Search

Background: The cellular and molecular changes during red blood cell (RBC) storage that affect posttransfusion recovery (PTR) remain incompletely understood. We have previously reported that RBCs of different storage biology cross-regulate each other when stored together (co-storage cross-regulation [CSCR]). However, the mechanism of CSCR is unclear. In the current study, we tested the hypothesis that CSCR involves acquisition of molecular signatures associated with PTR., Study Design and Methods: The whole blood compartment of either B6 or FVB mice was biotinylated in vivo prior to blood collection and storage. Bio-B6 or Bio.FVB were stored with RBCs from B6 mice transgenic for green florescent protein (GFP) (B6.GFP). After storage, avidin-magnetic beads were used to simultaneous purify Bio-RBCs (positive selection) and B6.GFPs (negative selection). Isolated populations were analyzed by transfusion to establish PTR, and subjected to metabolomic and proteomic analysis., Results: B6 RBCs acquired molecular signatures associated with stored FVB RBCs at both the metabolomic and proteomic level including metabolites associated with energy metabolism, oxidative stress regulation, and oxidative damage. Mitochondrial signatures were also acquired by B6 RBCs. Protein signatures acquired by B6 RBCs include proteins associated with vesiculation., Conclusion: The data presented herein demonstrate the appearance of multiple molecular changes from poor-storing RBCs in good-storing RBCs during co-storage. Whether this is a result of damage causing intrinsic molecular changes in B6 RBCs or if molecules of FVB RBC origin are transferred to B6 RBCs remains unclear. These studies broaden our mechanistic understanding of RBC storage (in particular) and potentially RBC biology (in general)., (© 2024 The Author(s). Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: Transgenic mice expressing RBC specific antigens are widely used in mechanistic studies of RBC alloimmunization. Existing RBC donor strains have random transgene integration, potentially disrupting host elements that can confound biological interpretation., Study Design and Methods: Integration site and genomic alterations were characterized by both targeted locus amplification and congenic backcrossing in the five most commonly used RBC alloantigen donor strains (KEL-K2 hi , KEL-K2 med , and KEL-K2 lo , and KEL-K1). A targeted transgenic approach was developed to allow RBC specific transgene expression from a safe harbor locus (ROSA26). Alloimmune responses were assessed by transfusing alloantigen expressing RBCs into wild-type recipients and measuring alloantibodies by flow cytometry., Results/findings: Four of the five analyzed strains had at least one gene disrupted by the transgene integration but none of the disrupted genes are known to be involved in RBC biology. The integration of KEL-K2 med potentially altered the immunological properties of RBCs, although the biological significance of the observed changes is unclear. The ROSA26 targeted approach resulted in a single copy of the transgene that maintains RBC specific expression without random disruption of genomic elements., Conclusion: These findings provide a detailed characterization of genomic disruption by transgene integration found in commonly used RBC donor strains that is relevant to numerous previous publications as well as future studies. With the possible exception of KEL-K2 med , transgene integration is not predicted to affect RBC biology in existing models, and new models can avoid this concern using the described targeted transgenic approach., (© 2022 AABB.)

Background: Genetically modified mice are used widely to explore mechanisms in most biomedical fields-including transfusion. Concluding that a gene modification is responsible for a phenotypic change assumes no other differences between the gene-modified and wild-type mice besides the targetted gene., Study Design and Methods: To test the hypothesis that the N-terminus of Band3, which regulates metabolism, affects RBC storage biology, RBCs from mice with a modified N-terminus of Band3 were stored under simulated blood bank conditions. All strains of mice were generated with the same initial embryonic stem cells from 129 mice and each strain was backcrossed with C57BL/6 (B6) mice. Both 24-h recoveries post-transfusion and metabolomics were determined for stored RBCs. Genetic profiles of mice were assessed by a high-resolution SNP array., Results: RBCs from mice with a mutated Band3 N-terminus had increased lipid oxidation and worse 24-h recoveries, "demonstrating" that Band3 regulates oxidative injury during RBC storage. However, SNP analysis demonstrated variable inheritance of 129 genetic elements between strains. Controlled interbreeding experiments demonstrated that the changes in lipid oxidation and some of the decreased 24-hr recovery were caused by inheritance of a region of chromosome 1 of 129 origin, and not due to the modification of Band 3. SNP genotyping of a panel of commonly used commercially available KO mice showed considerable 129 contamination, despite wild-type B6 mice being listed as the correct control., Discussion: Thousands of articles published each year use gene-modified mice, yet genetic background issues are rarely considered. Assessment of such issues are not, but should become, routine norms of murine experimentation., (© 2021 AABB.)

Background: Despite the significant adverse clinical consequences of RBC alloimmunization, our understanding of the signals that induce immune responses to transfused RBCs remains incomplete. Though RBC storage has been shown to enhance alloimmunization in the hen egg lysozyme, ovalbumin, and human Duffy (HOD) RBC alloantigen mouse model, the molecular signals leading to immune activation in this system remain unclear. Given that the nonclassical major histocompatibility complex (MHC) Class I molecule CD1D can bind to multiple different lysophospholipids and direct immune activation, we hypothesized that storage of RBCs increases lysophospholipids known to bind CD1D, and further that recipient CD1D recognition of these altered lipids mediates storage-induced alloimmunization responses., Study Design and Methods: We used a mass spectrometry-based approach to analyze the changes in lysophospholipids that are induced during storage of mouse RBCs. CD1D knockout (CD1D-KO) and wild-type (WT) control mice were transfused with stored HOD RBCs to measure the impact of CD1D deficiency on RBC alloimmunization., Results: RBC storage results in alterations in multiple lysophospholipid species known to bind to CD1D and activate the immune system. Prior to transfusion, CD1D-deficient mice had lower baseline levels of polyclonal immunoglobulin (IgG) relative to WT mice. In response to stored RBC transfusion, CD1D-deficient mice generated similar levels of anti-HOD IgM and anti-HOD IgG., Conclusion: Although storage of RBCs leads to alteration of several lysophospholipids known to be capable of binding CD1D, storage-induced RBC alloimmunization responses are not impacted by recipient CD1D deficiency., (© 2021 AABB.)

Background: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC., Study Design and Methods: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs., Results: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation., Conclusion: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio., (© 2021 AABB.)

Background: Platelet transfusions remain a mainstay of treatment for many patients with thrombocytopenia, but can lead to alloantibodies to Human Leukocyte Antigens (anti-HLA) resulting in inadequate responses to subsequent platelet transfusions (refractoriness), as well as complicate transplantation. Despite substantial decreases in alloimmunization with the implementation of leukoreduction, a significant percentage of patients still become alloimmunized following platelet transfusions. It remains unclear why some patients make anti-HLA antibodies, but others do not make anti-HLA antibodies even with chronic transfusion. Antecedent pregnancy correlates with risk of alloimmunization due to platelet transfusion in humans - however, isolation of pregnancy as a single variable is not possible in human populations., Study Design and Methods: A tractable murine model of pregnancy and transfusion was engineered by breeding C57BL/6 (H-2 b ) dames with BALB/c (H-2 d ) sires. After pregnancy, female mice were transfused with leukoreduced platelets from F1 (H-2 b/d ) donors that expressed the same paternal major histocompatibility complex (MHC) H-2 d alloantigens as the sires. Control groups allowed isolation of pregnancy or transfusion alone as independent variables. Alloimmunization was determined by testing serum for antibodies to H-2 d MHC alloantigens., Results: No alloantibodies were detected after pregnancy alone, or in response to transfusion of platelets alone; however, significant levels of alloantibodies were detected when pregnancy was followed by transfusion., Conclusions: These findings isolate antecedent pregnancy as a causal contribution to increased frequencies of alloimmunization by subsequent platelet transfusion in mice and provide a platform for ongoing mechanistic investigation., (© 2020 AABB.)

