Red Blood Cell Microparticles Research Articles

Significant hemolysis is present during irreversible electroporation of cardiomyocytes in vitro

BackgroundPulsed field ablation (PFA) of atrial fibrillation is a new method in clinical practice. Despite a favorable safety profile of PFA in atrial fibrillation ablation, rare cases of renal failure, probably due to hemolysis, have recently been reported. ObjectiveThe aim of this study was to determine the rate of hemolysis and cardiac cell death during in vitro PFA with different electric field intensities. MethodsBlood samples from healthy volunteers and mouse HL-1 cardiomyocyte cell lines were subjected to in vitro irreversible electroporation using 216 bipolar pulses, each lasting 2 μs with intervals of 5 μs, repeated 20 times at a frequency of 1 Hz. These pulses varied from 500 V to 1500 V. Cell-free hemoglobin levels were assessed spectrophotometrically, and red blood cell microparticles were evaluated by flow cytometry. Cardiomyocyte death was quantified with propidium iodide. ResultsPulsed field energy (1000 V/cm, 1250 V/cm, and 1500 V/cm) was associated with a significant increase in cell-free hemoglobin (0.32 ± 0.16 g/L, 2.2 ± 0.96 g/L, and 5.7 ± 0.39 g/L; P < .01) and similar increase in the concentration of red blood cell microparticles. Significant rates of cardiomyocyte death were observed at electric field strengths of 750 V/cm, 1000 V/cm, 1250 V/cm, and 1500 V/cm (26.5% ± 5.9%, 44.3% ± 6.2%, 55.5% ± 6.9%, and 74.5% ± 17.8% of cardiomyocytes; P < .01). ConclusionThe most effective induction of cell death in vitro was observed at 1500 V/cm. This intensity was also associated with a significant degree of hemolysis.

Procoagulant activity of red blood cell microparticles in stored packed red blood cell units and its relation to ABO blood grouping

BackgroundThroughout the storage of blood, the red cells undergo alterations known as “storage lesions,” which involve shape changes and the formation of microparticles (MPs). Studies of the formation of red cell microparticles (RMPs) emphasize the prospective application of RMPs as a quality control measure in the preparation and storage of blood components in the future. In the present study, twenty packed RBC units in citrate phosphate dextrose adenine-1 (CPDA1) were collected from volunteers and stored for 35 days. Over 35 days of storage, samples were collected at six distinct time points weekly and evaluated for the presence of RMPs. MPs were separated by the ultracentrifugation method. Electron microscopy was used to characterize the morphology and size of the isolated microparticles, and flow cytometry was performed to determine the percentage of RMPs that expressed glycophorin A (CD235a) and Annexin V antigens. RMPs' procoagulant activity (PCA) was assessed using a plasma recalcification test. RMP concentration in accordance with ABO blood grouping was assessed by using various types of donated blood groups.ResultsRMPs progressively increased over storage. The procoagulant activity (PCA) exhibited a significant increase during storage, as evidenced by a shorter plasma recalcification time (P value = 0.001). A significant negative correlation (P value = 0.001) between plasma recalcification time and Annexin V-positive microparticles, as well as a dual-positive Annexin V/CD235a population, was identified, indicating a strong correlation between the direct quantitative assay by flowcytometry and the functional assay through the PCA.ConclusionRMPs increase on storage with increased PCA. Finding ways to reduce these microparticles in packed RBC units is crucial for reducing the risk of transfusion-related coagulopathy.

Periprocedural Intravascular Hemolysis During Atrial Fibrillation Ablation: A Comparison of Pulsed Field With Radiofrequency Ablation

BackgroundHemolysis-related renal failure has been described after pulmonary vein isolation (PVI) with pulsed-field ablation (PFA). ObjectivesThis study sought to compare the potential for hemolysis during PVI with PFA vs radiofrequency ablation (RFA). MethodsIn consecutive patients, PVI was performed with PFA or RFA. Blood samples were drawn at baseline, immediately postablation, and 24 hours postablation. Using flow cytometry, the concentration of red blood cell microparticles (RBCμ) (fragments of damaged erythrocytes) in blood was assessed. Lactate dehydrogenase (LDH), haptoglobin, and indirect bilirubin were measured at baseline and 24 hours. ResultsSeventy patients (age: 64.7 ± 10.2 years; 47% women; 36 [51.4%] paroxysmal atrial fibrillation) were enrolled: 47 patients were in the PFA group (22 PVI-only and 36.4 ± 5.5 PFA applications; 25 PVI-plus, 67.3 ± 12.4 pulsed field energy applications), and 23 patients underwent RFA. Compared to baseline, the RBCμ concentration increased ∼12-fold postablation and returned to baseline by 24 hours in the PFA group (median: 70.8 [Q1-Q3: 51.8-102.5] vs 846.6 [Q1-Q3: 639.2-1,215.5] vs 59.3 [Q1-Q3: 42.9-86.5] RBCμ/μL, respectively; P < 0.001); this increase was greater with PVI-plus compared to PVI-only (P = 0.007). There was also a significant, albeit substantially smaller, periprocedural increase in RBCμ with RFA (77.7 [Q1-Q3: 39.2-92.0] vs 149.6 [Q1-Q3: 106.6-180.8] vs 89.0 [Q1-Q3: 61.2-123.4] RBCμ/μL, respectively; P < 0.001). At 24 hours with PFA, the concentration of LDH and indirect bilirubin increased, whereas haptoglobin decreased significantly (all P < 0.001). In contrast, with RFA, there were only smaller changes in LDH and haptoglobin concentrations (P = 0.03) and no change in bilirubin. ConclusionsPFA was associated with significant periprocedural hemolysis. With a number of 70 PFA lesions, the likelihood of significant renal injury is uncommon.

