BACKGROUND:Patients awaiting a solid organ transplant require pretransplant testing including high-resolution HLA typing and screening of HLA antibodies. However, the standard HLA antibody testing cannot encompass all the HLA alleles available in the Australian donor pool. With the advent of high-resolution HLA typing for organ allocation, there is an increased prevalence of detecting uncommon HLA alleles. It is challenging to manually determine whether patients have donor-specific HLA antibodies against these antigens. To address this issue, a bioinformatic pipeline, HLA-EP-RESOLVER, was developed to assess eplet reactivity for rare HLA unacceptable antigens (UAs). METHODS:The HLA-EP-RESOLVER pipeline was validated against sensitized patients, and a list of candidate UAs was identified for each patient. The candidate UAs must meet the following criteria: (1) match at the first field with existing UAs, (2) share eplets or serological types with the current UAs, (3) do not share the patient's self-eplets or eplets on negative beads, and/or (4) in the exception rules. The Australian deceased donor pool was used as the initial model. The predicted UAs were then confirmed with physical crossmatching. RESULTS:The pipeline proved to be highly accurate at predicting high-risk antigens by utilizing eplet analysis through HLA Matchmaker, and the HLA data extracted from the Australian donor population. The reactivity of several candidate UAs were confirmed by cellular verification. CONCLUSIONS:The HLA-EP-RESOLVER program was shown to be rapid, useful, and highly accurate in determining acceptable or unacceptable HLA alleles in the new era of rapid high-resolution donor typing before allocation.

The human Killer cell Immunoglobulin-like Receptor (KIR) genes, found on chromosome 19, encode for cell surface protein receptors that, through interaction with their ligand, modulate the action of Natural Killer (NK) cells and some subsets of T lymphocytes. KIR genes exhibit extensive variation through variable gene content, copy number, and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant, and infectious disease progression. KIR gene content has been used in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. With the introduction of massively parallel clonal next generation sequencing and single molecule long read third generation sequencing, allele level determination of KIR genotypes has become feasible. We describe a method for amplicon-based long read sequencing on the Oxford Nanopore Technologies platform that provides largely unambiguous allele level typing of KIR genes. The method was validated using DNA extracted from 48 10th International Histocompatibility Workshop (IHWS) cell lines with previously published allele level KIR genotypes and 176 Western Australian samples previously tested for the presence or absence of KIR genes. Our long-read sequencing method was able to accurately determine KIR alleles with an overall concordance of 97%-99% with the published data. Importantly, phasing ambiguity caused by the inability to phase heterozygous base positions over long stretches of gene sequence was resolved in several samples. Thus, our long read PCR sequencing strategy can be used to determine KIR genotypes at allele resolution level.

HLA-compatibility remains an important triage test for deceased donor kidney allocation. Low-intermediate resolution donor HLA-typing is typically available at allocation, but its accuracy in assigning pre-transplant donor-specific anti-HLA antibody (DSA) and HLA mismatches compared to 2-field high-resolution typing is poorly characterised. Consecutive deceased donor/recipient pairs from a single centre between 2016 and 2020 were included. Majority of donor typing at HLA-ABDRB1 loci were performed at low-intermediate resolution, with 2-field high-resolution NGS typing across extended loci performed by NGS-technique post-transplantation. We compared the two typing methods for (1) accuracy of pre-transplant DSA assignment; (2) misassignment of HLA-antigen/allele mismatches and performance of each model for acute rejection and (3) proportion of recipients who developed de novo DSA (dnDSA) when matched at antigen but mismatched at allele level. Of 179 deceased donor/recipient pairs, 157 donors had low-intermediate resolution typing and 22 with high-resolution ONT typing. Sixty-two recipients (35%) had potential pre-transplant DSAs, with incorrect assignment of allele-specific Class I and II actual DSAs in 31% and 53% of cases, respectively. NGS typing identified 59 (33%) additional HLA-DRB1 allele mismatches. ONT typing accurately assigned pre-transplant DSAs and allele mismatches in all cases. Seven (4%) recipients with antigen/allele level discordance developed dnDSAs, majority HLA-DQ antibodies. Two-field high-resolution donor HLA typing may provide a more accurate transplant immunological risk assessment and identify those at risk of developing dnDSA to matched HLA antigen.

