Ann Thorac Surg 1997;63:578-581
© 1997 The Society of Thoracic Surgeons
Current Review
Antimyosin Antibodies in Cardiac Rejection
Albert Schütz, MD,
Martin Breuer, MD,
Bernhard M. Kemkes, MD
Department of Cardiovascular Surgery, Klinikum Bogenhausen, Munich, Germany
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Abstract
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The antimyosin antibody is often applied to find out scintigraphically whether myocarditis, myocardial infarction, or (recently) cardiac rejection is present. In the past, a lot of experimental work and clinical studies were done to determine its position, especially for the noninvasive detection of cardiac transplant rejection. Efforts are focused on comparing its diagnostic benefit with that of endomyocardial biopsy. The feasibility of rejection grading and diagnostic reliability are essential parts of this discussion. On the basis of large prospective clinical studies and the information from several experimental animal trials, some important findings can be assumed. Antimyosin scintigraphy after the application of indium 111–labeled antimyosin antibodies is a reliable tool to detect or exclude noninvasively cardiac rejection in adults and children. A distinction among three rejection intensities is possible, as confirmed by immunohistologic examinations. Antimyosin scintigraphy is an important noninvasive method for detecting cardiac rejection, with considerable advantages compared with endomyocardial biopsy.
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Introduction
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Monoclonal antimyosin antibodies are in common use as diagnostic agents in cardiac diseases that are affected by myocytolysis. The monoclonal cell line used for the production of monoclonal antimyosin antibodies was produced by Khaw and associates [1]. Conversion of the whole immunoglobulin G antimyosin molecule into the Fab fragments was done by papain digestion. These large antimyosin immunoglobulins cannot penetrate the intact myocyte membrane, but bind readily to myofibrils exposed by the disrupted membranes of necrotic myocytes. After radiolabeling the antibody with the radionuclide indium 111, which has proven to be the most suitable agent for this technique, especially with regard to the antibody pharmacokinetics, the technical requirements for the scintigraphic detection of myocytolysis were achieved.
The specificity of the antibody for the cardiac myosin of necrotic myocytes could be detected by autoradiographic methods. The results were compared with triphenyltetrazolium chloride staining, which is specific for noninfarcted myocardium [2].
The encouraging experience using antimyosin antibodies in detection, description, and judging of patients with myocardial infarction has stimulated interest in the use of antimyosin in other cardiac diseases that result in myocyte necrosis.
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Use of Antimyosin Scintigraphy in Heart Transplantation
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Because heart transplantation has become a standard therapy for terminal heart failure, there is an increasing demand for new methods of reliable detection of cardiac allograft rejection. In 1987, the first studies were published about the use of antimyosin antibodies in rejecting hearts [3, 4]. Using an experimental heterotopic transplantation model in dogs, Addonizio and colleagues [3] reported an increased uptake in rejecting hearts and also found a positive correlation between the histopathologic rejection score and the scintigraphic results. These experimental data were confirmed in clinical studies by several groups during the following years [5–7]. Frist and co-workers [4] reported on 20 scintigraphic scans in 18 patients having routine histologic check or right ventricular biopsy for a suspected rejection episode. Planar images were performed 24 to 48 hours after indium 111 antimyosin Fab application. Among these 20 studies, there was a correspondence between histology and scintigraphy in 90% [4].
Carrio and associates [5] formulated the heart to lung ratio to quantitate the scintigraphic results more exactly by excluding interindividual variations in ground activity. However, they still used the conventional planar technique.
Our group compared 63 scintigraphic studies in patients who had heart transplantation with the corresponding results of right ventricular biopsy. There was a positive correlation of 90% and a negative correlation of 97.5% between biopsy and indium uptake. Moreover, we used the antimyosin scans to restudy patients who received rejection therapy for moderate or severe rejection (Fig 1
). All 13 control patients showed a negative scintigraphic scan and normal rejection-free histologic results [7] (Fig 2). We performed our scintigraphic examinations using the so-called single photon emission computed tomography technique, with the use of a rotating scintillation camera to facilitate a scintigraphic scan focused on the heart in a three-dimensional view. Thus, we were able to visualize the real radionuclide emission of the heart and to receive more differentiated results.
