Storage Of Red Blood Cell Units Research Articles

Loss of Oxygen Transport Potency As a Function of RBC Storage Duration

BACKROUND: Red blood cell (RBC) oxygen (O 2) capture/release is regulated via homeostatic modulation of hemoglobin oxygen (Hb-O 2) affinity in a manner that stabilizes O 2 delivery in the setting of reduced O 2 availability (i.e., hypoxia, anemia) or increased O 2 consumption (i.e., exercise, stress, disease). This is achieved via production of allosteric effectors (i.e., 2,3 DPG, ATP) and adjustment of the RBC biochemical milieu (i.e., pH, anion concentration) in response to regional stimuli including temperature and carbon dioxide (CO 2) abundance, encountered during circulatory transit (aka Bohr effect). Blood storage is known to affect Hb-O 2 affinity, because the environment to which the RBCs are exposed affects cellular metabolism, influencing [allosteric effector], pH, and [anion]. In addition, key proteins may either become depleted or impaired during storage, including anion exchange-1 (AE-1 or Band 3) and carbonic anhydrase, the two proteins that enable the Bohr effect. We therefore explored the novel hypothesis that RBC oxygen transport potency is diminished as a function of blood storage duration, due to loss of the Bohr effect. METHODS: Fresh (healthy control) or stored RBCs units (leuko-reduced in AS-1; Impact Life Davenport IA, USA), were analyzed weekly for up to 42 days. Hb-O 2 affinity was determined from the oxygen dissociation curve (ODC), which describes the relationship between the partial pressure of oxygen (pO 2) and the fraction of oxyhemoglobin (FHbO 2). ODCs were constructed at 2 fixed pH values (HEMOX analyzer, TSC Scientific Corp, PA, USA) or 2 fixed CO 2 levels (thin film rotating tonometer; Meon, Graz, Austria, in conjunction with a gas blender; Oxystreamer, Biospherix, Parish, NY, USA, with samples measured by arterial blood gas machine; ABL90Flex, Radiometer, Brea, CA, USA). Analysis included (1) standard p50 determination (i.e., pO 2 at which Hb is 50% saturated with O 2), (2) delta p50 between ODCs at fixed pH 7.6 and 7.2 - HEMOX, or ODCs at fixed pCO 2, eucarbia ~40 mmHg and hypercarbia ~70 mmHg - tonometer), (3) Area between the two curves measured (ABC), as described above. Additional analyses were performed from ODC data, including comparison of blood O 2 content per gram Hb (with SO 2 converted to blood O 2 content assuming 1-gram Hb binds 1.34 ml O 2), and blood O 2 capacitance (βO2), which quantifies the amount of O 2 unloaded for a given arteriovenous pO 2 gradient. RESULTS: As a function of blood storage duration, we found significant (1) reduction in p50 at 2 fixed pHs (HEMOX) and 2 fixed pCO 2s (tonometer) (2) reduction in delta p50 between ODCs at fixed pH 7.6 and 7.2 (HEMOX), or fixed pCO 2; eucarbia ~40 mmHg and hypercarbia ~70 mmHg (tonometer), i.e., significantly reduced Bohr effect (3) reduction in the area between ODC curves at different pH or CO 2 levels (ABC), a more comprehensive representation of the Bohr effect, across the full range of pO 2s encountered during circulatory transit (rather than the single point p50 measurement), (4) reduction in blood oxygen content per gram Hb, and (5) reduction in βO2. SUMMARY: We demonstrate that blood storage significantly impairs the Bohr effect. Consequently, compared to fresh RBCs, the oxygen transport potency of stored RBCs in AS-1 within one week is ~50-60% that of fresh RBCs and by the time the unit is outdated, is down to ~35%. Consequently, 2.86 the number of RBCs from an expiring stored transfusion unit are required to move the same amount of O 2 as a similar number of RBCs in fresh blood.

Sphingosine 1-phosphate has a negative effect on RBC storage quality.

Blood storage promotes the rapid depletion of red blood cell (RBC) high-energy adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (DPG), which are critical regulators of erythrocyte physiology and function, as well as oxygen kinetics and posttransfusion survival. Sphingosine-1-phosphate (S1P) promotes fluxes through glycolysis. We hypothesized that S1P supplementation to stored RBC units would improve energy metabolism and posttransfusion recovery. We quantified S1P in 1929 samples (n= 643, storage days 10, 23, and 42) from the REDS RBC Omics study. We then supplemented human and murine RBCs from good storer (C57BL6/J) and poor storer strains (FVB) with S1P (1, 5, and 10 μM) before measurements of metabolism and posttransfusion recovery. Similar experiments were repeated for mice with genetic ablation of the S1P biosynthetic pathway (sphingosine kinase 1 [Sphk1] knockout [KO]). Sample analyses included metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics, and analysis of end-of-storage posttransfusion recovery, under normoxic and hypoxic storage conditions. Storage promoted decreases in S1P levels, which were the highest in units donated by female or older donors. Supplementation of S1P to human and murine RBCs boosted the steady-state levels of glycolytic metabolites and glycolytic fluxes, ie the generation of ATP and DPG, at the expense of the pentose phosphate pathway. Lower posttransfusion recovery was observed upon S1P supplementation. All these phenomena were reversed in Sphk1 KO mice or with hypoxic storage. S1P is a positive regulator of energy metabolism and a negative regulator of antioxidant metabolism in stored RBCs, resulting in lower posttransfusion recoveries in murine models.

