Cell-free DNA is released into the bloodstream with cell death due to normal cell turnover or disease processes (21). Normally, healthy individuals have small amounts of cfDNA, most of which is released by hematopoietic cells with a small amount released from the heart (22). In organ transplant recipients, it was hypothesized that acute rejection leads to cell death in the allograft resulting in elevated levels of dd-cfDNA in the blood of transplant recipients (17). The clinical use of earlier techniques such as shot-gun sequencing and targeted quantification using polymerase chain reaction were impractical due to cost, complexity, requirement of previous genotyping of both donor and recipient, and sex-mismatch between the donor and recipient (23). These limitations were overcome with the advent of targeted amplification. Allosure (CareDx, Inc., Brisbane, CA) is a targeted amplification sequencing assay which has been analytically and clinically validated to quantify the percentage of dd-cfDNA in the transplant recipients’ blood (18). This technique was first utilized in the D-OAR study to target highly polymorphic single nucleotide polymorphisms (SNPs) to distinguish dd-cfDNA from recipient-derived cfDNA (rd-cfDNA) (24). The Allosure assay currently includes a panel of 405 SNPs. Another commercially available assay is the Prospera test (Natera, Inc., Austin, TX), validated for clinical use in the DEDUCE trial in 2021, and includes more than 13,000 SNPs (20). Table 1 summarizes the commercially available and research grade dd-cfDNA assays.

The advantages of dd-cfDNA testing include its noninvasive nature, potential for early detection, and opportunity for longitudinal monitoring. Elevated percentage of dd-cfDNA levels have been correlated with ACR and AMR, as well as other forms of allograft injury (25). Prior studies have demonstrated that an elevated dd-cfDNA fraction can precede biopsy-proven rejection by up to 3 months, highlighting its important role in screening allografts in routine surveillance, especially in the first year post-transplant (17, 19, 26). Apart from earlier detection, researchers from Genomic Research Alliance for Transplantation (GRAfT) consortium demonstrated that dd-cfDNA from patients with AMR were rich in guanosine-cytosine bases, quantitatively 5–11 times higher, and often detected sub-clinical AMR (19). By utilizing dd-cfDNA, up to 80% of the routine surveillance biopsies could be eliminated, highlighting the high NPV (>98%) of the test (19). Elevated dd-cfDNA fraction also offers prognostic information as higher levels have been linked to unfavorable outcomes including allograft dysfunction and death at one year (13). However, it is important to note that dd-cfDNA is non-specific marker of graft injury, therefore it is used as a screening tool for AR while EMB is the test of choice in cases of suspected rejection (27). Figure 1 shows the surveillance protocol for HT recipients at our center.

The lack of histological evidence of rejection on EMB has typically been reassuring to clinicians, but the interpretation of elevated dd-cfDNA fraction in the setting of a “negative” EMB is unclear. Plausible explanations for the discordance include subclinical rejection, non-rejection allograft injury, false positive dd-cfDNA, and false negative EMB. In instances where percentages of dd-cfDNA are elevated despite a negative biopsy, it is imperative to systematically consider these possibilities. This should include an initial assessment of technical reasons for dd-cfDNA fraction to be falsely elevated, such as improper handling of the sample, artifactual increase due to recent cardiac procedure such as biopsy or pacemaker implantation, or even after vigorous exercise (19, 28, 29). The absolute quantity of cfDNA levels is subject to large variations and have been reported in leukocytosis, systemic lupus erythematosus, myocardial infarction, and psychiatric disorders (30–33). Significant reduction in total rd-cfDNA can increase the dd-cfDNA fraction without cardiac injury. Pre-transplant considerations such as an autonomic storm in brain-dead donors or donor coronary artery disease can also lead to baseline cellular injury in the donor heart contributing to elevations in dd-cfDNA fraction in the recipient immediately post HT (34). Additionally, factors such as ischemic time, surgical complexity, pulmonary vascular resistance, and crossmatch status are speculated to lead to elevations in dd-cfDNA immediately post HT. To mitigate or reduce the likelihood of false positives, centers have adopted protocols where the timing of initial dd-cfDNA testing occurs 28 days following transplant (see Figure 1) (19).

If there are no such technical reasons, then a closer look at the immunosuppression trough levels and medication adherence is critical. Viral infections, most commonly cytomegalovirus (CMV), are associated with increased dd-cfDNA levels (27, 35, 36). CMV frequently causes leukopenia which may lead to reduction in rd-cfDNA and resulting increase in percentage of dd-cfDNA. Afzal et al. reported 4 clusters in their single center study of patients with elevated dd-cfDNA without biopsy evidence of AR: CMV viremia, non-CMV infections (which included SARS-CoV-2, Ebstein-Barr virus, and hepatitis B infection), females (with 40% having de novo DSA), and lastly patients with right ventricular dysfunction (27). Of note, ACR 1R was present in all patients with RV dysfunction, 80% of females, and none in the non-CMV infection group. It is unclear whether any patients with ACR 1R progressed to higher-grade rejection, but it will be important to study in future larger multicenter studies as currently most episodes of ACR 1R are not treated. However, in the D-OAR study by Kanwar et al., CMV infections were not associated with significantly higher percentages of dd-cfDNA as compared to those without CMV infection (36). Our group has observed transient elevations in dd-cfDNA immediately following vaccinations.

