Control RBCs Research Articles

Evaluation of the efficacy and safety of amustaline/glutathione pathogen-reduced RBCs in complex cardiac surgery: the Red Cell Pathogen Inactivation (ReCePI) study—protocol for a phase 3, randomized, controlled trial

BackgroundRed blood cell (RBC) transfusion is a critical supportive therapy in cardiovascular surgery (CVS). Donor selection and testing have reduced the risk of transfusion-transmitted infections; however, risks remain from bacteria, emerging viruses, pathogens for which testing is not performed and from residual donor leukocytes. Amustaline (S-303)/glutathione (GSH) treatment pathogen reduction technology is designed to inactivate a broad spectrum of infectious agents and leukocytes in RBC concentrates. The ReCePI study is a Phase 3 clinical trial designed to evaluate the efficacy and safety of pathogen-reduced RBCs transfused for acute anemia in CVS compared to conventional RBCs, and to assess the clinical significance of treatment-emergent RBC antibodies.MethodsReCePI is a prospective, multicenter, randomized, double-blinded, active-controlled, parallel-design, non-inferiority study. Eligible subjects will be randomized up to 7 days before surgery to receive either leukoreduced Test (pathogen reduced) or Control (conventional) RBCs from surgery up to day 7 post-surgery. The primary efficacy endpoint is the proportion of patients transfused with at least one study transfusion with an acute kidney injury (AKI) diagnosis defined as any increased serum creatinine (sCr) level ≥ 0.3 mg/dL (or 26.5 µmol/L) from pre-surgery baseline within 48 ± 4 h of the end of surgery. The primary safety endpoints are the proportion of patients with any treatment-emergent adverse events (TEAEs) related to study RBC transfusion through 28 days, and the proportion of patients with treatment-emergent antibodies with confirmed specificity to pathogen-reduced RBCs through 75 days after the last study transfusion. With ≥ 292 evaluable, transfused patients (> 146 per arm), the study has 80% power to demonstrate non-inferiority, defined as a Test group AKI incidence increase of no more than 50% of the Control group rate, assuming a Control incidence of 30%.DiscussionRBCs are transfused to prevent tissue hypoxia caused by surgery-induced bleeding and anemia. AKI is a sensitive indicator of renal hypoxia and a novel endpoint for assessing RBC efficacy. The ReCePI study is intended to demonstrate the non-inferiority of pathogen-reduced RBCs to conventional RBCs in the support of renal tissue oxygenation due to acute anemia and to characterize the incidence of treatment-related antibodies to RBCs.

3041 – CELLULAR MECHANISMS INVOLVED IN SERONEGATIVE HEMOLYTIC ANEMIAS

In the current dogma, immune hemolysis is considered an exclusively humorally-mediated state. However, in some cases, serological investigations are negative and without alternative explanations. In such a subset of unexplained seronegative hemolysis (SNH), we investigated for potentially attributable cellular mechanisms. Release of chromium-51(Cr51) was selected as the most sensitive method. Effector cells of the immune system (NK cells, neutrophils, monocytes) were purified by negative selection and evaluated as mediators of RBC lysis. Anti-D-opsonized R2R2 RBCs were selected as positive control RBCs for standardization of cytotoxicity assays and evaluation of the killing capacity of different immune cells. We observed that PBMCs were the least sensitive at lysing control RBC while NK cells, used at an effector/target ratio of 10:1, were the most efficient in killing control RBC, followed by monocytes and neutrophils. Neutrophils, used at an effector/target ratio of 25:1, are also capable of lysing control RBCs but required priming with GM-CSF. By informed consent, ten patients with unexplained SNH were tested for cellular-mediated hemolysis using autologous, purified immune cells (NK or neutrophils), against autologous RBCs with or without opsonization with autologous serum. Evidence of cell-mediated RBC lysis occurred in 60% (6/10) of the patients analysed (PBMCs, 1; NK, 4; neutrophils, 1). We also observed that RBC from patients with SNH display a significant reduction in the expression of CD47 when compared to healthy individuals. With CD47 being a "do not eat me" signal, its reduction is plausibly contributing to enhanced RBC clearance. These initial results suggest that cellular mechanisms beside extravascular antibody-mediated phagocytosis may play a role in seronegative hemolytic anemias.

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.

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.

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.

