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

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, myTAI HEART ® , 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 myTAI HEART 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

A Changing Paradigm in Heart Transplantation: An Integrative Approach for Invasive and Non-Invasive Allograft Rejection Monitoring

“…Dd-cf-DNA testing detected acute rejection with an area under the curve of 0.64 and provided an estimated NPV of 97.1% and positive predictive value of 8.9%. The limitation of genotyping donors was also addressed by North et al, who proposed a highly sensitive and quantitative multiplexed PCR test for 94 highly informative bi-allelic SNPs [ 6 ].…”

“…Using a specifically defined cut-off, the assay’s clinical performance characteristics included an NPV of 100% for grade 2R or higher ACR, with 100% sensitivity and 75.48% specificity. This test’s analytical validity facilitates conservative stratification of the probability of moderate to severe ACR as a potential companion tool for EMB in reducing the incidence of invasive biopsies, following the patients’ response to therapy [ 6 ].…”

“…Due to these drawbacks, numerous attempts have recently been made to explore the possibility of identifying a sensitive and non-invasive approach that might be used in combination with tissue histology to reduce the frequency of biopsies [ 5 ]. Omics approaches have expanded the number of relevant biomarkers for the diagnosis of allograft rejection [ 6 ] and surveillance mainly in asymptomatic patients [ 7 ].…”

The Organ Trail: A Review of Biomarkers of Organ Failure

“…DdcfDNA has also been utilized in the risk stratification of organ rejection post heart transplant in pediatrics and adults as reported by North et al. ( 40 ).…”

“…Thus, ddcfDNA can be used as a tissue specific biomarker to determine the likelihood of organ failure due to allograft rejection. DdcfDNA has also been utilized in the risk stratification of organ rejection post heart transplant in pediatrics and adults as reported by North et al (40).…”