Nonleukoreduced Red Blood Cell Research Articles

In vitro Evaluation of DINCH-Plasticized Blood Bags for Red Blood Cell Storage with CPDA-1 Anticoagulant

Introduction: Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer commonly used in blood bags. Despite its protective effects on red blood cell (RBC) storage, concerns about its reproductive toxicity exist. This study investigated the in vitro quality of RBC concentrates stored in bags using di(isononyl) cyclohexane-1,2-dicarboxylate (DINCH) as an alternative plasticizer. Methods: Using a pool-and-split study design, we produced 20 matched homogenous quintets of RBC concentrates in two DINCH bags and three DEHP bags with citrate phosphate dextrose adenine (CPDA-1) anticoagulant. RBC storage quality was assessed weekly for 35 days. Results: On day 35, the median hemolysis levels in the DINCH bags (0.297–0.342%) were marginally higher (p < 0.05) than the DEHP bags (0.204–0.240%). All DINCH bags showed <0.8% hemolysis. RBCs in the DINCH bags showed increased mean corpuscular volume and decreased eosin 5′ maleimide binding than in the DEHP bags. Higher pO₂ and lower pCO₂ levels in the DINCH bags indicated better gas permeability than in DEHP bags. Other metabolic parameters were comparable in both bags. Compared to DEHP, DINCH exhibited considerably lower levels of plasticizer leaching into blood bags. Conclusion: The quality of RBC concentrates stored for 35 days in DINCH-plasticized blood bags with CDPA-1 is generally comparable to those in DEHP bags. Hence, DINCH can be a viable alternative to DEHP in blood bags for nonleukoreduced RBC storage even without the use of next-generation additive solutions to improve RBC preservation quality.

Leukoreduction of red blood cell units decreases dysregulatory micro RNAs during routine storage: An observational study with In-silico analysis.

Red Blood cells (RBCs) bring about harmful consequences during storage. MicroRNA (miRNA) dysregulation in stored RBCs could represent potential biomarkers of storage lesions. Although leukoreduction prevents damage to RBCs, it is uncertain whether leukoreduction of RBCs would impact the dysregulation of miRNAs during storage. This study evaluated the potential role of miRNAs for any alteration of leukoreduced (LR) and non-leukoreduced (NLR) RBCs till 21 days of storage. In this prospective study, thirty male volunteers' blood was equally divided into leukoreduced RBCs (LR) and NLR RBC (NLR) bags and stored till Day 21 at 4-60c. Selected miRNAs were quantified on Days 0 and 21. Further, bioinformatic tools were used to analyze the selected miRNAs and their predicted target genes (mRNAs) and identify the miRNA-mRNA regulatory relationships. A significantly higher fold change values of three miRNAs (miR-96-5p, miR-197-3p, miR-769-3p) were observed in NLR RBCs (p < .05). A significantly higher (p < .05) expression levels of miR-150-5p and miR-197-3p were observed in NLR RBCs till 21 days of storage. Further, the correlation with mRNA quantification confirmed the regulatory role of these miRNAs upon functional pathway enrichment analysis. A higher level of dysregulation of miRNAs was observed in NLR RBCs. Validation from In-Silico analysis suggested the regulatory role of miRNAs in cell apoptosis, senescence, and RBC-related signaling pathways. This indicated that stored LR RBCs would likely have better in vivo survival and function following transfusion. However, an in vivo study of miRNA in RBCs is warranted for conclusive evidence.

Effect of leukoreduction on inflammation in critically ill dogs receiving red blood cell transfusions: A randomized blinded controlled clinical trial.

