Effect of Formalin Fixation for Near-Infrared Fluorescence Imaging with an Antibody-Dye Conjugate in Head and Neck Cancer Patients

Mol Imaging Biol. 2021 Apr;23(2):270-276. doi: 10.1007/s11307-020-01553-1. Epub 2020 Oct 19.


Purpose: This study evaluated the effect of formalin fixation for near-infrared (NIR) fluorescence imaging of an antibody-dye complex (panitumumab-IRDye800CW) that was intravenously administered to patients with head and neck squamous cell carcinoma (HNSCC) scheduled to undergo surgery of curative intent.

Procedures: HNSCC patients were infused with 25 or 50 mg of panitumumab-IRDye800CW followed by surgery 1-5 days later. Following resection, primary tumor specimens were imaged in a closed-field fluorescence imaging device, before and after formalin fixation. The fluorescence images of formalin-fixed specimens were compared with images prior to formalin fixation. Regions of interest were drawn on the primary tumor and on the adjacent normal tissue on the fluorescence images. The mean fluorescence intensity (MFI) and tumor-to-background ratios (TBRs) of the fresh and formalin-fixed tissues were compared.

Results: Of the 30 enrolled patients, 20 tissue specimens were eligible for this study. Formalin fixation led to an average of 10 % shrinkage in tumor specimen size (p < 0.0001). Tumor MFI in formalin-fixed specimens was on average 10.9 % lower than that in the fresh specimens (p = 0.0002). However, no statistical difference was found between the TBRs of the fresh specimens and those of the formalin-fixed specimens (p = 0.85).

Conclusions: Despite the 11 % decrease in MFI between fresh and formalin-fixed tissue specimens, the relative difference between tumor and normal tissue as measured in TBR remained unchanged. This data suggests that evaluation of formalin-fixed tissue for assessing the accuracy of fluorescence-guided surgery approaches could provide a valid, yet more flexible, alternative to fresh tissue analysis.

Trial registration: NCT02415881.

Keywords: Anti-EGFR antibody; Antibody-dye conjugate; Formalin fixation; Head and neck cancer; Near-infrared fluorescence imaging.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

Associated data

  • ClinicalTrials.gov/NCT02415881