacity, microcirculatory flow, and recipient response. Most reports in the human and veterinary literature support the concept that there are deleterious effects of the RBC storage lesion, but additional studies with improved experimental design are needed to identify compelling reasons to modify current blood banking and transfusion practices.

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 (Ehc), 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±0.63mM (median ± SD) and 0.032±0.107mM, respectively, over 42 days). Taking into account the decline in pH, Ehc became more positive (oxidized) during storage (median net increase of 35±16mV). In our study population heritability estimates for GSH, GSSG, tGSH, and Ehc 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.

An update on red blood cell storage lesions, as gleaned through biochemistry and omics technologies.

Red blood cell (RBC) aging in the blood bank is characterized by the accumulation of a significant number of biochemical and morphologic alterations. Recent mass spectrometry and electron microscopy studies have provided novel insights into the molecular changes underpinning the accumulation of storage lesions to RBCs in the blood bank. Biochemical lesions include altered cation homeostasis, reprogrammed energy, and redox metabolism, which result in the impairment of enzymatic activity and progressive depletion of high-energy phosphate compounds. These factors contribute to the progressive accumulation of oxidative stress, which in turn promotes oxidative lesions to proteins (carbonylation, fragmentation, hemoglobin glycation) and lipids (peroxidation). Biochemical lesions negatively affect RBC morphology, which is marked by progressive membrane blebbing and vesiculation. These storage lesions contribute to the altered physiology of long-stored RBCs and promote the rapid clearance of up to one-fourth of long-stored RBCs from the recipient's bloodstream after 24 hours from administration. While prospective clinical evidence is accumulating, from the present review it emerges that biochemical, morphologic, and omics profiles of stored RBCs have observable changes after approximately 14 days of storage. Future studies will assess whether these in vitro observations might have clinically meaningful effects.

Artificial oxygen carrier with pharmacologic actions of adenosine-5'-triphosphate, adenosine, and reduced glutathione formulated to treat an array of medical conditions.

Effective artificial oxygen carriers may offer a solution to tackling current transfusion medicine challenges such as blood shortages, red blood cell storage lesions, and transmission of emerging pathogens. These products, could provide additional therapeutic benefits besides oxygen delivery for an array of medical conditions. To meet these needs, we developed a hemoglobin (Hb)-based oxygen carrier, HemoTech, which utilizes the concept of pharmacologic cross-linking. It consists of purified bovine Hb cross-linked intramolecularly with open ring adenosine-5'-triphosphate (ATP) and intermolecularly with open ring adenosine, and conjugated with reduced glutathione (GSH). In this composition, ATP prevents Hb dimerization, and adenosine promotes formation of Hb polymers as well as counteracts the vasoconstrictive and pro-inflammatory properties of Hb via stimulation of adenosine receptors. ATP also serves as a regulator of vascular tone through activation of purinergic receptors. GSH blocks Hb's extravasation and glomerular filtration by lowering the isoelectric point, as well as shields heme from nitric oxide and reactive oxygen species. HemoTech and its manufacturing technology have been broadly tested, including viral and prion clearance validation studies and various nonclinical pharmacology, toxicology, genotoxicity, and efficacy tests. The clinical proof-of-concept was carried out in sickle cell anemia subjects. The preclinical and clinical studies indicate that HemoTech works as a physiologic oxygen carrier and has efficacy in treating: (i) acute blood loss anemia by providing a temporary oxygen bridge while stimulating an endogenous erythropoietic response; (ii) sickle cell disease by counteracting vaso-occlusive/inflammatory episodes and anemia; and (iii) ischemic vascular diseases particularly thrombotic and restenotic events. The pharmacologic cross-linking of Hb with ATP, adenosine, and GSH showed usefulness in designing an artificial oxygen carrier for multiple therapeutic indications.

