Platelet transfusions in haploidentical haematopoietic stem cell allograft candidates: Protecting HLA-A and HLA-B antigens through eplet analysis.

In patients awaiting an allogeneic haematopoietic stem cell transplantation, platelet transfusion is a risk factor for anti-HLA class I immunization because the resulting donor-specific antibodies complicate the allograft process. The objective of the present study was to determine the feasibility of a novel eplet-based strategy for identifying HLA class I mismatches between potential donors and the recipient when pre-allograft platelet transfusions were required. We included 114 recipient/haploidentical relative pairs. For each pair, we entered HLA-class I typing data into the HLA Eplet Mismatch calculator, defined the list of mismatched eplets (for the recipient versus donor direction) and thus identified the shared HLAs to be avoided. Using this list of HLAs, we defined the theoretical availability of platelet components (PCs) by calculating the virtual panel-reactive antibody (vPRA). We also determined the number of PCs actually available in France by querying the regional transfusion centre's database. The mean ± standard deviation number of highly/moderately exposed eplets to be avoided in platelet transfusions was 5.8 ± 3.3, which led to the prohibition of 38.5 ± 2 HLAs-A and -B. Taking into account the mismatched antigens and the eplet load, the mean ± standard deviation theoretical availability of PCs (according to the vPRA) was respectively 34.49% ± 1.95% for HLA-A and 80% ± 2.3% for HLA-B. A vPRA value below 94.9% for highly or moderately exposed eplets would predict that 10 PCs were actually available nationally. Although epitope protection of HLA molecules is feasible, it significantly restricts the choice of PCs.

[Challenges and solutions in current human leukocyte antigen confirmatory typing of hematopoietic stem cell transplantation].

The impact of human leukocyte antigen (HLA) on hematopoietic stem cell transplantation (HSCT) necessitates high precision in HLA genotyping. Confirmatory typing for patients and their related or unrelated donors before HSCT is critical. This study seeks to standardize HLA confirmatory typing in laboratories by examining the current state of HLA genotyping in the country, building upon the National Standards and Industrial Standards for HLA, and highlighting the significance of confirmatory typing for patients and potential donors prior to HSCT. A retrospective analysis over a decade reveals initial typing errors, indicating potential issues and critical considerations in pre-analytical, analytical, and post-analytical stages. Problems are attributed to three main causes: (1) random human errors, including technical mistakes, sample mix-up, and transcription inaccuracies; (2) limitations of technical methods, such as the varied sequence ranges between confirmatory and initial typing; (3) patient factors, involving high tumor burden, the influence of certain drugs on HLA genotyping results, and the second transplantation. Solutions are proposed for these problems, along with recommendations to standardize HLA confirmatory typing.

A Single-Center Analysis of How HLA Mismatch and Donor-Specific Antibodies Affect Short-Term Outcome After Lung Transplantation: A Pilot Study Before a Country-Wide Histocompatibility Study in Japan.

BACKGROUND: Analyzing HLA polymorphism in lung transplantation (LTx) is important, given its impact on LTx recipient survival and graft function. Accordingly, we conducted a retrospective study to examine the influence of HLA mismatch and donor-specific antibodies (DSA) on short-term outcomes and early-phase post-LTx complications. METHOD: HLA antigen or eplet mismatch in LTx patients at Tohoku University Hospital from 2018 to 2023 was determined, and DSA was measured on admission for surgery to identify preformed DSA and at weeks 4 to 12 post-LTx for de novo DSA, respectively. RESULTS: The participants were 45 LTx recipients, HLA-A/B/DR antigen mismatch (5-6 of 6) being identified in 57%, HLA-A/B/Cw/DR/DQ mismatch (8-10 of 10) in 57%, and HLA eplet mismatch (>61) in 46%. The prevalence of preformed DSA was 24%, and persistence (uncleared) was 16%. The incidence of de novo DSA was 16% after LTx. During the study,16 recipients experienced grade 3 primary graft dysfunction (PGD), 8 developed acute rejection, and 5 died. No HLA-related variables were significantly associated with post-LTx mortality and were not risk factors for high-grade PGD or acute rejection. CONCLUSION: Despite limitations in sample size, resulting in tentative findings, the study serves as a crucial pilot study for an ongoing multicenter prospective trial in Japan.

