When Blue Turns the Green Off: Implications of Methylene Blue Interference in Indocyanine Green Near-Infrared Fluorescence Imaging

Sentinel lymph node mapping is increasingly used in canine and feline oncology and often involves the combined use of visible dyes and fluorescent tracers. However, the effect of methylene blue on the fluorescence of indocyanine green during near-infrared imaging remains unclear. This explorative study aimed to quantitatively and qualitatively assess potential fluorescence quenching in solutions of methylene blue-indocyanine green at different ratios in three near-infrared imaging modalities (overlay, color map, contrast). Four solutions were prepared: 100%/0%, 75%/25%, 50%/50%, and 25%/75% indocyanine green/methylene blue. The fluorescence intensity of the four solutions was quantitatively measured in vitro using near-infrared imaging. Subsequently, four lymphographies, one for each solution, were performed from the metatarsal region of feline cadavers. Observers with varying levels of experience evaluated lymphographic images. Methylene blue caused a concentration-dependent reduction in fluorescence both at the quantitative evaluation and qualitative lymphography interpretation. Despite this reduction, fluorescence remained sufficient in cadavers for accurate identification of lymph nodes, and observer experience did not significantly affect interpretation, except for the color map mode. Because methylene blue-dominant solutions showed a greater quenching effect on indocyanine green fluorescence, clinicians should favor indocyanine green-dominant mixtures. This approach may preserve fluorescence performance, maintaining the surgical guidance benefits of methylene blue. Future confirmatory studies should include a substantially larger number of specimens to allow appropriate statistical comparisons and to better account for inter-individual variability.