Donor-derived cell-free DNA for reducing unnecessary biopsies in stable dnDSA-positive kidney transplant recipients: a multicenter, prospective study

Background: In clinically stable kidney transplant recipients with de novo donor-specific anti-HLA antibodies (dnDSA), only 30 ~ 40% exhibit biopsy-proven rejection, leading to a substantial number of unnecessary biopsies. Donor-derived cell-free DNA (dd-cfDNA) has emerged as a promising noninvasive biomarker of allograft injury. Nevertheless, its clinical utility for guiding biopsy decisions in dnDSA-positive stable kidney transplant recipients remains uncertain. This prospective multicenter cross-sectional study evaluated whether dd-cfDNA offers incremental predictive value for subclinical rejection in dnDSA-positive recipients with stable graft function. Methods: A total of 123 adult kidney transplant recipients with stable renal function were enrolled, comprising 46 dnDSA-negative and 77 dnDSA-positive patients. All participants underwent both dd-cfDNA and dnDSA testing, along with protocol or indication biopsies. Recipients with ABO-incompatible or preformed DSA-positive transplants were excluded. The diagnostic performance of dnDSA alone versus dnDSA combined with dd-cfDNA for predicting biopsy-proven subclinical rejection was assessed using receiver operating characteristic curve analysis. Results: Median dd-cfDNA levels were higher in dnDSA-positive patients (1.2% [IQR: 0.4–1.8]) compared with dnDSA-negative patients (0.3% [IQR: 0.2–0.4]). Subclinical rejection was histologically confirmed in 37 patients. Among dnDSA-positive recipients, combining dd-cfDNA ≥ 1.0 with dnDSA markedly improved diagnostic accuracy (AUC: 0.81; 95% CI: 0.74–0.88) compared with dnDSA alone (AUC: 0.74; 95% CI: 0.68–0.82). This combined approach achieved a positive predictive value of 46.2% and a negative predictive value of 97.8%. Moreover, elevated Banff microvascular inflammation (MVI) scores showed a strong correlation with higher dd-cfDNA levels ( P < 0.001). Conclusions: In dnDSA-positive kidney transplant recipients with stable renal function, dd-cfDNA provides incremental diagnostic value for identifying subclinical rejection and reflects the degree of microvascular injury. The integration of dd-cfDNA with dnDSA may enable more targeted and judicious use of biopsies, thereby reducing procedural burden while maintaining diagnostic precision.