Integrative HLA typing of tumor and adjacent normal tissue can reveal insights into the tumor immune response.

BACKGROUND: The HLA complex is the most polymorphic region of the human genome, and its improved characterization can help us understand the genetics of human disease as well as the interplay between cancer and the immune system. The main function of HLA genes is to recognize "non-self" antigens and to present them on the cell surface to T cells, which instigate an immune response toward infected or transformed cells. While sequence variation in the antigen-binding groove of HLA may modulate the repertoire of immunogenic antigens presented to T cells, alterations in HLA expression can significantly influence the immune response to pathogens and cancer. METHODS: RNA sequencing was used here to accurately genotype the HLA region and quantify and compare the level of allele-specific HLA expression in tumors and patient-matched adjacent normal tissue. The computational approach utilized in the study types classical and non-classical Class I and Class II HLA alleles from RNA-seq while simultaneously quantifying allele-specific or personalized HLA expression. The strategy also uses RNA-seq data to infer immune cell infiltration into tumors and the corresponding immune cell composition of matched normal tissue, to reveal potential insights related to T cell and NK cell interactions with tumor HLA alleles. RESULTS: The genotyping method outperforms existing RNA-seq-based HLA typing tools for Class II HLA genotyping. Further, we demonstrate its potential for studying tumor-immune interactions by applying the method to tumor samples from two different subtypes of breast cancer and their matched normal breast tissue controls. CONCLUSIONS: The integrative RNA-seq-based HLA typing approach described in the study, coupled with HLA expression analysis, neoantigen prediction and immune cell infiltration, may help increase our understanding of the interplay between a patient's tumor and immune system; and provide further insights into the immune mechanisms that determine a positive or negative outcome following treatment with immunotherapy such as checkpoint blockade.

[Genetic analysis of a Chinese pedigree with an allele dropout at the HLA-B locus].

OBJECTIVE: To delineate a deletional mutation of the HLA-B gene in a Chinese pedigree. METHODS: A female patient with acute myeloid leukemia who had visited Liuzhou People's Hospital in April 2022 was selected as the study subject. Routine human leukocyte antigen (HLA) was determined by using PCR-sequence specific oligonucleotide polymorphism (PCR-SSOP) and PCR-sequence-based typing (PCR-SBT) methods. Next generation sequencing (NGS) was used to validate the candidate variant in the HLA-B gene. RESULTS: The PCR-SBT and SSOP results for the HLA-B locus were inconsistent for the patient and her daughter. The SSOP results of the two individuals were HLA-B*35:01, 40:02 and HLA-B*35:01, 40:01, respectively. However, the PCR-SBT results has indicated a mismatch with the nearest HLA-B*35:01 at exon 4. NGS results showed that the HLA-B*35:01 had a 9 bp deletion in the intron 5. The patient's husband was HLA-B*40:01, 58:01, which was normal. CONCLUSION: The variant in intron 5 of the HLA-B gene in this pedigree has mapped to a primer-binding region for the SBT reagent, which has affected the accuracy of PCR-SBT results.

Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

Combined imputation of HLA genotype and self-identified race leads to better donor-recipient matching.

Allogeneic Hematopoietic Cell Transplantation (HCT) is a curative therapy for hematologic disorders and often requires human leukocyte antigen (HLA)-matched donors. Donor registries have recruited donors utilizing evolving technologies of HLA genotyping methods. This necessitates in-silico ambiguity resolution and statistical imputation based on haplotype frequencies estimated from donor data stratified by self-identified race and ethnicity (SIRE). However, SIRE has limited genetic validity and presents a challenge for individuals with unknown or mixed SIRE. We present MR-GRIMM "Multi-Race Graph IMputation and Matching" that simultaneously imputes the race/ethnic category and HLA genotype using a SIRE based prior. Additionally, we propose a novel method to impute HLA typing inconsistent with current haplotype frequencies. The performance of MR-GRIMM was validated using a dataset of 170,000 donor-recipient pairs. MR-GRIMM has an average 20 % lower matching error (1-AUC) than single-race imputation. The recall metric (sensitivity) of the race/ethnic category imputation from HLA was measured by comparing the imputed donor race with the donor-provided SIRE. Accuracies of 0.74 and 0.55 were obtained for the prediction of 5 broad and 21 detailed US population groups respectively. The operational implementation of this algorithm in a registry search could help improve match predictions and access to HLA-matched donors.

