Validation of a semi-quantitative scoring system and workflow for analysis of fluorescence quantification in companion animals.

Abstract

SIGNIFICANCE: Many commercially available near-infrared (NIR) fluorescence imaging systems lack algorithms for real-time quantifiable fluorescence data. Creation of a workflow for clinical assessment andanalysis may provide clinical researchers with a method for intraoperative fluorescence quantification to improve objective outcome measures. AIM: Scoring systems and verified image analysis are employed to determine the amount and intensity of fluorescence within surgical specimens both intra and postoperatively. APPROACH: Lymph nodes from canine cancer patients were obtained during lymph node extirpation following peritumoral injection of indocyanine green (ICG). First, a semi-quantitative assessment of surface fluorescence was evaluated. Images obtained with a NIR exoscope were analysed to determine fluorescence thresholds and measure fluorescence amount and intensity. RESULTS: fluorescence quantification (threshold of Hue&#x2009;=&#x2009;165-180, Intensity&#x2009;=&#x2009;30-255) displayed strong agreement with semi-quantitative scoring (&#x2009;=&#x2009;0.9734,&#x2009;<&#x2009;0.0001). Fluorescence intensity with either threshold of 35-255 or 45-255 were significant predictors of fluorescence and had high sensitivity and specificity (&#x2009;<&#x2009;0.05). Fluorescence intensity and quantification had a strong association (&#x2009;<&#x2009;0.001). CONCLUSION: The validation of the semi-quantitative scoring system by image analysis provides a method for objectiveobservation of tissue fluorescence. The utilization of thresholding for ICG fluorescence intensity allowsquantification of fluorescence when not built into the imaging system.