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This article discusses the use of intravenous immune globulin (IVIG) therapy as a preemptive treatment for de novo donor-specific anti-HLA antibodies (dnDSA) after lung transplantation. The development of dnDSA is common after lung transplantation and increases the risk of chronic lung allograft dysfunction (CLAD) and death. Different transplant centers have different approaches to managing asymptomatic dnDSA. This study reviews the use of high-dose IVIG monotherapy to reduce the strength of the antibodies and improve long-term outcomes. The results show that recipients who received IVIG treatment had a significant reduction in dnDSA strength and improved CLAD-free survival compared to those who did not receive treatment. Another study discussed in the article observed a trend towards a lower incidence of acute cellular rejection and antibody-mediated rejection in patients who cleared DSA after IVIG therapy. However, there was no significant difference in the incidence of CLAD or survival between those who cleared DSA and those with persistent DSA. The study suggests that preemptive therapy with IVIG may improve long-term outcomes in this high-risk group, but further research is needed to confirm its efficacy. [Extracted from the article]

Background and Objectives: This study describes the use of the Epvix platform for virtual cross‐matching (VC) of human leucocyte antigen (HLA)‐compatible platelets for patients with immune platelet refractoriness, and demonstrates effectiveness of the selected platelets. Materials and Methods: A prospective cohort of haematological patients was evaluated from 2018 to 2022. HLA‐typed donor bank profile was previously uploaded to the Epvix platform. Each patient's antibody reactivity panel (PRA) was included in the platform. Then, search, selection and VC were performed, and 24‐h‐corrected count increment (CCI) platelet transfusion was calculated (reference ≥2500). Results: Six patients were included (four female, two male), with mean age of 61 years. HLA antibodies were detected as the cause of immunity for all patients, whereas four patients also had non‐immune causes. High percentage of alloimmunization was detected in all studied patients (mean PRA: 85.7%). Thirty different donors were able to schedule and perform platelet donations. The mean 24‐h CCI count was 9882. All platelet transfusions achieved a satisfactory CCI count except for two transfusion events. Presence of non‐immune causes identified in these two cases could account for the unsatisfactory CCI. Conclusion: Epvix is a free application hosted on the Web and uses the HLAMatchmaker algorithm to generate histocompatibility reports. This study demonstrates the efficiency of VC performed by Epvix. However, physical cross‐matching will still be necessary in some instances, as the platform does not support human platelet antigen polymorphism. [ABSTRACT FROM AUTHOR]

The main reason for graft loss is the rejection of the donor organ, which may occur at different time after transplantation and may be caused by the recipient’s organism reaction against donor’s human leukocyte antigen (HLA) proteins. Donor-specific antibodies (DSA) are produced in patient’s organism as a response to foreign HLA antigens. Aim. The purpose of our study was to evaluate the effects of already existed and/or de novo generated DSAs in liver transplantation as predictors of graft rejection and to establish an interconnection between blood biochemical parameters (Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin level) with the level of DSA in patients with liver transplant. Methods. xMAP-Luminex next generation flow cytometry technology and LABScreen Single antigen beads reagent (Onelambda, USA) were used for antiHLA determination. Total bilirubin level was detected photometrically. The activity of ALT and AST was determined spectrophotometrically on the automatic analyzer COBAS C 111 (Roche, Switzerland) in accordance with the manufacture’s instruction. Results. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol. Conclusions. The DSA and PRA levels as well as total bilirubin and ALT and AST activity corresponded to each other and could be used for comprehensive both pre- and post-transplantation screening of patients requiring liver transplantation or re-transplantation. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol.

Background: Predicting whether a patient's platelet refractoriness (PR) is due to immune or nonimmune causes can be challenging. This study compared the demographics and clinical history of PR patients with human leukocyte antigen (HLA) antibodies (HLA‐PR) versus PR patients without HLA antibodies. Materials and Methods: A retrospective review of all patients with PR consults at a single institution over a 3‐year period was performed. Patient charts were reviewed for all patients with confirmed PR, and demographic information (e.g., sex, race and ethnicity, preferred language) and clinical history (e.g., pregnancy, transfusion, primary diagnosis) were collected. Patient characteristics were compared among the HLA and non‐HLA cohorts. Results: A total of 295 patients with confirmed PR were identified, of whom approximately 70% did not have HLA antibodies and 30% did. Approximately 84% of the HLA‐PR cohort was female. A history of transfusions was not associated with HLA‐PR (p =.1). A history of pregnancy was strongly associated with the occurrence of HLA‐PR (p <.001). Splenomegaly was associated with PR in the absence of HLA alloimmunization whereas infection, fever, bleeding, and disseminated intravascular coagulation were not. Conclusion: In this single‐institution retrospective review, a history of pregnancy was strongly associated with HLA‐PR, whereas a history of transfusion was not. [ABSTRACT FROM AUTHOR]

Introduction: In solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins. Materials: Allele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes. Results: For the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors.Discussion: In conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection. [ABSTRACT FROM AUTHOR]

Objective To explore the risk factors of transfusion-related acute lung injury (TRALI). Methods The clinical symptoms, signs, imaging examinations, and laboratory test results of two patients with TRALI after blood transfusion were retrospectively analyzed, and human leukocyte antigen (HLA) genotyping of the patient and HLA antibodies typing of the plasma donors were performed. Results The clinical manifestations and laboratory parameters of two patients were consistent with those of TRALI after blood transfusion. After timely clinical respiratory support treatment, all patients were improved. Blood donors produced high titers of HLA-Ⅱ antibodies after pregnancy, including antibodies that specifically recognize the patient′s HLA antigen. Conclusion Two patients developed TRALI after platelet transfusion from a female blood donor, which was caused by HLA-Ⅱ antibodies.

IntroductionWe previously reported the initial results of a phase II multicenter transplant trial using haploidentical parental donors for children and aolescents with high-risk sickle cell disease achieving excellent survival with exceptionally low rates of graft-versus-host disease and resolution of sickle cell disease symptoms. To investigate human leukocyte antigen (HLA) sensitization, graft characteristics, donor chimerism, and immune reconstitution in these recipients.MethodsCD34 cells were enriched using the CliniMACS® system with a target dose of 10 x 106 CD34+ cells/kg with a peripheral blood mononuclear cell (PBMNC) addback dose of 2x105 CD3/kg in the final product. Pre-transplant HLA antibodies were characterized. Donor chimerism was monitored 1-24 months post-transplant. Comprehensive assessment of immune reconstitution included lymphocyte subsets, plasma cytokines, complement levels, anti-viral T-cell responses, activation markers, and cytokine production. Infections were monitored.ResultsHLA antibodies were detected in 7 of 11 (64%) evaluable patients but rarely were against donor antigens. Myeloid engraftment was rapid (100%) at a median of 9 days. At 30 days, donor chimerism was 93-99% and natural killer cell levels were restored. By 60 days, CD19 B cells were normal. CD8 and CD4 T-cells levels were normal by 279 and 365 days, respectively. Activated CD4 and CD8 T-cells were elevated at 100-365 days post-transplant while naïve cells remained below baseline. Tregs were elevated at 100-270 days post-transplant, returning to baseline levels at one year. At one year, C3 and C4 levels were above baseline and CH50 levels were near baseline. At one year, cytokine levels were not significantly different from baseline.DiscussionThese results suggest that haploidentical transplantation with CD34-enriched cells and peripheral blood mononuclear cell addback results in rapid engraftment, sustained donor chimerism and broad-based immune reconstitution.

