An Exploratory dd-cfDNA Cutoff in a Chinese Heart Transplant Cohort: Results from a Prospective Study at Fuwai Hospital

Acute rejection after heart transplantation has long been detected by endomyocardial biopsy (EMB), an invasive procedure subject to sampling error and inter-observer variation. Donor-derived cell-free DNA (dd-cfDNA) measured in blood reflects graft injury and offers a non-invasive, repeatable route for monitoring.

On 5 October 2026, Professor Liu Sheng's team at Fuwai Hospital, Chinese Academy of Medical Sciences, published a prospective study in Transplant International that systematically evaluated the post-transplant dynamics of dd-cfDNA, its relationship with biopsy findings and its change after anti-rejection therapy in Chinese heart transplant recipients. The study included 80 recipients and 299 plasma samples; the authors describe it as the first exploratory dd-cfDNA cutoff (0.33%) established in a Chinese heart transplant cohort. AlloDx provided the dd-cfDNA testing service and methodological support for the study.

Figure 1 Graphical abstract of the study: cohort composition, post-transplant dynamics, rejection detection and post-treatment change. The two cohorts overlap, comprising 80 independent recipients. Source: Zheng et al., 2026, CC BY.

I. Background: most existing cutoffs derive from European and American cohorts

Most existing dd-cfDNA interpretation thresholds derive from cohorts made up largely of European and American patients. Genetic background, donor-recipient pairing and immunosuppressive regimens differ in Chinese recipients, so applying those thresholds directly may bias interpretation. Using EMB histology as the reference, this study explored the distribution of dd-cfDNA and its ability to identify acute rejection in Chinese recipients.

"We hope that prospective data from Chinese heart transplant recipients will provide a basis for using dd-cfDNA in East Asian populations," said Shanshan Zheng, first author of the paper.

II. Methods and cohorts: 48 Indel markers with ultra-deep NGS

The assay uses insertion-deletion (Indel) genetic markers: 48 Indel markers optimised for East Asian populations combined with ultra-deep next-generation sequencing (NGS) to quantify donor-derived DNA in plasma. It requires no molecular barcodes and is linearly quantitative from 0.05% to 1.5%.

Two complementary cohorts were enrolled: a reference cohort of 42 recipients and 243 plasma samples followed for almost one year after transplantation, and a biopsy cohort of 41 recipients and 56 samples used to analyse rejection-related findings and change around treatment. The cohorts overlap, comprising 80 independent recipients in total.

Figure 2 Study cohorts and sample composition. The reference cohort was followed longitudinally; the biopsy cohort includes samples taken at biopsy and, for some recipients, after treatment. Source: Zheng et al., 2026, CC BY.

III. Post-transplant dynamics: a relatively stable range from week 8

Longitudinal follow-up of the reference cohort showed that dd-cfDNA levels differed by post-transplant phase: median 0.29% at week 1, 0.27% at week 2, 0.21% at week 4, 0.19% at week 8, 0.21% at week 24 and 0.20% at week 48. Overall, levels began to stabilise from week 4 and reached a relatively stable range by week 8.

These changes indicate that dd-cfDNA results should be interpreted in the context of time since transplantation and of serial trends; signals related to the peri-operative period and ischaemia-reperfusion injury warrant cautious interpretation alongside the clinical picture.

Table 1 dd-cfDNA levels at each time point in the reference cohort. Compiled from the reference-cohort section of Table 2 of the paper.

Reference-cohort recipients were clinically stable but did not undergo routine surveillance biopsy, so the reference levels above are not equivalent to histologically confirmed absence of rejection.

Figure 3 Distribution of dd-cfDNA at different post-transplant time points; sample numbers and medians are retained in the figure. Source: Zheng et al., 2026, CC BY.