Background: Cigarette smoking is a frequent habit across blood donors (approx. 13% of the donor population), that could compound biologic factors and exacerbate oxidant stress to stored red blood cells (RBCs)., Study Design and Methods: As part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, a total of 599 samples were sterilely drawn from RBC units stored under blood bank conditions at Storage Days 10, 23, and 42 days, before testing for hemolysis parameters and metabolomics. Quantitative measurements of nicotine and its metabolites cotinine and cotinine oxide were performed against deuterium-labeled internal standards., Results: Donors whose blood cotinine levels exceeded 10 ng/mL (14% of the tested donors) were characterized by higher levels of early glycolytic intermediates, pentose phosphate pathway metabolites, and pyruvate-to-lactate ratios, all markers of increased basal oxidant stress. Consistently, increased glutathionylation of oxidized triose sugars and lipid aldehydes was observed in RBCs donated by nicotine-exposed donors, which were also characterized by increased fatty acid desaturation, purine salvage, and methionine oxidation and consumption via pathways involved in oxidative stress-triggered protein damage-repair mechanisms., Conclusion: RBCs from donors with high levels of nicotine exposure are characterized by increases in basal oxidant stress and decreases in osmotic hemolysis. These findings indicate the need for future clinical studies aimed at addressing the impact of smoking and other sources of nicotine (e.g., nicotine patches, snuff, vaping, secondhand tobacco smoke) on RBC storage quality and transfusion efficacy., (© 2020 AABB.)

Background: Coffee consumption is extremely common in the United States. Coffee is rich with caffeine, a psychoactive, purinergic antagonist of adenosine receptors, which regulate red blood cell energy and redox metabolism. Since red blood cell (purine) metabolism is a critical component to the red cell storage lesion, here we set out to investigate whether caffeine levels correlated with alterations of energy and redox metabolism in stored red blood cells., Study Design and Methods: We measured the levels of caffeine and its main metabolites in 599 samples from the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study via ultra-high-pressure-liquid chromatography coupled to high-resolution mass spectrometry and correlated them to global metabolomic and lipidomic analyses of RBCs stored for 10, 23, and 42 days., Results: Caffeine levels positively correlated with increased levels of the main red cell antioxidant, glutathione, and its metabolic intermediates in glutathione-dependent detoxification pathways of oxidized lipids and sugar aldehydes. Caffeine levels were positively correlated with transamination products and substrates, tryptophan, and indole metabolites. Expectedly, since caffeine and its metabolites belong to the family of xanthine purines, all xanthine metabolites were significantly increased in the subjects with the highest levels of caffeine. However, high-energy phosphate compounds ATP and DPG were not affected by caffeine levels, despite decreases in glucose oxidation products-both via glycolysis and the pentose phosphate pathway., Conclusion: Though preliminary, this study is suggestive of a beneficial correlation between the caffeine levels and improved antioxidant capacity of stored red cells., (© 2020 AABB.)

Background: Red blood cell (RBC) storage in the blood bank is associated with the progressive accumulation of oxidant stress. While the mature erythrocyte is well equipped to cope with such stress, recreative habits like alcohol consumption may further exacerbate the basal level of oxidant stress and contribute to the progress of the storage lesion., Study Design and Methods: RBC levels of ethyl glucuronide, a marker of alcohol consumption, were measured via ultra-high-pressure liquid chromatography coupled with high-resolution mass spectrometry. Analyses were performed on 599 samples from the recalled donor population at Storage Days 10, 23, and 42 (n = 250), as part of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study. This cohort consisted of the 5th and 95th percentile of donors with extreme hemolytic propensity out of the original cohort of 13,403 subjects enrolled in the REDS-III RBC Omics study. Ehtyl glucuronide levels were thus correlated to global metabolomics and lipidomics analyses and RBC hemolytic propensity., Results: Ethyl glucuronide levels were positively associated with oxidant stress markers, including glutathione consumption and turnover, methionine oxidation, S-adenosylhomocysteine accumulation, purine oxidation, and transamination markers. Decreases in glycolysis and energy metabolism, the pentose phosphate pathway and ascorbate system were observed in those subjects with the highest levels of ethyl glucuronide, though hemolysis values were comparable between groups., Conclusion: Though preliminary, this study is suggestive that markers of alcohol consumption are associated with increases in oxidant stress and decreases in energy metabolism with no significant impact on hemolytic parameters in stored RBCs from healthy donor volunteers., (© 2020 AABB.)

Background: Taurine is an antioxidant that is abundant in some common energy drinks. Here we hypothesized that the antioxidant activity of taurine in red blood cells (RBCs) could be leveraged to counteract storage-induced oxidant stress., Study Design and Methods: Metabolomics analyses were performed on plasma and RBCs from healthy volunteers (n = 4) at baseline and after consumption of a whole can of a common, taurine-rich (1000 mg/serving) energy drink. Reductionistic studies were also performed by incubating human RBCs with taurine ex vivo (unlabeled or 13 C 15 N-labeled) at increasing doses (0, 100, 500, and 1000 μmol/L) at 37°C for up to 16 hours, with and without oxidant stress challenge with hydrogen peroxide (0.1% or 0.5%). Finally, we stored human and murine RBCs under blood bank conditions in additives supplemented with 500 μmol/L taurine, before metabolomics and posttransfusion recovery studies., Results: Consumption of energy drinks increased plasma and RBC levels of taurine, which was paralleled by increases in glycolysis and glutathione (GSH) metabolism in the RBC. These observations were recapitulated ex vivo after incubation with taurine and hydrogen peroxide. Taurine levels in the RBCs from the REDS-III RBC-Omics donor biobank were directly proportional to the total levels of GSH and glutathionylated metabolites and inversely correlated to oxidative hemolysis measurements. Storage of human RBCs in the presence of taurine improved energy and redox markers of storage quality and increased posttransfusion recoveries in FVB mice., Conclusion: Taurine modulates RBC antioxidant metabolism in vivo and ex vivo, an observation of potential relevance to transfusion medicine., (© 2020 AABB.)

