Transfusion Of Stored Red Blood Cell Research Articles

Effects of Transfusion of Stored Red Blood Cells on Renal Ischemia-Reperfusion–Induced Hepatic Injury in Rats

Renal ischemia-reperfusion (IR) injures the liver as well as the kidneys. Transfusion of stored red blood cells (RBCs) triggers inflammatory responses, oxidative stress, and activation of innate immunity. In the present study, we investigated the effect of transfusion of stored RBCs on renal IR-induced hepatic injury. Sprague-Dawley rats were randomly divided into 3 groups based on the following treatments: rats subjected to sham operation (sham group), rats subjected to the induction of renal IR only (RIR group), and rats transfused with stored RBCs 1 hour after the start of reperfusion (RIR-TF group). Renal ischemia was induced for 1 hour, and reperfusion was allowed for 24 hours. After reperfusion, blood and liver tissue samples were obtained. Serum levels of aspartate and alanine aminotransferase were increased in the RIR-TF group compared with those in the RIR and sham groups. The hepatic mRNA expression levels of heme oxygenase-1 and neutrophil gelatinase-associated lipocalin were increased in the RIR-TF group compared with those in the RIR and sham groups. The mRNA expression level of high mobility group box-1 was also increased in the RIR-TF group compared with that in the RIR group. The transfusion of stored RBCs exacerbates renal IR-induced liver damage. Oxidative stress may be responsible for hepatic injury.

Storage differentially impacts alloimmunization to distinct red cell antigens following transfusion in mice.

The impact of blood storage on red blood cell (RBC) alloimmunization remains controversial, with some studies suggesting enhancement of RBC-induced alloantibody production and others failing to observe any impact of storage on alloantibody formation. Since evaluation of storage on RBC alloimmunization in patients has examined antibody formation against a broad range of alloantigens, it remains possible that different clinical outcomes reflect a variable impact of storage on alloimmunization to specific antigens. RBCs expressing two distinct model antigens, HEL-OVA-Duffy (HOD) and KEL, separately or together (HOD × KEL), were stored for 0, 8, or 14 days, followed by detection of antigen levels prior to transfusion. Transfused donor RBC survival was assessed within 24 h of transfusion, while IgM and IgG antibody production were assessed 5 and 14 days after transfusion. Stored HOD or KEL RBCs retained similar HEL or KEL antigen levels, respectively, as fresh RBCs, but did exhibit enhanced RBC clearance with increased storage age. Storage enhanced IgG antibody formation against HOD, while the oppositive outcome occurred following transfusion of stored KEL RBCs. The distinct impact of storage on HOD or KEL alloimmunization did not appear to reflect intrinsic differences between HOD or KEL RBCs, as transfusion of stored HOD × KEL RBCs resulted in increased IgG anti-HOD antibody development and reduced IgG anti-KEL antibody formation. These data demonstrate a dichotomous impact of storage on immunization to distinct RBC antigens, offering a possible explanation for inconsistent clinical experience and the need for additional studies on the relationship between RBC storage and alloimmunization.

Premature Macrophage Activation by Stored Red Blood Cell Transfusion Halts Liver Regeneration Post-Partial Hepatectomy in Rats.

Liver resection is a common treatment for various conditions and often requires blood transfusions to compensate for operative blood loss. As partial hepatectomy (PHx) is frequently performed in patients with a pre-damaged liver, avoiding further injury is of paramount clinical importance. Our aim was to study the impact of red blood cell (RBC) resuscitation on liver regeneration. We assessed the impact of RBC storage time on liver regeneration following 50% PHx in rats and explored possible contributing molecular mechanisms using immunohistochemistry, RNA-Seq, and macrophage depletion. The liver was successfully regenerated after PHx when rats were transfused with fresh RBCs (F-RBCs). However, in rats resuscitated with stored RBCs (S-RBCs), the regeneration process was disrupted, as detected by delayed hepatocyte proliferation and lack of hypertrophy. The delayed regeneration was associated with elevated numbers of hemorrhage-activated liver macrophages (Mhem) secreting HO-1. Depletion of macrophages prior to PHx and transfusion improved the regeneration process. Gene expression profiling revealed alterations in numerous genes belonging to critical pathways, including cell cycle and DNA replication, and genes associated with immune cell activation, such as chemokine signaling and platelet activation and adhesion. Our results implicate activated macrophages in delayed liver regeneration following S-RBC transfusion via HO-1 and PAI-1 overexpression.

Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

Hemolytic anemia blunts the cytokine response to transfusion of older red blood cells in mice and dogs.

Transfusion of red blood cells (RBCs) stored for longer durations induces hemolysis and inflammatory cytokine production in murine and canine models. Despite immune system activation by stored RBCs, human randomized trials suggest that fresher RBC transfusions do not improve clinical outcomes. We hypothesized that underlying recipient hemolysis may affect cytokine responses to older RBC transfusions. C57BL/6 mouse cohorts were infused with anti-TER119 antibody to induce hemolysis, rabbit anti-platelet antiserum to induce immune thrombocytopenia (ITP), or appropriate control antibodies. Two days later, mice were transfused with fresh or stored RBCs. Furthermore, in a prospective, randomized, blinded trial, 38 client-owned dogs with primary autoimmune hemolytic anemia (AIHA) and two dogs with ITP, requiring RBC transfusion, were enrolled and randomized to receive fresh (≤7 days) or old (≥21 days) stored RBC transfusions. Monocyte chemoattractant protein (MCP)-1 levels were assessed at defined times after transfusion. Prior immune-mediated hemolysis blunted the MCP-1 response to stored RBC transfusion in mice (361 ± 111 pg/ml vs. 6836 ± 1528 pg/ml in mice with immune hemolysis vs. ITP, respectively; mean ± SD; p< .0001). Although hemolysis markers increased after transfusion of older RBCs, the cytokine response was also muted in dogs with AIHA. No differences in morbidity or mortality were evident comparing dogs randomized to fresh or old RBCs. These data suggest that underlying hemolysis blunts inflammatory responses to old RBC transfusions. The canine data support randomized trial results suggesting a lack of clinical benefit with fresh RBC transfusions in subjects with underlying, baseline hemolysis.

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.

Transfusion of Storage-Damaged Red Blood Cells Induces Ferroptosis in Splenic Macrophages