Refractive Index Tomograms and Dynamic Membrane Fluctuations of Red Blood Cells from Patients with Diabetes Mellitus

Background: Diabetic effects on blood rheology are important topics and have been extensively investigated. In particular, it has been known that diabetic RBCs exhibit significantly deteriorated deformability compared to healthy RBCs. Despite the importance of previously measured mechanical properties of diabetic RBCs, several problems remain to be elucidated. First, conventional techniques have difficulties in measuring intrinsic properties of individual cells. Detailed quantification of diabetic alteration in RBCs at the individual cell level is also hard to achieve. Accordingly, previous approaches do not allow the systematical correlative analyses to be performed on simultaneously measured various cell parameters. As a consequence, there are still no general agreements on the integrated effects of diabetic complications or hyperglycemia on individual human RBCs.Methods: To effectively settle the arisen problems, we quantitatively and non-invasively measured the individual RBCs from diabetic and healthy blood donors to figure out the characteristics of diabetic RBCs by employing the quantitative phase imaging (QPI) technique. From the reconstructed three-dimensional (3-D) refractive index (RI) tomogram and the time-series of 2-D phase map of individual RBCs, the morphological (volume, surface area, and sphericity), biochemical (Hb concentration and Hb content), and mechanical (membrane fluctuation) parameters were retrieved. Systematic correlative analyses among measured six RBC parameters and independently determined HbA1c level, defined as the ratio of HbA1c to total Hb, were also performed. In measuring red cells, only gently sedimented RBCs of discocytes were selected. After excluding erroneously measured RBCs, 40, 40, 40, 38, 39, 40 control RBCs of six healthy donors and 40, 40, 40, 40, 40, 40 diabetic RBCs of six diabetic patients were finally analyzed respectively for the study of diabetic effects on characteristics of individual human RBCs.Results: HbA1c level is defined as a percentage of HbA1c, the majority of glycated Hbs in RBC, to total Hb contents in blood. Because HbA1c is formed through 120-days long, non-enzymatic glycation process of Hb, HbA1c level has been perceived as an effective mean to estimate mean glucose concentration of individuals for the last three months. In other words, HbA1c reflects the degree of hyperglycemia. The most noticeable alterations in diabetic RBCs can be found when mean RBC membrane fluctuations of individuals are rearranged in the increasing order of the measured HbA1c levels. The mean values of membrane fluctuations for RBCs are 59.7 ¡¾ 6.2, 58.3 ¡¾ 4.9, 55.0 ¡¾ 5.4, 57.1 ¡¾ 7.4, 51.0 ¡¾ 6.8, and 49.6 ¡¾ 4.6 nm for healthy volunteers, and 45.1 ¡¾ 4.0, 46.9 ¡¾ 3.4, 48.8 ¡¾ 4.0, 47.6 ¡¾ 4.2, 43.0 ¡¾ 4.1, and 48.0 ¡¾ 5.0 nm for diabetic patients, in order of increasing HbA1c. As shown in Fig. 4, mean membrane fluctuation of non-diabetic RBCs tends to decrease as the HbA1c level increases (Pearson correlation coefficient of -0.87 with a p-value of 0.02). Beyond this level of HbA1c, RBCs from diabetic patients exhibit significantly decreased and settled membrane fluctuation. This observed negative correlation between RBC membrane fluctuation and HbA1 is also conceptually in accordance with the report of the previous study.Conclusions: We quantitatively and non-invasively characterize the morphological, biochemical, and mechanical alterations of individual RBCs by diabetes mellitus. HbA1c levels of subjects, which reflect one's mean blood glucose concentration of last three months, were also determined using hematology analyzers. Accordingly, correlative analyses among the retrieved individual RBC parameters and HbA1c level were performed to search unique features which cannot be addressed by conventional techniques with incapability of simultaneous measurement of those parameters. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Murine tribbles homolog 2 deficiency affects erythroid progenitor development and confers macrocytic anemia on mice.

Tribbles homolog 2 (Trib2) is a member of Tribbles protein pseudokinases and involves in apoptosis, autoimmunity, cancer, leukemia and erythropoiesis, however, the physiological function of Trib2 in hematopoietic system remains to be elucidated. Here, we report that Trib2 knockout (KO) mice manifest macrocytic anemia and increase of T lymphocytes. Although Trib2 deficient RBCs have similar half-life as the control RBCs, Trib2 KO mice are highly vulnerable to oxidant-induced hemolysis. Endogenous Trib2 mRNA is expressed in early hematopoietic progenitors, erythroid precursors, and lymphoid lineages, but not in mature RBCs, myeloid progenitors and granulocytes. Consistently, flow cytometric analysis and in vitro colony forming assay revealed that deletion of Trib2 mainly affected erythroid lineage development, and had no effect on either granulocyte or megakaryocyte lineages in bone marrow. Furthermore, a genetic approach using double knockout of Trib2 and C/ebpα genes in mice suggested that Trib2 promotes erythropoiesis independent of C/ebpα proteins in vivo. Finally, ectopic expression of human Trib2 in zebrafish embryos resulted in increased expression of erythropoiesis-related genes and of hemoglobin. Taking all data together, our results suggest that Trib2 positively promotes early erythrocyte differentiation and is essential for tolerance to hemolysis.

