Noninvasive Assay for Donor Fraction of Cell-Free DNA in Pediatric Heart Transplant Recipients

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

“…DF increased across rejection grades: the median DF in 0R (n = 134) was 0.12% (IQR 0.09-0.23%), in 1R (n = 21) was 0.84% (IQR 0.21-4.64%), and in 2R (n = 3) was 1.04% (IQR 0.85-3.13%). Note that these DF mean values, and their interquartile ranges, are slightly different than previously reported for this sample set [66] due to application of the definitive, clinically validated myTAI HEART bioinformatics algorithm here and a developmental version previously.…”

“…Sensitivity for ACR 1R may also reflect the fact that patients with recurrent rejection classified as 1R according to the 2004 ISHLT classification, which combines grades 1A, 1B, and 2 of the 1990 classification, have decreased freedom from late ACR and poorer long-term outcomes when of 1990 ISHLT 1B/2 histology [117,118]. Also of note, within this validation sample set, presence of coronary artery vasculopathy (CAV) correlated with increased DF [66]. The exquisite sensitivity and rapid response of the test to myocardial injury is demonstrated by a median 7.5 fold increase in cfDNA genomic equivalents/ml across all patient ages and weights within 15 minutes postendomyocardial biopsy, this increase significantly higher in smaller and younger patients [119].…”

“…Patients were followed by biopsy and myTAI HEART testing through from June 2010 through Aug 2016. A preliminary account of this study population and initial results of application of the myTAI HEART test to it were reported by members of our group as a Clinical Letter in the Journal of the American College Cardiology in 2018 [66]. Mean subject age at blood sampling was 12.7 ± 8.1 years (range 0.1 to 30.2 years); additional clinical demographics are here reported (see Results and discussion).…”

“…Accordingly, some have targeted known donor-recipient discriminative loci, such as Y chromosome-specific sites (e.g., SRY) in sex-mismatched donor-recipient pairs, of clearly limited application, and HLA genes, limited by poor informativity of target sequences in some recipient-donor pairs [37,38,61,64,65]. More recently, advanced technologies such as multiplexed high-fidelity amplification combined with allele-specific real-time quantitative PCR [66] and newer versions of next generation sequencing (NGS) [39,67] have been leveraged. These new methods improve sensitivity by interrogating a large multiplicity of highly informative single nucleotide polymorphism (SNP) sites, empowering prospective clinical studies that correlate cfDNA DF with biopsy-documented transplant rejection grade in well-defined patient populations of heart [39,66,68,69], kidney [70][71][72], liver [73] and lung [67] transplant recipients.…”

“…More recently, advanced technologies such as multiplexed high-fidelity amplification combined with allele-specific real-time quantitative PCR [66] and newer versions of next generation sequencing (NGS) [39,67] have been leveraged. These new methods improve sensitivity by interrogating a large multiplicity of highly informative single nucleotide polymorphism (SNP) sites, empowering prospective clinical studies that correlate cfDNA DF with biopsy-documented transplant rejection grade in well-defined patient populations of heart [39,66,68,69], kidney [70][71][72], liver [73] and lung [67] transplant recipients. The foundational tenants of DF chimeric analysis are generally applicable to all organ transplantation categories, and bioinformatic advancements using these newer technologies no longer require donor sample availability to determine DF in unrelated and most related donor-recipient pairs [66,74].…”