The heritability of metabolite concentrations in stored human red blood cells

The degeneration of red blood cells (RBCs) during storage is a major issue in transfusion medicine. Family studies in the 1960s established the heritability of the RBC storage lesion based on poststorage adenosine triphosphate (ATP) concentrations. However, this critical discovery has not been further explored. In a classic twin study we confirmed the heritability of poststorage ATP concentrations and established the heritability of many other RBC metabolites. ATP concentrations and metabolomic profiles were analyzed in RBC samples from 18 twin pairs. On samples stored for 28 days, the heritability of poststorage ATP concentrations were 64 and 53% in CP2D- and AS-3-stored RBCs, respectively. Metabolomic analyses identified 87 metabolites with an estimated heritability of 20% or greater. Thirty-six metabolites were significantly correlated with ATP concentrations (p ≤ 0.05) and 16 correlated with borderline significance (0.05 ≤ p ≤ 0.10). Of the 52 metabolites that correlated significantly with ATP, 24 demonstrated 20% or more heritability. Pathways represented by heritable metabolites included glycolysis, membrane remodeling, redox homeostasis, and synthetic and degradation pathways. We conclude that many RBC metabolite concentrations are genetically influenced during storage. Future studies of key metabolic pathways and genetic modifiers of RBC storage could lead to major advances in RBC storage and transfusion therapy.

Measures of stored red blood cell quality

Blood banking underpins modern medical care, but blood storage, necessary for testing and inventory management, reduces the safety and efficacy of individual units of red blood cells (RBCs). Stored RBCs are damaged by the accumulation of their own waste products, by enzymatic and oxidative injury, and by metabolically programmed cell death. These chemical activities lead to a complex RBC storage lesion that includes haemolysis, reduced in vivo recovery, energy and membrane loss, altered oxygen release, reduced adenosine tri-phosphate and nitric oxide secretion, and shedding of toxic products. These toxic products include lysophospholipids that can cause transfusion-related acute lung injury, free iron that can potentiate infections and cause inflammation, and shed microvesicles that can scavenge nitric oxide and potentiate inflammation and thrombosis. However, most of the obvious negative outcomes of RBC storage are uncommon and appear to be related to exceptionally bad units. Generally, the quality of stored RBCs is highly related to the conditions of storage, so refrigerator temperature, intact bags, residual leucocyte counts and visible haemolysis remain excellent general measures. Specific biochemical measures, such as adenosine 5'-triphosphate (ATP) and 2,3-diphosphoglycerate (DPG) concentrations, calcium and potassium content or lipid breakdown products, require specialized measures that are not widely available, involve destructive testing and generally reflect only a part of the storage lesion. This review describes a number of components of the storage lesion and their measurement and attempts to access the utility of the measures.

From the Journal archives: The red blood cell storage lesion: past, present, and future

This retrospective observational study was performed to determine if the age of transfused units of red blood cells (RBCs) was related to mortality. The study included 31 transfused patients admitted to the intensive care unit during 1992 with severe sepsis (i.e., sepsis associated with organ dysfunction, hypoperfusion, or hypotension). Nineteen of 31 patients (61%) died. On average, non-survivors received older RBC units than survivors. In non-survivors, the median age of the units was 24 days (range 7-36 days), whereas the median age in survivors was 21 days (range 5-35 days). Analyzing outcomes according to different age categories revealed that survivors received 85% of units that were less than ten days old, whereas non-survivors received 76% of units that were more than 20 days old. These differences were statistically significant (P < 0.0001). The authors concluded that the duration of storage of RBCs is directly related to the risk of mortality among transfused patients with severe sepsis. Recognizing the limitations of their small retrospective study that did not adjust for confounders, they also stated that further studies are needed to confirm or refute this association.

Donor Variation In Biological Mediators During Storage Of Packed Red Blood Cells

IntroductionDuring routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. MethodsCytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). ResultsOf the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P<0.001, ANOVA). Of particular interest, the concentrations of these nine biological mediators varied greatly between the individual PRBC units. ICAM-1, VCAM-1 and IL-1α concentrations each varied 10 fold between units (range 1000 – 10 000 pg/mL for each), sCD14 varied 5 fold (range 20 000 - 100 000 pg/mL), sCD62L varied 4.4 fold (range 9000 – 40 000 pg/mL), and sCD62P varied 6.5 fold (range 200 -1300 pg/ml). In addition, it was apparent that a sub population (3/8) of the units assessed consistently had the highest levels of ICAM-1, sCD106 (VCAM-1), sCD14, sCD62L, IL-1α, sCD62P and angiogenin. For sCD62P, in particular, this “high” sub population had significantly different levels of sCD62P at each time point compared to the other five units (P<0.05 at each time point). The remaining BRMs studied were at the limits of detection (<20 pg/mL) for every unit at each time point, and no storage related changes were evident. ConclusionsThere was minimal change in the BRMs studied relevant to storage duration of the PRBC units. The most notable differences in the levels of biological mediators present in PRBC SN were due to donor-to-donor variation. These data suggest high levels of BRMs and potential immune modulation in transfusion recipients may be the result of donor-associated differences rather than storage-associated differences in blood components. Disclosures:No relevant conflicts of interest to declare.

