Detection Of anti-HLA Antibodies Research Articles

New approaches to kidney transplantation in Ukraine

The number of patients with end-stage renal disease in Ukraine, as in the whole world, increases. Kidney transplantation remains the most effective method of treatment for such patients. After 1933, when in Ukraine the world’s first kidney transplantation was performed, the number of there surgeries remained insignificant (up to 130 operations per year). In 2018–2020, the reform of transplantation in Ukraine began due to the change of some laws. And as a result, the number of such operations is gro­wing every year. Control over the transplantation process is carried out by a newly created body of the Ministry of Health of Ukraine, the Ukrainian Transplant Coordination Center. New transplant centers are being created, immunological support of transplant couples is being improved using polymerase chain reaction, flow cytometers and solid-phase methods due to HLA typing of the donor and recipient according to A, B, C, DR, DQ, DP loci, detection of anti-HLA antibodies, allele-specific anti-HLA antibodies using single antigen bead assay. Examinations of patients are carried out at the pre-transplantation stage and as part of monitoring after transplantation. Given the military actions, the lack of aviation and the long time of getting to the clinic, it became possible to preserve organs by methods of hypothermic or nomothermic perfusion. The number of organ transplantations is increasing. In 2023, several successful simultaneous heart and kidney, kidney and pancreas transplantations were performed. The problems of overcoming tissue incompatibility, choosing the optimal immunosuppressive therapy regimen for a given patient, training of nephrologists, wider and more effective use of perfusion machines, new protocols of immunosuppressive therapy, studying the peculiarities of kidney transplantation from marginal donors (the number of which in Ukraine prevails), kidney transplantation in highly sensitized recipients are still actual in Ukraine. The cooperation with our European and American colleagues is very important to improve the long-term results of such operations.

An Optimal Multi-Locus HLA-Typing in Potential Donors of Allogeneic Hematopoietic Stem Cells

Background. HLA-typing and matched donor selection as well as the detection of donor-specific anti-HLA antibodies are essential for allogeneic hematopoietic cell transplantation (allo-HSCT). In accordance with the guidelines of the Center for International Blood and Marrow Transplant Research (CIBMTR) optimal HLA-typing is performed on 11 HLA genes (-A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, and ‐DPB1) with an adequate coverage aiming to obtain the values at the two-field level.
 Aim. To assess the results of multi-locus HLA-typing in bone marrow/hematopoietic cell donors from the database at the National Research Center for Hematology in terms of their conformance with the CIBMTR guidelines for allo-HSCT and to analyze the frequency and distribution of HLA alleles and multi-locus HLA haplotypes.
 Materials & Methods. The study enrolled 3485 donors who were HLA-typed by next-generation sequencing.
 Results. In all donors, the alleles of HLA class I genes were identified at the fourth-field level (nucleotide sequence). When the results were reduced to the second-field level (amino acid sequence), 61 HLA-A, 92 HLA-B, and 49 HLA-C alleles were detected. The alleles of class II genes were discovered either at the two-field or high-resolution levels. Among the HLA-DRB locus genes, 57 DRB1, 11 DRB3, 6 DRB4, and 5 DRB5 alleles were identified. Also, 23 HLA-DQA1, 30 HLA-DQB1, 14 HLA-DPA1, and 33 HLA-DPB1 alleles were detected. There were reported 3289 different HLA haplotypes of A-B-C-DRB1-DQA1-DQB1-DPA1-DPB1 genes.
 Conclusion. The database created at the National Research Center for Hematology includes potential bone marrow/hematopoietic stem cell donors typed for 11 classical polymorphic genes HLA-A, ‐B, ‐C, ‐DRB1, ‐DRB3/4/5, ‐DQA1, ‐DQB1, ‐DPA1, and -DPB1, which is in line with the guidelines of CIBMTR. The frequency and distribution of HLA alleles and multi-locus HLA haplotypes in our donors correspond to those in populations of European origin. HLA-typing and donor selection with regard to 11 HLA genes will contribute to improving the outcomes of both unrelated and haploidentical HSCTs.

