Stored RBCs Research Articles

Is it useful to washing stored red blood cells in cardiopulmonary bypass priming fluid for neonatal cardiac surgery

Abstract Background The small blood volume and immature physiology of neonates necessitate careful consideration of the priming fluid composition during cardiopulmonary bypass (CPB) surgery, as it has a substantial impact on their physiologic stability. This study aimed to investigate the impact of allogeneic stored RBCs processed by an autotransfusion system into the CPB priming fluid for neonatal cardiac surgery. Methods This retrospective cohort study investigated the effects of incorporating unwashed (n=56) or washed (n=45) RBCs into the CPB priming fluid for elective neonatal cardiac surgery. We compared perioperative blood parameters, inflammatory mediators, coagulation indicators, vasoactive inotrope score (VIS), other relevant blood parameters and clinical outcomes between the two groups. The regression models were employed to evaluate the independent association between RBCs washing and prognostic outcomes. Results The autotransfusion system can effectively improve the quality of stored RBCs. During CPB, washed group exhibited higher peak hematocrit (P < 0.01), peak hemoglobin (P = 0.04), increased oxygen delivery index during rewarming (P < 0.05). The washed group exhibited significantly lower early postoperative lactate levels and VIS scores (P < 0.05). Despite demonstrating fluctuations in inflammatory (IL-6, IL-8, IL-10）and coagulation parameters(DD, FIB, FDP) compared to baseline, the two groups exhibited no significant divergence. Notably, the washed group experienced a lower incidence of hyperlactacidemia and delayed sternal closure at weaning from CPB. Conclusions The addition of washed allogeneic stored RBCs to neonatal CPB priming fluid significantly attenuated postoperative lactate elevation and lowered VIS without improving early alterations in the inflammatory and coagulation systems.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;Modeled PFKP effects relate to preventing loss of the total AXP pool in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Prony Analysis of Left Ventricle Pressure and Volume.

Direct measurement of cardiac pressure-volume (PV) relationships is the gold standard for assessment of ventricular hemodynamics, but few innovations have been made to "multi-beat" PV analysis beyond traditional signal processing. The Prony method solves the signal recovery problem with a series of dampened exponentials or sinusoids. It achieves this by extracting the amplitude, frequency, dampening, and phase of each component. Since its inception, application of the Prony method to biologic and medical signal has demonstrated a relative degree of success, as a series of dampened complex sinusoids easily generalizes to multifaceted physiological processes. In cardiovascular physiology, the Prony analysis has been used to determine fatal arrythmia from electrocardiogram signals. However, application of the Prony method to simple left ventricular function based on pressure and volume analysis is absent. We have developed a new pipeline for analysis of pressure volume signals recorded from the left ventricle. We propose fitting pressure-volume data from cardiac catheterization to the Prony method for pole extraction and quantification of the transfer function. We implemented the Prony algorithm using open-source Python packages and analyzed the pressure and volume signals before and after severe hemorrhagic shock, and after resuscitation with stored blood. Each animal (n=6 per group) underwent a 50% hemorrhage to induce hypovolemic shock, which was maintained for 30min, and resuscitated with 3-week-old stored RBCs until 90% baseline blood pressure was achieved. Pressure-volume catheterization data used for Prony analysis were 1s in length, sampled at 1000Hz, and acquired at the time of hypovolemic shock, 15 and 30min after induction of hypovolemic shock, and 10, 30, and 60min after volume resuscitation. We next assessed the complex poles from both pressure and volume waveforms. To quantify deviation from the unit circle, which represents deviation from a Fourier series, we counted the number of poles at least 0.2 radial units away from it. We found a significant decrease in the number of poles after shock (p=0.0072vs. baseline) and after resuscitation (p=0.0091vs. baseline). No differences were observed in this metric pre and post volume resuscitation (p=0.2956). We next found a composite transfer function using the Prony fits between the pressure and volume waveforms and found differences in both the magnitude and phase Bode plots at baseline, during shock, and after resuscitation. In summary, our implementation of the Prony analysis shows meaningful physiologic differences after shock and resuscitation and allows for future applications to broader physiological and pathophysiological conditions.

Innate and Adaptive Immunity to Transfused Allogeneic RBCs in Mice Requires MyD88.

RBC transfusion therapy is essential for the treatment of anemia. A serious complication of transfusion is the development of non-ABO alloantibodies to polymorphic RBC Ags; yet, mechanisms of alloantibody formation remain unclear. Storage of mouse RBCs before transfusion increases RBC immunogenicity through an unknown mechanism. We previously reported that sterile, stored mouse RBCs activate splenic dendritic cells (DCs), which are required for alloimmunization. Here we transfused mice with allogeneic RBCs to test whether stored RBCs activate pattern recognition receptors (PRRs) on recipient DCs to induce adaptive immunity. TLRs are a class of PRRs that regulate DC activation, which signal through two adapter molecules: MyD88 and TRIF. We show that the inflammatory cytokine response, DC activation and migration, and the subsequent alloantibody response to transfused RBCs require MyD88 but not TRIF, suggesting that a restricted set of PRRs are responsible for sensing RBCs and triggering alloimmunization.