BackgroundPrestorage leukoreduction of red blood cell (RBC) bags prevents accumulation of pro‐inflammatory mediators and experimentally attenuates post‐transfusion inflammation in healthy dogs. However, the effect of leukoreduction on post‐transfusion inflammation in critically ill dogs is unclear.HypothesisDogs transfused with leukoreduced (LR) RBC will have lower concentrations of leukocytes, interleukin (IL)‐6, IL‐8, monocyte chemoattractant protein‐1 (MCP‐1), and C‐reactive protein (CRP) within 24 hours of post‐transfusion compared to dogs transfused with nonleukoreduced (NLR) RBC.AnimalsSixty‐one RBC‐transfused dogs (LR = 34, NLR = 27).MethodsRandomized, blinded, controlled preliminary clinical trial. Blood bag processing was randomized to create identically appearing LR and NLR bags. Group allocation occurred with transfusion of the oldest compatible RBC bag. Blood samples were collected pretransfusion and at 8 and 24 hours post‐transfusion for leukocyte count, IL‐6, IL‐8, MCP‐1, and CRP. Data were analyzed on an intention‐to‐treat basis using linear mixed effects models. Significance was set at P < .05.ResultsNo significant differences were found between groups in concentrations of leukocytes (P = .93), IL‐6 (P = .99), IL‐8 (P = .75), MCP‐1 (P = .69), or CRP (P = .18) over time. Eleven LR dogs (32%) and 4 NLR dogs (15%) were euthanized in the hospital (P = .14). No natural deaths occurred.Conclusions and Clinical ImportanceNo differences in inflammation biomarker concentrations were detected over time between dogs transfused with LR or NLR RBC, but heterogeneity likely hampered the ability to detect a difference with this sample size. The novel randomization and enrollment protocol was successfully implemented across 2 participating institutions and will be easily scaled up for a future multicenter clinical trial.

Association between leukoreduced red blood cell transfusions and hospital‐acquired infections in critically ill children: A secondary analysis of the TRIPICU study

Hospital-acquired infections (HAIs) are an important problem in critically ill children. Studies show associations between the transfusion of non-leukoreduced red blood cell units (RBC) and increased HAI incidence rates (IRs). We hypothesize that transfusing pre-storage leukoreduced RBC is also associated with increased HAI IR. We aim to evaluate the associations between (1) a leukoreduced RBC restrictive transfusion strategy and HAI IR, (2) leukoreduced RBC transfusions and HAI IR, and (3) the number or volume of leukoreduced RBC transfusions and HAI IR in critically ill children. This post hoc secondary analysis of the "Transfusion Requirement in Paediatric Intensive Care Units" (TRIPICU) randomized controlled trial (637 patients) used quasi-Poisson multivariable regression models to estimate HAI incidence rate ratios (IRRs) and 95% confidence intervals (CI). A restrictive transfusion strategy yielded an IRR of 0.88 (95% CI 0.67, 1.16). The association between transfusing leukoreduced RBCs (IRR 1.25; 95% CI 0.73, 2.13) and HAI IR was not statistically significant. However, we observed significant associations between patients who received >20 cc/kg volume of leukoreduced RBC transfusions (IRR 2.14; 95% CI 1.15, 3.99) and ≥3 leukoreduced RBC transfusions (IRR 2.40; 95% CI 1.15, 4.99) and HAI IR. Exposing critically ill children to >20 cc/kg or ≥3 leukoreduced RBC transfusions were associated with higher HAI IR, suggesting dose-response patterns.

Red blood cell alloimmunization in sickle cell disease: assessment of transfusion protocols during two time periods.

Prevention of red blood cell (RBC) alloimmunization in patients with sickle cell disease (SCD) focuses on phenotypic RBC matching. We assessed alloimmunization among transfused patients with SCD after implementing leukoreduction and prophylactic antigen matching (PAM). Retrospective review of transfusion and medical records for SCD patients 18 months to 81 years of age was performed covering two 5-year periods: Period 1, no PAM, occasional leukoreduction, and Period 2, consistent leukoreduction and extended PAM (Rh, Kell, S, Fy, Jk) for patients already alloimmunized. Patients transfused in Period 1 were excluded from Period 2. A total of 293 patients were transfused in Period 1 and 183 in Period 2. Median time between first sample and last type and screen after transfusion was 2.12 years in Period 1 and 1.03 years in Period 2. Initial alloimmunization prevalence was lower in Period 2 (26.2%) versus Period 1 (37.5%) and after subsequent transfusions in Period 2 (23.8%) versus Period 1 (45.7%), although without significant difference after adjusting for number of units transfused, percentage of leukoreduced RBCs, sex, and age. Alloimmunized patients received more nonleukoreduced RBCs in Period 1 than nonalloimmunized. Patients transfused during inflammatory conditions were not significantly more likely to become alloimmunized. The prevalence of initial and subsequent RBC alloimmunization in Period 2 was lower than that in Period 1; however, overall prevalence remained high. We recommend leukoreduced, hemoglobin S-negative Rh and Kell PAM RBCs for transfusion of patients with SCD. Component and recipient factors affecting alloimmunization should be studied further.

