Medical Policy

This document addresses laboratory tests for the early detection of rejection following a heart transplant. This includes the Heartsbreath test, which measures the chemical byproducts of allograft rejection, AlloMap^® gene expression testing, AlloSure^® Heart, myTAI_HEART cell-free DNA (cfDNA), MMDx Heart and others.

Even with modern drug therapy, rejection remains a constant hazard, and transplant recipients must be tested repeatedly for signs of renewed rejection. Currently, the gold standard to detect heart transplant rejection is endomyocardial biopsy (EMB). This is typically performed weekly for the first 6 weeks, biweekly until the third month, monthly to 6 months and then every 1 to 3 months, as indicated. The use of EMB is not addressed in this document.

Medically Necessary:

AlloMap molecular expression testing is considered medically necessary as a non-invasive method of determining the risk of rejection in heart transplant recipients between 6 months and 5 years post-transplant.

Investigational and Not Medically Necessary:

Breath testing with the Heartsbreath test is considered investigational and not medically necessary for use as an aid in the diagnosis of heart transplant rejection.

AlloMap molecular expression testing is considered investigational and not medically necessary when the criteria above are not met.

Additional tests for detection of heart transplant rejection are considered investigational and not medically necessary including, but not limited, to AlloSure Heart, AlloSeq cell-free DNA, MMDx Heart and myTAI_Heart.

AlloMap Molecular Expression Testing

In 2008, FDA 510(k) clearance as a Class II approval was granted for AlloMap Molecular Expression Testing (CareDx, Inc., Brisbane, CA) as an in-vitro, diagnostic, multivariate, index assay of the gene expression profile of RNA isolated from peripheral blood mononuclear cells for the following indication: 

To aid in the identification of heart transplant recipients with stable allograft function who have a low probability of moderate/severe acute cellular rejection (ACR) at the time of testing in conjunction with standard clinical assessment. AlloMap is indicated for use in heart transplant recipients who are 15 years of age or older and at least 2 months (greater than or equal to 55 days) post-transplantation (FDA, 2008).

The test assesses the expression of 20 genes, about half of which are directly involved in rejection while the remainder provide other information needed for rejection risk assessment. It is hoped the results of this test will decrease the number of necessary endomyocardial biopsies (EMBs). Among the proposed benefits are the AlloMap test's ability to differentiate mild rejection, for which histologic findings may be the least accurate, and the potential for monitoring physiologic responses to steroid weaning.

The Cardiac Allograft Rejection Gene Expression Observation Study (CARGO) investigated these patterns of gene expression detected in peripheral blood by the AlloMap test. CARGO included eight U.S. cardiac transplant centers and 650 heart transplant (HT) recipients. Investigators tested the hypothesis that a gene expression test could distinguish between quiescence (International Society for Heart and Lung Transplantation [ISHLT] rejection grade 0) and moderate or severe rejection (ISHLT rejection grade greater than or equal to 3). In the initial gene discovery phase, leukocyte microarray testing covering 7370 genes was conducted on samples from 98 study participants with quiescent disease shown by EMB and from 285 participants whose biopsies showed rejection. Analysis of these tests showed 97 genes with the potential for assay development. Polymerase chain reaction (PCR) testing for these 97 genes was next conducted on 36 samples from participants experiencing rejection and on 109 samples from participants with EMB-determined quiescence. Analysis of data from this second round of testing identified 11 genes that best distinguished quiescence from rejection. Results from these 11 gene tests were combined into a rejection score in a range of 0-40. In the final validation phase of the study, samples from 63 individuals whose rejection status was known through EMB were prospectively tested in a blinded manner. The study concluded that the negative predictive value (NPV) of gene expression testing for grade 3A or greater rejection was 99.6% for individuals who were more than 1 year post-transplant and whose rejection score was below 30. The authors note that the test could not distinguish mild rejection from quiescence and that the positive predictive value (PPV) was low. They recommended further study to conclusively establish the utility of this test in the management of heart transplant recipients (Deng, 2006).

Subsequent validation studies and sub-study analyses of the CARGO results provide additional data regarding the potential utility of the AlloMap test in detecting transplant rejection (Bernstein, 2007; Mehra, 2007b; Mehra, 2008). More recent results of CARGO and the CARGO II trial reflect similar results. CARGO II was a European observational study conducted to further validate AlloMap’s gene expression profiling (GEP) test performance. For greater than or equal to 2 months and greater than 6 months post-transplantation, the CARGO II GEP score performance (Area Under the Curve-Receiver Operating Characteristic curve [AUC-ROC]=0.70 and 0.69) is similar to the CARGO study results (AUC-ROC=0.71 and 0.67). CARGO II’s published report does not show how many of the 91 participants began GEP monitoring prior to 6 months after transplant, and test results for this early-testing group were not separately analyzed. Trial investigators proposed that the low prevalence of ACR in the studied cohort contributed to the high NPV and limited the PPV of GEP testing. They concluded that the choice of threshold score for the practical use of GEP testing with AlloMap should consider the overall clinical assessment of the individual’s baseline risk for rejection (Crespo-Leiro, 2015; 2016).