A Study Protocol to Assess the Effects of Storage on Blood Components and Packed Red Blood Cells in Blood Bank

Background: For preservation of Packed Red blood cells (PRBCs), the recommended optimum conditions includes storage at 2-60 C in specialised blood storage refrigerators. PRBCs are required for blood transfusions in some life-saving situations and can be stored for 42 days. But during this storage period, continuous degradation of RBCs occurs and oxygen carrying capacity gets reduced. These changes inadvertently affect the lifespan of stored RBCs. Therefore during this storage period, routine evaluation is recommended. This study aimed to assess the storage effects on Blood Component and internal quality control of packed red cells.
 Methodology: This Prospective Cohort study includes analysis of 50 Packed Red blood cells bags for RBC count, Haemoglobin and RBC indices. The analysis will be done on Day 1, Day 21 and Day 42 from the date of blood collection. The study will be carried in Jawaharlal Nehru Medical College Blood Centre and Department of Pathology, JNMC, AVBRH, Sawangi Meghe, Wardha. The data collected will be analysed using appropriate statistical tools.
 Expected Results: Haemoglobin and Red cell indices with the help of cell counter will be analysed. Effects of storage on packed red blood cells will be drawn from the analysis.

The Post-Storage Performance of RBCs from Beta-Thalassemia Trait Donors Is Related to Their Storability Profile.

Blood donors with beta-thalassemia traits (βThal+) have proven to be good “storers”, since their stored RBCs are resistant to lysis and resilient against oxidative/proteotoxic stress. To examine the performance of these RBCs post-storage, stored βThal+ and control RBCs were reconstituted in plasma donated from transfusion-dependent beta-thalassemic patients and healthy controls, and incubated for 24 h at body temperature. Several physiological parameters, including hemolysis, were evaluated. Moreover, labeled fresh/stored RBCs from the two groups were transfused in mice to assess 24 h recovery. All hemolysis metrics were better in the group of heterozygotes and distinguished them against controls in the plasma environment. The reconstituted βThal+ samples also presented higher proteasome activity and fewer procoagulant extracellular vesicles. Transfusion to mice demonstrated that βThal+ RBCs present a marginal trend for higher recovery, regardless of the recipient’s immune background and the RBC storage age. According to correlation analysis, several of these advantageous post-storage characteristics are related to storage phenotypes, like the cytoskeleton composition, low cellular fragility, and enhanced membrane proteostasis that characterize stored βThal+ RBCs. Overall, it seems that the intrinsic physiology of βThal+ RBCs benefits them in conditions mimicking a recipient environment, and in the circulation of animal models; findings that warrant validation in clinical trials.

Hypoxic Storage of Red Blood Cells: Assessment of Adhesion Properties Using a Standardized Endothelium-on-a-Chip Microfluidic Platform