Supplementation with uric and ascorbic acid protects stored red blood cells through enhancement of non-enzymatic antioxidant activity and metabolic rewiring

Redox imbalance and oxidative stress have emerged as generative causes of the structural and functional degradation of red blood cells (RBC) that happens during their hypothermic storage at blood banks. The aim of the present study was to examine whether the antioxidant enhancement of stored RBC units following uric (UA) and/or ascorbic acid (AA) supplementation can improve their storability as well as post-transfusion phenotypes and recovery by using in vitro and animal models, respectively. For this purpose, 34 leukoreduced CPD/SAGM RBC units were aseptically split in 4 satellite units each. UA, AA or their mixture were added in the three of them, while the fourth was used as control. Hemolysis as well as redox and metabolic parameters were studied in RBC units throughout storage. The addition of antioxidants maintained the quality parameters of stored RBCs, (e.g., hemolysis, calcium homeostasis) and furthermore, shielded them against oxidative defects by boosting extracellular and intracellular (e.g., reduced glutathione; GSH) antioxidant powers. Higher levels of GSH seemed to be obtained through distinct metabolic rewiring in the modified units: methionine-cysteine metabolism in UA samples and glutamine production in the other two groups. Oxidatively-induced hemolysis, reactive oxygen species accumulation and membrane lipid peroxidation were lower in all modifications compared to controls. Moreover, denatured/oxidized Hb binding to the membrane was minor, especially in the AA and mix treatments during middle storage. The treated RBC were able to cope against pro-oxidant triggers when found in a recipient mimicking environment in vitro, and retain control levels of 24h recovery in mice circulation. The currently presented study provides (a) a detailed picture of the effect of UA/AA administration upon stored RBCs and (b) insight into the differential metabolic rewiring when distinct antioxidant “enhancers” are used.

Continuous-Flow Magnetic Fractionation of Red Blood Cells Based on Hemoglobin Content and Oxygen Saturation—Clinical Blood Supply Implications and Sickle Cell Anemia Treatment

Approximately 36,000 units of red blood cells (RBCs) are used every day in the U.S. and there is a great challenge for hospitals to maintain a reliable supply, given the 42-day expiration period from the blood donation date. For many years, research has been conducted to develop ex vivo storage solutions that limit RBC lysis and maintain a high survival rate of the transfused cells. However, little attention is directed towards potential fractionation methods to remove unwanted cell debris or aged blood cells from stored RBC units prior to transfusion, which could not only expand the ex vivo shelf life of RBC units but also avoid adverse events in transfused patients. Such fractionation methods could also limit the number of transfusions required for treating certain pathologies, such as sickle cell disease (SCD). In this work, magnetic fractionation is studied as a potential technology to fractionate functional and healthy RBCs from aged or sickle cells. It has been reported that during ex vivo RBC storage, RBCs lose hemoglobin (Hb) and lipid content via formation of Hb-containing exosomes. Given the magnetic character of deoxygenated- or met-Hb, in this work, we propose the use of a quadrupole magnetic sorter (QMS) to fractionate RBCs based on their Hb content from both healthy stored blood and SCD blood. In our QMS, a cylindrical microchannel placed inside the center of the quadrupolar magnets is subjected to high magnetic fields and constant field gradients (286 T/m), which causes the deflection of the paramagnetic, Hb-enriched, and functional RBCs from their original path and their collection into a different outlet. Our results demonstrated that although we could obtain a significant difference in the magnetic mobility of the sorted fractions (corresponding to a difference in more than 1 pg of Hb per cell), there exists a tradeoff between throughput and purity. Therefore, this technology when optimized could be used to expand the ex vivo shelf life of RBC units and avoid adverse events in transfused individuals or SCD patients requiring blood exchange therapy.

Tangential flow filtration facilitated washing of human red blood cells: A proof-of-concept study.

