Validation of next-generation sequencing-based chimerism testing for accurate detection and monitoring of engraftment in hematopoietic stem cell transplantation.

Allogenic hematopoietic stem cell transplantation (allo-HSCT) is a life-saving treatment for various hematological disorders. The success of allo-HSCT depends on the engraftment of donor cells and the elimination of recipient cells monitored through chimerism testing. We aimed to validate a next-generation sequencing (NGS)-based chimerism assay for engraftment monitoring and to emphasize the importance of including the most prevalent cell subsets in proficiency testing (PT) programs. We evaluated the analytical performance of NGS-based chimerism testing (AlloSeq-HCT and CareDx) with a panel of targeted 202 informative single-nucleotide polymorphisms (SNPs) (i.e., linearity and precision, analytical sensitivity and specificity, system accuracy, and reproducibility). We further compared the performance of our NGS panel with conventional short tandem repeat (STR) analysis in unfractionated whole blood and cell-subset-enriched CD3 and CD66. Our NGS-based chimerism monitoring assay has an impressive detection limit (0.3% host DNA) for minor alleles and analytical specificity (99.9%). Pearson's correlation between NGS- and STR-based chimerism monitoring showed a linear relationship with a slope of 0.8 and r = 0.973. The concordance of allo-HSCT patients using unfractionated whole blood, CD3, and CD66 was 0.95, 0.96, and 0.54, respectively. Utilization of CD3+ cell subsets for mixed chimerism detection yielded an average of 7.3 ± 7-fold higher donor percentage detection compared to their corresponding unfractionated whole blood samples. The accuracy of the NGS assay achieved a concordance of 98.6% on blinded external quality control STR samples. The reproducibility series showed near 100% concordance with respect to inter-assay, inter-tech, inter-instrument, cell flow kits, and AlloSeq-HCT software versions. Our study provided robust validation of NGS-based chimerism testing for accurate detection and monitoring of engraftment in allo-HSCT patients. By incorporating the cell subsets (CD3 and CD66), the sensitivity and accuracy of engraftment monitoring are significantly improved, making them an essential component of any PT program. Furthermore, the implementation of NGS-based chimerism testing shows potential to streamline high-volume transplant services and improve clinical outcomes by enabling early relapse detection and guiding timely interventions.

Development of a Population-Based Newborn Screening Method for Severe Combined Immunodeficiency in Manitoba, Canada.

The incidence of Severe Combined Immunodeficiency (SCID) in Manitoba, (1/15,000), is at least three to four times higher than the national average and that reported from other jurisdictions. It is overrepresented in two population groups: Mennonites (ZAP70 founder mutation) and First Nations of Northern Cree ancestry (IKBKB founder mutation). We have previously demonstrated that in these two populations the most widely utilized T-cell receptor excision circle (TREC) assay is an ineffective newborn screening test to detect SCID as these patients have normal numbers of mature T-cells. We have developed a semi-automated, closed tube, high resolution DNA melting procedure to simultaneously genotype both of these mutations from the same newborn blood spot DNA extract used for the TREC assay. Parallel analysis of all newborn screening specimens utilizing both TREC analysis and the high-resolution DNA procedure should provide as complete ascertainment as possible of SCID in the Manitoba population.