Utilizing principal component analysis in the identification of clinically relevant changes in patient HLA single antigen bead solid phase testing patterns.

BACKGROUND: HLA antibody testing is essential for successful solid-organ allocation, patient monitoring post-transplant, and risk assessment for both solid-organ and hematopoietic transplant patients. Luminex solid-phase testing is the most common method for identifying HLA antibody specificities, making it one of the most complex immunoassays as each panel contains over 90 specificities for both HLA class I and HLA class II with most of the analysis being performed manually in the vendor-provided software. Principal component analysis (PCA), used in machine learning, is a feature extraction method often utilized to assess data with many variables. METHODS & FINDINGS: In our study, solid organ transplant patients who exhibited HLA donor-specific antibodies (DSAs) were used to characterize the utility of PCA-derived analysis when compared to a control group of post-transplant and pre-transplant patients. ROC analysis was utilized to determine a potential threshold for the PCA-derived analysis that would indicate a significant change in a patient's single antigen bead pattern. To evaluate if the algorithm could identify differences in patterns on HLA class I and HLA class II single antigen bead results using the optimized threshold, HLA antibody test results were analyzed using PCA-derived analysis and compared to the clinical results for each patient sample. The PCA-derived algorithm had a sensitivity of 100% (95% CI, 73.54%-100%), a specificity of 75% (95% CI, 56.30%-92.54%), with a PPV of 65% (95% CI, 52.50%-83.90%) and an NPV of 100%, in identifying new reactivity that differed from the patients historic HLA antibody pattern. Additionally, PCA-derived analysis was utilized to assess the potential over-reactivity of single antigen beads for both HLA class I and HLA class II antibody panels. This assessment of antibody results identified several beads in both the HLA class I and HLA class II antibody panel which exhibit over reactivity from 2018 to the present time. CONCLUSIONS: PCA-derived analysis would be ideal to help automatically identify patient samples that have an HLA antibody pattern of reactivity consistent with their history and those which exhibit changes in their antibody patterns which could include donor-specific antibodies, de novo HLA antibodies, and assay interference. A similar method could also be applied to evaluate the over-reactivity of beads in the HLA solid phase assays which would be beneficial for lot comparisons and instructive for transplant centers to better understand which beads are more prone to exhibiting over-reactivity and impact patient care.

Imlifidase for Kidney Transplantation of Highly Sensitized Patients With a Positive Crossmatch: The French Consensus Guidelines.

Imlifidase recently received early access authorization for highly sensitized adult kidney transplant candidates with a positive crossmatch against an ABO-compatible deceased donor. These French consensus guidelines have been generated by an expert working group, in order to homogenize patient selection, associated treatments and follow-up. This initiative is part of an international effort to analyze properly the benefits and tolerance of this new costly treatment in real-life. Eligible patients must meet the following screening criteria: cPRA ≥ 98%, ≤ 65-year of age, ≥ 3 years on the waiting list, and a low risk of biopsy-related complications. The final decision to use Imlifidase will be based on the two following criteria. First, the results of a virtual crossmatch on recent serum, which shall show a MFI for the immunodominant donor-specific antibodies (DSA) > 6,000 but the value of which does not exceed 5,000 after 1:10 dilution. Second, the post-Imlifidase complement-dependent cytotoxicity crossmatch must be negative. Patients treated with Imlifidase will receive an immunosuppressive regimen based on steroids, rATG, high dose IVIg, rituximab, tacrolimus and mycophenolic acid. Frequent post-transplant testing for DSA and systematic surveillance kidney biopsies are highly recommended to monitor post-transplant DSA rebound and subclinical rejection.

