Using single antigen specificity magnetic beads for the isolation of specific antibodies against HLA antigens.

The presence of multiple donor-specific antibodies (DSAs) targeting HLA antigens poses a challenge to transplantation. Various techniques, including the use of recombinant cell lines and crossmatch cells have been developed to isolate DSAs. To simplify the extraction of HLA-specific DSAs from complex sera, we introduced magnetic beads with single HLA specificity (MagSort). Sera were treated with MagSort, allowing HLA-specific antibodies to bind to the beads, and these specific antibodies were subsequently eluted. MagSort beads, coated with 59 different HLA variants, underwent testing through 1329 adsorption/elution processes, demonstrating their effectiveness and specificity in adsorbing and eluting HLA-specific antibodies. The MagSort method proves comparable to the cell method, showing similar isolated antibody binding patterns. The isolated antibody binding patterns from MagSort reveal both known eplets and unknown patterns, suggesting its utility for eplet discovery. Additionally, MagSort proved effective in extracting signals for flow cytometry cross-matching, offering a means to assess the binding capability of isolated antibodies against specific donor cells.

Two-field resolution on-call HLA typing for deceased donors using nanopore sequencing.

The current practice of HLA genotyping in deceased donors poses challenges due to limited resolution within time constraints. Nevertheless, the assessment of compatibility between anti-HLA sensitized recipients and mismatched donors remains a critical medical need, particularly when dealing with allele-specific (second field genotyping level) donor-specific antibodies. In this study, we present a customized protocol based on the NanoTYPE® HLA typing kit, employing the MinION® sequencer, which enables rapid HLA typing of deceased donors within a short timeframe of 3.75 h on average at a three-field resolution with almost no residual ambiguities. Through a prospective real-time analysis of HLA typing in 18 donors, we demonstrated the efficacy and precision of our nanopore-based method in comparison to the conventional approach and without delaying organ allocation. Indeed, this duration was consistent with the deceased donor organ donation procedure leading to organ allocation via the French Biomedicine Agency. The improved resolution achieved with our protocol enhances the security of organ allocation, particularly benefiting highly sensitized recipients who often present intricate HLA antibody profiles. By overcoming technical challenges and providing comprehensive genotyping data, this approach holds the potential to significantly impact deceased donor HLA genotyping, thereby facilitating optimal organ allocation strategies.

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.

Single locus HLA sequencing with the nanopore technology for HLA disease association diagnosis.

Associations between HLA genotype and disease susceptibility encompass almost all the classic HLA loci. The level of typing resolution enabling a correct identification of an HLA disease susceptibility gene depends on the disease itself and/or on the accumulated knowledge about the molecular involvement of the HLA allele(s) engaged. Therefore, the application of Next Generation Sequencing technologies to HLA disease association, which would improve typing resolution, could prove useful to better understand disease severity. In the present study, we tested a nanopore sequencing approach developed by Omixon Biocomputing Ltd, dedicated to on-demand locus typing for HLA and disease, as an alternative to the conventional widely used sequence specific oligoprobe (SSO) approach. A total of 145 DNA samples used in routine diagnosis by SSO were retrospectively analyzed with nanopore technology, for HLA-A*02 immunotherapy decision for A*29, B*27, B*51, B*57 identification in class I, and DRB1, DQA1, and DQB1 for bullous dermatosis, rheumatoid arthritis, diabetes, and celiac disease requests in class II. Each locus was typed in a separate experiment, except for DQB1 and DQA1, which were analyzed together. Concordance between typings reached 100% for all the loci tested. Ambiguities by nanopore were only found for missing exon coverage. This approach was found to be very well adapted to the routine flow imposed by the SSO technique. This study illustrates the use of the new NanoTYPE MONO kit for single locus HLA sequencing for HLA and disease association diagnosis.

[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.

[Establishing and verifying the threshold value of HLA mixed antigen reagent screening test results].

