Faculty Collaboration Database

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. PLoS One 2020;15(1):e0227385

Date

01/14/2020

Pubmed ID

31929557

Pubmed Central ID

PMC6957190

DOI

10.1371/journal.pone.0227385

Scopus ID

2-s2.0-85077765307 (requires institutional sign-in at Scopus site)   25 Citations

Abstract

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.

Author List

North PE, Ziegler E, Mahnke DK, Stamm KD, Thomm A, Daft P, Goetsch M, Liang HL, Baker MA, Vepraskas A, Rosenau C, Dasgupta M, Simpson P, Mitchell ME, Tomita-Mitchell A

Authors

Donna K. Mahnke Research Scientist I in the Pediatrics department at Medical College of Wisconsin
Michael Edward Mitchell MD Chief, Professor in the Surgery department at Medical College of Wisconsin
Aoy Tomita Mitchell PhD Professor in the Surgery department at Medical College of Wisconsin
Paula E. North MD, PhD Professor in the Pathology department at Medical College of Wisconsin
Pippa M. Simpson PhD Adjunct Professor in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adolescent
Adult
Biomarkers
Cell-Free Nucleic Acids
Child
Child, Preschool
Female
Graft Rejection
Heart Transplantation
Humans
Infant
Male
Tissue Donors
Transplants
Young Adult