Extended genomic HLA typing identifies previously unrecognized mismatches in living kidney transplantation.

Introduction: Antibody mediated rejection (ABMR) is the most common cause of long-term allograft loss in kidney transplantation (KT). Therefore, a low human leukocyte antigen (HLA) mismatch (MM) load is favorable for KT outcomes. Hitherto, serological or low-resolution molecular HLA typing have been adapted in parallel. Here, we aimed to identify previously missed HLA mismatches and corresponding antibodies by high resolution HLA genotyping in a living-donor KT cohort. Methods: 103 donor/recipient pairs transplanted at the University of Leipzig Medical Center between 1998 and 2018 were re-typed using next generation sequencing (NGS) of the HLA loci -A, -B, -C, -DRB1, -DRB345, -DQA1, -DQB1, -DPA1, and -DPB1. Based on these data, we compiled HLA MM counts for each pair and comparatively evaluated genomic HLA-typing with pre-transplant obtained serological/low-resolution HLA (=one-field) typing results. NGS HLA typing (=two-field) data was further used for reclassification of de novo HLA antibodies as "donor-specific". Results: By two-field HLA re-typing, we were able to identify additional MM in 64.1% (n=66) of cases for HLA loci -A, -B, -C, -DRB1 and -DQB1 that were not observed by one-field HLA typing. In patients with biopsy proven ABMR, two-field calculated MM count was significantly higher than by one-field HLA typing. For additional typed HLA loci -DRB345, -DQA1, -DPA1, and -DPB1 we observed 2, 26, 3, and 23 MM, respectively. In total, 37.3% (69/185) of de novo donor specific antibodies (DSA) formation was directed against these loci (DRB345 ➔ n=33, DQA1 ➔ n=33, DPA1 ➔ n=1, DPB1 ➔ n=10). Conclusion: Our results indicate that two-field HLA typing is feasible and provides significantly more sensitive HLA MM recognition in living-donor KT. Furthermore, accurate HLA typing plays an important role in graft management as it can improve discrimination between donor and non-donor HLA directed cellular and humoral alloreactivity in the long range. The inclusion of additional HLA loci against which antibodies can be readily detected, HLA-DRB345, -DQA1, -DQB1, -DPA1, and -DPB1, will allow a more precise virtual crossmatch and better prediction of potential DSA. Furthermore, in living KT, two-field HLA typing could contribute to the selection of the immunologically most suitable donors.

[Using Next-Generation Sequencing Technology to Confirm the HLA Rare Alleles Detected by PCR-SSOP].

OBJECTIVE: To confirm the HLA genotypes of the samples including 4 cases of magnetic bead probe HLA genotyping result pattern abnormality and 3 cases of ambiguous result detected by PCR sequence-specific oligonudeotide probe (SSOP) method. METHODS: All samples derived from HLA high-resolution typing laboratory were detected by PCR-SSOP. A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality and 3 samples of ambiguous result were further confirmed by PCR sequence-based typing (SBT) technology and next-generation sequencing (NGS) technology. RESULTS: A total of 4 samples of magnetic bead probe HLA genotyping result pattern abnormality were detected by PCR-SSOP method. The results of SBT and NGS showed that the HLA-A genotype of sample 1 did not match any known genotypes. NGS analysis revealed that the novel allele was different from the closest matching allele A*31:01:02:01at position 154 with G>A in exon 2, which resulting in one amino acid substitution at codon 28 from Valine to Methionine (p.Val28Met). The HLA-C genotype of sample 2 was C*03:119, 06:02, sample 3 was C*03:03, 07:137, and sample 4 was B*55:02, 55:12. A total of 3 samples with ambiguous result were initially detected by PCR-SSOP method. The re-examination results of SBT and NGS showed that the HLA-B genotype of sample 5 was B*15:58, 38:02, sample 6 was DRB1*04:05, 14:101, and sample 7 was DQB1*03:34, 05:02. Among them, alleles C*03:119, C*07:137 and DRB1*14:101 were not included in the Common and Well-documented Alleles (CWD) v2.4 of the Chinese Hematopoietic Stem Cell Donor Database. CONCLUSION: The abnormal pattern of HLA genotyping results of magnetic probe by PCR-SSOP method suggests that it may be a rare allele or a novel allele, which needs to be verified by sequencing.