Non-invasive molecular biomarkers for monitoring solid organ transplantation: A comprehensive overview.

Solid organ transplantation is a life-saving intervention for individuals with end-stage organ failure. Despite the effectiveness of immunosuppressive therapy, the risk of graft rejection persists in all viable transplants between individuals. The risk of rejection may vary depending on the degree of compatibility between the donor and recipient for both human leucocyte antigen (HLA) and non-HLA gene-encoded products. Monitoring the status of the allograft is a critical aspect of post-transplant management, with invasive biopsies being the standard of care for detecting rejection. Non-invasive biomarkers are increasingly being recognized as valuable tools for aiding in the detection of graft rejection, monitoring graft status and evaluating the efficacy of immunosuppressive therapy. Here, we focus on the importance of molecular biomarkers in solid organ transplantation and their potential role in clinical practice. Conventional molecular biomarkers used in transplantation include HLA typing, detection of anti-HLA antibodies, killer cell immunoglobulin-like receptor genotypes, and anti-MHC class 1-related chain A antibodies, which are important for assessing the compatibility of the donor and recipient. Emerging molecular biomarkers include the detection of donor-derived cell-free DNA, microRNAs (regulation of gene expression), exosomes (small vesicles secreted by cells), and kidney solid organ response test, in the recipient's blood for early signs of rejection. This review highlights the strengths and limitations of these molecular biomarkers and their potential role in improving transplant outcomes.

Complement and Non-Complement Binding Anti-HLA Antibodies Are Differentially Detected with Different Antigen Bead Assays in Renal Transplant Recipients.

Two semi-quantitative, Luminex-based, single-antigen bead (SAB) assays are available to detect anti-HLA antibodies and evaluate their reactivity with complement binding. Sera from 97 patients with positive panel reactive antibody tests (>5%) were analyzed with two SAB tests, Immucor (IC) and One-Lambda (OL), for anti-HLA antibody detection and the evaluation of their complement-binding capacity. IC detected 1608/8148 (mean fluorescent intensity (MFI) 4195 (1995-11,272)) and 1136/7275 (MFI 6706 (2647-13,184)) positive anti-HLA class I and II specificities, respectively. Accordingly, OL detected 1942/8148 (MFI 6185 (2855-12,099)) and 1247/7275 (MFI 9498 (3630-17,702)) positive anti-HLA class I and II specificities, respectively. For the IC assay, 428/1608 (MFI 13,900 (9540-17,999)) and 409/1136 (MFI 11,832 (7128-16,531)) positive class I and II specificities bound C3d, respectively. Similarly, OL detected 485/1942 (MFI 15,452 (9369-23,095)) and 298/1247 (MFI18,852 (14,415-24,707)) C1q-binding class I and II specificities. OL was more sensitive in detecting class I and II anti-HLA antibodies than IC was, although there was no significant difference in the number of class II specificities per case. MFI was higher for complement vs. non-complement-binding anti-HLA antibodies in both assays. Both methods were equal in detecting complement-binding anti-HLA class I antibodies, whereas the C3d assay was more sensitive in detecting complement-binding anti-HLA class II antibodies.

The correlation of results of panel reactive antibody, identification, and single antigen beads in detection of anti-HLA antibodies: Istanbul Faculty of Medicine, tissue typing laboratory experience