The Invasive Monitoring Attenuation through Gene Expression (IMAGE) trial was published in 2010. This was a randomized, event-driven, noninferiority trial sponsored by the manufacturer of AlloMap (at that time, XDx, Inc.). IMAGE was conducted at 13 U.S. transplant centers between January 2005 and October 2009 with median follow-up of 19 months. This trial included 602 transplant recipients who had undergone a transplant more than 6 months prior and who were considered at low risk for rejection. The purpose of this study was to compare rejection outcomes between those who underwent routine EMB and those who were monitored with the AlloMap GEP test. The primary outcomes were the first occurrence of rejection with hemodynamic compromise, graft dysfunction due to other causes, death, or retransplantation. Results indicated that monitoring for rejection with GEP, as compared with routine biopsies, was not associated with an increased risk of serious adverse outcomes and resulted in the performance of significantly fewer biopsies. Subjects who were monitored with AlloMap and those who underwent routine EMB had similar 2-year cumulative rates of the composite primary outcome (14.5% and 15.3%, respectively; hazard ratio [HR] with GEP, 1.04; 95% confidence interval [CI]; 0.67 to 1.68). The 2-year rates of death from any cause were also similar in the two groups (6.3% and 5.5%, respectively; p=0.82). Although the limited power of the study did not allow for firm conclusions regarding the utility of AlloMap, the authors concluded that GEP of peripheral blood specimens may offer a reasonable alternative to routine EMB if the interval since transplantation is at least 6 months and the individual is considered to be at low risk for rejection (Pham, 2010).

Published in 2015, the E-IMAGE trial (Comparison of AlloMap Molecular Testing and Traditional Biopsy-based Surveillance for Heart Transplant Rejection Early Post-transplantation) was a single-center trial in which 60 subjects were randomized to GEP with AlloMap or to EMB started at 55 days post HT. The study examined results of both test methods for evidence of ACR in the first year post HT. A positive GEP ≥ 30 between 2 and 6 months, or ≥ 34 after 6 months, prompted a follow-up biopsy. The primary endpoint included a composite of death or retransplant, rejection with hemodynamic compromise or graft dysfunction at 18 months after HT. A coprimary endpoint included change in first-year maximal intimal thickness by intravascular ultrasound, which is a recognized surrogate for long-term outcome. The composite endpoint was similar between the AlloMap GEP and EMB groups (10% vs 17%; log-rank p=0.44). The coprimary endpoint of first-year intravascular ultrasound change demonstrated no difference in mean maximal intimal thickness (0.35 ± 0.36 vs 0.36 ± 0.26 mm; p=0.944). Steroid weaning was successful in both groups (91% vs 95%). The authors concluded that, in this pilot study, AlloMap GEP starting at 55 days post HT seemed comparable to EMB for rejection surveillance in selected HT recipients and does not result in increased adverse outcomes. However, it was noted that this study was underpowered to determine firm conclusions, and larger randomized trials are needed to confirm these findings (Kobashigawa, 2015).  

In 2010, the ISHLT issued guidelines for the care of HT recipients which included the following: 

• The standard of care for adult HT recipients is to perform periodic EMB during the first 6-12 months after transplant for rejection surveillance; (Class IIa, Level of Evidence: C)
• After the first year post-transplant, EMB surveillance every 4-6 months is recommended for patients at higher risk of late acute rejection; (Class IIa, Level of Evidence: C)
• GEP using the AlloMap test can be used to rule out the presence of ACR of grade 2R or greater in appropriate low-risk patients between 6 months and 5 years post-transplant (Class IIa, Level of Evidence: B) (Costanzo ISHLT, 2010). 

Another portion of the 2010 ISHLT guideline titled, “Task Force 2: Immunosuppression and Rejection” noted the following regarding the grading scale for risk of ACR in HT recipients:

Due to intra- and interobserver variability in the determination of the different grades of mild or moderate rejection and the observation that grades 1 and 2 were mostly self-limited, a revised heart allograft rejection grading system was published in 2005 as follows (Stewart, 2005):

• Grade 0 (no cellular rejection) was now named grade 0R (‘R’ added to reflect the revised 2005 scale);
• The intermediate grades of 1A, 1B, and 2 were re-classified as grade 1R, or mild ACR;
• Grade 3A was re-classified as grade 2R, moderate ACR; and
• Grades 3B and 4 were re-classified as grade 3R, severe ACR.
• In addition, AMR (antibody mediated rejection) was recognized as a clinical entity, and recommendation was issued for determination of its presence (AMR1) or absence (AMR0) (Taylor ISHLT, 2010).

In 2019, Moayedi and colleagues reported data from the Outcomes AlloMap Registry (OAR). OAR is an ongoing industry-sponsored observational, prospective, multicenter study including participants aged ≥ 15 years and ≥ 55 days post-cardiac transplant. The registry collects pre-transplant baseline clinical data and post-transplant information about clinical status, diagnostic tests (echocardiograms, coronary angiograms, EMB, AlloMap scores), donor-specific antibodies, immunosuppressive maintenance therapy and drug levels, interval hospitalizations, and development of post-transplant malignancies. The 2019 report included outcomes for 1504 HT recipients from 35 HT centers in the United States. Initial enrollment began in 2013. Severe ACR (≥ 2R) was observed in 2.0% of the cohort from 2 to 6 months and 2.2% after 6 months. The reported data showed significant overlaps in the interquartile ranges of AlloMap scores for individuals with and without biopsy-demonstrated ACR (ISHLT grade ≥ 2) between 2 and 6 months after transplantation.

