Search

Prior to transfusion of red blood cells (RBCs), recipients must be tested for the presence of alloantibodies to avoid immune complications. Liquid-preserved reagent RBCs with known blood group antigen phenotypes are used for testing. However, these reagents have practical constraints, including limited shelf-life and require constant refrigeration. To address these issues, we explore the effects of rapid freeze-drying conditions with trehalose cryoprotectant (0.1-1 M concentrations) on human RBCs and storage of freeze-dried RBCs (FDRBCs) at room temperature (RT) for up to 12 months. We report that rapid freeze-drying of RBCs for 2.5 hr with 0.5 M trehalose achieves recoverable cells with near-normal morphological shape, although size-reduced. The FDRBCs are metabolically active and functional in antibody-agglutination tests by the column agglutination test (CAT) for ABO and Rhesus-D blood group antigens. Expression of the Duffy blood group protein (CD234) decreases by 50% after freeze-drying RBCs. The initial recovery rate is ≤25%; however, 43% of these FDRBCs are still recoverable after RT storage for 12 months. In this proof-of-principle study, we show that rapid freeze-drying can stabilize RBCs. Further refinements to improve the recovery rate and preservation of antigenic epitopes will make FDRBCs a practical alternative source of reagent RBCs for pre-transfusion alloantibody identification., (© 2021 Wiley Periodicals LLC.)

Background: The screening of umbilical cord blood samples by the Direct Antiglobulin Test (DAT) is the reference tool for the identification of maternal erythrocyte alloantibodies present in erythrocytes; however, its diagnostic usefulness is controversial., Objective: To evaluate the diagnostic validity, safety, and efficiency of the eluate testing (detection of antibody in erythrocyte eluates by the Indirect Antiglobulin Test/IAT) in cord blood samples for detection of maternal erythrocyte alloantibodies in comparison with the DAT., Materials and Methods: Evaluation study of diagnostic tests. DAT and eluate testing were performed in 306 cord blood samples from neonates born to mothers admitted at Clínica Somer in Rionegro, Colombia; then, antibodies present in the eluates were identified with erythrocyte panels. Percentage of positive results by DAT and IAT were compared with the Pearson's chi-square test and the agreement between both assays with the Cohen's kappa coefficient. The diagnostic sensitivity, specificity, safety, and efficiency of the eluate testing were calculated, taking into account the use of DAT as an imperfect reference test., Results: The DAT detected alloantibodies in 6.21% of samples and the eluate testing in 14.1 %; the strength of agreement between both tests was moderate (k = 0.56) due to 25 discrepancies. The eluate testing showed sensitivity and specificity of 98.83 % and 92.31 % respectively, and a negative predictive value of 99.9 %. The diagnostic efficiency was sufficient for detection of maternal erythrocyte alloantibodies. The antibodies identified in the erythrocyte eluates were anti-A or anti-B (79.5 %), anti-D (136%), anti-C (2,3%), and anti-Fya (2,3%)., Conclusion: The eluate testing in cord blood samples is a valid, safe, and efficient test for the diagnosis of maternal erythrocyte alloantibodies., (Copyright © 2021. Published by Elsevier Ltd.)

Antibody-mediated rejection (ABMR) at early or late post-transplantation remains challenging. We performed a single-center single-arm study where four cases of acute ABMR and nine cases of chronic active ABMR (defined by Banff classification) were treated with double-filtration plasmapheresis (two cycles of three consecutive daily sessions with a 4-day gap between). At the end of the third and sixth DFPP sessions, the patients received rituximab 375 mg/m 2 . After a median follow-up of 1078 (61-1676) days, kidney-allograft survival was 50%. Before DFPP/rituximab therapy, the median donor-specific alloantibody (DSA) mean fluorescence intensity (MFI) was 9160 (4000-15 400); 45 days (D45) later it had significantly decreased to 7375 (215-18 100) (P = .018). In addition, at one-year (Y1) post-therapy, MFI had decreased further, that is, 4060 (400-7850) (P = .001). In two patients, DSA MFIs decreased and remained below 2000. The slope of estimated glomerular-filtration rate within the 6 months preceding intervention was -1.18 mL/min/month and remained unchanged at -1.29 mL/min/month within the year after intervention. Proteinuria remained unchanged. Baseline Banff scores on repeat allograft biopsies (post-therapy D45, Y1) did not show any improvement. Side-effects were mild to moderate. We conclude that the combined DFPP/rituximab significantly decreased DSAs in ABMR kidney-transplant recipients but did not improve renal function or renal histology at 1-year follow-up., (© 2021 Wiley Periodicals LLC.)

Hemophilia A (HA) is an inherited bleeding disorder which requires continuous replacement with factor (F) VIII concentrate. The main complication of HA is the development of neutralizing alloantibodies which inhibit FVIII activity (inhibitors). The objective of this study was to investigate the effect of the first FVIII infusions on immunological biomarkers in previously untreated patients with HA. Plasma samples were collected at enrollment before any FVIII infusion (T0) and at inhibitor development (INB +/T1) or up to 35 exposure days without inhibitors (INB -/T1). Anti-FVIII antibodies (immunoglobulin M, immunoglobulin G [IgG] 1, IgG3, and IgG4), chemokines (CCL2, CCL5, CXCL8, CXCL9, and CXCL10), and cytokines (interleukin [IL]-2, IL-4, IL-6, IL-10, interferon-γ, tumor necrosis factor, and IL-17) were assessed. A total of 71 children with severe HA were included, of whom 28 (39.4%) developed inhibitors. Plasma levels of anti-FVIII IgG4, IL-6, and CXCL8 were higher at INB +/T1 when compared with INB -/T1. This group presented a mixed cytokine profile and higher plasma levels of CXCL9 and CXL10 when compared with INB +/T1. We conclude that exposure to FVIII triggers a proinflammatory response mediated by IL-6 and CXCL8 in patients with HA who developed inhibitors. Regardless of inhibitor status, the immune system of all HA patients is stimulated after infusions of FVIII., Competing Interests: M.H.C. reports grants and personal fees from Novo Nordisk, Takeda, Biomarin, Pfizer, CSL Behring, and Roche. The other authors do not report conflicts of interest., (Thieme. All rights reserved.)

Background: Alloimmunization against the high-frequency Vel blood group antigen may result in transfusion reactions or hemolytic disease of fetus and newborn. Patients with anti-Vel alloantibodies require Vel-negative blood but Vel-negative individuals are rare (1:4000). Identification of Vel-negative donors ensures availability of Vel-negative blood; however, accurate Vel blood group typing is difficult due to variable Vel antigen expression and limited availability of anti-Vel typing sera. We report the production of a recombinant anti-Vel that also identifies weak Vel expression., Study Design and Methods: A recombinant anti-Vel monoclonal antibody was produced by cloning the variable regions from an anti-Vel-specific B cell isolated from an alloimmunized patient into a vector harboring the constant regions of immunoglobulin (Ig)G1-kappa or IgM-kappa. Antibody Vel specificity was tested by reactivity to SMIM1-transfected HEK293T cells and by testing various red blood cells (RBCs) of donors with normal, weak, or no Vel expression. High-throughput donor screening applicability was tested using an automated blood group analyzer., Results: A Vel-specific IgM class antibody was produced. The antibody was able to distinguish between Vel-negative and very weak Vel antigen-expressing RBCs by direct agglutination and in high-throughput settings using a fully automated blood group analyzer and performed better than currently used human anti-Vel sera. High-throughput screening of 13,288 blood donations identified three new Vel-negative donors., Conclusion: We generated a directly agglutinating recombinant anti-Vel IgM, M3F5S-IgM, functional in manual, automated agglutination assays and flow cytometry settings. This IgM anti-Vel will improve diagnostics by facilitating the identification of Vel-negative blood donors., (© 2019 AABB.)