BackgroundWe have performed a retrospective analysis of anti-HLA class I MHC and class II MHC antibodies measured using a single antigen bead (SAB) assay and a panel reactive antibody (PRA) assay. Material and methodsA group of 256 patients with end-stage renal disease (ESRD) was tested for anti-HLA antibodies in the tissue typing laboratory between 2017 and 2020. In the cohort, the serum samples of patients waiting for transplantation were tested. Both the PRA and SAB tests of these patients were analyzed using the Luminex (Immucor) method. The threshold of positivity was accepted as median fluorescence intensities (MFI) ≥1000 for PRA screening and MFI ≥750 for SAB screening. ResultsOverall, antibodies to HLA antigens were detected in 202 (78.9%) out of 256 patients in the PRA study. Antibodies against both class I/II antigens were detected only in 15.6% of these patients, whereas antibodies against only against class I HLA in 31.3% and only against class II HLA in 32.0%. By comparison, the SAB study found that 66.8% of patients were positive for HLA antigens. Furthermore, donor-specific antibodies (DSA) were detected in 52.0% of PRA-positive patients and 52.6% of SAB-positive patients. It was shown that 168 patients (83.2%) out of 202 PRA-positive patients were found to be SAB-positive. In addition, 51 patients negative in the SAB assay (94.4%) were also negative in the PRA assay. Statistical analysis established a significant correlation between the PRA and SAB positivity (p > 0.001).It was also shown that MFI ≥3000 PRA positivity for class I HLA antigens (p = 0.049) and MFI ≥5000 PRA positivity for class II antigens (p < 0.001) correlated with the SAB positivity in patients. ConclusionOur results showed the importance of both PRA and SAB assays to define the status of sensitization in patients.

Modes of assay interference and the effectiveness of serum pretreatment approaches in detection of anti-HLA antibodies

AimsSeveral modes of assay interference common to immunoassays affect solid-phase single-antigen bead-based immunoassays (SAB) used to detect antibodies against human leucocyte antigens (HLA). Best practice recommendations include methods to address...

Tvt CAR7: Phase 1 Clinical Trial of Base-Edited "Universal” CAR7 T Cells for Paediatric Relapsed/Refractory T-ALL

Background Genome editing can overcome HLA barriers to generate 'off-the-shelf’ CAR T cell therapies. Despite the success of CAR-T cell therapies in B-cell malignancies, the expression of shared T cell antigens has constrained the development of CAR T cells targeting T-cell malignancies, due to T cell fratricide. Targeted base editing using CRISPR guided cytidine deamination mediates highly precise C→U→T conversion which can directly disrupt gene expression without DNA breaks. This Phase I study will investigate the feasibility and safety of base edited (BE) allogeneic CAR T cells against CD7, disrupted for TCR, CD7 and CD52. The cells are used to secure remission ahead of allogeneic stem cell transplantation (allo-SCT). Investigational medicinal product (IMP) Two allogeneic healthy donor derived BE-CAR7 T cell banks were manufactured from steady state apheresis harvests using a semi-automated process under compliant conditions. Cells were activated with an anti-CD3/CD28 reagent (Transact) and electroporated with codon optimised base editor (coBE) mRNA and three single guide RNAs targeting TRBC, CD52 and CD7. Subsequent lentiviral transduction delivered CAR7 (59-62% efficiency) and magnetic bead processing depleted residual TCRαβ+ T cells (residual 0.1-0.3%). Highly efficient disruption of CD7 and CD52 (98-99%) was confirmed by sequencing. Cells were cryopreserved and QC tested by flow cytometry, ddPCR for copy number (3.1-3.6) and replication competent lentivirus was excluded. Anti-leukemic potency was confirmed in vitro and in human:murine chimera experiments. Trial sponsorship & approvals The TvT CAR7 study (ISRCTN15323014) is an open label, single centre, single arm and non-randomised clinical trial. The trial is sponsored by Great Ormond Street Hospital NHS trust and is supported by MRC, Wellcome Trust and NIHR. Clinical trial authorisation was provided by MHRA and ethical approval by a gene therapy advisory committee (GTAC) ahead of site initiation. Study aims and Objectives The study aims to establish the safety and feasibility of BE-CAR7 to induce molecular remission in children with relapsed/refractory (r/r) CD7-positive T-ALL, ahead of a planned allo-SCT. Assessments include incidence of adverse events, duration of remission, disease-free survival and overall survival. Expansion, persistence and elimination of BE-CAR7 cells and immune recovery after allo-SCT are monitored. Eligibility, treatment and recruitment The study opened on 14th April 2022 at Great Ormond Street Hospital recruiting children across the UK aged between 6 months and 16 years with r/r CD7+ T-cell malignancies quantifiable in bone marrow (>10-4 by flow or PCR). Exclusions include active uncontrolled infections, pre-existing GvHD or the detection of anti-HLA antibodies against the IMP. Eligible patients receive lymphodepletion with fludarabine (150 mg/sqm), cyclophosphamide (120 mg/kg) and alemtuzumab (1 mg/kg), followed by infusion of 0.2-2.0x106 BE-CAR7 T cells (maximum 5x104/kg TCRαβ T cells). Patients in molecular remission at day +28 proceed to allo-SCT, to remove BE-CAR 7 cells and promote immune reconstitution. The first subject was infused on 4th May 2022. Grade 2 CRS and grade 1 ICANS were observed, but no GvHD. Bone marrow assessment at d+28 revealed morphological remission without count recovery and with PCR-MRD <10-4 and the patient proceeded to reduced intensity allo-SCT as planned. This Phase 1 study aims to treat 10 children in the UK. Conclusion The study has demonstrated the feasibility of manufacturing "off-the-shelf” GMP-compliant base edited CAR7 T cells and is investigating their safety and efficacy against paediatric r/r T-ALL.