Multi-Omics Analysis of the Impact of Long Distance Trail Running on Red Blood Cells Highlights Acylcarnitine Mediated Lipid Damage Repair Mechanisms

Red blood cells (RBC) play critical roles in the maintenance of vascular tone and metabolite transfer in addition to their well characterized role in gas exchange. In this capacity, they play a central role in maintaining systemic activity during stressful physiological situations such as exercise. At the far end of the spectrum for endurance sports, the popularity of long-distance running has been increasing in recent years. However, literature on the impact of marathon and ultramarathon running on red blood cell physiology is limited. To characterize molecular responses in RBC to long-distance running, blood was sampled from 11 runners before and after the Martigny-Combes à Chamonix (MCC, distance: 40 km, elevation gain: 2300 m) or 12 runners before and after the Ultra - Trail du Mont Blanc (UTMB, distance: 171 km, elevation gain: 10,000 m) and separated into plasma and RBC components. Hematological parameters and RBC deformability were assessed before and after each race. 1 Here, the same samples were analyzed using mass spectrometry-based metabolomics, lipidomics, proteomics, and elemental analysis ( Figure 1A). While RBC after both races had decreased mean cell volume (MCV), RBC exhibited higher amounts of exposed phosphatidylserine (PS) on the outer membrane only after the MCC, which serves as a signal for eryptosis. However, hematocrit (Hct) remained unchanged after this race, but was significantly lower after the UTMB. 1 Although decreased Hct can result from hemodilution, the plasma levels of albumin, sodium, and potassium were not significantly altered in either race. In addition, RBC microparticles increased after the UTMB with concomitant decreases in the deformability of intact RBC at high shear stresses, effects of which were not observed after the MCC. Intracellular metabolic correlates with deformability were enriched with acylcarnitines (AC - Figure 1A), which inversely correlated with RBC deformability at 30 Pa. This compound class was also positively associated with the level of IL-6 in addition to kynurenine, an immunomodulatory metabolite which has also been linked to IL-6 level and RBC damage in COVID19. 2 Within RBC, that are largely devoid of mitochondria, AC play an intermediary role in membrane lipid repair ( Figure 1A). While AC and fatty acids were significantly higher after both races, the magnitude of increase was higher after the MCC ( Figure 1B). However, the ratios of lysophosphatidylcholines or lysophosphatidylethanolamines to their respective intact lipid counterparts were predominantly lower after the UTMB, indicating more active membrane lipid repair in these cells ( Figure 1B). In support, the coenzyme A precursor pantothenate was consumed after both races, though carnitine accumulated only after the UTMB possibly due to increased acyl-CoA utilization for lipid repair rather than interconversion into AC. Increased oxidative stress was also apparent in the proteome, which demonstrated that the proportion of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with irreversibly oxidized active site cysteine was significantly higher after the UTMB. As this protein plays a key regulatory role in glucose oxidation by mediating fluxes through glycolysis or the pentose phosphate pathway (PPP) in an oxidative stress-dependent fashion, 3 increased damage to its active site may partially explain a shift in glucose catabolism away from glycolysis and towards the PPP to generate NADPH reducing equivalents to cope with accumulating oxidative stress. In consideration of the varying temporal exposures to physiological stress between the MCC and UTMB, these results provide an in vivo snapshot into the accumulation of damage, repair, and removal of RBC as they circulate throughout the body during exercise. 1. Robert M, Stauffer E, Nader E, et al. Impact of Trail Running Races on Blood Viscosity and Its Determinants: Effects of Distance. Int. J. Mol. Sci. 2020;21(22):8531. 2. Thomas T, Stefanoni D, Dzieciatkowska M, et al. Evidence of Structural Protein Damage and Membrane Lipid Remodeling in Red Blood Cells from COVID-19 Patients. J. Proteome Res. 2020;19(11):4455-4469. 3. Reisz JA, Wither MJ, Dzieciatkowska M, et al. Oxidative modifications of glyceraldehyde 3-phosphate dehydrogenase regulate metabolic reprogramming of stored red blood cells. Blood. 2016;128(12):e32-e42.

Vascular pathophysiology of sickle cell disease

Sickle cell disease (SCD) is an hereditary disorder characterized by the production of an abnormal hemoglobin called hemoglobin S (HbS). HbS may polymerize in deoxygenated conditions, which leads to red blood cell (RBC) sickling. Sickled RBCs are more rigid and fragile, and prone to lysis. SCD patients exhibit various acute and/or chronic complications, which may affect several organs. The clinical presentation of SCD is highly variable from one patient to another and cannot be only explained by RBC sickling. Increased blood viscosity, caused by the presence of RBCs with abnormal deformability and aggregation, may increase vascular resistance and increase the risk of acute and chronic vascular complications. Chronic hemolysis results in decreased nitric oxide (NO) bioavailability which may compromise vasodilation and participate to the development of chronic vasculopathy. Furthermore, chronic hemolysis is responsible for increased inflammation and oxidative stress, which affect the vascular system and may promote the adhesion of circulating cells to endothelial cells. Extracellular vesicles and especially RBC microparticles (massively released in the context of SCD) are also at the origin of vascular damages and increased white blood cells adhesion to the endothelium, which may trigger vaso-occlusive crisis and other vascular-related complications. This review highlights the fact that SCD should not only be considered as a hematological disorder but also as a vascular disease.

Shear stimulated red blood cell microparticles: Effect on clot structure, flow and fibrinolysis.

