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

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... ased on signal-to-background 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 semi-quantitative 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.