Exploring the Impact of Humoral Immunogenicity with Tabelecleucel for the Treatment of EBV+ PTLD Following HCT and SOT

Background: Tabelecleucel is a non-genetically modified, allogeneic Epstein-Barr virus (EBV)-T-cell immunotherapy which targets and eliminates EBV-expressing cells in an HLA-restricted manner to treat patients (pts) with EBV+ post-transplant lymphoproliferative disease (PTLD) following hematopoietic stem cell transplantation (HCT) or solid organ transplantation (SOT). The theoretical risk of invoking a humoral or cell-mediated immune response against tabelecleucel is limited to the partially mismatched HLA allele. Tabelecleucel has proven to be safe and effective, with no confirmed evidence for clinical manifestation of immunogenicity in association with tabelecleucel. However, the theoretical consequences of eliciting an immune reaction against the mismatched HLA range from transient appearance of anti-HLA antibodies without any manifestation of clinically significant effects to the development of severe life-threatening conditions, which may include the potential for organ rejection. Therefore, evaluating the potential risk of immunogenicity against an allogeneic cell therapy like tabelecleucel is an important step in assessing the safety profile of the therapeutic agent. In this study we will assess the potential impact of humoral immunogenicity against tabelecleucel in pts with EBV+ PTLD after failure of rituximab or rituximab and chemotherapy enrolled in the ALLELE (NCT03394365) clinical study. Methods: Humoral immunogenicity was assessed through two methods of anti-HLA antibody detection that evaluated a total of 28 pts on study. The primary approach evaluated 9 pts by a pan anti-HLA detection method that provides a positive or negative result. In this approach evaluable pts must have a negative result in pre-treatment measurement and have at least one post-treatment measurement. Results are coupled with the pt's clinical safety profile to assess treatment related immunogenicity. The secondary approach evaluated 24 pts in a single-antigen bead method that provides not just a positive or negative result but also defines the specificity and strength of the positive result. Cross referencing the allele specificity of the Class I or Class II anti-HLA antibodies with the HLA type of the pt, the transplant and the infusion product aids in elucidating the source of the anti-HLA antibodies in these pts. Results: Two of the nine pts tested using the pan anti-HLA antibody detection approach had detection of anti-HLA antibodies in their pre-treatment sample. The remaining 7 evaluable pts had a median of 1 post-treatment measurement (min, max: 1, 3) and the median time post-treatment evaluated was 113 days (min, max: 40, 179). Six out of the 7 evaluable pts did not develop any anti-HLA antibodies post-treatment. One pt in the SOT cohort did develop anti-HLA antibodies 40 days after initial dosing on cycle 2 day 1. No treatment-emergent serious adverse events were reported for this pt, and only one adverse event of grade 1 fever was assessed as possibly related per investigator. The pt received additional doses of tabelecleucel yet did not manifest any subsequent events of fever or any signs of organ rejection. The second, single-antigen bead-based approach evaluated 24 pts. Four had anti-HLA antibodies present prior to tabelecleucel infusion. None of the pts evaluated had any incidence of de novo anti-HLA antibodies emerging post-treatment. For the 4 pts with pre-existing anti-HLA antibodies, evaluation of their safety profile and comparison to the total HLA immune profile of the pt is currently underway and will be reported at the time of presentation. Conclusions: To date, there has been no confirmed evidence for clinical manifestation of immunogenicity in association with tabelecleucel. Only 1 pt (tested using the pan anti-HLA method) out of 28 pts (tested using either method) had de novo emergence of anti-HLA antibodies post-infusion. This pt did not experience any severe events potentially reflecting immunogenicity such as hypersensitivity or SOT rejection. Taken together, these results reaffirm the low potential risk of developing humoral immunogenicity following tabelecleucel administration in HCT or SOT recipients with EBV+ PTLD after failure of rituximab or rituximab and chemotherapy.