Our understanding of the conformational and electrostatic determinants that underlie targeting of human leukocyte antigens (HLA) by anti-HLA alloantibodies is principally based upon in silico modelling. Here we provide a biochemical/biophysical and functional characterization of a human monoclonal alloantibody specific for a common HLA type, HLA-A*11:01. We present a 2.4 Å resolution map of the binding interface of this antibody on HLA-A*11:01 and compare the structural determinants with those utilized by T-cell receptor (TCR), killer-cell immunoglobulin-like receptor (KIR) and CD8 on the same molecule. These data provide a mechanistic insight into the paratope-epitope relationship between an alloantibody and its target HLA molecule in a biological context where other immune receptors are concomitantly engaged. This has important implications for our interpretation of serologic binding patterns of anti-HLA antibodies in sensitized individuals and thus, for the biology of human alloresponses.

Integrin α/β heterodimer adopts a compact bent conformation in the resting state, and upon activation undergoes a large-scale conformational rearrangement. During the inside-out activation, signals impinging on the cytoplasmic tail of β subunit induce the α/β separation at the transmembrane and cytoplasmic domains, leading to the extended conformation of the ectodomain with the separated leg and the opening headpiece that is required for the high-affinity ligand binding. It remains enigmatic which integrin subunit drives the bent-to-extended conformational rearrangement in the inside-out activation. The β 3 integrins, including α IIb β 3 and α V β 3 , are the prototypes for understanding integrin structural regulation. The Leu33Pro polymorphism located at the β 3 PSI domain defines the human platelet-specific alloantigen (HPA) 1a/b, which provokes the alloimmune response leading to clinically important bleeding disorders. Some, but not all, anti-HPA-1a alloantibodies can distinguish the α IIb β 3 from α V β 3 and affect their functions with unknown mechanisms. Here we designed a single-chain β 3 subunit that mimics a separation of α/β heterodimer on inside-out activation. Our crystallographic and functional studies show that the single-chain β 3 integrin folds into a bent conformation in solution but spontaneously extends on the cell surface. This demonstrates that the β 3 subunit autonomously drives the membrane-dependent conformational rearrangement during integrin activation. Using the single-chain β 3 integrin, we identified the conformation-dependent property of anti-HPA-1a alloantibodies, which enables them to differently recognize the β 3 in the bent state vs. the extended state and in the complex with α IIb vs. α V This study provides deeper understandings of integrin conformational activation on the cell surface., Competing Interests: The authors declare no conflict of interest.

Background: Rhesus (Rh) D antigen is the most important antigen in the Rh blood group system because of its strong immunogenicity. When RhD-negative individuals are exposed to RhD-positive blood, they may produce anti-D alloantibody, potentially resulting in delayed haemolytic transfusion reactions and Rh haemolytic disease of the foetus and newborn, which are difficult to treat. Inhibition of the binding of anti-D antibody with RhD antigens on the surface of red blood cells may effectively prevent immune haemolytic diseases., Materials and Methods: In this study, single-stranded (ss) DNA aptamers, specifically binding to anti-D antibodies, were selected via systematic evolution of ligands by exponential enrichment (SELEX) technology. After 14 rounds of selection, the purified ssDNA was sequenced using a Personal Genome Machine system. Haemagglutination inhibition assays were performed to screen aptamers for biological activity in terms of blocking antigen-antibody reactions: the affinity and specificity of the aptamers were also determined., Results: In addition to high specificity, the aptamers which were selected showed high affinity for anti-D antibodies with dissociation constant (K d ) values ranging from 51.46±14.90 to 543.30±92.59 nM. By the combined use of specific ssDNA aptamer 7 and auxiliary ssDNA aptamer 2, anti-D could be effectively neutralised at low concentrations of the aptamers., Discussion: Our results demonstrate that ssDNA aptamers may be a novel, promising strategy for the treatment of delayed haemolytic transfusion reactions and Rh haemolytic disease of the foetus and newborn.

Background: Single antigen beads (SAB) are used for monitoring HLA antibodies in pretransplant and posttransplant patients despite the discrepancy between virtual and actual crossmatch results and transplant outcomes. This discrepancy can be attributed to the presence of conformational variants of HLA-I on SAB, assessment of which would increase the concordance between SAB and flow cytometry crossmatch (FCXM) results, thus enabling improved organ accessibility for the waiting list patients and a better prediction of antibody-mediated rejection., Methods: The conformational variants were examined on HLA-I beads, iBeads, acid-/alkali-treated beads, and T cells using HLA-I monoclonal antibodies (W6/32, TFL-006, and heavy chain (HC)-10)., Results: The affinity of the monoclonal antibodies against HLA-I beads confirmed the presence and heterogeneous density of peptide-associated β2-microglobulin-associated HLA HC (pepA-β2aHC), peptide-free-β2aHC (pepF-β2aHC), and β2-free HC (β2fHC) on every single antigen-coated bead. In contrast, iBeads harbor a high density of pepA-β2aHC, low density of pepF-β2aHC, and are lacking β2fHC. The FCXM analyses confirmed the prevalence of pepA-β2aHC, but not pepF-β2aHC or β2fHC on resting T cells., Conclusions: The strength of a donor-specific antibody should be assessed with a bead-specific mean fluorescence intensity cutoff based on TFL-006 reactivity against HLA-I beads, and HC-10 against iBeads, where the β2fHC or pepF-β2aHC normalized donor-specific antibody level would reveal the true anti-pepA-β2aHC reactivity associated with positive FCXM.

Background: DSA are associated with reduced long-term transplant function and increased prevalence of chronic rejection in some patients, whereas others do not: our goal was to determine whether the sialylation of IgG and DSA could help to explain in these last cases their "non-aggressive" and/or "protective" biological activity., Methods: The sialylation level of total IgG in blood from two groups of kidney-transplant patients with de novo DSA, one with an AMR (DSA + AMR + ), and the other without were studied., Results: In the DSA + AMR - patients total IgG were more sialylated at time of transplant, and at the first detection of DSA, class I DSA were 2.6-fold more sialylated (mean 9.943±1.801 versus 3.898±2.475, p=0.058); DSA + AMR + patients exhibited higher levels of class II DSA., Conclusions: In our study, higher levels of sialylated IgG are detectable on day of transplant in patients who do not develop AMR, they have higher sialylated class I DSA at the initial detection of DSA, whereas class II DSA are significantly higher in patients who develop AMR. This is the first report suggesting that transplant outcome, and particularly AMR, is associated with levels of sialylated IgG antibodies. Our data suggest that DSA are functionally heterogeneous and that further studies with an enlarged cohort may improve our understanding of their clinical impact., (Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Fetal or neonatal alloimmune thrombocytopenia (FNAIT) is a potentially life-threatening disease where fetal platelets are destroyed by maternal anti-platelet IgG alloantibodies. The clinical outcome varies from asymptomatic, to petechiae or intracranial haemorrhage, but no marker has shown reliable correlation with severity, making screening for FNAIT impractical and highly inefficient. We recently found IgG Fc-glycosylation towards platelet and red blood cell antigens to be skewed towards decreased fucosylation, increased galactosylation and sialylation. The lowered core-fucosylation increases the affinity of the pathogenic antibodies to FcγRIIIa and FcγRIIIb, and hence platelet destruction. Here we analysed the N-linked glycans of human platelet antigen (HPA)-1a specific IgG1 with mass spectrometry in large series of FNAIT cases (n = 166) including longitudinal samples (n = 26). Besides a significant decrease in Fc-fucosylation after the first pregnancy (P = 0·0124), Fc-glycosylation levels remained stable during and after pregnancy and in subsequent pregnancies. Multiple logistic regression analysis identified anti-HPA-1a -fucosylation (P = 0·006) combined with galactosylation (P = 0·021) and antibody level (P = 0·038) correlated with bleeding severity, making these parameters a feasible marker in screening for severe cases of FNAIT., (© 2016 John Wiley & Sons Ltd.)