The ISHLT updated their Guidelines for the Care of Heart Transplant Recipients in 2023 (Velleca, 2023). They remarked that use of GEP to monitor for rejection after HT had increased since publication of their 2010 guidelines. The 2023 guidelines affirmed the 2010 definitions for grades of rejection. After referring to the results of the E-IMAGE and CARGO II trials, the guideline’s authors made the following recommendations:

Gene Expression Profiling (GEP) (i.e., Allomap) of peripheral blood can be used in low-risk patients between 2 months and 5 years after HT to identify adult recipients who have low risk of current ACR to reduce the frequency of EMB. Data in children does not allow a general recommendation of GEP as a routine tool at present (Class IIa, Level of Evidence: B).

After the first year, continued rejection surveillance (using a combination of noninvasive methods, GEP or EMB) is reasonable in patients at higher risk for late acute rejection (Class IIa, Level of Evidence: C).

The CARGO II and E-IMAGE trials found non-inferior outcomes for GEP compared to EMB monitoring of HT recipients who are between 6 months and 5 years post-transplant. Input from the transplant practice community supports GEP monitoring in this time period. Evidence for GEP monitoring beginning at 55 days after HT comes from the CARGO II and E-IMAGE trials. CARGO II did not provide details of test results in the 2-6 months post-transplant period. The E-IMAGE authors acknowledged that the trial was underpowered to draw firm conclusions about the noninferiority of early GEP use compared to EMB monitoring.

Breath testing

Heartsbreath (Breath test for Grade 3 heart transplant rejection), manufactured by Menssana Research, Inc., (Fort Lee, NJ) received U.S. Food and Drug Administration (FDA) clearance on February 24, 2004 under the Humanitarian Device Exemption (HDE)* program with the following indications for use:

The Heartsbreath test is indicated for use as an aid in the diagnosis of grade 3 heart transplant rejection in patients who have received heart transplants within the preceding year. The Heartsbreath test is intended for use as an adjunct to, and not as a substitute for, endomyocardial biopsy. The use of the device is limited to patients who have had endomyocardial biopsy (EMB) within the previous month (FDA, 2004).

The Heartsbreath test works on the principle that rejection of the transplanted heart is accompanied by oxidative stress that degrades membrane polyunsaturated fatty acids, generating alkanes and methylalkanes that are excreted in the breath as volatile organic compounds (VOCs). The individual breathes for 2 minutes through a disposable mouthpiece attached to a breath collecting device. The device then analyzes the VOCs in alveolar and room air and uses a proprietary algorithm to predict the probability of Grade 3 HT rejection.

The Heartsbreath test should not be used for individuals who have received an HT more than 1 year previously, or for those who have a Grade 4 HT rejection, because Heartsbreath has not been evaluated in these groups.

FDA clearance was based on the results of the Heart Allograft Rejection: Detection with Breath Alkanes in Low Levels (HARDBALL) Study sponsored by the National Heart Lung and Blood Institute (NHLBI). In this 3-year multicenter study, investigators evaluated a new marker of HT rejection, the breath methylated alkane contour (BMAC). In the HARDBALL study, 1061 breath VOC samples were collected from 539 HT recipients at seven sites on the day of scheduled EMB. The gold standard of rejection was the concordant set of ISHLT grades in biopsies read by two cardiac pathologists. Results showed the following percentages of concordant biopsies:

• Grade 0, 645 of 1061 (60.8%);
• Grade 1A, 197 (18.6%);
• Grade 1B, 84 (7.9%);
• Grade 2, 93 (8.8%);
• Grade 3A, 42 (4.0%).

A combination of 9 VOCs in the BMAC identified Grade 3 rejection (sensitivity 78.6%; specificity 62.4%; cross-validated sensitivity 59.5%; cross-validated specificity 58.8%; PPV 5.6%; NPV 97.2%). Site pathologists identified the same cases with sensitivity of 42.4%, specificity 97.0%, PPV 45.2% and NPV 96.7%. The authors concluded that a breath test for markers of oxidative stress was more sensitive and less specific for Grade 3 HT rejection than a biopsy reading by a single on-site pathologist, but the NPV of the two tests were similar. They concluded that a negative screening breath test could potentially identify transplant recipients at low risk of Grade 3 rejection and obviate the need for EMB in this group, thereby reducing the overall number of EMBs performed, which was estimated to be by as much as 50% (Phillips, 2004).

Currently, there is inadequate evidence in the published literature to demonstrate the safety, efficacy, and clinical utility of the Heartsbreath test in the management of rejection surveillance following HT. Large trials are needed to further define the role of this technology and demonstrate how use of this test will impact treatment management.

