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 C4d staining of capillary endothelia in formalin-fixed, paraffin-embedded right ventricular 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 formalin-fixed paraffin-embedded right ventricular tissue biopsies (14 positive for C4d and 16 negative for C4d) and 14 native heart biopsies were sectioned for IR analysis. Infrared images of entire sections were acquired and regions of interest from cardiomyocytes were identified. Extracted spectra were averaged across many pixels within each region of interest. Principal component analysis coupled with linear discriminant analysis 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. Principal component analysis coupled with linear discriminant analysis 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 with that of nonrejecting cardiac allografts and native hearts. Future study will focus on the predictive capabilities of this IR signature.