In Vivo Imaging of Acute Cardiac Rejection in Human Patients Using 99m Technetium Labeled Annexin V

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1 American Journal of Transplantation 2001; 1: Copyright C Munksgaard 2001 Munksgaard International Publishers ISSN In Vivo Imaging of Acute Cardiac Rejection in Human Patients Using 99m Technetium Labeled Annexin V Murray H. Kown a, H. William Strauss b, Francis G. Blankenberg c, Gerald J. Berry d, Sandy Stafford-Cecil b, Jonathan F. Tait e, Michael L. Goris b and Robert C. Robbins a, * a Department of Cardiothoracic Surgery, b Department of Nuclear Medicine, c Department of Radiology and d Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA; and e Department of Laboratory Medicine, University of Washington, USA * Corresponding author: Robert C. Robbins MD, Falk Research Building, 2nd Floor, Stanford University Medical School, Stanford, California , robbins@leland.stanford.edu Annexin V binds phosphatidylserine moieties on apoptotic cells. This study reports the initial experience at Stanford University Medical Center with 99m Tc-labeled annexin V imaging as a noninvasive measure of apoptosis in acute cardiac rejection. Ten cardiac transplant patients had 99m Tc Annexin V imaging and endomyocardial biopsy (EMB) performed within 24 h. No complications related to 99m Tc annexin V administration occurred. Eight patients had ISHLT grade of acute rejection of 1A or less. Five patients had two or more areas of uptake noted in the right ventricle on imaging studies. Two of these patients had positive biopsies: one patient had grade 2 rejection with two focal uptake areas and another had grade 3A rejection with three foci. An additional five patients had either one or zero hot spot areas and corresponding negative EMBs. 99m Tc-annexin V appears to be well tolerated and may identify patients with acute cardiac rejection. Key words: Apoptosis, acute rejection, heart transplantation, nuclear imaging Received 2 February 2001, revised and accepted for publication 21 May 2001 Introduction Acute cellular rejection is a principal cause of patient mortality during the first year following cardiac transplantation (1). Endomyocardial biopsies (EMBs) of rejecting hearts show a characteristic lymphocytic infiltrate, but the exact mechanism by which these cells mediate myocytolysis is disputed (2). Two forms of cellular death, apoptosis and necrosis, are postulated to occur (3 6). Apoptosis, otherwise known as programmed cell death, is an energy-requiring process where nuclear and cytoplasmic contents are condensed and phago- cytized without an inflammatory response (7,8). Necrosis, on the other hand, is characterized by myocyte swelling, disruption of cellular membranes, and marked inflammation (9). There is increasing evidence that myocyte apoptosis is widespread in acutely rejecting cardiac allografts. Laguens et al. (10) found a strong correlation between DNA fragmentation characteristic of apoptosis and evidence of acute cellular rejection. In an experimental study, apoptotic myocytes were found adjacent to areas of macrophage-rich inflammatory cells and were significantly higher in allografts when compared with native hearts or syngeneic transplants (11). Finally, the two major apoptotic pathways consisting of degranulation of proteases in conjunction with pore forming perforin and the interaction of fas fas ligand also appeared to be up-regulated in a rat model of acute cardiac allograft rejection (5). An early feature of apoptosis is the loss of the normal asymmetry of phosphatidylserines (PS) in the cellular membrane (12). This phospholipid is usually restricted to the inner surface of the lipid bilayer by an ATP-dependent enzyme, translocase. The rise in intracellular calcium during apoptosis, however, deactivates this enzyme, allowing PS to become exposed on the outer layer where it is an accessible target for various assays of apoptosis (13). Annexin V is a human protein that has a strong affinity for membrane-bound PS (14). It has been used conjugated to a fluorescein label to detect apoptotic cells in vitro with flow cytometry (15) as well as with radiolabeled 99m technetium ( 99m Tc) to measure in vivo apoptosis in a rat cardiac allograft model of rejection (16). Although endomyocardial biopsy remains the gold standard for diagnosis of acute rejection in heart