Antibody Mediated Rejection (AMR) following heart transplantation is increasingly recognized as a substantial complication that increases risk for graft failure, vasculopathy, and decreased recipient survival. While there has been improvement in the diagnosing of full-blown AMR, identification of potentially injurious early or subclinical injury due to antibody deposition remains a challenge. We evaluated 522 endomyocardial biopsies from 219 patients with a novel two-color immunohistochemistry panel that evaluated semiquantitatively graded (0-3) CD3/CD68, C3d, C4d and class II in formalin-fixed paraffin embedded tissue, and compared to cPRA, the presence of Donor Specific Antibodies (DSA) and other demographic variables at the time of the biopsy. Grading of cell mediated rejection (ACR) and other complications were also performed. Non-sensitized patients with cPRA= 0 at the time of biopsy (354 biopsies) had 66% ACR-0R, 30% ACR-1R, 4% ACR-2R; for C3d and C4d: 79% had neither immunoreactant, 8% only C4d, 3% only C3d and 10% had both. The grade of the C4d /C3d staining tended to be low. Allosensitized patients with positive cPRA (168 biopsies) had 53% ACR-0R, 42% ACR-1R, 4% ACR-2R, 1% ACR-3R; for C3d and C4d: 50% had neither immunoreactant, 37% had both, 8% only C4d, and 5% only C3d. The grade of the C4d /C3d staining ranged from 1-3. Of the positive cPRA patients, 47% had DSA and greater than 50% had C3d and C4d staining, along with increases in CD68 and class II expression. The incidence of morphological changes compatible with ongoing AMR (e.g., reactive endothelial cells) was significantly increased in the positive cPRA and DSA+ patients. Consecutive biopsies of positive cPRA patients revealed consistent staining profiles but variations in the levels of concomitant ACR. Our findings demonstrate that this antigen staining combination is useful in identifying minimal/subclinical to full blown AMR in both antibody positive and negative patients and that these patients are also at higher risk of having concomitant cellular rejection (“mixed rejection”). Serial monitoring with this combination will allow for appropriate rejection intervention and for identifying recipients at high risk for AMR complications.

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