The effects of storage of red blood cells on the development of postoperative infections after noncardiac surgery.

Prolonged storage of red blood cells (RBCs) is a potential risk factor for postoperative infections. The objective of this study was to examine the effect of age of RBCs transfused on development of postoperative infection. In this prospective, double-blind randomized trial, 199 patients undergoing elective noncardiac surgery and requiring RBC transfusion were assigned to receive nonleukoreduced RBCs stored for not more than 14 days ("fresh blood" group, n = 101) or for more than 14 days ("old blood" group, n = 98). The primary outcome was occurrence of infection within 28 days after surgery; secondary outcomes were postoperative acute kidney injury (AKI), in-hospital and 90-day mortality, admission to intensive care unit, and hospital length of stay (LOS). As older blood was not always available, an "as-treated" (AT) analysis was also performed according to actual age of the RBCs transfused. The median [interquartile range] storage time of RBCs was 6 [5-10] and 15 [11-20] days in fresh blood and in old blood groups, respectively. The occurrence of postoperative infection did not differ between groups (fresh blood 22% vs. old blood 25%; relative risk [RR], 1.17; confidence interval [CI], 0.71-1.93), although wound infections occurred more frequently in old blood (15% vs. 5%; RR, 3.09; CI, 1.17- 8.18). Patients receiving older units had a higher rate of AKI (24% vs. 6%; p < 0.001) and, according to AT analysis, longer LOS (mean difference, 3.6 days; CI, 0.6-7.5). Prolonged RBC storage time did not increase the risk of postoperative infection. However, old blood transfusion increased wound infections rate and incidence of AKI.

Where do Trypanosoma cruzi go? The distribution of parasites in blood components from fractionated infected whole blood.

Platelets (PLTs) are the blood component most frequently involved in Trypanosoma cruzi transfusion transmission cases reported in the literature, although whole blood (WB) and red blood cells (RBCs) have also been incriminated. However, there is little knowledge of the parasite distribution among blood components. The aim of this study was to investigate in which blood component T. cruzi parasites concentrate the most, after fractionating artificially T. cruzi-infected WB. The T. cruzi parasite load was studied by a specific quantitative real-time polymerase chain reaction (qPCR) in WB, buffy coat (BC), PLT concentrates, RBCs before and after leukoreduction, and plasma (PL). The parasite load in WB experimentally infected with 1.5 × 10(6) parasites (2.78 × 10(3) parasite equivalents/mL) was unevenly distributed among the separated blood components. The highest level was found in the BC (6.94 × 10(3) parasite equivalents/mL) and RBCs before leukoreduction by filtration (2.51 × 10(3) parasite equivalents/mL), after which RBCs presented a 99.9% reduction in parasite levels. Both PL and PLTs, partially leukoreduced by centrifugation but nonfiltered, had low parasite levels, the lowest concentration being in PL. The highest parasite concentration was detected in the BC, followed by RBCs before leukoreduction. There is a notable risk of transfusion-transmitted Chagas disease associated with nonleukoreduced RBCs. Leukoreduction may be an effective prevention strategy for transfusion-transmitted T. cruzi infection, especially in endemic countries and in nonendemic countries with a high rate of immigration from Latin America.

Bioactive Lipids Are Generated to Micromolar Levels during RBC Storage, Even in Leukoreduced Units