transplantation, it is an invasive and expensive procedure with an inherent risk of complications (17,18). While numerous investigators have suggested other less invasive tests, none have replaced the EMB as a means either to successfully detect acute rejection or to monitor the efficacy of treatment (19 21). This study was performed to determine the pharmacokinetic properties of 99m Tc-labeled annexin V in human cardiac transplant patients and to compare the results of imaging with this agent to endomyocardial biopsies. Patients and Methods Patient selection: inclusion/exclusion criteria Ten heart transplant patients (eight males and two females, mean age years) volunteered for this study. The tranplant technique con- 270

2 In Vivo Imaging of Acute Cardiac Rejection Table 1: Patient demographics Patient Image POD Gender Age 1 88 F M M F M M M M M M 62 The study was performed between 2 weeks and 1 year following heart transplantation. Eight male and two female patients participated, with mean age years. POD, post operative day. sisted of bi-caval anastomoses leaving the donor right atrium intact. The indications for transplantation were either idiopathic dilated cardiomyopathy or ischemic cardiomyopathy. The mean interval between transplantation and imaging was d (Table1). Patient inclusion criteria consisted of a postoperative time-point of 2weeks to 1year, minimum age of 18 years, and a life expectancy of at least 16 weeks. Because of the theoretical anticoagulant effects of annexin V (22), patients with any nonpharmacologic coagulation disorder were excluded. Other exclusion criteria included pregnancy or breast-feeding in female patients as well as patients unwilling or unlikely to be available for follow-up. All patients provided informed consent and the study was conducted under approval of the Human Subjects Committee at Stanford University School of Medicine. Preparation of 99m Tc-annexin V Annexin V was produced by expression in Escherichia coli as previously described (23) and labeled with 99m Tc by incubation with a technetium diamide dimercaptide N 2 S 2 chelate (TFP) just prior to intravenous administration (24). TFP is a bi-functional molecule capable of bonding lysine residues of various proteins on one moiety and 99m Tc on the other. A specific activity of mci/mg protein with a radiopurity of 92 97% was achieved. Imaging technique The administered dose of 99m Tc-annexin V ranged from 10 to 27mCi and depended upon radiopharmaceutical availability. The tracer was injected into a peripheral vein while blood pressure and pulse rate were monitored. Images were recorded as follows: (i) dynamic images of the heart (15 frames/s) for 10min in the anterior view; (ii) 10-min static images in both the anterior and LAO projections at 60 and 120min; (iii) single photon emission computed tomography (SPECT) images of the chest between 2 and 4h (30s/angle with 3æ increments for 60 views with a triple head camera for 30min total acquisition time); (iv) anterior and posterior whole body images between 2 and 4h; and (v) images of the heart in the anterior and LAO projections at 20 28h. All images were acquired using a high-resolution parallel collimater with a 15% window centered on the 140keV photopeak of 99m Tc. Dynamic and SPECT images were acquired on a Siemens MS3 camera on and matrices, respectively. Planar and whole body images were taken on a Siemens Body Scan Camera with and matrices, respectively (Siemens, Hoffman Estates, IL). Dynamic images were used to calculate the half-life of 99m Tc-annexin V blood clearance. Coronal sections of reconstructed SPECT images were reviewed by two investigators and scored for the number of sites with focal uptake in the right ventricle. Focal hot spots were defined as those areas with intensity greater than that of the surrounding blood pool. Vital signs were measured 10, 15, 30, 60min and 24h after intravenous injection of 99m Tc-annexin V. Complete blood counts, prothrombin time, partial thromboplastin time, bleeding time, comprehensive panel, and EKG were obtained before and after the study. Biopsy and tissue processing Endomyocardial biopsy was performed on each patient within 24 h of 99m Tc-annexin V imaging. A seven French Cordis Bioptome (Cordis Corporation, Miami, FL) was introduced percutaneously into the right internal jugular vein and advanced into the right