AlloSure Heart and other donor-derived cell free DNA (dd-cfDNA)

Elevated levels of donor-derived cell free DNA (dd-cfDNA) are shed from the donor graft when there is transplanted organ injury and rejection (Grskovic, 2016; Khush, 2019). The AlloSure Heart test (CareDx, Inc. Brisbane, CA) has been promoted as a noninvasive alternative to EMB as early as 14 days post transplant. This is a next generation sequencing (NGS)-based assay that quantifies dd-cfDNA relative to the total amount of cfDNA found in a plasma sample. It uses single-nucleotide polymorphisms (SNPs) to quantify dd-cfDNA in transplant recipients without requiring separate genotyping of the donor and recipient. Test results represent the percent of dd-cfDNA in the total cfDNA in an HT recipient. Changes in the percentage of dd-cfDNA over time provide further evaluation for HT rejection. This plasma test is only performed at a single CareDx CLIA laboratory. Results are expected to be reported within 3 days of blood draw. Early studies have consistently shown a correlation between elevated levels of cfDNA and organ rejection or cellular graft injury (Macher, 2019).

Clinical validity was investigated in two prospective observational studies, the Utility of Donor-Derived Cell Free DNA in Association with Gene Expression Profiling (D-OAR; NCT02178943) and the Cedars-Sinai single-center study. The D-OAR trial included 740 HT recipients at 26 transplant centers in the U.S. Plasma dd-cfDNA was quantified by targeted amplification and sequencing of a single nucleotide polymorphism panel. The dd-cfDNA levels were correlated to paired events of biopsy-based diagnosis of rejection. The median dd-cfDNA was 0.07% in reference HT recipients (2164 samples) and 0.17% in samples classified as acute rejection (35 samples; p=0.005). At a 0.2% threshold, dd-cfDNA had a 44% sensitivity to detect rejection and a 97% NPV. The Cedars-Sinai cohort study of 33 HT recipients considered at high risk for antibody-mediated rejection (AMR) found dd-cfDNA levels were elevated 3-fold in AMR, compared with subjects without AMR (99 samples; p=0.004). The authors concluded that reported test performance characteristics will guide the next stage of clinical utility studies of the dd-cfDNA assay (Khush, 2019).

Knight and colleagues conducted a systematic review of the literature for the use of cfDNA in monitoring of graft health after solid organ transplant (SOT). Electronic databases were searched for studies relating cfDNA fraction or levels to clinical outcomes, and data including measures of diagnostic test accuracy were extracted. Narrative analysis was performed. Ninety-five articles from 47 studies met the inclusion criteria (18 kidneys, 7 livers, 11 hearts, 1 kidney-pancreas, 5 lungs, and 5 multiorgans). The majority were retrospective or prospective cohort studies, with 19 reporting diagnostic test accuracy data. Multiple techniques for measuring dd-cfDNA were reported, including many not requiring a donor sample. It was noted that dd-cfDNA falls rapidly within 2 weeks post transplant and that baseline levels vary by organ type. Levels are elevated in the presence of allograft injury, including acute rejection and infection. Levels return to baseline after successful treatment. Elevation of cfDNA levels is seen in advance of clinically apparent organ injury. Discriminatory power was greatest for higher grades of T cell-mediated and antibody-mediated acute rejection, with high NPVs. The authors noted that cfDNA is a promising biomarker for monitoring the health of SOTs. Future study is needed to define clinical utility and benefit in routine prospective monitoring following SOT (Knight, 2019).

The myTAI_HEART (TAI Diagnostics, Inc., Milwaukee, WI) measures dd-cfDNA in blood plasma as a marker for ACR and transplanted organ injury. This test is conducted exclusively at the TAI Diagnostics clinical reference laboratory with proprietary software using quantitative PCR genotyping. It is proposed for use in HT recipients 2 months of age or older at least 1 week post HT (TAI, 2018). To date, observational studies with small sample size have suggested that dd-cfDNA monitoring of HT recipients may be a useful tool to detect and probably anticipate ACR (North, 2020). Further study is needed to inform about how test results should be interpreted in the context of the individual’s total clinical findings, history and other test results. TAI Diagnostics reported temporary suspension of production of the myTAI_HEART test in 2020. Production has not resumed at the time of this update.

In 2021, Agbor-Enoh and colleagues published results from the NIH-sponsored Genomic Research Alliance for Transplantion (GRAFT) study. Investigators followed 171 HT recipients from five transplant centers over a median post-transplant follow-up period of 17.7 months. Participants received EMBs and other standard post-transplant monitoring, as well as dd-cfDNA levels. They found that dd-cfDNA levels fell rapidly in the weeks following surgery. Rising dd-cfDNA was associated with acute rejection episodes. Elevation of dd-cfDNA ≥ 0.25% of total cfDNA detected acute rejection with 81% sensitivity, 85% specificity, and an NPV of 99.2%. Elevations of dd-cfDNA were greater in AMR than in ACR. Levels rose about 2 weeks before histologically-confirmed ACR and 3.2 months before AMR was detected on biopsy. Higher levels of dd-cfDNA were associated with greater degrees of graft dysfunction. Although the results of this observational study are promising, the authors concluded that, “This work paves the way for clinical utility and mechanistic studies in heart transplantation.”

Other laboratory tests

AlloSeq^® cfDNA (CareDx Inc. Brisbane, CA) also measures dd-cfDNA utilizing low DNA input, NGS technology, and streamlined analysis to assist in improved transplant surveillance. To date, this test is being used for research purposes only.