Background: The transfusion of stored red blood cells (RBCs) has been linked to negative outcomes, such as Transfusion-Related Acute Lung Injury (TRALI) and Transfusion Related Immunomodulation (TRIM). Bioactive lipids have been implicated as playing an important role in TRALI pathogenesis, both as a "second hit" when anti-HLA antibodies are present and also in anti-HLA independent TRALI. Likewise, bioactive lipids have been suggested to play a role in TRIM.Methods: To further study bioactive lipids in RBC storage, we developed a targeted metabolomics approach, with isotope dilution, to accurately and precisely quantify concentrations of multiple poly-unsaturated fatty acids (PUFAs) and their related oxidation products (oxylipins) in stored RBC units. Analytes in RBC samples were extracted using methanol with isotopically-labeled internal standards and analyzed by liquid chromatography-tandem mass spectrometry using multiple reaction monitoring (LC-MS/MS-MRM). Eight units of leukoreduced (LR) and non-leukoreduced (NLR) RBCs were analyzed longitudinally from 6 to 42 days of storage.Results: LR RBCs had a significant increase in multiple PUFAs over storage, including: arachidonic acid (AA), linoleic acid (LA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), linolenic acid (ALA), dihomo-γ-linolenic acid (DGLA), adrenic acid (22:4), and docosapentaenoic acid (22:5). Only a modest increase was observed over the first 20 days of storage, followed by a rapid increase from day 20 to day 42. AA showed the biggest increase in concentration, from 1.0±0.4 µM at day 6 to 13.4±5.2 µM at day 42 in LR RBC units. Oxidative products of PUFA metabolism were also analyzed. The concentration of hydroxyeicosatetraenoic acids (HETEs), oxidation products of AA, including 5-, 8-, 9-, 11-, 12- and 15-HETEs increased 5 to 13 fold from day 6 to day 42 of storage in LR RBCs. The concentration of other oxylipins including hydroxyoctadecadienoic acids(9- and 13-HODEs)and dihydroxyoctadecenoic acids (, 9, 10- and 12,13-DiHOMEs), hydroxyl-DHA(14-, 16- and 17-HDoHEs) were also significantly increased during storage. If present, prostaglandins (PGE2 and PGD2) and Leukotriene (LTB4), were below the lower limits of detection. Juxta position of LR and NLR RBCs, showed that the concentrations of 12-HETE, 15-HETE, 14-HDoHE, and 17-HDoHE in NLR units were significantly higher at day 42 (p<0.01). Of particular note, the average concentration of 12-HETE reached 1.1±0.8 µM in NLR at day 42, which was 45 fold higher than that in LR units. No differences in LR and NLR RBCs were noted at day 6.Conclusions: We report significant generation of PUFAs and products of lipid metabolism/oxidation over RBC storage, with a particular increase in generation after day 20 of storage. LR decreased the generation of some lipid species (12-HETE in particular); however, accumulation of many bioactive lipids occurs robustly despite LR. Because variation from unit to unit was small at day 6 but substantial at day 42, these findings also support significant donor-to-donor differences in the process of bioactive lipid generation during storage. Exactly how the generation of bioactive lipids effects medical outcomes of transfusion remains undetermined; however, the quantitation allowed by the current methods demonstrates the generation of bioactive lipids up to µM levels, which are known to be sufficient to have significant physiological and/or pathological impacts in other settings. DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.

Blood transmission studies of prion infectivity in the squirrel monkey (Saimiri sciureus): the Baxter study.

Four secondary transmissions of variant Creutzfeldt-Jakob disease (vCJD) infectivity have been associated with the transfusion of nonleukoreduced red blood cells collected from vCJD patients during the asymptomatic phase of the disease. Establishing efficient experimental models for assessing the risk of future transmissions of vCJD infectivity via blood transfusion is of paramount importance in view of a study of archived appendix samples in which the prevalence of asymptomatic vCJD infection in the United Kingdom was estimated at approximately 1 in 2000 of the population. In this study, we investigated transmission of vCJD and sporadic CJD (sCJD) infectivity from blood using the squirrel monkey, which is highly susceptible to experimental challenge with human prion disease. Whole blood collected from vCJD- and sCJD-infected squirrel monkeys was transfused at multiple time points into recipient squirrel monkeys. Blood recipients were euthanized approximately 7 years after their first blood transfusion. No clinical or pathologic signs of a prion disease were observed in either the sCJD- or the vCJD-transfused monkeys, and immunohistochemistry and biochemical investigations showed no PrP(TSE) in central nervous system or lymphoreticular tissues. Similarly, monkeys inoculated intracerebrally (IC) and intravenously (IV) with either buffy coat or plasma from vCJD and sCJD patients failed to develop disease. However, white blood cells from a chimpanzee-passaged strain of human Gerstmann-Sträussler-Scheinker (GSS) disease transmitted autopsy-proven disease to two IC-inoculated monkeys after incubation periods of 34 and 39 months. Blood transmits GSS but not sCJD or vCJD infectivity to IC- or IV-inoculated squirrel monkeys within a 7-year observation period.

