The following are key points to remember from a state-of-the-art review, which provides practical guidance for clinicians transitioning toward a less invasive acute rejection monitoring protocol after heart transplantation:

Background: Endomyocardial biopsy (EMB) has been used for surveillance and diagnosis of allograft rejection for patients after heart transplantation for many decades. However, this invasive form of testing has procedural risks and challenges associated with histopathological interpretation. Blood-based, noninvasive surveillance strategies have emerged, including gene expression profiling (GEP) and quantification of donor-derived cell-free deoxyribonucleic acid (dd-cfDNA), and are being adopted in many transplant centers to reduce frequency of EMBs. Uncertainty and challenges exist when considering a transition to a noninvasive rejection surveillance strategy. This review focuses on 10 key questions and offers practical guidance for making this transition.

1. Studies have shown that increased percentage of circulating dd-cfDNA (released in cell turnover) is correlated to EMB-detected rejection and has negative predictive value of 97% for acute cellular rejection (ACR) and antibody-mediated rejection (AMR). The dd-cfDNA tests are often used in conjunction with GEP testing. Stand-alone dd-cfDNA testing is available but studies are still being carried out to assess diagnostic utility without GEP testing. GEP assesses the expression of 11 genes in peripheral blood mononuclear cells and assigns an activation score. Studies have shown GEP reduces EMB use and is sensitive for the detection of ACR. However, GEP is not used for AMR detection and is not reflective of graft injury.
2. Several factors can affect GEP and dd-cfDNA interpretation. GEP scores may be falsely elevated in systemic inflammatory or infectious states (including CMV viremia). Confirmatory EMB for a high GEP score may not always be needed when considering other factors like alternate explanations, clinical status, dd-cfDNA level, and baseline patient risk for rejection. GEP results should be interpreted cautiously with recent pulse steroids or high baseline steroid doses and recent red blood cell transfusions. False elevations in dd-cfDNA can be seen with myocardial ischemia or other graft injury/trauma and should be interpreted cautiously in the setting of multiorgan transplant, bone marrow or stem cell transplant, pregnancy, cancer, sepsis, and systemic inflammation.
3. Defining thresholds for GEP scores and dd-cfDNA levels can be challenging as different cutoffs have been used to balance test sensitivity and specificity. Many centers have adopted specific absolute cutoffs but a more holistic approach which factors in a patient’s clinical status, baseline risk, and trajectory of testing is important when considering when to follow-up with confirmatory EMB for diagnosing rejection.
4. When considering implementing a schedule for a noninvasive surveillance, it is important to remember that high-dose steroids affect GEP results, so testing is not used until 55 days post-transplant at the earliest and if prednisone dosing is <20 mg/day. Levels of dd-cfDNA stabilize early at about 14 days post-transplant and testing can generally be started 3-4 weeks post-transplant. Practically speaking, when using a combined GEP and dd-cfDNA test, noninvasive surveillance monitoring starts at day 55 at many centers. Pairing the first noninvasive testing with a routine EMB is often done and helps to correlate findings. Frequency of testing following this is variable. Less data are available for GEP and dd-cfDNA testing after 2 years post-transplant. Routine testing is generally not recommended after 5 years post-transplant.
5. There is no consensus regarding concurrent cardiac imaging with blood-based noninvasive rejection screening. Some research study protocols included routine echocardiograms as part of standard of care. However, graft dysfunction post-transplant can be due to a variety of causes including rejection, primary graft dysfunction, and cardiac allograft vasculopathy (CAV). Also, graft dysfunction is often a late consequence of rejection, though assessment of other cardiac structures may be helpful in management.
6. While dd-cfDNA can detect both ACR and AMR, no currently commercially available test can distinguish between ACR, AMR, and allograft injury. Research studies suggest a different time course and degree of dd-cfDNA changes for ACR and AMR, but this was seen using research-grade assays. Further investigation is needed.
7. De novo donor-specific antibody (dnDSA) formation is common post-transplant, but only half of patients have AMR detected on EMB. Management for those with negative EMBs is uncertain. Small studies suggest that rising dd-cfDNA is associated with dnDSA development and associated graft injury, although more data are needed before defining a specific role of dd-cfDNA in this setting.
8. Beyond screening for acute rejection, dd-cfDNA may provide information about overall graft health. Elevated levels over time are associated with adverse outcomes and there may be a future role in CAV detection. The role for dd-cfDNA in modulating immunosuppression is unclear and is actively being studied.
9. Implementation challenges exist for beginning a less invasive rejection screening strategy. Important things to consider are the education of transplant faculty and staff on test interpretation, ensuring proper blood sample collection timing and processing, and reimbursement differences for outpatient and inpatient settings.
10. Selecting the appropriate patients for noninvasive rejection surveillance is important. This strategy is most often used for low-risk patients, although use in patients at higher risk for rejection can be done with appropriate experience and comfort level.

Clinical Topics: Cardiac Surgery, Cardiovascular Care Team, Diabetes and Cardiometabolic Disease, Heart Failure and Cardiomyopathies, Invasive Cardiovascular Angiography and Intervention, Noninvasive Imaging, Vascular Medicine, Cardiac Surgery and Heart Failure, Acute Heart Failure, Heart Failure and Cardiac Biomarkers, Heart Transplant, Interventions and Imaging, Interventions and Vascular Medicine

Keywords: Allografts, Biomarkers, Biopsy, Cell-Free Nucleic Acids, Cytomegalovirus Infections, Diagnostic Imaging, Erythrocyte Transfusion, Gene Expression Profiling, Graft Rejection, Heart Failure, Heart Transplantation, Immunologic Surveillance, Immunosuppression, Inflammation, Leukocytes, Mononuclear, Primary Graft Dysfunction, Stem Cell Transplantation, Steroids, Viremia

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