Significance of pretransplant and de novo anti-HLA antibody detection after simultaneous pancreas-kidney transplantation

Introduction. Nowadays, there are few studies concerning assessment of the clinical significance of determining the level of pretransplant and de novo anti-HLA antibodies in patients after simultaneous pancreas-kidney transplantation.Aim. The study of the incidence, timing of formation and specificity of pretransplant and de novo anti-HLA antibodies in patients after simultaneous pancreas-kidney transplantation.Material and methods. We conducted a prospective and retrospective research to study the incidence, timing of formation and specificity of pretransplant and de novo anti-HLA antibodies in 55 patients after simultaneous pancreas-kidney transplantation performed at the N.V. Sklifosovsky Research Institute for Emergency Medicine from 2008 to 2022.Results. There were 4 patients with preformed anti-HLA antibodies (7%). The formation of de novo anti-HLA antibodies after simultaneous pancreas-kidney transplantation was observed in 17 patients (31%). There were 5 patients with antiHLA class I, 3 patients with anti-HLA class II, 3 patients with anti-HLA class I and II, 5 patients with anti-MICA and 1 patient with both classes of anti-HLA and anti-MICA. The formation of de novo anti-HLA antibodies significantly increased the incidence of acute rejection (47% compared with 13%, p=0.014).Conclusion. The frequency of pretransplant and de novo anti-HLA antibody detection in the recipients at our Center is comparable to published data from other transplant centers. We obtained evidence that the formation of de novo antiHLA antibodies increases the incidence of acute rejection after simultaneous pancreas-kidney transplantation.

The allograft injury marker CXCL9 determines prognosis of anti-HLA antibodies after lung transplantation

Despite the common detection of non-donor specific anti-HLA antibodies (non-DSAs) after lung transplantation, their clinical significance remains unclear. In this retrospective single-center cohort study of 325lung transplant recipients, we evaluated the association between donor-specific HLA antibodies (DSAs) and non-DSAs with subsequent CLAD development. DSAs were detected in 30% of recipients and were associated with increased CLAD risk, with higher HRs for both de novo and high MFI (>5000) DSAs. Non-DSAs were detected in 56% of recipients, and 85% of DSA positive tests had concurrent non-DSAs. In general, non-DSAs did not increase CLAD risk in multivariable models accounting for DSAs. However, non-DSAs in conjunction with high BAL CXCL9levels were associated with increased CLAD risk. Multivariable proportional hazards models demonstrate the importance of the HLA antibody-CXCL9 interaction: CLAD risk increases when HLA antibodies (both DSAs and non-DSAs) are detected in conjunction with high CXCL9. Conversely, CLAD risk is not increased when HLA antibodies are detected with low CXCL9. This study supports the potential utility of BAL CXCL9measurement as a biomarker to risk stratify HLA antibodies for future CLAD. The ability to discriminate between high versus low-risk HLA antibodies may improve management by allowing for guided treatment decisions.

Research advances on haploidentical hematopoietic stem cell transplantation in the treatment of severe aplastic anemia in children.