Background: Anti-red blood cell (RBC) alloantibodies consisting of only the immunoglobulin G (IgG) 4 subtype are typically considered clinically insignificant. A US Food and Drug Administration-approved monoclonal anti-human globulin (16H8) is nonreactive with IgG4, which has been considered a benefit to avoid testing interference from IgG4. However, 16H8 also does not recognize two natural IgG3 variants (IgG3-03 and IgG3-13). Thus, 16H8 may miss clinically significant alloantibodies in some settings., Study Design and Methods: Novel mouse anti-human IgG hybridomas were generated and screened for reactivity with 32 human variants of anti-KEL1 across different IgG subtypes, as well as mutants to allow epitope mapping. Anti-IgG reactivity was determined using KEL1+ RBCs bound by each IgG variant as targets. Binding of anti-IgG was determined by flow cytometry., Results: 16H8 recognized an epitope involving amino acid 419, which is glutamate in IgG4, IgG3-03, and IgG3-13, explaining the lack of 16H8 reactivity with these subtypes/isoallotypes. A new monoclonal antibody (PUMA8) was isolated that, like 16H8, was nonreactive with IgG4 but without blind spots for known variants of IgG1, IgG2, or IgG3. PUMA8 recognized an epitope containing arginine at position 355, which is glutamine in IgG4. However, a recently described new IgG4 variant with an arginine at position 355 results in PUMA8 reactivity., Conclusion: PUMA8 represents an alternative to 16H8 that avoids IgG4 but without blind spots for IgG3 variants. However, PUMA8 reacts with one recently described IgG4 variant. In addition to relevance to immunohematology, these studies highlight the importance of patient variation with regards to assay performance in an era of personalized medicine., (© 2020 AABB.)

Background: Human immunoglobulin G (hIgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4). Due to genetic variations, each IgG subtype contains different isoallotypes. It was previously shown that a Food and Drug Administration-approved monoclonal anti-IgG failed to recognize 2 of 15 recombinant, human IgG3 anti-Kell (K1) isoallotypes (rIgG3-03 and rIgG3-13) by indirect antiglobulin test (IAT)., Study Design and Methods: We expressed and purified 15 recombinant human rIgG3 anti-K1 isoallotypes and investigated their antigen binding and ability to induce phagocytosis using homozygous (KK) and heterozygous (Kk) K1-positive red blood cells (RBCs) by gel IAT, flow cytometry, and a monocyte monolayer assay (MMA) with peripheral blood monocytes and cultured inflammatory (M1) and anti-inflammatory (M2) macrophages., Results: MMA results showed that differences in the Fc region of rIgG3 anti-K1 led to distinctive phagocytic activity with both monocytes and M1 macrophages. rIgG3-18 and rIgG3-19 showed an enhanced ability to induce phagocytosis. Differences in Fc regions also led to variations in the number of antibodies bound to KK RBCs. Despite the differences in phagocytic activity, all 15 rIgG3 clones are predicted to induce clinically significant hemolysis if K1-positive blood was transfused into patients., Conclusion: These results argue that antiglobulin reagents that fail to detect isoallotype rIgG3-03 or rIgG3-13 could present a transfusion risk or lack of detection of a potentially clinically significant anti-K1 in hemolytic disease of the fetus and newborn., (© 2020 AABB.)

Background: The chromium-51-labeled posttransfusion recovery (PTR) study has been the gold-standard test for assessing red blood cell (RBC) quality. Despite guiding RBC storage development for decades, it has several potential sources for error., Methods: Four healthy adult volunteers each donated an autologous, leukoreduced RBC unit, aliquots were radiolabeled with technetium-99m after 1 and 6 weeks of storage, and then infused. Subjects were imaged by single-photon-emission computed tomography immediately and 4 hours after infusion. Additionally, from subjects described in a previously published study, adenosine triphosphate levels in transfusates infused into 52 healthy volunteers randomized to a single autologous, leukoreduced, RBC transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage were correlated with PTR and laboratory parameters of hemolysis., Results: Evidence from one subject imaged after infusion of technetium-99m-labeled RBCs suggests that, in some individuals, RBCs may be temporarily sequestered in the liver and spleen immediately following transfusion and then subsequently released back into circulation; this could be one source of error leading to PTR results that may not accurately predict the true quantity of RBCs cleared by intra- and/or extravascular hemolysis. Indeed, adenosine triphosphate levels in the transfusates correlated more robustly with measures of extravascular hemolysis in vivo (e.g., serum iron, indirect bilirubin, non-transferrin-bound iron) than with PTR results or measures of intravascular hemolysis (e.g., plasma free hemoglobin)., Conclusions: Sources of measurement error are inherent in the chromium-51 PTR method. Transfusion of an entire unlabeled RBC unit, followed by quantifying extravascular hemolysis markers, may more accurately measure true posttransfusion RBC recovery., (© 2019 AABB.)

Red blood cell (RBC) storage in the blood bank is characterized by the progressive loss of metabolic regulation, a phenomenon that targets energy and antioxidant metabolism; While the progression of the storage lesion is inevitable, the rate at which this phenomenon occurs varies from donor to donor; Red blood cells from some donors at the end of storage are metabolically superior to RBCs from other donors at the beginning of storage, suggesting that the age of blood alone may not be a sufficiently accurate predictor of stored blood quality; and Animal studies and large-scale omics screening in blood donors have helped identify mechanistic contributors to the metabolic heterogeneity of stored blood units., (© 2019 AABB.)

Background: Humoral alloimmunization to human leukocyte antigen (HLA) can represent a barrier to solid-organ transplantation, can lead to a refractory state in patients requiring platelet transfusion, and can also contribute to transfusion-related acute lung injury (TRALI). While exposure to HLA-mismatched cells/tissues are generally required for HLA alloimmunization, the effect of the extent of major histocompatibility complex (MHC) mismatch between donor and recipient is poorly understood., Study Design and Methods: A novel mouse was generated that allows the expression of a single MHC Class I alloantigen, K d . Alloimmune responses to K d were studied in C57BL/6 mice transfused with splenocytes from different donor mice, allowing the analysis of responses to K d as an isolated alloantigen, or in the context of additional mismatched MHC molecules. Advanced tools were utilized to study responses to K d , including T-cell receptor transgenic mice that recognize the immunodominant K d peptide presented by C57BL/6 mice to CD4+ T cells., Results: A single MHC Class I alloantigen mismatch is less immunogenic than when the same alloantigen is encountered in the context of additional mismatched MHC alloantigens. This difference is due, at least in part, to induction of CD4+ helper T cells, as the effect is overcome by increasing either mature CD4+ T-cell help through immunization or by increasing the precursor frequency of naïve CD4+ T cells by adoptive transfer from T-cell receptor transgenic donors., Conclusion: These findings indicate that the immunogenicity of a single alloantigen can be affected by the context in which it is encountered, demonstrating the potential for cooperative effects between different mismatched MHC alloantigens., (© 2019 AABB.)