Microparticles (MPs) have activity in thrombus promotion and generation. Erythrocyte microparticles (ErMPs) have been reported to accelerate fibrinolysis in the absence of permeation. We hypothesized that shear induced ErMPs would affect fibrin structure of clots and change flow with implications for fibrinolysis. To determine the effect of ErMPs on clot structure and fibrinolysis. Plasma with elevated ErMPs was isolated from whole blood or from washed red blood cells (RBCs) resuspended in platelet free plasma (PFP) after high shear. Dynamic light scattering (DLS) provided size distribution of ErMPs from sheared samples and unsheared PFP controls. Clots were formed by recalcification for flow/lysis experiments and examined by confocal microscopy and SEM. Flow rates through clots and time-to-lysis were recorded. A cellular automata model showed the effect of ErMPs on fibrin polymerization and clot structure. Coverage of fibrin increased by 41% in clots formed from plasma of sheared RBCs in PFP over controls. Flow rate decreased by 46.7% under a pressure gradient of 10 mmHg/cm with reduction in time to lysis from 5.7 ± 0.7 min to 12.2 ± 1.1 min (p < 0.01). Particle size of ErMPs from sheared samples (200 nm) was comparable to endogenous microparticles. ErMPs alter the fibrin network in a thrombus and affect hydraulic permeability resulting in decelerated delivery of fibrinolytic drugs.

Ex Vivo Evaluation of Red Blood Cell Adhesion and Whole Blood Thrombosis in Pyruvate Kinase Deficiency

IntroductionPyruvate Kinase Deficiency (PKD) is an inherited glycolytic enzymopathy that is characterized by a life-long chronic hemolytic anemia with severe comorbidities. Hypercoagulability due to increased platelet activity caused by nitric oxide sequestration by cell free hemoglobin has been well-described not just in PKD, but in other hemolytic anemias as well, such as e.g., sickle cell disease (SCD). Hypercoagulability is often accompanied by a cascade of pathophysiological events leading to cell oxidative damage, endothelial activation, and changes in both cell stability and adhesive properties. Increased red blood cell (RBC) adhesion and hypercoagulability may impair microvascular blood flow. Despite these well-recognized rheological changes that are similar to those that occur in other hemolytic anemias, the relationship between baseline erythrocyte adhesion and thrombosis potential have not been well-studied in PKD.Methods10 PKD subjects and 5 healthy controls were recruited under the IRB-approved protocol from Wayne State University. Flow adhesion of whole blood to vascular cell adhesion molecule-1 (FA-WB-VCAM) was performed by flowing whole blood (1:1 dilution) through a microfluidic channel for 3 minutes (1 dyne/cm 2 shear stress, 1.67Hz pulse frequency). Flow adhesion avidity of the whole blood sample to VCAM-1 (FAAv-WB-VCAM), representing the strength of the RBC-VCAM-1 adhesive interactions, was assessed by quantifying adhesion following sequential increase in shear (5, 10, 20 dyne/cm 2). Thrombin generation assay was conducted using platelet poor plasma with and without thrombomodulin and microparticles (MP) as previously published [1]. Clotting time - reported as lag time (LT), time to peak (ttPeak) and peak height (velocity and amount of net thrombin production), and endogenous thrombin potential (ETP), representing number of substrates potentially convertible by thrombin, were measured. Significance was at p < 0.05.ResultsFA-WB-VCAM at baseline sample hematocrit was significantly elevated (Figure 1) in PKD subjects (808±377 cells/mm², n=10) compared to healthy controls (6±4 cells/mm², n=4) and even to our previously reported steady state levels in sickle cell samples (290±50 cells/mm² [2]. Thrombin generation profiles were similar between PKD subjects and healthy controls with the exception of the thrombin generation index (PPP+TP/PPP)*100ETP that was significantly (p<<0.01) elevated in citrated plasma of PKD subjects (92.9±6.8) as compared to healthy controls (68.6±11.9). For PKD subjects, FA-WB-VCAM correlated significantly with platelet counts (R²=0.81, p<0.05), and FAAv-WB-VCAM was negatively correlated with platelet (P=0.03, R 2=0.5), but not with erythrocyte-derived microparticles (MP). Platelet-derived MP strongly correlated with thrombin generation (ETP, p<0.01, R 2=0.76) but not with LT or ttPeak of thrombin generation. Red blood cell MP were significantly (p=0.02) decreased in splenectomized patients (200±170, n=7) vs. non-splenectomized subjects (2090±1860, n=3). LT and ttPeak were significantly longer in PKD subjects with thrombosis history than without.ConclusionsPKD subjects in this study had elevated RBC adhesive properties similar to that observed in SCD, confirming that pathologic RBC membrane damage resulting in increased adhesion is a common feature of hemolytic anemias. The hemoglobin level of 7.8±1.1 g/dL (mean±SD) for PKD patients was within 6 to 11 g/dl range of hemoglobin levels typical for SCD. There was no significant difference in any other measured parameters (thrombin generation, adhesion avidity, microparticles data). Thrombin generation in PKD subjects was not consistent with hypercoagulability. Based on these observations, pathologic RBC adhesion may be both a novel a mechanism driving hypercoagulability in individuals with PKD. Further studies to determine whether RBC-modifying therapies may decrease thrombosis risk in PKD are warranted.1. Zia A, Callaghan MU, Callaghan JH, et al. Hypercoagulability in adolescent girls on oral contraceptives - global coagulation profile and estrogen receptor polymorphisms. Am J Hematol, 2015;90:725-312. Pittman DD, Hines PC, Beidler D, et al. Evaluation of Longitudinal Pain Study in Sickle Cell Disease (ELIPSIS) by patient-reported outcomes, actigraphy, and biomarkers. Blood. 2021;137(15):2010-20 [Display omitted] DisclosuresHines: Functional Fluidics: Current holder of stock options in a privately-held company. Gao: Functional Fluidics: Current Employment. Herppich: Functional Fluidics: Ended employment in the past 24 months. Kwiatkowski: Imara: Consultancy, Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Sangamo: Research Funding; Bioverativ: Research Funding; Vertex: Research Funding; Silence Therapeutics: Consultancy; bluebird bio: Consultancy, Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Chiesi: Research Funding; CRISPR: Research Funding. Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company.

