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

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 and post hoc analysis 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: Post hoc fluorescence quantification (threshold of Hue = 165-180, Intensity = 30-255) displayed strong agreement with semi-quantitative scoring (k = 0.9734, p < 0.0001). Fluorescence intensity with either threshold of 35-255 or 45-255 were significant predictors of fluorescence and had high sensitivity and specificity (p < 0.05). Fluorescence intensity and quantification had a strong association (p < 0.001). Conclusion: The validation of the semi-quantitative scoring system by image analysis provides a method for objective in situ observation of tissue fluorescence. The utilization of thresholding for ICG fluorescence intensity allows post hoc quantification of fluorescence when not built into the imaging system.

Comparative aspects of targeted sentinel lymph node mapping in veterinary and human medicine: opportunities for future research.

There is a significant overlap in the genetic, metabolic and epigenetic alterations between human and companion animal cancers, including those of the oral cavity, breast, bladder, skin, lungs and pancreas. In many cancer types, the identification and removal of affected lymph nodes are essential for accurate cancer management, including treatment and prognosis. Historically, lymphadenectomy and subsequent radical resection based on regional anatomy, palpation and lymph node aspirates were considered sufficient; however, modern approaches with sentinel lymph node mapping (SLN) mapping have increased the accuracy of surgical decision-making. Preoperative and intraoperative SLN mapping techniques in veterinary patients parallel those used in human medicine. While many of these techniques are highly successful, the main challenges with current methodologies are their sensitivity and specificity for the presence of cancer, which can be overcome via precision medicine and targeted SLN mapping agents. Given the large population of dogs and cats with cancer, the crossover of knowledge between species can help to deepen our understanding of many of these cancers and can be useful in evaluating new drugs and/or therapies. In this review, we discuss SLN mapping techniques in veterinary medicine and the concept of precision medicine as it relates to targeted SLN mapping imaging agents. The large number of companion animals affected by cancer is an underutilized resource to bridge the translational gap and we aim to provide a reference for the use of dogs and cats as a comparative model for human SLN mapping.

Effect of volume and methylene blue on fluorescence intensity and transit of indocyanine green for sentinel lymph node mapping in a simulated feline tumor model.

OBJECTIVE: To compare the effect of volume and solution on transit time and fluorescence intensity (FI) using near-infrared fluorescence imaging (NIRF) in a simulated tumor model in cats. Secondarily, to describe SLN mapping with indocyanine green (ICG) NIRF and report any adverse effects of intradermally injected ICG in cats. ANIMALS: 7 healthy purpose-bred domestic shorthaired male cats. METHODS: Cats were randomly divided into 2 groups (ICG or ICG + methylene blue [MB]). Transit time and FI were determined for 1 or 2 mL solutions injected intradermally in 4 quadrants around a simulated tumor. Following massage, fluorescence intensity was quantified by calculating the corrected total ROI fluorescence using ImageJ software. Cats were monitored for adverse effects up to 4 weeks post-injection. RESULTS: A larger solution volume had decreased transit times to the SLN (P = .001). There was no significant difference in transit times between ICG and ICG-MB. ICG demonstrated a greater FI (P = .001) in the SLN compared to ICG-MB. Methylene blue did not percutaneously fluoresce under NIRF. The volume of the solution did not significantly affect FI. No adverse reactions were reported. CLINICAL RELEVANCE: Increased volume of ICG may aid in rapid percutaneous lymphatic tracking from tumor to SLN. Indocyanine green alone may be preferred over ICG-MB for greater visualization of the SLN. Intradermal injections of ICG and ICG-MB were well-tolerated in healthy cats with no significant complications. Clinical evaluation of this technique in an impaired lymphatic system, as seen in cancer patients, should be assessed in future research.

Quantitative and Semi-quantitative Methods for Assessing the Degree of Methylene Blue Staining in Sentinel Lymph Nodes in Dogs.