Tracking Syngeneic Leukocytes after Transfusion of Non-leukoreduced Blood in a Murine Model

Objective: Several Western European countries and Canada have introduced white blood cell reduction as a universal standard to prevent potential adverse effects of allogeneic blood transfusions. However, there is an ongoing controversy regarding its appropriateness in other countries such as North America. We were interested in the whereabouts of leukocytes following whole blood transfusion in mice to evaluate their potential sites of (adverse) action. Materials and methods: Wildtype C57BL/6 were transfused with non-leukoreduced blood from enhanced green fluorescent protein transgenic C57BL/6-Tg(CAGEGFP) 1 Osb/J mice. Cells were tracked on day 3 and day 5 after transfusion by flow cytometry. Results: Enhanced green fluorescent protein-labeled leukocytes were found in the blood circulation, the myocardium, bone marrow and lung tissue up until day 5, but not in the kidney, the liver or the spleen. Myeloperoxidase activity in the lungs was significantly higher on day 5 when compared to control animals (0.09 ± 0.01% vs. 0.26 ± 0.08%, p=0.047). There was no increase in cytokines. Conclusion: Syngeneic white blood cells survive in the recipient of non-leukoreduced red blood cell units. They circulate in the blood stream and can be found in the bone marrow and the lungs up to five days after transfusion.

First Demonstration of Transmissible Spongiform Encephalopathy-associated Prion Protein (PrPTSE) in Extracellular Vesicles from Plasma of Mice Infected with Mouse-adapted Variant Creutzfeldt-Jakob Disease by in Vitro Amplification

The development of variant Creutzfeldt-Jakob disease (vCJD) in three recipients of non-leukoreduced red blood cells from asymptomatic donors who subsequently developed the disease has confirmed existing concerns about the possible spread of transmissible spongiform encephalopathies (TSEs) via blood products. In addition, the presence of disease-associated misfolded prion protein (PrP(TSE)), generally associated with infectivity, has been demonstrated in the blood of vCJD patients. However, its origin and distribution in this biological fluid are still unknown. Various studies have identified cellular prion protein (PrP(C)) among the protein cargo in human blood-circulating extracellular vesicles released from endothelial cells and platelets, and exosomes isolated from the conditioned media of TSE-infected cells have caused the disease when injected into experimental mice. In this study, we demonstrate the detection of PrP(TSE) in extracellular vesicles isolated from plasma samples collected during the preclinical and clinical phases of the disease from mice infected with mouse-adapted vCJD and confirm the presence of the exosomal marker Hsp70 in these preparations.

Development of dose-response models of Creutzfeldt-Jakob disease infection in nonhuman primates for assessing the risk of transfusion-transmitted variant Creutzfeldt-Jakob disease.

Estimates for the risk of transmitting variant Creutzfeldt-Jakob disease (vCJD) via blood transfusion have relied largely on data from rodent experiments, but the relationship between dose (amount of infected blood) and response (vCJD infection) has never been well quantified. The goal of this study was to develop a dose-response model based on nonhuman primate data to better estimate the likelihood of transfusion-transmitted vCJD (TTvCJD) in humans. Our model used dose-response data from nonhuman primates inoculated intracerebrally (i.c.) with brain tissues of patients with sporadic and familial CJD. We analyzed the data statistically by using a beta-Poisson dose-response model. We further adjusted model parameters to account for the differences in infectivity between blood and brain tissue and in transmission efficiency between intravenous (i.v.) and i.c. routes to estimate dose-dependent TTvCJD infection. The model estimates a mean infection rate of 76% among recipients who receive one unit of whole blood collected from an infected donor near the end of the incubation period. The nonhuman primate model provides estimates that are more consistent with those derived from a risk analysis of transfused nonleukoreduced red blood cells in the United Kingdom than prior estimates based on rodent models. TTvCJD was recently identified as one of three emerging infectious diseases posing the greatest immediate threat to the safety of the blood supply. Cases of TTvCJD were reported in recipients of nonleukoreduced red blood cells and coagulation factor VIII manufactured from blood of United Kingdom donors. As the quantity of abnormal prions (the causative agent of TTvCJD) varies significantly in different blood components and products, it is necessary to quantify the dose-response relationship for a wide range of doses for the vCJD agent in transfused blood and plasma derivatives. In this paper, we suggest the first mechanistic dose-response model for TTvCJD infection based on data from experiments with nonhuman primates. This new model may improve estimates of the possible risk to humans.