Background: Biological and technical variability has been increasingly appreciated as a key factor impacting red blood cell (RBC) storability and, potentially, transfusion outcomes. Here, we performed metabolomics analyses to investigate the impact of factors other than storage duration on the metabolic phenotypes of stored RBC in a multicenter study., Study Design and Methods: Within the framework of the REDS-III (Recipient Epidemiology and Donor Evaluation Study-III) RBC-Omics study, 13,403 donors were enrolled from four blood centers across the United States and tested for the propensity of their RBCs to hemolyze after 42 days of storage. Extreme hemolyzers were recalled and donated a second unit of blood. Units were stored for 10, 23, and 42 days prior to sample acquisition for metabolomics analyses., Results: Unsupervised analyses of metabolomics data from 599 selected samples revealed a strong impact (14.2% of variance) of storage duration on metabolic phenotypes of RBCs. The blood center collecting and processing the units explained an additional 12.2% of the total variance, a difference primarily attributable to the storage additive (additive solution 1 vs. additive solution 3) used in the different hubs. Samples stored in mannitol-free/citrate-loaded AS-3 were characterized by elevated levels of high-energy compounds, improved glycolysis, and glutathione homeostasis. Increased methionine metabolism and activation of the transsulfuration pathway was noted in samples processed in the center using additive solution 1., Conclusion: Blood processing impacts the metabolic heterogeneity of stored RBCs from the largest multicenter metabolomics study in transfusion medicine to date. Studies are needed to understand if these metabolic differences influenced by processing/storage strategies impact the effectiveness of transfusions clinically., (© 2018 AABB.)

Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank., Study Design and Methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway., Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3., Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs., (© 2018 AABB.)

Background: Kell is a glycoprotein expressed on red blood cells (RBCs). Its K and k variants contain either Met (K antigen) or Thr (k antigen) at Position 193, respectively. Development of anti-K after K-mismatched antigen exposure via blood transfusions or pregnancy can destroy RBCs, leading to hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The immunogenicity of overlapping 15-mer Kell peptides with M193 or T193 at every possible position was investigated previously. Interestingly, Peptide W179 to M193, with the polymorphic M193T residue at the peptide's C-terminus, was the most effective at stimulating CD4 T cells from a series of K-immunized women., Study Design and Methods: This study investigates the basis for HLA restriction of anti-K immune responses. Major histocompatibility complex Class II (MHCII)-binding prediction algorithms and quantitative peptide-MHCII-binding assays were employed to determine the binding registers; anchor residues; and affinities of wild-type, truncated, and sequence-modified K and k peptides. Predictions were generated using Immune Epitope Database and ProPred algorithms. Competitive peptide-MHCII-binding assays utilized 12 recombinant HLA-DR proteins, K and k peptides, and high-affinity MHCII-restricted reference peptides., Results: The peptide-MHCII-binding assays identified a unique K peptide-binding register (W179-S187) restricted to HLA-DRB1*11:01, in addition to partially overlapping binding registers that included the K/k M193T polymorphic site and that bound promiscuously to multiple HLA-DR proteins., Conclusion: Three partially overlapping MHCII-binding motifs for HLA-DRB1*11:01 result in high-avidity K-peptide binding, which may contribute to HLA-DR11-restricted immunogenicity associated with the K allele., (© 2018 AABB.)

Background: Donor variability of red blood cell (RBC) storage has been observed in both humans and animal models. We utilized a strain of mice with RBCs known to store well (B6) and a strain known to store poorly (FVB) to test the hypothesis that RBCs affected the storage of other RBCs., Study Design and Methods: Five strains of mice were used: 1) transgenic B6 mice expressing green fluorescent protein (GFP) in their RBCs (GFP.B6), 2) wild-type B6 mice, 3) wild-type FVB mice, 4) F1 crosses between GFP.B6 and FVB mice (GFP.F1), and 5) the analogous wild-type (B6xFVB) F1 cross. GFP.B6 or GFP.F1 RBCs were mixed with wild-type (non-GFP) RBCs from B6 or FVB strains before storage. Twenty-four-hour RBC recoveries were determined for stored RBCs by enumerating circulating GFP+ RBCs by flow cytometry., Results: Twenty-four-hour recoveries of GFP.F1 RBCs was increased by co-storage with B6 RBCs but decreased by co-storage with FVB RBCs. This effect was dose dependent when tested with GFP.B6 RBCs; the more FVB blood added, the worse the 24-hour recoveries became. RBC cross-regulation did not occur when B6 and FVB RBCs were separated by a semipermeable membrane with a 0.4-µm size cutoff., Conclusion: These findings demonstrate that RBCs affect the storage of other RBCs, in both positive and negative directions, indicating not only that RBC storage is intrinsic to the RBC but that RBC-RBC communication occurs. Additional studies will be required to determine the nature of this effect and if these findings translate into human RBC storage., (© 2017 AABB.)

Background: Human immunoglobulin G (IgG) includes four different subtypes (IgG1, IgG2, IgG3, and IgG4), and it is also now appreciated that there are genetic variations within IgG subtypes (called isoallotypes). Twenty-nine different isoallotypes have been described, with 7, 4, 15, and 3 isoallotypes described for IgG1, IgG2, IgG3, and IgG4, respectively. The reactivity of anti-IgG with different isoallotypes has not been characterized., Study Design and Methods: A novel monoclonal anti-K antibody (PugetSound Monoclonal Antibody 1 [PUMA1]) was isolated and sequenced, and a panel of PUMA1 variants was expressed, consisting of the 29 known IgG isoallotypes. The resulting panel of antibodies was preincubated with K-positive red blood cells (RBCs) and then subjected to testing with currently approved anti-IgG by flow cytometry, solid phase systems, gel cards, and tube testing., Results: A US Food and Drug Administration (FDA)-approved monoclonal anti-IgG (gamma-clone) failed to recognize 2 of 15 IgG3 isoallotypes (IgG3-03 and IgG3-13) and 3 of 3 IgG4 isoallotypes (IgG4-01, IgG4-02, and IgG4-03). In contrast, an FDA-approved rabbit polyclonal anti-IgG recognized each of the known human IgG isoallotypes., Conclusion: These findings demonstrate "blind spots" in isoalloantibody detection by a monoclonal anti-IgG. If a patient has anti-RBC antibodies predominantly of an IgG3 subtype (the IgG3-03 and/or IgG3-13 variety), then it is possible that a clinically significant alloantibody would be missed. IgG-03 and IgG-13 have an estimated frequency of 1% to 3% in Caucasian populations and 20% to 30% in certain African populations. Nonreactivity with IgG4 is a known characteristic of this monoclonal anti-IgG, but IgG4 isoallotypes have not been previously reported., (© 2016 AABB.)