The Presage^® ST₂ assay (soluble suppression of tumorigenicity-2) (Critical Diagnostics, San Diego, CA) in serum is another noninvasive test which has been cleared by the FDA for use in the prognostic evaluation of chronic heart failure (HF). It has also been suggested for use as a prognostic biomarker post HT as a predictor of AMR (graft-versus-host disease). To date, published evidence has been limited to a few retrospective observational studies. Large well designed trials are needed (Januzzi, 2013; Pascual-Figal, 2011).

The Molecular Microscope^® MMDx—Heart (Kashi Clinical Laboratories, Portland, OR) is a microarray-based system that utilizes microRNA profiling (mRNA gene expression analysis) to assess EMB specimens following HT. It is proposed for use in prognostic evaluations for AMR. Further validation is needed in large well designed trials to confirm initial favorable findings (Halloran, 2017).

The Viracor TRAC^® Heart dd-cfDNA (Viracor Eurofins, Inc. Lee’s Summit, MO) is another assay that uses NGS to determine the percentage of circulating cfDNA in transplant recipients. The cfDNA is extracted from plasma isolated from whole blood. NGS and genome-wide recipient genotype data are then analyzed by a bioinformatics pipeline that calculates the percentage of dd-cfDNA present. This is proposed to correlate with allograft injury due to rejection. To date, this test has not been cleared for diagnostic use by the FDA. This test is not suitable for use during pregnancy, if the donor and recipient are identical twins, if the individual has received multiple transplants from different donors, or if the donor and recipient are siblings from a consanguineous marriage. According to the manufacturer, these clinical situations will cause the bioinformatics pipeline to generate an inaccurate result.

In summary, published scientific information does not show that use of laboratory tests, other than AlloMap molecular profiling testing, leads to improved health outcomes in clinical practice.

Although the current gold standard test for detecting rejection is EMB, this is limited in accuracy, has a high degree of inter-observer variability, and may yield tissue that is not representative of the overall pathology. It is also invasive and can lead to infections, arrhythmias, or ventricular perforation. Despite these limitations, the breath test is currently not established as a substitute for EMB.

According to a scientific statement about newer tests for HT rejection from the American Heart Association, the following is noted:

Standardization of management strategies for AMR is lacking in large part because of the absence of clinical trials that prospectively evaluate therapies for AMR. The definition of AMR is also in flux as more sensitive diagnostic modalities become available. Although the currently available gene expression profile test for rejection (Allomap) is useful in the prediction of ACR, there is evidence that the fraction of circulating cell-free donor DNA may be useful in detecting both ACR and AMR (Colvin, 2015).

*Note: A Humanitarian Use Device (HUD) is a device that has been given special approval by the FDA under the Humanitarian Device Exemption (HDE) regulations and is utilized in special circumstances where a condition is so rare (fewer than 4000 individuals in the U.S. per year) that testing of large numbers of subjects is not feasible. In these special situations, the FDA may grant an HDE provided that: the device does not pose an unreasonable or significant risk of illness or injury; and the probable benefit to health outweighs the risk of injury or illness from its use, taking into account the probable risks and benefits of currently available devices or alternative forms of treatment. Additionally, the FDA notes that the applicant must demonstrate that no comparable devices are available to treat or diagnose the disease or condition, and that they could not otherwise bring the device to market. The labeling for an HUD must state that the device is a Humanitarian Use Device and that, although the device is authorized by federal law, the effectiveness of the device for the specific indication has not been demonstrated (FDA, 2004).

Allograft rejection, also referred to as acute cellular rejection (ACR): The recipient’s immune system rejects the donor heart.

Antibody-mediated rejection (AMR): Refers to all allograft rejection caused by antibodies directed against donor-specific HLA molecules, blood group antigen (ABO)-isoagglutinins, or endothelial cell antigens. Antibody-mediated rejection causes chronic graft failure which is typically resistant to therapy and carries an ominous prognosis for the graft.

Endomyocardium: The innermost lining of the heart.

Endomyocardial biopsy (EMB): A tissue sample of the endomyocardium.

Heart transplant (HT): Removal of a human heart and replacing it with a donor heart.

The following codes for treatments and procedures applicable to this document are included below for informational purposes. Inclusion or exclusion of a procedure, diagnosis or device code(s) does not constitute or imply member coverage or provider reimbursement policy. Please refer to the member’s contract benefits in effect at the time of service to determine coverage or non-coverage of these services as it applies to an individual member.

When services may be Medically Necessary when criteria are met:

When services are Investigational and Not Medically Necessary:
For the procedure code listed above when criteria are not met.

When services are also Investigational and Not Medically Necessary:
For the procedure codes listed below, or when the code describes a procedure indicated in the Position Statement section as investigational and not medically necessary.