Aim. The purpose of our study was to evaluate the effects of already existed and/or de novo generated DSAs in liver transplantation as predictors of graft rejection and to establish an interconnection between blood biochemical parameters (Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin level) with the level of DSA in patients with liver transplant. Methods. Blood for testing was collected to tubes without anticoagulant, centrifugated at 1500 rpm per 10 minutes [5] and processed immediately (for biochemical parameters measurement) or stored at –70 °C (for antibody detection). xMAP-Luminex next generation flow cytometry technology and LABScreen Single antigen beads reagent (Onelambda, USA) were used for antiHLA determination. Analysis of results with further calculation of PRA percentage for antibodies to Class I & II separately was performed on HLAFusion 4.6 software. Briefly: patient’s serum was centrifugated at 10 000 rcf during 10 min, treated with 100 mM EDTA (Invitrogene, USA) and incubated with LABScreen beads, covered with HLA Class I &II antigens. Positive reaction was detected with second anti-human phycoerythrin (PE)-labelled IgG. The presence of antibodies to specific HLA antigen was determined by the numerical value of the mean fluorescence intensity (MFI). Serum was considered positive when PRA value was higher than 0% (PRA > 0%) and the mean MFI was higher than 600 (MFI ≥ 600). Total bilirubin level was detected photometrically. The activity of ALT and AST was determined spectrophotometrically on the automatic analyzer COBAS C 111 (Roche, Switzerland) in accordance with the manufacture’s instruction. Results and Discussion. The group of examined participants consisted patients diagnosed with different liver diseases (hepatic cirrhosis and biliary atresia). Patients therapy consisted of plasmapheresis (removal of blood plasma components, in particular anti-HLA antibodies), immunosuppressive drug “Tacrolimus” (decreases T-lymphocytes production), “Solu-Medrol” pulse therapy (synthetic glucocorticoid anti-inflammatory drug). It should be noted, that multiple transfusions before transplantation were associated with an increase in DSA and PRA levels, which increases the probability of the formation of DSA in the recipient and subsequent rejection of the transplanted organ (Figure, A). The use of the above drugs in various combinations depending on the patient’s medical history led to changes in levels of antibodies to HLA antigens and blood biochemical parameters. Before graft rejection we observed high values of PRA, DSA and increased level of key biochemical parameters in bloodstream, which indicated start of liver disfunction development. It should be noted, that levels of PRA and DSA grew earlier than ALT and AST activity (Figure, В) and total bilirubin concentration (Figure, С). We can speculate, that PRA increase as well as de novo generated DSA can be the reason of graft rejection, which was confirmed by the changes of key biochemical markers of liver functioning (Figure 1, B, C). We observed directly proportional dependence between biochemical blood parameters and PRA and DSA. They have the same trend of growth and decline depending on the effectiveness of the therapy. After immunosuppression therapy, DSA were not detected, blood biochemical parameters returned to normal reference values (total bilirubin = 15 μmol/L; AST = 40 IU/L; ALT = 45 IU/L), however, general anti-HLA antibodies level was still present therefore antibody monitoring should be continued to prevent the possibility of graft rejection and to adjust immunosuppression protocols. Conclusions. The DSA and PRA levels as well as total bilirubin and ALT and AST activity corresponded to each other and could be used for comprehensive both pre- and post-transplantation screening of patients requiring liver transplantation or re-transplantation. Detection of DSA and PRA was important at the same level as measurement of classical biochemical parameters of liver function (ALT, AST etc.) for monitoring of graft status and prevention of acute or chronical rejection and choosing correct immunosuppression protocol. [ABSTRACT FROM AUTHOR]

Proficiency testing (PT) surveys include data from laboratories across the world and are ideal for creating advanced educational content, beyond just consensus grading. Educational challenges provide a unique opportunity to probe common laboratory practices and risk assessment, especially in cases where there is no "analyte" tested. Human leukocyte antigen (HLA) compatibility evaluation between donor and recipient pairs has been traditionally assessed using T-cell and B-cell physical crossmatches. However, advancements in our ability to identify and characterize HLA antibodies using solid phase assays, in combination with changing deceased donor allocation schemes and improved HLA typing, have shifted the paradigm from performing physical crossmatches to the use of the virtual crossmatch (VXM). VXM is a compatibility assessment relying on the interpretation of pre-transplant HLA laboratory data and as such, it is not an "analyte". However, VXM results are used in clinical decision-making. The VXM assessment depends on patient characteristics as well as laboratory and transplant center practices but must ensure safe transplantation outcomes while maintaining equity in access to transplantation. In this manuscript, we describe the American Society for Histocompatibility and Immunogenetics (ASHI) PT Educational VXM Challenge, as a model for creating educational content using PT survey data. We discuss the different components of the VXM Challenge and highlight major findings and learning points acquired from ASHI VXM Challenges performed between 2018-2022, such as the lack of correlation between the VXM and the physical crossmatch in the presence of low level donor-specific antibodies (DSA), or when the DSA were aimed against donor alleles that are not present on the antibody panel, and in the presence of an antibody to a shared eplet. Finally, we show that the VXM Educational Challenge serves as a valuable tool to highlight the strengths and pitfalls of the VXM assessment and reveals differences in testing and result interpretation among participating HLA laboratories. [ABSTRACT FROM AUTHOR]

BackgroundCorrelation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.MethodsWe retrospectively assessed patients with transplant biopsies scored following Banff'15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.ResultsOne-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31-10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78-16.76], p = 0.003) were independent predictors of ABMRhDSApos.ConclusionsIn conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

