Packed Red Blood Cells Storage Research Articles

Upregulation of miR-20a-5p as the Potential MicroRNA Marker in Red Blood Cell Storage Lesion.

Packed red blood cells (PRBCs) can be preserved for 42 days, and stored PRBCs have slow, dangerous changes over time during storage. miRNA is approximately 22 nucleotides long, a small single-stranded noncoding RNA molecule. miRNA guides by pairing bases with their downstream target mRNA to regulate negative expression. They are essential in many life processes, including cell differentiation, proliferation, and apoptosis. Therefore, miRNA alterations may represent possible biomarkers of PRBC storage lesions. This study is aimed at validating the miR-20a-5p in PRBC storage. Study Design and Methods. A total of 20 PRBC samples were divided into day 1 and day 20 storage groups. Total miRNA was extracted and quantified by probe-based RT-qPCR assays to explore the potential role of miRNAs in PRBC storage lesions. Upregulated miR-20a-5p in PRBC storage on day 20 compared to day 1. MiR-20a-5p promoted cell survival, which may affect the downstream regulation and decrease PRBC viability in prolonged storage. On this basis, this detection may help to assess the quality of stored PRBCs.

Age of Red Cells for Transfusion and Outcomes in Patients with ARDS.

Packed red blood cells (PRBCs), stored for prolonged intervals, might contribute to adverse clinical outcomes in critically ill patients. In this study, short-term outcome after transfusion of PRBCs of two storage duration periods was analyzed in patients with Acute Respiratory Distress Syndrome (ARDS). Patients who received transfusions of PRBCs were identified from a cohort of 1044 ARDS patients. Patients were grouped according to the mean storage age of all transfused units. Patients transfused with PRBCs of a mean storage age ≤ 28 days were compared to patients transfused with PRBCs of a mean storage age > 28 days. The primary endpoint was 28-day mortality. Secondary endpoints included failure-free days composites. Two hundred and eighty-three patients were eligible for analysis. Patients in the short-term storage group had similar baseline characteristics and received a similar amount of PRBC units compared with patients in the long-term storage group (five units (IQR, 3–10) vs. four units (2–8), p = 0.14). The mean storage age in the short-term storage group was 20 (±5.4) days compared with 32 (±3.1) days in the long-term storage group (mean difference 12 days (95%-CI, 11–13)). There was no difference in 28-day mortality between the short-term storage group compared with the long-term storage group (hazard ratio, 1.36 (95%-CI, 0.84–2.21), p = 0.21). While there were no differences in ventilator-free, sedation-free, and vasopressor-free days composites, patients in the long-term storage group compared with patients in the short-term storage group had a 75% lower chance for successful weaning from renal replacement therapy (RRT) within 28 days after ARDS onset (subdistribution hazard ratio, 0.24 (95%-CI, 0.1–0.55), p < 0.001). Further analysis indicated that even a single PRBC unit stored for more than 28 days decreased the chance for successful weaning from RRT. Prolonged storage of PRBCs was not associated with a higher mortality in adults with ARDS. However, transfusion of long-term stored PRBCs was associated with prolonged dependence of RRT in critically ill patients with an ARDS.

Two-step process of cytoskeletal structural damage during long-term storage of packed red blood cells.

Storage of packed red blood cells (PRBC) for 42 days causes morphological, structural, and functional changes in the red cells. To assess the quality of stored PRBC, it is important to evaluate the main components of the product. The aim of this study was to evaluate the kinetics of the structural transformations in the cytoskeleton of red cells during long-term storage (up to 42 days). Bags of PRBC were stored with CPD/SAGM solution at +4 °C. Cytoskeletal parameters were measured on days 3, 12, 19, 21, 24, 28, 35, and 42 of storage to determine their changes. Atomic force microscopy was used to obtain images and analyse the parameters of the cytoskeletal network. As the storage time increased, a general PRBC test was performed. Membrane fixatives were not used at any stage of the preparation of the specimens for cytoskeletal imaging. When PRBC were stored for 42 days, the main changes to the cytoskeletal mesh included rupture of filaments, merger of small pores into larger ones, a decrease of the number of pores, thickening of filaments, and an increase of membrane stiffness. A process of irreversible changes to the cytoskeleton started on days 19-21. A kinetic model of changes in the parameters of the cytoskeletal mesh with time of PRBC storage was created. Two stages of impairment in cytoskeletal elements were found: rupture of filaments and clustering of protein components. The typical time of development and specifics of these stages are discussed. The consequences of the altered configuration of the cytoskeleton are also discussed. Destruction of the red cell cytoskeleton can have a negative effect on the efficacy of blood transfusion and increase the risk of post-transfusion complications. Our findings can be used in clinical medicine to evaluate the quality of PRBC for blood transfusion as well as for studies of the molecular organisation of red cells undergoing various types of physical and chemical treatment.