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Fig 1. . Antimyosin scintigraphy: single-photon emission computed tomographic image of radiolabeled antimyosin antibodies. The heart-to-lung ratio is increased up to 1.9 during moderate rejection.
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Fig 2. . Antimyosin scintigraphy: single-photon emission computed tomographic image after rejection therapy. The heart-to-lung ratio is in the normal range of 1.4, without signs of enriched antimyosin antibodies over the heart.
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Nardo and co-workers [6], in a similar study, followed up 10 patients with a total of 30 antimyosin scans for up to 2 years postoperatively. They corroborated the formerly reported excellent suitability of antimyosin scintigraphy for detection or exclusion of cardiac allograft rejection in man.
From the above listed experimental and clinical studies, one can conclude that antimyosin scintigraphy should become one of the keystones in cardiac rejection monitoring, in particular because of its noninvasive use compared with endomyocardial biopsy. However, there are some unanswered questions limiting the application and meaningfulness of this method in clinical use.
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Kinetics of Antimyosin Antibodies
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There is a 48-hour delay between tracer-labeled antibody injection and the scintigraphic examination. This time period depends on the radioisotope applied and on the pharmacokinetics of the specific antibody in the organism. The half-life of indium 111 is 68 hours, which is well in accordance with the pharmacokinetics of the Fab antibody fragments. Therefore, indium 111 became increasingly popular for radiolabeling diagnostic agents. But the question arises whether these technical aspects consider sufficiently the biologic mechanisms that determine the intramyocardial antibody accumulation during the rejection process.
To study this, we designed immunohistochemical and scintigraphic studies with antimyosin antibodies in an animal model. We performed heterotopic heart transplantation in 12 mongrel dogs and examined the biologic kinetics of the antibody during severe rejection by extraction of transmural biopsy cylinders every 2 hours until 48 hours after antimyosin application, followed by a 6-hour biopsy interval until day 5. For immunohistologic preparation of the antimyosin antibody, we used the indirect peroxidase method and immersion light microscopy after cryopreservation and cryosectioning of the myocardial tissue. We found that the maximum antibody accumulation during severe rejection occurs 20 to 90 hours after application. Only specimens obtained during this interval showed the complete immunohistology [8].
In a clinical study in patients having acute myocardial infarction, Khaw and associates [9, 10] detected the first positive scintigraphic results 7 hours after antibody injection. We detected the first positive immunohistologic staining 6 to 10 hours after antimyosin application [8]. The best scintigraphic results were seen after 18 hours. In our immunohistologic study, the morphologic maximum of antibody accumulation started in the 20th hour after antimyosin injection. Considering these biologic kinetics of the antibody in the context of Khaw's scintigraphic study in the ischemic heart, it seems advisable that scintigraphic examinations using antimyosin antibodies should be done at the earliest 20 hours after injection and no later than 90 hours after antibody application [8]. However, in clinical use for the patient who has had transplantation, this interval is an important disadvantage if acute rejection is the suspected diagnosis, and rejection therapy should be started as early as possible.
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Heart-to-Lung Ratio and Rejection Grading
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Since the first histologic gradation of acute cardiac rejection by Billingham [11], histologic results of endomyocardial biopsy have been considered the gold standard in rejection monitoring. The most important advantage of biopsy is the distinction of different rejection grades, offering detailed information about the extent of the rejection process. Because of its invasiveness and the suspicion that endomyocardial biopsy has a lack of sensitivity because of a certain number of false-negative results [12], often caused by a focal rejection type, it became necessary to look for another method of rejection monitoring that allows grading of the rejection intensity. Therefore, we started an experimental study to describe the feasibility of antimyosin scintigraphy for rejection grading. Antimyosin scintigraphy, in contrast to endomyocardial biopsy, has the advantage of assessing the extension of intracardiac myocytolysis in the entire heart.