Background: Blood transfusions are routine medical procedures in which stored blood or blood products (i.e., red blood cells, RBCs) are given to the patient to prevent adverse health outcomes due to acute or chronic anemia. The current regulations require RBCs to be stored at 4 ºC not more than 42 days before a transfusion. However, RBCs undergo extensive rheological changes during storage and may contribute to complications associated with transfusion. Hemanext has recently introduced an innovative storage system to ameliorate such storage lesions, in which RBCs are stored in a hypoxic environment, and thus they are exposed to much lower oxidative stress during storage. In this study, we report the changes in adhesion properties of stored RBCs to human endothelial cells following a 42-day storage in normoxia vs hypoxia, using a standardized endothelialized microfluidic platform: Endothelium-on-a-chip.Methods: Two units of 1-day old blood type O positive leukocyte reduced RBCs in additive solution were pooled and divided into equal aliquots (300mL each) A and B. Unit A was stored under conventional normoxic storage condition at 4°C for 42 days. Unit B was deoxygenated so that the percent oxygen saturation of the hemoglobin was less than 20%. The deoxygenated RBCs were stored in an oxygen impermeable storage bag for 42 days at 4°C. Adhesion levels were tested at the beginning (baseline, Week 1) and the end of the storage period (Week 6). To conduct the adhesion experiments, human umbilical vein endothelial cells (HUVECs) were first cultured within microfluidic channels under flow for at least 48 hours. Next, the HUVECs were treated with 40 µM heme for 4 hours at 37 ºC [1]. RBCs were centrifuged to remove the storage buffer and resuspended in basal cell culture medium (EGM, Lonza, Morristown, USA) supplemented with 10 mM HEPES at a hematocrit of 40%. A 15 µl of RBC solution was then injected over heme-activated HUVECs at a shear stress of 1 dyne/cm 2 followed by a 10-minute rinse with fresh basal culture medium to remove non-adherent RBCs. At the end of the experiment, adherent erythrocytes were manually quantified. Control experiments were conducted with non-activated HUVECs.Results: We found that RBCs that were stored under conventional normoxic condition displayed higher adhesion to heme-activated HUVECs than hypoxic condition as illustrated in Figure 1A and 1B. The baseline adhesion levels (Week 1) to non-activated HUVECs (control) were negligible (Fig. 1C, normoxia: 13±6; hypoxia: 18±10, p>0.05) while both cell populations had considerable adhesion levels to heme-activated HUVECs at baseline (Fig. 1C, normoxia: 372±59; hypoxia: 335±37). Following a 6-week storage, adhesion of RBCs stored in normoxia to heme-activated HUVECs was higher compared to those stored in hypoxic conditions while the p-value was not significant, which was likely due to the limited sample size (Fig. 1C, 1782±519 vs 594±55, p=0.08, N=3). These results suggest that storage-mediated RBC adhesion to heme-activated HUVECs may be ameliorated by the novel hypoxic-storage condition.Discussion: This study showed a decrease in the adhesion of hypoxic RBCs to heme-activated HUVECs when compared to conventionally stored RBCs for transfusion. This result suggests that hypoxic RBCs may reduce the risk of developing vaso-occlusion (VOC) after RBC transfusion in patients such as in sickle cell disease where adhesion to heme-activated HUVECs has been implicated in the pathogenesis of VOC [1].Acknowledgements: This work was funded by Hemanext. The authors would like to thank the Ohio Third Frontier Technology Validation and Start-up Fund (TVSF) and National Science Foundation Phase-I Small Business Technology Transfer (STTR) award, which supported this work in part. Stored RBCs were donated by Hemanext.Reference:1. Kucukal, E., et al., American Journal of Hematology, 2018. 93(8): p.1050-1060 [Display omitted] DisclosuresKucukal: BioChip Labs: Current Employment, Patents & Royalties. Kocevar: BioChip Labs: Current Employment. Nayak: BioChip Labs: Current Employment. Sowemimo-Coker: Hemanext: Current Employment. Zak: BioChip Labs: Current Employment, Current holder of stock options in a privately-held company; XaTek: Current Employment, Current holder of stock options in a privately-held company; TecTraum Inc: Current Employment, Current holder of stock options in a privately-held company. Omert: Hemanext: Current Employment. Gurkan: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Dx Now Inc.: Patents & Royalties; Xatek Inc.: Patents & Royalties.

Mouse background genetics in biomedical research: The devil's in the details.

Genetically modified mice are used widely to explore mechanisms in most biomedical fields-including transfusion. Concluding that a gene modification is responsible for a phenotypic change assumes no other differences between the gene-modified and wild-type mice besides the targetted gene. To test the hypothesis that the N-terminus of Band3, which regulates metabolism, affects RBC storage biology, RBCs from mice with a modified N-terminus of Band3 were stored under simulated blood bank conditions. All strains of mice were generated with the same initial embryonic stem cells from 129 mice and each strain was backcrossed with C57BL/6 (B6) mice. Both 24-h recoveries post-transfusion and metabolomics were determined for stored RBCs. Genetic profiles of mice were assessed by a high-resolution SNP array. RBCs from mice with a mutated Band3 N-terminus had increased lipid oxidation and worse 24-h recoveries, "demonstrating" that Band3 regulates oxidative injury during RBC storage. However, SNP analysis demonstrated variable inheritance of 129 genetic elements between strains. Controlled interbreeding experiments demonstrated that the changes in lipid oxidation and some of the decreased 24-hr recovery were caused by inheritance of a region of chromosome 1 of 129 origin, and not due to the modification of Band 3. SNP genotyping of a panel of commonly used commercially available KO mice showed considerable 129 contamination, despite wild-type B6 mice being listed as the correct control. Thousands of articles published each year use gene-modified mice, yet genetic background issues are rarely considered. Assessment of such issues are not, but should become, routine norms of murine experimentation.

RBC Transfusion Induced ST Segment Variability Following the Norwood Procedure.