Broadly Reactive Anti-HLA Antibodies after a Single Unit Transfusion with Nonleukoreduced Red Blood Cells

Broadly Reactive Anti-HLA Antibodies after a Single Unit Transfusion with Nonleukoreduced Red Blood Cells

Neutrophils release extracellular DNA traps during storage of red blood cell units

Blood transfusion is associated with an increased risk of organ damage, infection, and alloimmunity. Neutrophil extracellular traps (NETs) are extracellular chromatin fibers decorated with neutrophil granular proteins that have been linked to cytotoxicity, thrombosis, and autoimmunity. We questioned whether neutrophils in blood products release NETs during storage and thus could contribute to adverse reactions from blood transfusions. We analyzed supernatants and blood smears of human red blood cell (RBC) units that either were or were not leukoreduced before storage for markers of NETs. We identified extracellular DNA, which was associated with histones and myeloperoxidase, a marker of neutrophil granules, in supernatants and blood smears of nonleukoreduced RBC units. These markers of NETs were absent in leukoreduced RBC units. Importantly, NETs passed through blood transfusion filters and could therefore potentially be infused into patients. Our studies indicate that NETs are liberated during storage of nonleukoreduced RBC units. Future studies should address whether NETs in RBC units could potentially contribute to transfusion-associated complications.

The effects of non-leukoreduced red blood cell transfusions on microcirculation in mixed surgical patients

BackgroundThe impact of the storage process on oxygen-carrying properties of red blood cells and the efficacy of red blood cell (RBC) transfusions concerning tissue oxygenation remain an issue of debate in transfusion medicine. Storage time and leukocyte content probably interact since longer storage duration is thought to cause greater accumulation of leukocyte-derived cytokines and red blood cell injury. ObjectivesThe aim of this study was to investigate the effects of storage and the efficacy of fresh (stored for less than 1week) versus aged (stored for more than 3weeks) non-leukoreduced RBC transfusions on sublingual microvascular density and flow in mixed surgical patients. MethodsEighteen surgical patients were included in this study. Patients were randomly assigned into two groups receiving fresh (Group A) and aged (Group B) RBC transfusions. Sublingual microcirculatory functional capillary density (FCD) and microvascular flow index (MFI) were assessed using orthogonal polarization spectral (OPS) imaging. Measurements and collection of blood samples were performed after induction of general anesthesia, before RBC transfusion and 30min after the RBC transfusion ended. ResultsIn both groups RBC transfusions caused an increase in hemoglobin concentration (p<0.001). RBC transfusions increased FCD in Group A (p<0.001), while FCD remained unaffected in Group B. Changes in MFI following RBC transfusion in both groups remained unaltered. ConclusionsFresh non-leukoreduced RBC transfusions but not RBCs stored for more than 3weeks, were effective in improving microciruculatory perfusion by elevating the number of perfused microvessels in mixed surgical patients.

The Characterization of Microparticle Phenotypes in Stored Red Blood Cell Units.

Abstract 2282The transfusion of red blood cells (RBC) is associated with adverse outcomes in critically ill patients. One hypothesis explaining this observation involves the accumulation of potentially inflammatory/prothrombotic microparticles (MPs), released from cellular membranes during unit processing and storage. Despite their implication, the exact nature of MPs within RBC units remains incompletely defined. Here, we characterized the MP phenotypes present within stored RBC units and quantified the various populations. We directly sampled 11 O-positive non-leukoreduced RBC unit bags and isolated MPs by sequential centrifugation. We used a two-step process to detect MPs by flow cytometry. First, we used commercially available Megamix beads to define MPs according to their size during flow cytometric analysis and Trucount beads to quantify MP counts/mL (reported as median [IQR]). MPs ≤1 μm in size were subsequently classified into the following populations: total annexin V-positive, tissue factor-positive (CD142), and those staining for RBC- (intra/extracellular domain-specific glycophorin A, and band-3), leukocyte- (CD45), platelet- (CD42b), and endothelial-specific (CD144) markers. The total concentration of MPs was 1.52×109 MPs/mL [1.39×109]; however, only 4.3% were Annexin V-positive (5.16×107 MPs/mL [7.27×107]), and 11.7% were tissue factor-positive (4.24×107 MPs/mL [1.11×108]). Likewise, MPs with leukocyte-, platelet-, and endothelial-specific markers accounted for only 0.05% (4.44×105 MPs/mL [7.83×105]), 0.17% (1.99×106 MPs/mL [3.11×106]), and 0.06% (7.58×105 MPs/mL [8.04×105]), respectively (Figure 1). The amount of RBC-derived MPs varied depending on the antibody used. While only 4.2% of MPs (3.40×107 MPs/mL [2.91×107]) stained positive for the extracellular domain of glycophorin A, 41.3% (8.20×108 MPs/mL [4.06×108]) and 20.6% MPs (3.03×108 MPs/mL [3.53×108]) stained positive for the cytoplasmic domain of glycophorin A and band-3 respectively (Figure 1). The concentration of MPs positive for the cytoplasmic domain of glycophorin A was significantly greater their extracellular domain-specific counterparts (p<0.01). The results indicate that MPs in stored RBC units are primarily derived from RBCs, but also from other blood cells and vascular endothelial cells. A strong stain for the cytoplasmic domains of two RBC markers (cytoplasmic domain-specific glycophorin A and band 3) suggest the presence of a unique inside-out MP phenotype, potentially derived by RBC membrane “flipping” during microvesiculation. Ongoing studies are required to confirm these results and focus on 1) the mechanism of MP formation and (patho)physiological significance of inside-out RBC MPs and 2), the association between these MPs and the adverse effects seen in patients receiving RBC transfusions. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 370: Supernates from Stored Red Blood Cells Inhibit Collagen-Mediated Aggregation of Platelets Exposed to Arterial Shear Rates