IntroductionIn solid-organ transplantation, human leukocyte antigen (HLA) donor-specific antibodies (DSA) are strongly associated with graft rejection, graft loss, and patient death. The predominant tests used for detecting HLA DSA before and after solid-organ transplantation are HLA single antigen bead (SAB) assays. However, SAB assays may not detect antibodies directed against HLA epitopes that are not represented in the SAB. The prevalence and potential impact of unrepresented HLA epitopes are expected to vary by ethnicity, but have not been thoroughly investigated. To address this knowledge gap, HLA allele frequencies from seven ethnic populations were compared with HLA proteins present in SAB products from two manufacturers to determine unrepresented HLA proteins.MaterialsAllele frequencies were obtained from the Common, Intermediate, and Well Documented HLA catalog v3.0, and frequencies of unrepresented HLA types were calculated. Next-generation sequencing was used to determine HLA types of 60 deceased solid-organ donors, and results were used to determine if their HLA-A, -B, -C, and -DRB1 proteins were not present in SAB reagents from two vendors. Unrepresented HLA proteins were compared with the most similar protein in SAB assays from either vendor and then visualized using modeling software to assess potential HLA epitopes.ResultsFor the seven ethnic populations, 0.5% to 11.8% of each population had HLA proteins not included in SAB assays from one vendor. Non-European populations had greater numbers of unrepresented alleles. Among the deceased donors, 26.7% (16/60) had at least one unrepresented HLA-A, -B, -C, or -DRB1 protein. Structural modeling demonstrated that a subset of these had potential HLA epitopes that are solvent accessible amino acid mismatches and are likely to be accessible to B cell receptors.DiscussionIn conclusion, SAB assays cannot completely rule out the presence of HLA DSA. HLA epitopes not represented in those assays vary by ethnicity and should not be overlooked, especially in non-European populations. Allele-level HLA typing can help determine the potential for HLA antibodies that could evade detection.

BackgroundStudies prospectively monitoring de novo donor-specific antibodies (dnDSAs) and their clinical impact are sparse. This substudy of ATHENA was initiated to evaluate the effect of everolimus (EVR) or mycophenolic acid (MPA) in combination with reduced calcineurin inhibitor (CNI, tacrolimus [TAC] or cyclosporine [CsA]) on the formation of human leukocyte antibodies (HLA), including dnDSA, and the impact on clinical outcomes in kidney transplant (KTx) recipients.MethodsAll eligible patients were randomized 1:1:1 to receive either EVR + TAC, EVR + CsA or MPA + TAC, with basiliximab induction plus steroids after transplantation up to Month 12. The incidence of dnDSA by treatment group and the association with clinical events were evaluated descriptively as an exploratory objective in the intent-to-treat (ITT) and per-protocol (PP) populations with at least one antibody assessment.ResultsOverall, none of the patients in the EVR + TAC group had either dnDSA or antibody mediated rejection (PP or ITT population) and only one patient with dnDSA in the TAC + MPA group had antibody mediated rejection.ConclusionThe EVR regimen was comparable to MPA regimen with an extremely low incidence of dnDSA over 1 year of treatment.

Proficiency testing (PT) surveys include data from laboratories across the world and are ideal for creating advanced educational content, beyond just consensus grading. Educational challenges provide a unique opportunity to probe common laboratory practices and risk assessment, especially in cases where there is no “analyte” tested. Human leukocyte antigen (HLA) compatibility evaluation between donor and recipient pairs has been traditionally assessed using T-cell and B-cell physical crossmatches. However, advancements in our ability to identify and characterize HLA antibodies using solid phase assays, in combination with changing deceased donor allocation schemes and improved HLA typing, have shifted the paradigm from performing physical crossmatches to the use of the virtual crossmatch (VXM). VXM is a compatibility assessment relying on the interpretation of pre-transplant HLA laboratory data and as such, it is not an “analyte”. However, VXM results are used in clinical decision-making. The VXM assessment depends on patient characteristics as well as laboratory and transplant center practices but must ensure safe transplantation outcomes while maintaining equity in access to transplantation. In this manuscript, we describe the American Society for Histocompatibility and Immunogenetics (ASHI) PT Educational VXM Challenge, as a model for creating educational content using PT survey data. We discuss the different components of the VXM Challenge and highlight major findings and learning points acquired from ASHI VXM Challenges performed between 2018–2022, such as the lack of correlation between the VXM and the physical crossmatch in the presence of low level donor-specific antibodies (DSA), or when the DSA were aimed against donor alleles that are not present on the antibody panel, and in the presence of an antibody to a shared eplet. Finally, we show that the VXM Educational Challenge serves as a valuable tool to highlight the strengths and pitfalls of the VXM assessment and reveals differences in testing and result interpretation among participating HLA laboratories.

IntroductionWith the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies.Study designThe inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response.ResultsIn the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation.ConclusionsOur case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials.

Spherotech (SPT) microparticles capture non‐specific binding materials present in test serum, and EDTA removes the so called" prozone effect". This study presents a novel approach of combined SPT‐EDTA serum treatment prior to Luminex HLA antibody testing to remove high background, and prozone effect in a single step process, and compared the efficacy of SPT‐EDTA serum pre‐treatment with AdsorbOut (ADS) and Serum Cleaner (SC) to reduce background in solid phase immunoassays (SPI). A total of 21 serum samples with a history of elevated negative control (NC) values ≥500, and 20 samples with normal NC values were included to assess the potential adverse effects. A problem of high background was noted in 25% of our samples in SPI. We observed 80% effectiveness in reducing NC values <500 with SPT‐EDTA serum pre‐treatment compared to 72%, and 67% for ADS and SC‐treated sera, respectively. Interestingly, we found a strong correlation in antibody‐binding levels between SPT versus ADS; and ADS versus SC‐treated sera for both phenotype and single antigen bead assays (p < 0.001). No adverse effect was noted on NC, positive control (PC) values, PC/NC ratios in the upfront use of SPT‐EDTA as compared to EDTA alone. Our data revealed that combined SPT‐EDTA treated sera is more effective than ADS, and SC in reducing high background in SPI. Taken together, SPT‐EDTA serum treatment prior to Luminex HLA Ab testing is cost‐effective, our laboratory saves nearly 30% of the annual total cost for Ab testing and improved test turnaround time by two business days. [ABSTRACT FROM AUTHOR]

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It is still not fully elucidated which pretransplant donor‐specific HLA antibodies (DSA) are harmful after kidney transplantation. In particular, it needs to be clarified whether cumulative mean fluorescence intensities (MFI) against multiple HLA specificities have a predictive value for allograft function. Our retrospective single centre study analyzed preformed HLA antibodies determined by Luminex™ Single Antigen Bead (SAB) assay, including C1q addition, in relation to rejection and clinical outcome in 255 cross match negative kidney allograft recipients. Only 33 recipients (13%) of the total cohort showed early AMR during the first year posttransplant, but in patients with pre‐transplant DSA the rate was increased to 15 out of 40 (38%). Three year graft survival was significantly shorter in patients with histological signs of AMR compared with patients without AMR or with no biopsy (74%, 92%, and 97%, respectively, p < 0.0001). In patients with HLA‐DSA, a cumulative MFI value of all HLA antibodies of more than 103.000 indicated the highest risk for AMR posttransplant (p = 0.01). In conclusion, in patients with HLA‐DSA, the cumulative MFI value may help to further stratify the risk of AMR after kidney transplantation. [ABSTRACT FROM AUTHOR]