Retrospective case-control study. Nine-hundred seventy-three-bed pediatric hospital with a 54-bed cardiovascular ICU. Seventy-three neonates with hypoplastic left heart syndrome following the Norwood procedure, 38 with a Blalock-Taussig shunt and 35 with a right ventricle to pulmonary artery shunt. RBC transfusion. High-frequency physiologic data were captured 30 minutes prior to the initiation of (baseline) and during the 120 minutes of the transfusion. A rate pressure product was calculated for each subject and used as an indicator of myocardial oxygen demand. Electrocardiogram leads (aVL, V1, II) were used to construct a 3D ST segment vector to assess ST segment variability and functioned as a surrogate indicator of myocardial ischemia. One-hundred thirty-eight transfusions occurred in the Blalock-Taussig shunt group and 139 in the right ventricle to pulmonary artery shunt group. There was no significant change in the rate pressure product for either group; however, ST segment variability progressively increased for the entire cohort during the transfusion, becoming statistically significant by the end of the transfusion. Upon subgroup analysis, this finding was noted with statistical significance in the Blalock-Taussig shunt group and trending toward significance in the right ventricle to pulmonary artery shunt group. We found a significant increase in the ST segment variability and evidence of myocardial ischemia temporally associated with RBC transfusions in neonates following the Norwood procedure, specifically among those in the Blalock-Taussig shunt group, which may impact immediate and long-term outcomes.

Evaluation of the Functional Effects of an African American Glucose-6-Phosphate Dehydrogenase (G6PD) Polymorphism (Val68Met) on RBC Hemolytic Propensity and Post-Transfusion Recovery in a Humanized Mouse Model

Background: Growing evidence suggest that donor genetic variation is associated with RBCs storage integrity and post-transfusion recovery. In collaboration with the REDS III program, we performed a large-scale genome-wide association study (GWAS) study in ~13,000 healthy human blood donors, which demonstrated that RBCs with an African American G6PD-deficient A- variant (rs1050828, Val68Met) was associated with higher oxidative hemolysis after cold storage in normal volunteers. Despite a high prevalence of X-linked G6PD mutation in African American population (> 10%), blood donors are not routinely checked for G6PD deficiency and its importance in transfusion medicine is relatively understudied. We hypothesize that G6PD V68M SNP is associated with G6PD deficiency and modulates RBCs in vitro hemolytic propensity and in vivo post-transfusion recovery. Methods and Results: CRISPR-Cas9 technology was used to create non-synonymous human G6PD X-linked SNP (Val68Met) in C57B6 mice, and the desired genotypes were isolated by strategic back-crossing and sequential genotyping to ensure passage of SNP. G6PD enzymatic activity in erythrocytes was measured in fresh collected blood using a colorimetric assay kit. The predisposition of fresh and stored RBCs (after 11 days cold storage) to hemolysis was evaluated by subjecting washed mouse RBCs to selected stress assays, including osmotic fragility, mechanical fragility, and oxidative hemolysis using AAPH, diamide or H2O2. Hemolysis was measured by detection of supernatant cell-free hemoglobin using Drabkin's assay. Hematological values were measured using a Hemavet® 950 Hematology Analyzer System. Reticulocyte count was obtained using thiazole orange staining and analyzed by flow cytometry. We found severe disruption of G6PD enzymatic activity in erythrocytes from G6PD V68M SNP mice compared to WT mice (~5% residual activity in hemizygous male and ~60% in heterozygous female mice). Significant increased oxidative hemolysis was observed in both fresh and stored mouse RBCs with G6PD SNP, consistent with the GWAS study in human. G6PD V68M SNP hemizygous male mice had higher mean corpuscular volume (MCV) and lower mean corpuscular hemoglobin concentration (MCHC) compared to WT mice. However, no difference was observed in storage hemolysis, osmotic fragility, mechanical fragility and reticulocyte counts. Transfusion experiments with stored red blood cells from G6PD hemizygote males into GFP positive recipients will evaluate red blood cell recovery and half-life after standard cold storage and transfusion. Conclusions: We successfully generated a novel mouse strain carrying a "humanized" African American G6PD V68M variant which resembles the phenotype of humans with G6PD deficiency and increased oxidative hemolysis. Studies are undertaken to further investigate the effects of G6PD V68M SNP on RBCs structure, functions and in vivo post-transfusion recovery. Disclosures Gladwin: Bayer Pharmaceuticals: Other: Co-investigator; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning.

Differential Metabolomics and Lipidomics of Hypoxic/Hypocapnic Long-Term Stored Red Cells Associate with Improved 24-Hour Recovery after Transfusion

