Ann Thorac Surg 1996;62:1337-1340
© 1996 The Society of Thoracic Surgeons
Original Article: Cardiovascular
Noninvasive Diagnosis of Cardiac Allograft Rejection in an Orthotopic Canine Model
Jeffrey E. Everett, MD,
Mitchell N. Palmer, BS,
Jose Jessurun, MD,
Sara J. Shumway, MD
Division of Cardiovascular and Thoracic Surgery, Department of Surgery, and Department of Pathology, University of Minnesota, Minneapolis, Minnesota
Accepted for publication May 13, 1996.
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Abstract
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Background. A decline in the R wave voltage obtained from surface electrocardiograms once correlated with cardiac allograft rejection. With cyclosporine therapy, however, these electrocardiographic findings became inconsistent, occurring only during severe rejection episodes. Despite cyclosporine use, intramyocardial unipolar peak-to-peak amplitudes obtained from plunge electrodes are reported to be highly sensitive and specific for diagnosing rejection. These reports are based on heterotopic grafts, which atrophy over time, making long-term voltage changes during rejection difficult to interpret. The purpose of this study was to use analysis of unipolar peak-to-peak amplitudes as a prospective monitoring tool for diagnosing orthotopic cardiac allograft rejection.
Methods. Ten adult mongrel dogs underwent orthotopic heart transplantation with the attachment of four intramyocardial leads. The unipolar peak-to-peak amplitudes were measured daily and compared with endomyocardial biopsy results.
Results. We found that intramyocardial unipolar peak-to-peak amplitude analysis had a sensitivity and a specificity of 100% for diagnosing rejection. We also found that as the number of myocardial leads increased, the sensitivity of detecting rejection also increased.
Conclusions. We conclude that unipolar peak-to-peak amplitude analysis is an accurate noninvasive means for early detection of cardiac allograft rejection in an orthotopic model. Its success should allow less frequent, more selective use of endomyocardial biopsy.
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Introduction
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See also page 1340.
Evaluation of serial endomyocardial biopsy specimens, despite numerous disadvantages, remains the gold standard for diagnosing cardiac allograft rejection. Obtaining a specimen is an invasive procedure that exposes the patient to associated risks and complications. Biopsies can be done only at specialized centers, and account for considerable medical expense in the early posttransplantation period. Moreover, biopsies are not a practical screening tool; they cannot be done repeatedly to detect rejection before myocardial damage [1]. Clearly, noninvasive means for detecting cardiac allograft rejection are needed.
A variety of noninvasive techniques for detecting cardiac allograft rejection have been used, both clinically and experimentally, with highly variable results [1–15]. In the early years of cardiac transplantation, a decline in the R wave amplitude obtained from the surface 12-lead electrocardiogram correlated with graft rejection. With the advent of cyclosporine, this technique was no longer valid: the rejection process became less diffuse with less associated edema [1, 3, 15]. Several laboratories [1,7,9,16–18] reported that intramyocardial electrocardiographic (ECG) recordings were sensitive and specific for diagnosing rejection, but heterotopic models were used, often with nonstandard immunosuppression protocols. Heterotopic cardiac grafts atrophy over time, thus making long-term voltage changes during rejection difficult to interpret. In addition, not all groups used the four-lead configuration that is necessary for reliable sensitivity and specificity [1].
Our study used the four-lead intramyocardial electrocardiogram with unipolar peak-to-peak amplitude (UPPA) analysis to detect changes in cardiac electrophysiology during allograft rejection in a canine model of orthotopic heart transplantation.
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Material and Methods
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Ten adult mongrel dogs underwent orthotopic heart transplantation using a previously described technique [16]. All received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Donor Procedure
The selected donor dog was injected with Pentothal (sodium thiopental), placed on a ventilator, and maintained under general anesthesia using isoflurane and oxygen. A median sternotomy was done. The donor heart was harvested using potassium crystalloid cardioplegia and topical hypothermia. Four intramyocardial plunge electrodes (Medtronic Inc, Minneapolis, MN) were placed into the epicardium (one anteriorly and one posteriorly on each ventricle).