Various studies have shown that SARS-CoV-2 is a highly immunogenic virus. It is known that different types of immunogenic viral pathogens could trigger the formation of HLA antibodies. Therefore, there is a concern that the SARS-CoV-2 could also induce the development of HLA antibodies in volunteers, who donate convalescent plasma after their recovery from COVID-19. HLA antibodies have been identified as the main cause for transfusion-related acute lung injury (TRALI), a well-documented life-threatening complication of transfusions. The TRALI risk could be high in COVID-19 patients who need convalescent plasma, as such patients usually have already an impaired respiratory system affected by the SARS-CoV-2 infection. In this study, we screened 34 convalescent plasma donors on the presence of antibodies against HLA class I and II antigens. All included donors have no any history of sensitization events such as blood transfusions, pregnancy, or previous transplants. We found a high rate of HLA antibody formation in convalescent plasma donors. The frequency of positivity for HLA antibodies for class I, class II, class I and II, and the overall reactivity was 23%, 31%, 46%, and 76%, respectively. The presented data suggest a closed correlation between SARS-CoV-2 virus infection and the development of HLA antibodies in recovered convalescent plasma donors. This finding might have the potential to reduce the risk of TRALI and mortality rate in COVID-19 patients by implementing HLA diagnostic strategies before the administration of convalescent plasma.

Compromised detection of HLA specific antibodies due to complement mediated interference (CMI) is a well-recognized limitation of the single antigen bead (SAB) assay. Serum treatment with EDTA prior to SAB assay testing is a common strategy used to prevent CMI, however, treatment of individual sera, especially in large clinical runs, can extend assay turnaround time and increase the risk of a sample mix-up. In this study, we describe a simplified EDTA treatment strategy that can be applied simultaneously to all sera in a testing run. This strategy effectively prevents CMI without added pipetting steps and the increased turnaround time associated with other strategies. In the novel bead treatment (BT) method, EDTA solution and SAB suspension are combined and added into the wells of a testing tray together, patient sera are then added to the SAB/EDTA mixture. This eliminates the separate EDTA pre-treatment step used in the standard procedure (ST). Parallel testing using the BT, ST, and no EDTA treatment strategies followed by antibody identification using the Rapid Optimized SAB (ROB) protocol was performed for 19 well characterized sera with known CMI at two different laboratories. Both BT and ST methods were equally effective in preventing CMI. In addition, excellent MFI correlation was observed for specificities not affected by CMI. Both negative and pooled positive control sera performed as expected using the BT method and the pooled positive control serum, specifically designed to exhibit CMI, served well as an EDTA treatment control for all sera. The modified BT protocol can be easily implemented for clinical testing and eliminates extra pipetting steps, reduces the likelihood of pipetting error, and decreases turnaround time, while effectively preventing CMI and ensuring accurate detection of HLA antibodies. This protocol was recently implemented in the Halifax HLA Laboratory and has been very positively received by the laboratory team. [ABSTRACT FROM AUTHOR]

Summary: Background: 3% of kidney transplant recipients return to dialysis annually upon allograft failure. Development of antibodies (Ab) against human leukocyte antigens (HLA) is a validated prognostic biomarker of allograft failure. We tested whether screening for HLA Ab, combined with an intervention to improve adherence and optimization of immunosuppression could prevent allograft failure. Methods: Prospective, open-labelled randomised biomarker-based strategy (hybrid) trial in 13 UK transplant centres [EudraCT (2012-004308-36) and ISRCTN (46157828)]. Patients were randomly allocated (1:1) to unblinded or double-blinded arms and screened every 8 months. Unblinded HLA Ab+ patients were interviewed to encourage medication adherence and had tailored optimisation of Tacrolimus, Mycophenolate mofetil and Prednisolone. The primary outcome was time to graft failure in an intention to treat analysis. The trial had 80% power to detect a hazard ratio of 0.49 in donor specific antibody (DSA)+ patients. Findings: From 11/9/13 to 27/10/16, 5519 were screened for eligibility and 2037 randomised (1028 to unblinded care and 1009 to double blinded care). We identified 198 with DSA and 818 with non-DSA. Development of DSA, but not non-DSA was predictive of graft failure. HRs for graft failure in unblinded DSA+ and non-DSA+ groups were 1.54 (95% CI: 0.72 to 3.30) and 0.97 (0.54–1.74) respectively, providing no evidence of an intervention effect. Non-inferiority for the overall unblinded versus blinded comparison was not demonstrated as the upper confidence limit of the HR for graft failure exceeded 1.4 (1.02, 95% CI: 0.72 to 1.44). The only secondary endpoint reduced in the unblinded arm was biopsy-proven rejection. Interpretation: Intervention to improve adherence and optimize immunosuppression does not delay failure of renal transplants after development of DSA. Whilst DSA predicts increased risk of allograft failure, novel interventions are needed before screening can be used to direct therapy. Funding: The National Institute for Health Research Efficacy and Mechanism Evaluation programme grant (ref 11/100/34).

IntroductionWe previously reported the initial results of a phase II multicenter transplant trial using haploidentical parental donors for children and aolescents with high-risk sickle cell disease achieving excellent survival with exceptionally low rates of graft-versus-host disease and resolution of sickle cell disease symptoms. To investigate human leukocyte antigen (HLA) sensitization, graft characteristics, donor chimerism, and immune reconstitution in these recipients.MethodsCD34 cells were enriched using the CliniMACS® system with a target dose of 10 x 106 CD34+ cells/kg with a peripheral blood mononuclear cell (PBMNC) addback dose of 2x105 CD3/kg in the final product. Pre-transplant HLA antibodies were characterized. Donor chimerism was monitored 1-24 months post-transplant. Comprehensive assessment of immune reconstitution included lymphocyte subsets, plasma cytokines, complement levels, anti-viral T-cell responses, activation markers, and cytokine production. Infections were monitored.ResultsHLA antibodies were detected in 7 of 11 (64%) evaluable patients but rarely were against donor antigens. Myeloid engraftment was rapid (100%) at a median of 9 days. At 30 days, donor chimerism was 93-99% and natural killer cell levels were restored. By 60 days, CD19 B cells were normal. CD8 and CD4 T-cells levels were normal by 279 and 365 days, respectively. Activated CD4 and CD8 T-cells were elevated at 100-365 days post-transplant while naïve cells remained below baseline. Tregs were elevated at 100-270 days post-transplant, returning to baseline levels at one year. At one year, C3 and C4 levels were above baseline and CH50 levels were near baseline. At one year, cytokine levels were not significantly different from baseline.DiscussionThese results suggest that haploidentical transplantation with CD34-enriched cells and peripheral blood mononuclear cell addback results in rapid engraftment, sustained donor chimerism and broad-based immune reconstitution.