Transfusion of Soluble Target Antigens to Pre-Immunized Recipients: A Previously Overlooked Mechanism in Transfusion-Related Acute Lung Injury

Transfusion related lung injury (TRALI) is a leading cause for transfusion related morbidity and mortality in industrialized countries. The majority of cases is induced by antibodies present in donor plasma, and exclusion of antibody carriers from the donor pool has decreased the number of TRALI cases significantly. However, TRALI is still reported. We have identified a new and previously overlooked mechanism in which soluble antigens present in the blood component become the target of pre-existing antibodies present in the recipient.Some of us have recently reported a case of TRALI in a female patient precipitating after the transfusion of 120 mL of leuko-reduced packed red blood cells (PRBC). Immediate serologic work-up revealed no antibody in the donor material; but anti-CD177 was detected in the recipient's blood. The following transfusions with two units of PRBCs from CD177 negative donors were uneventful. CD177 is a glycosylphosphatidylinositol-anchored protein exclusively expressed on neutrophils. Only a small minority of Caucasians (<5%) do not express CD177 and can form antibodies when challenged with CD177 during pregnancy or blood transfusions. CD177 functions as a major surface carrier protein for proteinase-3 (PR3), one key contributor to PECAM-1 dependent neutrophil extravasation.Using a sandwich ELISA, we could demonstrate the presence of soluble CD177 in complex with PR3 (sCD177/PR3) in human plasma. As demonstrated by surface plasmon resonance analysis (SPR), PR3 (KD 4.98e-8 M) and the CD177/PR3 complex (KD 7.39e-9 M) specifically bind to immobilized PECAM-1, whereas CD177 does not interact with PECAM-1. In vitro, TNF-alpha pre-treated endothelial cells were negative with anti-CD177 or anti-PR3 antibodies by flow cytometry, but became positive after incubation with sCD177/PR3. Subsequent stimulation with monoclonal anti-CD177 or anti-PR3 induced the production of reactive oxygen species (ROS) in sCD177/PR3 positive (+EC), but not negative (-EC), endothelial cells. The same results were obtained after incubation with human anti-CD177 IgG (hIgG) isolated from plasma. If pre-treated ECs were incubated with recombinant PR3, only monoclonal anti-PR3, but not anti-CD177 or anti-CD177 hIgG induced ROS production. When experiments were repeated with F(ab)2 fragments, overall effects were still present, but diminished. To study the effects of this interaction on EC barrier integrity, ECs were grown on a transwell system, and pre-treated as outlined above. Monoclonal anti-CD177 or anti-PR3 as well as anti-CD177 hIgG significantly increased the albumin-FITC flux through +EC, but not -EC. In the presence of recombinant PR3 only, anti-PR3, but not anti-CD177, increased the albumin flux. Finally, we could demonstrate that the filtration process of buffy coat-reduced red blood cells by standard blood bank technology enhances the concentration of sCD177/PR3 in the storage solution almost 6-fold.In summary, our data demonstrate the presence of sCD177/PR3 in plasma and PRBC storage solution following filtration. When sCD177/PR3 is transfused, it has the capacity to bind to PECAM-1 on the EC surface of the transfusion recipient. The exposure of sCD177/PR3 positive endothelial cells to antibodies against CD177 or PR3 induces endothelial activation and barrier dysfunction in a partially Fcgamma-receptor dependent mechanism, comparable to the mechanism described for “classical” antibody-dependent TRALI. The relevance of soluble target proteins in transfused blood components has previously been overlooked. Furthermore, we assume that the identified new pathway might also be involved in endothelial dysfunction present in Wegener's granulomatosis. DisclosuresNo relevant conflicts of interest to declare.

Transfusion of Red Blood Cells Stored More Than 28 Days is Associated With Increased Morbidity Following Spine Surgery.

A retrospective study. The aim of this study was to describe the association between storage duration of packed red blood cells (PRBCs) and perioperative adverse events in patients undergoing spine surgery at a tertiary care center. Despite retrospective studies that have shown that longer PRBC storage duration worsens patient outcomes, randomized clinical trials have found no difference in outcomes. However, no studies have examined the impact of giving the oldest blood (28 days old or more) on morbidity within spine surgery. The surgical administrative database at our institution was queried for patients transfused with PRBCs who underwent spine surgery between December 4, 2008, and June 26, 2015. Patients undergoing spinal fusion, tumor-related surgeries, and other identified spine surgeries were included. Patients were divided into two groups on the basis of storage duration of blood transfused: exclusively ≤28 days' storage or exclusively >28 days' storage. The primary outcome was composite in-hospital morbidity, which included (1) infection, (2) thrombotic event, (3) renal injury, (4) respiratory event, and/or (5) ischemic event. In total, 1141 patients who received a transfusion were included for analysis in this retrospective study; 710 were transfused exclusively with PRBCs ≤28 days' storage and 431 exclusively with PRBCs >28 days' storage. Perioperative complications occurred in 119 patients (10.4%). Patients who received blood stored for >28 days had higher odds of developing any one complication [odds ratio (OR) = 1.82; 95% confidence interval (95% CI), 1.20-2.74; P = 0.005] even after adjusting for competing perioperative risk factors. Blood stored for >28 days is independently associated with higher odds of developing perioperative complications in patients transfused during spinal surgery. Our results suggest that blood storage duration may be an appropriate parameter to consider when developing institutional transfusion guidelines that seek to optimize patient outcomes. 3.

