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. Correspondence Information: Michael John Walsh. 840 S Wood St, Rm 130 CSN (MC847), 909 S Wolcott Ave, Chicago, Illinois. 606012. Email: walshm@uic.edu. Equal contribution, Vishal K Varma PhD Joint corresponding author, Aliya Husain MD, Michael J Walsh PhD AUTHORSHIP Imran Uraizee – Participated in research design, identifying patients, analyzing data and writing manuscript. Vishal K Varma - Participated in research design, collecting spectral data, analyzing spectral data and writing manuscript. Hari Sreedhar - Participated in analyzing spectral data and writing manuscript. Francesca Gambacorta – Participated in collecting spectral data Shaiju Nazeer - Participated in analyzing spectral data and writing manuscript. Aliya Husain - Participated in research design, identifying patients, and writing manuscript. Michael Walsh - Participated in research design, analyzing data and writing manuscript. DISCLOSURES The authors have no conflicts of interest to disclose. FUNDING This work was funded by the Chicago Biomedical Consortium with support from the Searle Funds at The Chicago Community Trust (Husain and Walsh joint PIs). Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.

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