Kidney donor allocation can occasionally be difficult in Australia given a small population spread over vast distances. Therefore, between 2017 and 2019 our service allowed transplantation from deceased donors into local (same-state) preemptive recipients, only if no well-matched dialysis-dependent transplant waitlist recipient was available. Transplantation using this novel allocation pathway was associated with good clinical and immunological outcomes.

The CDC crossmatch test is being phased out in solid organ donor allocation, and standard luminex single antigen bead assays do not differentiate complement activating function of HLA antibodies. The current study investigated the LIFECODES C3d‐binding assay to determine if it could accurately predict actual T and B cell CDC results in a cohort of highly sensitised patients. Nineteen serum samples from different highly sensitised solid organ patients were crossmatched against cells from 62 unique donors, with 174 total T and B cell crossmatches performed. The sera also underwent SAB assay using OLI and LC platforms, and C3d‐binding assay. Complement activating ability of each unique HLA antibody specificity detected using SAB was assigned based on the actual CDC results, which was then used to determine the accuracy of the C3d‐binding assay. The C3d‐binding assay was found to be highly accurate, with sensitivity of 95%, specificity 89% and negative predictive value 97% for class I DSA and the T cell CDC crossmatch results. Furthermore, we found 100% accuracy for prediction of the complement activating function of HLA‐C antibodies. Negative predictive value of above 90% was also found for HLA class II DSA. C3d‐binding proved more accurate than virtual crossmatch alone to predict CDC results. This study confirms that the C3d‐binding assay predicts actual CDC crossmatch results accurately. In particular, the high negative predictive value of the C3d‐binding assay may be extremely useful to define HLA antibodies that do not activate complement in highly sensitised recipients.

Human cytomegalovirus (HCMV) is carried lifelong by similar to 80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 amplified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three individuals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort.

Killer immunoglobulin-like receptors (KIR) regulate the function of natural killer cells through interactions with various ligands on the surface of cells, thereby determining whether natural killer (NK) cells are to be activated or inhibited from killing the cell being interrogated. The genes encoding these proteins display extensive variation through variable gene content, copy number and allele polymorphism. The combination of KIR genes and their ligands is implicated in various clinical settings including haematopoietic stem cell and solid organ transplant and infectious disease progression. The determination of KIR genes has been used as a factor in the selection of optimal stem cell donors with haplotype variations in recipient and donor giving differential clinical outcomes. Methods to determine KIR genes have primarily involved ascertaining the presence or absence of genes in an individual. With the more recent introduction of massively parallel clonal next-generation sequencing and single molecule very long read length third-generation sequencing, high-resolution determination of KIR alleles has become feasible. Determining the extent and functional impact of allele variation has the potential to lead to further optimisation of clinical outcomes as well as a deeper understanding of the functional properties of the receptors and their interactions with ligands. This review summarizes recently published high-resolution KIR genotyping methods and considers the various advantages and disadvantages of the approaches taken. In addition the application of allele level genotyping in the setting of transplantation and infectious disease control is discussed.

Understanding the basis of the immune determinants controlling disease outcome is critical to provide better care to patients and could be exploited for therapeutics and vaccine design. The discovery of the human immunodeficiency virus (HIV) virus as the causing agent of acquired immunodeficiency syndrome (AIDS) decades ago, led to a tremendous amount of research. Among the findings, it was discovered that some rare HIV+ individuals, called HIV controllers (HICs), had the ability to control the virus and keep a low viral load without the need of treatment. This ability allows HICs to delay or avoid progression to AIDS. HIV control is strongly associated with the expression of human leukocyte antigen (HLA) alleles in HICs. From the HIV protective HLAs described, HLA-B57 is the most frequent in HIC patients. HLA-B57 can present a large range of highly conserved Gag-derived HIV peptides to CD8+ T cells and natural killer (NK) cells, both the focus of this review. So far there are limited differences in the immune response strength, magnitude, or receptor repertoire towards HIV epitopes that could explain viral control in HICs. Interestingly, some studies revealed that during early infection the large breadth of the immune response towards HIV mutants in HLA-B57+ HIC patients, might in turn influence the disease outcome.