Red blood cell (RBC) transfusions are a common therapeutic, used in a wide range of settings (e.g., trauma). Therefore, potential outcomes, positive or negative, have special clinical relevance. Preparation of RBC units for transfusion involves refrigerated storage for an extended period of time, during which RBCs undergo multiple metabolic and physical changes, collectively known as the “storage lesion.” As a result, a proportion of the stored RBCs are rapidly cleared from the circulation post-transfusion by macrophages in the liver and spleen. However, the subsequent effects on macrophages of an acute, robust erythrophagocytosis event are still unclear, especially given the substantial amount of iron that is introduced into the cell in a short amount of time. Therefore, we sought to study the effects of substantial erythrophagocytosis on splenic macrophages, which normally play a key role in RBC-derived iron recycling. One possible outcome is macrophage cell death due to ferroptosis, an iron-dependent form of non-apoptotic cell death originally identified in cancer cells, but not yet demonstrated in macrophages.Using a mouse model of storage-damaged RBC transfusion and clearance, wild-type C57BL/6 mice were transfused with fresh (i.e., <24 hr after collection) or 12-14-day stored RBCs, and splenocytes were analyzed at defined time points post-transfusion. By 2 hours post-transfusion, a significant portion of splenic red pulp macrophages (RPMs; VCAM-1+, F4/80hi, CD11blo) had ingested stored RBCs (38.2%; p < 0.0001; Figure A), but not fresh RBCs (2.1%; p < 0.05). Other phagocytes, such as Ly6C+ monocytes (Ly6Chi, CD11b+, CD115+, Ly6G-) and granulocytes (Ly6G+, Ly6Chi, CD11b+) cleared far fewer RBCs: 7.02% (p < 0.0001) and 0.66% respectively. By 2 hours post-transfusion, the number of RPMs dramatically decreased (from 2.4 x 106 to 0.5 x 106 cells per spleen; p < 0.001; Figure B), before returning to steady-state levels by 2 days post-transfusion; thus, transfusion of stored RBCs markedly depletes the available macrophage population in the spleen. Nonetheless, Ki67 staining revealed that RPMs in mice transfused with stored RBCs substantially increased their cell division by 1 day post-transfusion (5.8% to 14.8%; p < 0.01), indicating that these tissue resident cells can self-regenerate locally after transfusion-mediated depletion.Further studies into the mechanism of RPM cell death uncovered ferroptosis-specific characteristics. Thus, by 2 hours after transfusion of old RBCs, increased levels of DCF fluorescence, indicative of reactive oxygen species (16.1% to 24.1%; p < 0.01; Figure C) and C11-Bodipy fluorescence, indicative of lipid peroxidation (2.0% to 19.8%; p < 0.0001; Figure D) were seen. In addition, qPCR analysis of RPMs revealed a 7.9-fold increased expression of PTGS2, which encodes COX-2 (p < 0.001); PTGS2 mRNA is reported to be induced in cells undergoing ferroptosis, downstream of lipid peroxidation. Using bone-marrow-derived macrophages (BMMs) exhibiting a uniform population of F4/80hi and VCAM-1+ cells, which are characteristic of RPMs, we demonstrated that ferrostatin-1 (a ferroptosis inhibitor; 100 µM) rescued cell death in BMMs that had ingested antibody-coated RBCs (30.8% to 13.7%, p < 0.0001; Figure E). Furthermore, ferrostatin-1 decreased the levels of reactive oxygen species (61.4% to 12.6%, p < 0.0001) and lipid peroxides (25.7% to 13.6%; p < 0.001) in BMMs that had ingested RBCs, without decreasing the overall proportion of BMMs that ingested RBCs. In addition, BMMs and J774.1 cells (a murine macrophage cell line) were sensitive to erastin (25% cell death; 1 µM, p < 0.0001), which induces ferroptosis in numerous cancer cell lines.In conclusion, substantial erythrophagocytosis after transfusion of storage-damaged RBCs induces ferroptosis in splenic RPMs. To date, ferroptosis has been described in cancer cells, kidney tubule cells, fibroblasts, neurons, and T cells. However, to our knowledge, this is the first report in macrophages, which play important roles in RBC clearance and iron recycling. This finding sheds light on potential adverse effects on macrophage populations from transfusions of “old” RBCs and also highlights a potential clinical use for ferroptosis inhibitors for preventing substantial losses in an important phagocyte population. [Display omitted] DisclosuresSpitalnik:New Health Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Autologous transfusion of "old" red blood cells-induced M2 macrophage polarization through IL-10-Nrf2-HO-1 signaling complexes.

Red blood cell (RBC) transfusion is associated with systemic inflammation and immune suppression as adverse outcomes. To investigate the immunomodulatory function of the transfused autologous RBC in altering pro-inflammatory and immunosuppressive effects. A total of 24 Sprague Dawley male rats were randomly divided into 3 groups (n = 8 in each group). Group 1 did not receive blood transfusions, while the other 2 groups of rats separately received transfusion of RBC stored for 14 days (group 2) and 35 days (group 3). The rats were treated with HO-1 inhibitor, HO-1 inducer and nuclear factor erythroid 2-related factor 2 (Nrf2) activator after they separately received autologous transfusion of RBC that were cryopreserved for 14 days or 35 days. The blood samples of the rats were collected 12 h after the transfusion, and the macrophage phenotype of M1 and M2 were analyzed with flow cytometry (FCM). Also, the surface protein expression of CD68 and CD200R in macrophages were analyzed and the inflammatory signals in the serum were measured with enzyme-linked immunosorbent assay (ELISA). Moreover, the location and expression of proteins heme oxygenase 1 (HO-1), arginine 1 (Arg-1) and nitric oxide synthase 2 (NOS2) in macrophage were detected with immunofluorescence (IF). Autologous transfusion of long-time stored ("old") RBC promoted macrophage polarization to M2 phenotype and upregulated the expression of its surface proteins CD68 and CD200R. The pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-6, IL-1β, and IL-18 were inhibited, and the secretion of NOS isoforms (iNOS) in serum was reduced with blood transfusion; contrarily, the production of IL-10 and CCL22 was increased. Additionally, HO-1, Arg-1 and NOS2 proteins were located in the cytoplasm, and HO-1 and Arg-1 proteins were highly expressed in macrophage, while the expression of protein NOS2 was low. Moreover, Nrf2, HO-1 and Arg-1 proteins were upregulated in macrophage after receiving "old" RBC transfusion. Autologous transfusion of "old" RBC drove the macrophage phenotype toward M2 macrophages and induced immunosuppressive effects through the IL-10-NRF2-HO-1 signals.

