The SweD-HTx-study is an observational, multicenter cohort study including adult HTx recipients from Skåne University Hospital (Lund), Sahlgrenska University Hospital (Gothenburg), and Karolinska University Hospital (Stockholm). The aim of the study is to establish a robust, nationwide protocol for dd-cfDNA-based rejection monitoring in Swedish HTx recipients and to enhance our understanding of rejection mechanisms and immune activation. All participants undergo endomyocardial biopsies as part of routine post-transplant surveillance. Heart transplantations are performed at Skåne University Hospital or Sahlgrenska University Hospital, with post-transplant follow-up conducted at the outpatient transplant clinics at the participating centers.

Eligible participants are adults (≥18 years) who undergo orthotopic HTx. Exclusion criteria include pregnancy, multi-organ transplantation, or a history of other solid organ or hematopoietic stem cell transplantation.

Peripheral blood samples are collected according to standardized operating procedures and transported at ambient temperature to the immunology laboratory for processing. A subset of samples is immediately cryopreserved upon collection. Clinical and demographic data are extracted from the electronic medical record (EMR) using standardized data collection forms. The main outcome is the correlation between analyzed dd-cfDNA fraction levels and biopsy-proven rejection, defined by International Society of Heart and Lung Transplantation (ISHLT)'s histopathological criteria. The longitudinal changes in dd-cfDNA fraction and DSA will be analyzed as well as the association between DSA and dd-cfDNA fraction.

Introduction:

Heart transplantation (HTx) and left ventricular assist device (LVAD) therapy are the primary treatment options for advanced heart failure. While HTx improves survival, it presents both short- and long-term risks, including immune-mediated injury and adverse effects from immunosuppressants. Cardiac allograft rejection affects 15-20% of recipients and often necessitates increased immunosuppression. Early detection is crucial, and serial endomyocardial biopsies (EMBs) remain the standard surveillance method.

Recently, biomarkers such as donor-derived cell-free DNA (dd-cfDNA) have emerged for detecting inflammation and cellular damage. Dd-cfDNA reflects apoptosis and necrosis in the transplanted organ and can be measured as a fraction of total cell-free DNA via PCR-based assays. It is a sensitive marker of early antibody-mediated rejection (AMR) and acute cellular rejection (ACR), often rising before histopathological changes are seen. In the US, dd-cfDNA assays are common post-transplant, but in Europe, their use is limited by cost and access.

In Sweden, 60-70 heart transplants are performed each year, with national protocols guiding post-transplant care. Frequent EMBs are performed in the first year. There is a need for non-invasive monitoring to reduce patient discomfort and improve diagnostics. This study aims to establish a national dd-cfDNA testing protocol using next-generation sequencing and to advance understanding of rejection and immune activation.

Materials and Methods:

Study Design:

The SweD-HTx study is a multicenter, observational cohort involving adult HTx recipients from Skåne University Hospital (Lund), Sahlgrenska University Hospital (Gothenburg), and Karolinska University Hospital (Stockholm). Transplants are performed at Skåne or Sahlgrenska, with follow-up at all three sites. Eligible patients are adults (≥18 years) undergoing orthotopic HTx. Exclusion criteria: pregnancy, multi-organ transplantation, or prior solid organ/hematopoietic stem cell transplant.

Blood samples are collected per standard protocols and sent to immunology labs. Some samples are cryopreserved. Clinical and demographic data are gathered from electronic records.

Study Population:

Patients are recruited after being accepted for HTx, aiming to enroll 120 participants nationwide, with recruitment expected to conclude within 2 years (first patient: Feb 2025). Written informed consent is required.

Study Visits and Sample Collection:

Blood for dd-cfDNA and DSA is collected prospectively at set intervals, coordinated with routine EMBs: weeks 1-4, months 2, 2.5, 3, 4, 5, 6, 8-9, 10-12, 16, 20, and 24 post-transplant, varying by center protocol. Samples are processed and plasma stored at -80 °C. DSA analysis is performed at each visit. Patients are monitored for five years with annual data collection.

The primary outcome is the correlation between dd-cfDNA levels and biopsy-proven rejection (per ISHLT criteria). Longitudinal changes in dd-cfDNA and DSA, as well as their association, will be analyzed.

Planned Substudies:

1. Incidence of graft dysfunction (GDF) and CAV over five years, examined in relation to DSA and dd-cfDNA.
2. Association between dd-cfDNA and circulating biomarkers of graft injury or inflammation.

dd-cfDNA Analysis: Dd-cfDNA quantification is done with a next-generation sequencing (NGS) assay. The dd-cfDNA fraction (%) is calculated as: (donor-derived cfDNA / total cfDNA) × 100. All analyses follow validated protocols and manufacturer instructions.

Histopathological Evaluation:

EMBs are graded locally by cardiovascular pathologists per ISHLT guidelines (2005 for ACR, 2013 for AMR). Rejection is defined as pAMR1(H+), pAMR1(I+), pAMR2, pAMR3, ACR grade 2R or 3R, or combinations thereof. Non-rejection: pAMR0, ACR grades 0 or 1R. Results are documented in electronic records.

Donor-Specific Antibody Analysis:

DSAs are analyzed at each center using the Luminex xMAP platform, reported as mean fluorescence intensity (MFI). Relationships among DSA, dd-cfDNA, and biopsy findings will be explored.

Ethical Considerations:

The study has ethics approval (Reference: 2024-03357-01, 2025-04085-02) and follows the Declaration of Helsinki, ISHLT ethics guidelines, Good Clinical Practice, and relevant regulations. Written informed consent is obtained from all participants.

Data Management and Statistical Analysis:

Data are stored in a secure, password-protected database with coded identifiers. Descriptive statistics summarize baseline characteristics. Associations among dd-cfDNA, DSA, and biopsies will be analyzed with non-parametric tests and multivariable regression models. Analyses are performed using R.

Trial Governance:

The SweD-HTx is an investigator-initiated study. DEVYSER supplies the DNA test kits, with testing performed at local immunology labs using NGS and standardized controls. The investigators and an independent steering committee oversee the trial. DEVYSER has no role in data collection, analysis, interpretation, or publication decisions. The study has received funding from Region Stockholm (ALF grants).

Results:

As of 1 January 2026, 72 participants have been enrolled from all centers, with 436 samples collected. Data include demographics, lab results, and immunosuppressive regimens at each sampling time point. DSA and dd-cfDNA levels will be correlated and analyzed longitudinally, with planned substudies on imaging and long-term follow-up.

Inclusion Criteria:

• Adults (≥18 years) who undergo orthotopic HTx.

Exclusion Criteria:

• Pregnancy,
• Multi-organ transplantation
• Solid organ
• Hematopoietic stem cell transplantation.

This study will include adult HTx recipients from Skåne University Hospital (Lund), Sahlgrenska University Hospital (Gothenburg), and Karolinska University Hospital (Stockholm). All participants undergo endomyocardial biopsies as part of routine post-transplant surveillance. Heart transplantations are performed at Skåne University Hospital or Sahlgrenska University Hospital, with post-transplant follow-up conducted at the outpatient transplant clinics at the participating centers.

Technical and operational constraints