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.

Allele-specific quantification of human leukocyte antigen transcript isoforms by nanopore sequencing.

Introduction: While tens of thousands of HLA alleles have been identified by DNA sequencing, the contribution of alternative splicing to HLA diversity is not well characterized. In this study, we sought to determine if long-read sequencing could be used to accurately quantify allele-specific HLA transcripts in primary human lymphocytes.

Methods: cDNA libraries were prepared from peripheral blood lymphocytes from 12 donors and sequenced by nanopore long-read sequencing.

Shared graft-versus-leukemia minor histocompatibility antigens in DISCOVeRY-BMT.

T-cell responses to minor histocompatibility antigens (mHAs) mediate graft-versus-leukemia (GVL) effects and graft-versus-host disease (GVHD) in allogeneic hematopoietic cell transplantation. Therapies that boost T-cell responses improve allogeneic hematopoietic cell transplant (alloHCT) efficacy but are limited by concurrent increases in the incidence and severity of GVHD. mHAs with expression restricted to hematopoietic tissue (GVL mHAs) are attractive targets for driving GVL without causing GVHD.

Unique Molecular Identifier-Based High-Resolution HLA Typing and Transcript Quantitation Using Long-Read Sequencing.

HLA typing provides essential results for stem cell and solid organ transplants, as well as providing diagnostic benefits for various rheumatology, gastroenterology, neurology, and infectious diseases. It is becoming increasingly clear that understanding the expression of patient HLA transcripts can provide additional benefits for many of these same patient groups. Our study cohort was evaluated using a long-read RNA sequencing methodology to provide rapid HLA genotyping results and normalized HLA transcript expression.

Bilineal evolution of a U2AF1-mutated clone associated with acquisition of distinct secondary mutations.

Rare hematologic malignancies display evidence of both myeloid and lymphoid differentiation. Here, we describe such a novel bilineal event discovered in an adult woman with B-lymphoblastic leukemia (BLL). At the time of BLL diagnosis, the patient had a normal karyotype and a bulk sequencing panel identified pathogenic variants in BCOR, EZH2, RUNX1, and U2AF1, a genotype more typical of myeloid neoplasia.

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.

The receptor binding domain of the viral spike protein is an immunodominant and highly specific target of antibodies in SARS-CoV-2 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA.

The RBD Of The Spike Protein Of SARS-Group Coronaviruses Is A Highly Specific Target Of SARS-CoV-2 Antibodies But Not Other Pathogenic Human and Animal Coronavirus Antibodies.

A new Severe Acute Respiratory Syndrome Coronavirus variant (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus present with clinically inapparent, mild or severe disease. Currently, the presence of the virus in individual patients and at the population level is being monitored by testing symptomatic cases by PCR for the presence of viral RNA.

Performance of a multiplexed amplicon-based next-generation sequencing assay for HLA typing.

Background: Next-generation sequencing (NGS) has enabled efficient high-resolution typing of human leukocyte antigen (HLA) genes with minimal ambiguity. Most commercially available assays amplify individual or subgroup of HLA genes by long-range PCR followed by library preparation and sequencing. The AllType assay simplifies the workflow by amplifying 11 transplant-relevant HLA genes in one PCR reaction.

Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes () and the ATP-binding cassette B1 gene () may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability.

Using Nanopore Whole-Transcriptome Sequencing for Human Leukocyte Antigen Genotyping and Correlating Donor Human Leukocyte Antigen Expression with Flow Cytometric Crossmatch Results.

Transplant centers are increasingly using virtual crossmatching to evaluate recipient and donor compatibility. However, the current state of virtual crossmatching fails to incorporate donor human leukocyte antigen (HLA) expression in the assessment, despite numerous studies that have demonstrated the impact of donor HLA expression on physical crossmatch outcomes. Whole-transcriptome sequencing (RNA-Seq) for HLA enables simultaneous determination of HLA genotyping and relative HLA expression.

Development of data-driven models for the flow cytometric crossmatch.

HLA laboratories use virtual crossmatching (VXM) to predict recipient and donor compatibility using HLA antibody data and donor HLA type. Increasingly, transplant centers are utilizing VXM as the final compatibility determination prior to transplant. However, the VXM interpretation is based on HLA experience of individual transplant centers.

HLA alleles and haplotypes observed in 263 US families.