A Fatal Case of Cefazolin-Induced Immune Hemolytic Anemia in Iran

Introduction: Drug-induced immune hemolytic anemia (DIIHA) is known as an unpredictable and in some cases serious complication occurring upon pharmacotherapy. Cefazolin, as a first generation cephalosporin used against gram-negative bacteria, has rarely been reported to cause hemolytic anemia. Here, we report the first fatal case of hemolytic anemia associated with cefazolin treatment. Materials and methods: A 24-year-old woman undergoing operative hysteroscopy for resection of the uterine septum developed hemolytic anemia upon receiving cefazolin immediately before and after the operation. A few hours after surgery, clinical signs of disseminated intravascular coagulation (DIC) appeared. Despite all supportive medical measures that were taken, the patient died of multiple organ failure about 24 hours after admission to ICU. The patient had no history of transfusion or allergies to any types of drugs including antibiotics. Direct anti-globulin test (DAT) was conducted, and the presence of antibody and complement on patient’s RBCs was investigated. In addition, the reaction between patient’s RBC eluate and cefazolin-coated control RBCs was explored. Results: DAT was strongly positive (4+); in addition, the presence of IgG4 and C3d on the patient’s RBCs was shown. Moreover, the eluate from patient’s RBCs reacted (3+) with RBCs pre-coated with cefazolin. Discussion: The results confirmed the presence of cefazolin-dependent antibodies. This case reveals the importance of taking precaution before using cefazolin even in cases where there is no previous evidence of drug sensitization.

Engineering a "Self-Healing" Hydrogel-Based Microvasculature-on-a-Chip for Investigating the Effects of Cellular and Biomolecular Interactions on Endothelial Permeability in Sickle Cell Disease

Background: Endothelial activation and dysfunction play critical roles in vaso-occlusive crises and vasculopathy in sickle cell disease (SCD). However, it remains unclear how the myriad of cellular and biomolecular interactions that occur in SCD directly affect endothelial cell activation and injury, due largely in part to the lack of robust in vitro models for studying these complex biophysical processes. To this end, we engineered the first perfusable hydrogel-based microfluidic device comprised of "endothelialized" channels at the microvascular sizescale. Unlike typical microfluidic devices, which are silicone(PDMS)-based, this device is comprised of a collagen-based hydrogel that is not only more physiologic but also enables real-time monitoring of the endothelium permeability while allowing the user to tightly control hemodynamic conditions and the cellular and molecular components of the perfusate. Interestingly, in this system, upon removal of injurious stimuli, the endothelial cells are able to "self-heal" after injury and fully establish their barrier function. Using this system, we first tested whether interaction of SCD patient RBCs with endothelium under flow can directly cause endothelial permeability. We then studied how shear stress promotes endothelial cell activation and injury caused by free hemin, a byproduct of hemolysis in SCD.Results and Discussions: After seeding into the hydrogel-based device, human endothelial cells formed a monolayer that covered the entire inner surface of the microchannels and can be maintained for >1 month under flow conditions. Establishing that cultured endothelial cells are functional in this system, the cells appropriately formed continuous adherens junctions under flow as indicated by VE-cadherin staining (Figure 1A) and also deposited their own subendothelial extracellular matrices, including collagen IV (Figure 1B) and laminin (Figure 1C). Finally, perfusion of fluorescently-tagged albumin (BSA) sufficient endothelial barrier function of our system as all fluorescence signal was contained within the "vascular" space (Figure 1D and 1E).RBCs isolated from SCD patients were perfused into the endothelialized channels for 4 hours. Strikingly, the direct interaction of the perfused SCD RBCs with the engineered endothelium, in and of itself, was sufficient to induce endothelial permeability (Figure 2A), a phenomenon that did not occur with perfusion of control RBCs isolated from healthy volunteers. Interestingly, impermeability was reestablished as the endothelium "healed" 1 day post-interaction with patient RBCs (Figure 2B). We then perfused hemin in the channels under two different flow rates and the flow velocity profile, wall shear stress, shear rate, and pressure were characterized using COMSOL (Figure 3A and 3B). Interestingly, 1 hour of hemin exposure (10 µM) at higher shear stress compared to lower shear stress not only caused increased endothelial permeability and loss of endothelial cells at 1 day post-treatment, but also dampened the "healing" of injured endothelial cells and reestablishment of impermeability after removal of hemin from the system (Figure 3C and D).Conclusions and on-going work: Our physiologic hydrogel-based microvasculature-on-a-chip system enables investigation of how cell/molecular interactions directly affect endothelial permeability in real time and represents a milestone in the use of microfluidic devices for SCD research. In addition, the "self-healing" capability mimics the in vivo microvasculature and is a unique capability of this system as compared to current in vitro models. With this novel system, we determined that RBC-endothelial interactions under physiologic flow are sufficient to induce endothelial barrier dysfunction. We also demonstrated the additive role of hemodynamics in promoting hemin-induced endothelial activation and dysfunction. Ongoing work investigating the mechanisms of our observations will unveil insight into SCD pathogenesis and our system can be applied to other vascular diseases as well. [Display omitted] [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Vascular Geometry and Flow Profile Mediate Pathological Cell-Cell Interactions in Sickle Cell Disease As Measured with “Do-It-Yourself” “Endothelial-Ized” Microfluidics