Background: Uncontrolled bleeding is a leading cause of death on the battlefield. We demonstrated previously that supernatant fluid from stored non-leukoreduced (NLR) red blood cells (RBC) inhibited aggregation of platelets in whole blood from healthy donors as measured by impedance aggregometry and decreased GPIIb/IIIa expression. In the current study, we examine stored NLR RBC supernatant fluid inhibition of platelet aggregation in a physiological model of arterial flow. Methods: Fresh citrated blood (500 ul) was mixed with supernate from stored NLR RBC (median age 44 days, 250 ul) and tyrodes buffer (250 ul) for impedance aggregometry. Flow model experiments utilized blood prepared with calcein-labeled platelet rich plasma, RBCs (40% hematocrit), and supernate (25% total volume). Arterial simulated flow (shear rate 1000/sec) over collagen I was carried out in a 75μm x 350μm x 5mm microchamber (Bioflux). GPIIb/IIIa expression was assessed by flow cytometry. Results: Supernates from nine stored NLR RBC units decreased platelet aggregation amplitude in fresh blood from eleven donors by an average of 40±12% vs Tyrode’s buffer controls while one supernate did not exhibit significant inhibition. In concordance with previously reported results, supernates decreased GPIIb/IIIa expression on platelets in whole blood by 53±1.2%. Linear regression analysis of a single flowed experiment showed a 60.5% decrease in aggregation rate (NLR RBC supernate vs HEPES buffered saline control). Inhibition of the rate of aggregation observed in the flow model vs aggregation amplitude by impedance correlated well (sup1: 85.1% vs. 52%; sup 2: 46.6% vs. 41%). Conclusions: Stored NLR RBC units significantly inhibit collagen-stimulated aggregation of platelets in the presence of high (arterial) and low (stirring) shear rates, and reduce expression of the fibrinogen-binding glycoprotein, IIb/IIIa, on the platelet surface. The substantial decrease in platelet aggregation in a physiologically relevant model of whole blood flow suggests massive transfusion of NLR RBC units may decrease the hemostatic capacity of platelets in combat casualties and contribute to uncontrolled hemorrhage. [apc1]unclear

Lysophosphatidylcholines: Bioactive Lipids Generated During Storage of Blood Components

Transfusion-related acute lung injury (TRALI) is suggested to be a "two hit" event, resulting from priming and activation of pulmonary neutrophils. It is known that neutrophil activation may result from infusion of lysophosphatidylcholines (LysoPCs) accumulated during storage of blood components. The aim of our study was to verify whether the LysoPCs are released into the storage medium of blood components. We measured the LysoPCs concentration in the supernatants from stored apheresis platelet concentrates (PLTs), packed non-leukoreduced red blood cell concentrates (RBCs), leukoreduced red blood cell concentrates (L-RBCs), fresh frozen plasma (FFP) and donor plasma (control). Lipids were separated on high-performance thin-layer chromatography, detected by primulin spray and quantified by photodensitometric scanning. The LysoPCs concentration in donor plasma was similar to that in FFP. During storage the LysoPCs content in PLTs increased almost two-fold as compared to the fresh isolated platelets. In RBCs and L-RBCs the LysoPC level was very low or below detection limit and did not increase throughout the storage period. According to our observations bioactive LysoPCs may be considered a neutrophil-activating factor only following PLT transfusions but not RBCs transfusions.

