Prospective evaluation of donor-derived cell-free DNA for noninvasive detection of acute rejection in an Asian heart transplant population.

Endomyocardial biopsy (EMB) is the gold standard for diagnosing post-heart transplant acute rejection (AR) but is invasive. Donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive biomarker, yet thresholds for Asian recipients remain undefined. This prospective study enrolled 80 patients, with 299 dd-cfDNA specimens analyzed via a novel Chinese-developed next-generation sequencing assay targeting 48 Indel sites. A reference cohort (n = 42) established post-transplant dd-cfDNA kinetics, while a biopsy cohort (n = 41, 18 acute cellular rejection [ACR], 9 antibody-mediated rejection [AMR], 14 no rejection [NR]) paired with EMB evaluated diagnostic performance. dd-cfDNA peaked at week 1, with a stable trend emerging from week 4 and stable levels reached by week 8. AR patients had significantly higher dd-cfDNA levels than NR patients (0.87% vs. 0.23%, P < 0.001). The optimal composite AR threshold was 0.33% (AUC = 0.874, sensitivity 81.0%, specificity 85.7%). dd-cfDNA decreased post-therapy (P = 0.011) and showed an inverse association with left ventricular ejection fraction (LVEF) (P = 0.004). This study provides initial evidence for an assay- and cohort-specific dd-cfDNA threshold (0.33%) for AR detection in Chinese recipients. Given the modest NPV (75.0%), this threshold is better suited for a "rule-in" strategy, and low values should not be used in isolation to defer biopsy. External validation in multicenter cohorts is required before routine clinical implementation.