Storage lesion accumulates during refrigerated storage of RBC, and development of novel methods and/or additive solutions are needed for more efficacious RBC with reduced burden of harmful byproducts. Autologous 24-hr post-transfusion recovery (PTR24) at the end of storage measured in healthy volunteers is the only in vivo practical metrics to evaluate consequences of RBC storage. PTR24 studies are logistically and economically demanding and as such, the identification of easier biomarkers of stored blood quality can expedite the development and testing of novel storage strategies. We performed a prospective, randomized, dual‐arm, crossover, two‐center radio labeled 24-hr PTR24 study was conducted as a part of the pivotal study to evaluate Hemanext® hypoxic/hypocapnic processing system for meeting standard FDA acceptability criteria for RBC storage. Subjects donated CP2D whole blood (500 ± 50 mL). The Test arm consisted of whole blood‐derived RBC, leukoreduced, and processed to achieve hypoxic/hypocapnic state in AS-3 additive within 12 hours of phlebotomy then stored for 42 days at 1‐6°C under hypoxic conditions (Test). Unprocessed normal atmosphere stored units (Control) were stored within 8 hours under conventional storage condition. PTR24 data was obtained from 19 test subjects and 21 control subjects (14 paired) by infusion of 51‐Cr/99‐Tc(m) radiolabeled RBCs*. At day 0, 21 and 42, in vitro metrics, including conventional quality parameters (%hemolysis, ATP, 2,3-DPG, morphology, microparticles and deformability), as well as a large panel of metabolites and lipids were analyzed for correlations with PTR24. Bioactive lipids were quantified by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM). Pearson's correlation coefficients for PTR24 and analytes were examined; correlation pattern differed significantly between normal air Control and hypoxic Test RBCs and thus analyzed separately. Among the conventional RBC quality metrics, ATP as well as ability to rejuvenate ATP and 2,3-DPG at end of storage correlated with PTR24. RBC deformability correlated only with Control, while reduced oxidative damage in Test removed its dependency. Test showed stronger correlations with ATP and other metabolic parameters. 302 metabolites and lipids were analyzed by qualitative metabolomics workflow, and significant correlations were observed with |R| ≥ 0.5 for 37 compounds in normal air stored RBCs (15 with p ≤ 0.05), and 41 compounds in hypoxic RBCs (19 with p ≤ 0.05). The top 5 correlating and inversely correlating compounds for each condition are reported along with p-values in Table 1. For RBC stored in normal air, strong correlations were primarily seen with membrane lipids and associated fatty acids, amino acids, and the Pentose Phosphate Pathway intermediates. Hypoxic storage PTR24 values primarily correlated with alternative membrane components, amino acids, and Pentose Phosphate Pathway intermediates (Table 2). Among 67 bio-reactive lipids analyzed quantitatively, negative correlations of oxylipins (9- or 13-HODEs, 5-, 8-, 11-, 12-HETEs) to PTR24 were observed under normal conditions, but not under hypoxic condition (Table 3). In summary, RBC deformability correlated only with PTR24 of normal air stored RBC, while reduced oxidative damage in hypoxic/hypocapnic conditions removed this dependency. Although normal air and hypoxic stored RBCs show differing metabolite correlates with PTR24, significant associations tend to occur within similar overall pathways. Comparing the PTR24-metabolite correlations between normal air and hypoxic/hypocapnic RBC storage reveals that intermediates of oxidative stress management and membrane homeostasis are potential predictors of post-transfusion recovery. Detailed analysis of lipids supports that hypoxic/hypocapnic storage may reduce membrane lipid degradation and generates the hypothesis that the metabolism of bioactive lipids under hypoxia/hypocapnia may be different from the one found in RBC stored under normal air storage conditions. * Radio-labeling for 14 subjects occurred without prior RDRC approval; one site did not meet GMP standards. Disclosures Yoshida: New Health Sciences Inc.: Employment, Equity Ownership. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder. Nemkov:Omix Technologies, Inc.: Equity Ownership, Other: Founder. Dioguardi:Hemanext: Employment. Iselin:Hemanext: Employment. Dunham:Hemanext: Employment. Cancelas:Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding.

Transition to 37°C reveals importance of NADPH in mitigating oxidative stress in stored RBCs.

The RBC storage lesion is a multiparametric response that occurs during storage at 4°C, but its impact on transfused patients remains unclear. In studies of the RBC storage lesion, the temperature transition from cold storage to normal body temperature that occurs during transfusion has received limited attention. We hypothesized that multiple deleterious events might occur in this period of increasing temperature. We show dramatic alterations in several properties of therapeutic blood units stored at 4°C after warming them to normal body temperature (37°C), as well as febrile temperature (40°C). In particular, the intracellular content and redox state of NADP(H) were directly affected by post-storage incubation at 37°C, as well as by pro-oxidant storage conditions. Modulation of the NADPH-producing pentose phosphate pathway, but not the prevention of hemoglobin autoxidation by conversion of oxyhemoglobin to carboxyhemoglobin, provided protection against storage-induced alterations in RBCs, demonstrating the central role of NADPH in mitigating increased susceptibility of stored RBCs to oxidative stress. We propose that assessing RBC oxidative status after restoration of body temperature constitutes a sensitive method for detecting storage-related alterations that has the potential to improve the quality of stored RBCs for transfusion.

Characterizing red blood cell age exposure in massive transfusion therapy: the scalar age of blood index (SBI).

