Fresh Red Blood Cells Research Articles

Dielectrophoretic force measurement of red blood cells exposed to oxidative stress using optical tweezers and a microfluidic chip.

Red blood cell (RBC) dysfunction is often associated with a pathological intervention, and it has been proposed as a critical risk factor for certain lethal diseases. Examining the cell viability of RBCs under various physiological conditions is essential and of importance for precise diagnosis and drug discovery in the field of medicine and pharmacy. In this paper, we report a new analytical method that employs dielectrophoretic (DEP) force measurements in absolute units to assess the viability, and potentially the functionality of RBCs. We precisely quantify the frequency-dependent DEP forces of the RBCs by using a micro-electrode embedded chip combined with optical tweezers. DEP characteristics are known to be well-correlated with the viability of biological cells, and DEP forces are measured in both fresh and long-term stored RBCs to investigate the effect that the storage period has on the cell viability. Moreover, we investigate the DEP behavior of RBCs when exposed to oxidative stress and verify whether EDTA protects the RBCs from an oxidant. From the experiments, it is found that the fresh RBCs without oxidative stress display very high DEP forces over the entire frequency range, exhibiting two cutoff frequencies. However, both the RBCs stored for the long-term period and exposed to oxidative stress reveals that there exist no significant DEP forces over the frequency range. The results indicate that the DEP forces can serve as a useful parameter to verify whether the RBCs in certain blood are fresh and not exposed to oxidative stress. Therefore, it is believed that our system can be applied to a diagnostic system to monitor the cell viability of the RBCs or other types of cells.

Optimal hematocrit in an artificial microvascular network.

Higher hematocrit increases the oxygen-carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist. We used viscometry and an artificial microvascular network device to determine the optimal hematocrit in vitro. Suspensions of fresh red blood cells (RBCs) in plasma, normal saline, or a protein-containing buffer and suspensions of stored red blood cells (at Week 6 of standard hypothermic storage) in plasma with hematocrits ranging from 10 to 80% were evaluated. For viscometry, optimal hematocrits were 10, 25.2, 31.9, 37.1, and 37.5% for fresh RBCs in plasma at shear rates of 3.2 or less, 11.0, 27.7, 69.5, and 128.5 inverse seconds. For the artificial microvascular network, optimal hematocrits were 51.1, 55.6, 59.2, 60.9, 62.3, and 64.6% for fresh RBCs in plasma and 46.4, 48.1, 54.8, 61.4, 65.7, and 66.5% for stored RBCs in plasma at pressures of 2.5, 5, 10, 20, 40, and 60 cm H2 O. Although exact optimal hematocrit values may depend on specific microvascular architecture, our results suggest that the optimal hematocrit for oxygen delivery in the microvasculature depends on perfusion pressure. Therefore, anemia in chronic disorders may represent a beneficial physiological response to reduced perfusion pressure resulting from decreased heart function and/or vascular stenosis. Our results may help explain why a therapeutically increasing hematocrit in such conditions with RBC transfusion frequently leads to worse clinical outcomes.

The dielectric spectroscopy of human red blood cells: the differentiation of old from fresh cells

Objective: The objective of the study was to gauge the effect of storage lesions on the dielectric response of red blood cells (RBC), in particular those processes linked to deformations of the cellular membrane known as the β-dispersion. Approach: The dielectric response of RBC suspensions, exposed to blood-bank cold storage, was studied using time-domain dielectric spectroscopy (TDDS) in the frequency range of 500 kHz up to 1 GHz. The measured dielectric processes are characterized by their dielectric strength (Δε) and relaxation time (τ). Changes in the dielectric properties of the RBC suspensions due to storage-related lesions were evaluated. For a quantitative characterization of RBC lesions, we measured the deformability of fresh and stored RBC as expressed by their elongation ratio (ER), which was achieved under a shear stress of 3.0 Pa. Main Result: The results show that the storage of RBC induced a statistically significant decrease of dielectric relaxation times. In addition, a sound correlation between the mean values of ER and the relaxation times was observed (Spearman’s correlation coefficient ρ = 0.847). We draw the conclusion that those alterations in the relaxation time are induced by changes in the shape of the RBC that happen during cold-storage. Significance: The evolution of the β-dispersion of RBC opens new possibilities in the blood bank inventory management.

Evidence-Based Red Blood Cell Transfusion Practices in Cardiac Surgery.

