The Application of Confocal Microscopy in the Grading of Rejection after Cardiac Transplant

Abstract

Background: Cardiac allograft surgery (heart transplant) can lead to the rejection of the donor organ by the host’s immune system. Rejection is mitigated by targeted immunosupressant drugs, doses of which are determined by the degree of histologically-determined rejection present in endomyocardial biopsies (EMBs) taken routinely from the transplanted heart. Given the low number of organs avaliable for transplant, the severe consequences of a non-successful transplant and the burden of additional biopsies on the health system and the patient, it is essential that the rejection status/grade of EMBs be assessed as accurately and consistently as possible. Current assessment protocols using light microscopy have shortcomings regarding intra- and inter-assessor reliability, and are limited by the stain used for assessment. Therefore this project aimed to develop an improved protocol for EMB rejection grading using fluorescent confocal microscopy. Methods: EMBs were obtained from patients 1) over the spectrum of rejection grades, as determined by trained pathologists, 2) healthy patients (as negative controls for rejection) and 3) patients with Idiopathic Dilated Cardiomyopathy (as positive controls for rejection). Following preliminary experiments to determine the most appropriate method of tissue processing, biopsies were cryosectioned and fluorescently stained for elements of rejection, then imaged using confocal microscopy. The best staining method was determined using Fast Fourier Transforms (FFTs). Rejection was determined using i) counts of the number of infiltrating immune cells and ii) assessment of the loss of cardiac troponin-T from cardiomyocytes. Results: FFT data indicated that staining was best with paraformaldehyde, rather than formalin or RNAlater®. Tissue graded 2R was associated with the highest amount of inflammatory cell infiltration, and the lowest troponin proportion, indicating myocyte injury. Conversely, tissue with the lowest rejection grade (0R) was associated with the lowest level of infiltration and myocyte damage. Conclusion: The present study has demonstrated that fluorescent confocal microscopy could be used in addition to the current system of endomyocardial biopsy assessment. This technique holds much potential for future improvements in this area.