Effect of parachute delivery on red blood cell (RBC) and plasma quality measures of blood for transfusion.

BackgroundParachute airdrop offers a rapid transfusion supply option for humanitarian aid and military support. However, its impact on longer‐term RBC survival is undocumented. This study aimed to determine post‐drop quality of RBCs in concentrates (RCC), and both RBCs and plasma in whole blood (WB) during subsequent storage.Study design and methodsTwenty‐two units of leucodepleted RCC in saline, adenine, glucose, mannitol (SAGM) and 22 units of nonclinical issue WB were randomly allocated for air transportation, parachute drop, and subsequent storage (parachute), or simply storage under identical conventional conditions (4 ± 2°C) (control). All blood products were 6–8 days post‐donation. Parachute units were packed into Credo Cubes, (Series 4, 16 L) inside a PeliCase (Peli 0350) and rigged as parachute delivery packs. Packs underwent a 4‐h tactical flight (C130 aircraft), then parachuted from 250 to 400 ft before ground recovery. The units were sampled aseptically before and after airdrop at weekly intervals. A range of assays quantified the RBC storage lesion and coagulation parameters.ResultsBlood units were maintained at 2–6°C and recovered intact after recorded ground impacts of 341–1038 m s−2. All units showed a classical RBC storage lesion and increased RBC microparticles during 42 days of storage. Fibrinogen and clotting factors decreased in WB during storage. Nevertheless, no significant difference was observed between Control and Parachute groups. Air transportation and parachute delivery onto land did not adversely affect, or shorten, the shelf life of fresh RBCs or WB.DiscussionAppropriately packaged aerial delivery by parachute can be successfully used for blood supply.

Red Blood Cells Microparticles Are Associated with Hemolysis Markers and Leg Ulceration in Patients with Sickle Cell Disease

Microparticles are sub-micron, unilamellar vesicles possessing protein and other materials derived from the plasma membrane of their parent cells and literature data suggest that they may have a role in the pathophysiology of SCD. Red blood cell microparticles (RMP) are derived from the red blood cells (RBC) as a result of shedding of unwanted surface features during reticulocyte maturation, apoptosis, and activation by endogenous triggers. Upon release, RMP carry trapped hemoglobin and heme along with them.The contribution of RMP to hemolysis, endothelial cells lesions and clinical phenotypes of SCD is largely unknown. There are some data that suggest that, due to the repeated sickling-unsickling cycles and polymerization of hemoglobin S, patients with SCD are likely to generate more RMP in their blood than healthy subjects, and the generated RMP could contribute to the vascular lesions and vaso-occlusion. Hence, the current study aimed to study some aspects of MP in patients with SCD.This study involved 118 adults with SCD (89 HbSS and 29 HbSC) in steady state, and on regular follow up and 39 healthy matched controls. Participants had their plasma RMP quantified by flow cytometry, plasma hemoglobin and heme by colorimetric assay kits and haptoglobin and hemopexin by ELISA kits. Since the identification of RMP is a procedure with several different approaches, in this work a very strict methodology was used (Splitter et al., 2016). RMP were measured by number per ml of blood. Automated analyzers were used to carry out hematologic and biochemical analyses and records of the SCD patients were examined for clinical events. Institutional review board approval was obtained and the study protocols followed the Declaration of Helsinki as revised in 2000.We found that the SCD patients had higher levels of RMP than the controls (HbSS > HbSC > controls), 531,685.0±204,495.0 vs 171,034.0±54,144.0 vs 1,128.0±387.0/mL, respectively, P <0.0001). Similar patterns were observed with plasma hemoglobin (87.7±37.7 vs 60.19±19.7 vs 46.52±14.0mg/dL, P <0.0001), heme (62.5±32.4 vs 48.8±27.0 vs 35.2±16.4µM, P <0.0001), and HbF (14.6±8.2 vs 1.6±2.5 vs 0.29±0.2%, p<0.0001). On the contrary, a reverse order i.e HbSS < HbSC < controls was observed with haptoglobin (25.3±36.6 vs 135.5±160.0 vs 1205.0±553.0µg/mL, P <0.0001) and hemopexin (542.7±349.7 vs 669.0±296.3 vs 1029.0±146.6µg/mL, P <0.0001) and hemoglobin concentration (8.59±1.4 vs 11.3±1.4 vs 13.8±1.2g/dL, P <0.0001). When we tested the association between RMP and other parameters, we found that RMP strongly correlated positively with total bilirubin (r=0.49, p<0.0001), unconjugated bilirubin (r=0.509, P <0.0001), LDH (r=0.61,p<0.0001), reticulocyte counts (r=0.41, p<0.0001), plasma hemoglobin (r=0.46, P <0.0001), and heme (r=0.59, P <0.0001) in SCD patients. On the contrary, a modest negative correlation was observed between RMP and HbF (r= -0.31, P=0.0025), and hemopexin (r= -0.26, P=0.035). These data suggest that RMP is associated with hemolysis in the studied SCD cohorts.We found no differences between SS patients with and without hydroxyurea n=68 & 50 respectively, (572,353.0±265,112.0 vs 400,000.0±127,511.0/ml, P=0.807). We also identified association between RMP and history of leg ulceration. SCD patients who had leg ulcer (n=17), had significantly higher levels of RMP compared with patients without leg ulceration (590,588±709,441/ml vs 422,000±1,809390/ml, P=0.0044). Similarly, they had higher LDH 461.5±186.9U/l vs 377.0±140.0U/l, P=0.04 and heme 82.6±45.5µM vs 55.8±27.7µM, P=0.0096.In conclusion, these data suggest that RMP may be associated with hemolysis and leg ulceration in patients with SCD thus supporting the notion that RMP may have an important role in the pathophysiology of SCD. It is possible that RMP contribute to endothelial lesion and leg ulcer phenotype through the toxic injuries mediated by hemoglobin and heme trapped in them. Hence, therapies targeting RMP could be another strategy to combat SCD. DisclosuresFertrin:Alexion Pharmaceuticals: Consultancy.