Background: Red blood cell (RBC) hemolysis represents an intrinsic mechanism for human vascular disease. Intravascular hemolysis releases hemoglobin and other metabolites that inhibit nitric oxide signaling and drive oxidative and inflammatory stress. Although these pathways are important in disease pathogenesis, genetic and population modifiers of hemolysis, including sex, have not been established., Study Design and Methods: We studied sex differences in storage or stress-induced hemolysis in RBC units from the United States and Canada in 22 inbred mouse strains and in patients with sickle cell disease (SCD) using measures of hemolysis in 315 patients who had homozygous SS hemoglobin from the Walk-PHASST cohort. A mouse model also was used to evaluate posttransfusion recovery of stored RBCs, and gonadectomy was used to determine the mechanisms related to sex hormones., Results: An analysis of predisposition to hemolysis based on sex revealed that male RBCs consistently exhibit increased susceptibility to hemolysis compared with females in response to routine cold storage, under osmotic or oxidative stress, after transfusion in mice, and in patients with SCD. The sex difference is intrinsic to the RBC and is not mediated by plasmatic factors or female sex hormones. Importantly, orchiectomy in mice improves RBC storage stability and posttransfusion recovery, whereas testosterone repletion therapy exacerbates hemolytic response to osmotic or oxidative stress., Conclusion: Our findings suggest that testosterone increases susceptibility to hemolysis across human diseases, suggesting that male sex may modulate clinical outcomes in blood storage and SCD and establishing a role for donor genetic variables in the viability of stored RBCs and in human hemolytic diseases., Competing Interests: Dr. James C. Zimring has a sponsored research agreement with Immucor, and serves on the scientific advisory board for Rubious Therapeutics. The rest of the authors declare no competing financial interests., (© 2016 AABB.)

Background: Accumulation of bioactive lipids during red blood cell (RBC) storage has been identified as a potential source of posttransfusion sequelae in vulnerable populations. Typically, white blood cells (WBCs) have been implicated in the generation of bioactive lipids, and leukoreduction has been seen as a solution to this issue., Study Design and Methods: We developed a targeted metabolomics approach with isotope dilution to quantify a panel of bioactive lipids in both leukoreduced (LR) and nonleukoreduced (NLR) RBC units over the course of storage., Results: Leukoreduction greatly attenuated the production of 12-hydroxyeicosatetraenoic acid (HETE), 12-hydroxyeicosapentaenoic acid, and 14-hydroxydocosahexaenoic acid (HDoHE), all three of which are mediated by 12-lipoxygenase present in WBCs and platelets. However, despite leukoreduction, micromolar levels of linoleic acid (LA), arachidonic acid (AA), and docosahexaenoic acid (DHA) were observed in the RBC units stored for 42 days. These major polyunsaturated fatty acids (PUFAs) and their oxidation products (oxylipins) also significantly increased with storage time, including 5-, 8-, 9-, 11-, 12-, and 15- HETEs from AA; 9- and 13-hydroxyoctadecadienoic acid (HODE); 9-, 10-, and 12,13-dihydroxyoctadecenoic acids from LA; and 14-, 16-, and 17-HDoHEs from DHA. The majority of PUFAs and oxylipins accumulated in the supernatant fraction. Large donor-to-donor variations were observed in both LR-RBC and NLR-RBC units., Conclusion: While the exact role the accumulation of PUFAs and oxylipins plays in RBC unit quality and transfusion medical outcomes remains undetermined, the analytes of interest in this study may serve as biomarkers for lipid degradation and oxidation during storage and may induce changes in human physiology upon transfusion., (© 2016 AABB.)

Background: Transfusion of platelets (PLTs) is a common therapy in a number of clinical settings. However, it is well understood that there is substantial donor-to-donor variation in how well PLTs store and thus the quality of the products that are transfused. The basis of such variation is poorly understood, and there are limited metrics by which units of PLTs can be assessed for their posttransfusion performance. It has repeatedly been demonstrated that myriad biologic changes take place during PLT storage; however, which of the changes correlate with quality of the stored PLTs and/or are mechanistically involved in PLT function remains undetermined., Study Design and Methods: The current study tested stored PLTs from 21 normal subjects, combining high-resolution metabolomics of stored PLTs with in vivo PLT recoveries and survivals. Both individual analytes and metabolic pathways that correlate with posttransfusion PLT viability were identified., Results: Caffeine metabolites were associated with poor PLT recovery; caffeine metabolism was not ongoing in the PLT bag and remained at prestorage levels. Acylcarnitines, particular fatty acid metabolites, and oxidized fatty acids were associated with poor PLT survivals. Of the myriad metabolic changes during PLT storage, these are the first reported metabolic findings to begin distinguishing which changes are of functional importance regarding posttransfusion PLT performance., Conclusions: Together, these findings provide novel mechanistic insights into the functional biology of the PLT storage lesion as well as identifying potential targets for modifying donor environment (e.g., caffeine consumption) and also metrics of quality assessment for stored human PLTs., (© 2016 AABB.)

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.)

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.)

Intravenous immune globulin (IVIG) was developed initially as an immunoglobulin replacement therapy for primary humoral immunodeficiency, but is now widely used in the treatment of autoinflammatory and autoimmune pathologies. In a small number of patients, hemolytic sequelae have been observed after IVIG administration. The lack of a simple one-to-one correlation between measurable hemagglutinins and hemolysis has led to complicated hypotheses involving coincident necessary variables (e.g., a two-hit hypothesis) and also to the positing of causal factors other than hemagglutinins. One such hypothesis is that immune complexes (ICs) contained within IVIG lead to hemolysis. IVIG-mediated hemolysis was addressed at a recent meeting sponsored by the Food and Drug Administration; the Plasma Protein Therapeutics Association; and the National Heart, Lung, and Blood Institute. The primary literature was reviewed at this meeting followed by detailed discussion. Participants concluded that there is both a theoretical basis by which ICs could contribute to hemolysis after IVIG administration and some published data in support of such a possibility. However, the reported data contain substantial caveats, and the existing evidence does not rise to a level sufficient to either confirm or reject a role for ICs. More detailed and focused human studies will be required to further assess the potential role of ICs in IVIG induced hemolysis. This paper summarizes the relevant literature and expands upon the conclusions of this workshop., (© 2015 AABB.)

Background: In mice, refrigerator-stored red blood cells (RBCs) are cleared by extravascular hemolysis and induce cytokine production. To enhance understanding of this phenomenon, we sought to model it in vitro., Study Design and Methods: Ingestion of refrigerator-stored murine RBCs and subsequent cytokine production were studied using J774A.1 mouse macrophage cells and primary murine splenic macrophages. Wild-type and Ccl2-GFP reporter mice were used for RBC clearance in vivo., Results: Although J774A.1 cells and primary macrophages preferentially ingested refrigerator-stored RBCs in vitro, compared to freshly isolated RBCs, neither produced increased cytokines after erythrophagocytosis. In contrast, phagocytosis of refrigerator-stored RBCs in vivo induced increases in circulating monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC) and correspondingly increased mRNA levels in mouse spleen and liver. In the spleen, these were predominantly expressed by CD11b+ cells. Using Ccl2-GFP reporter mice, the predominant splenic population responsible for MCP-1 mRNA production was tissue-resident macrophages (i.e., CD45+, CD11b+, F4/80+, Ly6c+, and CD11c(low) cells)., Conclusion: J774A.1 cells and primary macrophages selectively ingested refrigerator-stored RBCs by phagocytosis. Although cytokine expression was not enhanced, this approach could be used to identify the relevant receptor-ligand combination(s). In contrast, cytokine levels increased after phagocytosis of refrigerator-stored RBCs in vivo. These were primarily cleared in the liver and spleen, which demonstrated increased MCP-1 and KC mRNA expression. Finally, in mouse spleen, tissue-resident macrophages were predominantly involved in MCP-1 mRNA production. The differences between cytokine production in vitro and in vivo are not yet well understood., (© 2014 AABB.)