Ischaemic brain damage induces autoimmune responses, including the production of autoantibodies with potential neuroprotective effects. Platelets share unexplained similarities with neurons, and the formation of anti-platelet antibodies has been documented in neurological disorders. The aim of this study was to investigate the presence of anti-platelet antibodies in the peripheral blood of patients after ischaemic stroke and determine any clinical correlations. Using a flow cytometry-based platelet immunofluorescence method, we detected platelet-reactive antibodies in 15 of 48 (31%) stroke patients and two of 50 (4%) controls (p < 0.001). Western blotting revealed heterogeneous reactivities with platelet proteins, some of which overlapped with brain proteins. Stroke patients who carried anti-platelet antibodies presented with larger infarcts and more severe neurological dysfunction, which manifested as higher scores on the National Institutes of Health Stroke Scale (NIHSS; p = 0.009), but they had a greater recovery in the NIHSS by the time of hospital discharge (day 7 ± 2) compared with antibody-negative patients (p = 0.043). Antibodies from stroke sera reacted more strongly with activated platelets (p = 0.031) and inhibited platelet aggregation by up to 30.1 ± 2.8% (p < 0.001), suggesting the potential to interfere with thrombus formation. In conclusion, platelet-reactive antibodies can be found in patients soon after ischaemic stroke and correlate with better short-term outcomes, suggesting a potential novel mechanism limiting thrombosis.

HLA eplet matching is a novel approach to define acceptable HLA mismatches for transplant recipients. We performed an eplet analysis of three different transplant case‐series to determine if the available software programs gave accurate results. Eplet analysis was performed for three different transplant case‐series typed by NGS for all HLA class I and II loci. The three different HLA datasets were entered into both the HLAMatchmaker program (v2.1) and OLI Fusion MatchMaker (v4.2) software tools. Eplet results which were discordant were cross referenced against eplet registry and published HLA allele sequence data to determine the correct assignments. The comparison reveals that there was poor concordance between the two eplet programs. Analysis of the same donor/recipient pair often gave rise to different total eplet scores, incorrect eplet mismatches and antibody verification status, and both programs have eplets assigned to incorrect HLA alleles. Overall, the OLI Fusion MatchMaker eplet tool gave more accurate and useful eplet results. Eplet matching is still primarily a research tool. Before eplet matching can enter routine clinical practice further work is required to validate the accuracy of available eplet software programs. Incorrect eplet assignment could have serious adverse consequences in the clinical transplant setting.

The 17th International HLA and Immunogenetics Workshop (IHIW) organizers conducted a Pilot Study (PS) in which 13 laboratories (15 groups) participated to assess the performance of the various sequencing library preparation protocols, NGS platforms and software in use prior to the workshop. The organizers sent 50 cell lines to each of the 15 groups, scored the 15 independently generated sets of NGS HLA genotyping data, and generated "consensus" HLA genotypes for each of the 50 cell lines. Proficiency Testing (PT) was subsequently organized using four sets of 24 cell lines, selected from 48 of 50 PS cell lines, to validate the quality of NGS HLA typing data from the 34 participating IHIW laboratories. Completion of the PT program with a minimum score of 95% concordance at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci satisfied the requirements to submit NGS HLA typing data for the 17th IHIW projects. Together, these PS and PT efforts constituted the 17th IHIW Quality Control project. Overall PT concordance rates for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 were 98.1%, 97.0% and 98.1%, 99.0%, 98.6%, 98.8%, 97.6%, 96.0%, 99.1%, 90.0% and 91.7%, respectively. Across all loci, the majority of the discordance was due to allele dropout. The high cost of NGS HLA genotyping per experiment likely prevented the retyping of initially failed HLA loci. Despite the high HLA genotype concordance rates of the software, there remains room for improvement in the assembly of more accurate consensus DNA sequences by NGS HLA genotyping software.