The 17th International HLA and Immunogenetics Workshop (IHIW) conducted a project entitled "The Study of Haplotypes in Families by NGS HLA". We investigated the HLA haplotypes of 1017 subjects in 263 nuclear families sourced from five US clinical immunogenetics laboratories, primarily as part of the evaluation of related donor candidates for hematopoietic stem cell and solid organ transplantation. The parents in these families belonged to five broad groups - African (72 parents), Asian (115), European (210), Hispanic (118) and "Other" (11).

Quality control project of NGS HLA genotyping for the 17th International HLA and Immunogenetics Workshop.

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.

Suitability of dried DNA for long-range PCR amplification and HLA typing by next-generation sequencing.

Storage and stable shipment of genomic DNA are of great concern to laboratories that may need to perform testing off archived samples. There are some dry-state storage methods that are available that have the potential to provide a way to store samples at room temperature for long periods of time as well as offer a means to ship DNA to other facilities without the same safety concerns that come with shipping liquid samples. The recovered DNA should be of sufficient integrity such that downstream applications can be performed without concern of the sample quality.

Characteristics, properties, and potential applications of circulating cell-free dna in clinical diagnostics: a focus on transplantation.

The initial discovery of cell-free DNA (cfDNA) in 1948 by Mandel and Metais has led to numerous investigations evaluating the role of cfDNA in various disease states. cfDNA has been characterized in various patient populations with similar results. cfDNA are typically 150 bp of double-stranded DNA that are thought to be released from nucleosomes during apoptosis and necrosis.

The Past, Present, and Future of HLA Typing in Transplantation.

The HLA region is the most polymorphic genes in the human genome and is associated with an increasing number of disease states. Historically, HLA typing methodology has been governed by phenotypic determination. This practice has evolved into the use of molecular methods such as real-time PCR, sequence-specific oligonucleotides, and sequencing-based methods.

HLAProfiler utilizes k-mer profiles to improve HLA calling accuracy for rare and common alleles in RNA-seq data.

Background: The human leukocyte antigen (HLA) system is a genomic region involved in regulating the human immune system by encoding cell membrane major histocompatibility complex (MHC) proteins that are responsible for self-recognition. Understanding the variation in this region provides important insights into autoimmune disorders, disease susceptibility, oncological immunotherapy, regenerative medicine, transplant rejection, and toxicogenomics. Traditional approaches to HLA typing are low throughput, target only a few genes, are labor intensive and costly, or require specialized protocols.

C1q Test for Identification of Sensitized Liver Recipients at Risk of Early Acute Antibody-Mediated Rejection.

BACKGROUND The majority of liver recipients transplanted with positive crossmatch have a post-transplant course free of acute antibody-mediated rejection (AMR). However, 10% of sensitized recipients develop early severe AMR. There is no clear strategy that would allow for predicting the postoperative course in sensitized liver recipients.

Buccal swab genomic DNA fragmentation predicts likelihood of successful HLA genotyping by next-generation sequencing.

Many clinical human leukocyte antigen (HLA) laboratories are adopting next-generation sequencing (NGS) technology for HLA genotyping. There have been several reports of the cost-benefit and reduction in turn-around-time provided by NGS. Ninety-six percent of buccal swabs and peripheral blood samples had reportable HLA genotyping by NGS.

Glomerular C4d deposits can mark structural capillary wall remodelling in thrombotic microangiopathy and transplant glomerulopathy: C4d beyond active antibody-mediated injury: a retrospective study.

Peritubular capillary C4d (ptc-C4d) usually marks active antibody-mediated rejection, while pseudolinear glomerular capillary C4d (GBM-C4d) is of undetermined diagnostic significance, especially when seen in isolation without concurrent ptc-C4d. We correlated GBM-C4d with structural GBM abnormalities and active antibody-mediated rejection in 319 renal transplant and 35 control native kidney biopsies. In kidney transplants, ptc-C4d was associated with GBM-C4d in 97% by immunofluorescence microscopy (IF) and 61% by immunohistochemistry (IHC; P < 0.

Performance Characteristics and Validation of Next-Generation Sequencing for Human Leucocyte Antigen Typing.

High-resolution human leukocyte antigen (HLA) matching reduces graft-versus-host disease and improves overall patient survival after hematopoietic stem cell transplant. Sanger sequencing has been the gold standard for HLA typing since 1996. However, given the increasing number of new HLA alleles identified and the complexity of the HLA genes, clinical HLA typing by Sanger sequencing requires several rounds of additional testing to provide allele-level resolution.