Background: Red blood cell (RBC) alloantibodies to nonself antigens may develop after transfusion or pregnancy, leading to morbidity and mortality in the form of hemolytic transfusion reactions or hemolytic disease of the newborn. A better understanding of the mechanisms of RBC alloantibody induction, or strategies to mitigate the consequences of such antibodies, may ultimately improve transfusion safety. However, such studies are inherently difficult in humans., Study Design and Methods: We recently generated transgenic mice with RBC-specific expression of the human KEL glycoprotein, specifically the KEL2 or KEL1 antigens. Herein, we investigate recipient alloimmune responses to transfused RBCs in this system., Results: Transfusion of RBCs from KEL2 donors into wild-type recipients (lacking the human KEL protein but expressing the murine KEL ortholog) resulted in dose-dependent anti-KEL glycoprotein immunoglobulin (Ig)M and IgG antibody responses, enhanced by recipient inflammation with poly(I:C). Boostable responses were evident upon repeat transfusion, with morbid-appearing alloimmunized recipients experiencing rapid clearance of transfused KEL2 but not control RBCs. Although KEL1 RBCs were also immunogenic after transfusion into wild-type recipients, transfusion of KEL1 RBCs into KEL2 recipients or vice versa failed to lead to detectable anti-KEL1 or anti-KEL2 responses., Conclusions: This murine model, with reproducible and clinically significant KEL glycoprotein alloantibody responses, provides a platform for future mechanistic studies of RBC alloantibody induction and consequences. Long-term translational goals of these studies include improving transfusion safety for at-risk patients., (© 2013 American Association of Blood Banks.)

Background: Red blood cell (RBC) transfusion is a lifesaving therapy, the logistic implementation of which requires RBC storage. However, stored RBCs exhibit substantial donor variability in multiple characteristics, including hemolysis in vitro and RBC recovery in vivo. The basis of donor variability is poorly understood., Study Design and Methods: We applied a murine model of RBC storage and transfusion to test the hypothesis that genetically distinct inbred strains of mice would demonstrate strain-specific differences in RBC storage. In vivo recoveries were determined by monitoring transfused RBCs over 24 hours. Timed aliquots of stored RBCs were subjected to tandem chromatography/mass spectrometry analysis to elucidate metabolic changes in the RBCs during storage., Results: Using independent inbred mouse strains as donors, we found substantial strain-specific differences in posttransfusion RBC recovery in vivo after standardized refrigerated storage in vitro. Poor posttransfusion RBC recovery correlated with reproducible metabolic variations in the stored RBC units, including increased lipid peroxidation, decreased levels of multiple natural antioxidants, and accumulation of cytidine. Strain-dependent differences were also observed in eicosanoid generation (i.e., prostaglandins and leukotrienes)., Conclusion: These findings provide the first evidence of strain-specific metabolomic differences after refrigerated storage of murine RBCs. They also provide the first definitive biochemical evidence for strain-specific variation of eicosanoid generation during RBC storage. The molecules described that correlate with RBC storage quality, and their associated biochemical pathways, suggest multiple causal hypotheses that can be tested regarding predicting the quality of RBC units before transfusion and developing methods of improved RBC storage., (© 2013 American Association of Blood Banks.)

Background: The human Kell blood group system currently contains 35 antigens determined by allelic polymorphisms in the Kell glycoprotein, a single-pass Type II transmembrane protein. The Kell glycoprotein was initially cloned through screening of a cDNA library; however, direct amino acid sequencing of most of the Kell glycoprotein has not been reported. The N-terminus of the Kell glycoprotein contains two potential translational start sites, which result in differences in the cytoplasmic tail., Study Design and Methods: Protein extracts were isolated from human red blood cell membranes and were digested with trypsin. The resulting peptides were subjected to liquid chromatography-tandem mass spectrometry, allowing resolution of peptides from the N-terminus of the Kell glycoprotein., Results: Peptides were isolated and sequenced that correspond to the upstream methionine start site predicted by the full cDNA sequence. No evidence of internal translation initiation at Methionine 20 was detected., Conclusions: These findings identify the translational start site and define the full cytoplasmic tail of the human Kell glycoprotein., (© 2013 American Association of Blood Banks.)

Background: The storage of red blood cells (RBCs) results in numerous changes, which over time result in decreased recovery of transfused RBCs. In addition (at least in animal models), stored RBCs can be more immunogenic and also stimulate the systemic release of inflammatory cytokines in transfusion recipients. One component of the RBC storage lesion is the accumulation of oxidative damage. We tested the hypothesis that adding a chemical antioxidant (ascorbic acid) to stored RBCs would improve the quality of the stored RBCs., Study Design and Methods: RBCs were harvested from FVB.HOD mice that express an RBC-specific model transgene (HOD) and stored for 14 days with either ascorbic acid in saline or saline alone. Twenty-four-hour posttransfusion recovery of RBCs was tracked by flow cytometry. Alloimmunization was monitored by flow cytometry crossmatch. Cytokines were monitored by multiplex bead arrays., Results: RBCs stored under standard conditions had decreased 24-hour posttransfusion recovery and increased induction of both alloantibodies and interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1 secretion in the mouse recipients. Addition of ascorbic acid from 3.6 to 10.8 mmol/L resulted in a significant decrease in microparticle formation, an improved RBC 24-hour posttransfusion recovery (p<0.01), and a decrease in recipient alloimmunization (p=0.0001). Induction of MCP-1 and IL-6 secretion was not decreased by ascorbic acid., Conclusions: These data indicate that the addition of ascorbic acid solution to RBCs during storage has a beneficial effect on recovery and immunogenicity of RBCs, but not cytokine induction. The addition of ascorbic acid (or other antioxidants) to human RBCs may have beneficial effects., (© 2013 American Association of Blood Banks.)

Background: Alloimmunization to antigens on transfused red blood cells (RBCs) represents a major barrier to chronic transfusion. In extreme cases of multiple alloimmunization, clinicians may be faced with the decision of transfusing incompatible RBCs or risking death from lack of transfusion. The disastrous results of hemolytic transfusion reactions are well understood, and major pathways of clearance have been described. However, well described but poorly understood is the survival of a subset of incompatible donor RBCs during hemolysis, despite antibody binding., Study Design and Methods: We utilize a tractable murine model of incompatible transfusion in which RBCs from transgenic donor mice expressing human glycophorin A (hGPA) are transfused into recipients passively immunized with anti-hGPA., Results: As in humans, the majority of RBCs are cleared but a subset of incompatible donor RBCs persist in circulation, despite being bound by antibodies. Data contained herein reject the hypothesis that lack of clearance is due to insufficient antibody or overwhelming of phagocytic machinery; rather, we establish that surviving RBCs represent a distinct population resistant to clearance., Conclusions: These studies demonstrate that surviving RBCs during incompatible transfusion can represent a population that is resistant to clearance., (© 2012 American Association of Blood Banks.)