Background and Significance: Cell-cell interactions between blood cells and endothelial cells play an important role in sickle cell disease (SCD) pathophysiology. While in vivo transgenic animal models and in vitro systems have both contributed to our understanding of these pathologic cell-cell interactions in SCD, isolating the causes and effects of cellular interactions is exceedingly difficult in the former and recapitulating the complex vascular geometries found in vivo is not readily available with current systems in the latter. The vascular system comprises diverse geometries that range from normal (e.g. curves and bifurcations) to pathologic (e.g. aneurysms and stenoses) and as blood flows from one vascular geometry to another, the local shear stress profile acutely changes. Interestingly, changes in shear stress are known to alter endothelial pro-inflammatory signaling pathways and expression of cell adhesion molecules, especially vascular cell adhesion molecule-1(VCAM-1) (Tzima, Nature, 2005), which is implicated in SCD vasculopathy. Here we present a rapid and inexpensive method using only off-the-shelf materials to create “do-it-yourself” (DIY) microfluidic devices that incorporate endothelial cells and clinically relevant vascular geometries; this system effectively and bridges current in vitro and in vivo models to study SCD. Using this technique, we developed a vascularized bifurcation, and observed that shear stress changes can be extremely localized, affecting only several 10s of cells, and are associated with changes in VCAM1 expression. We used this in vitro vascularized bifurcation to test the hypothesis that SS RBC-endothelial cell adhesion occurs primarily at bifurcations, which are difficult to visualize in vivo (Nagel,Arterioscler Thromb Vasc Biol, 1999). We demonstrate that SCD RBCs do primarily aggregate at bifurcations, specifically in locations where the shear stress has decreased and VCAM-1 is upregulated.Methods: In order to bridge in vitro data with the complex vascular geometric environments found in vivo, we developed a “DIY” endothelialized microfluidic model (Figure 1A). A strand of 500um diameter polymethylmethacrylate (PMMA) optical fiber is laid flat on top of a layer of polydimethylsiloxane (PDMS) and covered with a second, thin layer of PDMS. After curing, the optical fiber is pulled out, exposing a hollow, circular, channel that can be used as a microchannel to seed endothelial cells. A wide variety of endothelial cells can be successfully seeded in these devices, such as human umbilical vein endothelial cells, human aortic endothelial cells, and human microvascular endothelial cells. Slight alterations to this fabrication method result in the creation of multiple vascular geometries, such as curved or bifurcated channels with or without aneurysms or stenoses.Results: Curved channels & bifurcations (Figure 1B-C) are seeded with endothelial cells (Figure 1E-F). Computational fluidic dynamics calculations show that the shear varies by 2.5 fold within the bifurcation. As shear affects endothelial expression, we tested if the extremely localized shear changes created in this system were sufficient to alter local endothelial expression of VCAM-1 Indeed, in our system, VCAM1 expression significantly correlated with shear variation (Figure1G), and was highest near the bifurcation point. Noting this localized variation in adhesion molecule expression, we tested whether the bifurcations are implicated in SCD RBC adhesion to the endothelium. With our vascularized bifurcation model and custom image analysis software that quantifies RBC aggregation, we observed that SCD RBC adhesion predominantly occurred at the point of bifurcation where the shear is lowest and VCAM1 expression is greatest, and minimal endothelial adhesion occurred with healthy control RBCs (Figure 2). This phenomenon persisted with tumor necrosis factor-stimulation of the endothelium.Conclusion: This DIY system represents an easily accessible technique that allows any researcher to bridge the gap between in vitro and in vivo models of pathological cell-cell interactions in SCD. We demonstrate that recapitulating the complex vascular geometries in vivo is vital to understanding blood cell-endothelial interactions and this system will not only be useful for studying SCD, but a myriad of hematologic and vascular diseases as well. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