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless, detection of spurious reactivities remains a recognized assay limitation. In addition, the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique, which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed, with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly, negligible reactivity was detected in the last wash control, confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly, antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis, AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has led to significant morbidity and mortality in lung transplant recipients. Respiratory viral infections may be associated with de-novo HLA donor-specific antibody production and impact lung transplant outcome. Since one of the immunomodulation strategies post-SARS-CoV-2 infection in lung transplant recipients include decreasing or holding anti-metabolites, concerns have been raised for higher incidence of de-novo HLA donor specific antibody production in lung transplant recipients. We performed a retrospective chart review of 24 consecutive lung transplant recipients diagnosed with COVID-19 to investigate this concern. We observed no significant differences in the CPRA or MFI levels of HLA class I and II antibodies pre- COVID-19 compared to 1 and 6 months post-COVID-19 diagnosis in 11/24 (45.8 %) LTR (p = 0.98 and p = 0.63 respectively). HLA class I and II DSA were detected in 5/24 LTR pre-COVID-19 diagnosis and persisted with no significant differences in the median MFI levels at 1 and 6 months post-COVID-19 diagnosis (p = 0.89). De-novo HLA class I and II DSA were detected in 1/24 (4.2 %) LTR at one month post-COVID-19 diagnosis and persisted with no significant differences in the median MFI levels at 1 and 6 months post-COVID-19 diagnosis (p = 0.54). Our results suggest that there was no significant association between SARS-CoV-2 infection and immunomodulation on pre-existing or de novo HLA donor specific antibodies. [ABSTRACT FROM AUTHOR]

Hematopoietic Stem Cell Transplantation (HSCT) is a curative treatment for several malignant and non-malignant diseases. Access to this life saving therapy was limited by the requirement of an HLA matched donor. Introduction of platforms allowing transplantation with haploidentical and partially mismatched donors has enlarged the donor pool so that most candidates have a possible donor. HLA circulating antibodies specific for the donor's mismatched antigens may cause delayed engraftment and primary graft failure. The role of the HLA laboratory supporting HSCT has expanded to provide HLA antibody detection and monitoring for selection of compatible donors and for monitoring of desensitization treatments. This review gives an outline of the evolution of HSCT and HLA and the current tools available to the HLA team to support donor selection and desensitization treatments in this new era of transplantation. [ABSTRACT FROM AUTHOR]

Introduction: HLA antigens are highly polymorphic, and their immunogenicity is dependent on the configurations of polymorphic amino acids. Monitoring anti‐HLA immunization is essential in organ transplantation, as antibodies directed against HLA molecules are a major cause of rejection. Anti‐HLA antibodies are not specific for HLA antigens, but recognize B‐cell epitopes present on HLA molecules. Methods: To better understand antibody reactivity patterns, we calculated the Spearman correlation of the mean fluorescence intensity (MFI) of anti‐HLA antibodies identified by a single‐antigen assay performed using a Luminex® immunobeads assay on a large number of samples. Then, we built a computer tool analyzing antibody reactivity patterns with an accessibility by a web browser linked to the International Epitope Registry. We also extended our model to Onelambda® and Lifecodes® single‐antigen class I and class II assays. Results and Discussion: The resulting MFI correlations reflect HLA antibody cross‐reactivity and eplets similarity. We built HLA Graph, a computer tool that analyzes the eplets involved in antibody reactivity profiles. HLA Graph is usable with Onelambda® and Lifecodes® single‐antigen class I and class II assays. The interpretation of reactivity against alleles not tested by the antibody assays and against the alpha and beta chains of HLA‐DQ and HLA‐DP loci were also developed. Conclusion: HLA Graph is a free and ready‐to‐use bioinformatics tool that can be used by all laboratories performing anti‐HLA antibody identification by immunobead assay. [ABSTRACT FROM AUTHOR]

Human leukocyte antigen (HLA) sensitization remains an impediment to successful solid organ transplantation, whether it be chances of receiving a transplant offer or subsequent transplant longevity. Current treatments targeting HLA antibodies lack long-term effectiveness; therefore, preventing HLA sensitization should remain a priority in all potential wait-list candidates and transplant recipients. Recent advances in the management of anemia in patients with chronic kidney disease may reduce the need for red cell transfusions. However, data from several anemia intervention studies of novel therapeutic agents have shown that a need for transfusion will remain. It has also been increasingly recognized that blood transfusions following kidney transplantation, especially in the peri-operative period, are common. Routine data on transfusion incidence, indications, and outcomes are not captured by most kidney and transplant registries across the globe. This restricts the evidence to inform both clinicians and patients on the clinical effects of transfusion, which have been considered both an allogeneic stimulus and to be immunomodulatory.This review aims to provide an update on what is currently known about transfusion-induced HLA sensitization in wait-list candidates and transplant recipients, summarizes where evidence is lacking, and demonstrates the distinct need for patient blood management guidelines in the field of kidney transplantation., (Copyright © 2024 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

The single antigen bead (SAB) assay is the most used test for the identification of HLA specific antibodies pre- and post-transplant. Nevertheless, detection of spurious reactivities remains a recognized assay limitation. In addition, the presence of weak reactivity patterns can complicate unacceptable antigen assignment. This work presents the evaluation of the adsorption with crossmatch cells and elution (AXE) technique, which was designed to help differentiate weak HLA specific antibodies targeting native antigens from spurious and background SAB assay reactivity. The AXE protocol uses selected donor cells to adsorb HLA specific antibodies from sera of interest. Bound antibodies are then eluted off washed cells and identified using the SAB assay. Only antibodies targeting native HLA are adsorbed. Assay evaluation was performed using five cell donors and pooled positive control serum. AXE efficiency was determined by comparing SAB reactivity of adsorbed/eluted antibody to that of the antibodies in unadsorbed sera. A robust efficiency was seen across a wide range of original MFI for donor specific antibodies (DSA). A higher absorption/elution recovery was observed for HLA class I antigens vs. class II. Locus-specific variation was also observed, with high-expression HLA loci (HLA-A/B/DR) providing the best recovery. Importantly, negligible reactivity was detected in the last wash control, confirming that AXE eluates were not contaminated with HLA antibody carry-over. Donor cells incubated with autologous and DSA-containing allogeneic sera showed that AXE selectively adsorbed HLA antibodies in a donor antigen-specific manner. Importantly, antibodies targeting denatured epitopes or other non-HLA antigens were not detected by AXE. AXE was particularly effective at distinguishing weak HLA antibodies from background reactivity. When combined with epitope analysis, AXE enhanced precise identification of antibody-targeted eplets and even facilitated the characterization of a potential novel eplet. Comparison of AXE to flow cytometric crossmatching further revealed that AXE was a more sensitive technique in the detection of weak DSA. Spurious reactivities on the current SAB assay have a deleterious impact on the assignment of clinically relevant HLA specificities. The AXE protocol is a novel test that enables users to interrogate reactive patterns of interest and discriminate HLA specific antibodies from spurious reactivity.