The mortality of trauma patients requiring massive transfusion to treat hemorrhagic shock approaches 17% at 24 hours and 26% at 30 days. The use of stored RBCs is limited to less than 42 days, so older RBCs are delivered first to rapidly bleeding trauma patients. Patients who receive a greater quantity of older RBCs may have a higher risk for mortality. Characterizing blood age exposure requires accounting for the age of each RBC unit and the quantity of transfused units. To address this challenge, a novel Scalar Age of Blood Index (SBI) that represents the relative distribution of RBCs received is introduced and applied to a secondary analysis of the Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) randomized controlled trial (NCT01545232, https://clinicaltrials.gov/ct2/show/NCT01545232). The effect of the SBI is assessed on the primary PROPPR outcome, 24-hour and 30-day mortality. The distributions of blood storage ages successfully maps to a parameter (SBI) that fully defines the blood age curve for each patient. SBI was a significant predictor of 24-hour and 30-day mortality in an adjusted model that had strong predictive ability (odds ratio, 1.15 [1.01-1.29], p = 0.029, C-statistic, 0.81; odds ratio, 1.14 [1.02-1.28], p = 0.019, C-statistic, 0.88, respectively). SBI is a simple scalar metric of blood age that accounts for the relative distribution of RBCs among age categories. Transfusion of older RBCs is associated with 24-hour and 30-day mortality, after adjustment for total units and clinical covariates.

Haematological and morphological evaluation of feline whole blood units collected for transfusion purposes.

Despite the increasing availability of feline blood collected and stored for transfusion purposes, few studies have been performed on feline blood units. The aim of this prospective in vitro study was to evaluate haematological and morphological changes in feline blood cells in whole blood units between collection and end of storage. Haematological examination (red blood cells [RBCs], haemoglobin, haematocrit, red cell distribution width, mean cell volume, mean cell haemoglobin concentration, mean cell haemoglobin, white blood cells [WBCs] and platelet [PLT] count) was performed on 40 non-leukoreduced feline whole blood units at the time of collection (day[D]0) and after storage (D35). The blood was collected into citrate-phosphate-dextrose-adenine anticoagulant-preservative solution using an open system in a veterinary blood bank and stored for 35 days at 4 ± 2°C. Twenty of these feline whole blood units were also analysed for blood cell morphology (normal RBCs, macrocytes, echinocytes, spherocytes, schistocytes, lysed RBCs, RBCs with Heinz bodies and recognisable WBC and PLT count). Differences between the two examination times were statistically analysed. There was a statistically significant decrease in WBC and PLT counts after storage at D35 (P <0.0001 for both). The most significant cellular morphological changes after storage were an increase in echinocyte count (P = 0.0001), and lysed RBCs (P <0.0001), and a decrease in normal RBCs (P <0.0001). Recognisable WBCs - mainly lymphocytes - were present at the end of storage. This study showed that significant morphological changes occur in RBCs in feline blood units during storage for 35 days. In vivo studies are required to establish if these changes could affect the ability of stored RBCs to circulate and provide adequate oxygen delivery after transfusion.

Glucose-6-Phosphate Dehydrogenase Deficiency in Blood Donors Is Associated with Decreased Post-Transfusion Red Cell Recovery