Orthotopic Heart Transplantation
An unmatched recipient mongrel dog was placed under general anesthesia as already described. A left lateral thoracotomy was done. The superior and inferior venae cavae were mobilized and cannulated. A right groin dissection was done to isolate and cannulate the femoral artery. The dog was systemically heparinized and placed on total cardiopulmonary bypass. Flows were adjusted to maintain stable mean arterial pressure and venous blood gases. The aorta was cross-clamped, and the native heart was excised. The donor heart with the attached intramyocardial leads was placed into the thoracic cavity. The graft was sewn in place using the technique of Lower and Shumway. The heart was deaired, and perfusion was restored by releasing the cross-clamp. The heart was defibrillated if necessary. The dog was weaned from cardiopulmonary bypass. Inotropic support was used as needed over the ensuing 24 hours. The ECG leads were brought through the chest wall and attached to a subcutaneous block. The block could be accessed percutaneously after operation, allowing direct electric connection to the heart. A pleural catheter was placed, and the chest was closed in layers. The dog was then allowed to awaken from general anesthesia.
Electrodes
Sutureless intramyocardial screw-in electrodes were placed into the dorsal and ventral aspects of each ventricle of the donor dog. The electrodes were brought through the chest wall into a subcutaneous pocket and attached to a block that allowed direct connection with the epicardial pacing electrodes. Each electrode had a corresponding small circular grid on the block, which could be accessed percutaneously.
Immunosuppression Protocol
Beginning the day of transplantation, each dog received cyclosporin A (18 mg•kg-1•day-1), azathioprine (2 mg•kg-1•day-1), and methylprednisolone (500 mg/day). Cyclosporin A levels were monitored using high-pressure liquid chromatography; the dose was adjusted to maintain a drug level of 200 to 250 µg/L. On day 4 after transplantation, methylprednisolone was discontinued, and prednisone (1 mg•kg-1•day-1) was initiated. After voltages stabilized, a baseline biopsy of the allograft was done.
To allow allograft rejection, prednisone was withdrawn first. If rejection did not occur within 5 days, azathioprine was withdrawn. Cyclosporin A was maintained at therapeutic levels throughout the study period. Surveillance biopsies were done weekly and whenever voltage criteria for rejection were present. Once histologic rejection was detected, dogs were treated with methylprednisolone (500 mg/day intravenously) for 2 days, and full immunosuppression was resumed. When voltages stabilized and biopsy specimens showed resolution of the rejection process, the immunosuppressive drugs were tapered as described, and the cycle was repeated.
Electrocardiographic Analysis and Endomyocardial Biopsy
The UPPA, defined as the magnitude of the first fast negative deflection of the QRS complex, was calculated daily from each lead. Data were acquired using a variable-gain signal amplifier with filter settings of 1,000 Hz (high pass) and 0.05 Hz (low pass). Electrocardiograms were recorded using an analog-to-digital converter installed on a personal computer. Voltage analysis for each lead was obtained by averaging the UPPAs of ten representative beats. The daily average was compared with the previous day's average and with the average from the previous 3 days. A decline of 9% from the previous day's average or 15% from the previous 3-day average indicated rejection and necessitated endomyocardial biopsy for tissue confirmation. The UPPA results were then compared with the endomyocardial biopsy results. In a previous study [1], our laboratory detected 13 of 14 biopsy-confirmed episodes of rejection using a 15% reduction in UPPAs compared with the greatest reference voltage of the previous 3 days or a 9% decrease from the previous day.
In the current study, allograft endomyocardial biopsies were done under sterile conditions using standard cardiac biopsy forceps. The dog was sedated with Pentothal, and a small incision was made to expose the right internal jugular vein. The forceps were introduced through a venotomy. Several endomyocardial specimens were obtained from the right ventricle and septum using fluoroscopic guidance. The venotomy was repaired and the skin incision, closed. Tissue was placed in formalin and sectioned for microscopic examination. All biopsy specimens were evaluated by a cardiac pathologist who was unaware of the cardiac voltage results.
Statistical Analysis
Correlation between endomyocardial histology and voltage was determined using Fisher's 2 x 2 cross-table 
2 analysis, which takes into account small sample sizes (usually n < 20). Significance was defined as a p value of less than 0.05.
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Results
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Of the 10 dogs undergoing transplantation, only 4 had prolonged allograft survival that allowed meaningful interpretation of voltages and endomyocardial biopsy specimen. The other 6 dogs died as a result of technical failure, not rejection. The mean duration of follow-up was 35 days after transplantation.