Peer Reviewed Publications:

1. Agbor-Enoh S, Jackson AM, Tunc I, et al. Late manifestation of alloantibody-associated injury and clinical pulmonary antibody-mediated rejection: evidence from cell-free DNA analysis. J Heart Lung Transplant. 2018; 37(7):925-932.
2. Agbor-Enoh S, Shah P, Tunc I, et al. GRAfT Investigators. Cell-free DNA to detect heart allograft acute rejection. Circ. 2021; 143(12):1184-1197.
3. Agbor-Enoh S, Tunc I, De Vlaminck I, et al. Applying rigor and reproducibility standards to assay donor-derived cell-free DNA as a non-invasive method for detection of acute rejection and graft injury after heart transplantation. J Heart Lung Transplant. 2017; 36(9):1004-1012.
4. Bernstein D, Williams GE, Eisen H, et al. Gene expression profiling distinguishes a molecular signature for grade 1B mild acute cellular rejection in cardiac allograft recipients. J Heart Lung Transplant. 2007; 26(12):1270-1280.
5. Cadeiras M, Shahzad K, John MM, et al. Relationship between a validated molecular cardiac transplant rejection classifier and routine organ function parameters. Clin Transplant. 2010; 24(3):321-327.
6. Cadeiras, M, von Bayern M, Sinha A, et al. Noninvasive diagnosis of acute cardiac allograft rejection. Curr Opin Organ Transplant. 2007; 12(5):543-550.
7. Crespo-Leiro MG, Stypmann J, Schulz U, et al. Performance of gene-expression profiling test score variability to predict future clinical events in heart transplant recipients. BMC Cardiovasc Disord. 2015; 15:120.
8. Crespo-Leiro MG, Stypmann J, Schulz U, et al. Clinical usefulness of gene-expression profile to rule out acute rejection after heart transplantation: CARGO II. Eur Heart J. 2016; 37(33):2591-2601.
9. Crespo-Leiro MG, Zuckermann A, Bara C, et al. Concordance among pathologists in the second Cardiac Allograft Rejection Gene Expression Observational Study (CARGO II). Transplantation. 2012; 94(11):1172-1177.
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16. Halloran PF, Potena L, Van Huyen JD, et al. Building a tissue-based molecular diagnostic system in heart transplant rejection: The heart Molecular Microscope Diagnostic (MMDx) System. J Heart Lung Transplant. 2017; 36(11):1192-1200.
17. Hidestrand M, Tomita-Mitchell A, Hidestrand PM, et al. Highly sensitive non-invasive cardiac transplant rejection monitoring using targeted quantification of donor specific cell free DNA. J Am Coll Cardiol. 2014; 63(12):1224-1226.
18. Januzzi JL, Horne BD, Moore SA, et al. Interleukin receptor family member ST2 concentrations in patients following heart transplantation. Biomarkers. 2013; 18(3):250-256.
19. Jarcho JA. Fear of rejection--monitoring the heart-transplant recipient. N Engl J Med. 2010; 362(20):1932-1933.
20. Kamath M, Shekhtman G, Grogan T, et al. Variability in donor-derived cell-free DNA scores to predict mortality in heart transplant recipients - A proof-of-concept study. Front Immunol. 2022; 13:825108.
21. Keller M, Bush E, Diamond JM, et al. Use of donor-derived-cell-free DNA as a marker of early allograft injury in primary graft dysfunction (PGD) to predict the risk of chronic lung allograft dysfunction (CLAD). J Heart Lung Transplant. 2021; 40(6):488-493.
22. Khush KK, Patel J, Pinney S, et al. Noninvasive detection of graft injury after heart transplant using donor‐derived cell‐free DNA: A prospective multicenter study. Am J Transplant. 2019; 19:2889-2899.
23. Knight SR, Thorne A, Lo Faro ML. Donor-specific cell-free DNA as a biomarker in solid organ transplantation. A systematic review. Transplant. 2019; 103(2):273-283.
24. Kobashigawa J, Patel J, Azarbal B, et al. Randomized pilot trial of gene expression profiling versus heart biopsy in the first year after heart transplant: early invasive monitoring attenuation through gene expression trial (EIMAGE). Circ Heart Fail. 2015; 8(3):557-564.
25. Loupy A1, Duong Van Huyen JP2, et al. Gene expression profiling for the identification and classification of antibody-mediated heart rejection. Circ. 2017; 135(10):917-935.