The study involved heterotopic cervical heart transplantation in dogs. Acute rejection of the transplants was monitored by daily transmural biopsy. The biopsy cylinders were examined by a pathologist and graded according to the Billingham classification [11]. Moreover, we examined the morphologic antimyosin antibody accumulation within the myocardial tissue by immunohistology. When daily histologic studies demonstrated acute rejection, antimyosin antibodies were applied, and antimyosin scintigraphy was performed 48 hours later. For immunohistologic control of the antimyosin antibody within the myocardium, we took transmural specimens from both ventricles. In total, we examined 61 biopsy cylinders extracted 48 hours after antimyosin application. The conventional histologic evaluation, according to the Billingham classification [11] for acute rejection, showed no rejection in 8 cases, mild rejection in 19 cases, and moderate rejection in 21 biopsy cylinders. In 13 samples, severe cardiac rejection was detected. In nonrejecting hearts, no antimyosin antibody could be observed. In most of the hearts with mild rejection, antibody deposition was mainly intercellular, close to the myocyte membrane. This observation is important because we did not expect intercellular deposition of an antibody specific for an intracellular protein, but this finding concurs with the positive antimyosin scintigraphy scans during mild rejection found in former clinical studies [7]. Intracellular antimyosin deposition was the typical localization pattern during moderate rejection. During severe cardiac rejection with widespread myocytolysis, the antimyosin antibodies were found mostly intracellularly and in particular around the nucleus.
The described immunohistologic differences in the intracardiac antibody localization depending on the rejection grade are important for our current understanding of the rejection process at the microscopic level. It seems that antimyosin antibodies are not, as mentioned so far, exclusively specific for the necrotic, destroyed myocyte. It rather became apparent that even in that earliest stages of cardiac transplant rejection, which are characterized by intercellular lymphatic infiltration without any signs of myocytolysis, the antimyosin antibody can be detected intracardially, close to the histologically intact myocytes. This observation was confirmed by positive scintigraphic scans during mild rejection [7].
During the rejection process, an escape of intracellular proteins such as myosin seems to be induced by cellular effector mechanisms or by means of lymphokine liberation. Thus, the antimyosin antibody would be able to bind its specific epitope, the cardiac myosin within the intercellular space. Henney [13] investigated the immunologic mechanisms of cytotoxic lymphocytes and described the existence of so-called pore-forming proteins initiating the cytotoxic lymphocyte–mediated cell lysis. These proteins (polyperforin 1 and 2) seem to be able to create transmembranous channels into the target cell membrane, facilitating communication between the intra- and intercellular space [13]. The histologic detection of antimyosin antibodies during mild rejection is important for the use of antimyosin scintigraphy in the early detection of allograft rejection. With respect to these results, antimyosin scintigraphy in the early detection of allograft rejection. With respect to these results, antimyosin scintigraphy is the only noninvasive method for rejection monitoring that allows the distinction of various rejection grades.
Comparing these two different methods for antimyosin antibody description within the rejecting heart with the histologic rejection grade from transmural biopsy, it is clear that the immunohistologic antimyosin antibody accumulation is ongoing, with an accompanying elevation of the heart to lung ratio dependent on the histologic grade of rejection.
Simultaneous with these engaging experimental results, clinical examinations have been started to assess the use of antimyosin scintigraphy for the grading of cardiac rejection [7]. Former clinical examinations concerning rejection grading with antimyosin scintigraphy could not detect any reliable relation between rejection grade and heart-to-lung ratio [4, 14, 15]. The retrospectively performed clinical investigation in 58 patients having in total 89 imaging studies showed a clear difference in the heart-to-lung ratio of patients with histologically absent rejection and patients who had cardiac rejection. In mild rejection, the heart to lung ratio ranged from 1.7 to 1.8. Moderate-rejecting hearts showed a heart-to-lung ratio of more than 1.8. We observed severe rejection in only 1 case, with a ratio of 2.7. In 91%, a positive endomyocardial biopsy finding correlated with a heart-to-lung ratio of more than 1.6. Among nonrejecting transplanted hearts and patients with cardiomyopathy, 98% had a heart-to-lung ratio of less than 1.6 [7].