We observed six episodes of histologically confirmed rejection. All were detected using an amplitude decline of 9% from the previous day's average or 15% from the previous 3-day average. A representative example of the recorded electrocardiograms used to calculate the UPPAs is shown in Figure 1
. Rejection episodes were graded Ib to II in severity. No evidence of rejection was detected in any of the 13 biopsy specimens obtained when voltages were normal; when there was a voltage decline, the six biopsy samples were positive for rejection (p < 0.01). Thus, no false-negative or false-positive amplitudes were obtained, which represents an overall sensitivity of 100% and a specificity of 100% for diagnosing cardiac allograft rejection. Importantly, all dogs maintained therapeutic cyclosporin A levels throughout the study.
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Fig 1. . Example of a single tracing recorded from an intramyocardial electrode and generated using the electrocardiogram isolation unit with filter settings of 0.05 Hz (low pass) and 1,000 Hz (high pass).
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Three dogs underwent pulse steroid therapy for histologically confirmed rejection. Interestingly, voltage stabilized within 24 hours after methylprednisolone administration, with no further decline. Voltages slowly recovered over the next 3 to 5 days and approached prerejection baseline (Fig 2). When biopsy specimens showed resolution of the rejection process, the immunosuppressive drugs were again tapered. Two dogs had second episodes of rejection, each detected by voltage decline.
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Fig 2. . Changes in unipolar peak-to-peak amplitude (UPPA) that indicated two episodes of biopsy-proven rejection (* = UPPA decline > 15% compared with previous 3-day average) in 1 transplant recipient. Weekly biopsy specimens obtained during stable voltages did not reveal these rejection episodes.
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Comment
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Intramyocardial ECG recordings were first reported in 1984 as an index of irreversible myocardial ischemic injury [17]. This technique was later used to noninvasively diagnose cardiac allograft rejection [7]. Early heterotopic models showed a strong correlation between voltage decline and graft rejection [1, 7, 9, 10, 18]. Further studies [1, 18] found that this technique remained valid during cyclosporin A therapy. Enthusiasm led to clinical trials in several European centers [4, 20], but the results varied widely. One center [4] reported a 100% negative predictive value of intramyocardial ECG recordings, a finding allowing a 90% reduction in endomyocardial biopsies. Measurements consisted of continuous long-term (overnight) recording of a two-channel signal, which was achieved with an implanted telemetric dual-chambered pacemaker. In contrast, another center [20] found no correlation between voltage decline and graft rejection. Voltages were measured within 2 hours after the biopsy. Myocardial biopsies were done weekly in the first 3 months and then at various times depending on the individual patient's course.
Given these inconsistent results and concerns over instrument-related infection, interest soon declined. However, these clinical trials used only one or two electrodes for monitoring. Previous work in animal models established that four leads are necessary for reliable sensitivity and specificity [1]. In addition, infection has not been a problem in reported applications [1, 4]. Although our average follow-up after transplantation was only 35 days, we found no infectious complications related to hardware.
To further assess this technique, we used four-lead intramyocardial electrocardiograms in an orthotopic canine model. We found the four-lead technique to be both sensitive and specific for diagnosing cardiac allograft rejection. Using two to four electrodes, we retrospectively looked at the sensitivity of the various lead combinations. As the number of leads increased, so did the sensitivity: the four-lead combination was 100% sensitive (Table 1). Admittedly, our sample size of 19 is not large. However, using Fisher's 2 analysis (which takes into account small sample sizes), the p value was less than 0.01. It is interesting that all of the rejection episodes were mild. It might be necessary to change the voltage criteria for clinical applicability in the orthotopic setting.
Our current model relied on percutaneous access to the subcutaneously placed ECG block. Other centers [4, 9, 21] have used telemetric signals to achieve similar results. For clinical application, a telemetric pacemaker would allow ECG recordings over standard phone lines. Signals could easily be recorded daily and analyzed by computer at a remote site.
In summary, four-lead ECG voltage analysis is sensitive and specific for diagnosing cardiac allograft rejection in an orthotopic model. It detects rejection in its earliest stages and therefore eliminates unnecessary biopsies, especially in the early posttransplantation period. Biopsy could be reserved until clinically indicated or directed by voltage analysis, thus resulting in less risk to the patient, lower costs, and expeditious treatment if rejection should occur.
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Footnotes
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Address reprint requests to Dr Shumway, UMHC, Box 207, 420 Delaware St SE, Minneapolis, MN 55455.
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References
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Abstract
Introduction