Pre-Transfusion Rejuvenation of RBCs for Red Blood Cell Exchange: Report of Feasibility

Background: Patients with Sickle Cell Disease (SCD) often require transfusions to minimize disease burden, increase oxygen carrying capacity, and prevent tissue hypoxia. Furthermore, patients with significant complications such as stroke or acute chest syndrome depend on red blood cell exchange transfusion (RBCX) to maintain a decreased percentage of hemoglobin S in circulation and prevent further SCD complications. Red blood cells (RBCs) stored for transfusion develop a storage lesion over time. This is in part due to depleted 2,3-diphosphoglycerate (2,3-DPG) and ATP, which can be reversed by biochemical rejuvenation of RBCs. The FDA-approved rejuvenation process has been shown to normalize hemoglobin oxygen affinity, decrease in-bag hemolysis, and increase RBC deformability. Therefore, we hypothesized that rejuvenation of RBC units prior to RBCX would be associated with diminished loss of hemoglobin A between treatment sessions and increased post-RBCX p50 (the partial pressure of oxygen at 50% hemoglobin saturation, a measure of oxygen affinity).Methods: Following approval by the Duke University Health System IRB we recruited 4 SCD patients maintained crisis-free on chronic RBCX for the past 3 months. The study had an open-label, crossover design of standard (S) and rejuvenated (R) RBCX sessions in the following sequence: S-R-R-R-S-S. To assess rejuvenation benefit to chronic RBCX SCD patients, we first developed a calculation of perseverance of RBCs given during RBCX using an exponential decay model, and then applied rejuvenation to the final 2-4 donor units given in RBCX, which ensured that approximately 30-40% of post-RBCX circulating RBCs would be rejuvenated. Rejuvenation was performed by trained blood bank personnel according to the rejuvenation solution manufacturer's instructions on RBC units between 14-21 days old. The primary outcome for the study was time-normalized change in Hemoglobin A between RBCX treatments (derived from hemoglobin electrophoresis results), with our secondary outcome being change in p50 before and after RBCX. This study is partially funded by Zimmer Biomet. We report preliminary results demonstrating feasibility of this approach.Results: At this time we have completed 8 standard RBCX and 3 rejuvenated RBCX sessions. Preliminary data are presented in Table 1. A total of 10 rejuvenated units have been transfused with no adverse events reported. Despite the low sample size, it was clear that rejuvenation attenuated the p50 decrease seen after RBCX. Hemoglobin electrophoresis data is not yet available as no subjects have had a scheduled RBCX session since their initial rejuvenated RBCX session. RBC rejuvenation was feasible but logistically complicated due to the time and personnel required for the rejuvenation process, and complex coordination between the subjects, the apheresis service, and the blood bank was required for successful implementation. One subject did not present for a scheduled RBCX, resulting in the discard of 4 units of rejuvenated RBCs.Conclusion: Red cell rejuvenation holds promise as a way of maximizing the exchange transfusion benefit to the most at risk SCD patients. We additionally plan to evaluate change in RBC microparticles before and after standard and rejuvenated RBCX sessions in these patients. Rejuvenation as currently performed provides some technical challenges, such as increased RBC unit processing time and greater required coordination with clinical service. [Display omitted] DisclosuresLandrigan:Zimmer Biomet: Employment, Research Funding. Shah:Novartis: Other: Speaker; Alexion: Other: Speaker. Welsby:Zimmer Biomet: Research Funding.

Production of erythrocyte microparticles in a sub-hemolytic environment.

Microparticles are produced by various cells due to a number of different stimuli in the circulatory system. Shear stress has been shown to injure red blood cells resulting in hemolysis or non-reversible sub-hemolytic damage. We hypothesized that, in the sub-hemolytic shear range, there exist sufficient mechanical stimuli for red blood cells to respond with production of microparticles. Red blood cells isolated from blood of healthy volunteers were exposed to high shear stress in a microfluidic channel to mimic mechanical trauma similar to that occurring in ventricular assist devices. Utilizing flow cytometry techniques, both an increase of shear rate and exposure time showed higher concentrations of red blood cell microparticles. Controlled shear rate exposure shows that red blood cell microparticle concentration may be indicative of sub-hemolytic damage to red blood cells. In addition, properties of these red blood cell microparticles produced by shear suggest that mechanical trauma may underlie some complications for cardiovascular patients.

Association of endothelial and red blood cell microparticles with acute myocardial infarction in Chinese: a retrospective study.