ventricle under fluoroscopic guidance. Four to six biopsy specimens were obtained from each patient and immediately fixed in 10% buffered formalin, processed in paraffin, and cut into 5-mm sections. Slides were stained with hematoxylin and eosin (H&E), analyzed by an experienced pathologist, and assigned a histologic grade of acute rejection using the ISHLT working formulation (25) (ISHLT grade 0 4). Separate slides prepared from the same specimens were assessed for DNA fragmentation consistent with apoptosis using Apoptag, a commercially available TUNEL staining kit (Intergen, Purchase, NY) (Figure 1). For TUNEL staining, 5-mm sections were de-paraffinized using xylene and rehydrated in decreasing dilutions of ethanol. Slides were treated with proteinase K(20mg/mL) for 10min at room temperature and washed in distilled water. Endogenous peroxidase was quenched with 3.0% H 2 O 2 for 10min. Slides were then washed three times in TdT buffer and covered in working strength TdT enzyme. Following stop buffer wash for 30min, Table 2: Laboratory values Patient PT PTT Bleeding Time WBC HCT PLT (10.7) 23.7 (25.5) 6 (4.5) 5.3 (7.4) 26.1 (28.6) 141 (147) (10.7) 32.2 (25.5) 20 (13) 4.6 (5.0) 34.6 (34.5) 157 (157) (10.6) 27.9 (27.9) 7.5 (6) 5.0 (4.1) 34.8 (34.7) 231 (219) (12.6) 35.4 (35.0) 4.5 (5.0) 5.3 (6.5) 28.5 (29.7) 154 (151) (12.4) 29.7 (31.6) 20 (18) 5.9 (4.7) 28.6 (29.6) 292 (305) (11.2) 37.9 (38.1) 8 (9) 9.7 (9.5) 20.4 (28.3) 111 (118) (11.5) 25.8 (27.2) 7 (4.5) 8.8 (8.3) 43.1 (40.4) 138 (138) (10.7) 28.4 (20.2) 4.5 (6) 10.1 (7.9) 30.1 (27.9) 252 (103) (10.7) 31.3 (29.0) NA 9.7 (8.3) 35.0 (32.6) 431 (319) (10.8) 25.0 (25.6) NA 9.9 (9.6) 27.1 (28.2) 312 (327) Laboratory values showed no significant differences both before and 24 h after (in parentheses) radiolabeled annexin V administration. PT, prothrombin time; PTT, partial thromboplastin time; WBC, white blood cells, HCT, hematocrit; PLT, platelets. American Journal of Transplantation 2001; 1:

3 Kown et al. Figure 1: Hematoxylin and eosin (H&E) and TUNEL staining in endomyocardial biopsy specimens. A. High-power magnification showing no acute rejection (ISHLT grade 0, 400 ). B. TU- NEL slide with absence of darkened nuclei denoting apoptosis. C. Solitary focus of mononuclear cells in a biopsy with focal mild acute rejection (ISHLT grade 1A). D. TUNEL staining showing scattered apoptotic nuclei within the inflammatory infiltrate. E. One of two foci of inflammation with myocyte damage in grade 3A rejection specimen. F. Numerous apoptotic nuclei of inflammatory cells in grade 3A rejection sample. Other foci of rejection also showed apoptotic inflammatory cells. antidigoxigenin conjugate was applied for 30min. Slides were developed with diaminobenzidine (Sigma Chemical, St Louis, MO) for 1 min and counterstained with hematoxylin. Presence of TUNEL-positive nuclei was assessed in infiltrating inflammatory cells, myocytes, and endothelial cells by an experienced pathologist at 400 magnification and assigned scores as follows:, none; π, scattered; ππ, many or multifocal distribution. Thyroid tissue was used as a positive control, as a basal rate of apoptotic cellular turnover is normally present within this organ (26). Results Toxicities and adverse reactions No adverse events, reactions, or complications occurred. Despite earlier reports of the potential anticoagulant effects of annexin V (22), no prolongations of bleeding times or coagulation parameters were noted (Table 2). Pharmacokinetics 99m Technetium-annexin V exhibited typical multicompartment volume of distribution following intravenous administration with rapid mixing in the blood followed by diffusion into the extracellular space. The half-life of blood clearance was min in this study, similar to the approximate 10-min time-point found in rats by Ohtsuki et al. (27). Images at 24 h revealed only residual activity within the bowel lumen. Images Whole-body images at 2 h after injection of 99m Tc-annexin V demonstrated characteristic uptake within the liver, kidneys, and urinary bladder similar to that seen in the experimental studies (16). In addition, the spleen, paranasal sinuses, and testicles had consistent uptake. The ovaries of the two female patients were difficult to discern because of overlying bowel (Figure 2). 272 American Journal of Transplantation 2001; 1:

4 In Vivo Imaging of Acute Cardiac Rejection Uptake in the hearts was variable and was not nearly the same degree of intensity as that seen in the rat heart allografts. Unlike the diffuse pattern of 99m Tc-annexin V binding seen in untreated rejecting rat hearts, the uptake in human patients receiving immunosuppression was limited to discreet hot spots in the myocardium. SPECT coronal sections through the right ventricular wall demonstrated between zero and three sites of focal activity (Figure 3). Histology and 99m Tc-annexin V image scores Acute rejection occurred infrequently in the 10 volunteer subjects. Eight had either no evidence of acute rejection (ISHLT grade 0) or focal mild acute rejection (ISHLT grade 1A) in the biopsy specimens obtained within 24 h of 99m Tc-annexin V imaging. The remaining two patients had either focal moderate acute rejection (grade 2) or multifocal moderate acute rejection (grade 3A) (Table 3). This final patient had myocyte damage in just two foci, the minimum needed to receive the 3A classification. Comparisons between histology and 99m Tc-annexin V imaging demonstrated a potential for the latter to overestimate rejection (Table 4). Of the eight patients with either ISHLT grade 0 or 1A, four had a scan with either zero or one focal area of uptake. The remaining four patients, however, had between two and three focal hot spots. Of the two patients with moderate rejection, each had a scan with relatively high numbers of uptake with two and three right ventricular foci noted. Table 3 also shows the histologic results of EMBs conducted both before and after that which was conducted at the time of 99m Tc-annexin V imaging. Although the intervening time between biopsies varied from patient to patient, there was no obvious indication that the intensity of 99m Tc-annexin V images either preceded or lagged biopsy results. TUNEL staining TUNEL positive staining was most prevalent in infiltrating inflammatory cells and in occasional myocytes. No endothelial cells had evidence of apoptosis (Table 5). The number of TU- NEL-positive myocytes did not appear to have a strong correlation with either 99m Tc-annexin V imaging or histologic grade of acute rejection. Discussion This study describes our initial experience with radiolabeled annexin V as an imaging tool for the diagnosis of acute rejection in human heart transplant patients. Experiments in heterotopic rat heart transplants initially showed a strong correlation between the histologic grade of allograft rejection and the percentage uptake of 99m Tc-annexin V (16). Unlike control rat hearts, however, where severe rejection produced images with intense diffuse uptake, scans in immunosuppressed human patients, who by and large presented with minimal acute rejection, had much smaller regions of focal activity. Only two of the 10 patients in this study had moderate acute rejection. While both of these patients had relatively high numbers of focal hot spots (two or more) seen on coronal SPECT images, the data regarding the remaining eight patients indicated that 99m Tc-annexin V might document a level of cellular death that is not recognized by EMB histology alone. Four of the eight patients with low acute rejection scores (ISHLT IA or less) also had higher numbers of foci seen on their 99m Tc-annexin images. One explanation for this was suggested in a study by Puig et al. (28), in which apoptotic cells were found in the absence of histologic evidence of acute rejection (ISHLT grade 0) despite documentation of myocardial injury. The authors hypothesized that apoptosis may be the additional mechanism of myocardial damage in cardiac transplantation that was not indicated by the EMB histology alone. Figure 2: Whole body 99m technetium-annexin V image: anterior (A) and posterior (B) views. Images acquired 2 h postintravenous 99m Tc-annexin V administration show characteristic uptake within the kidneys, urinary tract, liver, spleen, paranasal sinuses, and testes, and focal uptake within the heart. American Journal of Transplantation 2001; 1: The utility of an apoptosis assay for the diagnosis of acute rejection in heart transplant patients remains to be established. It is unclear from the data in this study whether the 99m Tc-annexin V images are truly picking up apoptotic areas of the heart based on its lack of strong correlation with the TUNEL assay results. However, TUNEL also appears to have 273

