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.

Validating a semi-quantitative method for assessment of degree of methylene blue staining in sentinel lymph nodes Abstract Purpose: To develop a digital algorithm and validate a semi-quantitative scoring method for surface methylene blue (MB) staining in whole lymph nodes (LN). Methods: Lymph nodes from canine models undergoing sentinel lymph node (SLN) mapping were prospectively assessed ex vivo and photographed. Two blinded observers evaluated all images and assigned a score based on surface staining (0 -no blue stain, 1 -1-50% stained, 2 -51-100% stained). A standard reference for degree of blue staining was based on signal-tobackground ratios using computer-based imaging software with an output measurement of percentage of staining of the LN. Agreement between observers was assessed using the Kappa coefficient. Results: 124 lymph nodes were included and demonstrated strong agreement (K = 0.8007, p < 0.0001) between results of semi-quantitative scoring and image analysis. Also, strong interobserver and intraobserver agreement was observed for the scoring system (K = 0.8051, p < 0.0001 and K = 0.9493, p < 0.0001, respectively). Discussion: Agreement between scoring system and imaging software illustrates a validated method in assessing MB staining, without the need for analysis software. The use of a semiquantitative scoring system shows promise for a simple, objective assessment of MB staining in surgery and for future study. Lymph nodes can have variable surface colour, which can make assessment of blue staining challenging for novice observers in certain cases. This study describes a digital algorithm for quantitative analysis of blue staining in LN thereby providing a novel and objective reporting mechanism in scientific research involving SLN mapping.

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.

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 &lt; 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 &lt; 0.0001 and k = 0.8983, p &lt; 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.

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 minutes, 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 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.