Mounting evidence suggests that endothelial cell-derived microparticles (EMPs) and red blood cell-derived microparticles (RMPs), which have procoagulant and vasoconstriction effects, are involved in the development of vascular acute cardiovascular events. The aim of this study was to analyze the circulating levels of EMPs and RMPs in patients with acute myocardial infarction (AMI), and to explore the correlations between EMPs and RMPs and the severity of coronary artery disease. Plasma samples from 110 patients with AMI and 57 non-coronary artery disease group (nonCAD) were collected in the present study. The flow cytometry was used to measure the EMPs (CD31) and RMPs (CD235a) qualitatively and quantitatively. The levels of EMPs and RMPs in the AMI group were higher than that in the Non-CAD group, yet no significant difference was found between STEMI and non-STEMI subjects. The levels of EMPs and RMPs in multi-vessel were higher than in the single-vessel l disease. In the Thrombolysis in Myocardial Infarction (TIMI) risk assessment, the levels of EMPs in the high-risk group were higher than that in both intermediate- and low-risk group. The low-risk group had the lowest EMP levels, the difference between the groups being statistically significant (P=0.001). No significant difference in RMP levels was noted upon TIMI stratification. According to the ROC curve analysis, the areas under the curve (AUC) of EMPs and RMPs were 0.706 and 0.668, respectively. The circulating levels of EMPs and RMPs in patients with AMI are elevated, and the level of EMPs is related to the degree of coronary artery disease and the prognosis risk. The EMPs are more likely to be potential biomarkers than RMPs to provide diagnostic value for AMI.

Effects of repleting organic phosphates in banked erythrocytes on plasma metabolites and vasoactive mediators after red cell exchange transfusion in sickle cell disease.

Red blood cell (RBC) exchange (RCE) transfusion therapy is indicated for certain patients with sickle cell disease (SCD). Although beneficial, this therapy is costly and inconvenient to patients, who may require it monthly or more often. Identification of blood and plasma biomarkers that could improve or help individualise RCE therapy is of interest. Here we examined relevant blood and plasma metabolites and biomarkers of vasoactivity and RBC fragility in a pilot study of SCD patients undergoing RCE using either standard RBC units or RBC units treated with a US Food and Drug Administration (FDA)-approved additive solution containing phosphate, inosine, pyruvate, and adenine ("PIPA"). In this prospective, single-blind, cross-over pilot clinical trial, patients were randomised to receive either standard RBC exchange or PIPA-treated RBC exchange transfusion with each RCE session over a 6-month treatment period. Pre- and post-transfusion blood samples were obtained and analysed for RBC O2 affinity, ATP, purine metabolites, RBC microparticles, and cell free haemoglobin. Red blood cell O2 affinity was maintained after PIPA-RCE in contrast to standard RCE, after which P50 fell (net O2 affinity rose). Plasma ATP did not change significantly after RCE using either of the RBC unit types. Exchange transfusion with PIPA-treated RBC units led to modest increases in plasma inosine and hypoxanthine. Plasma cell free haemoglobin fell after either standard or PIPA-treated RBC exchange transfusion (novel findings), and to a similar extent. RBC-derived microparticles in the plasma fell significantly and similarly after both standard and PIPA-treated RCE transfusion. In summary, treatment of RBCs with PIPA prior to RCE elicited favourable or neutral changes in key metabolic and vascular biomarkers. Further study of its efficacy and safety is recommended and could ultimately serve to improve outcomes in chronically transfused SCD patients.

Impact of storage, leukofiltration, and ascorbic acid fortification on red cell-derived microparticles in stored packed red blood cells: A flow cytometric and procoagulant study

INTRODUCTION: During blood storage, red blood cells (RBCs) undergo changes termed “storage lesion” including shape modification and microparticles (MPs) production, as well as oxidative injury to lipids and proteins. OBJECTIVE: We studied the impact of storage on RBCs by quantification of red cell MPs and their potential procoagulant activity (PCA). The antioxidant effect of ascorbic acid (AA) was assessed, as a proposed additive, to improve stored RBC quality. METHODS: Forty randomly selected packed red cell units (filtered and nonfiltered) were sampled weekly over 35 days of storage. Flow cytometric and coagulation tests were performed on MPs isolated from erythrocyte concentrates. RBCs were either in citrate phosphate dextrose adenine-1 (CPDA1) only (standard storage) or CPDA1 with AA (Vitamin C) in three concentrations. RESULTS: Red cell MPs progressively increased over 35 days of storage. Filtration efficiently reduced the production of MPs (P < 0.001). PCA increased significantly on storage assessed by the shortened coagulation time (P < 0.001). A strong inverse correlation between coagulation time and Annexin V positive MPs was found, (r = −0.723, P < 0.001), and the dual-positive population. This highlights the strong correlation between the functional assay (PCA) and the direct quantitative assay (flow cytometry). The red cells fortified with AA demonstrated a reduction in the accumulation of RBC MPs by almost 50% compared to standard storage in CPDA-1 alone in an evident dose-dependent manner. CONCLUSION: We concluded that MPs increase on storage with increased PCA. AA reduced MPs production which can be possible additive for further research in the future

Does endurance training improve red blood cell aging and hemorheology in moderate-trained healthy individuals?

ObjectiveTo examine the impact of a 6-week endurance training on red blood cell (RBC) aging and deformability of healthy participants to detect possible improved hemorheological and performance-related adaptations. MethodsA total of 31 participants (17 females and 14 males) performed a 6-week moderate training protocol (three 1-h running sessions per week at 70% of maximal heart rate). Blood was sampled before and after the training. RBCs from each participant were fractioned according to density and age into 4 RBC subfractions. Subfractions were examined for changes of RBC properties, including aging distribution, RBC deformability, RBC microparticles, and phosphatidylserine concentrations. RBC and plasma nitrite levels were measured as indicators of nitric oxide metabolism. ResultsAerobic performance, peak oxygen consumption, ventilatory thresholds, velocity at the aerobic–anaerobic threshold, and lactate at exhaustion improved after training. The relative amount of both young RBCs and old RBCs increased, and the amount of the main RBC fraction decreased. Phosphatidylserine externalization and RBC-derived microparticles decreased. Overall deformability expressed as shear stress required to achieve half-maximum deformation to theoretical maximal elongation index at infinite shear stress improved in unfractioned RBCs (p < 0.001). Nitrite decreased in total (p = 0.001), young (p < 0.001), main (p < 0.001), and old (p = 0.020) aged RBCs and in plasma (p = 0.002), but not in very old RBCs. ConclusionThese results indicate that non-endurance-trained healthy participants benefit from a regular moderate running training program because performance-related parameters improve and a younger RBC population with improved RBC properties is induced, which might support oxygen supply in the microcirculation.

