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Richards A.L.,Bloodworks NW Research Institute | Hendrickson J.E.,Yale University | Zimring J.C.,Bloodworks NW Research Institute | Zimring J.C.,University of Washington | Hudson K.E.,Bloodworks NW Research Institute
Transfusion | Year: 2016

BACKGROUND Generation of antibodies against red blood cell (RBC) antigens can be a clinically significant problem. The underlying mechanisms that regulate the production of RBC antibodies are only partially understood; however, factors such as inflammation significantly increase the rates of RBC antibody generation. Humoral alloimmunization begins with consumption of transfused RBCs by antigen-presenting cells (APCs). Recently, it has become appreciated that there are multiple different types of APCs. The relative contribution of APC subsets to RBC antibodies has not been described in either the quiescent or the inflamed states. STUDY DESIGN AND METHODS To evaluate the types of APCs that consume RBCs, and how inflammation affects this process, C56Bl/6 mice were treated with polyinosinic-polycytidylic acid (poly(I:C)) to induce an inflammatory response and/or were transfused with 3,3′-dihexadecyloxacarbocyanine perchlorate-labeled syngeneic RBCs. Erythrophagocytosis (both at baseline and during inflammation) was analyzed for different subsets of macrophages (MΦ), dendritic cells (DCs), B cells, and monocytes, by a combined approach using flow cytometry and fluorescent microscopy technology. RESULTS In four independent experiments, erythrophagocytosis at baseline was predominately performed by red pulp MΦ; however, during inflammation both plasmacytoid DCs (pDCs) and monocytes increased RBC consumption. Furthermore, pDCs up regulated MHC-II and activation markers CD80 and CD86. In addition to changing patterns of erythrophagocytosis, inflammation also led to a significant decrease in CD11c+ conventional DC populations and an increase in granulocytes. CONCLUSIONS The nature of APCs that consume transfused RBCs is changed by inflammation. Given that APCs initiate humoral immune responses, these findings provide potential mechanistic insight into how inflammation regulates RBC alloimmunization. © 2016 AABB. Source

de Wolski K.,Bloodworks NW Research Institute | Fu X.,Bloodworks NW Research Institute | Fu X.,University of Washington | Roback J.D.,Emory University | And 5 more authors.
Haematologica | Year: 2016

Transfusion of red blood cells is a very common inpatient procedure, with more than 1 in 70 people in the USA receiving a red blood cell transfusion annually. However, stored red blood cells are a non-uniform product, based upon donor-to-donor variation in red blood cell storage biology. While thousands of biological parameters change in red blood cells over storage, it has remained unclear which changes correlate with function of the red blood cells, as opposed to being co-incidental changes. In the current report, a murine model of red blood cell storage/transfusion is applied across 13 genetically distinct mouse strains and combined with high resolution metabolomics to identify metabolic changes that correlated with red blood cell circulation post storage. Oxidation in general, and peroxidation of lipids in particular, emerged as changes that correlated with extreme statistical significance, including generation of dicarboxylic acids and monohydroxy fatty acids. In addition, differences in anti-oxidant pathways known to regulate oxidative stress on lipid membranes were identified. Finally, metabolites were identified that differed at the time the blood was harvested, and predict how the red blood cells perform after storage, allowing the potential to screen donors at time of collection. Together, these findings map out a new landscape in understanding metabolic changes during red blood cell storage as they relate to red blood cell circulation. © 2016 Ferrata Storti Foundation. Source

Waterman H.R.,Bloodworks NW Research Institute | Kapp L.M.,Bloodworks NW Research Institute | Howie H.L.,Bloodworks NW Research Institute | Hod E.A.,Columbia University | And 3 more authors.
Vox Sanguinis | Year: 2015

Background and Objectives: Human studies have demonstrated substantial donor-to-donor variation in refrigerated RBC storage with respect to several variables, including 24-h post-transfusion RBC recovery. However, the human studies leading to these observations are mostly performed using autologous transfusions of stored RBCs, thereby avoiding issues of infectious disease transmission and alloimmunization. Accordingly, one cannot distinguish whether variability in 24-h RBC recovery is due to alterations in RBC storage, differences in phagocytic activity of the recipient's reticuloendothelial system or both. Similar to humans, genetically distinct inbred mouse strains have substantial differences in RBC storage biology, including 24-h post-transfusion RBC recovery. Materials and Methods: In this report, we juxtaposed 24-h recoveries in 15 distinct inbred strains of mice, holding the RBC donor constant to isolate transfusion recipient variation as an independent variable. Strains were chosen for differences in baseline reticulocyte count and haemoglobin, which may correlate to RBC life span and turnover. Results: Unlike large differences observed in storage of RBCs obtained from different strains of mice, only subtle strain-to-strain differences were observed regarding 24-h post-transfusion RBC recoveries. Conclusions: These findings indicate that the murine strains examined are not likely to be useful in sorting out mechanisms of clearance of stored RBCs, and suggest that such mechanisms may be generally conserved in the strains of mice analysed. © 2015 International Society of Blood Transfusion. Source

Waterman H.R.,University of Washington | Kapp L.M.,University of Washington | Munday A.,University of Washington | Odem-Davis K.,University of Washington | And 2 more authors.
Transfusion | Year: 2016

BACKGROUND Platelet (PLT) transfusions can be an essential therapy for patients with thrombocytopenia to maintain hemostasis. However, some patients become alloimmunized to antigens on PLTs (typically HLA), which can prevent efficacy of PLT transfusion due to antibody-mediated clearance. In extreme cases, patients with alloimmunization to multiple HLAs can become "refractory" to PLT transfusion, such that insufficient compatible PLT units can be found to meet transfusion needs. MATERIALS AND METHODS An in vivo murine model of PLT-induced alloimmunization was refined so as to include both transfusion with allogeneic leukoreduced PLTs and studies of posttransfusion PLT recoveries, allowing assessment of alloimmunization and refractoriness. Basic mechanisms of antibody-mediated PLT clearance were investigated using recipients missing either the C3 complement gene or the common gamma chain for Fc receptors. In addition, the efficacy of using costimulatory blockade as a therapeutic intervention was assessed by testing CTLA4-Ig administration before PLT transfusion. RESULTS Fcγ receptors (but not complement C3) are required for alloantibody-mediated PLT refractoriness. In addition, levels of anti-MHC predict the extent of refractoriness in a given animal. Finally, costimulatory blockade as a therapeutic modality prevents transfusion-induced PLT refractoriness. CONCLUSIONS Together these findings introduce new experimental methods, basic mechanistic understanding, and a potential therapeutic intervention for alloimmunization to MHC-based antigens on transfused PLTs. © 2015 AABB. Source

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