The development of inhibitors, i.e. neutralizing alloantibodies against factor (F) VIII or FIX, is the most significant complication of protein replacement therapy for patients with hemophilia, and is associated with both increased mortality and substantial physical, psychosocial and financial morbidity. Current management, including bypassing agents to treat and prevent bleeding, and immune tolerance induction for inhibitor eradication, is suboptimal for many patients. Fortunately, there are several emerging gene therapy approaches aimed at addressing these unmet clinical needs of patients with hemophilia and inhibitors. Herein, we review the mounting evidence from preclinical hemophilia models that the continuous uninterrupted expression of FVIII or FIX delivered as gene therapy can bias the immune system towards tolerance induction, and even promote the eradication of pre-existing inhibitors. We also discuss several gene transfer approaches that directly target immune cells in order to promote immune tolerance. These preclinical findings also shed light on the immunologic mechanisms that underlie tolerance induction., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: Preexisting, donor-specific antibodies (DSAs) are culprits of hyperacute rejection. Donor-specific antibodies are also formed de novo, and their role in acute and chronic rejection is increasingly appreciated. However, it is difficult to assess damage inflicted exclusively by DSAs when alloreactive T cell and B cell responses coincide. We reasoned that allosensitization with "costimulation-deficient" cells should induce DSA synthesis but not naive cytotoxic T lymphocyte (CTL) precursors' priming via direct allorecognition. Accordingly, we have developed a novel model to quantify DSA-mediated cytotoxicity in vivo., Methods: C57BL/6 (H-2b) mice were sensitized with H-2 kidney epithelial cells, and a cytofluorimetric killing assay was tailored to the measurement of allocytotoxicity. We took cell/complement depletion, costimulation blockade, and serum transfer approaches to reveal the mediators of cytotoxicity. "Third-party" controls and a skin allotransplantation model were used to confirm DSAs' specificity for allo-major histocompatibility complex. We validated our experimental approach in other mouse strains primed with different allogeneic cell types, including endothelial cells. To demonstrate the usefulness of our model/method for drug efficacy testing, we examined the effect of CTLA4-Ig and rapamycin on DSA-mediated cytolysis., Results: Allosensitization of MHC-disparate mouse strains with costimulation-deficient cells led to robust cytotoxicity mediated by complement-fixing DSAs and phagocytic cells. This response was independent of CTLs, natural killer or natural killer T cells. It required CD4 T cell help, CD40 signaling and CD28-based costimulation during allosensitization and could be reversed by sustained rapamycin treatment., Conclusions: The unique model described herein should enable mechanistic studies on sensitization and effector phases of humoral alloreactivity as well as efficacy testing of future immunotherapies to prevent DSA-induced pathology.

Intravenous immunoglobulin (IVIG) is made from thousands of donors having a variety of blood groups. All of the donors being used for IVIG production, with the exception of group AB donors, have in their plasma antibodies of variable titer commonly known as isohemagglutinins or ABO antibodies. As blood groups O and A are the most commonly found in the world population, most of the plasma used in IVIG production is from donors having these blood groups, with group B and group AB donors being fewer in number. Consequently, all batches of IVIG contain antibodies that are reactive with individuals of group A, group B, and group AB. These antibodies were originally discovered by Dr Karl Landsteiner in the early 1900s and are now known to consist of immunoglobulin (Ig)M, IgG, and IgA classes. As the process for producing IVIG results in almost exclusively IgG, isohemagglutinins contained in IVIG are of this immunoglobulin class. ABO antibodies are highly clinically significant and, because of this, blood bank cross-matching is done to ensure that blood of the correct type is transfused into recipients to avoid a so-called major mismatch or major incompatibility that can cause significant morbidity and often death. Administration of IVIG, which contains ABO antibodies, is often infused into individuals who have the corresponding ABO antigens, commonly called a minor mismatch, and although not as significant as a major mismatch, the isohemagglutinins contained in the IVIG have some risk for a significant transfusion reaction due to the ABO incompatibility. Indeed, currently there is no way to match IVIG to recipients according to blood type, so when IVIG is administered to group A, B, or AB recipients, there is potential for transfusion reactions analogous to a blood transfusion mismatch. For this reason, strict guidelines have been put into place to restrict the titers of the ABO antibodies contained in IVIG. This review will provide background information about the discovery and biochemistry of the ABO antigens and discuss the various isohemagglutinins that are found in plasma of the different ABO blood types and their potential clinical significance. In addition, a brief discussion of the controversial topic of the origins of these antibodies will conclude this review., (© 2015 AABB.)

Background: High-dose intravenous immunoglobulin (IVIG) treatments are implicated in hemolytic events in some patients receiving treatment. The passive transfer of IgG anti-A and anti-B agglutinin is thought to play a role in the development of these events. The purpose of this study was to determine the prevalence of high-titer IgG anti-A and anti-B in plasma donors and investigate if there is any advantage of excluding these donors from the donor pool to limit anti-A and anti-B content in IVIG product., Study Design and Methods: IgG anti-A and anti-B levels were assessed from group O donor plasma, manufacturing IgG plasma pools, and finished IVIG product (Gammagard Liquid). Antibody level in group O donors was also assessed by sex and age for their relative contribution of antibody to the plasma pool., Results: The majority of group O donors (80%) had antibody titers of less than 1000. Of those with titers of at least 1000, theoretical estimates provide further evidence that the effects of high-titer donors are minimal. Antibody levels in plasma pools both during the manufacturing process and from the final IVIG product also support that anti-A and anti-B levels are low. In general, there were more females than males with higher antibody titer levels, with significantly more females than males with anti-A., Conclusion: Excluding donors with high anti-A and anti-B titers has minimal impact on the finished IVIG product titers due to ABO antibody neutralization and the dilution factor in the manufacturing pool., (© 2015 AABB.)

Background: Increased reporting of intravenous immunoglobulin (IVIG)-related hemolytic reactions (HRs) triggered an investigation by the German and Swiss health authorities to identify potential risk factors., Study Design and Methods: From the EudraVigilance database HRs reported between 2008 and 2013 were retrieved for seven IVIG preparations. HRs were classified as mild to moderate (hemoglobin [Hb] decline < 2 g/dL)] or severe (Hb decline > 2 g/dL) and separately analyzed for IVIG doses of less than 2 g/kg body weight and 2 g/kg body weight or more. It was assessed whether HR reporting rates correlate with the isoagglutinin content of the different preparations., Results: Of 569 HR cases retrieved, 103 cases were excluded due to insufficient data, leaving 466 for analysis. Ninety-three cases were classified as mild to moderate and 373 as severe. Approximately 80% of the severe HRs concerned patients with blood group A and only three patients with blood group O. Testing of isoagglutinin titers revealed substantial differences between the seven preparations. IVIG products with high anti-A/anti-B titers (≥32) had elevated HR reporting rates, particularly when cumulative doses at least 2 g/kg were administered., Conclusion: The isoagglutinin content of IVIGs correlates with the risk for HRs. Exclusion of high-titer donations and manufacturing steps that deplete isoagglutinins should be considered for risk mitigation. In patients with blood groups A or AB receiving doses of at least 2 g/kg, the use of IVIG batches with low isoagglutinin titers should be considered to prevent HRs., (© 2015 AABB.)