Protective Effects of Carbon Monoxide Delivered By Corm-401 in Hyperhemolysis in Patients with Sickle Cell Disease

IntroductionHyperhemolysis has been well described in delayed hemolytic transfusion reaction (DHTR), which is one of the most serious complications of transfusion especially in patients with sickle cell disease (SCD). In the most severe cases, this life-threatening syndrome may reach 10% lysis of total red blood cells (RBCs) (Habibi A et. al. Am J Hematol 2016). DHTR is characterized by an acute anemia with increased plasma free Hb and free heme. Hyperhemolysis can also be found in other hemoglobinopathies and extracorporeal circulation procedures.Most in vitro studies on hyperhemolysis used only purified Hb or heme which did not allow to investigate all deleterious effects on the endothelium. We have developed a global and physiopathologic approach to assess the mechanism of hyperhemolysis-induced endothelial dysfunction. For this purpose, we created a microfluidic model reproducing hyperhemolysis observed in the most severe DHTR but it's also applicable for other pathogenesis of hyperhemolysis.Among the potential therapeutic approaches, carbon monoxide (CO) is beneficial on endothelial cells by its anti-inflammatory, antioxidant and vasodilator effect which reduce the toxicity of Hb and improve tissue perfusion (Motterlini R et.al. Nat. Rev. Drug Discov 2010). Here we investigated the therapeutic effects of a CO-releasing molecule, CORM-401, in hyperhemolysis-induced endothelial dysfunction.Materials and methodsFlow culture Human Umbilical Vein Endothelial Cells (HUVECs) in fibronectin-coated µ-Slides were preconditioned for 4 hours under shear stress of 1dyn/cm² with sonicated RBCs of healthy donors (AA) reconstituted in serum of either AA or SCD patients (SS) at 7 g/L of free Hb. In negative control conditions, cells were pretreated similarly with either AA serum or culture medium. Cells were then collected for analyses of actin network, membrane markers and endothelial function. Treatment with CORM-401 or inactive CORM (iCORM) was performed at fixed concentration during the preconditioning and perfusion steps.ResultsHemolysis induces multiple endothelial damage and dysfunctionAfter exposure to hemolysate, we noted a significant increase of subendothelial matrix exposition (fibronectin-coated surfaces) due to actin network reorganization and cells detachment. Immunofluorescence staining showed a moderate activation of HUVECs induced by hemolysate with membrane expression of adhesion molecules such as ICAM-1, E-Selectin and VCAM-1, but not VWF and P-Selectin. At a similar free Hb level, preconditioning with hemolysate without microparticles (MPs) had the lowest deleterious impact on HUVECs suggesting a role of MPs in hyperhemolysis.Perfusion of AA whole blood on HUVECs pretreated with hemolysate resulted in aggregation and activation of platelets in a GPIIbIIIa-dependent manner at injury sites. We also observed a significant increase of adhered RBCs (14-fold compared to control).Compared to whole blood RBC lysis, purified Hb induced similar subendothelial exposure and RBC adherence but did not lead to significant HUVECs activation.To study the DHTR in RBC transfusion of SCD patients, SS serum was used to perform hemolysis preconditioning of HUVECs. Adherence of RBCs was similar to condition using AA serum suggesting that endothelial damage in DHTR of SCD patients depend on hemolysate compositions and RBC MPs, rather than on patients' serum.Effects of CORM-401 on hemolysis-induced endothelial dysfunctionAdding CORM-401 (50 to 100 µM) to the hemolysate produced 15% free COHb leading to a concentration-dependent decrease in hemolysis-induced HUVECs activation and RBC adherence (Figure 1 C, E). While no significant effects at the subendothelial exposure were observed, the aggregation of platelets at injury sites was decreased after CORM-401 treatment compared to controls (Figure 1 A, B, D), confirming the anti-platelet aggregation effect of this molecule (Chlopicki S et. al. Naunyn Schmiedebergs Arch Pharmacol 2012).ConclusionOur in vitro model enabled the reproduction of endothelial damage by hyperhemolysis and to determine the deleterious effects of each hemolysate component. The in vitro beneficial effects of CORM-401 were also demonstrated and an in vivo study on SCD mice is underway to explore the therapeutic effects of this molecule against hyperhemolysis-induced endothelial damage. [Display omitted] DisclosuresBartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

Pharmacokinetics of Human Red Blood Cell Microparticles Prepared Using High-Pressure Extrusion Method.

Red blood cell microparticles (RMPs) is a high potency hemostatic agent, which may serve as a viable therapeutic approach. They generate thrombin in vitro and effective in arresting bleeding in animal bleeding models. However, prior to ascertaining the clinical efficacy of RMPs, detailed preclinical evaluation is necessary. Therefore, we aimed to characterize RMPs, ascertain their stability, and determine their pharmacokinetics in rats. RMPs were prepared from human RBCs by a high-pressure extrusion method. Pharmacokinetic parameters were computed from groups receiving various RMPs dosing regimens. Volume of distribution, elimination rate constant, and clearance for RMPs were also assessed. Major portion of prepared microparticles were RMPs and a very small portion of particles were from platelets and leukocytes. RMPs were stable when stored at 5 and -20°C for at least 12 months. In vivo half-life was found to vary for each paradigm, but in general, was less than 2 min for most of the paradigms evaluated. Our results demonstrate that RMPs are stable during prolonged storage and have a short half-life. Therefore, the clinical use of RMPs as a hemostatic agent, within a tailored treatment paradigm, may be advantageous in achieving prolonged systemic therapeutic benefit without provoking any thrombotic complications.