Red blood cell (RBC) units in hypothermic storage degrade over time, commonly known as the RBC storage lesion. These older RBC units can cause adverse clinical effects when transfused, as older RBCs in the unit lyse and release cell-free haemoglobin (Hb), a potent vasodilator that can elicit vasoconstriction, systemic hypertension and oxidative tissue injury after transfusion. In this study, we examined a novel method of washing ex vivo stored single RBC units to remove accumulated cellular waste, specifically cell-free Hb, using tangential flow filtration (TFF) driven by a centrifugal pump. The TFF RBC washing system was run under hypothermic conditions at 4°C, at a constant system volume with 0.9wt% saline as the wash solution. The RBC washing process was conducted on 10 separate RBC units. For this proof-of-concept study, RBC units were expired at the time of washing (60-70 days old). Cell-free Hb was quantified by UV-visible absorbance spectroscopy and analysed via the Winterbourn equations. Pre- and post-wash RBC samples were analysed by Hemox Analyser, Coulter counter and Brookfield rheometer. The RBC volume fraction in solution was measured throughout the wash process by standard haematocrit (HCT) analysis. No substantial decrease in the HCT was observed during the TFF RBC washing process. However, there was a significant decrease in RBC concentration in the first half of the TFF RBC wash process, with no significant change in RBC concentration during the second half of the TFF cell wash process with an 87% overall cell recovery compared with the total number of cells before initiation of cell washing. Utilization of the extinction coefficients and characteristic peaks of each Hb species potentially present in solution was quantified by Winterbourn analysis on retentate and permeate samples for each diacycle to quantify Hb concentration during the washing process. Significant cell-free Hb reduction was observed within the first four diacycles with a starting cell-free Hb concentration in the RBC unit of 0.105 mM, which plateaus to a constant Hb concentration of 0.01 mM or a total extracellular Hb mass of 0.2g in the resultant washed unit. The oxygen equilibrium curve showed a significant decrease in P50 between the initial and final RBC sample cell wash with an initial P50 of 15.6 ± 1.8 mm Hg and a final P50 of 14 ± 1.62 mm Hg. Cooperativity increased after washing from an initial Hill coefficient of 2.37 ± 0.19 compared with a final value of 2.52 ± 0.12. Overall, this study investigated the proof-of-concept use of TFF for washing single RBC units with an emphasis on the removal of cell-free Hb from the unit. Compared with traditional cell washing procedures, the designed system was able to more efficiently remove extracellular Hb but resulted in longer wash times. For a more complete investigation of the TFF RBC washing process, further work should be done to investigate the effects of RBC unit storage after washing. The designed system is lightweight and transportable with the ability to maintain sterility between uses, providing a potential option for bedside ex vivo transfusion in clinical applications.

Storage Differentially Impacts Immunization to Red Cell Antigens

Introduction: The impact of red blood cell (RBC) storage on alloimmunization rates after transfusion remains controversial, with clinical experience demonstrating conflicting results. Indeed, some reports suggest increased rates of alloimmunization associated with longer storage of RBC units, while others suggest no association between alloimmunization rates and length of storage. We hypothesized that this discrepancy may reflect the differential impact of storage on alloimmunization related to each unique antigen and the immune response elicited. Here we define the effect of red cell storage on two distinct antigens, including the Hel-Ova-Duffy (HOD) and KEL antigens, using a preclinical murine model of RBC storage and transfusion.Methods: RBCs singly expressing HOD (HOD RBC) or KEL (KEL RBC) antigens, in addition to those dually expressing both HOD and KEL (HOD x KEL RBC) antigens, were collected from respective transgenic donor mice into CPDA. Units of HOD, KEL or HOD x KEL RBC were generated and used immediately (fresh RBC), or stored for 8 (D8) or 14 (D14) days at 4C before transfusion into wildtype recipients. Recipient cytokine profiles were assessed 2 hours post-transfusion using a multiplex inflammation panel. Donor RBC survival at 24 hours post-transfusion was assessed by flow cytometry. Serum was collected from recipients on days 5 and 14 post-transfusion and analyzed for IgM or IgG development by flow-cytometric crossmatch using HOD or KEL RBC targets.Results: Post-transfusion RBC survival was decreased with increased storage, but was not significantly different between donor cell types (HOD, KEL or HOD x KEL). Recipient cytokine profiles demonstrated a distinct inflammatory profile associated with storage. Development of IgM antibodies against HOD in mice transfused fresh versus D8 or D14 HOD or HOD x KEL RBCs were not significantly different; however, significantly increased (p<0.05) IgG antibodies were detected in recipients transfused with D8 and D14 HOD or HOD x KEL RBCs as compared to those transfused fresh HOD or HOD x KEL RBCs. In contrast, and while the development of anti-KEL IgM antibodies was not significantly different in mice transfused fresh versus D8 or D14 KEL or HOD x KEL RBCs, the IgG response to KEL was abrogated with longer storage, with recipients generating significantly less (p<0.05) anti-KEL IgG following transfusion with stored KEL or HOD x KEL RBCs as compared to fresh HOD or HOD x KEL RBCs.Conclusions: Although the impact of RBC storage on various measures of transfusion efficacy have been determined, the impact of storage on alloimmunization remains incompletely understood. Using a murine model of transfusion, we demonstrate that storage differentially impacts immunization to red cell antigens, increasing the response to certain antigens, like HOD, while decreasing the response to others, like KEL. Although it is unclear whether these findings apply to other RBC antigens and what relevance this holds for human patients, our study provides insight into the impact of storage on red cell alloimmunization and may enlighten future storage protocols. DisclosuresStowell: Argenx: Speakers Bureau; Grifols: Speakers Bureau; Alexion: Consultancy.

