Label-free Identification of Antibody-mediated Rejection in Cardiac Allograft Biopsies Using Infrared Spectroscopic Imaging.

BACKGROUND: Antibody-mediated rejection (AMR) in cardiac allograft recipients remains less well-understood than acute cellular rejection, is associated with worse outcomes, and portends a greater risk of developing chronic allograft vasculopathy. Diffuse immunohistochemical (IHC) C4d staining of capillary endothelia in formalin-fixed, paraffin-embedded (FFPE) right ventricular (RV) endomyocardial biopsies is diagnostic of immunopathologic AMR but serves more as a late-stage marker. Infrared (IR) spectroscopy may be a useful tool in earlier detection of rejection. We performed mid-IR spectroscopy to identify a unique biochemical signature for AMR.

METHODS: A total of 30 posttransplant FFPE RV tissue biopsies (14 positive for C4d and 16 negative for C4d) and 14 native heart biopsies were sectioned for IR analysis. IR images of entire sections were acquired and regions of interest (ROI) from cardiomyocytes were identified. Extracted spectra were averaged across many pixels within each ROI. Principal component analysis coupled with linear discriminant analysis (PCA-LDA) and predictive classifiers were applied to the data.

RESULTS: Comparison of averaged mid-IR spectra revealed unique features among C4d-positive, C4d-negative, and native heart biopsies. PCA-LDA and classification models demonstrated that spectral features from the mid-IR fingerprint region of these 3 groups permitted accurate automated classification into each group.

CONCLUSIONS: In cardiac allograft biopsies with immunopathologic AMR, IR spectroscopy reveals a biochemical signature unique to AMR compared to that of nonrejecting cardiac allografts and native hearts. Future study will focus on the predictive capabilities of this IR signature.