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.

Intraosseous transfusion of hemoglobin vesicles in the treatment of hemorrhagic shock with collapsed vessels in a rabbit model.

Intravenous transfusion sometimes encounters difficulty under prehospital conditions when peripheral vessels are collapsed and inaccessible. We investigated whether the cellular type hemoglobin-based oxygen carriers (Hemoglobin Vesicles: HbVs) allow intraosseous administration into blood circulation for the resuscitation of rabbits with severe hemorrhagic shock. New Zealand white rabbits (2.5 kg average) were set in severe hemorrhagic shock [mean arterial pressure (MAP): 21 ± 2 mm Hg, Hb 5.1 ± 0.8 g/dL]. Immediately thereafter, 12 mL/kg of HbVs, 5% human serum albumin (HSA), autologous whole blood (WB), stored red blood cells (RBCs) or 36 mL/kg of Lactated Ringer's (LR) were intraosseously transfused, followed by an additional intraosseous transfusion with 8 mL/kg of HSA (following HbV, HSA or stored RBC transfusion), or WB or 24 mL/kg of LR (following LR transfusion), respectively. Intraosseous transfusion of HbVs increased MAP (48 ± 9 mm Hg) and improved hypohemoglobinemia (7.1 ± 0.6 g/dL) as well as WB or RBC transfusion. In contrast, neither HSA nor LR improved hemodynamics or Hb levels. Seven out of 10 rabbits receiving HbVs survived for 24 hours, while only one out of 10 rabbits receiving LR survived (WB and RBC; 100% survivals, HSA; 30% survival). Intraosseous infusion of HbVs might be an effective initial treatment to maintain hemodynamics during acute hemorrhagic shock. This approach could be used in emergency situations in which access to peripheral vessels is difficult.

Numerical Model for the Determination of Erythrocyte Mechanical Properties and Wall Shear Stress in vivo From Intravital Microscopy.

The mechanical properties and deformability of Red Blood Cells (RBCs) are important determinants of blood rheology and microvascular hemodynamics. The objective of this study is to quantify the mechanical properties and wall shear stress experienced by the RBC membrane during capillary plug flow in vivo utilizing high speed video recording from intravital microscopy, biomechanical modeling, and computational methods. Capillaries were imaged in the rat cremaster muscle pre- and post-RBC transfusion of stored RBCs for 2-weeks. RBC membrane contours were extracted utilizing image processing and parametrized. RBC parameterizations were used to determine updated deformation gradient and Lagrangian Green strain tensors for each point along the parametrization and for each frame during plug flow. The updated Lagrangian Green strain and Displacement Gradient tensors were numerically fit to the Navier-Lame equations along the parameterized boundary to determined Lame's constants. Mechanical properties and wall shear stress were determined before and transfusion, were grouped in three populations of erythrocytes: native cells (NC) or circulating cells before transfusion, and two distinct population of cells after transfusion with stored cells (SC1 and SC2). The distinction, between the heterogeneous populations of cells present after the transfusion, SC1 and SC2, was obtained through principle component analysis (PCA) of the mechanical properties along the membrane. Cells with the first two principle components within 3 standard deviations of the mean, were labeled as SC1, and those with the first two principle components greater than 3 standard deviations from the mean were labeled as SC2. The calculated shear modulus average was 1.1±0.2, 0.90±0.15, and 12 ± 8 MPa for NC, SC1, and SC2, respectively. The calculated young's modulus average was 3.3±0.6, 2.6±0.4, and 32±20 MPa for NC, SC1, and SC2, respectively. o our knowledge, the methods presented here are the first estimation of the erythrocyte mechanical properties and shear stress in vivo during capillary plug flow. In summary, the methods introduced in this study may provide a new avenue of investigation of erythrocyte mechanics in the context of hematologic conditions that adversely affect erythrocyte mechanical properties.