This guideline, from a European Society of Organ Transplantation (ESOT) working group, concerns the management of kidney transplant patients with HLA antibodies. Sensitization should be defined using a virtual parameter such as calculated Reaction Frequency (cRF), which assesses HLA antibodies derived from the actual organ donor population. Highly sensitized patients should be prioritized in kidney allocation schemes and linking allocation schemes may increase opportunities. The use of the ENGAGE 5 ((Bestard et al., Transpl Int, 2021, 34: 1005–1018) system and online calculators for assessing risk is recommended. The Eurotransplant Acceptable Mismatch program should be extended. If strategies for finding a compatible kidney are very unlikely to yield a transplant, desensitization may be considered and should be performed with plasma exchange or immunoadsorption, supplemented with IViG and/or anti-CD20 antibody. Newer therapies, such as imlifidase, may offer alternatives. Few studies compare HLA incompatible transplantation with remaining on the waiting list, and comparisons of morbidity or quality of life do not exist. Kidney paired exchange programs (KEP) should be more widely used and should include unspecified and deceased donors, as well as compatible living donor pairs. The use of a KEP is preferred to desensitization, but highly sensitized patients should not be left on a KEP list indefinitely if the option of a direct incompatible transplant exists. [ABSTRACT FROM AUTHOR]

Luminex single antigen bead (SAB) assays used to detect HLA antibodies may artificially increase sensitisation in highly sensitised patients (HSP). The presence of denatured HLA (dHLA) within the assay enables antibodies specific to cryptic HLA epitopes to bind, such antibodies are not clinically relevant. We sought to exclude dHLA reactivity in a cohort of very HSP, calculated reaction frequency (cRF) 95%–100% and determine the effect upon sensitisation. Such patients have limited access to suitable donors and small changes in their HLA antibody profile, particularly where their cRF is 100%, can increase their opportunity of a transplant. We determined the presence of dHLA by aligning antibody reactivity which did not correspond to known HLA class I epitope mismatches with the results of assays modified to detect class I dHLA. 130 class I dHLA reactions were identified within 11 HSP, all of whom had clear sensitising events. cRF was corrected for dHLA, mean cRF 98.2% (93–100) pre and 95.5% (87–100) post correction (p = 0.0156). An increase in the number of predicted compatible donors (p = 0.0078) after dHLA correction was demonstrated. Two manufacturers SAB assays were used. A reduction of patients with 100% cRF was observed for both manufactures. dHLA is contributing to sensitisation in HSP and is detrimental to their chances of receiving a compatible transplant. The observed dHLA reactivity varied according to kit manufacturers (p = 0.0001), this is potentially a useful finding for laboratories wishing to discriminate between nHLA and dHLA, but without the resources required to regularly perform dHLA assay and epitope analyses. [ABSTRACT FROM AUTHOR]

With the introduction of the virtual allocation crossmatch in the Eurotransplant (ET) region in 2023, the determination of unacceptable antigen mismatches (UAM) in kidney transplant recipients is of utmost importance for histocompatibility laboratories and transplant centers. Therefore, a joined working group of members from the German Society for Immunogenetics (Deutsche Gesellschaft für Immungenetik, DGI) and the German Transplantation Society (Deutsche Transplantationsgesellschaft, DTG) revised and updated the previous recommendations from 2015 in light of recently published evidence. Like in the previous version, a wide range of topics is covered from technical issues to clinical risk factors. This review summarizes the evidence about the prognostic value of contemporary methods for HLA antibody detection and identification, as well as the impact of UAM on waiting time, on which these recommendations are based. As no clear criteria could be determined to differentiate potentially harmful from harmless HLA antibodies, the general recommendation is to assign all HLA against which plausible antibodies are found as UAM. There is, however, a need for individualized solutions for highly immunized patients. These revised recommendations provide a list of aspects that need to be considered when assigning UAM to enable a fair and comprehensible procedure and to harmonize risk stratification prior to kidney transplantation between transplant centers. [ABSTRACT FROM AUTHOR]

Complement‐mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti‐HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre‐treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE‐conjugated secondary Ab (IgGPE), an Alexa Fluor 532‐conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE‐conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays. [ABSTRACT FROM AUTHOR]

Several polymorphisms within Fc receptors (FCR) have been described, some of which correlate with allograft function. In the current study, we determined three Fcγ receptor and five Fcα receptor dimorphisms in 47 kidney transplant recipients who had been vaccinated against Streptococcus pneumoniae. We analyzed if FCR genotypes correlated with pneumococcal antibodies and their serotype-specific opsonophagocytic function, tested prior to and at months 1 and 12 post-vaccination. In parallel, we assessed antibodies against HLA and MICA and determined kidney function. We observed that IgG2 antibodies against pneumococci at months 1 and 12 after vaccination and IgA antibodies at month 1 differed significantly between the carriers of the three genotypes of FCGR3A rs396991 (V158F, p = 0.02; 0.04 and 0.009, respectively). Moreover, the genotype of FCGR3A correlated with serotype-specific opsonophagocytic function, reaching statistical significance (p < 0.05) at month 1 for 9/13 serotypes and at month 12 for 6/13 serotypes. Heterozygotes for FCGR3A had the lowest antibody response after pneumococcal vaccination. On the contrary, heterozygotes tended to have more antibodies against HLA class I and impaired kidney function. Taken together, our current data indicate that heterozygosity for FCGR3A may be unfavorable in kidney transplant recipients. [ABSTRACT FROM AUTHOR]