Cardiac surgical patients are among the highest consumers of allogeneic red blood cells (RBCs) due to the prevalence of anemia and bleeding. Up until recently, there was a paucity of high-quality evidence informing transfusion decisions in this patient group which led to wide variability in transfusion decision making. The article reviews and critically analyzes the available evidence for RBC transfusion in cardiac surgery, focusing on trials of transfusion triggers and age of blood, and provides suggestions for future research. Observational studies analyzing outcomes in patients transfused vs those not transfused have consistently shown RBC transfusion to be associated with adverse outcomes. However, multiple sources of bias in these studies invalidate their conclusions. The best available evidence comes from randomized controlled trials which compare liberal vs restrictive transfusion thresholds. To date, 6 randomized controlled trials have been reported in cardiac surgical patients, and pooled analyses have shown no differences in clinical outcomes between the 2 strategies. Similarly, research into age of RBCs and adverse outcomes has failed to demonstrate a pathological effect attributable to the storage lesion; the recent multicenter Red Cell Storage Duration Study (RECESS) trial has demonstrated no difference in outcomes between patients receiving fresh or old RBCs. Future research needs to identify what a safe transfusion threshold may be, and how this differs for different patient groups and different stages of the perioperative journey. There is also a need to evaluate other physiological parameters which, coupled with hemoglobin concentration, can better inform those patients who need an RBC transfusion.

Red blood cells for transfusion in patients with sepsis: respective roles of unit age and exposure to recipient plasma

Red blood cell (RBC) storage in blood banks is not exempt from cellular injury. Alterations not observed on RBCs freshly isolated from units can rapidly appear in circulation. The transfusion of old blood units, even if this is a controversial issue, could therefore have adverse effects on the recipient. We wanted to determine the respective effects of storage duration and recipient plasma on RBCs for transfusion into patients with severe sepsis. Eleven stored RBC units were sampled at various time points, approximately Days 3 to 8 (referred to as fresh RBCs) and Days 38 to 42 (old RBCs) and tested in coincubation experiments with plasma obtained from 13 patients with severe sepsis and 17 healthy donors as controls. RBCs were tested after 24 or 48 hours at 37°C for the detection of senescence markers (phosphatidylserine exposure, calcium influx, and reactive oxygen species detection and decrease in size) with or without exposure to plasma. We confirmed that a 42-day refrigerated storage of RBCs alone (without any incubation in plasma) had no significant effect on RBCs and no senescence marker detected. By contrast, ex vivo exposure to plasma samples altered both fresh and old RBCs, with a much larger effect for old RBCs, regardless of the plasma used (sepsis vs. control). We show that the main factor affecting the senescence of RBCs for transfusion into patients with severe sepsis is the age of the stored units rather than the clinical status of the recipient.

Response to Assuring hospital supply of fresh red blood cells for critically ill patients

Response to Assuring hospital supply of fresh red blood cells for critically ill patients

Transfusion of 35-day-stored red blood cells does not alter lipopolysaccharide tolerance during human endotoxemia.

Transfusion-related immunomodulation (TRIM) encompasses immunosuppressive and proinflammatory effects induced by red blood cell (RBC) transfusion. Changes that occur during storage in the RBC product have been hypothesized to underlie TRIM, mediated by tolerance of toll-like receptors (TLR). We investigated whether transfusion of 35-day-stored autologous RBCs alters cytokine production in response to stimulation with lipopolysaccharide (LPS) or lipotheic acid (LTA), in a clinically relevant model of endotoxemia. Eighteen volunteers received 2ng/kg LPS intravenously, followed by normal saline or 2- or 35-day-stored autologous RBC transfusion. Before LPS, before transfusion, and 6 hours after transfusion blood was collected to measure cytokine gene expression. Whole blood was used for ex vivo stimulation with LPS and LTA, after which cytokine levels were measured with enzyme-linked immunosorbent assay. In vivo LPS induced a biphasic response in cytokine mRNA with peak values 2 hours after LPS infusion. Storage time of RBC transfusion did not influence cytokine mRNA levels. In vivo infusion of LPS resulted in tolerance for ex vivo stimulation with LPS and LTA. However, transfusion of either fresh or stored RBCs did not further affect the capacity to produce cytokines after ex vivo stimulation. In a clinically relevant model of human endotoxemia, autologous transfusion of 35-day-stored RBCs does not influence cytokine mRNA levels nor does it change the capacity of white blood cells in whole blood to produce cytokines after ex vivo stimulation with LPS or LTA.