Paradoxical Effects Of Tumor Necrosis Factor-Alpha On Endothelial Adhesion Of Stored Human Red Blood Cells

IntroductionThe duration of red blood cell (RBC) storage is currently being examined as a possible determinant of clinical outcomes in transfused patients in large randomized trials in several patient populations, as well as in preclinical models. Biochemical and functional changes coincident with blood storage, referred to as the “storage lesion,” are numerous and complex. Longer storage periods have, for example, been linked to increased tendency of RBCs to adhere to endothelium. How the proadhesive effect of storage influences the transfusion recipient might depend in part on preexisting comorbidities, consistent with a “two-hit” model of transfusion morbidity. We therefore developed a two-hit in vitro model using older stored RBCs and TNF-α pretreatment (activation) of HUVECs in order to better understand mechanisms of post-transfusion complications related to RBC adhesion. MethodsRed blood cell (RBC) preparation and labeling: Using an IRB-approved protocol, stored packed RBC segments stored in additive solution (AS)-3 were obtained at 35-42 days storage from the Transfusion Service at Duke Hospital. The stored RBCs were removed aseptically and washed 3 times with PBS, labeled with fluorescent dye PKH26, and allowed to incubate for 3 minutes. BSA (1%) in DPBS was added, and after one minute, the labeled cells were washed three times in PBS with Ca++ and Mg++ while avoiding exposure to light. Finally 5 µL of labeled RBCs was suspended in 3 mL of PBS for adhesion assays.Adhesion assaysHuman umbilical vein endothelial cells (HUVECs), grown to confluence on glass slides precoated with a 2% gelatin solution in a 5% CO2 incubator at 37.1°C, were placed in a graduated height-flow chamber. The height was measured at 7 different points along the chamber. After 15 minutes of PBS flow (2 mL min-1), the RBC sample was introduced to the chamber slowly at a rate of 1.5 mL min-1. The RBCs were then allowed to incubate without flow for 5 minutes, and the number of adhered cells at each location (height) was recorded. After 5 minutes of fluidic flow with PBS, the number of adherent cells at each location, corresponding to varying shear stresses, was counted. Percent adhesion and shear stress were calculated at each height.TNF-α treatment of HUVECs: Where indicated, TNF-α (20 µL) was added to 20 mL of cell media to yield a final concentration of 10 ng mL-1, and allowed to incubate overnight (16 hours). ResultsAdhesion of 35-42-days stored RBCs to non-activated HUVECs varied widely, as reported, and at shear stresses of 1, 2, and 5 dynes/cm2 was 42.92%, 10.77%, and 2.157% respectively, with a typical inverse relation to shear stress (p<0.005). Surprisingly, adhesion of stored RBCs to TNF-α-pretreated HUVECs was consistently lower than that to non-pretreated HUVECs. Mean adhesion of stored RBCs to TNF-α-pretreated HUVECs at 1, 2, and 5 dynes/cm2 was 15.43%, 6.120%, and 1.819%. [Display omitted] SummaryIn summary, we find that endothelial adhesion of stored RBCs nearing expiration tends to be inhibited rather than increased by prior treatment of HUVECs with the cytokine TNF-α. Since these stored RBCs had been pre-storage leukoreduced, it is unlikely that there are leukocyte-related confounders. It is possible, therefore, that TNF-α confers a protective effect against RBC adhesion in the microcirculation in the setting of transfusion of older stored RBCs. The mechanism of the paradoxical TNF-α effect is currently under investigation and may involve nitric oxide, which is deficient in stored RBCs. Adverse events related to the RBC storage lesion may depend in a critical and complex manner on the underlying pathophysiology. Disclosures:No relevant conflicts of interest to declare.