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is characterized by red blood cell (RBC) destruction in response to oxidative stress. Although blood donors are not routinely screened for G6PD deficiency, the transfusion of stored G6PD-deficient RBCs may have serious adverse outcomes. By measuring G6PD enzyme activity of RBC units from a large metropolitan hospital transfusion service, we sought to determine 1) the prevalence of G6PD-deficient RBC units, 2) if G6PD activity changes during storage, and 3) if G6PD activity in segments correlates with its activity in the bags., Study Design and Methods: Quantitative G6PD activity was measured in 301 randomly selected RBC units and 73 D+C-E- (i.e., R r or R R ) RBC units, all stored in additive solutions. G6PD deficiency was defined as activity less than 60% of the normal mean., Results: The frequency of G6PD-deficient units in the general inventory was 0.3% (1/301; 95% confidence interval [CI], <0.01%-2.1%). In contrast, its frequency in D+C-E- RBC units was 12.3% (9/73; 95% CI, 6.4%-22.0%). G6PD activity did not significantly change during the 42-day storage period, and G6PD activity measured in RBC storage bags and attached segments correlated well (r=0.7-0.9, p ≤ 0.001, Spearman rank correlation)., Conclusions: Although the frequency of G6PD-deficient RBC units in the transfusion service general inventory was relatively low, it was significantly higher among a subset of R r or R R units. The latter are preferentially allocated for transfusion to patients with sickle cell disease to decrease the risk of RBC alloimmunization, possibly allowing more of these units to be inadvertently targeted to these patients., (© 2012 American Association of Blood Banks.)

Background: KEL1, also known as "K", is one of the most immunogenic red blood cell (RBC) antigens. KEL2, also known as "k," differs from KEL1 by a single amino acid. Anti-Kell system antibodies can lead to significant adverse clinical outcomes in humans, including hemolytic complications in alloimmunized transfusion recipients or in infants of alloimmunized mothers. To provide a platform for in-depth immunologic studies of alloimmunization and subsequent sequelae, we generated transgenic mice expressing the human KEL1 or KEL2 antigens., Study Design and Methods: Vectors were created in which cDNAs encoding either KEL1 or KEL2 were regulated by an erythroid specific β-globin promoter and enhancer. Pronuclear microinjections were carried out into a C57BL6 background, and founder pups were identified by polymerase chain reaction and screened for expression by flow cytometry. RBC life span and antigen stability were assessed by dye labeling RBCs, transfusing into agammaglobulinemic (µMT) recipients, and tracking by flow cytometry., Results: The expression of either KEL1 or KEL2 is RBC specific and first occurs on early RBC precursors. Both KEL1 and KEL2 RBCs have a normal circulatory life span and stable antigen expression. Expression of KEL1 or KEL2 does not result in altered levels of murine Kell, and resulting RBCs have normal hematologic variables., Conclusion: The KEL1 and KEL2 mice represent the first murine system of RBC immunity with antithetical antigens, allowing a more precise modeling of human RBC immunology in general and also a platform for development of novel therapeutics to prevent or minimize the dangers of RBC alloimmunization to the KEL1 and KEL2 antigens in particular., (© 2012 American Association of Blood Banks.)

Background: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA., Study Design and Methods: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy., Results: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice., Conclusion: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs., (© 2012 American Association of Blood Banks.)

Background: Platelet (PLT) transfusions can induce humoral and cellular alloimmunity. HLA antibodies can render patients refractory to subsequent transfusion, and both alloantibodies and cellular alloimmunity can contribute to subsequent bone marrow transplant (BMT) rejection. Currently, there are no approved therapeutic interventions to prevent alloimmunization to PLT transfusions other than leukoreduction. Targeted blockade of T-cell costimulation has shown great promise in inhibiting alloimmunity in the setting of transplantation, but has not been explored in the context of PLT transfusion., Study Design and Methods: We tested the hypothesis that the costimulatory blockade reagent CTLA4-Ig would prevent alloreactivity against major and minor alloantigens on transfused PLTs. BALB/c (H-2(d)) mice and C57BL/6 (H-2(b)) mice were used as PLT donors and transfusion recipients, respectively. Alloantibodies were measured by indirect immunofluorescence using BALB/c PLTs and splenocytes as targets. BMTs were carried out under reduced-intensity conditioning using BALB.B (H-2(b) ) donors and C57BL/6 (H-2(b)) recipients to model HLA-identical transplants. Experimental groups were given CTLA4-Ig (before or after PLT transfusion) with control groups receiving isotype-matched antibody., Results: CTLA4-Ig abrogated both humoral alloimmunization (H-2(d) antibodies) and transfusion-induced BMT rejection. Whereas a single dose of CTLA4-Ig at time of transfusion prevented alloimmunization to subsequent PLT transfusions, administration of CTLA4-Ig after initial PLT transfusion was ineffective. Delaying treatment until after PLT transfusion failed to prevent BMT rejection., Conclusions: These findings demonstrate a novel strategy using an FDA-approved drug that has the potential to prevent the clinical sequelae of alloimmunization to PLT transfusions., (© 2012 American Association of Blood Banks.)

Background: Alloantibodies are a clinically significant sequelae of platelet (PLT) transfusion, potentially rendering patients refractory to ongoing PLT transfusion support. These antibodies are often IgG class switched, suggesting the involvement of CD4+ T-cell help; however, PLT-specific CD4+ T cells have not been visualized in vivo, and specifics of their stimulation are not completely understood., Study Design and Methods: A murine model of alloimmunization to transfused PLTs was developed to allow in vivo assessment and characterization of CD4+ T cells specific for PLT major histocompatibility complex (MHC) alloantigen. PLTs were harvested from BALB/c mice, filter leukoreduced, and transfused into C57BL/6 recipients. PLT-specific CD4+ T-cell responses were visualized by using a T-cell receptor transgenic mouse that detects peptide from donor MHC I presented on recipient MHC II. Antibody responses were determined by indirect immunofluorescence using BALB/c donor targets., Results: C57BL/6 recipients of BALB/c leukoreduced PLT transfusions produced BALB/c antibodies, with proliferation of antigen-specific CD4+ T cells seen in the spleen but not lymph nodes or liver. Depletion of recipient CD4+ cells or splenectomy independently abrogated the alloantibody response., Conclusion: We report a novel model to study antigen-specific CD4+ T cells during alloimmunization to PLT transfusion. The presented data support a critical role for CD4+ T-cell help in the humoral response to PLT transfusion and establish the spleen as a required microenvironment for effective CD4+ T-cell priming against donor PLT-derived MHC I., (© 2011 American Association of Blood Banks.)