Objective: To establish the threshold value of human leukocyte antigen (HLA) mixed antigen reagent screening test results, and to verify it by HLA single antigen reagent confirmation test results. Methods: The results of 2 255 serum samples tested for HLA antibodies by HLA mixed antigen reagent in the department of HLA Laboratory, the First Affiliated Hospital of Soochow University from October 2017 to December 2021 were retrospectively analyzed. Among them, 1 139 samples were also tested by single antigen HLA Class-Ⅰ reagent and 1 116 samples were also tested by single antigen HLA Class-Ⅱ reagent. Based on the same antigens coated with both reagents, the Mean Fluorescence Intensity (MFI) and Nomalized Background ratio (NBG ratio) of 12 HLA Class-Ⅰ beads and 5 HLA Class-Ⅱ beads in the HLA mixed antigen reagent and the MFI of 77 anti-HLA class-Ⅰ antibodies and 35 anti-HLA class-Ⅱ antibodies detected by HLA single antigen reagent were recorded. The MFI and NBG ratio of HLA mixed antigen reagent beads in 1 139 or 1 116 samples were segmented according to the positive rate of antibodyies detected by the single antigen reagent corresponding to the antigens coated with each HLA mixed antigen reagent bead, and the results of the HLA mixed antigen screening test were verified by the HLA single antigen reagent confirmation test. Results: The threshold values of MFI and NBG ratio of HLA mixed antigen reagent's 17 beads were established. The MFI of No. 1 to No. 17 beads of HLA mixed antigen reagent ranged from 26.86 to 21 925.58, and the NBG ratio ranged from 0 to 434.65. According to the positive detection rate of HLA single antigen reagent corresponding to the coated antigens, the MFI and NBG ratio of the beads of HLA mixed antigen reagent were divided into positive interval, suspicious positive interval, suspicious negative interval and negative interval. The positive rates of anti-HLA class-Ⅰ antibodies by HLA mixed antigen reagent and single antigen HLA Class-Ⅰ reagent were 87.5% (997/1 139) and 66.3% (755/1 139). The positive rates of anti-HLA class-Ⅱ antibodies were 63.4% (707/1 116) and 44.9% (501/1 116). In the samples with suspicious negative, suspicious positive and positive results of HLA class-Ⅰ、Ⅱ antibodies detected by HLA mixed antigen reagent, the positive detection rates of single antigen HLA Class-Ⅰ reagent were 14.9% (17/114), 41.3% (145/351) and 91.3% (590/646), respectively. The positive detection rates of single antigen HLA Class-Ⅱ reagent were 15.5% (58/375), 26.5% (81/306) and 88.8% (356/401), respectively. Conclusions: In this study, the threshold values of MFI and NBG ratio of HLA mixed antigen reagent screening test are established, and the threshold values are verified by the results of HLA single antigen reagent confirmation test. HLA mixed reagent screening test can be used for screening of HLA antibodies, and if necessary, it should be combined with HLA single antigen confirmatory test for clinical detection of HLA antibodies.

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.

Predicting flow cytometry crossmatch results from single-antigen bead testing.

The aim of this study was to devise an algorithm that would predict flow cytometry crossmatch (FCXM) results using single-antigen bead (SAB) mean fluorescent intensity (MFI) levels using samples received through the National External Quality Assurance Scheme (NEQAS) 2B external proficiency testing scheme between 2019 and 2023. A total of 159 serum samples were retrospectively screened using LABScreen Single Antigen Class I and II (SAB), and 40 peripheral blood samples were human leucocyte antigen (HLA) typed with LABType SSO. Donor-specific antibodies were identified for each cell-serum combination tested, and cumulative MFI values were calculated for each test before correlating the screening result with the consensus crossmatch results for this scheme. HLA Class I MFIs were combined to predict the T cell crossmatch. For the B cell crossmatch prediction, two options were considered: (i) HLA Class II MFI values alone and (ii) HLA Class I + Class II MFIs. Receiver operating characteristic analysis was carried out to identify the combined MFI threshold that predicted NEQAS consensus results with the greatest sensitivity and specificity. HLA Class I combined MFI >5000 predicted T cell crossmatch results with 96% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 92% negative predictive value (NPV). For B cell results, HLA Class I + Class II combined MFIs >11,000 gave the best model, showing 97% sensitivity, 82% specificity, 96% PPV and 85% NPV. However, for samples with only HLA Class II sensitization, combined MFIs >13,000 improved the B cell crossmatch predictions: 92% sensitivity, 95% specificity, 96% PPV and 91% NPV. Using this model, combined MFI can be used to predict the immunological risk posed by donor-specific antibodies when it is not possible to carry out an FCXM.

