Targeted Next-Generation Sequencing for Human Leukocyte Antigen Typing in a Clinical Laboratory: Metrics of Relevance and Considerations for Its Successful Implementation.

CONTEXT: - Numerous feasibility studies to type human leukocyte antigens (HLAs) by next-generation sequencing (NGS) have led to the development of vendor-supported kits for HLA typing by NGS. Some clinical laboratories have introduced HLA-NGS, and many are investigating the introduction. Standards from accrediting agencies form the regulatory framework for introducing this test into clinical laboratories.

OBJECTIVES: - To provide an assessment of metrics and considerations relevant to the successful implementation of clinical HLA-NGS typing, and to provide as a reference a validated HLA-NGS protocol used clinically since December 2013 at the Children's Hospital of Philadelphia (Philadelphia, Pennsylvania).

DATA SOURCES: - The HLA-NGS has been performed on 2532 samples. The initial 1046 and all homozygous samples were also typed by an alternate method. The HLA-NGS demonstrated 99.7% concordance with the alternate method. Ambiguous results were most common at the DPB1 locus because of a lack of phasing between exons 2 and 3 or the unsequenced exon 1 (533 of 2954 alleles; 18.04%) and the DRB1 locus because of not sequencing exon 1 (75 of 3972 alleles; 1.89%). No ambiguities were detected among the other loci. Except for 2 false homozygous samples, all homozygous samples (1891) demonstrated concordance with the alternate method. The article is organized to address the critical elements in the preanalytic, analytic, and postanalytic phases of introducing this assay into the clinical laboratory.

CONCLUSIONS: - The results demonstrate that HLA typing by NGS is a highly accurate, reproducible, efficient method that provides more-complete sequencing information for the length of the HLA gene and can be the single methodology for HLA typing in clinical immunogenetics laboratories.