Background: The mechanism of action of anti-D in ameliorating immune thrombocytopenia (ITP) remains unclear. The monoclonal antibody (MoAb) Ter119, which targets murine red blood cells (RBCs), has been shown to mimic the effect of anti-D in improving antibody-mediated murine ITP. The mechanism of Ter119-mediated ITP amelioration, especially the role of the antigen-binding and Fc domains, remains untested. A functional Fc domain is crucial for many therapeutic MoAb activity; therefore, the requirement of Ter119 Fc domain in ITP amelioration is investigated using outbred CD-1 mice., Study Design and Methods: Ter119 variants, including Ter119 F(ab')2 fragments, deglycosylated Ter119, and afucosylated Ter119, were generated to test their effect in ameliorating antibody-induced murine ITP. In vivo inhibition of FcγRIII and FcγRIIB was achieved using the Fab fragment of the FcγRIII/FcγRIIB-specific MoAb 2.4G2., Results: Ter119 F(ab')2 fragments and deglycosylated Ter119 were unable to ameliorate murine ITP or mediate phagocytosis of RBCs by RAW264.7 macrophages in vitro. Inhibition of FcγRIII and FcγRIIB, as well as Ter119 defucosylation, do not affect Ter119-mediated ITP amelioration., Conclusion: The Fc domain of Ter119, as well as its Fc glycosylation, is required for Ter119-mediated ITP amelioration. Moreover, both Fc and Fc glycosylation are required for Ter119-mediated phagocytosis in vitro. These findings demonstrate the importance of the Fc domain in a therapeutic MoAb with anti-D-like activity., (© 2015 AABB.)

Background: RhIG is obtained from hyperimmunized healthy anti-D donors (HIDs) boosted with D+ red blood cells (RBCs). One hypothesis for its mechanism of action is fast clearance of opsonized D+ RBCs through Fcγ receptor (FcγR)III. Levels of immunoglobulin (Ig)G Fc-fucosylation influence interactions with FcγRIII, with less Fc-fucosylation strengthening the interaction., Study Design and Methods: Anti-D IgG1 Fc-glycosylation patterns in 93 plasma samples from 28 male and 28 female Dutch HIDs and RhIG were analyzed with mass spectrometry. The Fc-glycosylation profiles of HIDs were evaluated with regard to their immunization history., Results: HID sera demonstrated clearly lowered anti-D Fc-fucosylation compared to normal IgG fucosylation (93%); this was more pronounced for female than for male HIDs (47% vs. 65%, p = 0.001). RhIG preparations from seven manufacturers varied greatly in the level of Fc-fucosylation (56%-91%). The level of fucosylation slightly increased upon repeated immunization, although it remained fairly constant over time. The RhIG from the different manufacturers all demonstrated increased Fc-galactosylation (64%-82%) compared to total IgG (38%-51%)., Conclusion: RhIG preparations vary in Fc-fucosylation and all demonstrate increased galactosylation. Despite not knowing the exact working mechanism, immunoprophylaxis could perhaps be optimized by selection of donors whose anti-D have low amounts of Fc-fucose, to increase the clearance activity of anti-D preparations, as well as high amounts of galactosylation, for anti-inflammatory effects. Implementing a biologic assay in the standardization of RhIG preparations might be considered., (© 2014 AABB.)

Background: Pooling of plasma of different blood groups before large scale manufacturing of Uniplas(®) results in the formation of low levels of soluble immune complexes (CIC). The aim of this study was to investigate the level and removal of CIC during Uniplas(®) manufacturing. In addition, an in vitro hemolysis assay should be developed and investigate if Uniplas(®) does induce complement-mediated hemolysis of human red blood cells (RBC)., Materials and Methods: In-process samples from Uniplas(®) (universal plasma) and Octaplas(LG)(®) (blood group specific plasma) routine manufacturing batches were tested on CIC using commercially available ELISA test kits. In addition, CIC was produced by admixing heat-aggregated immunoglobulins or monoclonal anti-A/anti-B antibodies to plasma and removal of CIC was followed in studies of the Uniplas(®) manufacturing process under down-scale conditions. The extent of RBC lysis was investigated in plasma samples using the in-house hemolysis assay., Results: Levels of CIC in Uniplas(®) are within the normal ranges for plasma and comparable to that found in Octaplas(LG)(®). Down-scale experiments showed that both IgG/IgM-CIC levels are significantly removed on average by 40-50% during Uniplas(®) manufacturing. Uniplas(®) does not induce hemolysis of RBCs in vitro. Hemolysis occurs only after spiking with high titers of anti-A/anti-B antibodies and depends on the antibody specificity (i.e. titer) in the plasma sample., Conclusion: The results of this study confirm the safety of Uniplas(®) regarding transfusion to patients of all ABO blood groups., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A common lament is that long-term kidney transplant outcomes remain the same despite improvements in early graft survival. To be fair, progress has been made-in both our understanding of chronic injury and modestly, graft survival. However, we are still a long way from actually solving this important and difficult problem. In this review, we outline recent data supporting the existence of several causes of renal allograft loss, the incidences of which peak at different time points after transplantation. On the basis of this broadened concept of chronic renal allograft injury, we examine the challenges of clinical trial design in long-term studies, including the use of surrogate end points and biomarkers. Finally, we suggest a path forward that, ultimately, may improve long-term renal allograft survival., (Copyright © 2015 by the American Society of Nephrology.)

Replacement therapy is currently used to prevent and treat bleeding episodes in coagulation factor deficiencies. However, structural differences between the endogenous and therapeutic proteins might increase the risk for immune complications. This study was aimed at identifying factor (F)VII variants resistant to inhibitory antibodies developed after treatment with recombinant activated factor VII (rFVIIa) in a FVII-deficient patient homozygous for the p.A354V-p.P464Hfs mutation, which predicts trace levels of an elongated FVII variant in plasma. We performed fluorescent bead-based binding, ELISA-based competition as well as fluorogenic functional (activated FX and thrombin generation) assays in plasma and with recombinant proteins. We found that antibodies displayed higher affinity for the active than for the zymogen FVII (half-maximal binding at 0.54 ± 0.04 and 0.78 ± 0.07 BU/ml, respectively), and inhibited the coagulation initiation phase with a second-order kinetics. Isotypic analysis showed a polyclonal response with a large predominance of IgG1. We hypothesised that structural differences in the carboxyl-terminus between the inherited FVII and the therapeutic molecules contributed to the immune response. Intriguingly, a naturally-occurring, poorly secreted and 5-residue truncated FVII (FVII-462X) escaped inhibition. Among a series of truncated rFVII molecules, we identified a well-secreted and catalytically competent variant (rFVII-464X) with reduced binding to antibodies (half-maximal binding at 0.198 ± 0.003 BU/ml) as compared to the rFVII-wt (0.032 ± 0.002 BU/ml), which led to a 40-time reduced inhibition in activated FX generation assays. Taken together our results provide a paradigmatic example of mutation-related inhibitory antibodies, strongly support the FVII carboxyl-terminus as their main target and identify inhibitor-resistant FVII variants.

The International Registry of Antibody-Defined HLA Epitopes (http://www.epregistry.com.br) has been recently established as a tool to understand humoral responses to HLA mismatches. These epitopes can be structurally defined as eplets by three-dimensional molecular modeling and amino acid sequence differences between HLA antigens. A major goal is to identify HLA eplets that have been verified experimentally with informative antibodies. This report addresses class II epitopes encoded by genes in the HLA-D region. Our analysis included reviews of many publications about epitope specificity of class II reactive human and murine monoclonal antibodies and informative alloantibodies from HLA sensitized patients as well as our own antibody testing results. As of July 1, 2014, 24 HLA-DRB1/3/4/5, 15 DQB, 3 DQA and 8 DPB antibody-verified epitopes have been identified and recorded. The Registry is still a work-in-progress and will become a useful resource for HLA professionals interested in histocompatibility testing at the epitope level and investigating antibody responses to HLA mismatches in transplant patients., (Copyright © 2014. Published by Elsevier Inc.)