Similar donors—similar blood?

Red blood cell (RBC) storage lesions have been suggested as contributing factors to suboptimal clinical outcomes. While undesirable effects of storage are well documented, their clinical relevance is still debated. Focus on storage time as the sole determinant of RBC quality ignores the variability in cell properties that may depend on factors other than age. Mechanical fragility (MF) aggregately reflects many storage-related functional and structural changes. This study evaluates interdonor versus intradonor variability, throughout storage, of both MF and autohemolysis (AH). Thirteen uniformly manufactured RBC units were collected initially as whole blood from nonsmoking, group A+, male Caucasian research donors. Mechanical stress was applied using a bead mill with oscillation at 50 Hz over durations varying from 0.5 to 60 minutes. MF profiles were described in terms of percent hemolysis after stresses of specified durations. Two months later, 11 of the 13 donors returned and assays were performed using the same protocol to allow comparison of intradonor versus interdonor variation. At 5 days postcollection, RBC MF profiles exhibited marked interdonor variability (up to twofold) overall. Both autolysis and MF across all units increased during storage-with rates of these increases varying by up to 10-fold for certain MF variables. Especially high AH and MF were observed for an outlier donor (with p < 0.05), for whom follow-up revealed previously undisclosed hereditary hypertriglyceridemia (levels exceeding approx. 1000 mg/dL). RBCs, even from similar donors, vary significantly in levels and changes of both AH and MF, the clinical significance of which must still be ascertained. While further study is needed, donors with severe hypertriglyceridemia may not be appropriate as blood donors due to the unacceptable level of hemolysis observed during storage of our affected study subject.

The level of complement activation fragments is higher in red blood cell units than segments

Recent studies have indicated that segments from red blood cell (RBC) units are poorly representative of the quality of the units themselves [1–3]. In these studies, there were significant differences in hematocrit, hemoglobin and ATP levels between the two sources. This finding raises the possibility that similar differences occur in other blood or plasma parameters as well. In the course of studies to determine the effect of the RBC storage lesion on adverse outcomes post-transfusion in trauma patients, we sampled segments and units and measured levels of several complement activation fragments by ELISA (Quidel Corp, San Diego, CA).

Effects of gamma irradiation and 15 days of subsequent ex vivo storage on the cytosolic red blood cell proteome analyzed by 2D‐DIGE and Orbitrap MS

Gamma irradiation of red blood cell (RBC) concentrates is routinely used to prevent transfusion-associated graft-versus-host disease. So far, the effects of ionizing radiation on RBC structure and function and especially the proteome are not fully understood. RBC concentrates were irradiated with 30 Gy and stored for 1 or 15 days at 4 ± 2°C. Following cell lysis and hemoglobin depletion, 2D-DIGE was used to examine the changes of the cytosolic RBC proteome. Significantly altered spots were analyzed using bottom-up proteomic approaches and selected marker proteins validated by western blotting. Gamma irradiation was found to enhance conventional RBC storage lesions. Following 15 days of postirradiation storage, the abundances of a total of 27 spots were significantly altered and 3 out of 13 identified proteins were selected and validated as potential marker proteins for the assessment of irradiation-induced cytosolic RBC lesions. Gamma irradiation and subsequent ex vivo storage according to the Council of Europe guidelines were found to affect RBC protein structures. The validated marker proteins can serve as a basis for the development of a screening assay to monitor the quality of irradiated RBC concentrates during ex vivo storage.