Elevated dd-cfDNA fraction may reflect ischemic injury secondary to cardiac allograft vasculopathy (CAV) (34). Therefore, further ischemic evaluation with coronary angiography or noninvasive techniques should be considered. Finally, myocarditis is a possible etiology of non-rejection myocardial injury. Elevated percentage of dd-cfDNA due to toxoplasmosis has been described (37). Recurrent sarcoidosis and giant cell myocarditis following transplant are well-established, presumably they could also cause an elevated dd-cfDNA fraction. A high index of suspicion is required to make these diagnoses along with close collaboration with pathologists. To evaluate these possibilities, the incorporation of supplemental diagnostic techniques is warranted, as outlined in the central illustration (Figure 2). We, briefly discuss the role of multimodal diagnostics in evaluation of such cases when dd-cfDNA fraction is elevated in the absence of biopsy-proven rejection.

Additional biomarkers have been investigated to predict AR. Early work using troponin T and I to diagnose AR was not encouraging and recent efforts with high sensitivity assays have been shown to be more promising (38). Similarly, B type natriuretic peptide and its fragments correlated poorly with AR, though patient level trending was noted to be more informative (38). Current ISHLT guidelines state these biomarkers may have a role in a comprehensive strategy for rejection surveillance (38). The combination of dd-cfDNA fraction and gene expression profiling (GEP) has been extensively studied with incremental improvement in predicting AR on biopsy (39). A recent publication combined dd-cfDNA with a readily available biomarker NT-proBNP to improve accuracy in predicting rejection (40). Both these studies relied on EMB as the gold standard for diagnosing rejection, despite its known limitations, and neither study addressed the clinical dilemma of elevated dd-cfDNA levels in the context of reassuring biopsy findings.

The Molecular Microscope Diagnostic System (MMDx, Thermo Fisher Scientific, Waltham, MA) analyzes EMBs using microarrays to quantify mRNA transcripts associated with rejection and injury (41). It is unique in that is uses machine-based learning algorithms to compare each biopsy against a reference database of samples and follows the principle that depending on the type and severity of transplanted organ injury, specific genes are activated and produce unique mRNA patterns (42). While MMDx does not replace histology, it can provide additional information to reconcile discordant results. A contemporary study included more than 3,200 EMBs to identify 7 rejection archetypes as well as non-rejection injury and atrophy-fibrosis (43). The ongoing Trifecta-Heart study seeks to incorporate all three tools (dd-cfDNA, MMDx, and EMB histopathology) to improve accuracy in diagnosing AR (NCT04707872) (44).

Donor specific antibodies (DSAs), primarily targeting donor human leukocyte antigen molecules, are associated with AMR and poor graft survival (45). DSAs may be present at the time of transplant, or may develop following transplant termed de novo. The development of de novo DSA(s) or significant increase in the strength of known DSA(s) coupled with allograft dysfunction even in the presence of a negative EMB may warrant treatment. Moreover, antibodies against non-HLA antigens such as angiotensin II type-1 receptor and others have been linked to AMR and BNR (46, 47); however, monitoring is highly variable across centers, and standardized treatment protocols remain poorly defined.

Cardiac MRI is another promising non-invasive alternative surveillance tool for AR in HT. Besides assessing graft function and valve lesions, parametric mapping with T1 and T2 sequences can potentially enable detection of myocardial edema, fibrosis, and interstitial expansion. In a small study of 60 HT patients, T2 relaxation has been shown to be an independent predictor of AR (48). Another small randomized trial comparing EMB and CMR for AR surveillance demonstrated high diagnostic accuracy of CMR achieving 92% sensitivity and 92% NPV for AR (49). However, CMR is not able to distinguish between different types of AR. CMR can also aid in the diagnosis of myocarditis, including toxoplasmosis, giant cell, and sarcoidosis.

As the clinical utility of dd-cfDNA continues to evolve in HT surveillance, several avenues hold promise for advancing the field and addressing current diagnostic limitations. Transitioning from relative quantification or donor fraction to absolute quantification of dd-cfDNA, may enhance diagnostic specificity and reduce variability across diverse clinical scenarios. However, absolute level of dd-cfDNA has also been correlated to AR providing a stable, reproducible measure of donor-specific injury (18, 50–52). This approach may be particularly beneficial in patients with high baseline cfDNA due to concurrent illness or those with multi-organ transplants, where interpreting percentage of dd-cfDNA can be challenging. The primary results of the DEFINE-HT (Development of Non-Invasive Cell-Free DNA to Supplant Invasive Biopsy in Heart Transplantation) study recently reported that Prospera (Natera, Inc, Austin, TX) with donor quantity score (DQS) incorporating both absolute dd-cfDNA and donor fraction, predicted allograft dysfunction 3 times more than EMB and demonstrated strong correlation with clinical outcomes at one year (53).

The use of dd-cfDNA in multiorgan transplant recipients is not well-described. Dd-cfDNA fractions of healthy allografts vary by the transplanted organ, the heart is typically the lowest at < 0.20%, kidney < 0.5% (54), lung < 1% (55), and liver the highest ranging from 5 to 30% (56), which makes selecting a threshold for multiorgan recipients challenging. Intriguingly, dd-cfDNA can be fractionated if recipients have organs from multiple donors, permitting one to follow each organ independently (57). If the transplanted organs are from a single donor however, setting an appropriate threshold must be done on a case-by-case basis, which has been reported in the literature (58) and we have done at our center.

Integration of dd-cfDNA with other emerging diagnostics, such as MMDx, CMR, and DSA profiling, represents an important step toward a precision medicine approach in HT. By combining molecular, immunological, and imaging data, clinicians may be able to generate a more comprehensive and nuanced risk profile for each patient, enabling more timely and targeted interventions. Lastly, longitudinal studies are needed to understand the kinetics of dd-cfDNA over time, establish optimal surveillance intervals, and determine the clinical significance of transient elevations in the absence of rejection.