Several high-throughput multiplex blood group molecular typing platforms have been developed to predict blood group antigen phenotypes. These molecular systems support extended donor/patient matching by detecting commonly encountered blood group polymorphisms as well as rare alleles that determine the expression of blood group antigens. Extended molecular typing of a large number of blood donors by high-throughput platforms can increase the likelihood of identifying donor red blood cells that match those of recipients. This is especially important in the management of multiply-transfused patients who may have developed several alloantibodies. Nevertheless, current molecular techniques have limitations. For example, they detect only predefined genetic variants. In contrast, target enrichment next-generation sequencing (NGS) is an emerging technology that provides comprehensive sequence information, focusing on specified genomic regions. Target enrichment NGS is able to assess genetic variations that cannot be achieved by traditional Sanger sequencing or other genotyping platforms. Target enrichment NGS has been used to detect both known and de novo genetic polymorphisms, including single-nucleotide polymorphisms, indels (insertions/deletions), and structural variations. This review discusses the methodology, advantages, and limitations of the current blood group genotyping techniques and describes various target enrichment NGS approaches that can be used to develop an extended blood group genotyping assay system., (Published by Elsevier Inc.)

Hemolytic transfusion reactions due to red blood cell (RBC) alloantibodies are a leading cause of transfusion-associated death. In addition to reported deaths, RBC alloantibodies also cause significant morbidity in the form of delayed hemolytic transfusion reactions. These alloantibodies may also cause morbidity in the form of anemia, with compatible RBC units at times being unable to be located for highly alloimmunized patients, or in the form of hemolytic disease of the newborn. Thus, preventing RBC alloantibodies from developing in the first place, or mitigating the dangers of existing RBC alloantibodies, would decrease transfusion-associated morbidity and mortality. A number of human studies have evaluated the impact on RBC alloimmunization rates of providing partially phenotypically or genotypically matched RBCs for transfusion, and a number of animal studies have evaluated the impact of single variables on RBC alloimmunization. The goal of this review is to take a comprehensive look at existing human and animal data on RBC alloimmunization, focusing on strategies that may mitigate this serious hazard of transfusion. Potential factors that impact initial RBC alloimmunization, on both the donor and recipient sides, will be discussed. These factors include, but are not limited to, exposure to the antigen and an ability of the recipient's immune system to present that antigen. Beyond these basic factors, coexisting "danger signals," which may come from the donor unit itself or which may be present in the recipient, also likely play a role in determining which transfusion recipients may become alloimmunized after RBC antigen exposure. In addition, to better understanding factors that influence the development of RBC alloantibodies, this review will also briefly discuss strategies to decrease the dangers of existing RBC alloantibodies., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Alloantibody can be a major barrier to successful organ transplantation; however, therapy to control antibody production or to alter its impact on the allograft remains limited. The goal of this review is to examine the regulatory steps that are involved in the generation of alloreactive B cells, with a specific emphasis on how known mechanisms relate to clinical situations in transplant recipients. Thus, we will examine the process of activation of mature, naïve B cells and how this relates to de novo antibody production. The role of long-lived plasma cells in persistent antibody production and the factors regulating their longevity will be explored. The regulation of memory B cells and their possible roles in alloimmunity also will be assessed. Finally, we will review current therapeutic approaches aimed at controlling alloantibody and assess their efficacy. By examining the pathways to antibody production mechanistically, we hope to identify important gaps in our current knowledge and gain insight into possible new therapeutic approaches to overcoming antibody in transplant patients.

There has been increasing interest in the role played by B cells and their associated antibody in the immune response to an allograft, driven by the need to undertake antibody-incompatible transplantation and evidence suggesting that B cells play a role in acute T-cell-mediated rejection and in acute and chronic antibody-mediated rejection. This review focuses on the molecular events, both activating and inhibitory, which control B-cell activation, and considers how this information might inform therapeutic strategies. Potential targets include the BAFF (B-cell-activating factor belonging to the tumour necrosis factor family) and CD40-CD40L pathways and inhibitory molecules, such as CD22 and FcγRIIB. B cells can also play an immunomodulatory role via interleukin (IL)10 production and may contribute to transplant tolerance. The expansion of allograft-specific IL10-producing B cells may be an additional therapeutic goal. Thus, the treatment paradigm required in transplantation has shifted from that of simple B-cell depletion, to that of a more subtle, differential manipulation of different B-cell subsets., (© 2013 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

We analyzed 60 patients with idiopathic early allograft loss (defined as death or retransplantation at <90 days) to determine the relative contribution of preformed donor-specific human leukocyte antigen alloantibodies (DSAs) to this endpoint, and we defined strict criteria for the diagnosis of antibody-mediated rejection (AMR) in liver allografts. The inclusion criteria encompassed the availability of a pretransplant serum sample and both postreperfusion and follow-up tissue specimens for a blinded, retrospective re-review of histology and complement component 4d (C4d) staining. AMR was diagnosed on the basis of the presence of all 4 of the following strict criteria: (1) DSAs in serum, (2) histopathological evidence of diffuse microvascular injury/microvasculitis consistent with antibody-mediated injury, (3) diffuse C4d staining in the portal microvasculature with or without staining in the sinusoids or central veins in at least 1 sample, and (4) the exclusion of other causes of a similar type of injury. Patients thought to be experiencing definite AMR on the basis of routine histopathology alone showed the highest levels of DSA sensitization. Forty percent of patients with pretransplant DSAs with a pattern of bead saturation after serial dilutions developed AMR. Another multiparous female developed what appeared to be a strong recall response, which resulted in combined AMR and acute cellular rejection (ACR) causing graft failure. A contribution of DSAs to allograft failure could not be excluded for 3 additional patients who received marginal grafts. In conclusion, liver allograft recipients with preformed DSAs with a high mean fluorescence intensity despite dilution seem to be at risk for clinically significant allograft injury and possibly for loss from AMR, often in combination with ACR., (© 2013 American Association for the Study of Liver Diseases.)

Antibody-mediated rejection (AMR) is difficult to diagnose after ABO-compatible or ABO-identical (ABO-C) liver transplantation. To determine whether complement component 4d (C4d) immunostaining would be useful for diagnosing AMR, we compared the results of C4d immunohistochemistry for allograft biopsy samples with assays for anti-donor antibodies performed at the time of biopsy. One hundred fourteen patients with ABO-C grafts and 29 patients with ABO-incompatible (ABO-I) grafts were included. Linear C4d endothelial staining (identifiable with a 4× objective lens) or staining seen in 50% or more of the portal tracts was considered positive. Five of the 114 patients (4%) with ABO-C grafts and 15 of the 29 patients (52%) with ABO-I grafts showed C4d positivity. In the ABO-C cases, C4d positivity in late biopsy samples (≥30 days after transplantation) was associated with stage 2 or higher fibrosis (METAVIR score; P = 0.01) and with the presence of donor-specific anti-human leukocyte antigen DR antibodies (HLA-DR DSAs) with a mean fluorescence intensity > 5000 according to the Luminex single-antigen bead assay (P = 0.04). Conversely, the presence of HLA-DR DSAs was associated with the presence of stage 2 or higher fibrosis, acute cellular rejection, and C4d positivity. During the 2-year follow-up, neither C4d positivity nor HLA-DR DSAs were related to graft loss. Among ABO-I patients, C4d positivity was not associated with allograft dysfunction or fibrosis. Only 3 of the 15 C4d-positive patients (20%) showed periportal hemorrhagic edema, which could be a histological sign of AMR in ABO-I grafts, and they were the only cases associated with elevations in anti-donor A/B antibody titers. In conclusion, C4d endothelial positivity among ABO-C patients is an uncommon event that could be associated with chronic graft damage with or without clinical AMR. C4d positivity is common among ABO-I patients and may not be associated with allograft dysfunction if alloantibody titers are not elevated., (© 2013 American Association for the Study of Liver Diseases.)

In this issue of Blood, Kapur et al show that maternal human platelet-specific antigen 1a (HPA 1a)-specific antibodies causing neonatal alloimmune thrombocytopenia (NAIT) possess oligosaccharides that are deficient in "core fucose" residues and appear to be more effective than fucosylated antibodies in promoting phagocytosis of antibody-coated platelets.

