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

## TL;DR

This study explores how methylene blue affects the fluorescence of indocyanine green used in imaging lymph nodes in pets, finding that while methylene blue reduces fluorescence, it still allows for effective lymph node mapping.

## Contribution

The study is the first to quantitatively and qualitatively assess methylene blue's interference with indocyanine green fluorescence in sentinel lymph node mapping in veterinary oncology.

## Key findings

- Higher concentrations of methylene blue reduced indocyanine green fluorescence intensity in vitro.
- Fluorescence remained sufficient for accurate lymph node identification in cadavers despite the reduction.
- Observer experience did not significantly affect interpretation except in color map mode.

## Abstract

The accurate identification of the lymph node that drains a tumor (sentinel lymph node) is essential for cancer staging in pets. To this end, dyes are injected to identify lymph nodes and guide surgeons in their removal. Two commonly used tracers are methylene blue and indocyanine green. Indocyanine green emits fluorescence that can be detected with near-infrared cameras, helping surgeons visualize lymphatic vessels and lymph nodes. These agents are sometimes used together, but it is unclear whether methylene blue could reduce the fluorescence intensity of indocyanine green, which could make lymph nodes harder to detect with lymphography. This exploratory study investigated whether methylene blue affects the fluorescence of indocyanine green in sentinel lymph node mapping. This study quantified fluorescence intensity in different mixtures of these two tracers in vitro and then qualitatively interpreted lymphography in cat cadavers. Observers with varying levels of experience evaluated fluorescence intensity and the lymphographic images. The results showed that higher concentrations of methylene blue reduced fluorescence, but the signal remained sufficient to correctly interpret the lymphography. These findings indicate that combining methylene blue with indocyanine green may be effective for sentinel node mapping in pets. Future confirmatory studies should include a substantially larger number of specimens to allow appropriate statistical comparisons and to better account for inter-individual variability.

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.