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The gene complex located on chromosome 19q13.4 encodes the Killer-cell Immunoglobulin-like Receptors (KIRs), which exhibit remarkable polymorphism in both gene content and sequences. Further, the repertoire of KIR genes varies within and between populations, creating a diverse pool of KIR genotypes. This study was carried out to characterize KIR genotypes and haplotypes among 379 Arab Kuwaiti individuals including 60 subjects from 20 trio families, 49 hematopoietic cell transplantation (HCT) recipients and 270 healthy Kuwaiti volunteer HCT donors. KIR Genotyping was performed by a combination of reverse sequence specific oligonucleotide probes (rSSO) and/or Real Time PCR. The frequencies of KIR genes in 270 healthy Kuwaiti volunteer donors were compared to previously reported frequencies in other populations. In addition, we compared the differences in KIR repertoire of patients and healthy donors to investigate the reproducibility of previously reported significant differences between patients with hematological malignancies and healthy donors. The observed frequencies in our cohort volunteer HCT donors was comparable to those reported in neighboring Arab populations. The activating genes KIR2DS1, KIR2DS5 and KIR3DS1 and the inhibitory gene KIR2DL5 were significantly more frequent in patients compared to healthy donors, however, none of the previously reported differences were reproducible in our Kuwaiti cohort. This report is the first description of KIR gene carrier frequency and haplotype characterization in a fairly large cohort of the Kuwaiti population, which may have implications in KIR based HCT donor selection strategies., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Many clinical laboratories supporting solid organ transplant programs use multiple HLA genotyping technologies, depending on individual laboratory needs. Sequence-specific primers and quantitative polymerase chain reaction (qPCR) serve the rapid turnaround necessary for deceased donor workup, while sequence-specific oligonucleotide probe (SSOP) technology is widely employed for higher volumes. When clinical need mandates high-resolution data, Sanger sequencing-based typing (SBT) has been the "gold standard." However, all those methods commonly yield ambiguous typing results that utilize valuable laboratory resources when resolution is required. In solid organ transplantation, high-resolution typing may provide critical information for highly sensitized patients with donor-specific anti-HLA antibodies (DSA), particularly when DSA involve HLA alleles not discriminated by SSOP typing. Arguments against routine use of SBT include assay complexity, long turnaround times (TAT), and increased costs. Here, we compare a next generation sequencing (NGS) technology with SSOP for accuracy, effort, turnaround time, and level of resolution for genotyping of 11 HLA loci among 289 specimens from five clinical laboratories. Results were concordant except for SSOP misassignments in eight specimens and 21 novel sequences uniquely identified by NGS. With few exceptions, SSOP generated ambiguous results while NGS provided unambiguous three-field allele assignments. For complete HLA genotyping of up to 24 samples by either SSOP or NGS, bench work was completed on day 1 and typing results were available on day 2. This study provides compelling evidence that, although not viable for STAT typing of deceased donors, a single-pass NGS HLA typing method has direct application for solid organ transplantation., (© 2019 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd.)

Current high-resolution HLA typing technologies frequently produce ambiguous results that mandate extended testing prior to reporting. Through multiplex sequencing of individual amplicons from many individuals at multiple loci, next generation sequencing (NGS) promises to eliminate heterozygote ambiguities and extend the breadth of genetic data acquired with little additional effort. We report here on assessment of a novel NGS HLA genotyping system for resequencing exons 2 and 3 of DRB1/B3/B4/B5, DQA1 and DQB1 and exon 2 of DPA1 and DPB1 on the MiSeq platform. In a cohort of 2605 hematopoietic cell transplant recipients and donors, NGS achieved 99.6% accuracy for DRB1 allele assignments and 99.5% for DQB1, compared to legacy genotypes generated pretransplant. NGS provided at least single 4-digit allele resolution for 97% of genotypes at DRB1 and 100% at DQB1. Overall, NGS typing identified 166 class II alleles, including 9 novel sequences with greater than 99% accuracy for DRB1 and DQB1 genotypes and elimination of diploid ambiguities through in-phase sequencing demonstrated the robust reliability of the NGS HLA genotyping reagents and analysis software employed in this study., (Copyright © 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Clinical immunogenetics laboratories performing routine sequencing of human leukocyte antigen (HLA) genes in support of hematopoietic cell transplantation are motivated to upgrade to next-generation sequencing (NGS) technology by its potential for cost savings as well as testing accuracy and flexibility. While NGS machines are available and simple to operate, there are few systems available that provide comprehensive sample preparation and data analysis workflows to complete the process. We report on the development and testing of the Integrated Genotyping System (IGS), which has been designed to specifically address the challenges associated with the adoption of NGS in clinical laboratories. To validate the system for a variety of sample DNA sources, we have tested 336 DNA specimens from whole blood, dried blood spots, buccal swabs, and lymphoblastoid cell lines. HLA class I and class II genotypes were derived from amplicon sequencing of HLA-A, -B, -C for exons 1–7 and HLA-DPA1, -DPB1, -DQA1, -DQB1, -DRB1, -DRB3, -DRB4, -DRB5 for exons 1–4. Additionally, to demonstrate the extensibility of the IGS to other genetic loci, KIR haplotyping of 93 samples was carried out in parallel with HLA typing using a workflow based on the HLA system. These results are discussed with respect to their applications in the clinical setting and consequent potential for advancing precision medicine. [ABSTRACT FROM AUTHOR]