Immunoglobulin G (IgG) formed during pregnancy against human platelet antigens (HPAs) of the fetus mediates fetal or neonatal alloimmune thrombocytopenia (FNAIT). Because antibody titer or isotype does not strictly correlate with disease severity, we investigated by mass spectrometry variations in the glycosylation at Asn297 in the IgG Fc because the composition of this glycan can be highly variable, affecting binding to phagocyte IgG-Fc receptors (FcγR). We found markedly decreased levels of core fucosylation of anti-HPA-1a-specific IgG1 from FNAIT patients (n = 48), but not in total serum IgG1. Antibodies with a low amount of fucose displayed higher binding affinity to FcγRIIIa and FcγRIIIb, but not to FcγRIIa, compared with antibodies with a high amount of Fc fucose. Consequently, these antibodies with a low amount of Fc fucose showed enhanced phagocytosis of platelets using FcγRIIIb(+) polymorphonuclear cells or FcγRIIIa(+) monocytes as effector cells, but not with FcγRIIIa(-) monocytes. In addition, the degree of anti-HPA-1a fucosylation correlated positively with the neonatal platelet counts in FNAIT, and negatively to the clinical disease severity. In contrast to the FNAIT patients, no changes in core fucosylation were observed for anti-HLA antibodies in refractory thrombocytopenia (post platelet transfusion), indicating that the level of fucosylation may be antigen dependent and/or related to the immune milieu defined by pregnancy.

Elution is a procedure for recovery of antibody attached to intact,immunoglobulin-coated red blood cells (RBCs) by disrupting the antigen-antibody bonds. The recovered antibody is collected in an inert diluent and is referred to as an eluate. Testing of an eluate may be desired to identify antibody(ies) coating the RBCs of patients with a positive direct antiglobulin test. Many types of elution procedures have been developed and described; however,·an acid elution is suitable for antibody recovery in most cases, such as recovery of alloantibodies and warm-reactive autoantibodies.Studies have compared methods such as xylene, chloroform, digitnin acid, dichloromethane, citric acid, and Immucor Elu-KitII (cold acid elution). The ELU-Kit II has been shown to be quick and effective at eluting a wide range of alloantibodies as well as autoantibodies without the use of hazardous chemicals or costly reagent preparation time that some methods use. It is for these reasons that the ELU-Kit II is a very popular method for the elution of immunoglobulin G (IgG) antibodies.

Classic immunohematology approaches, based on agglutination techniques, have been used in manual and automated immunohematology laboratory routines. Red blood cell (RBC) agglutination depends on intermolecular attractive forces (hydrophobic bonds, Van der Walls, electrostatic forces and hydrogen bonds) and repulsive interactions (zeta potential). The aim of this study was to measure the force involved in RBC aggregation using double optical tweezers, in normal serum, in the presence of erythrocyte antibodies and associated to agglutination potentiator solutions (Dextran, low ionic strength solution [LISS] and enzymes). The optical tweezers consisted of a neodymium:yattrium aluminium garnet (Nd:YAG) laser beam focused through a microscope equipped with a minicam, which registered the trapped cell image in a computer where they could be analyzed using a software. For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities, was determined. The median of the RBC aggregation force measured in normal serum (control) was 1 × 10(-3) (0.1-2.5) poise.cm. The samples analyzed with anti-D showed 2 × 10(-3) (1.0-4.0) poise.cm (p < 0.001). RBC diluted in potentiator solutions (Dextran 0.15%, Bromelain and LISS) in the absence of erythrocyte antibodies, did not present agglutination. High adherence was observed when RBCs were treated with papain. Results are in agreement with the imunohematological routine, in which non-specific results are not observed when using LISS, Dextran and Bromelain. Nevertheless, false positive results are frequently observed in manual and automated microplate analyzer using papain enzyme. The methodology proposed is simple and could provide specific information with the possibility of meansuration regarding RBC interaction.

c-Met, the high affinity receptor for hepatocyte growth factor (HGF), is one of the most frequently activated tyrosine kinases in many human cancers and a target for cancer therapy. However, inhibitory targeting of c-Met with antibodies has proven difficult, because most antibodies have intrinsic agonist activity. Therefore, the strategy for reducing the agonism is critical for successful development of cancer therapies based on anti-c-Met antibodies. Here we developed a mechanism-based assay method for rapid screening of anti-c-Met antibodies, involving the determination of Akt phosphorylation and c-Met degradation for agonism and efficacy, respectively. Using the method, we identified an antibody, F46, that binds to human c-Met with high affinity (Kd = 2.56 nM) and specificity, and induces the degradation of c-Met in multiple cancer cells (including MKN45, a gastric cancer cell line) with minimal activation of c-Met signaling. F46 induced c-Met internalization in both HGF-dependent and HGF-independent cells, suggesting that the degradation of c-Met results from antibody-mediated receptor internalization. Furthermore, F46 competed with HGF for binding to c-Met, resulting in the inhibition of both HGF-mediated invasion and angiogenesis. Consistently, F46 inhibited the proliferation of MKN45 cells, in which c-Met is constitutively activated in an HGF-independent manner. Xenograft analysis revealed that F46 markedly inhibits the growth of subcutaneously implanted gastric and lung tumors. These results indicate that F46, identified by a novel mechanism-based assay, induces c-Met degradation with minimal agonism, implicating a potential role of F46 in therapy of human cancers.

Large scale red blood cell (RBC) antigen genotyping of donors is currently well developed. There is scarce information, however, to select patients who might benefit from preemptive extended RBC antigen-matched transfusions. Female sex has been proposed as a risk factor for RBC alloimmunization after transfusion. To asses whether females respond differently to RBC alloantigens compared with males, we conducted a literature review on RBC alloimmunization. Clinical studies on RBC alloimmunization incidence were searched for in databases from 1950 through 2011. Studies were included when data were available to calculate the female-to-male risk ratio for alloimmunization. Based on the reported age, adult patients (>18 years) were distinguished from pediatric patients (≤18 years), and articles were analyzed according to disease categories. Thirty articles fulfilled the inclusion criteria. The Mantel-Haenszel risk ratio estimate of combined adult studies showed that women with sickle cell disease had an increased relative risk (27%) on RBC alloantibodies compared with men. Other groups showed equal alloimmunization risk in women and men. Women slightly more often than men possess RBC antibodies. This is likely explained by more exposure to immunizing events through pregnancy and/or transfusions in females with sickle cell disease. The results support the current policy implemented in many countries for Rhesus/Kell matching in patients with a hemoglobinopathy irrespective of sex. Thus, based solely on sex difference, the results do not justify recommending additional matching for women, besides preemptive K and c antigen matching for women during the (pre-) fertile age, as already applied in many European countries for the prevention of fetal morbidity., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: HNA-3 is a diallellic system located on choline transporter-like protein 2 (CTL2), defined by a polymorphism at Amino Acid 154. HNA-3a antibodies are of clinical importance in transfusion-related acute lung injury but antibody detection requires labor-intensive granulocyte isolation from HNA-typed donors and the use of techniques such as the granulocyte agglutination test or granulocyte immunofluorescence test. Also, there is no commercial test for detection of HNA-3 antibodies., Study Design and Methods: HEK293 cells were transfected to generate stable cell lines expressing CTL2 fragments (Amino Acids 55-230) and full-length membrane bound CTL2 with HNA-3a and -3b epitopes. Soluble fragments were used in enzyme-linked immunosorbent assays to detect HNA-3 antibodies. The cell lines expressing full-length proteins were trypsin treated to remove HLA antigens and frozen at -80°C. Thawed cells were then used to detect HNA-3 antibodies by flow cytometry., Results: Glycosylated and soluble CTL2 fragments were correctly recognized by 15 of 31 anti-HNA-3a sera and by both available anti-HNA-3b sera. Twenty-one anti-HLA sera reacted variably with untreated cell lines expressing full-length CTL2. After trypsin treatment of the cell lines, reactivity with HLA antisera was abrogated and all 31 anti-HNA-3a and two anti-HNA-3b sera bound to the corresponding cell line., Conclusion: Whereas soluble, glycosylated CTL2 fragments cannot be used for the detection of HNA-3 antibodies, the HEK293 cells expressing full-length CTL2 proteins were useful in the detection of HNA-3 antibodies even in the presence of HLA antibodies. Moreover, the cell lines can be stored for at least 6 months before use., (© 2011 American Association of Blood Banks.)