Background:Pre-transfusion refrigerated storage of red cells (RBCs) induces a series of metabolic and structural changes in stored RBCs (i.e., the “storage lesion”). The resulting, progressive, time-dependent decrease in RBC quality leads to decreased post-transfusion RBC recovery (PTR). Markers of oxidative stress increase during storage, suggesting the possibility that oxidative stress may be a key contributor to the storage lesion. Glucose-6-phosphate dehydrogenase (G6PD) deficiency, an X-linked condition, decreases the ability of RBCs to withstand oxidative stress by compromising their ability to synthesize NADPH. Therefore, we hypothesized that RBCs from G6PD-deficient donors would have inferior storage quality, as compared to RBCs from G6PD-normal (i.e., control) donors.Aims:This study examined whether G6PD deficiency, the most common enzyme deficiency worldwide, which impairs the RBC response to oxidative stress, also decreases RBC storage quality.Methods:Male volunteers were screened for G6PD deficiency by enzymatic assay, and next generation sequencing was performed to identify the G6PD variant in deficient subjects. In addition, participants were screened for the presence of hemoglobin variants and thalassemia by high performance liquid chromatography and complete blood count. Twenty seven control and 10 G6PD-deficient volunteers each received informed consent, donated 1 unit of leukoreduced RBCs that was stored in AS-3 under standard blood banking conditions, and completed an autologous 51-Chromium-labeled PTR study after 40-42 days of storage. Hemolysis rate in the transfused unit was measured on the day of the PTR study using a Drabkin assay. Results for control and G6PD-deficient PTRs and hemolysis rates were compared using unpaired t-tests.Results:A total of 145 males were screened for G6PD deficiency, yielding 15 deficient (10%) and 130 G6PD normal control (90%) subjects. Twenty seven control (G6PD normal) and 10 deficient participants who did not have hemoglobin variants or microcytosis completed the study. All blood donors met the hemoglobin acceptance criteria for autologous whole blood donation of at least 11.5 g/dL. G6PD gene sequencing of the 10 enrolled G6PD-deficient subjects identified the A- variant in 9 volunteers and the Mediterranean variant in 1. The autologous mean 24-hour PTR for control RBC units was 86.8 ± 2.8% (mean ± SD). The autologous mean 24-hour PTR for G6PD-deficient RBC units was 81.0 ± 7.2% (mean ± SD), which was significantly lower than that for control RBCs (p=0.001). None of the control volunteers (0/27) had PTR results below 75%, one of the key FDA acceptability criteria for stored RBCs. In contrast, two G6PD-deficient volunteers (2/10) had PTR results below 75% (i.e., 68.8% and 70.1%). There was no association between enzymatic G6PD activity and PTR when adjusted for G6PD status (i.e., among control or G6PD-deficient subjects, the enzymatic G6PD activity did not predict PTR). Finally, there was no difference between control and deficient RBC units in mean in vitro hemolysis rates at the end of storage (0.29 ± 0.18% vs. 0.24 ± 0.07%, respectively; p=0.38)Conclusions:This is the first report of the effect of G6PD deficiency on the storage quality at outdate of transfused donor RBCs stored in AS-3 additive solution as assessed by PTR, the FDA gold standard. These results demonstrate that the storage quality of G6PD-deficient RBCs is inferior to G6PD-normal RBCs. In particular, based on the FDA criteria that acceptable RBC units should have a 24-hour PTR of ≥75% at the end of storage, RBC units from G6PD-deficient donors had a 20% failure rate. The clinical consequences of the mean 5.8% decreased PTR of G6PD-defcient RBC units remain to be determined. Patients with sickle cell disease often require lifetime transfusions and up to 12.3% of phenotypically-matched RBC units utilized in their care may be G6PD-deficient. Thus, these data raise the question of whether there is value in screening blood donors for G6PD deficiency, particularly in the setting of chronic transfusion therapy, such as for patients with sickle cell disease and thalassemia. DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Iron accelerates hemoglobin oxidation increasing mortality in vascular diseased guinea pigs following transfusion of stored blood.

Non-transferrin-bound iron (NTBI) and free hemoglobin (Hb) accumulate in circulation following stored RBC transfusions. This study investigated transfusion, vascular disease, and mortality in guinea pigs after stored RBC transfusion alone and following cotransfusion with apo-transferrin (apo-Tf) and haptoglobin (Hp). The effects of RBC exchange transfusion dose (1, 3, and 9 units), storage period (14 days), and mortality were evaluated in guinea pigs with a vascular disease phenotype. Seven-day mortality and the interaction between iron and Hb as cocontributors to adverse outcome were studied. Concentrations of iron and free Hb were greatest after transfusion with 9 units of stored RBCs compared with fresh RBCs or stored RBCs at 1- and 3-unit volumes. Nine units of stored RBCs led to mortality in vascular diseased animals, but not normal animals. One and 3 units of stored RBCs did not cause a mortality effect, suggesting the concomitant relevance of NTBI and Hb on outcome. Cotransfusion with apo-Tf or Hp restored survival to 100% following 9-unit RBC transfusions in vascular diseased animals. Our data suggest that increases in plasma NTBI and Hb contribute to vascular disease-associated mortality through iron-enhanced Hb oxidation and enhanced tissue injury.

Temperature-dependent haemolytic propensity of CPDA-1 stored red blood cells vs whole blood - Red cell fragility as donor signature on blood units.

To preserve cellular integrity and avoid bacterial growth, storage and transfer of blood and blood products follow strict guidelines in terms of temperature control. We evaluated the impact of ineligible warming of whole blood donations on the quality of blood components. One-hundred and twenty units of whole blood (WB) from eligible blood donors were collected in CPDA-1 and stored at 4±2 °C. During shipment to the blood processing centre, a gradual warming up to 17 °C was recorded within a period of less than eight hours. The warmed units were processed to packed red blood cells (PRBCs) or stored as WB units at 4±2 °C. In-bag haemolysis, osmotic fragility (mean corpuscular fragility, MCF) and bacterial growth were assessed in blood and blood components throughout the storage period. Normal basal and early storage levels of haemolysis were recorded in both PRBC and WB units. Thereafter, PRBCs exhibited higher average in-bag haemolysis and MCF index compared to the WB units throughout the storage. Moreover, 14.3 and 52.4% of the PRBC units exceeded the upper permissible limit of 0.8% haemolysis at the middle (1.220±0.269%) or late (1.754±0.866%) storage period, respectively. MCF index was similar in all PRBCs at the middle of storage but significantly lower in the non-haemolysed compared to the haemolysed units of PRBCs on the last days. The fragility of stored RBCs was proportional to the donor-related values of day 2 samples (r=0.861, p<10-32). In the qualified PRBCs, MCF was correlated with haemolysis at every time point of the storage period (r=0.332, p<0.050). Bacterial growth was detected by blood culture in two units of PRBCs. Transient, gradient warming of whole blood from 4 to 17 °C led to increased incidence of in-bag haemolysis in PRBC but not in WB units. Haemolysis is a multi-parametric phenotype of stored blood, and MCF is a donor-related and highly dynamic measure that can, in part, predict the storage lesion.