Changes in Erythrocyte Morphology at Initiation of Cardiopulmonary Bypass Without Blood Transfusion Were Not Associated With Less Deformability During Cardiac Surgery

During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). Prospective study. Tertiary care center affiliated with a university hospital. Adults who underwent elective cardiac surgery requiring CPB. Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.

Transfusion with Stored Antigen-Negative Blood Impairs T Cell Cross Priming to Red Cell Alloantigen in a Subsequent Transfusion

BACKGROUND: Donor red blood cell (RBC) alloantigens are efficiently cross-presented by recipient antigen presenting cells (APCs), thereby priming recipient naive CD8 T cells; however, this process and its downstream immunological consequences are poorly understood. RBC antigen cross presentation has been linked to cytotoxic T cell responses driving bone marrow graft rejection, but in other systems it causes tolerance. The conventional dendritic cell (cDC) subset, cDC1, is required for cross-presentation of RBC antigen. Macrophages are insufficient for cross-presentation in the absence of cDC1s, but do promote RBC antigen cross-presentation by cDCs in vitro. After prolonged storage, a portion of transfused RBCs are rapidly cleared by the spleen. In murine models, transfused stored RBCs have multiple effects on splenic APCs, including architectural distortion, dendritic cell activation, and macrophage depletion via ferroptosis due to increased erythrophagocytosis. Thus, the antigen presentation machinery controlling alloimmunization to RBC antigens may be influenced by the storage duration of previously transfused RBCs.AIMS: To better understand how recent transfusions may influence the immunogenicity of future transfusions, we used a mouse model to determine whether prior transfusion of stored, antigen-matched RBCs influences CD8 T cell cross priming to antigen mismatched RBCs in a subsequent transfusion.METHODS: In an in vivo cross-priming assay, OVA-expressing RBCs from transgenic HOD mice (with RBC surface expression of hen egg lysozyme, ovalbumin (OVA), and Duffy antigens) were the transfused antigen source and naïve OT-1 CD8 T cells, which express an MHC class I restricted OVA-specific TCR transgene, were the responders. Magnetically-selected CD8+ OT-1 cells, labeled with CellTrace Far Red (CTFR), were adoptively transferred into MHC identical (H-2Kb) UBC-GFP C57BL6/J recipients. After 16 hours, these mice were transfused with fresh or stored (12 days in CPDA-1), leukoreduced UBC-GFP RBCs or PBS. Five hours after the initial transfusion, recipients were transfused again with fresh leukoreduced HOD RBCs. To determine the effect of stored RBC transfusion on T cell priming to a non-RBC associated antigen, groups of mice carrying adoptively transferred OT-1 T cells were transfused with fresh or stored RBCs and then challenged 5 hours later with soluble OVA protein (10 µg, IV). After 4 days, splenic OT-1 T cells were analyzed for proliferation (by CTFR dye dilution) and activation (by CD44 and CD122 expression).RESULTS: Proliferation and activation of OT-1 T cells in response to challenge with soluble OVA protein were significantly increased in mice that had been previously transfused with stored RBCs compared to those transfused with fresh RBCs (Fig. 1A). In contrast, OT-1 T cell activation in response to HOD RBC transfusion was decreased in mice that had previously received a stored RBC transfusion, as compared to those receiving fresh RBCs or PBS (Fig. 1B).CONCLUSION: The increased T cell priming after soluble OVA challenge in the mice previously transfused with stored vs. fresh RBCs may be related to the increased expression of co-stimulatory molecules on DCs after stored RBC transfusion. However, T cell priming to the RBC-associated antigen requires additional factors, which are disrupted by previous transfusion of stored, but not fresh, RBCs. Splenic red pulp macrophages potentiate OT-1 T cell cross priming by dendritic cells in vitro and become severely depleted after stored RBC transfusion in vivo. Thus, macrophage help may be needed for T cell cross priming to an RBC antigen in vivo, and stored RBC transfusions may disrupt macrophage function, thereby affecting the immunogenicity of subsequent RBC alloantigen exposure.Figure 1. Effect of fresh and stored RBC transfusion on priming after antigenic challenge with soluble OVA or HOD RBCs.Representative contour plots show proliferation-induced dye dilution and CD44 expression on OT-1 cells in the spleen 4 days after transfusion of fresh (left) or stored (right) RBCs followed 5 hours later with IV injection of soluble OVA protein (A) or HOD RBCs (B). Dot plots at the right of representative contour plots show compiled data from two independent experiments quantifying the percentage of activated and proliferated cells (CTFR diluted, CD44hi) in the OT-1 subgate (mean ± SEM, n = 4-9). *P < 0.05, **P < 0.01, unpaired t test. DisclosuresNo relevant conflicts of interest to declare.