IHP-Loaded Red Blood Cells Efficacy in Sickle Transgenic Mice

Abstract 2119Homozygous patients (SS) display the most severe form of sickle cell disease (SCD) with high risk of painful complications during their lifetime including vaso-occlusions (VOC), acute chest syndromes (ACS) and strokes. Even though multiple factors seem to be involved in those sickle related events, hypoxia is the most important triggering factor. Transfusing patients with IHP-RBCs (homologous RBCs loaded with Inositol Hexaphosphate) could potentiate existing transfusion therapy. Indeed, IHP by reducing the oxygen-hemoglobin affinity improves the capacity of transfused RBCs to release more oxygen in the blood stream and to the tissues. In a previous work, we showed that IHP-RBCs were 7-fold more effective than normal RBCs to reduce in vitro sickling of homozygous patient RBCs (Bourgeaux et al, Transfusion, 2010). We hypothesized that in vivo, IHP-RBCs with increased oxygenation properties may reduce the severity of SCD pathophysiology in transgenic mice.In a first study, BERK transgenic mice which are more representative of SCD in childhood (splenomegaly and RBC sequestration crisis) were used to evaluate the effect of chronic IHP-RBCs transfusions. Mice (n=5-7/group) were subjected to 4 repeated RBC exchanges (every 2 weeks) using IHP-RBCs or control RBCs. One week after the last exchange, mice were challenged with hypoxia and sacrificed for organ and hematological analysis. It was observed an improved survival rate and a prevention of anemia under hypoxic conditions (Hb=7.0±0.6 g/dl vs 5.9±0.4 g/dl for mice receiving control RBCs). On the contrary to control RBCs, IHP-RBCs treatment also corrected impaired brain development observed in female BERK mice with higher brain weight to total body weight ratio (2.12±0.07% versus 1.89±0.08% for untreated mice, p<0.05). IHP-RBCs by modulating cellular blood components and improving the vascular tone also lowered the risk of VOC in BERK mice with decreased proportion of reticulocytes (26±6 % vs 34±4% for treatment with normal RBCs and 42±2 % for untreated mice), reduced levels of soluble thrombospondin-1 involved in adhesion processes (104±22 pg/mL vs 314±56 pg/mL for treatment with normal RBCs and 223±50 pg/mL for untreated mice) as well as endothelin-1, a peptide promoting vaso-constriction.In a second study, SAD transgenic mice which were shown to be a pertinent model for acute sickle events were used to investigate the efficiency of a simple transfusion of IHP-RBCs. Mice (n=10–12/group) partially exchanged with IHP-RBCs or control RBCs were subjected to an acute hypoxic-reoxygenation stress 24h post-transfusion in order to induce specific lung damages. The therapeutic benefice of IHP-RBCs over classical transfusion to reduce lung injury was evidenced by significantly decreased mRNA levels of molecules involved in intravascular pathology (7-fold and 8-fold reduction in VCAM-1 and Tissue factor mRNA levels as compared to mice receiving control RBCs (p<0.05). AOPP used as specific biomarker of oxidative stress was significantly reduced with IHP-RBCs treatment as compared to untreated mice (2.64±0.27 μmol/L vs 3.87±0.30 μmol/L respectively).From a safety point of view, one single intravenous administration of IHP-RBCs showed no apparent toxicity in healthy mice and seems to be as safe as classical transfusion. Only moderate hemolysis was observed after transfusion with increased serum bilirubin level but this parameter recovered normal values within 24h.In conclusion, entrapping IHP into transfused RBCs is a powerful tool to prevent acute or chronic manifestations of SCD in transgenic mice. For therapeutic use in human, IHP-RBCs may potentiate chronic transfusions by reducing the number of RBC units to be transfused but also the frequency of transfusions thus improving patient quality of life. In addition, with the recent improvements in scoring acute manifestations of SCD, IHP-RBCs may also potentiate classical transfusion in the treatment of chest syndromes by acting more rapidly and more efficiently. Disclosures:Bourgeaux:ERYtech Pharma: Employment. Campion:ERYTECH Pharma: Employment. Chevrier:ERYtech Pharma: Employment. Godfrin:ERYTECH Pharma: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

New antimalarial indolone- N-oxides, generating radical species, destabilize the host cell membrane at early stages of Plasmodium falciparum growth: role of band 3 tyrosine phosphorylation

Although indolone- N-oxide (INODs) genereting long-lived radicals possess antiplasmodial activity in the low-nanomolar range, little is known about their mechanism of action. To explore the molecular basis of INOD activity, we screened for changes in INOD-treated malaria-infected erythrocytes ( Pf-RBCs) using a proteomics approach. At early parasite maturation stages, treatment with INODs at their IC 50 concentrations induced a marked tyrosine phosphorylation of the erythrocyte membrane protein band 3, whereas no effect was observed in control RBCs. After INOD treatment of Pf-RBCs we also observed: (i) accelerated formation of membrane aggregates containing hyperphosphorylated band 3, Syk kinase, and denatured hemoglobin; (ii) dose-dependent release of microvesicles containing the membrane aggregates; (iii) reduction in band 3 phosphorylation, Pf-RBC vesiculation, and antimalarial effect of INODs upon addition of Syk kinase inhibitors; and (iv) correlation between the IC 50 and the INOD concentrations required to induce band 3 phosphorylation and vesiculation. Together with previous data demonstrating that tyrosine phosphorylation of oxidized band 3 promotes its dissociation from the cytoskeleton, these results suggest that INODs cause a profound destabilization of the Pf-RBC membrane through a mechanism apparently triggered by the activation of a redox signaling pathway rather than direct oxidative damage.