The effect of red blood cell transfusion on tissue oxygenation and microcirculation in severe septic patients.

BackgroundMicrocirculation plays a vital role in the development of multiple organ failure in severe sepsis. The effects of red blood cell (RBC) transfusions on these tissue oxygenation and microcirculation variables in early severe sepsis are not well defined.MethodsThis is a prospective, observational study of patients with severe sepsis requiring RBC transfusions of one to two units of non-leukoreduced RBCs for a hemoglobin < 7.0, or for a hemoglobin between 7.0 and 9.0 with lactic acidosis or central venous oxygen saturation < 70%. This study took place in a 54-bed, medical-surgical intensive care unit of a university-affiliated hospital. Thenar tissue oxygen saturation was measured by using a tissue spectrometer on 21 patients, and a vaso-occlusive test was performed before and 1 hour after transfusion. The sublingual microcirculation was assessed with a Sidestream Dark Field device concomitantly on 11 of them.ResultsRBC transfusion resulted in increase in hemoglobin (7.23 (± 0.87) to 8.75 (± 1.06) g/dl; p < 0.001). RBC transfusion did not globally affect near-infrared spectrometry (NIRS)-derived variables. However, percent change in muscle oxygen consumption was negatively correlated with baseline (r = - 0.679, p = 0.001). There was no statistically significant correlation between percent change in vascular reactivity and baseline (p = 0.275). There was a positive correlation between percent change in oxygen consumption and percent change in vascular reactivity (r = 0.442, p = 0.045). In the 11 patients, RBC transfusion did not globally affect NIRS-derived variables or SDF-derived variables. There was no statistically significant correlation between percent change in small vessel perfusion and baseline perfusion (r = -0.474, p = 0.141), between percent change in small vessel flow and baseline flow (r = -0.418, p = 0.201), or between percent change in small vessel perfusion and percent change in small vessel flow (r = 0.435, p = 0.182).ConclusionsIn a small sample population, muscle tissue oxygen consumption, microvascular reactivity and sublingual microcirculation were globally unaltered by RBC transfusion in severe septic patients. However, muscle oxygen consumption improved in patients with low baseline and deteriorated in patients with preserved baseline. Future research with larger samples is needed to further examine the association between RBC transfusion and outcomes of patients resuscitated early in severe sepsis, with an emphasis on elucidating the potential contribution of microvascular factors.

Estimation of variant Creutzfeldt‐Jakob disease infectivity titers in human blood

Blood of individuals with variant Creutzfeldt-Jakob disease (vCJD) is infectious but the titer is unknown. Current estimates of possible vCJD infectivity titers in blood have largely relied on an assumption that the titers of vCJD agent in human blood are likely to be similar to those in blood of rodents infected with model transmissible spongiform encephalopathy agents, assayed by intracerebral inoculations of rodents of the same species. We analyzed published descriptions of experimental transfusion-transmitted (TT) bovine spongiform encephalopathy and scrapie in sheep and reports of TTvCJD in humans, applying statistical approaches to estimate the probable number of intravenous infectious doses (ID(iv) ) per unit of transfused blood (ID(iv) /unit). For humans, ID(iv) /unit of nonleukoreduced red blood cells (NLR-RBCs) were estimated by two statistical models. Sheep blood collected at or near onset of clinical illness contained a mean of 0.80 ID(iv) /unit. Estimates of infectivity in NLR-RBCs from donors incubating vCJD indicated a probable mean infectivity of 0.29 ID(iv) /unit (Model 1) and 0.75 ID(iv) /unit (Model 2). The analysis predicted a mean of 21 vCJD-infected recipients expected in a cohort transfused with vCJD-implicated NLR-RBCs in the United Kingdom. Our analysis suggested that, while less than one ID(iv ) is likely to be present in a given unit of NLR-RBCs collected from a donor incubating vCJD, there is a high probability of TT infection among recipients of vCJD-implicated blood components. The analysis supports continuing measures currently recommended to reduce the risk of TTvCJD.