A two-threshold algorithm using donor-derived cell-free DNA fraction and quantity to detect acute rejection after...

Area of Science:

• Transplantation immunology
• Molecular diagnostics
• Biomarker discovery

Background:

• Donor-derived cell-free DNA (dd-cfDNA) is a key biomarker for acute rejection (AR) in heart transplantation (HTx).
• Total cell-free DNA (cfDNA) levels can influence dd-cfDNA fraction measurements.
• dd-cfDNA quantity offers a complementary metric to fraction for AR assessment.

Purpose of the Study:

• To evaluate a 2-threshold algorithm (2TA) combining dd-cfDNA fraction (dd-cfDNA%) and donor-quantity score (DQS) for AR detection in HTx.
• To assess if the 2TA improves upon dd-cfDNA% alone in diagnosing AR.
• To determine the diagnostic performance metrics of the 2TA.

Main Methods:

• Prospective collection of 808 plasma samples from 187 adult HTx patients with contemporaneous endomyocardial biopsies.
• Analysis of cfDNA using a single nucleotide polymorphism-based next-generation sequencing workflow.
• Measurement of dd-cfDNA% and DQS, with AR diagnosis based on biopsy results.

Main Results:

• Both dd-cfDNA% and DQS were significantly elevated in AR compared to non-AR samples (P < 10^-14).
• The 2TA (dd-cfDNA% > 0.26% or DQS > 18 copies/mL) achieved 86.5% sensitivity and 83.6% specificity for AR detection (AUC = 0.881).
• The 2TA demonstrated improved performance over dd-cfDNA% alone, with a mean net reclassification index of 16.4% and a 37.3% reduction in false positives.

Conclusions:

• The 2TA effectively detects acute rejection in heart transplant recipients.
• Combining dd-cfDNA fraction and quantity enhances AR detection accuracy compared to fraction alone.
• This algorithmic approach may improve non-invasive AR monitoring in HTx patients.