Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability

Autoři: Paula E. North aff001;  Emily Ziegler aff003;  Donna K. Mahnke aff003;  Karl D. Stamm aff003;  Angela Thomm aff003;  Paul Daft aff003;  Mary Goetsch aff004;  Huan ling Liang aff004;  Maria Angeles Baker aff003;  Adam Vepraskas aff003;  Chris Rosenau aff003;  Mahua Dasgupta aff005;  Pippa Simpson aff005;  Michael E. Mitchell aff002;  Aoy Tomita-Mitchell aff004
Působiště autorů: Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America aff001;  Childrens Hospital of Wisconsin, Milwaukee, Wisconsin, United States of America aff002;  TAI Diagnostics, Inc., Wauwatosa, Wisconsin, United States of America aff003;  Department of Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America aff004;  Department of Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin, United States of America aff005
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0227385


Lifelong noninvasive rejection monitoring in heart transplant patients is a critical clinical need historically poorly met in adults and unavailable for children and infants. Cell-free DNA (cfDNA) donor-specific fraction (DF), a direct marker of selective donor organ injury, is a promising analytical target. Methodological differences in sample processing and DF determination profoundly affect quality and sensitivity of cfDNA analyses, requiring specialized optimization for low cfDNA levels typical of transplant patients. Using next-generation sequencing, we previously correlated elevated DF with acute cellular and antibody-mediated rejection (ACR and AMR) in pediatric and adult heart transplant patients. However, next-generation sequencing is limited by cost, TAT, and sensitivity, leading us to clinically validate a rapid, highly sensitive, quantitative genotyping test, myTAIHEART®, addressing these limitations. To assure pre-analytical quality and consider interrelated cfDNA measures, plasma preparation was optimized and total cfDNA (TCF) concentration, DNA fragmentation, and DF quantification were validated in parallel for integration into myTAIHEART reporting. Analytical validations employed individual and reconstructed mixtures of human blood-derived genomic DNA (gDNA), cfDNA, and gDNA sheared to apoptotic length. Precision, linearity, and limits of blank/detection/quantification were established for TCF concentration, DNA fragmentation ratio, and DF determinations. For DF, multiplexed high-fidelity amplification followed by quantitative genotyping of 94 SNP targets was applied to 1168 samples to evaluate donor options in staged simulations, demonstrating DF call equivalency with/without donor genotype. Clinical validation studies using 158 matched endomyocardial biopsy-plasma pairs from 76 pediatric and adult heart transplant recipients selected a DF cutoff (0.32%) producing 100% NPV for ≥2R ACR. This supports the assay’s conservative intended use of stratifying low versus increased probability of ≥2R ACR. myTAIHEART is clinically validated for heart transplant recipients ≥2 months old and ≥8 days post-transplant, expanding opportunity for noninvasive transplant rejection assessment to infants and children and to all recipients >1 week post-transplant.

Klíčová slova:

Biopsy – Blood – Blood plasma – Cardiac transplantation – DNA fragmentation – Genotyping – White blood cells – Alu elements


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