Numerical Determination of Wall Shear Stress in Red Blood Cell Membranes

Red cells in microvessels deform extensively in order to flow, thus resulting in high observed apparent viscosities and high stress concentrations localized to the red cell membrane. Flow through microvessels is consequently aided by the high compliance in the red cell membrane. However, changes in the mechanical properties of Red Blood Cells (RBC) as a result of storage lesions, can increase stress concentrations in the membrane, impairing oxygen transport and resulting in increased vesiculation and likely cell lysis. The objective of this study is to determine the mechanical properties and thus wall shear stress experienced by a RBC membrane during capillary flow. The RBC membrane is modeled as a two dimensional Hookean Isotropic Solid. Videos of capillary flow of both fresh RBCs and RBCs stored for 2 weeks were taken in the rat cremaster muscle. The mechanical properties, including the elastic modulus, shear stress, and shear strain, of both conditions were compared for both groups. In order to determine strain, each RBC membrane will be parameterized by N (X,Y) points determined by the Moore Neighbor Tracing algorithm of videos of capillary flow obtained by intravital microscopy of the rat cremaster muscle, from which N deformation gradient tensors, [F], can be obtained numerically for each frame of the video. From these deformation gradient tensors, the updated Cauchy Strain Tensor will be determined as a function of position on the Cartesian plane relative to the centroid of the RBC. The strains determined from the updated Langrangian Green Strain are numerically fit to the Navier‐Lame Equations along the boundary of the RBC to experimentally fit the two Lame's Constants during capillary flow. These constants will then be used to determine the Cauchy Stress Tensor, using Hooke's Law, along the boundary of the RBC, allowing for the calculation of the Wall Shear Stress. The average Elastic Modulus of fresh cells was found to be 5.4 ± 0.4 × 106 N/m2, while of that of RBCs stored for two weeks was found to be statistically larger and was calculated to be 6.0 ± 1.0 × 106 N/m2. Additionally, stored RBCs experienced higher shear strains and wall shear strains over larger areas of the membrane as compared with stored cells. Additionally, stress concentrators in the membrane were observed at the points of contact with the capillary wall. However, for stored cells, the high stress acted on a larger segment of the membrane. This algorithm for determining wall shear stress experienced by RBC membranes in vivo during capillary flow was thus able to detect statistically significant differences in the both the mechanical properties and stress concentrations between fresh cells and stored cells.Support or Funding InformationThis work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL110900, R56‐HL123015, and R01‐HL126945.

Efficacy of fresh packed red blood transfusion in organophosphate poisoning.

The mortality rate caused by organophosphate (OP) poisoning is still high, even the standard treatment such as atropine and oxime improves a lot. To search for alternative therapies, this study was aimed to investigate the effects of packed red blood cell (RBC) transfusion in acute OP poisoning, and compare the therapeutic effects of RBCs at different storage times.Patients diagnosed with OP poisoning were included in this prospective study. Fresh RBCs (packed RBCs stored less than 10 days) and longer-storage RBCs (stored more than 10 days but less than 35 days) were randomly transfused or not into OP poisoning patients. Cholinesterase (ChE) levels in blood, atropine usage and durations, pralidoxime durations were measured.We found that both fresh and longer-storage RBCs (200–400 mL) significantly increased blood ChE levels 6 hours after transfusion, shortened the duration for ChE recovery and length of hospital stay, and reduced the usage of atropine and pralidoxime. In addition, fresh RBCs demonstrated stronger therapeutic effects than longer-storage RBCs.Packed RBCs might be an alternative approach in patients with OP poisoning, especially during early stages.

Abstract WMP105: Human Monocyte Derived Macrophage Phagocytize Eryptotic Erythrocytes in a Phosphotidylserine Dependent Mechanism

Introduction: After intracerebral hemorrhage (ICH), erythrocytes contribute to secondary injury by releasing toxic hemoproteins. Our lab has previously shown that blood derived macrophages play an important role in ICH clearance but mechanisms of phagocytosis by human macrophages are unknown. This study aims to quantify eryptotic (phosphatidylserine (PtdSer)-expressing) red blood cells (RBCs) in an in vivo model of ICH, and to investigate the mechanisms that play a role in autologous eryptotic phagocytosis by human monocyte derived macrophages (huMDMs). Methods: ICH was induced in mice by autologous blood injection. The mice were sacrificed at 1 day after ICH. The brains were separated into hemispheres and digested into a single cell suspension for analysis by flow cytometry. Cells were stained with antibodies to cell surface markers and annexin V to quantify externalized PtdSer expression. Human monocytes were cultured with M-CSF for 7 days to generate huMDMs. Autologous RBCs were heat shocked (HS) to induce eryptosis. The huMDMs were cocultured with HS RBCs, HS RBCs treated with annexin V, or control RBCs. After 1 hour of coculture, the huMDMs were washed, stained and erythrophagocytosis quantified by microscopy. Results: The proportion of cells that externalized PtdSer increased by almost 20 fold at day 1 after ICH. Control brains mixed with fresh RBCs and subjected to tissue prep did not show PtdSer expression, ensuring that the PtdSer expression detected was induced in vivo (Fig A). HS RBCs increased PtdSer expression and were efficiently phagocytosed by huMDMs. Treatment of HS RBCs with annexin V to antagonize PtdSer-receptor interactions decreased RBC phagocytosis to levels comparable to control RBCs (Figs B and C). Conclusions: In vivo after ICH, erythrocytes externalize PtdSer, a cue to be engulfed by macrophages. Human macrophages phagocytose RBCs in a PtdSer-dependent mechanism. These findings highlight potential targets to enhance ICH clearance.

A comparative study showing the potency of anti-C3d and anti-immunoglobulin G in polyspecific anti-human globulin using fresh and cryopreserved red blood cells.