Effect of Red Blood Cell Storage Duration on Outcome After Paediatric Cardiac Surgery: A Prospective Observational Study

Retrospective reviews have found the use of stored packed red blood cells (PRBCs) in priming to be associated with increased risk of postoperative complications. The purpose of study was to prospectively investigate the influence of duration of storage of PRBCs used in priming the cardiopulmonary bypass (CPB) circuit on the metabolic profile of the patients, and postoperative outcome after paediatric cardiac surgery. Between January 2015 and December 2015, 198 consecutive children operated for cardiac surgery using CPB and received blood for priming the circuit were included. Patients were divided into two groups based on the duration of storage of the blood, newer PRBCs group who received blood stored for ≤14days and the older PRBCs group who received blood stored for >14 days. Mean duration of blood storage used for priming in newer PRBCs blood group (n=103) was 8.4±3.7days while it was 21.9±4.5days in older PRBCs group (n=95). Metabolic parameters of the PRBCs improved to physiological limits in both the groups after initiation of CPB. Postoperative hepatic, pulmonary, haematological complications, sepsis and multi-organ failure were more in the old PRBCs group. However, the difference was not significant. Similarly, there was no significant difference in incidence of prolonged mechanical ventilation, intensive care unit stay and hospital stay and mortality between the two groups. Metabolic parameters of the stored blood become normal after initiation of CPB irrespective of duration of storage. In paediatric patients without significant co-morbidity, undergoing cardiac surgery, transfusion of washed stored blood up to 28days in CPB priming is safe especially if lesser amount of transfusion is required.

Morphology, membrane nanostructure and stiffness for quality assessment of packed red blood cells

Transfusion of packed red blood cells (PRBC) to patients in critical states is often accompanied by post-transfusion complications. This may be related with disturbance of properties of PRBC and their membranes during long-term storage in the hemopreservative solution. The purpose of our work is the study of transformation of morphology, membranes stiffness and nanostructure for assessment of PRBC quality, in vitro. By atomic force microscopy we studied the transformation of cell morphology, the appearance of topological nanodefects of membranes and by atomic force spectroscopy studied the change of membrane stiffness during 40 days of storage of PRBC. It was shown that there is a transition period (20–26 days), in which we observed an increase in the Young’s modulus of the membranes 1.6–2 times and transition of cells into irreversible forms. This process was preceded by the appearance of topological nanodefects of membranes. These parameters can be used for quality assessment of PRBC and for improvement of transfusion rules.

Procoagulant role of microparticles in routine storage of packed red blood cells: potential risk for prothrombotic post-transfusion complications

During routine storage, packed red blood cells (PRBC) undergo biochemical and morphological changes including loss of red blood cell (RBC) membrane asymmetry and release of microparticles (MPs) bearing phosphatidylserine (PS), a procoagulant phospholipid. This study investigated the association between PRBC storage duration, MP profile and procoagulant activity. Leukodepleted PRBC-supernatant (PRBC-SN; n=13) was prepared at weekly intervals throughout storage. Phospholipid-dependent procoagulant activity, assessed using a factor X-activated clotting time (XACT) assay, decreased throughout storage (p<0.0001), corresponding with increased procoagulant phospholipid content. As determined by flow cytometry, total numbers of MPs and of PS-bearing MPs increased by Day 28 of storage (p<0.01 and p<0.05, respectively, versus D1), and these MPs were predominantly RBC-derived (CD235+). Depletion of MPs from stored (Day 42) PRBC-SN using 0.22μm filters reduced the number of PS-bearing MPs (p<0.01) but did not increase XACT clotting times. Furthermore, the reduction in procoagulant activity when lactadherin was used to block PS was not altered pre- or post-filtration of PRBC-SN. In conclusion, routine PRBC storage was associated with accumulation of MPs (particularly RBC-derived PS-bearing MPs) and ofprocoagulant phospholipids; however, depletion of PS-bearing MPs by 0.22μm filtration did not reduce phospholipid-dependent procoagulant activity.