Expression of the Rh/RhAG complex is reduced in Mi.III erythrocytes

Miltenberger blood group antigen subtype III (Mi.III) is characterized by expression of a glycophorin B-A-B hybrid (Gp.Mur) on the erythrocyte surface. The two alleles of glycophorin B are substituted with the B-A-B hybrid alleles in homozygous Mi.III (Mi.III(+/+)), and thus, Mi.III(+/+) erythrocytes lack glycophorin B (GPB) and express Gp.Mur only. Because GPB is a major component of the Rh complex on RBCs, in this study, we explored how the absence of GPB might affect Rh expression in Mi.III RBCs. (1) Mi.III+ RBCs were serologically identified and further differentiated their homozygosity or heterozygosity by immunoblot or direct sequencing. (2) RhD and RhCcEe mRNA was cloned, and their sequences analysed. (3) The expression levels of Rh antigen, Rh-associated glycoprotein (RhAG) and the U antigen in MI.III vs. non-Mi.III RBCs were assessed by flow cytometry and Western blot. Compared with the non-Mi.III samples, the surface expression of the Rh antigen was reduced to 76·4% in Mi.III(+/+) RBCs and 93·6% in Mi.III(+/-). RhAG expression was also significantly reduced in Mi.III(+/+), but not in Mi.III(+/-). The U antigen expression in Mi.III(+/-) was only 14·9% relative to the control RBCs, while GPB was half the level of the controls. The mRNA sequences of Rh polypeptides from Mi.III+ samples were identical to the NCBI reference sequences. Substitution of GPB with Gp.Mur significantly reduced the expression of Rh antigen and RhAG on the Mi.III(+/+) erythrocyte membrane. The Mi.III phenotype is predicted to induce considerable structural variations within the band 3/Rh-associated macrocomplexes.

Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients

An increased oxidative stress and a decreased life span of erythrocytes (RBCs) are reported in patients with diabetes. Aim of this study was to assess in RBCs from patients with type 2 diabetes whether downstream effector mechanisms of apoptosis, such as activation of caspase-3, is operative, and whether an iron-related oxidative imbalance, occurring inside RBCs and in plasma, could be involved in caspase-3 activation. In 26 patients with type 2 diabetes and in 12 healthy subjects, oxidative stress was evaluated by means of different markers; non-protein-bound iron, methemoglobin and glutathione were determined in RBCs, and non-protein-bound iron was also determined in plasma. Erythrocyte caspase-3 activation was evaluated by an immunosorbent enzyme assay. Arterial hypertension, demographic and standard biochemical data were also evaluated. The results show, for the first time, that type 2 diabetic RBCs put into motion caspase-3 activation, which is significantly higher than in control RBCs. Such an effector mechanism of "eryptosis" was positively correlated to blood glucose levels and to the increased plasma NPBI level. Caspase-3 activation was also positively correlated to occurrence of arterial hypertension. The results suggest that an extracellular oxidative milieu can be responsible for erythrocyte caspase-3 activation in patients with type 2 diabetes. In turn, caspase-3 activation can be envisaged as a novel mechanism which, by impairing the maintenance of erythrocyte shape and function, might contribute to the shortened life span of RBCs from patients with type 2 diabetes and to hemorheological disorders observed in these patients.

Irreversible AE1 Tyrosine Phosphorylation Leads to Membrane Vesiculation in G6PD Deficient Red Cells

BackgroundWhile G6PD deficiency is one of the major causes of acute hemolytic anemia, the membrane changes leading to red cell lysis have not been extensively studied. New findings concerning the mechanisms of G6PD deficient red cell destruction may facilitate our understanding of the large individual variations in susceptibility to pro-oxidant compounds and aid the prediction of the hemolytic activity of new drugs.Methodology/Principal FindingsOur results show that treatment of G6PD deficient red cells with diamide (0.25 mM) or divicine (0.5 mM) causes: (1) an increase in the oxidation and tyrosine phosphorylation of AE1; (2) progressive recruitment of phosphorylated AE1 in large membrane complexes which also contain hemichromes; (3) parallel red cell lysis and a massive release of vesicles containing hemichromes. We have observed that inhibition of AE1 phosphorylation by Syk kinase inhibitors prevented its clustering and the membrane vesiculation while increases in AE1 phosphorylation by tyrosine phosphatase inhibitors increased both red cell lysis and vesiculation rates. In control RBCs we observed only transient AE1 phosphorylation.Conclusions/SignificanceCollectively, our findings indicate that persistent tyrosine phosphorylation produces extensive membrane destabilization leading to the loss of vesicles which contain hemichromes. The proposed mechanism of hemolysis may be applied to other hemolytic diseases characterized by the accumulation of hemoglobin denaturation products.