The identification and cloning of tumor antigen-specific T cell receptors (TCRs) and the production of the soluble form of the TCR (sTCR) contributed to the development of diagnostic and therapeutic tools for cancer. Recently, several groups have reported the development of technologies for the production of sTCRs. The native sTCR has a very low binding affinity for the antigenic peptide/MHC (p/MHC) complex. In this study, we established a technology to produce high affinity, functional sTCRs. We generated a novel sTCR-Fc fusion protein composed of the TCR V and C regions of the TCR linked to the immunoglobulin (Ig) Fc region. A Western blot analysis revealed that the molecular weight of the fusion protein was approximately 60 kDa under reducing conditions and approximately 100-200 kDa under non-reducing conditions. ELISAs using various antibodies showed that the structure of each domain of the TCR-Fc protein was intact. The TCR-Fc protein immobilized by an anti-Cβ antibody effectively bound to a p/MHC tetramer. An SPR analysis showed that the TCR-Fc protein had a low binding affinity (KD; 1.1 × 10(-5)M) to the p/MHC monomer. Interestingly, when the TCR-Fc protein was pre-incubated with an anti-Cβ antibody, its binding affinity for p/MHC increased by 5-fold (2.2 × 10(-6)M). We demonstrated a novel method for constructing a functional soluble TCR using the Ig Fc region and showed that the binding affinity of the functional sTCR-Fc was markedly increased by an anti-Cβ antibody, which is probably due to the stabilization of the Vα/Vβ region of the TCR. These findings provide new insights into the binding of sTCRs to p/MHCs and will hopefully be instrumental in establishing functional sTCR as a diagnostic and therapeutic tool for cancer., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During pregnancy, women are tolerant of their semi-allogeneic fetus whilst not being immunosuppressed and indeed readily form alloantibodies. This 'Immunological Paradox of Pregnancy' may be explained by an understanding of placental anatomy and immunology. Trophoblast cells form the interface between the fetus and maternal tissues and blood and escape allorecognition because they lack classical human leucocyte antigen (HLA) class I and II molecules. Local immunoregulation, or tolerance, in the decidua is mediated partly by HLA-G(+) extravillous trophoblasts (EVT) that invade the tissue and prevent killing by maternal natural killer cells, cytotoxic T cells and macrophages. Placental hormones orchestrate the composition and regulatory function of maternal immune cells. In contrast, syncytiotrophoblast cells at the surface of chorionic villi, in contact with maternal blood, maintain a state of mild maternal systemic immunity via activation of innate immunity and skewing towards humoral immunity. This enables maintenance of a healthy immune system in pregnant women and robust protective antibody responses to pathogens whilst enabling survival of the fetus. However, this has the unfortunate consequence that pregnant women readily form alloantibodies to incompatible alloantigens on fetal red cells, platelets and leucocytes if fetomaternal haemorrhage (FMH) occurs. The antibodies are initially low affinity but after re-immunization with further FMH become functionally effective, high-titre IgG., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

Background and Objectives: Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells., Materials and Methods: We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004-2008) and period-2 (2009-2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE(™) transformed red cells (kodecytes) were used., Results: Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2., Conclusion:   Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

Neonatal alloimmune thrombocytopenia (NAIT) is caused by fetomaternal platelet incompatibility with maternal antibodies crossing the placenta and destroying fetal platelets. Antibodies against human platelet antigen-1a (HPA-1a) and HPA-5b are responsible for the majority of NAIT cases. We observed a suspected NAIT in a newborn with a platelet count of 25 G/l and petechial haemorrhages. Serological analysis of maternal serum revealed an immunisation against αIIbβ3 on paternal platelets only, indicating the presence of an antibody against a new rare alloantigen (Sec(a)) residing on αIIbβ3. The location of Sec(a) on αIIbβ3 was confirmed by immunoprecipitation. Nucleotide sequence analysis of paternal β3 revealed a single nucleotide exchange (G(1818)T) in exon 11 of the β3 gene (ITGB3), changing Lys(580) (wild-type) to Asn(580) (Sec(a)). Two additional members of the family Sec were typed Sec(a) positive, but none of 300 blood donors. Chinese hamster ovary cells expressing Asn(580), but not Lys(580) αIIbβ3, bound anti-Sec(a), which was corroborated by immunoprecipitation. Adhesion of transfected cells onto immobilised fibrinogen showed reduced binding of the Asn(580) variant compared to wild-type αIIbβ3. Analysis of transfected cells with anti-LIBS and PAC-1 antibody showed reduced binding when compared to the wild-type. No such effects were observed with Sec(a) positive platelets, which, however, are heterozygous for the Lys(580)Asn mutation. In this study, we describe a NAIT case caused by maternal alloimmunisation against a new antigen on αIIbβ3. Analysis with mutant transfected cells showed that the Lys(580)Asn mutation responsible for the formation of the Sec(a) antigenic determinant affects αIIbβ3 receptor function.

The detection of donor-specific antibodies after organ transplantation might provide an incisive way to monitor allo-specific immunity and predict graft outcome. Still, the availability of new assays for these antibodies prompts us to pose some questions about results that might be observed. These questions include whether the antibodies detected in the blood are a sensitive measure of alloimmunity, whether the detected antibodies are truly specific for the donor and whether they are noxious for the graft. Here, we explain why answers to these questions might interest the basic scientist and clinician., (© 2011 John Wiley & Sons A/S.)

Background: Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown., Study Design and Methods: For epitope mapping, a library of 23 different HNA-3a (R(154)) and three HNA-3b (Q(154)) peptides covering different parts of the first extracellular loop of CTL2 (aa(55-231)) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples., Results: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R(154) peptides in the EIA nor with R(154) or Q(154) peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests., Conclusion: Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies., (© 2011 American Association of Blood Banks.)

Background: Antibodies specific for the neutrophil antigen HNA-3a cause severe, sometimes fatal transfusion-related acute lung disease (TRALI) when transfused, but it has not been possible to screen blood donors for anti-HNA-3a because using neutrophils as targets was impractical and molecular properties of the antigen were unknown. Recently it was shown that HNA-3a is carried on choline transporter-like protein-2 (CTL2) and that the HNA-3a/b phenotype is closely correlated with an R154Q amino acid polymorphism in CTL2. However, it has not been shown by direct experiment that R154 is essential for the HNA-3a epitope., Study Design and Methods: Preliminary attempts to express recombinant full-length CTL2 (R154) recognized by anti-HNA-3a were unsuccessful. We therefore tested HNA-3a-specific antibodies from donors implicated in TRALI reactions for reactivity against chemically synthesized linear and cyclic CTL2 peptides containing R154 or Q154., Results: Nine of 20 HNA-3a antibodies recognized the R154, but not the Q154 version of a cyclic 36-residue CTL2 peptide (D131-K166). However, 11 others failed to distinguish between the two versions of this peptide., Conclusion: The findings provide direct evidence that R154 in the context of CTL2 D131-K166 is necessary to create the HNA-3a epitope but, in the context of cyclic CTL2 peptide D131-K166, is sufficient to detect only about one-half of the HNA-3a-specific antibodies implicated in TRALI. It is likely that fragments of CTL2 longer than can be made on a large scale with an automated synthesizer will be needed to produce a target capable of detecting all examples of anti-HNA-3a in donated blood., (© 2011 American Association of Blood Banks.)