5 Kown et al. Figure 3: SPECT images. A. Representative coronal section of 2 h SPECT image with liver (a), right atrium (b), right ventricle (c), and spleen (d) delineated. B. Representative coronal sections of 2 h SPECT images with zero (a), one (b), two (c), and three (d) hot spot areas visible within the right ventricle. Variable number of foci are also visible in the region of the right atrium. 274 American Journal of Transplantation 2001; 1:

6 In Vivo Imaging of Acute Cardiac Rejection Table 3: Histologic scores of acute rejection Patient Pre-study Study Post-study 1 2 (ª14) 1A 1A (14) 2 0 (ª15) 0 0 (105) 3 0 (ª38) 1A 1A (35) 4 0 (ª14) 0 1B (14) 5 1A (ª59) 2 1B (64) 6 0 (ª27) 1A 0 (14) 7 1A (ª7) 1A n/a 8 2 (ª8) 1A 1A (14) 9 1A (ª14) 3A 1A (14) 10 0 (ª7) 0 1A (7) The study column exhibits ISHLT rejection grades at the time of 99m Tc-annexin V imaging. Pre- and post-study biopsy results are also listed, with the number of interval days from 99m Tc-annexin V imaging shown in parentheses. limitations as an accurate assay for programmed cell death. Because DNA fragmentation is a transient phenomenon and represents a late execution phase of apoptosis, it may underestimate this process (29). Low specificity and possibility of Table 4: Interpretation of 99m Tc-annexin V images Patient ISHLT rejection score RV 1 1A A A 0 7 1A 1 8 1A 2 9 3A Number of focal areas of uptake within the right ventricle (RV) are listed with corresponding ISHLT rejection grades, respectively. Although both ISHLT grade 2 and 3A hearts had positive 99m Tc-annexin V images, there was also a propensity for overscoring, with half of the patients with grade IA or less having two foci of uptake on imaging. Table 5: TUNEL staining Patient IC M EC 1 π 2 π π 6 7 π 8 ππ π 9 ππ π 10 TUNEL-positive nuclei were assessed in regions of infiltrating cells (IC), myocytes (M), and endothelial cells (EC):, none; π, scattered; ππ, many or multifocal distribution. There was a relative scarcity of TUNEL-positive cells within cardiac myocytes regardless of ISHLT grade or 99m Tc-annexin V image scores. American Journal of Transplantation 2001; 1: tissue sectioning artefacts are additional limitations of this study (30 32). There is also disagreement among investigators regarding which cell types are truly undergoing apoptosis. While some have documented TUNEL-positive myocytes in their animal and human models of cardiac rejection (5,10,11,13), others, including this laboratory, have noted that apoptosis appears to be occurring predominantly within the infiltrating immune cells (30,33). It is unknown, therefore, whether the 99m Tcannexin V images are measuring apoptosis in cardiac myoctes, in infiltrating immune cells, or perhaps indicating a degree of apoptosis that is not suggested by the TUNEL staining alone. Because of these questions regarding TUNEL, it is unclear what significance to place on the lack of a strong correlation between histologic evidence of TUNEL-positive cells and annexin V images in this study. The role of apoptosis in the true pathogenesis of myocardial damage is also not yet universally accepted and there is no current experience using this form of cell death as a criterion for diagnosing acute rejection. Even the gold standard endomyocardial biopsy procedure possesses certain limitations of sampling error and misinterpretation of specimens that call into question its accuracy in truly reflecting the extent of myocardial damage (34 40). It is possible, therefore, that studies such as 99m Tc-annexin V imaging that potentially highlight additional mechanisms of cellular death may help further elucidate the process of acute rejection. This was suggested by Ciliberto et al. (41) who found that although echocardiography did not have sufficient sensitivity to replace EMB completely as a diagnostic assay of acute rejection, it could be considered a useful adjunct. Apoptosis imaging may in fact be more useful in patients who are at later time-points from their transplantation procedures. Most acute rejection occurs within the first year. Many centers may thus be reluctant to forego the EMB during this high-risk period and yet might question the need for continued biopsies once the patient survives beyond this 275