Nanodefects of membranes cause destruction of packed red blood cells during long-term storage

Packed red blood cells (PRBC) are used for blood transfusion. PRBC were stored for 30 days under 4°С in hermetic blood bags with CPD anticoagulant-preservative solution. Hematocrit was 50–55%. The distortions of PRBC membranes nanostructure and cells morphology during storage were studied by atomic force microscopy. Basic measurements were performed at the day 2, 6, 9, 16, 23 and 30 of storage and additionally 2–3 days after it.Topological defects occurred on RBC membranes by day 9. They appeared as domains with grain-like structures (“grains”) sized up to 200nm. These domains were appeared in almost all cells. Later these domains merged and formed large defects on cells. It was the formation of domains with the “grains” which was onset process leading eventually to destruction of PRBC. Possible mechanisms of transformation of PRBC and their membrane are related to the alterations of spectrin cytoskeleton. During this storage period potassium ions and lactat concentrations increased, pH decreased, intracellular concentration of reduced glutathione diminished in the preservative solution. Changes of PRBC morphology were detected within the entire period of PRBC storage. Discocytes predominated at the days 1 and 2. By day 30 PRBC transformed into irreversible echinocytes and spheroechinocytes. Study of defects of membranes nanostructure may form the basis of assessing the quality of the stored PRBC. This method may allow to work out the best recommendations for blood transfusion.

Changes in gene expression following trauma are related to the age of transfused packed red blood cells.

Transfusion of packed red blood cells (PRBCs) is associated with an increased incidence of nosocomial infections and an increased risk of death. The duration of storage before transfusion may influence these outcomes. Here, we explore the association between the age of transfused PRBCs and specific patterns of inflammatory gene expression in severely injured trauma patients. Severely injured trauma patients requiring intensive care unit treatment and receiving transfusion of PRBCs within 24 hours of the injury were recruited. Blood samples were obtained within 2 hours of the trauma, at 24 hours, and at 72 hours. Messenger RNA was extracted from whole blood, and gene expression was quantified using quantitative polymerase chain reaction. The median age of the units of PRBCs transfused to each patient was recorded. The primary outcome measure was the change in candidate gene expression over the initial 72 hours. Sixty-four patients were studied. Fifty-three patients (83%) were male, and the median age was 40.5 years (interquartile range [IQR], 31-59). Median Injury Severity Score (ISS) was 31.5 (IQR, 23-43), and 55 patients (86%) experienced a blunt injury. Forty-one patients (64%) developed a nosocomial infection, and 15 patients (23%) died before hospital discharge. Each patient received a median of 5 U of PRBCs (IQR, 4-9.8 U) during the first 24 hours of hospital admission. The median age of the units of PRBCs transfused in each patient was 20 days (IQR, 17-22 days). Older blood was associated with greater decreases in interleukin 12 (IL-12), IL-23, and RORγt (all p's < 0.05) gene expression over the initial 24 hours, greater decreases in IL-12 gene expression over 72 hours, and a rise in transforming growth factor β gene expression over the first 72 hours. A multivariate analysis confirmed the independence of these associations. Increasing the duration of storage of PRBCs before transfusion is associated with a pattern of gene expression consistent with more severe immunosuppression. Epidemiologic study, level III.

Effect of duration of packed red blood cell storage on morbidity and mortality in dogs after transfusion: 3,095 cases (2001-2010).

BackgroundAccumulating evidence suggests that transfusion of packed red blood cells (PRBCs) stored for >14 days is associated with increased rates of sepsis, multiple organ dysfunction, and mortality in human patients.ObjectiveTo determine if duration of PRBC storage has an effect on morbidity and mortality in dogs after transfusion.AnimalsDogs admitted to the Matthew J Ryan Veterinary Hospital of the University of Pennsylvania.MethodsA retrospective case review of dogs identified through blood bank logbooks that received PRBC transfusions (minimum, 5 mL/kg) between 2001 and 2010. Dogs were categorized according to major cause of anemia (eg, hemorrhage, hemolysis, ineffective erythropoiesis) for analysis.ResultsA total of 3,095 dogs received 5,412 PRBC units. Longer duration of PRBC storage was associated with development of new or progressive coagulation failure (P = .001) and thromboembolic disease (P = .005). There was no association between duration of PRBC storage and survival for all dogs overall. However, a logistic regression model indicated that for dogs with hemolysis, 90% of which had immune‐mediated hemolytic anemia, longer duration of PRBC storage was a negative risk factor for survival. For every 7 day increase in storage, there was a 0.79 lesser odds of 30 day survival (95% CI, 0.64–0.97; P = .024).Conclusions and Clinical ImportanceDuration of PRBC storage does not appear to be a major contributing factor to mortality in the overall canine population. However, longer duration of PRBC storage may negatively impact outcome in dogs with immune‐mediated hemolytic anemia, thus warranting further investigation with prospective studies.