PH modulation ameliorates the red blood cell storage lesion in a murine model of transfusion

BackgroundProlonged storage of packed red blood cells (pRBCs) induces a series of harmful biochemical and metabolic changes known as the RBC storage lesion. RBCs are currently stored in an acidic storage solution, but the effect of pH on the RBC storage lesion is unknown. We investigated the effect of modulation of storage pH on the RBC storage lesion and on erythrocyte survival after transfusion. MethodsMurine pRBCs were stored in Additive Solution-3 (AS3) under standard conditions (pH, 5.8), acidic AS3 (pH, 4.5), or alkalinized AS3 (pH, 8.5). pRBC units were analyzed at the end of the storage period. Several components of the storage lesion were measured, including cell-free hemoglobin, microparticle production, phosphatidylserine externalization, lactate accumulation, and byproducts of lipid peroxidation. Carboxyfluorescein-labeled erythrocytes were transfused into healthy mice to determine cell survival. ResultsCompared with pRBCs stored in standard AS3, those stored in alkaline solution exhibited decreased hemolysis, phosphatidylserine externalization, microparticle production, and lipid peroxidation. Lactate levels were greater after storage in alkaline conditions, suggesting that these pRBCs remained more metabolically viable. Storage in acidic AS3 accelerated erythrocyte deterioration. Compared with standard AS3 storage, circulating half-life of cells was increased by alkaline storage but decreased in acidic conditions. ConclusionsStorage pH significantly affects the quality of stored RBCs and cell survival after transfusion. Current erythrocyte storage solutions may benefit from refinements in pH levels.

Pleomorphic Structures in Human Blood Are Red Blood Cell-Derived Microparticles, Not Bacteria.

BackgroundRed blood cell (RBC) transfusions are a common, life-saving therapy for many patients, but they have also been associated with poor clinical outcomes. We identified unusual, pleomorphic structures in human RBC transfusion units by negative-stain electron microscopy that appeared identical to those previously reported to be bacteria in healthy human blood samples. The presence of viable, replicating bacteria in stored blood could explain poor outcomes in transfusion recipients and have major implications for transfusion medicine. Here, we investigated the possibility that these structures were bacteria.ResultsFlow cytometry, miRNA analysis, protein analysis, and additional electron microscopy studies strongly indicated that the pleomorphic structures in the supernatant of stored RBCs were RBC-derived microparticles (RMPs). Bacterial 16S rDNA PCR amplified from these samples were sequenced and was found to be highly similar to species that are known to commonly contaminate laboratory reagents.ConclusionsThese studies suggest that pleomorphic structures identified in human blood are RMPs and not bacteria, and they provide an example in which laboratory contaminants may can mislead investigators.

Transfusion of 35-Day Stored RBCs in the Presence of Endotoxemia Does Not Result in Lung Injury in Humans.

Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. Preclinical studies have shown that aged RBCs can induce transfusion-related acute lung injury in the presence of a "first hit" (e.g., sepsis). Clinical studies, however, show conflicting results on this matter. We tested whether maximally stored RBCs are able to induce lung injury in the presence of a "first hit" in humans (Dutch Trial Register: NTR4455). Open-label, randomized controlled trial. Healthy male volunteers. Eighteen healthy male volunteers donated one unit of autologous RBCs 2 or 35 days before the experiment. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). After 2 hours, volunteers received normal saline (n = 6), 2-day stored transfusion (n = 6), or 35-day stored transfusion (n = 6) ("second hit"). Blood was sampled hourly. Six hours after transfusion, the diffusion capacity of the lungs for carbon monoxide was tested and volunteers underwent spirometry, chest x-ray study, and a bronchoalveolar lavage. All volunteers fulfilled sepsis criteria after lipopolysaccharide injection. The stored blood transfusion did not result in significant changes in either hemodynamic or respiratory variables compared with the control groups. Furthermore, chest x-rays, lung function, and PaO2/FIO2 ratios did not differ between groups. Transfusion of stored autologous RBCs did not result in an increased level of protein in the lungs or neutrophil influx. Transfusion of 35-day stored autologous RBCs in the presence of endotoxemia does not result in lung injury in humans.