HLA-DQ eplet mismatch load may identify kidney transplant patients eligible for tacrolimus withdrawal without donor-specific antibody formation after mesenchymal stromal cell therapy.

Recently, the randomized phase-II Triton study demonstrated that mesenchymal stromal cell (MSC) therapy facilitated early tacrolimus withdrawal in living donor kidney transplant recipients. The current sub-study analyzed formation of de novo donor-specific HLA antibodies (dnDSA) in the context of the degree of HLA eplet mismatches. At the time of protocol biopsy at 6 months, 7/29 patients (24%) in the MSC group and 1/27 patient (3.7%) in the control group had developed dnDSA. In the MSC group, all dnDSA were anti-HLA-DQ; two patients had anti-DQ alone and five patients combined with anti-class I, HLA-DR or -DP. Despite excess dnDSA formation in the MSC-arm of the study, the evolution of eGFR (CKD-EPI) and proteinuria were comparable 2 years posttransplant. All dnDSA were complement-binding and three patients had antibody-mediated rejection in the protocol biopsy, but overall rejection episodes were not increased. Everolimus had to be discontinued in nine patients because of toxicity, and tacrolimus was reintroduced in six patients because of dnDSA formation. The HLA-DQ eplet mismatch load independently associated with dnDSA (adjusted hazard ratio = 1.07 per eplet mismatch, p = 0.008). A threshold of ≥11 HLA-DQ eplet mismatches predicted subsequent dnDSA in all 11 patients in the MSC group, but specificity was low (44%). Further research is warranted to explore HLA molecular mismatch load as a biomarker to guide personalized maintenance immunosuppression in kidney transplantation.

Benchmarking freely available HLA typing algorithms across varying genes, coverages and typing resolutions.

Identifying the specific human leukocyte antigen (HLA) allele combination of an individual is crucial in organ donation, risk assessment of autoimmune and infectious diseases and cancer immunotherapy. However, due to the high genetic polymorphism in this region, HLA typing requires specialized methods. We investigated the performance of five next-generation sequencing (NGS) based HLA typing tools with a non-restricted license namely HLA*LA, Optitype, HISAT-genotype, Kourami and STC-Seq. This evaluation was done for the five HLA loci, HLA-A, -B, -C, -DRB1 and -DQB1 using whole-exome sequencing (WES) samples from 829 individuals. The robustness of the tools to lower depth of coverage (DOC) was evaluated by subsampling and HLA typing 230 WES samples at DOC ranging from 1X to 100X. The HLA typing accuracy was measured across four typing resolutions. Among these, we present two clinically-relevant typing resolutions (P group and pseudo-sequence), which specifically focus on the peptide binding region. On average, across the five HLA loci examined, HLA*LA was found to have the highest typing accuracy. For the individual loci, HLA-A, -B and -C, Optitype's typing accuracy was the highest and HLA*LA had the highest typing accuracy for HLA-DRB1 and -DQB1. The tools' robustness to lower DOC data varied widely and further depended on the specific HLA locus. For all Class I loci, Optitype had a typing accuracy above 95% (according to the modification of the amino acids in the functionally relevant portion of the HLA molecule) at 50X, but increasing the DOC beyond even 100X could still improve the typing accuracy of HISAT-genotype, Kourami, and STC-seq across all five HLA loci as well as HLA*LA's typing accuracy for HLA-DQB1. HLA typing is also used in studies of ancient DNA (aDNA), which is often based on sequencing data with lower quality and DOC. Interestingly, we found that Optitype's typing accuracy is not notably impaired by short read length or by DNA damage, which is typical of aDNA, as long as the DOC is sufficiently high.

A national proficiency scheme for human leucocyte antigen typing by next-generation sequencing.