A Novel Connection Between Stress Erythropoiesis and Coagulation Activation in Sickle Cell Disease.

Abstract 905 Introduction:Sickle cell disease (SCD) is associated with a hypercoagulable state. Multiple studies show that plasma from these patients exhibit: 1) increased thrombin generation; 2) decreased levels of natural anticoagulant proteins; and 3) a defect in the activation of fibrinolysis. The mechanism of coagulation activation in SCD is presumed to be multi-factorial, with contributions from abnormal erythrocyte phospholipid asymmetry and induction of tissue factor (TF) following hemolysis. In addition, hemolysis in SCD leads to elevated levels of erythropoietin (EPO) in patients, increased reticulocyte counts and the presence of stress (or shift) reticulocytes in circulating blood. These stress reticulocytes retain expression of the α4b1 integrin and are demonstrably adhesive to vascular factors in SCD. We have previously reported that these stress reticulocytes bind to blood monocytes in SCD patients via the α4b1 integrin, but the effect of this interaction on either cell remained unknown in SCD. Objective:With the increasing evidence that hemolysis and subsequent stress erythropoiesis associates with coagulation activation, we sought to evaluate the role of erythropoietin and the effect of stress reticulocyte adhesion to monocytes on coagulation activation in SCD patients. Methods:Coagulation activation in plasma samples was examined by evaluating TF activity on microparticles derived from patients with SCD. Stress reticulocytes were visualized and enumerated from these same patients using Wright Giemsa stained blood smears counter stained with new methylene blue to detect reticulocytes. Reticulocytes were scored as a stress reticulocytes based on the amount of punctuate reticular material, cell size, and presence of nuclear material. Stress reticulocyte induction of monocyte tissue factor expression was measured by flow cytometry after incubation of THP-1 monocytic cells with purified SS RBCs or control RBCs. To determine if induced THP-1 TF expression was due stress reticulocyte binding, THP-1 TF expression was examined in the presence or absence of known inhibitors of the monocyte/stress reticulocyte interaction. TF expression on CD14+ monocytes was examined in whole blood from SCD patients using flow cytometry. Plasma erythropoietin levels were quantified by ELISA. Results:We found that direct binding of the stress reticulocyte increased THP-1 TF expression 2.5 fold. This increase in TF expression was completely ablated by function blocking antibodies against the α4 integrin, but not by an isotype-matched control IgG. In whole blood samples, we also found increased TF expression on CD14+ monocytes with stress reticulocytes directly bound, compared to those monocytes in the same patient without stress reticulocytes bound (p = 0.002, n =3).We noted a strong correlation between stress reticulocyte count and TF activity on plasma microparticles in SCD (rspearman = 0.8656, CI = 0.5382 – 0.9660, p = 0.0006, n=11). Furthermore, we found that EPO induced α4b1 activation on the stress reticulocyte. This activation may promote both adhesion to the monocyte and an increase in TF expression. Consequently, we noted a strong trend towards an association of EPO with microparticle TF activity in SCD (rspearman= 0.5740, CI=-0.06 – 0.8780, p=0.068, n= 11) suggesting that EPO, by promoting the interaction between the stress reticulocyte and the monocyte, may contribute to TF activity in SCD. Conclusion:Taken together, we find that stress reticulocyte adhesion to monocytes and monocytic cells induces TF expression and may promote TF activity in patients. These data suggest a novel connection between stress erythropoiesis and coagulation activation in SCD. Disclosures:No relevant conflicts of interest to declare.

Anticoagulant‐dependent in vitro hemagglutination in a cat

A 17-year-old domestic shorthaired cat was presented to the Cornell University Hospital for Animals for recheck of hyperthyroidism previously treated with radioiodine. Marked agglutination was noted in a blood sample collected into EDTA for a CBC; no other clinical or hematologic evidence of hemolysis was observed and none developed despite persistent agglutination in additional EDTA samples collected over a 2-month period. Blood drawn into heparin and sodium citrate tubes did not have grossly or microscopically detectable agglutination, unless EDTA was added. Plasma from the cat induced agglutination of washed RBCs from a control cat in the presence of EDTA but not in the presence of heparin. Flow cytometric analysis of samples created by mixing plasma from the patient with washed RBCs from a control cat showed immunoglobulin coating of the control RBCs, predominantly by IgM. These findings suggested an anticoagulant-dependent antibody-mediated mechanism for the agglutination. EDTA-dependent hemagglutination has not been reported previously in cats, although rare cases have been described in humans. The phenomenon needs to be recognized as an in vitro occurrence in order to prevent erroneous diagnosis of immune-mediated hemolytic anemia.

