Abrogating biologics interference in flow cytometric crossmatching.

The flow cytometric crossmatch is currently the gold standard for evaluating donor and recipient histocompatibility. The assay however does have limitations and is sensitive to false positive reactions resulting from the presence of non-HLA antibodies or therapy related immune biologics. Such false positive reactions can lead to the inappropriate decline of an acceptable donor organ or unnecessary therapeutic intervention. Here we describe the successful validation of anti-idiotype blocking antibodies in prevention of false positive flow crossmatch results caused by biologic therapy. Blocking antibodies specific for the Fab portion of Rituximab and/or Alemtuzumab were incubated with biologic containing patient serum prior to use in flow cytometric crossmatching. Biologic blocking successfully negated false positive crossmatch results with Rituximab (B cell ave. % change = -97%) or Alemtuzumab (T cell ave. % change = -99%, B cell ave. % change = -95%) infused sera respectively. Simultaneous blocking of these biologics was also successful. A complex case is presented to demonstrate the application of this procedure.

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. Ultimately the RNA-Seq needs to be faster to be incorporated into the virtual crossmatching process. However, to demonstrate feasibility, the utility of the MinION sequencer (Oxford Nanopore Technologies, Oxford, UK) was evaluated in combination with RNA-Seq to generate HLA genotypes and to determine HLA class I expression. Although HLA class I expression varied among individuals, the pattern of HLA expression remained relatively consistent (HLA-B > HLA-A = HLA-C). HLA-A and -C had similar expression profiles. The impact of donor HLA expression was evaluated using serum samples containing a single donor-specific antibody (DSA). By making DSA consistent, donor HLA expression variability could be assessed. With consistent DSA mean fluorescence intensity, there was a direct relationship between the donor HLA expression to which the DSA is against and flow cytometric crossmatch median channel shifts.

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. The HLA loci most likely to fail genotyping from buccal swabs were DQB1, DPB1, and DPA1. Successful buccal swab samples had significantly less genomic DNA fragmentation compared to buccal swab samples that were unsuccessful. Increasing sequencing depth of coverage for heavily fragmented samples rescued HLA genotyping. This information provides laboratories with a quality assurance parameter that reduces the amount of repeat NGS needed to achieve high-resolution HLA genotyping. This information should further reduce laboratory and patient costs for HLA genotyping.

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. Although next-generation sequencing (NGS) is routinely used in molecular genetics, few clinical HLA laboratories use the technology. The performance characteristics of NGS HLA typing using TruSight HLA were determined using Sanger sequencing as the reference method. In total, 211 samples were analyzed with an overall accuracy of 99.8% (2954/2961) and 46 samples were analyzed for precision with 100% (368/368) reproducibility. Most discordant alleles were because of technical error rather than assay performance. More important, the ambiguity rate was 3.5% (103/2961). Seventy-four percentage of the ambiguities were within the DRB1 and DRB4 loci. HLA typing by NGS saves approximately $6000 per run when compared to Sanger sequencing. Thus, TruSight HLA assay enables high-throughput HLA typing with an accuracy, precision, ambiguity rate, and cost savings that should facilitate adoption of NGS technology in clinical HLA laboratories.