Role of heme in lung bacterial infection after trauma hemorrhage and stored red blood cell transfusion: A preclinical experimental study.

BackgroundTrauma is the leading cause of death and disability in patients aged 1–46 y. Severely injured patients experience considerable blood loss and hemorrhagic shock requiring treatment with massive transfusion of red blood cells (RBCs). Preclinical and retrospective human studies in trauma patients have suggested that poorer therapeutic efficacy, increased severity of organ injury, and increased bacterial infection are associated with transfusion of large volumes of stored RBCs, although the mechanisms are not fully understood.Methods and findingsWe developed a murine model of trauma hemorrhage (TH) followed by resuscitation with plasma and leukoreduced RBCs (in a 1:1 ratio) that were banked for 0 (fresh) or 14 (stored) days. Two days later, lungs were infected with Pseudomonas aeruginosa K-strain (PAK). Resuscitation with stored RBCs significantly increased the severity of lung injury caused by P. aeruginosa, as demonstrated by higher mortality (median survival 35 h for fresh RBC group and 8 h for stored RBC group; p < 0.001), increased pulmonary edema (mean [95% CI] 106.4 μl [88.5–124.3] for fresh RBCs and 192.5 μl [140.9–244.0] for stored RBCs; p = 0.003), and higher bacterial numbers in the lung (mean [95% CI] 1.2 × 107 [−1.0 × 107 to 2.5 × 107] for fresh RBCs and 3.6 × 107 [2.5 × 107 to 4.7 × 107] for stored RBCs; p = 0.014). The mechanism underlying this increased infection susceptibility and severity was free-heme-dependent, as recombinant hemopexin or pharmacological inhibition or genetic deletion of toll-like receptor 4 (TLR4) during TH and resuscitation completely prevented P. aeruginosa–induced mortality after stored RBC transfusion (p < 0.001 for all groups relative to stored RBC group). Evidence from studies transfusing fresh and stored RBCs mixed with stored and fresh RBC supernatants, respectively, indicated that heme arising both during storage and from RBC hemolysis post-resuscitation plays a role in increased mortality after PAK (p < 0.001). Heme also increased endothelial permeability and inhibited macrophage-dependent phagocytosis in cultured cells. Stored RBCs also increased circulating high mobility group box 1 (HMGB1; mean [95% CI] 15.4 ng/ml [6.7–24.0] for fresh RBCs and 50.3 ng/ml [12.3–88.2] for stored RBCs), and anti-HMGB1 blocking antibody protected against PAK-induced mortality in vivo (p = 0.001) and restored macrophage-dependent phagocytosis of P. aeruginosa in vitro. Finally, we showed that TH patients, admitted to the University of Alabama at Birmingham ER between 1 January 2015 and 30 April 2016 (n = 50), received high micromolar–millimolar levels of heme proportional to the number of units transfused, sufficient to overwhelm endogenous hemopexin levels early after TH and resuscitation. Limitations of the study include lack of assessment of temporal changes in different products of hemolysis after resuscitation and the small sample size precluding testing of associations between heme levels and adverse outcomes in resuscitated TH patients.ConclusionsWe provide evidence that large volume resuscitation with stored blood, compared to fresh blood, in mice increases mortality from subsequent pneumonia, which occurs via mechanisms sensitive to hemopexin and TLR4 and HMGB1 inhibition.

Endotoxemia Results in Trapping of Transfused Red Blood Cells in Lungs with Associated Lung Injury.