Background: Increased rates of red blood cell (RBC) alloimmunization in patients with sickle cell disease may be due to transfusion frequency, genetic predisposition, or immune dysregulation. To test the hypothesis that sickle cell pathophysiology influences RBC alloimmunization, we utilized two transgenic mouse models of sickle cell disease., Study Design and Methods: Transgenic sickle mice, which express human α and β(S) globin, were transfused with fresh or 14-day-stored RBCs containing the HOD (hen egg lysozyme, ovalbumin, and human Duffy(b) ) antigen; some recipients were inflamed with poly(I : C) before transfusion. Anti-HOD alloantibody responses were subsequently measured by enzyme-linked immunosorbent assay and flow crossmatch; a cohort of recipients had posttransfusion serum cytokines measured by bead array., Results: Both Berkeley and Townes homozygous (SS) and heterozygous (AS) mice had similar rates and magnitude of anti-HOD RBC alloimmunization after fresh HOD RBC transfusion compared with control animals; under no tested condition did homozygous SS recipients make higher levels of alloantibodies than control animals. Unexpectedly, homozygous SS recipients had blunted cytokine responses and lower levels of anti-HOD alloantibodies after transfusion of 14-day stored RBCs, compared with control animals., Conclusions: In sum, homozygous β(S) expression and the ensuing disease state are not alone sufficient to enhance RBC alloimmunization to transfused HOD RBCs in two distinct humanized murine models of sickle cell disease under the conditions examined. These data suggest that other factors may contribute to the high rates of RBC alloimmunization observed in humans with sickle cell disease., (© 2012 American Association of Blood Banks.)

Background: Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh-plus-stored RBCs., Study Design and Methods: Fresh, 14-day-stored or fresh plus 14-day-stored leukoreduced RBCs from HOD-transgenic donors (with RBC-specific expression of hen egg lysozyme, ovalbumin, and human Duffy(b)) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated after transfusion., Results: In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05)., Conclusions: These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform "old blood" groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units., (© 2011 American Association of Blood Banks.)

Background: Fourteen-day stored red blood cells (RBCs) containing an RBC-specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes., Study Design and Methods: HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured after transfusion., Results: Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine- or heat-treated RBCs had elevated circulating levels of keratinocyte-derived chemokine/CXCL-1, monocyte chemoattractant protein-1, and interleukin-6 after transfusion. Furthermore, phenylhydrazine- or heat-treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1- and 10.3-fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch., Conclusions: Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic., (© 2011 American Association of Blood Banks.)

In April 2010, a working group sponsored by the National Heart, Lung, and Blood Institute was assembled to identify research strategies to improve our understanding of alloimmunization caused by the transfusion of allogeneic blood components and to evaluate potential approaches to both reduce its occurrence and manage its effects. Significant sequelae of alloimmunization were discussed and identified, including difficulties in maintaining chronic transfusion of red blood cells and platelets, hemolytic disease of the newborn, neonatal alloimmune thrombocytopenia, and rejection of transplanted cells and tissues. The discussions resulted in a consensus that identified key areas of future research and developmental areas, including genetic and epigenetic recipient factors that regulate alloimmunization, biochemical specifics of transfused products that affect alloimmunization, and novel technologies for high-throughput genotyping to facilitate extensive and efficient antigen matching between donor and recipient. Additional areas of importance included analysis of unappreciated medical sequelae of alloimmunization, such as cellular immunity and its effect upon transplant and autoimmunity. In addition, support for research infrastructure was discussed, with an emphasis on encouraging collaboration and synergy of animal models biology and human clinical research. Finally, training future investigators was identified as an area of importance. In aggregate, this communication provides a synopsis of the opinions of the working group on the above issues and presents both a list of suggested priorities and the rationale for the topics of focus. The areas of research identified in this report represent potential fertile ground for the medical advancement of preventing and managing alloimmunization in its different forms and mitigating the clinical problems it presents to multiple patient populations., (© 2011 American Association of Blood Banks.)

Background: Red blood cell (RBC) alloimmunization can be a serious complication of blood transfusion, but factors influencing the development of alloantibodies are only partially understood. Within FDA-approved time limits, RBCs are generally transfused without regard to length of storage. However, recent studies have raised concerns that RBCs stored for more than 14 days have altered biologic properties that may affect medical outcomes. To test the hypothesis that storage time alters RBC immunogenicity, we utilized a murine model of RBC storage and alloimmunization., Study Design and Methods: Blood from transgenic HOD donor mice, which express a model antigen (hen egg lysozyme [HEL]) specifically on RBCs, was filter leukoreduced and stored for 14 days under conditions similar to those used for human RBCs. Fresh or 14-day-stored RBCs were transfused into wild-type recipients. The stability of the HOD antigen and posttransfusion RBC survival were analyzed by flow cytometry. RBC alloimmunization was monitored by measuring circulating anti-HEL immunoglobulin levels., Results: Transfusion of 14-day-stored, leukoreduced HOD RBCs resulted in 10- to 100-fold higher levels of anti-HEL alloantibodies as detected by enzyme-linked immunosorbent assay than transfusion of freshly collected, leukoreduced RBCs. RBC expression of the HOD antigen was stable during storage., Conclusions: These findings demonstrate that HOD murine RBCs become more immunogenic with storage and generate the rationale for clinical trials to test if the same phenomenon is observed in humans. Length of storage of RBCs may represent a previously unappreciated variable in whether or not a transfusion recipient becomes alloimmunized.

Background: Several differences exist between antigens on transfused red blood cells (RBCs) and other immunogens, including anatomical compartmentalization. Whereas antigens from microbial pathogens and solid organ transplants drain into local lymph nodes, circulating RBCs remain segregated in the peripheral circulation, where they are consumed by antigen-presenting cells (APCs) in the spleen and liver. Accordingly, it was hypothesized that the splenic APCs play a central role in primary alloimmunization to transfused RBCs., Study Design and Methods: Recipient mice were splenectomized and transfused with transgenic RBCs expressing the membrane-bound hen egg lysozyme (mHEL) model RBC antigen. In some experiments, mHEL-specific CD4+ T cells were adoptively transferred into recipient mice to allow investigation of helper T-cell responses. Unmanipulated or sham-splenectomized mice served as controls. Recombinant murine cytomegalovirus expressing mHEL (mHEL-MCMV) was used as a control non-RBC immunogen. Humoral responses were measured by mHEL-specific enzyme-linked immunosorbent assay and flow cytometric–based RBC cross-match., Results: Control animals synthesized detectable anti-HEL immunoglobulin (Ig)G after a single mHEL RBC transfusion. mHEL-specific CD4+ T cells underwent robust expansion, and adoptive transfer of CD4+ T cells resulted in a 1000-fold increase in anti-HEL IgG. In contrast, minimal anti-HEL IgG was detectable in splenectomized mice, mHEL-specific CD4+ T cells did not proliferate, and adoptive transfer did not increase anti-HEL IgG. However, anti-HEL IgG response after exposure to mHEL-MCMV was equivalent in control and splenectomized mice., Discussion: Together, these findings illustrate the distinct properties of transfused RBCs as immunologic stimuli, with the spleen playing a critical role in primary RBC alloimmunization at the level of CD4+ T-cell activation.

Background: Storage of red blood cells (RBCs) is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in posttransfusion survival. A mouse model of RBC storage and transfusion was developed to assess survival kinetics of mouse RBCs., Study Design and Methods: RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and chromium-51 labeling. Phosphatidylserine externalization and CD47 expression was also evaluated., Results: Mean 24-hour survivals of transfused RBCs were 99, 91, 64, 54, 30, and 18% after 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression were also observed., Conclusions: Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiologic consequences of transfusing stored RBCs.