BACKGROUND:The antiglobulin test is used to determine red blood cells (RBCs) having surface-bound immunoglobulin G (IgG) and/or complement (C3b, C3d, C4b, and C4d) free in serum or attached to antigens on RBCs. In such circumstances, the quality of the anti-human globulin (AHG) which is used in routine cross-match/indirect agglutination test plays a vital role in blood transfusion medicine. For potency testing of polyspecific AHG, it is recommended by the Food and Drug Administration and the Centre for Biologics Evaluation and Research to use fresh O +ve RBCs within 1 hr of collection for sensitization with anti-C3d.AIM AND OBJECTIVE:Freshly collected red cells were cryopreserved and stored at a temperature of −70°C for 30 days. These cells were then sensitized with C3d and IgG after deglycerolization. Their viability was checked by potency testing using polyspecific AHG-containing anti-C3d and anti-IgG.MATERIALS AND METHODS:Anonymous left over fresh whole blood samples were collected from the Indian Red Cross Society, New Delhi, with CPDA as an anticoagulant, and the samples were treated within 1 h of collection. ABO and Rh (D) phenotyping was performed by test tube method. RBCs and plasma from the same donor were used throughout the study.RESULTS:In this comparative study, fresh RBCs (O +ve) maintained their viability after cryopreservation and were also found to be suitable for sensitization with C3d and IgG. The potency of polyclonal AHG did not differ significantly with fresh RBCs and cryopreserved RBCs for 30 days.CONCLUSION:This result suggests that the sensitization of fresh RBCs with IgG and C3d is not affected by using cryopreserved cells for 30 days.

Dendritic Cell Cross Presentation of RBC Antigens in-Vivo Is Not Affected By RBC Storage Duration and Requires Red Pulp Macrophage "Help" in-Vitro

BACKGROUND: Chronic transfusion is associated with an increased risk of bone marrow graft rejection. In prior studies with mouse models, minor histocompatibility antigens in red blood cell (RBC) and platelet products were presented in the context of recipient MHC class I to prime recipient cytotoxic T cells; these represent cross presentation and cross priming, respectively. Once primed in the recipient, these T cells may attack allogeneic bone marrow grafts. Thus, understanding the mechanism of transfusion-induced T cell cross priming may lead to new methods to reduce bone marrow graft rejection in chronically transfused patients. Cross presentation is performed by a specialized subset of antigen presenting cells (APCs) - CD11b lo, XCR1+ dendritic cells (XCR DCs). Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Fluorescent tracking of transfused RBCs showed that splenic red pulp macrophages (RPMs) ingest the majority of damaged RBCs, but RPMs are weak APCs. Splenic dendritic cells, including XCR DCs, also display increased uptake of stored vs. fresh RBCs. These data suggested that refrigerated storage may increase cross presentation of RBC antigens, thereby enhancing T cell cross priming.AIMS: To compare T cell cross priming after transfusion of fresh and refrigerator-stored RBCs, a mouse model was used with transgenic OVA-carrying RBCs as the antigen source and transgenic naive MHC class I-restricted (H-2Kb) OVA-specific T cells (OT-1) as responders. In parallel, an in-vitro system was established to determine the cellular elements required for cross presentation of RBC antigens.METHODS: Purified CD8 T cells from OT-1 mice were loaded with the cell proliferation tracking dye Cell Trace Far Red (CTFR) and adoptively transferred (4 x 10e6 per mouse) into cohorts of 8-12 week old C57BL/6 GFP+ mice (n=3 per group). The next day mice were transfused with 400 uL of fresh (<24 hours old), or stored RBCs (14 days old) from transgenic HOD mice (RBCs express surface HEL, OVA, and Duffy antigens). After 96 hours, flow cytometry was used to assess OT-1 cell proliferation by dye dilution and the expression of activation markers CD44, CD122 and CD62L. Control transfusions with non-HOD GFP+ RBCs confirmed the antigen specificity of the response, and transfusions of HOD blood that had undergone RBC lysis ruled out direct antigen presentation by MHC identical white cells in the transfusate. For the in-vitro cross presentation assay, 1x10e5 CTFR-loaded OT-1 cells were plated in U-bottom 96 well plates with all possible combinations of fresh or stored HOD RBCs (50 x 10e6 per well), bone marrow Flt3L-derived C57BL/6 murine dendritic cells (Flt3L-DCs, 5 X 10e4 per well), and magnetically selected BALB/c (H-2Kd) RPMs (5 x 10e4 per well). MHC mismatch rules out antigen presentation by BALB/c RPMs (H-2Kd) to OT-I T cells (H-2Kb).RESULTS: Transfusion of both fresh and stored HOD RBCs induced vigorous proliferation and activation of OT-1 cells. After 4 days, no differences were seen in the proliferation and activation profiles of OT-1 cells in mice receiving fresh (78±4% CD44hi, CTFR diluted) vs. stored (79±5%) HOD RBCs. The in-vitro cross presentation assay showed weak to absent OVA-specific OT-1 proliferation with co-cultures including fresh or stored HOD RBCs alone, and fresh or stored HOD RBCs plus Flt3L-DCs. In contrast, cultures including RPMs and Flt3L-DCs showed enhanced OVA-specific OT-1 proliferation with fresh (29±10% CTFR diluted) and stored (25±2%) HOD RBCs.CONCLUSION: Cross priming by fresh RBCs was stronger than expected based on prior studies showing minimal RBC uptake by XCR DCs after fresh GFP + RBC transfusion. Cross priming in this system may be more sensitive due to the high frequency of antigen specific T cells and, thus, is saturated by the small antigen load delivered by fresh transfusion. It is also possible that direct uptake of RBCs by DCs harms the cross-presentation machinery and offsets the effect of increased RBC antigen delivery. Optimal cross-presentation of RBC antigen may require cooperation with cells functionally specialized for metabolizing RBC by-products. Indeed, the in-vitro study shows RPMs promote DC-mediated cross presentation of antigens on fresh and stored RBCs. Thus, RPMs may share ingested RBC antigen with adjacent DCs and increase DC activation by secreting inflammatory cytokines.. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Dose-Dependent Effect of Stored Red Blood: Results of a Sub-Group Analysis of the Age of Blood Evaluation (ABLE) Trial