Red blood cell (RBC) transfusion is associated with organ failure, in particular in the critically ill. We hypothesized that endotoxemia contributes to increased trapping of RBCs in organs. Furthermore, we hypothesized that this effect is more pronounced following transfusion of stored RBCs compared with fresh RBCs. Adult male Sprague-Dawley rats were randomized to receive injection with lipopolysaccharide from E coli or vehicle and transfusion with fresh or stored biotinylated RBCs. After 24 h, the amount of biotinylated RBCs in organs was measured by flow cytometry, as well as the 24-h post-transfusion recovery. Markers of organ injury and histopathology of organs were assessed. Endotoxemia resulted in systemic inflammation and organ injury. Following RBC transfusion, donor RBCs were recovered from the lung and kidney of endotoxemic recipients (1.2 [0.8-1.6]% and 2.2 [0.4-4.4]% of donor RBCs respectively), but not from organs of healthy recipients. Trapping of donor RBCs in the lung was associated with increased lung injury, but not with kidney injury. Stored RBCs induced organ injury in the spleen and yielded a lower 24-h post-transfusion recovery, but other effects of storage time were limited. Endotoxemia results in an increased percentage of donor RBCs recovered from the lung and kidney, which is associated with lung injury following transfusion.

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.

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.

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.

Effect of Transfusion of Red Blood Cells With Longer vs Shorter Storage Duration on Elevated Blood Lactate Levels in Children With Severe Anemia: The TOTAL Randomized Clinical Trial.

Although millions of transfusions are given annually worldwide, the effect of red blood cell (RBC) unit storage duration on oxygen delivery is uncertain. To determine if longer-storage RBC units are not inferior to shorter-storage RBC units for tissue oxygenation as measured by reduction in blood lactate levels and improvement in cerebral tissue oxygen saturation among children with severe anemia. Randomized noninferiority trial of 290 children (aged 6-60 months), most with malaria or sickle cell disease, presenting February 2013 through May 2015 to a university-affiliated national referral hospital in Kampala, Uganda, with a hemoglobin level of 5 g/dL or lower and a lactate level of 5 mmol/L or higher. Patients were randomly assigned to receive RBC units stored 25 to 35 days (longer-storage group; n = 145) vs 1 to 10 days (shorter-storage group; n = 145). All units were leukoreduced prior to storage. All patients received 10 mL/kg of RBCs during hours 0 through 2 and, if indicated per protocol, an additional 10 mL/kg during hours 4 through 6. The primary outcome was the proportion of patients with a lactate level of 3 mmol/L or lower at 8 hours using a margin of noninferiority equal to an absolute difference of 25%. Secondary measures included noninvasive cerebral tissue oxygen saturation during the first transfusion, clinical and laboratory changes up to 24 hours, and survival and health at 30 days after transfusion. Adverse events were monitored up to 24 hours. In the total population of 290 children, the mean (SD) presenting hemoglobin level was 3.7 g/dL (1.3) and mean lactate level was 9.3 mmol/L (3.4). Median (interquartile range) RBC unit storage was 8 days (7-9) for shorter storage vs 32 days (30-34) for longer storage without overlap. The proportion achieving the primary end point was 0.61 (95% CI, 0.52 to 0.69) in the longer-storage group vs 0.58 (95% CI, 0.49 to 0.66) in the shorter-storage group (between-group difference, 0.03 [95% CI, -0.07 to ∞], P < .001), meeting the prespecified margin of noninferiority. Mean lactate levels were not statistically different between the 2 groups at 0, 2, 4, 6, 8, or 24 hours. Kaplan-Meier analysis and global nonlinear regression revealed no statistical difference in lactate reduction between the 2 groups. Clinical assessment, cerebral oxygen saturation, electrolyte abnormalities, adverse events, survival, and 30-day recovery were also not significantly different between the groups. Among children with lactic acidosis due to severe anemia, transfusion of longer-storage compared with shorter-storage RBC units did not result in inferior reduction of elevated blood lactate levels. These findings have relevance regarding the efficacy of stored RBC transfusion for patients with critical tissue hypoxia and lactic acidosis due to anemia. clinicaltrials.gov Identifier: NCT01586923.