26. Macher HC, García-Fernández N, Adsuar-Gómez A, et al. Donor-specific circulating cell free DNA as a noninvasive biomarker of graft injury in heart transplantation. Clin Chim Acta. 2019; 495:590-597.
27. Marboe CC, Lal PG, Chu K, et al. Distinctive peripheral blood gene expression profiles in patients forming nodular endocardial infiltrates (Quilty lesions) following heart transplantation. J Heart Lung Transplant. 2005;  24(2 suppl):S97.
28. Mehra MR, Kobashigawa JA, Deng MC, et al.; CARGO Investigators. Transcriptional signals of T-cell and corticosteroid-sensitive genes are associated with future acute cellular rejection in cardiac allografts. J Heart Lung Transplant. 2007b; 26(12):1255-1263.
29. Mehra MR, Kobashigawa JA, Deng MC, et al.; CARGO Investigators. Clinical implications and longitudinal alteration of peripheral blood transcriptional signals indicative of future cardiac allograft rejection. J Heart Lung Transplant. 2008; 27(3):297-301.
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31. North PE, Ziegler E, Mahnke DK, et al. Cell-free DNA donor fraction analysis in pediatric and adult heart transplant patients by multiplexed allele-specific quantitative PCR: Validation of a rapid and highly sensitive clinical test for stratification of rejection probability. PLoS One. 2020; 15(1).
32. Pascual-Figal DA, Garrido IP, Blanco R, et al. Soluble ST2 is a marker for acute cardiac allograft rejection. Ann Thorac Surg. 2011; 92(6): 2118-2124.
33. Pham MX, Deng MC, Kfoury AG, et al. Molecular testing for long-term rejection surveillance in heart transplant recipients: design of the Invasive Monitoring Attenuation through Gene Expression (IMAGE) trial. J Heart Lung Transplant. 2007; 26(8):808-814.
34. Pham MX, Teuteberg JJ, Kfoury AG, et al.; IMAGE Study Group. Gene-expression profiling for rejection surveillance after cardiac transplantation. N Engl J Med. 2010; 362(20):1890-1900.
35. Phillips M, Boehmer JP, Cataneo RN, et al. Heart allograft rejection: detection with breath alkanes in low levels (the HARDBALL study). J Am Coll Cardiol. 2002; 40(1):12-13.
36. Phillips M, Boehmer JP, Cataneo RN, et al. Heart allograft rejection: detection with breath alkanes in low levels (the HARDBALL study). J Heart Lung Transplant. 2004a; 23(6):701-708.
37. Phillips M, Boehmer JP, Cataneo RN, et al. Prediction of heart transplant rejection with a breath test for markers of oxidative stress. Am J Cardiol. 2004b; 94(12):1593-1594.
38. Regalie W, Stamm K, Hidestrand P. Novel assay to calculate donor fraction of cell-free DNA in heart transplant. J Am Coll Cardiol. 2018; 71(11):supplement A764.
39. Regalie W, Stamm K, Mahnke D, et al. Noninvasive assay for donor fraction of cell-free DNA in pediatric heart transplant recipients. J Am Coll Cardiol. 2018; 71(25):2982-2983.
40. Richmond ME, Zangwill SD, Kindel SJ, et al. Donor fraction cell-free DNA and rejection in adult and pediatric heart transplantation. J Heart Lung Transplant. 2020; 39(5): 454-463.
41. Sobotka PA, Gupta DK, Lansky DM, et al. Breath pentane is a marker of acute cardiac allograft rejection. J Heart Lung Transplant. 1994; 13(2):224-229.
42. Starling RC, Pham M, Valantine H, et al.; Working Group on Molecular Testing in Cardiac Transplantation.  Molecular testing in the management of cardiac transplant recipients: initial clinical experience. J Heart Lung Transplant. 2006; 25(12):1389-1395.
43. Strecker T, Rösch J, Weyand M, Agaimy A. Endomyocardial biopsy for monitoring heart transplant patients: 11-years-experience at a German heart center. Int J Clin Exp Pathol. 2013; 6(1):55-65.
44. Van Huyen JPD, Tible M, Gay A, et al. MicroRNAs as non-invasive biomarkers of heart transplant rejection. Eur Heart J. 2014; 35(45):3194-3202.
45. Yamani MH, Taylor DO, Haire C, et al. Post-transplant ischemic injury is associated with up-regulated AlloMap gene expression. Clin Transplant. 2007a; 21(4):523-525.
46. Yamani MH, Taylor DO, Rodriguez ER, et al. Transplant vasculopathy is associated with increased AlloMap gene expression score. J Heart Lung Transplant. 2007b; 26(4):403-406.