▪The red blood cell (RBC) storage lesion reflects cumulative damage to stored erythrocytes. Randomized clinical trials have yet to identify clinical consequences associated with prolonged RBC storage, however the majority of these trials have not incorporated dose-response analyses. In one of the subgroup analyses undertaken in the ABLE trial, a potential volume-dependent effect associated with storage was identified1.In this secondary analysis of the ABLE study, we conducted detailed analyses aiming to better characterize whether the number of RBC units administered to patients receiving either fresh or standard-issue blood is associated with mortality in critically-ill adults.We undertook a secondary evaluation of the 2510 patients enrolled in the ABLE trial. This randomized, controlled, clinical trial compared mortality rates and several secondary outcomes in mechanically-ventilated adults, who received either "fresh" RBC units (stored for ≤7 days) or "standard-issue" RBC units (first-in, first-out). Our secondary analysis consisted of comparing short- and long-term hazards of death between fresh and standard arms within pre-specified subgroups of patients that received ≤3 RBC units and those that received >3 units. Additional analyses using subgroups defined by 2-unit, and 1-unit increments were performed. Cox proportional hazards regression was used to model the hazard of death at the specified endpoints, adjusting for possible confounders. We examined interactions between randomization to fresh RBCs and number of RBC units transfused, as a means of establishing a dose-dependent relationship. We also undertook adjusted and unadjusted survival analyses between treatment arms within subgroups.In the subgroup that received ≤3 RBCs, there were 754 patients in the fresh arm and 789 in the standard arm; among patients transfused >3 RBCs, there were 457 in the fresh arm and 430 in the standard arm. When stratified by 2 unit increments, in the fresh and standard arms, respectively, there were 601 and 632 patients given ≤2 units, 279 and 272 given 3-4 units, 108 and 107 given 5-6 units, and 223 and 208 given ≥7 units.Baseline characteristics were comparable between treatment arms within major subgroups, aside from a slightly higher proportion of fresh RBC patients admitted due to trauma in the most transfused subgroups (>3 units: 11.4% of standard, 16.4% of fresh; ≥7 units: 11.5% of standard, 19.3% of fresh) and fewer patients admitted with medical causes in the same subgroups (>3 units: 77.7% of standard, 69.6% of fresh; ≥7 units: 77.9% of standard, 67.7% of fresh).There was significant interaction between fresh RBC unit transfusion and the volume of transfused RBC units on long-term (90 and 180 days) but not short-term (28-day, in-ICU, in-hospital) hazards of death for all subgroup analyses. This observation suggests that the effect of RBC storage differs with different transfusion volumes.In subgroups of ≤3 and >3 units, a trend was observed of increased hazard of death with fresh RBCs in the less-transfused subgroup, and decreased hazard of death with fresh RBCs in the more-transfused subgroup at all endpoints (Figure 1). Similar trends were observed with smaller RBC unit increments. At all endpoints, the hazard of death was higher with fresh RBCs in patients given 3-4 units and, after a threshold of 5 units, the trend reversed: patients given ≥7 fresh units had a lower hazard (Figure 2).Overall survival was not significantly different between treatment arms in any of the subgroups.This secondary analysis suggests a volume-dependent, threshold effect of prolonged red cell storage. Fresh RBCs were associated with a trend of decreased hazard of mortality when more than 5 units were transfused. The findings support current transfusion practice for patients requiring transfusion of <5 RBC units. Further study on the effect of RBC unit storage age should be focused on patients anticipated to require more than 5 RBC transfusions.The ABLE trial was funded by the Canadian Institutes of Health (grant #177453) and the Établissement français du sang. Dr. Mack is supported by a Fonds de recherche du Québec - Santé ’Resident Physician Health Research Career Training Program’ award.1. Lacroix J, Hébert PC, Fergusson DA, Tinmouth A, Cook DJ, Marshall JC, Clayton L, McIntyre L, Callum J, Turgeon AF, et al. Age of Transfused Blood in Critically Ill Adults. New England Journal of Medicine. 2015;372(15):1410-1418. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rejuvenation of Stored Red Blood Cells Increases Oxygen Release Capacity

Introduction:The goal of a red blood cell (RBC) transfusion is to treat anemia and improve oxygen delivery to tissues (Sharma 2011). RBC metabolic changes during liquid storage increases the affinity of hemoglobin for oxygen by depletion of 2,3-diphosphoglycerate (2,3-DPG). This change reduces the partial pressure of O2 where the oxygen tension of hemoglobin is 50% saturated (p50). Transfusion of stored RBCs manifests immediate deficits in patient 2,3-DPG concentration after surgery with incomplete in vivo restoration 72 hours post-surgery (Scott 2016). This change may bring into question the efficiency of peripheral oxygen unloading of liquid stored RBCs following transfusion.Ex-vivo rejuvenation of allogeneic RBCs increases the levels of ATP and 2,3-DPG and increases the p50 of stored RBCs by right-shifting the Oxyhemoglobin Dissociation Curve (ODC) (Dennis 1979). RBC Oxygen Release Capacity (ORC) is determined by the percent of oxygen removed from hemoglobin across the arterial (100 mmHg O2) - venous (40 mmHg O2) pressure gradient (Li 2016). The objective was to evaluate the changes in 2,3-DPG and p50 during routine blood bank storage for 35 days and the impact on ORC after RBC rejuvenation.Methods:Five (5) units of human whole blood were collected in CPD, processed into leukocyte reduced RBC units and stored in an additive solution (AS-1). Nearly fresh RBC were obtained from a local blood center after days 3 - 6 of storage at 1-6 °C and then stored up to 35 days at 1-6 °C. A ten (10) mL aliquot was withdrawn from each unit on the day of receipt, then on Days 7, 14, 21, 28, and 35. Each aliquot was split equally by volume into Control (untreated) and Rejuvenated Groups (n=5 per group). The Rejuvenated samples (5 mL) were incubated with 0.8 mL rejuvesol™ Solution (Zimmer Biomet) in a dry air blood warmer (Sarstedt SAHARA-III) for one hour at 37 °C. Complete blood counts (CELL-DYN 3700), ODC (TCS Scientific Corp Hemox-Analyzer), and 2,3-DPG (Roche) on perchloric acid extracts were collected. The ORC was calculated from the ODC as previously described (Li 2016).Results:Five (5) units of CPD/AS-1 RBC units were received less than one week post-donation (5.0 ± 1.2 Days). As expected in the Control Group aliquots (n = 5), 2,3-DPG concentration and the p50 value declined significantly (p < 0.001, ANOVA) from Day 7 through Day 35 (Figure 1). Rejuvenated Group aliquots exhibited a significant increase in 2,3-DPG concentration and improved p50 (p < 0.001, t-test) at each storage interval after incubation with rejuvesol Solution compared to untreated Control aliquots (Figure 1). RBC rejuvenation shifted the ODC to the right (Figure 2) and significantly increased the ORC compared to Control aliquots (Figure 3). The ORC of Rejuvenated aliquots did not decline significantly with storage duration (p = 0.11, ANOVA) while Control aliquots were significantly impacted with storage duration (p < 0.001, ANOVA).Conclusion:Reduction in ORC with storage duration of unrejuvenated RBCs suggests impaired oxygen tissue delivery occurs with stored RBCs to the tissue microenvironment. Transfusion practices designed to increase hemoglobin concentration may be less effective with increased RBC age because of reduced oxygen release capacity. These in vitro results confirm previous reports regarding 2,3-DPG changes during storage and treatment with rejuvenation (Valeri 2000). Additional research is proposed to confirm these observations on full RBC units, the clinical impact of reduced oxygen release capacity, and what impact RBCs with a superphysiological ORC have on the tissue microenvironment. [Display omitted] [Display omitted] [Display omitted] DisclosuresInglut:Zimmer Biomet: Employment. Kausch:Zimmer Biomet: Employment. Gray:Zimmer Biomet: Employment. Landrigan:Zimmer Biomet: Employment.

Functional Heterogeneity of Red Cell-Derived Microparticles from Different Sources: Calcium Ionophore Vs. Storage Vs. Extrusion

BACKGROUND. Cell derived microparticles (MP), are small vesicle (<1um) released in cell activation or apoptosis. Depending on stimulus, MPs can be heterogeneous in phenotype and functional activities such as hemostatic vs thrombogenic vs proinflammatory. In this study, we compared RMP produced in three ways: (1) induced by calcium ionophore (RMP-CaI), (2) released from packed cells (PC) during storage up to 42 days (RMP-Stor) and (3)by high-pressure extrusion of washed RBC (RMP-HPE). The aim of the study was to elucidate how the functional property of these species vary with mode of production.METHODS. For RMP-Stor, samples were taken at intervals up to 42 days of PC stored at 4oC, then centrifuged to recover the RMP released. For RMP-CaI, supernatants of fresh RBC exposed to Ca2+/ionophore (A23187) for 30 min were centrifuged. For RMP-HPE, washed RBC were subjected to high-pressure extrusion, and the resulting RMP were collected by centrifugation and washed. Flow cytometric counts were by CD235a (glycophorin A) and annexinV (AnV) binding. Thromboelastography (TEG) compared equal counts of each sample type, based on CD235a, diluted as needed with particle-free pooled plasma (PFP).RESULTS: (1) Yield per mL of initial packed RBC: The highest yield of RMP was seen in RMP-HPE, as expected since ≈100% of RBC convert to RMP-HPE. This was followed by RMP-CaI, which was about 3x more than RMP-Stor at 42 days. The RMP-Stor increased linearly from day 21 to 42. (2) AnV binding was expressed as ratio AnV+/CD235+, and is a marker of procoagulant phospholipid. This ratio was least in RMP-HPE (0.38) and more than doubled in both RMP-Stor (0.92) and RMP-CaI (1.0). (3) Size of RMP by forward scatter (FS) increased steadily with time in RMP-Stor, from day 21 (0.56 um) to day 42 (0.79 um). These values are somewhat larger than RMP-CaI (0.47 um) or RMP-HPE (0.52 um). The side-scatter signal correlated with the FS values. (4) Acetylcholinesterase (AChE) activity was by far highest in RMP-Stor (56 nM/min, per 106 particles) vs. 4.5 for RMP-CaI, and 1.5 for RMP-HPE. (5) Procoagulant activity: In TEG, the increase in MA (maximum amplitude) by addition of RMP-Stor was 64 mm, but was not increased by RMP-HPE or RMP-CaI. The R-time (lag) showed greatest shortening with RMP-Stor (10.2 min), followed by RMP-CaI (6.7 min), and RMP-HPE (3.6 min) (6)Anti-fibrinolytic activity: In presence of 0.4 ug/mL of tissue plasminogen activator (tPA), for the purpose of making fibrinolytic activity more detectable in a short time, all 3 species of RMP effectively inhibit t-PA-mediated fibrinolysis measured by TEG parameters, including MA, Ly30, and TTG (total thrombus formation). The RMP-HPE was most effective followed by RMP-Stor, and RMP-CaI.CONCLUSION/DISCUSSION. Results demonstrate clear and distinctive differences in functional and phenotypic properties of the 3 species compared. RMP-Stor and RMP-CaI are lager in size, higher in PS and AChE expression, and higher in procoagulant activity, compared to RMP-HPE. On the other hand, RMP-HPE are higher in number generated per mL packed RBC and exhibit stronger inhibition of tPA-mediated fibrinolysis. It is well known that RMP generated by storage and calcium ionophore are enriched in lipid rafts. In contrast, the RMP-HPE are not enriched in lipid rafts, as reflected in low AChE. We conjecture that the contents of lipid raft on RMP may play an important role in determining the functional properties of RMP. DisclosuresNo relevant conflicts of interest to declare.

Monocyte-mediated activation of endothelial cells occurs only after binding to extracellular vesicles from red blood cell products, a process mediated by β-integrin.

Red blood cell (RBC) transfusion is associated with organ failure. The mechanism remains unknown, but may include adherence of blood cells to the microvasculature. We hypothesized that RBC-derived extracellular vesicles (EVs) interact with monocytes to activate endothelial cells. Human umbilical vein endothelial cells were incubated with supernatant from fresh and stored RBC units either containing EVs or depleted from EVs, with or without the addition of immune cells. We measured expression of adhesion markers by flow cytometry and markers of coagulation and inflammation in the culture medium. We studied phagocytosis of EVs by monocytes by using confocal microscopy and flow cytometry. Incubation of endothelial cells with monocytes alone did not induce up regulation of adhesion markers. The addition of both monocytes and supernatant from RBCs containing EVs resulted in up regulation of endothelial expression of intercellular adhesion molecule 1 and E-selectin when compared to baseline. Up regulation was absent when stimulated with RBC supernatant depleted from EVs. EVs are phagocytosed by monocytes, which was partly abrogated after coincubation with two different complement receptor 3 (CR3)-blocking antibodies. Addition of RBC-derived EVs also increased levels of von Willebrand factor (VWF). There were no differences between groups related to storage time. EVs from RBC transfusion bags activate monocytes with subsequent up regulation of endothelial cell adhesion markers. EVs are phagocytosed by monocytes through CR3. Furthermore, these EVs proved to be a source of VWF. These effects are unrelated to storage time. Thereby, EVs from RBC transfusion bags induce a proinflammatory and procoagulant endothelial cell response.