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.

Single Nucleotide Variations In Spectrin-1β Accentuate The Red Blood Cell Storage Lesion

IntroductionDuring routine storage packed red blood cells (PRBC) undergo biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. Donor-to-donor variability in the severity of the storage lesion has been reported. The extent to which donor-associated differences in blood component storage affect blood product quality and post-transfusion outcome remains unknown. Murine models with single nucleotide variants (SNV) in gene encoding spectrin-1β were used to investigate the impact of mutations on the RBC storage lesion. MethodsTwo murine lineages with N-ethyl-N-nitrosourea (ENU) generated single SNV in Spnb1, encoding spectrin-1β (Table 1), were selected from the Australian Phenomics Facility library (http://databases.apf.edu.au/mutations). Using genetic selection, homozygous (HOM), heterozygous (HET) and unaffected (WT) mice from each strain were generated (C57BL/6 background strain). Murine blood was leucoreduced, prepared in SAGM (0.4 HCT) and stored at 4°C for time course assessment of RBC characteristics. At day (D), D2, D7, D14 and D21 of storage, RBC integrity and evidence of storage-related changes were investigated using RBC osmotic fragility and flow cytometric analysis of CD44, CD47, TER119 and phosphatidylserine (PS). Data were generated from analysis of blood from Spnb1 (pedigree spectrin-1β a) homozygous (HOM, n=3), heterozygous (HET, n=3) and unaffected (WT, n=2 ); Spnb1 (pedigree spectrin-1β b) HOM (n=4), HET( n=4); C57BL/6 (n=4). The Mann-Whitney Test and ANOVA were utilised for statistical analyses (95% CI).Table 1Pedigrees selected for mutations in RBC structural genes (Build 38)Gene (Protein)ChromosomeBase changeAmino acid changeAmino acid positionMutation genomic position (bp)DomainPolyPhen scoreSpnb1 (Spectrin-1β) (a)12A - GS - P85776,620,753Spectrin binding0.24 benignSpnb1 (Spectrin-1β) (b)12T - CD - G66476,621,331Spectrin binding0.2 benign ResultsAt D2 of storage SNV in Spnb1 did not alter RBC characteristics, with all mice studied demonstrating a similar resistance to osmotic lysis and levels of CD44, CD47, TER119 and PS. By D7 of storage, clear pedigree-related differences in RBC characteristics were evident. At D7, RBC from spectrin-1β(a) HOM mice had significantly increased osmotic fragility and exposure of PS as well as significantly reduced CD44 and TER119 expression compared to unaffected siblings and background strain. Of note, these changes were not evident in the spectrin-1β(b) HOM mice at D7. For both strains at D7, heterozygous SNV did not exhibit altered storage parameters. By D14 both HOM and HET spectrin-1β(a) mice demonstrated a phenotype consistent with an exacerbated RBC storage lesion, characterised by significantly increased osmotic fragility and exposure of PS, and reduced CD44 and CD47 compared to background strain. At D14 there was also evidence of exacerbation of the storage lesion in stored RBC from HOM spectrin-1β(b) mice (significantly increased PS), though this was not to the extent observed in the spectrin-1β(a) mice. By D21 all murine RBC were substantially degraded under these storage conditions. ConclusionsSNV in Spnb1,encoding RBC structural protein spectrin-1β, resulted in both early onset and exacerbation of the RBC storage lesion. Further, the degree of storage lesion and the point at which RBC degradation was observed was not only dependent on the homozygous or heterozygous status, but the mutation itself. These data demonstrate that minor genetic variation in genes encoding important RBC proteins contribute to donor related differences in PRBC storage. Disclosures:No relevant conflicts of interest to declare.

Lipids: Free Fatty Acids, Eicosanoids, and Lysophospholipids and the Pro-Inflammatory Effects of Transfusion

