Heart Allograft Rejection: Detection With Breath Alkanes in Low Levels (the HARDBALL Study) Michael Phillips, MD, FACP, a,b John P. Boehmer, MD, c Renee N. Cataneo, MA, a Taseer Cheema, MD, a Howard J. Eisen, MD, d John T. Fallon, MD, PhD, FAHA, e Peter E. Fisher, MD, f Alan Gass, MD, g Joel Greenberg, BS, a Jon Kobashigawa, MD, h Donna Mancini, MD, f Barry Rayburn, MD, i and Mark J. Zucker, MD j Background: We evaluated a new marker of heart transplant rejection, the breath methylated alkane contour (BMAC). Rejection is accompanied by oxidative stress that degrades membrane polyunsaturated fatty acids, evolving alkanes and methylalkanes, which are excreted in the breath as volatile organic compounds (VOCs). Methods: Breath VOC samples (n = 1,061) were collected from 539 heart transplant recipients before scheduled endomyocardial biopsy. Breath VOCs were analyzed by gas chromatography and mass spectroscopy, and BMAC was derived from the abundance of C4 –C20 alkanes and monomethylal- kanes. The “gold standard” of rejection was the concordant set of International Society for Heart and Lung Transplantation (ISHLT) grades in biopsies read by 2 reviewers. Results: Concordant biopsies were: Grade 0, 645 of 1,061 (60.8%); 1A, 197 (18.6%); 1B, 84 (7.9%); 2, 93 (8.8%); and 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%, positive predictive value 5.6%, negative predictive value 97.2%). Site pathologists identified the same cases with sensitivity of 42.4%, specificity 97.0%, positive predictive value 45.2% and negative predictive value 96.7%. Conclusions: A breath test for markers of oxidative stress was more sensitive and less specific for Grade 3 heart transplant rejection than a biopsy reading by a site pathologist, but the negative predictive values of the 2 tests were similar. A screening breath test could potentially identify transplant recipients at low risk of Grade 3 rejection and reduce the number of endomyocardial biopsies. J Heart Lung Transplant 2004;23:701– 8. More than 61,000 heart transplant operations have been performed since 1967; at least 23,000 of the recipients are presently known to be alive, although the actual number of survivors may exceed 30,000. 1 All these recipients require periodic screening for rejection, a condition that is difficult to detect clinically. Symptoms such as malaise, fatigue, dyspnea, edema and anorexia are uncommon because ventricular function is usually not affected. Right ventricular endomyocardial biopsy is the current “gold standard” for diagnosis of heart transplant rejection, and post-operative biopsies are generally performed weekly for the first 6 weeks, biweekly until the third month, monthly until the sixth month, then every 1 to 3 months depending on clinical indications. However, most biopsies yield normal or near-normal results that elicit no changes in treatment. Although considered safe, the procedure is invasive and may cause complications such as hematoma, infection, arrhythmia, ventricular perforation and fistulas. a From Menssana Research, Inc. Fort Lee, New Jersey; b Department of Medicine, New York Medical College, Valhalla, New York; c Depart- ment of Medicine, M.S. Hershey Medical Center of the Pennsylvania State University School of Medicine, Hershey, Pennsylvania; d Temple University Hospital, Philadelphia, Pennsylvania; e Department of Pa- thology, Mount Sinai Medical Center, New York; f Columbia Presby- terian Medical Center, New York, New York; g Department of Medi- cine, Mount Sinai Medical Center, New York, New York; h University of California Los Angeles Medical Center, Los Angeles, California; i University of Alabama at Birmingham, Birmingham, Alabama; and j Newark Beth Israel Medical Center, Newark, New Jersey. Submitted January 22, 2003; revised July 18, 2003; accepted July 23, 2003. Michael Phillips is president of Menssana Research, Inc., and holds patents issued and pending on the breath test employed in this study. None of the other authors has any conflict of interest or financial interest with regard to this article. Supported by a grant from the NHLBI (2R44HL59715). This study was presented as a Late Breaking Clinical Trial at the American College of Cardiology 51st Annual Scientific Session, At- lanta, GA, March 19, 2002. Reprint requests: Michael Phillips, MD, FACP, Menssana Research, Inc., 1 Horizon Road, Suite 1415, Fort Lee, NJ 07024. E-mail: menssana@bellatlantic.net Copyright © 2004 by the International Society for Heart and Lung Transplantation. 1053-2498/04/$–see front matter. doi:10.1016/ j.healun.2003.07.017 701