Very high resolution typing of HLA genes by Next Generation Sequencing (NGS) methods is becoming mainstream in histocompatibility laboratories for the workup of haematopoietic stem cell and solid organ transplant patients and donors. Determining the full sequence for class I and extended exon coverage for class II genes produces HLA types with far fewer ambiguities than traditional Sanger sequencing based typing (SBT) and other lower resolution typing methods. In addition NGS brings benefits of higher throughput and lower cost. We have validated the Illumina TruSight HLAv2 typing kit for HLA-A, B, C, DRB1/3/4/5, DQA1, DQB1, DPA1 and DPB1. A total of 151 samples were tested following the method described by the vendor. All method steps were carried out manually. Testing comprised 6 batches of 24 samples and 1 batch of 12 samples, which were made up of 73 local DNA samples typed previously by a combination of SBT, SSP and SSO, 54 DNA samples from the UCLA reference panel and 24 samples from the 17th International HLA Workshop Study blinded proficiency panel. Reference types for all eleven loci were not available for all local samples, four digit types were available for all eleven loci for the UCLA samples. Sequence analysis and genotype calling was performed using Illumina Assign for Trusight HLA v2.1 and GenDX NGSEngine. HLA type concordance rate, ambiguity rate and accuracy were examined. Total percent concordance between the reference type (n = 130) and genotype determined by Assign was as follows: A: 100; B: 99.2; C: 99.2; DRB1: 97.6.5; DRB345: 98.4; DQA1: 99.2; DQB1: 100; DPA1: 100; and DPB1: 97.6. No allele (1st, 2nd and 3rd field) or genotype ambiguity was found for HLA-A, B, C, DRB345 DQB1 or DPA1. An ambiguous type was determined in 4 cases for DRB1, 3 cases for DQA1 and 29 cases for DPB1. In the case of DRB1 and DPB1 this was due to lack of part of the gene sequence and for DQA1 it was due to poor read depth in those samples. Average read depth across all loci was 223. Repeat rates varied between 1.3% (A & B) and 10.9% (DPA1). The Illumina TruSight HLAv2 typing kit provides a suitable first pass high resolution typing system for stem cell and solid organ transplant patients and donors. Accuracy is excellent and the ambiguity levels are very low. We intend to introduce this method for routine HLA typing.

The interaction of membrane-bound platelet-specific glycoproteins (GP) with the extracellular matrix plays a significant role in hemostasis. Human Platelet Antigens (HPA) are glycoproteins expressed on platelet membranes. Polymorphisms in Human Platelet Antigens (HPA) can stimulate production of antibodies in recipients of transfused platelets from donors with different HPAs. Platelet incompatibility is associated with various forms of thrombopenias, post-transfusion purpura and other blood disorders. HPA typing is typically performed with serological techniques that are currently being replaced by molecular methods. Linkage Biosciences has developed and validated a test consisting of 24 reactions that identifies both variants of the 12 relevant SNPs located within HPA genes. Of these 24 variants, 18 alleles have been shown to produce alloantigens. The LinkSe¯q system overcomes the major challenges of HPA molecular typing by providing a robust and automated approach resulting in increased laboratory productivity and decreased turn-around time. The analysis is mediated by SureTyper™ software which generates rapid typing results. With less than 10 minutes of hands-on set-up and no further operator intervention with the reagents, LinkSe¯q uses state of the art real-time PCR detection to provide complete molecular genotyping results in approximately 90 minutes. Further, since amplified products are never handled, the risk of laboratory contamination is reduced. The LinkSe¯q system can provide a simple, effective and robust method for determining the HPA typing profile of DNA samples.