7 Kown et al. time-point (42). 99m Tc-annexin V could therefore be used in this latter group of patients to help identify those who would benefit from the invasive biopsy procedure for more definitive diagnosis. Current studies using 99m Tc-annexin V have increased inclusion criteria to include all heart transplant recipients who are beyond 1-year post-transplant and will thus allow study of this subset of patients. Another consideration that will be assessed in those patients surviving past the first year is the ability of 99m Tc-annexin V to predict the development of allograft vasculopathy. The study by Bergese et al. (30) found higher numbers of apoptotic cells in tolerant grafts as well as a preponderance of TUNEL-positive cells in the areas around vessels with neointimal hypertrophy. Future studies will thus assess whether higher 99m Tc-annexin V uptake may correlate with the development of graft coronary artery disease. The main questions that remain are the relevance of apoptosis in acute cardiac rejection and the ability for 99m Tcannexin V imaging to measure this process. Currently, 99m Tcannexin V appears to be well tolerated and the results of this initial feasibility study suggest that it may be potentially useful as a noninvasive predictor of acute allograft rejection. The results of this study are preliminary, however, given the small number of patients, the lack of significant rejection episodes, and the absence of follow-up images. Future studies will attempt to delineate further the prospective natural history of 99m Tc-annexin V imaging by serially following the effects of immunosuppressive treatment in those patients diagnosed with acute cardiac rejection. Follow-up images in these patients should help to establish whether this assay can reliably indicate ongoing rejection or resolution as was demonstrated in the previous animal models. Acknowledgments The investigators thank Ms Bonnie Bell for processing the histologic sections, and Allan Green, PhD MD, and the Theseus Imaging Corporation (Cambridge, MA) for their generous support of this project. References 1. Hosenpud JD, Bennett LE, Keck BM, Fiol B, Boucek MM, Novick RJ. The Registry of the International Society for Heart and Lung Transplantation: sixteenth official report J Heart Lung Transplant 1999; 18: Barry WH. Mechanisms of immune-mediated myocyte injury. Circulation 1994; 89: Hetts SW. To die or not to die: an overview of apoptosis and its role in disease [see comments]. J Am Med Assoc 1998; 279: Kabelitz D. Apoptosis, graft rejection, and transplantation tolerance. Transplantation 1998; 65: Kageyama Y, Li XK, Suzuki S et al. Apoptosis is involved in acute cardiac allograft rejection in rats. Ann Thorac Surg 1998; 65: Shaddy RE. Apoptosis in heart transplantation. Coron Artery Dis 1997; 8: Akiyama K, Gluckman TL, Terhakopian A et al. Apoptosis in experi- mental myocardial infarction in situ and in the perfused heart in vitro. Tissue Cell 1997; 29: Gopal S, Narasimhan U, Day JD et al. The Quilty lesion enigma: focal apoptosis/necrosis and lymphocyte subsets in human cardiac allografts. Pathol Int 1998; 48: Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death [see comments]. Am J Pathol 1995; 146: Laguens RP, Meckert PM, Martino JS, Perrone S, Favaloro R. Identification of programmed cell death (apoptosis) in situ by means of specific labeling of nuclear DNA fragments in heart biopsy samples during acute rejection episodes. J Heart Lung Transplant 1996; 15: Szabolcs M, Michler RE, Yang X et al. Apoptosis of cardiac myocytes during cardiac allograft rejection. Relation to induction of nitric oxide synthase. Circulation 1996; 94: Zwaal RF, Schroit AJ. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 1997; 89: Blankenberg FG, Strauss HW. Non-invasive diagnosis of acute heartor lung-transplant rejection using radiolabeled annexin V. Pediatr Radiol 1999; 29: van Engeland M, Nieland LJ, Ramaekers FC, Schutte B, Reutelingsperger CP. Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry 1998; 31: Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 1994; 84: Vriens PW, Blankenberg FG, Stoot JH et al. The use of technetium Tc 99m annexin V for in vivo imaging of apoptosis during cardiac allograft rejection. J Thorac Cardiovasc Surg 1998; 116: Valantine HA, Schroeder JS. Cardiac transplantation. Intensive Care Med 1989; 15: Billingham ME. Endomyocardial biopsy diagnosis of acute rejection in cardiac allografts. Prog Cardiovasc Dis 1990; 33: Moidl R, Chevtchik O, Simon P et al. Noninvasive monitoring of peak filling rate with acoustic quantification echocardiography accurately detects acute cardiac allograft rejection. J Heart Lung Transplant 1999; 18: Hoff SJ, Stewart JR, Frist WH et al. Noninvasive detection of acute rejection in a new experimental model of heart transplantation. Ann Thorac Surg 1993; 56: Carrier M. Noninvasive assessment of cardiac transplant rejection. A critical look at the approach to acute rejection. Can J Surg 1991; 34: Ravanat C, Archipoff G, Beretz A, Freund G, Cazenave JP, Freyssinet JM. Use of annexin-v to demonstrate the role of phosphatidylserine exposure in the maintenance of haemostatic balance by endothelial cells. Biochem J 1992; 282: Tait JF, Smith C. Site-specific mutagenesis of annexin V: role of residues from Arg-200 to Lys-207 in phospholipid binding. Arch Biochem Biophys 1991; 288: Kasina S, Rao TN, Srinivasan A et al. Development and biologic evaluation of a kit for preformed chelate technetium-99m radiolabeling of an antibody Fab fragment using a diamide dimercaptide chelating agent. J Nucl Med 1991; 32: Billingham ME, Cary NR, Hammond ME et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Heart Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 1990; 9: Phelps E, Wu P, Bretz J, Baker JRJ. Thyroid cell apoptosis. A new understanding of thyroid autoimmunity. Endocrinol Metab Clin North Am 2000; 29: Ohtsuki K, Akashi K, Aoka Y et al. Technetium-99m HYNIC-annexin V: a potential radiopharmaceutical for the in-vivo detection of apoptosis. Eur J Nucl Med 1999; 26: American Journal of Transplantation 2001; 1:

8 In Vivo Imaging of Acute Cardiac Rejection 28. Puig M, Ballester M, Matias-Guiu X et al. Burden of myocardial damage in cardiac allograft rejection: scintigraphic evidence of myocardial injury and histologic evidence of myocyte necrosis and apoptosis. J Nucl Cardiol 2000; 7: van Heerde WL, Robert-Offerman S, Dumont E et al. Markers of apoptosis in cardiovascular tissues: focus on Annexin V. Cardiovasc Res 2000; 45: Bergese SD, Klenotic SM, Wakely ME, Sedmak DD, Orosz CG. Apoptosis in murine cardiac grafts. Transplantation 1997; 63: Kanoh M, Takemura G, Misao J et al. Significance of myocytes with positive DNA in situ nick end-labeling (TUNEL) in hearts with dilated cardiomyopathy: not apoptosis but DNA repair. Circulation 1999; 99: Sloop GD, Roa JC, Delgado AG, Balart JT, Hines IIM, Hill JM. Histologic sectioning produces TUNEL reactivity. Arch. Pathol Lab Med 1999; 123: Jollow KC, Sundstrom JB, Gravanis MB, Kanter K, Herskowitz A, Ansari AA. Apoptosis of mononuclear cell infiltrates in cardiac allograft biopsy specimens questions studies of biopsy-cultured cells. Transplantation 1997; 63: Brunner-La Rocca HP, Sutsch G, Schneider J, Follath F, Kiowski W. Natural course of moderate cardiac allograft rejection (International Society for Heart Transplantation grade 2) early and late after transplantation. Circulation 1996; 94: Frigerio M, Bonacina E, Gronda E et al. A semiquantitative approach to the evaluation of acute cardiac allograft rejection at endomyocardial biopsy. J Heart Lung Transplant 1997; 16: Hausen B, Rohde R, Demertzis S, Albes JM, Wahlers T, Schafers HJ. Strategies for routine biopsies in heart transplantation based on 8- year results with more than 13,000 biopsies. Eur J Cardiothorac Surg 1995; 9: Hutter JA, Wallwork J, English TA. Management of rejection in heart transplant recipients: does moderate rejection always require treatment? J Heart Transplant 1990; 9: Novitzky D, Rose AG, Cooper DK, Reichart B. Interpretation of endomyocardial biopsy after heart transplantation. Potentially confusing factors. S Afr Med J 1986; 70: Winters GL, Loh E, Schoen FJ. Natural history of focal moderate cardiac allograft rejection. Is treatment warranted? Circulation 1995; 91: Winters GL. The challenge of endomyocardial biopsy interpretation in assessing cardiac allograft rejection. Curr Opin Cardiol 1997; 12: Ciliberto GR, Mascarello M, Gronda E et al. Acute rejection after heart transplantation: noninvasive echocardiographic evaluation. J Am Coll Cardiol 1994; 23: White JA, Guiraudon C, Pflugfelder PW, Kostuk WJ. Routine surveillance myocardial biopsies are unnecessary beyond one year after heart transplantation. J Heart Lung Transplant 1995; 14: American Journal of Transplantation 2001; 1: