Manuscript edited by all authors. Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Footnotes Funding. resection, the tumor specimen was imaged using a closed-field fluorescent imaging device. Relevant pathological data was available for five patients on retrospective review. For each of these five patients, two regions of highest fluorescence intensity at the peripheral margin and one region of lowest fluorescence intensity were identified, and results were correlated with histology to determine if the region of highest fluorescence intensity along the mucosal margin (i.e., the sentinel margin) was truly the closest margin. Results: Imaging acquisition of the mucosal surface of the specimen immediately after surgery took 30 s. In all of the specimens, the region AZ 23 of highest fluorescence at the specimen edge had AZ 23 a significantly smaller margin distance than other sampled regions. The average margin distance at the closest, sentinel, margin was 3.2 mm compared to a margin distance of 8.0 mm at other regions ( 0.0001). Conclusions: This proof-of-concept study suggests that, when combined with routine FSA, fluorescent specimen imaging can be used to identify the closest surgical margin on the specimen. This approach may reduce sampling error of intraoperative evaluation, which should ultimately improve the ability of the surgeon to identify the sentinel margin. This rapid sentinel margin identification improves the surgeon’s orientation to areas most likely to be positive in the surgical wound bed and may expedite pathology workflow. 0.05 were considered statistically significant. Results Subjects Between December 2015 and June 2018 a total of 29 patients underwent infusion of panitumumab-IRDye800 for intraoperative fluorescent imaging including fluorescence imaging of their tumor specimen directly after resection. Of these patients, only five had sufficient pathological data to be included in the study. Patient and tumor characteristics are presented in Table 1. Imaging acquisition of the peripheral surface of the specimen took ~30 s, after which the specimen was sent to pathology and processed for standard of care assessment. As part of the retrospective analysis, the sentinel margin was identified by determining the region of WNT-4 highest fluorescence intensity along the specimen edge. Each serial cross-section of the specimen was also assessed by fluorescence imaging, and the sentinel margin distance was compared to all the other margin distances with low AZ 23 fluorescence signal obtained in the tissue sections (~8C18 analyzable margins per specimen). We chose to evaluate two margins as potential sentinel margins (where the fluorescence was highest and second highest at the specimen edge). Table 1 Patient and tumor characteristics. sitestagegrade 0.0001). Open in a separate window Figure 2 Margin distance by fluorescent signal. (A) Graph showing increase in margin distance at control regions when compared to sampled regions of highest fluorescence intensity. Representative brightfield image of resected tumor specimen (B) taken from buccal region in patient, seen in AZ 23 (D). (C) Corresponding closed-field fluorescent image of resected tumor specimen with black dotted line indicating overlaid circumferential mask, white dashed line indicating slice from which H&E slide (E) was taken, highlighting the difference in margin distance at the periphery between control AZ 23 region and region of highest fluorescence intensity. (F) High resolution image taken from Odyssey demonstrating fluorescence distribution within microscopic section. *** 0.0001. Comparison of Margin Distances at the Fluorescence Extremes Next, we sought to determine if margin distance would increase linearly in the regions of highest to lowest fluorescence intensity along the periphery of the mucosal surface. A significant difference was found for margin distance when comparing each group (first sentinel margin, second sentinel margin, and low-fluorescence control). The sentinel margin (highest fluorescence region at the cut edge of the specimen) measured on average 2.4 mm, compared to 4.0 mm for the second sentinel margin and 8.0 mm for control regions ( 0.0001). As shown in Figure 3, in all the imaged specimens, the margin distance was closest at the point of highest fluorescence signal, the sentinel margin, compared to the second, with the largest margin at the low fluorescence intensity region. The average increase in margin.