Performance characteristics of next-generation sequencing–based engraftment monitoring and microchimerism detection in allogeneic hematopoietic cell transplantation: A practical approach for clinical assay validation Journal Article
| Authors: | Blouin, A. G.; Nelson, W.; Geraghty, D.; Askar, M.; Ye, F. |
| Article Title: | Performance characteristics of next-generation sequencing–based engraftment monitoring and microchimerism detection in allogeneic hematopoietic cell transplantation: A practical approach for clinical assay validation |
| Abstract: | Chimerism analysis by next-generation sequencing (NGS) is an emerging method for engraftment monitoring after allogeneic hematopoietic cell transplantation. A high-sensitivity method is required for the detection of microchimerism (<1% chimerism), which may have clinical utility in early relapse detection, allograft monitoring in organ transplantation, and other allogeneic cellular therapies (such as microtransplantations). As more clinical laboratories adopt this method, a thorough assessment of performance is needed. This study evaluated one such NGS-based assay that uses both single-nucleotide polymorphisms and insertions/deletions as genetic markers. An assessment of accuracy, linearity, sensitivity, and reproducibility was performed. Analytical sensitivity was 0.2% donor for single donor and 0.5% donors for double donors. The assay showed a high degree of reproducibility over a full range of chimerism. Comparison to short-tandem-repeat (STR) PCR showed high concordance; yet <5% chimerism was consistently detected by NGS, but not by STR-PCR. Comparison to real-time quantitative PCR showed high concordance, but with lower correlation in the midrange (40% to 60% chimerism). Overall, the assay showed consistent performance with high sensitivity and accuracy compared with STR-PCR and real-time quantitative PCR across a full range of chimerism in the setting of single-donor and multidonor transplantations. In addition, criteria for quality metrics were established for sequencing performance and data analysis and considerations made for clinical laboratory validation of NGS-based chimerism assay and analysis software. © 2024 Association for Molecular Pathology and American Society for Investigative Pathology |
| Keywords: | controlled study; allograft; major clinical study; single nucleotide polymorphism; validation process; sensitivity and specificity; sensitivity analysis; allele; chimera; engraftment; amplicon; clinical evaluation; data analysis; hematopoietic cell; benchmarking; allogeneic hematopoietic stem cell transplantation; intermethod comparison; real time polymerase chain reaction; genetic marker; cell transplantation; sample; molecular pathology; unrelated donor; short tandem repeat; organ transplantation; indel mutation; dna library; microchimerism; high throughput sequencing; measurement accuracy; human; article; related donor |
| Journal Title: | Journal of Molecular Diagnostics |
| Volume: | 26 |
| Issue: | 11 |
| ISSN: | 1525-1578 |
| Publisher: | Elsevier Science, Inc. |
| Date Published: | 2024-11-01 |
| Start Page: | 995 |
| End Page: | 1006 |
| Language: | English |
| DOI: | 10.1016/j.jmoldx.2024.07.003 |
| PUBMED: | 39181323 |
| PROVIDER: | scopus |
| PMCID: | PMC11524324 |
| DOI/URL: | |
| Notes: | Article -- MSK Cancer Center Support Grant (P30 CA008748) acknowledged in PubMed and PDF -- MSK corresponding authors are Amanda Blouin and Fei Ye -- Source: Scopus |
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