Transfused Stored or Antibody-Coated Red Blood Cells Are Internalized By and Activate Splenic Professional Antigen Presenting Cells

BACKGROUND: Alloimmunization to antigens on transfused red blood cells (RBCs) is a major problem in transfusion medicine. Development of alloantibodies limits the availability of compatible RBC units for chronically transfused patients. In addition, chronic RBC transfusion therapy is an important risk factor for cell-mediated bone marrow transplant rejection. To develop anti-RBC alloantibodies, professional antigen presenting cells (APCs) are required to digest and load RBC antigens into MHC class II for presentation to CD4 helper T cells. Mouse studies suggest that RBC-associated minor histocompatibility antigens can also be cross-presented into recipient MHC class I; thereby contributing to CD8 T-cell mediated immune responses against bone marrow allografts. Transfusion of RBCs after prolonged refrigerated storage induces erythrophagocytosis and pro-inflammatory gene expression in the spleen. Splenic red pulp macrophages (RPMs) ingest damaged erythrocytes, catabolize heme, and recycle iron but they are weak APCs. Conversely, dendritic cells (DCs) are the most potent of all APCs. A rare and genetically distinct subset of CD8+ DCs can efficiently ingest dead or dying cells and is uniquely capable of activating CD4 and CD8 T cell responses by presenting antigen via MHC class II and by cross-presenting cell-associated antigens via MHC class I. The RBC storage lesion, in addition to promoting phagocytosis by RPMs, may also induce RBC uptake by splenic DCs. Inflammation induced by stored RBC transfusions, or by antibody-mediated RBC clearance, may enhance DC activation and RBC uptake, thereby triggering humoral and cell-mediated immune responses to RBC antigens.AIMS: A mouse model of green fluorescent protein (GFP)+ RBC transfusion and flow cytometry were used to track the uptake of fresh, antibody-coated and stored RBCs into splenic professional APC subsets and the effects of these RBCs on APC activation.METHODS: Cohorts of 8-12 week old C57BL/6 male mice (n=4 per group) were transfused with one mouse-equivalent unit of syngeneic leukoreduced and citrate-phosphate-dextrose-adenine suspended fresh GFP+ RBCs (<24 hours old), stored GFP+ RBCs (11 days old), fresh GFP+ RBCs coated with anti-RBC polyclonal antibodies, or saline. Two hours after transfusion, spleens were dissociated (Liberase™ DL plus DNAse), FcBlocked, and stained with 7-color antibody panels to define CD8+ DCs (CD11c+ MHC-II+ CD8+ DEC205+), CD8- DCs (CD11c+ MHC-II+ CD8-CD11b+), and RPMs (VCAM-1+ CD11blo). Activation markers included MHC class II and CD86. Data were analyzed using FlowJo and Excel.RESULTS: RBC transfusion after prolonged storage markedly increased GFP+ RBC uptake by RPMs (5.8±0.91% fresh vs. 47±2.3% stored) and CD8+ DCs (1.3±0.27% fresh vs. 13±1.0% stored). A smaller increase in RBC uptake was seen in CD8- DCs (0.6±0.068% fresh vs 1.5±0.083% stored). Antibody-coated RBCs showed two-fold increased uptake by CD8+DCs (25±1.5%) compared with stored RBCs and they showed similar uptake into RPMs (50±1.2%) and CD8- DCs (1.1±0.047%). Lack of GFP+ DC staining with the Ter119 RBC marker confirmed that the GFP signal was internalized and not adsorbed onto the DC surface. CD8+ DCs that internalized GFP+ RBCs showed significantly increased CD86 and MHC class II expression consistent with activation.CONCLUSION: Taken together, these results suggest that transfusion of stored or antibody-coated RBCs increases RBC uptake by, and activation of, professional splenic APCs. CD8+ DC uptake of stored or antibody-coated RBCs may contribute to humoral and cellular anti-RBC immune responses via antigen presentation in MHC class II and class I, respectively. Thus, these mechanisms may increase the risk of alloimmunization to RBC antigens. [Display omitted] DisclosuresZimring:Immucor Inc.: Research Funding; BloodworksNW: Patents & Royalties: Patent Application filed on technology in this abstract - no royalties.