Immunology Status of Patients During Kidney Transplant.

OBJECTIVES: The immunology status of a patient has a crucial role in kidney transplant. We investigated the effectiveness of a desensitization protocol, guided by the immunology status of patients, for kidney transplant candidates. MATERIALS AND METHODS: Antibody screening for human leukocyte antigens was conducted with the Luminex single-antigen microsphere bead assay method for 34 patients from June 2021 to June 2022. Donor human leukocyte antigen genotypes at 8 loci (A*, B*, С*, DRB1*, DQA1*, DQB1*, DPA1*, and DPB1*) were determined, to correlate the specificities of recipient human leukocyte antigen antibodies with donor antigens and identify unacceptable donor antigen combinations. Specialized immunology studies measured panel reactive antibody levels and human leukocyte antigen class I and class II antibodies. A crossmatch compatibility test using complementdependent cytotoxicity was conducted. RESULTS: Of the 34 patients, 10 completed all 3 stages of the desensitization therapy. Most patients experienced decreased sensitization to human leukocyte antigen class I and class II antibodies. Two patients achieved complete clearance of A1 and DQ5 antibodies, respectively, whereas 1 patient exhibited an increase in donor-specific antibody mean fluorescence intensity. Prior to desensitization therapy, the crossmatch compatibility test yielded positive results with T and B lymphocytes. After completing the therapy, the crossmatch test showed negative results in 4 cases with T lymphocytes and positive results with B lymphocytes. Plasmapheresis sessions effectively reduced circulating antibodies. However, the combination of rituximab and plasmapheresis alone did not achieve a negative crossmatch test required for kidney transplant. CONCLUSIONS: It is crucial to assess the reduction of donor-specific antibody quantity, considering both the percentage and the mean fluorescence intensity. To avoid false-positive results in crossmatch analysis, drug half-life must be considered. Laboratories should have various crossmatch techniques, such as flow cytometry and single-antigen microsphere bead assay technology, available for research and urgent cases that require crossmatch analysis.

Superiority of Single-Antigen Bead Study in Donor-Specific Antibodies: Determination in Highly Sensitized Patients.

OBJECTIVES: The presence of donor-specific antibodies against HLA before kidney transplant has been variably associated with decreased long-term graft survival. Data on the association between pretransplant donor-specific antibodies and rejection and cause of graft failure in recipients of donor kidneys are scarce. MATERIALS AND METHODS: For this study of HLA antibody levels, we analyzed serum samples from 76 patients (48 women and 28 men) who were prepared for kidney transplant at the Baskent University Istanbul Hospital between 2017 and 2022. Levels were determined by using Lifecodes panel reactive antibody class I and II identification kits and Lifecodes LSA class I and II identification kits by the Luminex assay method. RESULTS: Multiple antigen tests showed more than 70% sensitization detected against both class I and class II antigens in our patient group. When some samples were reevaluated with the single-antigen bead method, desensitization values were shown to be considerably reduced compared with values from multiple antigen methods. CONCLUSIONS: The single-antigen-coated bead method can be useful in determining the risk of donor-specific antibodies in highly sensitized patients.

Recognition of the HLA-B*15:01:17 allele in a Taiwanese individual.

Nucleotide substitution in codon 129 of HLA-B*15:01:01:01 results in a novel allele, HLA-B*15:01:17.

[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.

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.

Characterization of novel HLA-A*24:608N allele discovered in Koreans.