Abstract SCI-48Stored packed red blood cells (PRBCs) have been implicated in increased morbidity and mortality in critically ill patients, patients requiring cardiothoracic surgery, and injured patients following blunt and/or penetrating trauma. Lipids are generated during the routine storage of cellular blood components and have been implicated in transfusion-related acute lung injury (TRALI) and postinjury multiple organ failure (MOF). These lipids are comprised of two classes, as denoted by their retention times on normal-phase HPLC: 1) nonpolar lipids, including arachidonic acid, 5-hydroxy-eicosotetranoic acid (HETE), 12-HETE, 15-HETE, and 5-oxo-eicosotetranoic acid (5-oxo-ETE); and 2) a mixture of lysophosphatidylcholines (lyso-PCs), including stearoyl-, oleoyl-, or palmitoyl-lyso-PCs as well as C16 or C18 lyso-platelet activating factor. Importantly, all of these lipids were identified by quadrupole LC/MS/MS. The former are derived from red blood cells, because they are generated during the routine storage of prestorage leuko- and platelet-reduced PRBCs, while the latter are platelet-derived, and are generated during storage of platelet concentrates (PC) and unmodified PRBCs but not leukocyte-reduced PRBCs (LR-PRBCs). Generation of the nonpolar lipids requires an active phospholipase, such as peroxiredoxin-6, which accumulates during PRBC and LR-PRBC storage and appears to be active and T-phosphorylated. The nonpolar lipids do not accumulate during PC storage. These lipids are released into the plasma fraction of the stored component and have been shown to: 1) prime the polymorphonuclear leukocytes (PMN) oxidase; 2) activate primed PMNs; and 3) serve as the second event in a two-event model of TRALI in both rodents and sheep. This lipid priming of PMNs occurs through activation of specific receptors, for example G2A, for lyso-PCs, and causes activation of G-protein-linked cellular receptor, G-protein release, and stimulation of kinase cascades. This results in translocation of the cytosolic oxidase components and a change from a resting PMN phenotype to a hyperactive, adherent phenotype. Conversely, stored PRBCs have also been implicated in MOF. However, acute lung injury does not occur until 72 hours postinjury, implicating PRBCs as a first event(s). The nonpolar lipids that accumulate during routine storage activate primary human pulmonary microvascular endothelial cells (HMVECs) and primary human liver sinusoidal endothelial cells (LSECs), as quantified by increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and the synthesis and release of chemokines, for example IL-8. This proinflammatory activation results in PMN adherence, and such HMVEC activation occurs through activation of the BLT2 receptor and activation of a PKC-dependent kinase cascade. Stored PRBCs (day 42) may serve as the first event in a two-event rodent model of acute lung injury. We conclude that lipids generated during the routine storage of cellular blood components have proinflammatory effects in vitro and in vivo, which may explain some of the adverse events of transfusion, including TRALI and postinjury MOF, and that inhibition and avoidance of these compounds may make transfusions safer. Further research is already under way in several prospective trials, including: 1) Age of Blood Components transfused in the PICU (ABC-PICU); 2) red cell storage duration and outcomes in cardiac surgery at the Cleveland Clinic; 3) Red Cell Storage duration Study (RECESS); and 4) the Age of Blood Evaluation trial (ABLE). Disclosures:Silliman:Pall Corporation: Honoraria, Research Funding.

The storage lesion of packed red blood cells affects coagulation

The storage lesion of packed red blood cells (PRBCs) consists of biochemical changes associated with increased inflammatory mediators and decreased oxygen-carrying capacity. The effect of storage on the coagulation system is less well studied. The purpose of this study is to determine how PRBC storage time affects the activated coagulation time (ACT), using an invitro model. Type-matched PRBCs and fresh-frozen plasma were mixed, warmed to 37°C, and recalcified. The microsample ACT was repeatedly determined by 2 methods (ACT+ and ACT-LR) at 1 week, 3 weeks, and 5 weeks after date of donation. Hematocrit, pH, and ionized calcium were measured to assess how the model compared to physiologic circumstances. Statistical analysis was performed using the Student 2-tailed t-test with unequal variance. Hematocrit, pH, and ionized calcium were successfully maintained within narrow ranges. The model was notably acidotic, consistent with circumstances of shock and massive transfusion. There was no significant difference in the ACT+ between week 1 and week 3 (P = .183), but there was a significant difference between week 3 and week 5 (P = .029) and between week 1 and week 5 (P = .007). The ACT-LR showed a significant difference between week 1 and week 3 (P = .001), but not between week 3 and week 5 (P = .286). There was again a strong difference between week 1 and week 5 results (P = .011). In an isolated invitro model, the storage lesion of PRBCs is associated with decreased coagulation. This may have relevance for transfusion practices in coagulation-sensitive circumstances such as trauma.

Apoptotic Microrna Profiling of Packed Red Blood Cells During Storage.