Effects of trehalose-loaded liposomes on red blood cell response to freezing and post-thaw membrane quality

We are investigating the use of liposomes, which are synthetic, microscopic vesicles, for the intracellular delivery of trehalose into mammalian cells. This study focuses on the effects trehalose-containing liposomes improve the recovery and membrane quality of human RBCs following cryopreservation. Unilamellar liposomes consisting of a lipid bilayer composed of DPPC, PS and cholesterol (60:30:10mol%) were synthesized using an extrusion method. Liposome-treated RBCs (l-RBCs) were resuspended in either physiological saline, 0.3M trehalose or liposome solution, then cooled with slow (0.95±0.02°C/min), medium (73±3°C/min) and fast (265±12°C/min) cooling rates and storage in liquid nitrogen, followed by a 37°C thawing step. RBC post-thaw quality was assessed using percent recovery, RBC morphology, PS and CD47 expression. Liposome treatment did not adversely affect the RBC membrane. Post-thaw recovery of l-RBCs was significantly higher (66%±5% vs 29%±4%) compared to control RBCs (c-RBC, p=0.003). Medium and high cooling rates resulted in significantly higher cell recovery compared to a slow cooling rate (p=0.039 and p=0.041, respectively). The recovery of l-RBCs frozen in liposome solution and trehalose solution was significantly higher than that of l-RBCs frozen in NaCl solution for all three cooling rates (p=0.021). Flow cytometry and morphology assessment showed that liposome treatment resulted in improved post-thaw membrane quality. There was no statistically significant difference in the post-thaw recovery between RBCs treated with liposomes containing trehalose in their aqueous core and RBCs treated with liposomes containing saline in their aqueous core (p=0.114). Liposome treatment significantly improves the recovery and membrane integrity of RBCs following low temperature exposure.

Comparative assessment of normal and methoxypolyethylene glycol–modified murine red cells on swimming endurance and hippocampal injury in hypoxic mice

Membrane grafting of methoxypolyethylene glycol (mPEG) provides a unique strategy in preventing the immunologic recognition in blood transfusion. mPEG-modified red blood cells (mPEG-RBCs) have acceptable in vitro properties and provide a useful solution to problems with clinical blood matching. The aim of this study was to demonstrate the physiologic normality of mPEG-RBCs in mice. Mouse RBCs were withdrawn via cardiac bleed and modified with 1.0 mmol per L mPEG with succinimidyl propionate linker. The fluorescein-labeled mPEG-RBCs were then transfused into recipient mice for in vivo survival analysis. At the same time, the exsanguine mouse model was produced, and mice were transfused with mPEG-RBCs. The effects of mPEG-RBC transfusion on the hemoglobin (Hb) level, swimming endurance capacity, and hypoxic-ischemic injury in hippocampal pyramidal cells of exsanguine mice were investigated. mPEG-RBCs showed the same in vivo survival curve and t((1/2)) as those of untreated RBCs. Transfusion of mPEG-RBCs could elevate Hb level of exsanguine mice and improve their swimming endurance capacity, and histologic studies showed that mPEG-RBCs could also restore the hypoxic-ischemic injury of hippocampal pyramidal cells in exsanguine mice, which were similar with control RBCs. That is, mPEG-RBCs functioned in a similar fashion to untreated RBCs in exsanguine mice. Therefore, these results revealed that mPEG-RBCs had normal oxygen-carrying capacity. In conclusion, the results confirmed that mPEG-RBCs could perform their in vivo function of carrying O(2) and improve some physiologic indexes of exsanguine mice, and the physiologic normality of mPEG-RBCs provides new findings for clinical use.

Increased procoagulant activity of red blood cells in the presence of cisplatin

Cisplatin based chemotherapy is a well recognized risk factor for coagulation disorders and thrombosis. The pathophysiological mechanisms by which cisplatin promote thrombosis are not well understood. Red blood cells (RBCs) were separated from peripheral blood of patients with breast cancer (n = 10) and healthy adults (n = 6) and treated with cisplatin. Coagulation time of RBCs was assessed by one step recalcification time and the productions of thrombin, intrinsic and extrinsic factor Xa were measured in the presence or absence of various concentrations of lactadherin. Exposed phosphatidylserine was stained with lactadherin and observed by confocal microscopy and flow cytometry. Neither fresh RBCs nor RBCs treated without cisplatin had potent procoagulant activity. Cisplatin treatment increased procoagulant activity of RBCs in a cell number- and concentration-dependent manner. Exposed phosphatidylserine was stained with lactadherin and after cisplatin treatment, strong fluorescence was revealed by confocal microscopy. Lactadherin bound RBCs from patients with breast cancer increased from (1.9 +/- 0.5)% on control RBCs to (68.0 +/- 3.5)% on RBCs treated with 10 micromol/L cisplatin for 24 hours. Cisplatin treatment increases procoagulant activity of RBCs, which have a strong association with exposure of phosphatidylserine. The increased procoagulant activity may contribute to the pathogenesis of thrombophilia during cisplatin based chemotherapy in breast cancer patients.