Kleinhans [16] restudied the data of clinical scintigraphic investigations in heart transplant recipients in their center and related them to the corresponding biopsy results. A total of 200 studies in more than 90 patients were discussed. Using at least four biopsy specimens to exclude false-negative histologic results, they found a sensitivity of the heart-to-lung ratio of 81% and a specificity of 87%. The sensitivity of endomyocardial biopsy was described as 80% when taking four biopsy specimens. To reach a sensitivity of about 95%, they calculated the need for at least ten specimens, provided that two or more invasive biopsy procedures were performed.
Concerning the results of the presented studies, it seems possible to differentiate various rejection grades by antimyosin scintigraphy, as confirmed by experimental immunohistologic examinations. The differentiation of rejection grades similar to endomyocardial biopsy is the most important advantage of antimyosin scintigraphy in comparison with other noninvasive methods that have been developed for the detection of cardiac rejection [17].
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Antimyosin Scintigraphy in Children
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Since heart transplantation in children became more common, the lack of a suitable method for rejection monitoring also became obvious. Because of the technical difficulty of endomyocardial biopsy in children, a noninvasive technique for rejection monitoring is in great demand. Up to now, there have been only a few studies using antimyosin scintigraphy in children [18]. Good feasibility was reported. Only mild sedation was needed, and given the small thoracic diameter, the short time planar method was sufficient; imaging with single photon emission computed tomography was not necessary. The sensitivity of 83% with a specificity of 100% after 16 examinations in 8 children may be of lower statistical value, but is well in accordance with the results obtained previously in large clinical studies in adults [4, 16]. However, because of the total-body radiation dose of 74 mBq, this technique should be used sparingly, especially in children.
The theoretically possible interference of human antimouse antibodies due to repeated exposures of the patient to foreign antigens with diagnostically or therapeutically applied murine antimyosin antibodies is, in clinical use, of secondary importance. In a large multicenter trial reporting on 1,742 serum samples, none of the samples showed any evidence of antimyosin-induced human antimouse antibodies, and no allergic or anaphylactic response has been reported [19].
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Conclusion
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In summary, it has become evident that antimyosin antibody scintigraphy after heart transplantation has a meaningful position in rejection diagnosis. Its sensitivity seems to be at least as good as that of the gold standard, endomyocardial biopsy. Histologic rejection monitoring is an invasive diagnostic procedure and has become the subject of increasing discussion because of the recently described occurrence of false-negative biopsy results due to focal rejection. Antimyosin scintigraphy, however, allows a noninvasive examination of the entire heart. Moreover, because of the specificity of antimyosin scintigraphy of nearly 90%, some transplantation centers base their indication for rejection therapy on a positive antimyosin scintigraphy scan alone. A positive scintigraphic result should be reason enough, even in the case of negative biopsy results, to initiate rejection therapy. In addition, the technique allows easy noninvasive control of the heart after rejection therapy. The use of antimyosin scintigraphy in children after transplantation seems to be more practicable and easier for the young patient than histologic monitoring, and its reliability is at least comparable.
Rejection grading seems to be possible, but continuing studies are required to define the real status of antimyosin scintigraphy for this purpose. Quantification of different rejection intensities should be reserved for the experienced investigator.
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Footnotes
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Address reprint requests to Dr Schütz, Department of Cardiovascular Surgery, Klinikum Bogenhausen, Englschalkingerstr 77, 81925 Munich, Germany.
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References
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- Addonizio LJ, Michler RE, Marboe C, et al. Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antibodies Fab. J Am Coll Cardiol 1987;9:555–64.[Abstract]
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Abstract
Introduction