Toxic masculinity in red blood cell units? Testosterone therapy in blood donors revisited.

FDA guidelines limit the use of blood from donors taking testosterone replacement therapy (TRT) to red blood cell (RBC) concentrates, whereas plasma and platelets are discarded. The purpose of this study is to bring awareness to above-average free testosterone concentrations in RBC units from TRT donors. We quantified the concentrations of free (bioavailable; pg/ml) and total (protein bound and free; ng/dl) testosterone in plasma (frozen within 24 h) and supernatants from 42-day stored leukocyte-reduced RBC units from 17 TRT male donors and 17 matched controls (no TRT). Total testosterone concentrations were determined by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Free testosterone concentrations were quantified in the same samples using equilibrium dialysis/LC-MS/MS. Plasma free and total testosterone concentrations in TRT donors were 2.9 and 1.8 times higher than that of controls. Total testosterone concentrations in RBC supernatants were about 30% of that of plasma. In contrast, free testosterone concentrations in RBC supernatants were 80%-100% of that of plasma and were significantly (p=.005) higher in TRT compared with controls (252.3 ± 245.3 vs. 103.4 ± 88.2pg/ml). Supraphysiological free testosterone concentrations (>244 pg/ml) in RBC supernatants were observed in five TRT donors and two control donors. RBC units from TRT donors may contain supraphysiological concentrations of free testosterone. This may be resolved by avoiding blood collections soon after testosterone dosing and by enhanced screening of TRT donors. These data establish a rationale for new studies and reexamination of the current guidelines concerning the utilization of blood components from TRT donors.

A New Centrifugation-Free Approach for Reducing IgA Levels in Units of Stored Red Blood Cells

Introduction: Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients when RBCs from IgA-deficient donors are not available. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA to levels suitable for IgA-deficient recipients in a single washing cycle, hence double washing is frequently required. Additionally, cell processors are designed to wash one RBC unit at a time, making washing several units for a multiple-unit transfusion a laborious, lengthy and logistically difficult process. Importantly, these conventional cell washers are complex, expensive machines that may not be available in smaller medical facilities, thus limiting availability of washed RBCs on demand. To address these limitations, we developed a simple, portable, disposable system capable of washing stored RBC units at practical volumetric throughput without the use of centrifugation, while reducing IgA below 0.05 mg/dL in a single run. This new washing system consists of two core modules: (i) a coil of narrow-bore plastic tubing with three sequential bifurcations for high-throughput separation of washed RBCs from washing solution, and (ii) a highly-engineered membrane-hydrogel composite for concentrating the suspension of washed RBCs to the desired level of hematocrit.Materials and Methods: Several units of stored RBCs (n = 4, leukoreduced, 4-6 weeks storage duration) were purchased from the Gulf Coast Regional Blood Center (Houston, TX). Samples of stored RBCs were withdrawn from the units and diluted with 0.9% saline to a hematocrit (Hct) of 10%, to match the dilution typically used in the conventional centrifugation-based washing procedure (2000 mL of saline per typical 350 mL RBC unit with a 60-65% initial Hct). The diluted RBC samples were then washed either by our new washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics.Results and Discussion: Stored RBCs were diluted to 10% Hct with normal saline and passed through the new washing system at 5.0 mL/min to produce washed RBCs with output Hct of about 42%, and Hct of washing solution (waste) of about 2%. Overall, washing performed by either of the two methods substantially reduced hemolysis, lowered the concentration of free hemoglobin (by >4-fold) and total free protein (by >10-fold), while keeping the intracellular ATP unchanged (Table 1). Washing increased the fraction of well-preserved cells (discocytes, echinocytes 1) from 62.9 ± 14.3% in the initial RBC units to 74.1 ± 6.9% for our experimental device, which was a more pronounced improvement than for conventional centrifugation (67.8 ± 5.1%), although the difference was not statistically significant. Importantly, our new washing system reduced the level of IgA to well below 0.05 mg/dL required by AABB in a single pass, significantly outperforming the traditional, centrifugation-based washing procedure.Conclusions: This study demonstrated the feasibility of washing stored RBCs in normal saline for transfusion to patients with selective IgA deficiency without the use of centrifugation. Our inexpensive, portable, disposal system was significantly better than centrifugation in reducing the IgA level, and performed on par with the conventional procedure for all other key in vitro metrics of RBC quality. The ability of this new washing system to operate in line with existing infusion equipment at bedside could be particularly attractive in remote or resource-limited settings, when conventional centrifugation-based cell washers are unavailable.Acknowledgments: This work was supported by the 2012 NIH Director's Transformative Research Award (R01HL117329). [Display omitted] DisclosuresShevkoplyas:Halcyon Biomedical Incorporated: Employment, Equity Ownership, Patents & Royalties: U.S. Patent Appl. 61/929,357, Research Funding; New Health Sciences, Inc.: Consultancy, Research Funding.

Rejuvenated RBCs Increase Oxygen Consumption in a Simulated Exchange Transfusion Model of Sickle Cell Disease Patients

Introduction: Exchange transfusion of red blood cells (RBCs) in patients with Sickle Cell Disease (SCD) may require 6 to 8 units to reduce sickle hemoglobin (Hb-S) to less than 30 percent of total hemoglobin (NIH No. 02-2117). Low oxygen affinity (high p50) in patients with SCD is manifested by impaired blood viscosities and reduced hemoglobin concentration. Well characterized RBC metabolic changes during liquid storage of donor RBC include an increase in the affinity of hemoglobin for oxygen (low p50) by depletion of 2,3-diphosphoglycerate (2,3-DPG). Transfusion of stored RBCs (Standard RBC) suggests inefficient and impaired O2 delivery to tissues compared to fresh circulating RBCs (Hasan 1994, Scott 2016) or after exchange transfusion in SCD (Uchida 1998). Extracorporeal manipulation of p50 through RBC rejuvenation increases the level of 2,3-DPG and increases the p50 of stored RBCs by right-shifting the Oxyhemoglobin Dissociation Curve (ODC) (Dennis 1979). Rejuvenation occurs by incubating RBCs at 37 °C for 60 minutes with a rejuvenation solution (Citra Labs). An exchange transfusion simulation model was developed to evaluate the changes in oxygen release capacity of rejuvenated RBCs and the potential impact on estimated oxygen consumption (VO2) compared to Standard RBCs.Methods: Oxyhemoglobin dissociation curves were generated from (5) RBC units (leukocyte-reduced), CPD/AS-1, on Day 0, after 21 days of storage (Standard RBC), and after rejuvenation on day 21 (REJ RBC) which were used to estimate p50 values of the donor/recipient mixtures after each exchange transfusion and to determine VO2 as previously described (Li 2016, Srinivasan 2017). An elevated p50 of 30.4 mm Hg, typical for SCD patients (Uchida 1998), was used to estimate the baseline VO2 (200 mL O2/min). VO2 was determined after each simulated RBC exchange with Standard or REJ RBCs assuming a constant blood volume of 5L, cardiac output of 5 L/min, total hemoglobin concentration of 10 g/dL. The model assumed an exchange rate of one apheresis unit of circulating RBCs to transfusion of one unit of Standard or REJ RBCs.Results: Donor RBC p50 was 26.9 ± 2.0 mm Hg on Day 0 and declined with storage to 17.6 ± 1.5 mm Hg at 21 days (p < 0.001, t-test). Rejuvenation restored p50 to 34.8 ± 2.7 mm Hg which was significantly higher than Day 0 (p < 0.001, t-test). The model predicted a reduction in Hb-S from 70 % to 22%, a decrease in p50 after the 8 unit exchange transfusion to 21.6 mm Hg with Standard RBC, but an increase in p50 to 33.3 mm Hg with REJ RBC (Figures 1a & 1b). Similarly, VO2 increased in the REJ RBC group but decreased sequentially with Standard RBC upon each additional exchange transfusion (Figure 1c). The model predicted a decrease in VO2 to 118 mL O2/min for the Standard RBC group and an increase to 233 mL O2/min with REJ RBC group after the exchange transfusion.Conclusions: The model predicted a baseline VO2 of 200 mL O2/min; however, in fresh whole blood from healthy volunteers with Hb-A, normal VO2 was estimated at 244 mL O2/min (Srinivasan 2017). This simulation suggested that performing an exchange transfusion with REJ RBCs could achieve 95% of normal oxygen consumption assuming a constant cardiac output even though the hemoglobin was maintained at 10 g/dL. While exchange transfusion has been a successful therapy to reduce the Hb-S percentage in chronically transfused SCD patients, improving tissue oxygen exchange may not yet be optimized as evidenced by post transfusion declines in p50 (Miller 1980, Uchida 1998). The model is limited because it does not account for improved rheological properties and blood viscosity following exchange transfusion, which was previously reported to improve exercise capacity even though p50 declined (Miller 1980). The model also did not account for any influence from irreversibly sickled cells. Transfusion practices to correct anemia and improve tissue oxygenation may be less effective than intended due to the diminished oxygen release capacity of standard stored RBC units. Transfusion strategies should consider whether the use of RBCs with increased p50 may be physiologically advantageous.Figure 1: Predicted ODC (a) before and after the simulated exchange transfusion with Standard or REJ RBCs and predicted Hb-A vs Hb-S ratios (b) and VO2 (c) after each unit of the exchange transfusion with Standard or rejuvenated RBCs. [Display omitted] DisclosuresKausch:Zimmer Biomet: Employment, Research Funding. Gray:Zimmer Biomet: Employment, Research Funding. Landrigan:Zimmer Biomet: Employment, Research Funding.

Optimization of red blood cell unit storage during SARS-COV-2 Pandemic: adopting new strategies to ensure supply in a decentralized blood bank in Brazil.

As coronavirus disease-2019 (COVID-19) spread worldwide and social restrictions were intensified, difficulties in blood supply were expected to result in a shortage of blood donors, logistic issues and a change in blood consumption. Consequences could be detrimental to the meeting of the blood supply demand, especially in a decentralized blood bank in the State of São Paulo responsible for providing blood to more than 100 hospitals, mostly of the public health system. Aiming to minimize negative effects and focusing on maintenance of the blood supply, a different approach was discussed and adopted. Briefly, strategies were related to monitoring and promoting measures to achieve a positive RBC unit balance. Thus, the number of donors, transfusions, RBC unit inventory, RBC unit loss and RBC units within up to 5 days from the expiration date were evaluated. Several strategies were adopted to ensure sufficient availability of RBC units: blood donation was improved with social media and extra blood collections, a restrictive transfusion protocol was implemented, a new logistic process to use RBC units closer to the expiration date was established and non-isogroup transfusions were avoided. Altogether, described strategies were crucial to optimize blood storage during the pandemic. Investing in monitoring and logistics contributed to a positive RBC unit balance and conserving these strategies could be useful.

Extracellular ubiquitin promotes hepatoma metastasis by mediating M2 macrophage polarization via the activation of the CXCR4/ERK signaling pathway.

BackgroundStored red blood cell (RBC) transfusion has been shown to enhance the risk of cancer recurrence. However, the underlying mechanism remains unknown. At our lab, we have demonstrated that the extracellular ubiquitin (eUb) released by aged RBCs could promote tumor metastasis in a melanoma mouse model. This study aimed to confirm the pro-tumor effect of eUb on hepatocellular carcinoma (HCC) and explore the related immunoregulatory mechanisms.MethodsForty HCC tissue specimens and the corresponding adjacent nontumor and normal liver tissues were collected. Two human hepatoma cell lines (MHCC-97H and HepG2.2.15), one murine hepatoma cell line (Hepa1-6), and one human monocyte cell line (THP-1) were adopted in this study. The coculture of hepatoma cells with macrophages was initiated with Transwell inserts. Cell migration in vitro was detected by Transwell and wound-healing assays, while in vivo tumor metastasis was measured by luciferase assay and H&E staining. Macrophage polarization was measured by flow cytometry, immunofluorescence, ELISA, qPCR, and Western blot. Protein expression was detected by Western blot, and immunoprecipitation was used to confirm the interaction between Ub and CXCR4 (CXC chemokine receptor type 4).ResultsUb and CXCR4 were significantly upregulated in HCC tissues, and a positive correlation existed between them. In vitro, the migration of hepatoma cells was not affected by eUb directly, but their metastatic abilities were enhanced after coculture with the macrophages pretreated with eUb. Meanwhile, eUb promoted hepatoma cell metastasis in the lung in vivo and increased the ratio of M2 macrophages in the lung tissues and peripheral blood of tumor-bearing mice. Furthermore, the eUb-induced M2 macrophage polarization was related to the activation of the CXCR4/ERK (extracellular regulated protein kinase) signaling pathway.ConclusionsExtracellular ubiquitin promoted hepatoma metastasis through M2 macrophage polarization via the activation of the CXCR4/ERK signaling pathway, indicating that a personalized transfusion strategy is needed for the treatment of HCC patients. Neutralizing Ub in stored RBC units could lessen the detrimental clinical outcomes induced by the transfusion of stored RBCs.

Clearance and phenotype of extracellular vesicles after red blood cell transfusion in a human endotoxemia model

BackgroundIn the critically ill, extracellular vesicles (EV) from red blood cells (RBC) have been related to adverse effects of blood transfusion. Stored RBC units contain high concentrations of RBC- EVs, thereby increasing the concentration of EVs in the circulation after transfusion. The mechanisms underlying the clearance of donor RBC-EVs after transfusion are unknown. This study investigates whether membrane markers that are associated with clearance of RBCs are also implicated in clearance of RBC-EVs in human endotoxemic recipients of a transfusion. MethodsSix volunteers were injected with Escherichia coli lipopolysaccharide, and after two hours transfused with an autologous RBC unit donated 35 days earlier. Samples were collected from the RBC unit and the volunteers before and after transfusion. RBC-EVs were labeled with (anti) glycophorin A, combined with (anti) CD44, CD47, CD55, CD59, CD147, or lactadherin to detect phosphatidylserine (PS) and analyzed on a A50 Micro flow cytometer. ResultsIn the RBC unit, RBC-EVs solely exposed PS (7.8%). Before transfusion, circulating RBC-EVs mainly exposed PS (22%) and CD59 (9.1%), the expression of the other membrane markers was much lower. After transfusion, the concentration of RBC- EVs increased 2.4-fold in two hours. Thereafter, the EV concentration decreased towards baseline levels. The fraction of EVs positive for all tested membrane markers decreased after transfusion. ConclusionBesides a minor fraction of PS-exposing EVs, RBC-EVs produced during storage do not expose detectable levels of RBC membrane markers that are associated with clearance, which is in contrast to the EVs produced by the circulating RBCs.

MicroRNA Profiling of Exosomes Derived from Red Blood Cell Units: Implications in Transfusion-Related Immunomodulation.

Purpose To elucidate the microRNAs existent in exosomes derived from stored red blood cell (RBC) unit and their potential function. Materials and Methods Exosomes were isolated from the supernatant derived from stored RBC units by sequential centrifugation. Isolated exosomes were characterized by TEM (transmission electron microscopy), western blotting, and DLS (dynamic light scattering). MicroRNA (miRNA) microarray was performed to detect the expression of miRNAs in 3 exosome samples. Results revealed miRNAs that were simultaneously expressed in the 3 exosome samples and were previously reported to exist in mature RBCs. Functions and potential pathways of some detected miRNAs were illustrated by bioinformatic analysis. Validation of the top 3 abundant miRNAs was carried out by qRT-PCR (quantitative reverse transcription‐polymerase chain reaction). Results TEM and DLS revealed the mean size of the exosomes (RBC-derived) as 64.08 nm. These exosomes exhibited higher abundance of short RNA than the long RNA. 78 miRNAs were simultaneously detected in 3 exosome samples and mature RBCs. Several biological processes might be impacted by these miRNAs, through their target gene(s) enriched in a particular signalling pathway. The top 3 (abundant) miRNAs detected were as follows: miR-125b-5p, miR-4454, and miR-451a. qRT-PCR revealed higher abundance of miR-451a than others. Only miR-4454 and miR-451a abundance tended to increase with increasing storage time. Conclusion Exosomes derived from stored RBC units possessed multiple miRNAs and, hence, could serve various functions. The function of exosomes (RBC-derived) might be implemented partly by the predominantly enriched miR-451a.

Total plasma heme concentration increases after red blood cell transfusion and predicts mortality in critically ill medical patients.

Relationships between red blood cell (RBC) transfusion, circulating cell-free heme, and clinical outcomes in critically ill transfusion recipients are incompletely understood. The goal of this study was to determine whether total plasma heme increases after RBC transfusion and predicts mortality in critically ill patients. This was a prospective cohort study of 111 consecutive medical intensive care patients requiring RBC transfusion. Cell-free heme was measured in RBC units before transfusion and in the patients' plasma before and after transfusion. Total plasma heme levels increased in response to transfusion, from a median (interquartile range [IQR]) of 35 (26-76) μmol/L to 47 (35-73) μmol/L (p < 0.001). Posttransfusion total plasma heme was higher in nonsurvivors (54 [35-136] μmol/L) versus survivors (44 [31-65] μmol/L, p = 0.03). Posttransfusion total plasma heme predicted hospital mortality (odds ratio [95% confidence interval] per quartile increase in posttransfusion plasma heme, 1.76 [1.17-2.66]; p = 0.007). Posttransfusion total plasma heme was not correlated with RBC unit storage duration and weakly correlated with RBC unit cell-free heme concentration. Total plasma heme concentration increases in critically ill patients after RBC transfusion and is independently associated with mortality. This transfusion-associated increase in total plasma heme is not fully explained by RBC unit storage age or cell-free heme content. Additional studies are warranted to define mechanisms of transfusion-related plasma heme accumulation and test prevention strategies.

Donor-specific individuality of red blood cell performance during storage is partly a function of serum uric acid levels.