Abstract 3145Poster Board III-82 IntroductionDuring storage, RBCs undergo physiological changes often termed as storage lesions that adversely affect their survival in vivo, following transfusion. MicroRNAs (miRs), the negative regulators of cellular mRNAs, control cellular expression of genes relevant to differentiation and apoptosis via mRNA degradation or inhibition of translation. Recent reports indicate that matured red blood cells (RBC) contain diverse population of miRs in abundance. Understanding the role of miRs in RBC during storage would perhaps provide insights into the mechanisms associated with storage lesions. MethodsIn this study, we utilized a membrane-based array to obtain differential miR profiles of 52-apoptosis-associated miRs in packed RBCs during storage. The packed RBCs were obtained from the National Institutes of Health (NIH) blood bank and stored at appropriate storage condition (4-8°C) for up to 40 days. Samples were collected at days 0, 10, 20, 30 and day 40 and subjected to miR analysis. Our rationale is that since miRs are regulators of apoptosis, profiling of apoptosis associated miRs in packed RBCs during storage would provide the first step towards understanding the underlying mechanisms associated with storage lesions. ResultsOur miR analysis identified perturbation of six miRNAs during packed RBC storage. Two miRs remain at high levels throughout the RBC storage period studied while four miRs demonstrated an upward trend from day 0 to day 40 of storage. TarMir bioinformatics-based target gene identification for miR-96 identified CASPN1 mRNA as its target. The presence of CASPN1 mRNA was confirmed by RT-PCR. Although this observation tempts us to speculate that an interaction of CASPN1 with miR-96 in stored RBC is possible, further experimental verification of this interaction is warranted. ConclusionThe differential microarray analysis presented here suggest that further refinement of miR profiling of stored red blood cells could be of value as predictive markers of ‘cellular status of RBC' during storage. Future experimental analysis of selected mRNA-miR interactions in packed RBC during storage would provide insights into the mechanisms of storage lesionsThe findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. DisclosuresNo relevant conflicts of interest to declare.

The effect of packed red blood cell storage on arachidonic acid and advanced glycation end-product formation

The transfusion of packed red blood cells (PRBCs) is a significant risk to blood recipients. Blood banking procedures permit the storage of PRBCs for up to 42 days. Storage of PRBCs can cause polymorphonuclear granulocytes (PMNs) activation and the development of neutrophil-mediated transfusion-related acute lung injury. The aim of our study was to determine if PRBC storage has an influence on the formation of arachidonic acid (AA) and advanced glycation end products (AGEs). Twenty units of PRBCs were used to measure AA and AGE levels. The samples were taken on the 0th, 14th, 28th, and 42nd days of PRBC storage. The AA level was analyzed by gas-liquid chromatography-mass spectrometry and AGE level by an immunoenzymatic test. During the first 14 days of PRBC storage, the AA level significantly increased and then slowly decreased. The AGE level increased continuously during the whole time of the study. In a model experiment, the AA glycoxidation product trans-2-nonenal (T2N) formed adducts in reaction with hemoglobin which were detectable with the test for AGE. It is highly probable that the observed increase in AGE level is related to the decrease in AA in PRBCs, which can be associated with the formation of toxic aldehydes, especially T2N and 4-hydroxynonenal (HNE), from AA. Glucose in the PRBCs (preservative solution) can contribute to AGE formation as well. The formation of AGEs, HNE, and T2N in PRBCs, their influence on PMNs in vitro, and confirmation of our assumption need further studies.

Effects of fresh versus old stored blood in the priming solution on whole blood lactate levels during paediatric cardiac surgery

It has been suggested that lactate levels may predict morbidity and mortality in paediatric patients during corrective open-heart surgery. Packed red blood cells (PRBC) are frequently necessary for priming the reservoir used in cardiopulmonary bypass (CPB). The storage of PRBC might cause a significant increase in lactate levels. The purpose of the present study was to quantify the increase in lactate levels in stored red blood cells over time and to compare lactate levels after transfusions of fresh (< or =12 days) versus old blood (>12 days) in 20 patients. We found an increase in lactate levels from 6.0 to 44.7mmol/L (mean 17.0+/-7.8 mmol/L) during storage. Lactate levels were also significantly higher after the onset of CPB in paediatric patients transfused with old blood than in patients transfused with fresh blood (1.43+0.36 versus 3.46+/-0.63, p=0.0006). Our results suggest that the higher lactate levels found after the initiation of CPB should be used with caution when assessing tissue hypoxia and predicting outcome.

Hypotension and acute pulmonary insufficiency following transfusion of autologous red blood cells during surgery: a case report and review of the literature

Transfusion of autologous blood is associated with fewer complications, although all untoward events of transfusion may not be negated with this strategy. We report a case of acute pulmonary insufficiency and hypotension following transfusion of autologous packed red blood cells (PRBCs) in a patient, who was undergoing major surgery. Anti-HLA class-I and class-II and anti-granulocyte antibodies were measured in the unit and in the recipient. Neutrophil (PMN)-priming activity was measured as the augmentation of the formyl-Met-Leu-Phe-activated respiratory burst. No immunoglobulins were identified; however, significant lipid-priming activity was present in the implicated, autologous PRBC unit that primed PMNs from both healthy people and the recipient. In addition, lipids, identical to those that accumulate during PRBC storage, caused significant hypotension when infused into rats at similar concentrations found in stored PRBCs. We conclude that the observed transfusion-related acute lung injury reaction with significant hypotension may be the result of two independent events: the first is related to inherent host factors, in this case major surgery, and the second is the infusion of lipids that accumulate during the routine storage of PRBCs.