Haploidentical hematopoietic stem cell transplantation is a recommended alternative therapy for children with severe aplastic anemia who lack a human leukocyte antigen (HLA)-identical sibling donor and do not respond well to immunosuppressive therapy; however, due to non-identical HLA, the patients may have donor-specific anti-HLA antibody, which may lead to a relatively high incidence rate of poor graft function. Compared with HLA-identical transplantation, conditioning regimen for haploidentical transplantation still needs to be explored. This article reviews the detection and treatment of donor-specific anti-HLA antibody, the selection of conditioning regimen, and the mechanism and treatment of poor graft function in haploidentical hematopoietic stem cell transplantation.

Hidden Patterns of Anti-HLA Class I Alloreactivity Revealed Through Machine Learning.

Detection of alloreactive anti-HLA antibodies is a frequent and mandatory test before and after organ transplantation to determine the antigenic targets of the antibodies. Nowadays, this test involves the measurement of fluorescent signals generated through antibody–antigen reactions on multi-beads flow cytometers. In this study, in a cohort of 1,066 patients from one country, anti-HLA class I responses were analyzed on a panel of 98 different antigens. Knowing that the immune system responds typically to “shared” antigenic targets, we studied the clustering patterns of antibody responses against HLA class I antigens without any a priori hypothesis, applying two unsupervised machine learning approaches. At first, the principal component analysis (PCA) projections of intra-locus specific responses showed that anti-HLA-A and anti-HLA-C were the most distantly projected responses in the population with the anti-HLA-B responses to be projected between them. When PCA was applied on the responses against antigens belonging to a single locus, some already known groupings were confirmed while several new cross-reactive patterns of alloreactivity were detected. Anti-HLA-A responses projected through PCA suggested that three cross-reactive groups accounted for about 70% of the variance observed in the population, while anti-HLA-B responses were mainly characterized by a distinction between previously described Bw4 and Bw6 cross-reactive groups followed by several yet undocumented or poorly described ones. Furthermore, anti-HLA-C responses could be explained by two major cross-reactive groups completely overlapping with previously described C1 and C2 allelic groups. A second feature-based analysis of all antigenic specificities, projected as a dendrogram, generated a robust measure of allelic antigenic distances depicting bead-array defined cross reactive groups. Finally, amino acid combinations explaining major population specific cross-reactive groups were described. The interpretation of the results was based on the current knowledge of the antigenic targets of the antibodies as they have been characterized either experimentally or computationally and appear at the HLA epitope registry.

Precision medicine in transplantation and hemodialysis.

In kidney transplantation, precision medicine has already entered clinical practice. Donor and recipient human leucocyte antigen (HLA) regions are genotyped in two class 1 and usually three class 2 loci, and the individual degree of sensitization against alloimmune antigens is evaluated by the detection of anti-HLA donor-specific antibodies. Recently, the contribution of non-HLA mismatches to outcomes such as acute T- and B-cell-mediated rejection and even long-term graft survival was described. Tracking of specific alloimmune T- and B-cell clones by next generation sequencing and refinement of the immunogenicity of allo-epitopes specifically in the interaction with HLA and T- and B-cell receptors may further support individualized therapy. Although the choices of maintenance immunosuppression are rather limited, individualization can be accomplished by adjustment of dosing based on these risk predictors. Finally, supplementing histopathology by a transcriptomics analysis allows for a biological interpretation of the histological findings and avoids interobserver variability of results. In contrast to transplantation, the prescription of hemodialysis therapy is far from precise. Guidelines do not consider modifications by age, diet or many comorbid conditions. Patients with residual kidney function routinely receive the same treatment as those without. A major barrier hitherto is the definition of ‘adequate’ treatment based on urea removal. Kt/Vurea and related parameters neither reflect the severity of uremic symptoms nor predict long-term outcomes. Urea is poorly representative for numerous other compounds that accumulate in the body when the kidneys fail, yet clinicians prescribe treatment based on its measurement. Modern technology has provided the means to identify other solutes responsible for specific features of uremic illness and their measurement will be a necessary step in moving beyond the standardized prescription of hemodialysis.