Previous investigations in leukoreduced units of red blood cells (RBCs) in mannitol additive solution revealed the close association of uric acid (UA) levels in vivo with the susceptibility of RBCs to storage lesion markers. In this study, we examined whether UA has a similar correlation with the capability of RBCs to cope with the oxidative provocations of storage under different conditions, namely, in CPDA-1 and in the absence of leukoreduction. The UA-dependent antioxidant capacity of the supernatant was measured in nonleukoreduced units of RBCs in CPDA (n=47). The possible effect of UA variability on the storage lesion profile was assessed by monitoring several physiologic properties of RBCs and supernatant, including cell shape, reactive oxygen species, and size distribution of extracellular vesicles, in units exhibiting the lowest or highest levels of UA activity (n=16) among donors, throughout the storage period. In stored RBC units, the UA-dependent antioxidant activity of the supernatant declined as a function of storage duration but always in strong relation to the UA levels in fresh blood. Contrary to units of poor-UA activity, RBCs with the highest levels of UA activity exhibited better profile of calcium- and oxidative stress-driven modifications, including a significant decrease in the percentages of spherocytes and of 100- to 300-nm-sized vesicles, typically associated with the exovesiculation of stored RBCs. The antioxidant activity of UA is associated with donor-specific differences in the performance of RBCs under storage in nonleukoreduced CPDA units.

Effects of red blood cell storage time on transfused patients in the ICU-protocol for a systematic review.

Patients in the intensive care unit (ICU) are often anaemic due to blood loss, impaired red blood cell (RBC) production and increased RBC destruction. In some studies, more than half of the patients were treated with RBC transfusion. During storage, the RBC and the storage medium undergo changes, which lead to impaired transportation and delivery of oxygen and may also promote an inflammatory response. Divergent results on the clinical consequences of storage have been reported in both observational studies and randomised trials. Therefore, we aim to gather and review the present evidence to assess the effects of shorter vs. longer storage time of transfused RBCs for ICU patients. We will conduct a systematic review with meta-analyses and trial sequential analyses of randomised clinical trials, and also include results of severe adverse events from large observational studies. Participants will be adult patients admitted to an ICU and treated with shorter vs. longer stored RBC units. We will systematically search the Cochrane Library, MEDLINE, Embase, BIOSIS, CINAHL and Science Citation Index for relevant literature, and we will follow the recommendation by the Cochrane Collaboration and the Preferred Reporting Items for Systemtic Review and Meta-Analysis (PRISMA)-statement. We will assess the risk of bias and random errors, and we will use the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) approach to evaluate the overall quality of evidence. We need a high-quality systematic review to summarise the clinical consequences of RBC storage time among ICU patients.

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.

Wnt Signaling in Red Blood Cells

Introduction: Red blood cells (RBCs) are the most common cell type in the human body.These cells deliver oxygen to the body's tissues and are composed of a cytoplasm that is rich in hemoglobin and is surrounded by a membrane that is essential for the cells function by providing properties such as stability and deformability. The membrane is composed of a lipid bilayer, transmembrane proteins, and a filamentous meshwork of proteins such as actin and adducin that forms the cells cytoskeleton along the entire cytoplasmic surface of the membrane. RBCs lack a nucleus and other cellular organelles that enable gene expression. Thus, it has been assumed, that signaling cascades such as the Wnt signaling pathway are not active in RBCs.The Wnt pathways are fundamental for normal development and homeostasis and regulate, among other, cell growth, motility and differentiation. Although there is no functional data connecting between Wnt signaling and RBCs, previous proteomic studies have shown that some of the non-canonical Wnt signaling components, such as specific small GTPases and kinases are present in RBCs. In this study, we show for the first time that Wnt ligands activate signaling cascades in RBCs.Methods: RBCs were collect from healthy donors and treated with different Wnt ligands. A large number of methods were used to evaluate the RBCs morphological properties, life span, vitality, flexibility and protein expression patterns.Results: Our results clearly show that different Wnt ligands can dramatically increase the live span of RBCs by affecting the cells membrane cytoskeleton. Some of the Wnts affects include activation of GTPases such as Rac, JNK and RhoA, which lead to actin modification. These changes in the actin cytoskeleton result in increased membrane stability. In parallel, treating RBCs with Wnts leads to activation of PKC and RhoA resulting in phosphorylation of adducin which in turn increases the cells membrane flexibility and improves the vitality of the cell.Summary: Our novel findings indicate that the non-canonical Wnt signaling pathway in RBCs is active and can stabilize the RBCs cytoskeleton enhancing the vitality, deformability and life span of the cell.These novel findings may help in the development of new therapeutic strategies for people suffering from different hemolytic disease that effect the RBCs cytoskeleton and provide new insights into the improvement of stored RBC units. 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.

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.