Antibodies against HLA antigens are ubiquitous in the sera of transplant patients. Analysis of anti-HLA antibodies specificity has gone through a long history of development using assays like agglutination and lymphocytotoxicity, which utilize lymphocytes, and flow cytometry, which utilize multiplex beads coupled with single antigens. Hundreds of HLA antigens are identified to date, and the realization that antibody reactivity against the antigens is multispecific presented difficulties in accurately defining antibody specificity. Although Cross Reacting Groups (CREG), describing cross reactivity among HLA antigens, were helpful with determining specificity, they proved to be inadequate for the highly sensitized patients. Amino acid sequencing and three-dimensional modeling of the HLA molecules significantly advanced our understating of the HLA antigens and their epitopes. Although sensitive assays for antibody testing advanced analysis, they unmasked additional specificities undetectable by traditional methods, and the presence of naturally occurring anti-HLA antibodies in sera further complicated analysis and underscored the need to understand antibody reactivity and their epitopes. Hundreds of HLA class-I and class-II epitopes were defined by the Tekasaki and Duquesnoy groups and their usefulness in organ transplants were further advanced by a great number of transplant centers. Alloantibody specificities, CREGs, and nondonor specific antigens (NDSA) are now explained by public epitopes [ABSTRACT FROM AUTHOR]

Purpose: Currently 80% of lung transplant centers use induction immunosuppression. However, there is a lack of standardization of induction protocols within and across lung transplant centers. This study explores the association of two different induction immunosuppression strategies used at our center [single dose rabbit antithymocyte globulin (rATG) vs. alemtuzumab] compared to no induction with immunologic and clinical outcomes after lung transplantation., Methods: A total of 174 consecutive lung transplant recipients (LTR) between 2016 and 2019 were included in the analysis. Twenty nine LTR (16.7%) received no induction, 22 LTR (12.6%) received alemtuzumab, 123 LTR (70.6%) received a single dose of rATG; 1.5 mg/kg within 24 h of transplant for induction. All LTR had a negative flow cytometry crossmatch on the day of the transplant. All LTR were assessed for de novo HLA donor-specific antibodies (DSA) development and clinical outcomes, including the risk of acute cellular rejection (ACR), antibody-mediated rejection (AMR), chronic lung allograft dysfunction (CLAD), and overall survival post-transplant., Results: The median lung allocation score (LAS) was significantly higher in LTR that did not receive Induction immunosuppression (76; range = 35.3-94.3) compared to induction with rATG (41.6; range = 31.6-91) and alemtuzumab (51; range = 33.1-88.2) (p < 0.001). De novo HLA DSA were detected in 50/174 (28.7%) LTR within 12 months post-transplant. They were detected in 13/29 (44.8%) LTR without induction immunosuppression compared to 28/123 (22.8%) and 9/22 (40.9%) LTR with rATG and alemtuzumab induction, respectively (p = 0.02). The percent freedom from ACR rates between LTR who received alemtuzumab induction was significantly higher compared to LTR who received rATG or no induction at 1 (p = 0.02), 2 (p = 0.01) and 3 (p = 0.05) years post-transplant. In addition, the overall 1-year survival rates were significantly higher in LTR who received rATG or alemtuzumab induction compared to LTR without induction immunosuppression (p = 0.02)., Conclusion: Induction immunosuppression strategies utilizing rATG or Alemtuzumab have unique and contrasting benefits in LTR. Combination of alemtuzumab induction and a lower dose of maintenance immunosuppression may reduce the incidence of ACR in LTR. Single-dose rATG or alemtuzumab induction immunosuppression may also improve the 1 year overall LTR survival compared to no induction., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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Human leukocyte antigen (HLA)-induced signaling in endothelial and smooth muscle cells causes dramatic cytoskeletal rearrangement, increased survival, motility, proliferation, adhesion molecule and chemokine expression, and adhesion of leukocytes. These mechanisms are directly related to endothelial activation, neointimal proliferation, and intragraft accumulation of leukocytes during antibody-mediated rejection (AMR) and chronic rejection. Clustering of HLA by ligands in trans, such as in antigen-presenting cells at the immune synapse, triggers physiological functions analogous to HLA antibody-induced signaling in vascular cells. Emerging evidence has revealed previously unknown functions for HLA beyond antigen presentation, including association with coreceptors in cis to permit signal transduction, and modulation of intracellular signaling downstream of other receptors that may be relevant to HLA signaling in the graft vasculature. We discuss the literature regarding HLA-induced signaling in vascular endothelial and smooth muscle cells, as well as under endogenous biological conditions, and how such signaling relates to functional changes and pathological mechanisms during graft injury.

The pathophysiology of antibody-mediated rejection (AMR) in solid organ transplants is multifaceted and predominantly caused by antibodies directed against polymorphic donor human leukocyte antigens (HLAs). Despite the clearly detrimental impact of HLA antibodies (HLA-Abs) on graft function and survival, the prevention, diagnosis, and treatment of AMR remain a challenge. The histological manifestations of AMR reflect the signatures of HLA-Ab-triggered injury, specifically endothelial changes, recipient leukocytic infiltrate, and complement deposition. We review the interconnected mechanisms of HLA-Ab-mediated injury that might synergize in a 'perfect storm' of inflammation. Characterization of antibody features that are critical for effector functions may help to identify HLA-Abs that are more likely to cause rejection. We also highlight recent advances that may pave the way for new, more effective therapies.

BackgroundCorrelation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved.MethodsWe retrospectively assessed patients with transplant biopsies scored following Banff’15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA.ResultsOne-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31–10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78–16.76], p = 0.003) were independent predictors of ABMRhDSApos.ConclusionsIn conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

Background: Donor‐specific antibodies (DSA) to HLA have been associated with graft loss in hematopoietic progenitor cell (HPC) transplantation. Limited data associate therapeutic plasma exchange (TPE) with desensitization and successful engraftment. We report an attempt of desensitization and observed overshooting of DSA during transplantation. Case report and results: A 27‐year‐old female with cutaneous T cell lymphoma was scheduled for HPC transplantation from her HLA‐haploidentical half‐sister, who carried the HLA‐DRB1*13:03:01 allele. The patient had the corresponding DSA. Lacking an alternative donor option at the time, we attempted a desensitization approach by immunosuppression with tacrolimus and mycophenolate mofetil (MMF). Unexpectedly, DSA increased from a mean fluorescence intensity (MFI) of 1835 on day −63 to 9008 on day −7. The MFI increased further during 3 TPE procedures and intravenous immunoglobulin (IVIG) until day −1. After transplantation, the DSA remained elevated despite 2 more TPE/IVIG procedures and graft‐versus‐host disease prophylaxis with high‐dose cyclophosphamide, sirolimus, and MMF. Flow cytometric crossmatch, initially negative, turned positive after transplantation. Primary graft failure occurred and was attributed to antibody‐mediated rejection. A second transplantation from a 7/8 HLA‐matched unrelated donor, not carrying DRB1*13:03 allele, resulted in successful engraftment. Conclusion: Unexpected and rapid increases of a DSA can occur despite the use of current desensitization approaches. This is problematic when conditioning has already started, as such increases are unlikely to be overcome by TPE or other interventions for desensitization. Overshoot of DSA in HPC transplantation has rarely been reported. Its cause remains unclear and can include underlying disease, immunotherapy, chemotherapy, or TPE. [ABSTRACT FROM AUTHOR]