Long-Term Stored Hemoglobin-Vesicles, a Cellular Type of Hemoglobin-Based Oxygen Carrier, Has Resuscitative Effects Comparable to That for Fresh Red Blood Cells in a Rat Model with Massive Hemorrhage without Post-Transfusion Lung Injury.

Hemoglobin-vesicles (HbV), encapsulating highly concentrated human hemoglobin in liposomes, were developed as a substitute for red blood cells (RBC) and their safety and efficacy in transfusion therapy has been confirmed in previous studies. Although HbV suspensions are structurally and physicochemically stabile for least 1-year at room temperature, based on in vitro experiments, the issue of whether the use of long-term stored HbV after a massive hemorrhage can be effective in resuscitations without adverse, post-transfusion effects remains to be clarified. We report herein on a comparison of the systemic response and the induction of organ injuries in hemorrhagic shock model rats resuscitated using 1-year-stored HbV, freshly packed RBC (PRBC-0) and by 28-day-stored packed RBC (PRBC-28). The six-hour mortality after resuscitation was not significantly different among the groups. Arterial blood pressure and blood gas parameters revealed that, using HbV, recovery from the shock state was comparable to that when PRBC-0 was used. Although no significant change was observed in serum parameters reflecting liver and kidney injuries at 6 hours after resuscitation among the three resuscitation groups, results based on Evans Blue and protein leakage in bronchoalveolar lavage fluid, the lung wet/dry weight ratio and histopathological findings indicated that HbV as well as PRBC-0 was less predisposed to result in a post-transfusion lung injury than PRBC-28, as evidenced by low levels of myeloperoxidase accumulation and subsequent oxidative damage in the lung. The findings reported herein indicate that 1-year-stored HbV can effectively function as a resuscitative fluid without the induction of post-transfused lung injury and that it is comparable to fresh PRBC, suggesting that HbV is a promising RBC substitute with a long shelf-life.

Synthesis, carbonic anhydrase I and II isoenzymes inhibition properties, and antibacterial activities of novel tetralone-based 1,4-benzothiazepine derivatives.

Benzothiazepine compounds have a wide range of applications such as antibacterial, antidepressants, anticonvulsants, antihypertensives, antibiotics, antifungal, hypnotic, enzyme inhibitors, antitumor, anticancer and anti-HIV agents. In this study, the synthesis of novel tetralone-based benzothiazepine derivatives (1-16) and their in vitro antibacterial activity and human carbonic anhydrase isoenzymes I and II (hCA I and II) inhibitory effects were investigated. Both isoenzymes were purified by sepharose-4B-l-tyrosine-sulfanilamide affinity chromatography from fresh human red blood cells. All compounds demonstrated the low nanomolar inhibitory effects on both isoenzymes using esterase activity. Benzothiazepine derivative 2 demonstrated the best hCA I inhibitory effect with Ki value of 18.19nM. Also, benzothiazepine derivative 7 showed the best hCA II inhibitory effect with Ki value of 11.31nM. On the other hand, acetazolamide clinically used as CA inhibitor, showed Ki value of 19.92nM against hCA I and 33.60nM against hCA II, respectively.

Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods.

Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF) or whole blood filtration (WBF) methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs) have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA) is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days) and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF) were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p = 0.0010); fresh WBF units had higher cfDNA than fresh RCF units (p = 0.0093). Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p = 0.0031); fresh WBF RBCs had higher cfDNA than older RCF RBCs (p = 0.024). Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes.

Analysis of red blood cells' dynamic status in a simulated blood circulation system using an ultrahigh-speed simultaneous framing optical electronic camera.

Alterations in the morphologic and mechanical properties of red blood cells (RBCs) are considered direct indicators of blood quality. Current measures of characterizing these properties in vivo are limited by the complicated hemodynamic environment. To better evaluate the quality of fresh and stored blood, a new research platform was constructed to evaluate the hemodynamic characteristics of RBCs. The research platform consists mostly of a microfluidic chip, microscope, and ultrahigh-speed simultaneous framing optical electronic camera (USFOEC). The microfluidic chip was designed to simplify the complicated hemodynamic environment. The RBCs were diluted in erythrocyte preservative fluid and infused into the microfluidic channels. After approximately 600× magnification of using the microscope and camera, the RBCs' dynamic images were captured by the USFOEC. Eight sequential and blur-free images were simultaneously captured by the USFOEC system. Results showed that RBC deformation changed with flow velocity and stored RBCs were less sensitive to deformation (Kfresh < Kstored ). The frozen-stored RBCs were better able to sustain hydrodynamic stress (DI49day = 0.128 vs. DIfrozen = 0.118) than cold-stored RBCs but more sensitive to variations in flow speed (K49day = 1626.2 vs. Kfrozen = 1318.2). Results showed that the stored RBCs had worse deformability than fresh RBCs, but frozen-stored RBCs may incur less damage during storage than those stored at merely cold temperatures. This USFOEC imaging system can serve as a platform for direct observation of cell morphological and mechanical properties in a medium similar to a physiologic environment. © 2016 International Society for Advancement of Cytometry.