Interleukin-33 (IL-33) stimulates the generation of cells and cytokines characteristic of a Th2 immune response. We examined the effects of IL-33 on allografted heart tissue in a chronic cardiac rejection model, including analysis of the peripheral myeloid and lymphoid compartments. B6.C-H2bm12/KhEg hearts were transplanted into MHC class II-mismatched C57Bl/6J mice; IL-33 was administered daily. Cells from allografts and spleens were isolated for flow cytometry and cultured for cytokine production; some tissues were used for immunohistochemistry. Animals treated with IL-33 showed significantly longer allograft survival, which was associated with a distinct cytokine profile produced by graft-infiltrating cells. Proinflammatory IL-17A production was decreased with IL-33 treatment, while increased levels of IL-5, IL-10, and IL-13 were observed. After IL-33 therapy, flow cytometry showed a direct induction of CD4(+) Foxp3(+) Treg, whereas the number of B220(+) CD19(+) B cells, and circulating, as well as allograft deposited, alloantibodies was reduced. Following IL-33 treatment, a significant decrease in graft-infiltrating CD11b(high) Gr1(high) granulocytes coincided with a significant increase in CD11b(high) Gr1(intermediate) myeloid-derived suppressor cells (MDSC). In conclusion, IL-33 treatment in the setting of chronic rejection promotes the development of a Th2-type immune response that favors MDSC and Treg expansion, reduces antibody-mediated rejection (AMR), and ultimately, prolongs allograft survival., (© 2011 The Authors. Transplant International © 2011 European Society for Organ Transplantation.)

Background: Type 3 von Willebrand disease (VWD) is an autosomal recessive bleeding disorder, characterized by virtually undetectable plasma von Willebrand factor (VWF) and consequently reduced plasma factor VIII levels. Genetic mutations responsible for type 3 VWD are very heterogeneous, scattered throughout the VWF gene and show high variability among different populations., Methods: Twenty-five severe VWD patients were studied by direct sequencing of the 51 coding exons of the VWF gene. The total number of VWD type 3 families in Hungary is 24, of which 23 were investigated., Results: Fifteen novel mutations were identified in 31 alleles, five being nonsense mutations (p.Q1238X, p.Q1898X, p.Q1931X, p.S2505X and p.S2568X), four small deletions and insertions resulting in frame shifts (c.1992insC, c.3622delT, c.5315insGA and c.7333delG), one a large partial deletion (delExon1-3) of the 5'-region, four candidate missense mutations (p.C35R, p.R81G, p.C295S, p.C623T) and one a candidate splice site mutation (c.1730-10C>A). Six previously described mutations were detected in 17 alleles, including the repeatedly found c.2435delC, p.R1659X and p.R1853X. Only one patient developed alloantibodies to VWF, carrying a homozygous c.3622delT., Conclusion: We report the genetic background of the entire Hungarian type 3 VWD population. A large novel deletion, most probably due to a founder effect, seems to be unique to Hungarian type 3 VWD patients with high allele frequency. In contrast to previous reports, none of the five patients homozygous for the large partial deletion developed inhibitors to VWF. This discrepancy raises the possibility of selection bias in some of the reports., (© 2011 International Society on Thrombosis and Haemostasis.)

Purpose: To establish a cornea transplant model in a pigmented rat strain and to define the immunologic reaction toward corneal allografts, by studying the cellular and humoral immune response after keratoplasty., Methods: Full thickness penetrating keratoplasty was performed on Brown Norway (RT1n) recipients using fully major histocompatibility complex (MHC)-mismatched Piebald-Viral-Glaxo (PVG; RT1c) donors. Using multicolor flow cytometry (FACS) we quantified and compared the cellular composition of draining versus non-draining lymph nodes (LN). Furthermore, we developed an isolation method to release viable graft infiltrating lymphocytes (GIL) and subjected them to phenotypic analysis and screened serum from transplanted animals for allo-antibodies., Results: Assessing ipsi-lateral submandibular LN we find ample evidence for post surgical inflammation such as elevated absolute numbers of cluster of differentiation (CD)4+, CD8+, B-cells, and differential expression of CD134. However, we could not unequivocally identify an allo-antigen-specific immune response. FACS analysis of lymphocytes isolated from collagenase digested rejected corneas revealed the following six distinct subpopulations: MHC-2+ cells, CD4+ T-cells, CD8+ T-cells, CD161(dull) large granular lymphocytes, CD3+ CD8+ CD161(dull) natural killer (NK)-T-cells and CD161(high) CD3⁻ NK cells. At post-operation day (POD)-07 only CD161(dull) MHC-2(neg) large granular lymphocytes (LGLs) were detected in syngeneic and allo-grafts. In concordance with an increase in B-cell numbers we often detected copious amounts of allo-antibodies in serum of rejecting animals, in particular immunoglobulin (Ig) M (IgM), immunoglobulin (Ig) G1 (IgG1), and IgG2a., Conclusions: Our results demonstrate that despite its immune privileged status and low-responder characteristics of the strain combination, allogeneic corneal grafts mount a full fledged T helper1 (Th1) and Th2 response. The presence of NK-T-cells and NK-cells in rejecting corneas shows the synergy between innate and adaptive immunity during allograft destruction.

Carbohydrate-antibody interactions mediate many cellular processes and immune responses. Carbohydrates expressed on the surface of cells serve as recognition elements for particular cell types, for example, in the ABO(H) blood group system. Antibodies that recognize host-incompatible ABO(H) system antigens exist in the bloodstream of all individuals (except AB individuals), preventing blood transfusion and organ transplantation between incompatible donors and recipients. A similar barrier exists for cross-species transplantation (xenotransplantation), in particular for pig-to-human transplantation. All humans express antibodies against the major carbohydrate xenoantigen, Galalpha (1,3)Gal (alphaGal), preventing successful xenotransplantation. Although antibody binding sites are precisely organized so as to selectively bind a specific antigen, many antibodies recognize molecules other than their native antigen. A range of peptides have been identified that can mimic carbohydrates and inhibit anti-alphaGal antibodies. However, the structural basis of how the peptides achieved this was not known. Previously, we developed an in silico method which we used to investigate carbohydrate recognition by a panel of anti-alphaGal antibodies. The method involves molecular docking of carbohydrates to antibodies and uses the docked carbohydrate poses to generate maps of th antibody binding sites in terms of prevalent hydrogen bonding and van der Waals interactions. We have applied this method to investigate peptide recognition by the anti-alphaGal antibodies. It was found that the site maps of the peptides and the carbohydrates were similar, indicating that the peptides interact with the same residues as those involved in carbohydrate recognition. This study demonstrates the potential for "design by mapping" of anti-carbohydrate antibody inhibitors.

Memory T cells are known to play a key role in prevention of allograft tolerance in alloantigen-primed mice. Here, we used an adoptively transferred memory T cell model and an alloantigen-primed model to evaluate the abilities of different combinations of monoclonal antibodies (mAb) to block key signaling pathways involved in activation of effector and memory T cells. In the adoptively transferred model, the use of anti-CD134L mAb effectively prevented activation of CD4(+) memory T cells and significantly prolonged islet survival, similar to the action of anti-CD122 mAb to CD8(+) memory T cells. In the alloantigen-primed model, use of anti-CD134L and anti-CD122 mAbs in addition to co-stimulatory blockade with anti-CD154 and anti-LFA-1 prolonged secondary allograft survival and significantly reduced the proportion of memory T cells; meanwhile, this combination therapy increased the proportion of regulatory T cells (Tregs) in the spleen, inhibited lymphocyte infiltration in the graft, and suppressed alloresponse of recipient splenic T cells. However, we also detected high levels of alloantibodies in the serum which caused high levels of damage to the allogeneic spleen cells. Our results suggest that combination of four mAbs can significantly suppress the function of memory T cells and prolong allograft survival in alloantigen primed animals., (© 2010 The Authors. Journal compilation © 2010 European Society for Organ Transplantation.)