Red Blood Cell Lipidomics analysis through HPLC-ESI-qTOF: application to red blood cell storage

Recent developments in mass spectrometry (MS) have enabled fast and sensitive detection of lipid species in different biological matrices. In the present study we performed an on-line HPLC-microTOF-Q MS approach to the red blood cell (RBC) lipidome. We thus exploited bioinformatic tools for the interrogation of novel databases, such as LIPID MAPS. By means of ad hoc software suites for mass spectrometry-based metabolomics analyses, we could address the key biological issue of the RBC lipidome, within the framework of RBC storage for transfusion purposes. Samples were collected from subjects living in the province of Viterbo, where olive oil consumption represents a central aspect of the diet. On this ground, we could postulate a diet specific effect on the accumulation of lipid-specific storage lesions. The analyses yielded the tentative identification of a huge number of lipid molecules on the basis of accurate intact mass values and retention times, and MS/MS validation. This analytical workflow was exploited to consolidate existing knowledge on the RBC lipid composition and individuate statistically significant fluctuations of lipids throughout storage duration of RBC concentrates under blood bank conditions. Our analysis indicated ceramides, glycerophospholipids and sterols as key targets of RBC storage lesions to the lipidome, that will deserve further targeted investigations in the future. It also emerged how compositional analyses of the RBC lipidome might end up yielding different results on the basis of the background of the blood donor (i.e. diet), which might translate into region-specific lipidomic alterations over storage progression of RBC concentrates.

23. The challenges and limitations of current clinical methods for the hypothermic storage and cryopreservation of human red blood cells

One of the most important advances that led to the creation of blood banks and the widespread therapeutic use of blood products was the development of techniques for ex vivo blood preservation. All efforts to extend the storage of blood past a few days had failed until the discovery of the protective effects of glucose on citrated blood in 1916. Since then, the hypothermic storage of viable red blood cells (RBC) at 4 °C has advanced to the point where these cells can be preserved ex vivo for up to 45 days with modern additive and extender solutions. Cryopreservation of RBCs for transfusion is one of the most successful applications of the cryobiology research that followed the discovery of glycerol as a cryoprotectant. This presentation will review the challenges and limitations of the current methods used in blood banks for the hypothermic storage and cryopreservation of human RBC used in transfusion medicine. Emerging data on the RBC storage lesion and RBC cryoinjury will be discussed with a focus on changes to cell metabolism, oxidative injury and membrane microvesiculation. Current RBC quality control methods and international regulations will be briefly described and details on the Canadian Blood Services RBC Quality Monitoring program will be presented. Recent clinical data on the effects of hypothermic red blood cell storage and cryopreservation on recipient outcomes will be reviewed. While RBC transfusions have been used in the treatment of the anemic patient for almost 100 years, clinical data indicates that there is a need for additional improvements. Applying emerging principles and techniques in the preservation sciences would have important clinical impact on the quality and efficacy of hypothermic stored and cryopreserved RBC.

The effects of red blood cell preparation method on in vitro markers of red blood cell aging and inflammatory response

Studies are currently under way examining whether the age of stored red blood cells (RBCs) affects clinical outcome in transfusion recipients. The effects of storage duration on the RBC storage lesion are well documented, while fewer studies are available regarding the effect of RBC production method. In this study, we compared in vitro RBC quality variables and markers of inflammatory response in apheresis and whole blood (WB)-derived RBCs, specifically those prepared after an overnight room temperature hold (RTH) of WB. SAGM RBCs, prepared from WB after overnight RTH (n = 10), were compared to SAGM RBCs prepared using an apheresis device (Alyx, n = 10). As a control, SAGM RBCs were also prepared within 2 hours of WB collection (2-hr WB, n = 10). All RBCs were stored at 4°C for 42 days with weekly assay of in vitro variables, cytokines and/or chemokines, and neutrophil activation after incubation with RBC supernatant. RTH WB RBCs exhibited decreased levels of 2,3-diphosphoglycerate acid (2.3 μmol/g hemoglobin [Hb] ± 2.1 vs. 13.7 ± 1.3 μmol/g Hb) and morphology (160 ± 10 vs. 192 ± 5) on Day 1 and increased hemolysis (0.45 ± 0.21% vs. 0.31 ± 0.09%) and microparticles (6.1 ± 2.8/10(3) RBCs vs. 3.9 ± 1.1/10(3) RBCs) on Day 42 compared to apheresis RBCs. Gro-α and ENA-78 cytokine levels were significantly higher in RTH WB than Alyx RBCs during storage. CD11b expression was highest in neutrophils exposed to supernatant from RTH WB RBCs (p < 0.05). RBC preparation method has a meaningful effect on the RBC storage lesion, which should be taken into account in addition to length of storage.