Correlation analysis between virtual and Complement-Dependent-Cytotoxicity crossmatch in a monocenter retrospective series of 118 allografted patients

Purpose of the StudyThe detection of patients' anti-HLA antibodies before allogeneic hematopoietic stem cell transplantation (HSCT) may affect post-transplant outcome, due to a potential detrimental impact on engraftment or toxicity-related issues. Crossmatch (XM) techniques provide support to physicians during the pre-transplant phase but the role of Complement-Dependent Cytotoxicity XM (CDC-XM) is not well-defined when performed routinely and in parallel with the virtual XM. Patients and MethodsWe report here our experience with both virtual and CDC-XM tests on n = 118 patients undergoing search for a donor other than HLA-identical sibling from July 2013 to June 2018 at our Institution. When anti-HLA antibodies (Abs) were present, they were classified as donor-specific Abs (DSA) or non-DSA. ResultsOn the n = 118 patients, n = 35 (29.7 %) had a positive virtual XM test (of which one of more DSA were found in n = 8; 6.8 %) and n = 5 had a positive CDC-XM test. These latter, positive for HLA class II only, were interpreted as false-positive results due to prior administration of anti-CD20 to the patients, all affected by lymphoma; none of them had a positive virtual XM for anti-HLA Abs of class II. Importantly, all these patients successfully engrafted, further supporting the lack of significant impact of CDC-XM positive results in this series. ConclusionsAccording to our data on more than a hundred patients, routinely performed CDC-XM does not seem to add significant information with respect to virtual XM. We cannot exclude the usefulness of CDC-XM in specific situations, although a positive CDC-XM result was an unfrequent event.

Platelet transfusion refractoriness and anti-HLA immunization.

Platelet transfusion refractoriness (PTR), defined as an unsatisfactory post-transfusion platelet count increment, is a common complication of patients receiving multiple transfusions. Different strategies are described in the management of PTR. In this work, we demonstrate the efficacy of the detection and identification of anti-HLA antibodies in the recipient using a threshold of 3000 mean fluorescence intensity (MFI), and the seek of donors not expressing HLA antigens against which the patient is immunized.

Выявление донор-специфических анти-HLA-антител методом индивидуальной перекрестной пробы де-факто у педиатрических реципиентов перед трансплантацией гемопоэтических стволовых клеток от гаплосовместимого донора

Выявление донор-специфических анти-HLA-антител методом индивидуальной перекрестной пробы де-факто у педиатрических реципиентов перед трансплантацией гемопоэтических стволовых клеток от гаплосовместимого донора

Detection of antibodies to denatured human leucocyte antigen molecules by single antigen Luminex.

The anti-HLA antibody detection has been improved in sensitivity and specificity with solid-phase antigen bead (SAB) assays based on Luminex. However, false positive results due to denatured HLA (dHLA) may arise after single antigen test. The aim of this study was to compare the performance of the two Luminex technology-based anti-HLA detection kits available in the market in showing undesired anti-HLA antibody results. A prospective cohort was assessed for anti-HLA antibodies with single antigen A manufacturer (AM) kit and a comparison cohort with single antigen B manufacturer (BM) kit. A total of 11 out of 90 patients in a prospective cohort presented monospecific HLA-I antibodies with AM, and 5 out of 11 confirmed monospecific reaction with BM. Despite the confirmation of monospecific reaction with both manufacturers, 80% were assigned as dHLA reaction by specific crossmatch. Further comparative cohorts detected four out of six monospecific reactions with BM that were confirmed as possible dHLA reactions. A positive SAB test should rule out a reaction against a dHLA molecule, thus avoidance of prolonged waitlist periods or misattribution of anti-HLA reactions after transplantation.

The challenge of using the virtual crossmatch as a singular tool for the detection of Anti-HLA antibodies- A study from a tertiary care institute from South India

IntroductionDetection of donor specific antibodies (DSA) is critical in both solid organ and mismatched haematopoietic stem cell transplants. The single antigen bead assay (SAB) is widely used as a virtual crossmatch in these settings. However, HLA allele variation across ethnicities and differing genetic backgrounds is a well-known and acknowledged fact and representation of alleles prevalent in a population is key while using a virtual crossmatch as a sole decision making tool. Against this background, this study was performed to assess the feasibility of using the SAB as a single tool to identify DSA in our population. Materials and methodsThe HLA alleles identified in the study population were analysed to assess their representation on SAB panels from two different vendors. ResultsThe study population comprised of a total of 966 subjects for whom 6 loci high resolution HLA typing was done. A total of 241 different alleles were assigned in the population. Among the 241 alleles identified in our study population, 48.55% (n = 117) alleles were represented in the SAB A panel and 48.13% (n = 116) represented in the SAB B panel. Unrepresented alleles were 51.45% (n = 124) in panel A and 51.87% (n = 125) in panel B. All the twelve alleles were represented for 16.05% (n = 155) and 16.25% (n = 157) of study population in panel A and in panel B respectively. The remaining individuals (83.95%, (n = 811) in panel A and 83.75%, (n = 809) in panel B) had at least one allele unrepresented. ConclusionOur study addresses an important limitation in utilizing the SAB as a single tool to identify DSA, owing to non-representation of locally prevalent / unique alleles in our population. More than 50% of alleles were unrepresented in both the SAB assays we studied, which included alleles from both Class I and Class II. We recommend therefore that, until a comprehensive coverage of alleles is provided, or epitope matching becomes robust, that the SAB be combined with a physical crossmatch when mismatched alleles are not represented.

Human Leukocyte Antigen Examination Using Luminex®-Based Assays for Donor-Recipient Compatibility Assessment in Kidney Transplantation: Our Preliminary Experience

Background: Detection of anti-HLA antibodies (HLAabs) in organ transplantation recipients is vital to determine whether the recipient has specific HLAabs against the donor’s HLA molecules (donor-specific anti-HLA antibodies [DSA]). Methods: This preliminary study involved seven subjects: five without prior HLA sensitization and two with a history of possible HLA sensitization (kidney transplantation and pregnancy). Two of the subjects were siblings, two of the subjects were mother and son, and two of them were first cousins once removed. All the subjects underwent HLA typing, and four of the subjects underwent HLAabs assays using the sequence-specific oligonucleotide probes (SSOP) method. Results: There were 10/16 HLA matches between subjects who were siblings and between subjects who were mother and son, and 6/16 HLA matches were found between subjects who were first cousins once removed. Subjects with previous kidney transplantation developed 59 HLA-Abs toward class I and class II HLA (the mean fluorescence intensity (MFI) value range: 1,003.36 to 12,404.77). HLAabs were also found among subjects without prior HLA sensitization, albeit with relatively lower MFI values. Conclusions: HLA typing and HLAabs detection for donor-recipient compatibility assessment using the SSOP method offers more detailed, semi-quantitative results. Further research involving larger subjects in cohort settings will be useful for profiling MFI cut-off values for HLAabs.

Quadritherapy vs standard tritherapy immunosuppressant regimen after heart transplantation: A propensity score-matched cohort analysis.

After heart transplant, adding everolimus (EVL) to standard immunosuppressive regimen mostly relies on converting calcineurin inhibitors (CNIs) into EVL. The aim of this study was to describe the effects of combining low-dose EVL and CNIs in maintenance immunosuppression regimen (quadritherapy) and compare it with standard tritherapy associating standard-dose CNIs, mycophenolate mofetil, and corticosteroids. In the 3-year registry cohort of heart transplanted patients, those who received quadritherapy were compared with those who received tritherapy. EVL was added after 3months posttransplant. Three analyses were performed to control for confounders: propensity score matching, multivariable survival, and inverse probability score weighting analyses. Among 213 patients who were included (75 with quadritherapy), propensity score matching selected 64 unique pairs of patients with similar characteristics. In the matched cohort (n=128), quadritherapy was associated with fewer deaths (3 [4.7%] vs 17 [21.9%], P=.007) and biopsy-proven acute rejections (15 [23.4%] vs 31 [48.4%], P=.002). These results were confirmed in the overall cohort (n=213), after multivariable and inverse probability score weighting analyses. Renal function and donor-specific HLA-antibodies remained similar in both groups. Low-dose combination quadritherapy was associated with fewer deaths and rejections, compared with standard immunosuppression tritherapy.