Endocytosis of Red Blood Cell Microparticles by Pulmonary Endothelial Cells is Mediated By Rab5

Microparticles are submicron vesicles shed from aging erythrocytes as a characteristic feature of the red blood cell (RBC) storage lesion. Exposure of pulmonary endothelial cells to RBC-derived microparticles promotes an inflammatory response, but the mechanisms underlying microparticle-induced endothelial cell activation are poorly understood. In the present study, cultured murine lung endothelial cells (MLECs) were treated with microparticles isolated from aged murine packed RBCs or vehicle. Microparticle-treated cells demonstrated increased expression of the adhesion molecules ICAM and E-selectin, as well as the cytokine, IL-6. To identify mechanisms that mediate these effects of microparticles on MLECs, cells were treated with microparticles covalently bound to carboxyfluorescein succinimidyl ester (CFSE) and cellular uptake of microparticles was quantified via flow cytometry. Compared with controls, there was a greater proportion of CFSE-positive MLECs from 15 min up to 24 h, suggesting endocytosis of the microparticles by endothelial cells. Colocalization of microparticles with lysosomes was observed via immunofluorescence, indicating endocytosis and endolysosomal trafficking. This process was inhibited by endocytosis inhibitors. SiRNA knockdown of Rab5 signaling protein in endothelial cells resulted in impaired microparticle uptake as compared with nonsense siRNA-treated cells, as well as an attenuation of the inflammatory response to microparticle treatment. Taken together, these data suggest that endocytosis of RBC-derived microparticles by lung endothelial cells results in endothelial cell activation. This response seems to be mediated, in part, by the Rab5 signaling protein.

Microparticles as prognostic biomarkers in dengue virus infection

Promising biomarkers which may help predict the risk of developing severe dengue virus infection (DVI) are lacking and will be helpful. Thus the main aim of this study was to analyze the role of cell-derived microparticles (MP) in DVI. Sixty patients with DVI i.e. 18: dengue with warning signs (DWS); 1: DSS and 41: dengue without warning signs (DWOS); along with 15 controls (other febrile illness) were included in the study. The following MPs were assessed: annexinV, platelet (CD41a), red blood cell (RBC) (CD235a) and activated endothelial (CD62e) MPs. Patients with profound thrombocytopenia without bleeding had statistically elevated platelet MP (PMP) levels when compared to patients with profound thrombocytopenia with bleeding (p < .001). RBC MPs were found to be significantly elevated in the 2nd phase in patient with DWS which was seen earliest on day 4 of infection with a cut off of ≥2200 MPs/μl when compared to patients with DWOS (p < .0001). PMPs may prove to be a promising novel biomarker which helps discriminate patients in need of prophylactic platelet transfusion from those who do not. RBC MPs, on the other hand could be potential biomarkers capable of identifying potentially severe patients who require immediate care. Thus, MPs seem to be a promising important biomarker in many aspects of DVI.

Microparticles from stored red blood cells promote a hypercoagulable state in a murine model of transfusion

BackgroundRed blood cell-derived microparticles are biologically active, submicron vesicles shed by erythrocytes during storage. Recent clinical studies have linked the duration of red blood cell storage with thromboembolic events in critically ill transfusion recipients. In the present study, we hypothesized that microparticles from aged packed red blood cell units promote a hypercoagulable state in a murine model of transfusion. MethodsMicroparticles were isolated from aged, murine packed red blood cell units via serial centrifugation. Healthy male C57BL/6 mice were transfused with microparticles or an equivalent volume of vehicle, and whole blood was harvested for analysis via rotational thromboelastometry. Serum was harvested from a separate set of mice after microparticles or saline injection, and analyzed for fibrinogen levels. Red blood cell-derived microparticles were analyzed for their ability to convert prothrombin to thrombin. Finally, mice were transfused with either red blood cell microparticles or saline vehicle, and a tail bleeding time assay was performed after an equilibration period of 2, 6, 12, or 24 hours. ResultsMice injected with red blood cell-derived microparticles demonstrated an accelerated clot formation time (109.3 ± 26.9 vs 141.6 ± 28.2 sec) and increased α angle (68.8 ± 5.0 degrees vs 62.8 ± 4.7 degrees) compared with control (each P < .05). Clotting time and maximum clot firmness were not significantly different between the 2 groups. Red blood cell-derived microparticles exhibited a hundredfold greater conversion of prothrombin substrate to its active thrombin form (66.60 ± 0.03 vs 0.70 ± 0.01 peak OD; P < .0001). Additionally, serum fibrinogen levels were lower in microparticles-injected mice compared with saline vehicle, suggesting thrombin-mediated conversion to insoluble fibrin (14.0 vs 16.5 µg/mL, P < .05). In the tail bleeding time model, there was a more rapid cessation of bleeding at 2 hours posttransfusion (90.6 vs 123.7 sec) and 6 hours posttransfusion (87.1 vs 141.4 sec) in microparticles-injected mice as compared with saline vehicle (each P < .05). There was no difference in tail bleeding time at 12 or 24 hours. ConclusionRed blood cell-derived microparticles induce a transient hypercoagulable state through accelerated activation of clotting factors.