A novel null HLA-A*24 allele, HLA-A*24:608N, was identified in five Korean subjects including three from a family and two separate individuals. This study was performed to discern its immunological function in transplantation settings. Because this null variant had deletions of approximately 12 k base pairs from intron 3 to 3' end of the HLA-A gene, low resolution HLA typing and amplicon-based next generation sequencing (NGS) typing methods had failed to assign it. Hybrid capture-based NGS method confirmed that this novel variant had a large deletion. T-lymphocyte crossmatching by complement-dependent lymphocytotoxicity and flow cytometry with a serum consisting anti-HLA-A24 antibody revealed negative results, implying that an individual with this allele would not carry a functioning A24 antigen. These findings highlight the importance of identifying a null HLA allele by employing appropriate molecular method and providing expected crossmatching outcomes in a real-world transplantation setting.

Discovery of the novel HLA-A*33:256 allele, a variant of HLA-A*33:03:01:01, by next-generation sequencing.

One nucleotide substitution in codon 280 of HLA-A*33:03:01:01 results in the novel allele, HLA-A*33:256.

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.

Complement-dependent cytotoxicity crossmatch in solid organ transplantation: The gold standard or golden history?

Complement-dependent cytotoxicity crossmatch (CDC-XM) has been considered for many years the standard of practice for determining compatibility in solid organ transplantation (SOT). However, as this method is laborious, time intensive and lacks sensitivity and specificity, it has been replaced in many laboratories worldwide by flow cytometry crossmatch (FCXM) and/or virtual crossmatch (vXM). With this study we intend to show the relevance of performing CDC-XM in the era of virtual crossmatching. We retrospectively analyzed 1,007 consecutive T and B cell deceased donor (DD) CDC-XMs performed in parallel using non-treated and dithiothreitol (DTT) treated sera between May 2022 and January 2023 in waitlisted patients with no donor specific antibodies (DSA) against HLA-A, B and/or DR antigens. Thirty five of 1,007 (3.5%) T cell crossmatches and 132 of 1,007 (13.1%) B cell crossmatches were positive with non-treated sera. Correlation with the vXM demonstrated no DSA in any of the positive T cell crossmatches. DSA were also absent in 126/132 positive B cell crossmatches, indicating a high rate of false positive CDC-XM. Indeed, only 4/35 T cell and 13/132 B cell CDC-XM remained positive after treatment with DTT, confirming that false positive reactivity with non-treated sera is high. Class I HLA DSA against C locus antigens were present in 17/1,007 T cell crossmatches and none were detected by CDC-XM (sensitivity = 0%). Similarly, only 6/77 B cell crossmatches with DSA targeting HLA-C, DQ and/or DP antigens were CDC-XM positive (sensitivity = 7.8%). Furthermore, only 4/6 positive B cell CDC-XM were confirmed to have complement binding potential using the C1q assay, suggesting additional false positive reactivity in 2/6 of the positive CDC-XM. Our study demonstrates that CDC-XM exhibits poor sensitivity, high false positive reactivity (especially without DTT treatment) and does not meaningfully contribute to pre-transplant compatibility testing in the context of vXM based allocation. Furthermore, the use of CDC-XM can unnecessarily delay or even prevent safe and appropriate transplant allocation.

Crossmatch assays in transplantation: Physical or virtual?: A review.

The value of the crossmatch test in assessing pretransplant immunological risk is vital for clinical decisions, ranging from the indication of the transplant to the guidance of induction protocols and treatment with immunosuppressants. The crossmatch tests in transplantation can be physical or virtual, each with its advantages and limitations. Currently, the virtual crossmatch stands out for its sensitivity and specificity compared to the physical tests. Additionally, the virtual crossmatch can be performed in less time, allowing for a reduction in cold ischemia time. It shows a good correlation with the results of physical tests and does not negatively impact graft survival. Proper communication between clinicians and the transplant immunology laboratory will lead to a deeper understanding of each patient's immunological profile, better donor-recipient selection, and improved graft survival.

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.