Government Agency, Medical Society, and Other Authoritative Publications:

1. Berry GJ, Burke MM, Andersen C, et al. The 2013 International Society for Heart and Lung Transplantation Working formulation for the standardization of nomenclature in the pathologic diagnosis of antibody-mediated rejection in heart transplantation. J Heart Lung Transplant. 2013; 32(12):1147-1162.
2. CareDx. Outcomes AlloMap Registry: The long-term management and outcomes of heart transplant recipients with AlloMap Testing (OAR). NLM Identifier: NCT01833195. Last updated Feb. 5, 2020. Available at: https://clinicaltrials.gov/ct2/show/NCT01833195?term=AlloMap. Accessed on May 15, 2023.
3. CareDx. Utility of Donor-Derived Cell Free DNA in Association With Gene Expression Profiling (D-OAR). NLM Identifier: NCT02178943. Last updated January 30, 2019. Available at: https://www.clinicaltrials.gov/ct2/show/NCT02178943?term=NCT02178943&draw=2&rank=1. Accessed on May 15, 2023.
4. CareDx. AlloSeq cfDNA, a donor-derived cell-free DNA measurement solution for on-site monitoring of solid organ transplants. Available at: https://caredx.com/wp-content/uploads/2020/10/P198-Poster-AQ-cfDNA-1.pdf. Accessed on May 15, 2023.
5. Centers for Medicare and Medicaid Services. National Coverage determination. Heartsbreath Test for heart transplant rejection. NCD #260.10. December 8, 2008. Available at: http://www.cms.gov/medicare-coverage-database/details/ncd-details.aspx?NCDId=325&ncdver=1&DocID=260.10&from2=index_chapter_list.asp&list_type=&bc=gAAAAAgAAAAAAA%3d%3d&. Accessed on May 15, 2023.
6. Colvin MM, Cook JL, Chang P, et al. Antibody-mediated rejection in cardiac transplantation: emerging knowledge in diagnosis and management: A Scientific Statement from the American Heart Association. Circ. 2015; 131(18):1608-1639.
7. Costanzo MR, Dipchand A, Starling R. et al. The International Society of Heart and Lung Transplantation guidelines for the care of heart transplant recipients. J Heart Lung Transplant. 2010; 29(8):914-956. Available at: http://www.jhltonline.org/article/S1053-2498(10)00358-X/abstract. Accessed on May 15, 2023.
8. Francis GS, Greenberg BH, Hsu DT, et al. ACCF/AHA/ACP/HFSA/ISHLT 2010 clinical competence statement on management of patients with advanced heart failure and cardiac transplant: a report of the ACCF/AHA/ACP Task Force on Clinical Competence and Training. Circulation. 2010; 122(6):644-672. Available at: http://circ.ahajournals.org/content/122/6/644.full.pdf. Accessed on May 15, 2023.
9. Moayedi Y, Foroutan F, Miller RJH, et al. Risk evaluation using gene expression screening to monitor for acute cellular rejection in heart transplant recipients. J Heart Lung Transplant. 2019; 38(1):51-58.
10. National Heart, Lung and Blood Institute (NHLBI). Genome Transplant Dynamics. NLM Identifier: NCT02423070. Last updated March 3, 2023. Available at: https://www.clinicaltrials.gov/ct2/show/NCT02423070?term=NCT02423070&draw=2&rank=1. Accessed on May 15, 2023.
11. Society for Cardiovascular Pathology. International Society for Heart and Lung Transplantation (ISHLT) revised grading criteria. 2014.
12. Stewart S, Winters GL, Fishbein MC, et al. Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J Heart Lung Transplant. 2005; 24(11):1710-1720.
13. Taylor D, Meiser B, Webber, S, et al. The International Society of Heart and Lung Transplantation (ISHLT). Guidelines for the Care of Heart Transplant Recipients, Task Force 2: Immunosuppression and Rejection. 2010 Nov. 8; 1-41.
14. Tice JA. California Technology Assessment Forum (CTAF). Gene expression profiling for the diagnosis of heart transplant rejection. A Technology Assessment.  San Francisco, CA: CTAF; October 13, 2010.
15. U.S. Food and Drug Administration (FDA). Center for Devices and Radiological Health (CDRH). New Humanitarian Device Approval. Heartsbreath No. H030004. Rockville, MD:FDA. February 24, 2004. Available at: https://www.accessdata.fda.gov/cdrh_docs/pdf3/h030004c.pdf. Accessed on May 15, 2023.
16. U. S. Food and Drug Administration (FDA). Center for Devices and Radiological Health (CDRH). Nucleic Acid based tests. AlloMap^® Molecular Expression Testing. Available at: https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleic-acid-based-tests. Accessed on March 28, 2023.
17. U.S. Food and Drug Administration (FDA). Center for Devices and Radiological Health. Humanitarian Use Device Exemptions. Available at: http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/DeviceApprovalsandClearances/HDEApprovals/ucm161827.htm. Accessed on May 15, 2023.
18. Velleca A, Shullo MA, Dhital K, et al. The International Society for Heart and Lung Transplantation (ISHLT) Guidelines for the Care of Heart Transplant Recipients. J Heart Lung Transplant. 2023; 42(5):e1-e141.
19. XDx, Inc. A Comparison of AlloMap Molecular Testing and Traditional Biopsy-based Surveillance for Heart Transplant Rejection Early Post-transplantation (EIMAGE). NLM Identifier: NCT00962377. Last updated December 21, 2010. Available at: http://www.clinicaltrials.gov/ct2/show/NCT00962377?term=AlloMap&rank=1. Accessed on May 15, 2023.
20. XDx, Inc. Cardiac Allograft Rejection Gene Expression Observational (CARGO) II STUDY (CARGOII). NLM Identifier: NCT00761787. Last updated March 9, 2009. Available at: http://www.clinicaltrials.gov/ct2/show/NCT00761787?term=AlloMap&rank=2. Accessed on May 15, 2023.
21. XDx, Inc. IMAGE: A Comparison of AlloMap Molecular Testing and Traditional Biopsy-based Surveillance for Heart Transplant Rejection. NLM Identifier: NCT00351559.  Last updated November 20, 2009. Available at: http://www.clinicaltrials.gov/ct2/show/NCT00351559?term=00351559&rank=1. Accessed on May 15, 2023.

AlloMap
AlloSure Heart
AlloSeq cfDNA
Breath Test as an Aid for Diagnosis of Heart Transplant Rejection
Donor-derived cell free DNA, dd-cfDNA
Gene Expression Molecular Profiling
Heartsbreath
MMDx
myTAI _HEART
Presage ST₂
Viracor TRAC Heart dd-cfDNA

The use of specific product names is illustrative only.  It is not intended to be a recommendation of one product over another, and is not intended to represent a complete listing of all products available.