IV. Rejection-related change: 0.87% versus 0.23%

In the biopsy cohort, median dd-cfDNA was 0.87% in the acute rejection (AR) group and 0.23% in the non-rejection group, a statistically significant difference (P<0.001). Subtype analysis showed that moderate-to-severe acute cellular rejection (ACR) and higher-grade antibody-mediated rejection (AMR) were both associated with higher dd-cfDNA levels.

Figure 4 dd-cfDNA in the non-rejection and acute rejection groups. AR, acute rejection; NR, non-rejection. Source: Zheng et al., 2026, CC BY.

Table 2 Median dd-cfDNA by rejection subtype versus control. P values refer to each two-group comparison; compiled from the results section and Figure 3 of the paper.

No significant difference was observed between mild acute cellular rejection (ACR 1R) and ACR 0R, indicating that the ability of dd-cfDNA to discriminate rejection severity requires further study. As a molecular marker of graft injury, its results must be interpreted together with histology and other clinical findings.

V. Diagnostic performance: 0.33% as the exploratory cutoff in this cohort

Against biopsy as the reference, the area under the receiver operating characteristic curve (AUC) of dd-cfDNA for acute rejection was 0.874; at the 0.33% cutoff derived in this cohort, sensitivity was 81.0%, specificity 85.7%, positive predictive value 89.5% and negative predictive value (NPV) 75.0%.

Table 3 Diagnostic metrics for acute rejection. Compiled from the main diagnostic analysis of the paper; AUC follows the overall ROC analysis.

Figure 5 ROC curves for acute rejection and its subtypes. AUC was 0.874 for AR, 0.832 for ACR and 0.875 for AMR. Source: Zheng et al., 2026, CC BY.

The 0.33% value is an exploratory result tied to this assay, population and study design, and cannot yet serve as a uniform standard for all East Asian recipients. Because the biopsy cohort consisted mainly of recipients with clinically suspected rejection, NPV was 75.0%: a value below the cutoff cannot by itself justify excluding rejection or deferring biopsy, and external validation in larger multicentre cohorts is required.

VI. Treatment monitoring: from 0.98% to 0.38% within two weeks

Fourteen recipients who received anti-rejection therapy and had paired samples were analysed. Median dd-cfDNA fell from 0.98% before treatment to 0.38% two weeks after treatment, a statistically significant change (P=0.011). In subgroup analysis the decrease was significant in ACR recipients but not in the AMR subgroup.

Figure 6 dd-cfDNA before and after anti-rejection therapy in 14 recipients; post-treatment samples were collected at 2 weeks. Source: Zheng et al., 2026, CC BY.

Limited by sample size, these findings offer a research lead for monitoring treatment response and cannot yet guide drug adjustment on their own.

VII. AlloDx's role: dd-cfDNA testing and methodological support

AlloDx provided dd-cfDNA testing on the IndelTrace™ 2.0 platform together with methodological support. The platform combines multiplex PCR targeted amplification with Indel markers, is quantitative from 0.05% to 1.5%, and does not require molecular barcodes or donor genotype.

This collaboration applied a domestically developed Indel-based assay in a prospective clinical study at Fuwai Hospital, building local evidence for non-invasive monitoring in heart transplantation. Fuwai Hospital has extensive experience in heart transplantation and post-operative management: according to public reports, more than 1,500 heart transplants had been performed by June 2025, with a 5-year recipient survival rate of 88.4%. Professor Liu Sheng's team undertakes the National Key R&D Program and several National Natural Science Foundation projects and continues to advance clinical research in heart transplantation.

Detection services and technical notes for non-invasive monitoring of transplant injury are described in Non-invasive Transplant Injury Monitoring。

References

1. Zheng S, Liu S, Zhu Z, et al. Prospective evaluation of donor-derived cell-free DNA for noninvasive detection of acute rejection in an Asian heart transplant population. Transplant International. 2026;39:16415. DOI: 10.3389/ti.2026.16415 (open access, CC BY).

Disclaimer: This article reports scientific progress and is intended for medical and scientific information exchange only. It does not constitute diagnostic or treatment advice, nor a claim of product performance.