BACKGROUND: Accurate human leucocyte antigen (HLA) typing is essential for solid organ transplantation (SOT) and hematopoietic stem cell transplantation (HSCT). Next-generation sequencing (NGS) is increasingly adopted by clinical laboratories performing HLA typing. To ensure reliable NGS-based HLA typing throughout China, a HLA-NGS proficiency testing (PT) scheme was set up. METHODS: A panel of 10 DNA reference materials for 11 HLA loci were distributed to 24 participants. They were asked to submit NGS typing results for at least six loci including HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1, and HLA-DPB1. Information regarding individual laboratory's sample preparation method, NGS platform, and genotyping software was also collected. RESULTS: Thirteen participants correctly identified HLA alleles in all samples. The total concordance rate for all HLA alleles typed was 99.2% (3390/3420). The overall discordance was 0.2% (3/1440) for Class I, and 1.4% (27/1980) for Class II loci. Further investigation of the typing mistakes pointed to poor sequencing coverage and wrong allele calls. The errors identified reflect the deficient aspect of NGS in HLA typing, which may have implications for quality improvement for relevant laboratories. CONCLUSIONS: The results of this PT underscore the necessity of regular participation in external quality assessment for clinical laboratories that perform HLA-NGS typing.

Heart transplantation across preformed donor-specific antibody barriers using a perioperative desensitization protocol.

Heart transplantation across preformed donor-specific HLA-antibody barriers is associated with impaired short- and long-term survival. Therefore, in recipients with preformed anti-HLA antibodies, waiting for crossmatch-negative donors is standard practice. As an alternative strategy, recipients with preformed anti-HLA donor specific antibodies have been managed at our institutions with a perioperative desensitization regimen. A retrospective analysis was performed comparing heart transplant recipients with preformed donor-specific HLA-antibodies to recipients without donor-specific antibodies. Recipients with a positive virtual crossmatch received a perioperative desensitization protocol including tocilizumab intraoperatively, plasma exchange and rituximab followed by a six-month course of IgGAM. Among the 117 heart-transplanted patients, 19 (16%) patients underwent perioperative desensitization, and the remaining 98 (84%) patients did not. Cold ischemic time, posttransplant extracorporeal life support for primary graft dysfunction, and intensive care unit stay time did not differ between groups. At 1-year follow-up, freedom from pulsed steroid therapy for presumed rejection and biopsy-confirmed acute cellular or humoral rejection did not differ between groups. One-year survival amounted to 94.7% in the treated patients and 81.4% in the control group. Therefore, heart transplantation in sensitized recipients undergoing a perioperative desensitization appears safe with comparable postoperative outcomes as patients with a negative crossmatch.

Development of Poland's Central Bone Marrow Donor Registry Affects Donor Searches for Patients With Hematological Diseases.

In the last 10 years, numbers of donors in the case of Polish potential unrelated hematopoietic stem cells (HSC) have increased dynamically, reaching 1,900,000 in 2020 (including donors presented in World Marrow Donor Association as PL6). On the world scale, the share of Polish donors in the global registry increased from 1% in 2010 to about 5% in 2020. The level and range of typing of potential donors has also been improving steadily toward the international "gold standard." At the end of 2020, 92% of Polish resources were at least HLA-A, B, C, DR, DQ high- or intermediate-resolution typed, mostly by way of genomic typing techniques, mostly by way of genomic typing techniques. The Central Bone Marrow Donor Registry (CBMDR) also stands out in terms of the young ages of potential donors. As of 2020, 27.2% of those registered were younger than 30 years of age, and 36.3% were aged between 30 and 40 years. Sixty percent of registered donors were female. The data in question were presented at the World Marrow Donor Association Search & Match Service, ensuring their visibility and accessibility to Polish and international search units and registries. In 2020, donors in Poland were the subject of almost 18,000 search requests from 40 countries, with 271 extended typing requests and more than 7600 confirmatory typing requests. The total number of donations from Polish donors also increased. In 2020, HSC of bone marrow, peripheral blood, or lymphocytes were collected from 1391 donors, as opposed to 94 donors in 2010. The growing number of donors available in the CBMDR means a better chance of a donor being found among Polish resources, without any need to resort to international registers. Although in 2010, just 24% of Polish recipients received HSC from Polish donors, by 2019 the figure was as high as 67%, and reached 63% in 2020. The CBMDR is an example of proper strategy on registry development being implemented in Poland.