Aim Increasingly, laboratories that support hematopoietic cell transplantation programs are implementing NGS technology for high resolution HLA genotyping. Extension of NGS to other laboratory services can provide efficiency in staffing and equipment usage. We have evaluated NGS of SNP and InDel polymorphisms for engraftment monitoring. Beyond workplace efficiencies, NGS of InDel polymorphism is not subject to stutter artifacts that can compromise results from the STR technology currently in wide use for chimerism analysis. Methods A panel of 49 polymorphisms on 20 chromosomes, comprising 37 SNPs on 18 chromosomes and 12 InDels on 10 chromosomes, was assembled. Initial amplification for the panel was reduced to 2 multiplexed PCRs with 5 ng of sample DNA per reaction. Total time for library preparation is 6 h, followed by a 7 h sequencing run on the MiSeq platform, with 1 h for chimerism analysis. Hands-on technologist time consists of just 3 of the 14 h time frame. Preliminary evaluation involved 4 sets of artificial mixtures of DNA from 2 unrelated individuals, with mixtures ranging from 10% to 0.1%, see Table. Results On average, 30 informative markers were identified between unrelated individuals. NGS achieved 0.5% sensitivity in all 4 sets of mixtures. The table shows the experimental values and standard deviation, SD, compared with the expected ratios among informative the polymorphisms. Informative InDel polymorphisms showed no false positive artifacts in unmixed specimens. Conclusions The results show that NGS technology provides informative, accurate and sensitive detection of a minority species in mixed specimens. With the 6 h library preparation and 7 h sequencing run, NGS offers rapid turnaround for result reporting. Additional testing is underway to assess the NGS technology in low cell count specimens and in mixtures of 3 or more unrelated individuals, intended to simulate retransplant and double cord donor transplant. [ABSTRACT FROM AUTHOR]

Introduction Accuracy and precision are critically important in the assessment of minimal residual disease (MRD) and chimerism following hematopoietic cell transplantation. Reliable and sensitive detection of changes in engraftment status can impact patient care by early detection of potential failure of engraftment or relapse. We evaluated a commercially available NGS technology for engraftment monitoring for sensitivity, accuracy and reproducibility. Objective Introduction of next generation sequencing technology for engraftment monitoring. Methods Samples analyzed by NGS at Baylor Transplant Immunology Laboratory included archived clinical specimens with historic results by STR, n=19, or qPCR, n=15, test results; 72 artificial mixtures of volunteer donors, and 5 ASHI external proficiency specimens. NGS analysis assessed 182 SNPs validated in the 1000 Genomes HapMap project and distributed across all 23 chromosomes, including the x chromosome. The rapid workflow allows a TAT of 2 days for up to 24 samples, with a less than 6 hour NGS library preparation protocol, followed by a 6 hour sequencing run time and 30 minute analysis. The low sample requirement, 1 ng DNA per PCR, facilitates chimerism assessment in multiple cell subsets including low frequency populations such as CD34-positive cells. Results NGS showed close concordance with historic results from STR and qPCR (figure 1), providing validation of NGS technology. Figure 2 compares NGS results versus the reference values of artificial mixtures and demonstrates NGS accuracy, while the low tech to tech variation data reveals assay precision. Limit of detection experiments showed reliable 0.1% sensitivity, as illustrated in Figure 3, with NGS analysis of 4 mixtures at 0.1%.. In addition, NGS reported results for the external proficiency samples correlated closely with the mean % donor reported throughout the full range of donor/recipient proportions including concordance with a sample of 100% donor. Conclusions Application of NGS technology to chimerism analysis in HCT provides accurate and precise test results. With the reliable 0.1% sensitivity and low input DNA that allows testing of multiple critical cell subsets, NGS is particularly relevant to early detection of MRD, graft failure, and relapse. [ABSTRACT FROM AUTHOR]