Background: To develop a digital algorithm for quantitative assessment of surface methylene blue staining in whole lymph nodes and validate a semi-quantitative visual scoring method for patient-side use. Methods: Lymph nodes from canine patients with spontaneous tumors undergoing sentinel lymph node mapping were prospectively assessed ex vivo and photographed. Using an open-source computer-based imaging software, an algorithm was developed for quantification of staining based on a signal-to-background ratio. Next, two blinded observers evaluated images and assigned a semi-quantitative visual score based on surface staining (0-no blue stain, 1-1-50% stained, and 2-51-100% stained) and those results were compared to the established quantitative standard. Results: Forty-three lymph nodes were included. Image analysis successfully quantified blue staining and differentiated from normal lymph node tissue in all cases. Agreement between observers using the Kappa coefficient demonstrated strong agreement (k = 0.8581, p < 0.0001) between semi-quantitative visual scoring and image analysis. There was substantial interobserver and intraobserver agreement for the scoring system (k = 0.7340, p < 0.0001 and k = 0.8983, p < 0.0001, respectively). Conclusion: A digital algorithm using an open-source software was simple and straightforward to use for quantification of blue staining. The use of a semi-quantitative visual scoring system shows promise for a simple, objective, repeatable assessment of methylene blue staining at the time of surgery. This study demonstrates reliable and repeatable methods for blue staining quantification thereby providing a novel and objective reporting mechanism in scientific research involving sentinel lymph node mapping.

Evaluating the Feasibility and Efficacy of a Dual-Modality Nanoparticle Contrast Agent (Nanotrast-CF800) for Image-Guided Sentinel Lymph Node Mapping in the Oral Cavity of Healthy Dogs.

A combination of pre and intraoperative sentinel lymph node (SLN) mapping techniques have been suggested to optimize SLN detection. A novel liposomal nanoparticle, Nanotrast-CF800 (CF800), utilizes computed tomography lymphography (CTL) and near infrared fluorescence imaging (NIRF) for image-guided surgery and SLN mapping. This novel tracer agent has not been evaluated in companion animals. The objective of this study was to evaluate the feasibility and efficacy of CF800 for SLN mapping in the oral cavity of healthy dogs and to report any local adverse effects. Six healthy adult purpose-bred research dogs randomly received either 1 mL (group 1) or 2 mL (group 2) of CF800 injected into the submucosa at the level of the right canine maxillary tooth. CTL and percutaneous NIRF were performed at 1, 3, and 10 min, then 1, 2, 4, 7, and 10 days post-injection (p.i). Overall, both CTL and NIRF identified SLNs in all dogs. The overall peak mean contrast enhancement of the SLNs was 73.98 HU (range 63.45-86.27 HU) at 2 days p.i. Peak fluorescence of the SLN occurred at 1 day p.i. The agent was retained within the SLN for at least 7 days for CTL and 4 days for percutaneous NIRF. No adverse effects were observed. Local administration of CF800 was simple and feasible for the detection of SLNs using CTL+NIRF in the head and neck of healthy dogs and was not associated with significant local adverse events.

Determining agreement between preoperative computed tomography lymphography and indocyanine green near infrared fluorescence intraoperative imaging for sentinel lymph node mapping in dogs with oral tumours.

Lymphatic drainage from the head and neck is variable with significant crossover, therefore sentinel lymph node (SLN) mapping can help ensure the appropriate lymph node(s) are sampled. To improve sensitivity, SLN mapping utilizing multiple modalities and a combination of preoperative computed tomography lymphography (CTL) and intraoperative near infrared fluorescence imaging (NIRF) with indocyanine green (ICG) +/- methylene blue (MB) dye has been suggested. The aim of this study was to describe a method for intraoperative ICG lymphography and determine agreement for SLN detection using preoperative CTL and intraoperative ICG NIRF + MB lymphography (IOL) in dogs with oral tumours. Fourteen client-owned dogs were included. All dogs had preoperative CTL with iodinated contrast and intraoperative IOL with an exoscope. Lymph nodes with CTL contrast-enhancement, blue staining or fluorescence were considered sentinel. The overall SLN identification rate was 100% when CTL and IOL were combined. A total of 57 SLNs were identified. Indocyanine green NIRF identified a greater proportion of SLNs (91%; 52/57) compared with MB (50.8%; 29/57) and CTL (42.1%; 24/57). Eighteen SLNs were identified by all three modalities with a fair level of agreement using Fleiss kappa. These findings suggest a combination of preoperative CTL with intraoperative SLN mapping techniques may greatly improve the ability to accurately detect the SLN in dogs with oral tumours.