Objectives: In our study group of Thai PLT apheresis donors, we assessed the prevalence of anti‐leucocyte antibodies. Background: Antibodies against human leucocyte antigens (anti‐HLA), neutrophil antigens (anti‐HNA), and major histocompatibility complex class I related chain A (anti‐MICA) in blood products can lead to transfusion‐related acute lung injury (TRALI). To reduce the risk of TRALI, some blood centres are implementing strategies based on screening platelet (PLT) apheresis donors for the presence of anti‐leucocyte antibodies. Methods/Materials: Blood samples were collected from non‐transfused individuals, 340 males and 63 females (50 nulliparous and 13 parous). Anti‐HLA class I and II and anti‐MICA were analysed using the Luminex assay, and anti‐HNA‐3 was detected using the granulocyte agglutination test. Results: Anti‐HLA was found in 14 of 403 subjects (3.5%). Ten subjects (2.5%) tested positive for HLA class I, 2 (0.5%) for HLA class II, and 2 (0.5%) for both HLA class I and HLA class II. Anti‐HLA class I or II were detected in 2 of 13 (15.4%) parous females and only anti‐HLA class I was found in 4 (8.0%) nulliparous females. Six of 327 subjects tested (1.8%), all males, were positive for anti‐MICA. Anti‐HNA‐3 was not found in any of the 403 individuals. Conclusions: Screening for anti‐HLA class I and II should be implemented for Thai PLT apheresis donors. Although immunisation against HNA and MICA seems to be a rare event in Thais, further work is necessary to decide whether our PLT apheresis donors should be screened for HNA and MICA antibodies. [ABSTRACT FROM AUTHOR]

Abstract Background Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. Methods Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. Results The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. Conclusion Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

Delayed graft function (DGF) occurs in a significant proportion of deceased donor kidney transplant recipients and was associated with graft injury and inferior clinical outcome. The aim of the present multi-center study was to identify the immunological and non-immunological predictors of DGF and to determine its influence on outcome in the presence and absence of human leukocyte antigen (HLA) antibodies. 1,724 patients who received a deceased donor kidney transplant during 2008–2017 and on whom a pre-transplant serum sample was available were studied. Graft survival during the first 3 post-transplant years was analyzed by multivariable Cox regression. Pre-transplant predictors of DGF and influence of DGF and pre-transplant HLA antibodies on biopsy-proven rejections in the first 3 post-transplant months were determined by multivariable logistic regression. Donor age ≥50 years, simultaneous pre-transplant presence of HLA class I and II antibodies, diabetes mellitus as cause of end-stage renal disease, cold ischemia time ≥18 h, and time on dialysis >5 years were associated with increased risk of DGF, while the risk was reduced if gender of donor or recipient was female or the reason for death of donor was trauma. DGF alone doubled the risk for graft loss, more due to impaired death-censored graft than patient survival. In DGF patients, the risk of death-censored graft loss increased further if HLA antibodies (hazard ratio HR=4.75, P < 0.001) or donor-specific HLA antibodies (DSA, HR=7.39, P < 0.001) were present pre-transplant. In the presence of HLA antibodies or DSA, the incidence of biopsy-proven rejections, including antibody-mediated rejections, increased significantly in patients with as well as without DGF. Recipients without DGF and without biopsy-proven rejections during the first 3 months had the highest fraction of patients with good kidney function at year 1, whereas patients with both DGF and rejection showed the lowest rate of good kidney function, especially when organs from ≥65-year-old donors were used. In this new era of transplantation, besides non-immunological factors, also the pre-transplant presence of HLA class I and II antibodies increase the risk of DGF. Measures to prevent the strong negative impact of DGF on outcome are necessary, especially during organ allocation for presensitized patients.

Objective: to evaluate the effects of pre-existing HLA antibodies on the posttransplantation period and to study changes in the generation of donor-specific antibodies in recipients after allogeneic cadaveric kidney transplantation.Materials and methods. One hundred and ten serum samples from patients on the kidney transplantation waiting list (WL) were tested for pre-existing HLA antibodies. To evaluate the effect of preantibodies, the investigators examined 55 patients (32 males and 23 females, whose age was 28 to 63 years) who had undergone kidney transplantation at the N.V. Sklifosovsky Research Institute of Emergency Care in October 2009 to September 2010. In the second part of the investigation, HLA antibody levels were monitored in the posttransplantation period. A total of 27 patients (15 males and 12 females aged 26 to 61 years) were examined after kidney transplantations made at the N.V. Sklifosovsky Research Institute of Emergency Care in March to September 2010.Results and discussion. Since November 2009, the serum level of pre-existing HLA antibodies had been measured in all the patients registered on the kidney transplantation WL at the N.V. Sklifosovsky Research Institute of Emergency Care. One hundred and ten patients have been examined today; of them 69 males and 41 females are waiting for kidney transplantation.Conclusion. The level of pre-existing HLA antibodies must be estimated in the recipients to be prepared for kidney transplantation since this makes it possible to identify in practice a group of patients requiring a more thorough selection of donor organs and preoperative preparation that involves the methods of extracorporeal blood correction and medical therapy.

Seeking to develop immunosuppression regimens that would take into account the patient's level of sensitization to the antigens of the main histocompatibility complex, we studied 123 patients after kidney transplantation. Depending on the choice of immunosuppressive therapy, two groups were formed. The study group included 55 patients who received the immunosuppression regimen adapted to their HLA sensitization level. In the comparison group, 68 patients received baseline immunosuppression, including calcineurin inhibitors, mycophenolic acid preparations, and corticosteroids. AntiHLA antibody detection was performed by assessing the mean fluorescence intensity (MFI) on the Luminex platform when patient's placing on the transplant waiting list. It was found that highly HLA-sensitized recipients should receive antithymocyte polyclonal antibodies with or without plasmapheresis immediately after surgery in order to prevent the rejection reaction. The moderately HLA-sensitized patients should receive the baseline immunosuppression in combination with administration of monoclonal antibodies (simulect); the polyclonal antibodies should be administered only if necessary (in decreased diuresis rate, increased creatinine, etc.). In unsensitized patients, the baseline immunosuppression is enough to induce tolerance. Thus, the administration of immunosuppressive therapy adapted to the pre-existing HLA-sensitization level can significantly improve the treatment oucomes in kidney transplant recipients in the post-transplant period.