Erythrocyte Transfusion and Postsurgical Morbidity in Cardiac Surgery Patients: Is It the Storage Time or the Number of Transfused Concentrates that Really Matters?

To the Editor:—We read with interest the article about the influence of erythrocyte concentrate storage time on postoperative morbidity in cardiac surgery patients published by Leal-Noval et al. in a recent issue of the Journal, 1which was accompanied by an editorial view. 2The main finding of this study was an association between storage time of packed red blood cells (PRBC) and the acquisition of postoperative nosocomial pneumonia, but not between the former and prolonged mechanical ventilation time. We would like to comment regarding two points. First, we wish to review some mechanisms by which the relationship between the number of units and pneumonia/ventilation might be connected. Second, we want to point out that the problem may not simply be the age of the transfused blood, but also the absolute numbers of units transfused.The current authors suggested that allogeneic transfusion may cause some deleterious effects that have not been recognized and which may explain the association between the number of PRBC transfusions and prolonged mechanical ventilation, but not between the duration of storage and prolonged mechanical ventilation. 1Induction of a subclinical transfusion-related acute lung injury (TRALI) could be one of those frequently underdiagnosed deleterious effects. It has been proposed that TRALI is the result of two clinical events, the first being a predisposing clinical condition (e.g. , a postoperative systemic inflammatory response syndrome) and the second being the transfusion of biologically active molecules in stored PRBC and platelets that have neutrophil priming activity. The concentration of some of these molecules (e.g. , cytokines, neutral lipids, lysophosphatidylserine) in the stored blood component increases with the product age, whereas that of other molecules (e.g. , human lymphocyte antibody class I, class II, and granulocyte antibodies) does not. To this regard, Silliman et al. 3have found that TRALI was associated with the transfusion of older blood products, especially platelets, and that patients with cardiac disease who required cardiopulmonary bypass were at particular risk to develop TRALI reactions. However, although a significant proportion of patients (135, 16.9%) received platelet transfusion, Leal-Noval et al. 1have not studied the possible association between either the number or the age of platelet concentrates and prolonged mechanical ventilation time. Despite that, because the number of PRBC transfused was significantly higher in patients with mechanical ventilation time greater than 24 h, the development of a certain degree of TRALI, in a dose-dependent manner, may result in a worsening of the pulmonary dysfunction induced by cardiopulmonary bypass with a prolonged mechanical ventilation time and, perhaps, contributes to a higher incidence of postoperative nosocomial pneumonia. 4Regarding PRBC storage time, there are several storage-dependent alterations in erythrocytes that, together with transfusion-related immunomodulation, may contribute to the increased risk of nosocomial pneumonia in the transfused patients. The decrease in the ability of erythrocytes to deform and unload oxygen in the peripheral tissues, possibly favoring the degree of visceral ischemia, have been mentioned, 1,2but other alterations may account for when erythrocytes are stored together with white blood cells. Leukocyte enzymes, including neuraminidase, are definitive contributors to the desialylation of erythrocytes during storage and to the exposure of phosphatidylserine residues in the membrane outer leaflet. 5These changes make the erythrocytes more adherent to the inflamed endothelium and convert it into a procoagulant surface, thus contributing to the alteration of microvascular blood flow. In addition, these changes are also signals of senescence and enhance erythrocyte phagocytosis by resident macrophages. 5All methods in current use for erythrocyte storage result in the maintenance of 90% or more of the in vivo recovery for about 2–3 weeks; later an increasing proportion of erythrocytes become nonviable. When old PRBC are transfused, a substantial number of cells (some 1012erythrocytes in a four-unit transfusion) must be phagocytosed, which obviously uses part of the capacity of the phagocytic mononuclear system and may compete with infection defense with possible serious consequences, such as postoperative nosocomial pneumonia. 6We agree with Leal-Noval et al. 1with regard to the need for continued research of the possible deleterious effects of old PRBC transfusion. However, in previous work, 4this group showed an increase in the incidence of postoperative nosocomial pneumonia in those patients transfused with more than 4 units. In the present work, 1patients who developed postoperative nosocomial pneumonia received more PRBC units than those who did not (8.8 ± 4.7 vs. 5.4 ± 4.7, respectively; P &lt; 0.05). Because the authors found that each transfused PRBC unit increased the risk of postoperative pneumonia by 11%, 1we believe that the implementation of a perioperative blood-saving program, including several alternatives to allogeneic erythrocyte transfusion (e.g. , preoperative treatment of anemia, aprotinin in pump-prime, perioperative autologous salvage), together with a restrictive transfusion strategy, 7–9would probably be a better option to reduce postoperative morbidity in cardiac surgery patients.