Confocal Scanning Microscopy in Assessment of Cardiac Allograft Rejection – A Pilot Study

• Confocal microscopy was investigated for its ability to identify features of cardiac rejection.
• Confocal microscopy was able to identify features of cardiac rejection similar to those found in hematoxylin and eosin.
• Features identified included perivascular and/or interstitial infiltrate, myocyte damage, and Quilty lesions.
• We observed distortion in myofilament organization in damaged myocytes that could potentially assist in defining the boundary between 1R or 2R rejection grading.

Cardiac allograft rejection is typically diagnosed on the basis of hematoxylin and eosin (H&E) histology of endomyocardial biopsies. This diagnosis is made based on the degree of immune cell infiltrate and associated myocyte damage. However, considerable variability in rejection grading between pathologists can occur. Confocal microscopy provides high contrast and high resolution imaging that has the potential to provide detailed views of pathological features of allograft rejection. In this pilot study we sought to determine if confocal microscopy could be used to detect features of cardiac rejection. This was achieved by collection of additional sample at 30 biopsy procedures from 15 heart transplant patients. Routine pathological grading of H&E histology identified 5 gradings of 0R, 21 gradings of 1R, and 3 gradings of 2R. From these gradings, 3 samples for 0R, 9 samples for 1R, and 3 samples for 2R were imaged by confocal microscopy. This was achieved by fluorescently labeling sections with DAPI, wheat germ agglutinin, and phalloidin, to visualize the cell nuclei, cell border and extracellular matrix, and muscle cell actin, respectively. Labeling with these fluorescent markers was of high contrast. However, we did note variability in DAPI and phalloidin labeling of tissue sections. Confocal imaging of these labels revealed the following features at high resolution: perivascular and/or interstitial infiltrate, myocyte damage, and Quilty lesions. In particular increased detail of damaged myocytes reveals distortion in myofilament organization that could be exploited to distinguish between 1R and 2R grades. In conclusion, confocal microscopy provided high contrast and resolution imaging of cardiac biopsies that could be explored further to aid assessment of cardiac allograft rejection.