Detection of the novel HLA-B*55:123 allele in a Taiwanese bone marrow donor.

One nucleotide substitution in exon 3 of HLA-B*55:02:01:01 results in a novel allele, HLA-B*55:123.

Personalized HLA typing leads to the discovery of novel HLA alleles and tumor-specific HLA variants.

Accurate and full-length typing of the HLA region is important in many clinical and research settings. With the advent of next generation sequencing (NGS), several HLA typing algorithms have been developed, including many that are applicable to whole exome sequencing (WES). However, most of these solutions operate by providing the closest-matched HLA allele among the known alleles in IPD-IMGT/HLA Database. These database-matching approaches have demonstrated very high performance when typing well characterized HLA alleles. However, as they rely on the completeness of the HLA database, they are not optimal for detecting novel or less well characterized alleles. Furthermore, the database-matching approaches are also not adequate in the context of cancer, where a comprehensive characterization of somatic HLA variation and expression patterns of a tumor's HLA locus may guide therapy and clinical outcome, because of the pivotal role HLA alleles play in tumor antigen recognition and immune escape. Here, we describe a personalized HLA typing approach applied to WES data that leverages the strengths of database-matching approaches while simultaneously allowing for the discovery of novel HLA alleles and tumor-specific HLA variants, through the systematic integration of germline and somatic variant calling. We applied this approach on WES from 10 metastatic melanoma patients and validated the HLA typing results using HLA targeted NGS sequencing from patients where at least one HLA germline candidate was detected on Class I HLA. Targeted NGS sequencing confirmed 100% performance for the 1st and 2nd fields. In total, five out of the six detected HLA germline variants were because of Class I ambiguities at the third or fourth fields, and their detection recovered the correct HLA allele genotype. The sixth germline variant let to the formal discovery of a novel Class I allele. Finally, we demonstrated a substantially improved somatic variant detection accuracy in HLA alleles with a 91% of success rate in simulated experiments. The approach described here may allow the field to genotype more accurately using WES data, leading to the discovery of novel HLA alleles and help characterize the relationship between somatic variation in the HLA region and immunosurveillance.

Obinutuzumab in Kidney Transplantation: Effect on B-cell Counts and Crossmatch Tests.

BACKGROUND: Resistance to the action of rituximab (RTX) has been documented in several diseases. More recently, obinutuzumab (OBZ) has shown promise where RTX has failed in oncology and lupus nephritis. Unlike RTX, OBZ is a weak activator of complement, which may avoid the false-positive complement-dependent cytotoxicity (CDC) crossmatch tests after RTX infusions. METHODS: The aim of this study was to explore the effect of OBZ on B-cell depletion in kidney-transplant candidates and its impact on crossmatch test results. We included 12 patients, who were either highly sensitized kidney-transplant candidates or kidney-transplant recipients presenting with antibody-mediated rejection. Six received OBZ, and 6 received RTX. CD-19 counts, flow cytometry, and CDC crossmatch tests were run immediately before and at 2 wk after drug infusion. RESULTS: OBZ reduced CD-19 counts: median reduction was 98%. B-cell CDC crossmatch test results became positive following RTX infusion but were not affected by OBZ infusion. CONCLUSIONS: OBZ effectively depleted B-cell counts in sensitized kidney-transplant candidates and, unlike RTX, had no effect on CDC crossmatch results.

Next-generation sequencing and clinical histocompatibility testing.

Histocompatibility testing is essential for donor identification and risk assessment in solid organ and hematopoietic stem cell transplant. Additionally, it is useful for identifying donor specific alleles for monitoring donor specific antibodies in post-transplant patients. Next-generation sequence (NGS) based human leukocyte antigen (HLA) typing has improved many aspects of histocompatibility testing in hematopoietic stem cell and solid organ transplant. HLA disease association testing and research has also benefited from the advent of NGS technologies. In this review we discuss the current impact and future applications of NGS typing on clinical histocompatibility testing for transplant and non-transplant purposes.