Methylene blue may blunt ICG fluorescence in sentinel node imaging

CURRENT FULL VERSION: A new study in Animals is raising a practical question for veterinary oncologic surgery: when clinicians combine methylene blue with indocyanine green for sentinel lymph node mapping, are they making nodes easier to find, or making the fluorescent signal harder to see? The paper, “When Blue Turns the Green Off,” examined whether methylene blue quenches ICG fluorescence during near-infrared imaging and found that interference is real enough to warrant attention in veterinary workflows that rely on dual tracers. (pmc.ncbi.nlm.nih.gov)

That question lands at a time when sentinel lymph node mapping is becoming more embedded in companion animal oncology. In both human and veterinary surgery, ICG and methylene blue are familiar tools for fluorescence-guided procedures and lymphatic mapping, and reviews of fluorescence-guided abdominal surgery describe both dyes as established clinical fluorophores with different strengths, limitations, and imaging behavior. In dogs, methylene blue remains attractive because it is visible, inexpensive, and easy to use intraoperatively, while ICG offers near-infrared signal that can improve tissue penetration and node detection. (pmc.ncbi.nlm.nih.gov)

The broader veterinary literature shows why this matters. A 2025 JAVMA study of 38 client-owned dogs with oral neoplasms found that CT lymphangiography and intraoperative methylene blue together identified all metastatic cases and achieved a 97.4% sentinel lymph node detection rate, but agreement between the two modalities was only moderate, with discrepancies in nine cases, including one metastatic node. The authors concluded that using at least two mapping modalities is advisable because metastasis and technique can affect identification rates. That makes any dye-dye interaction more than a bench detail: if one tracer weakens another, the “second modality” may not be as independent as surgeons assume. (pubmed.ncbi.nlm.nih.gov)

The new Animals paper appears to add exactly that caution. Based on the abstracted study description, Gariboldi and colleagues quantitatively and qualitatively assessed methylene blue–ICG mixtures at different ratios using three near-infrared imaging modalities, including fluorescence-lifetime imaging microscopy, to test for quenching. Even without full procedural details available here, the central signal is clear: methylene blue can diminish ICG fluorescence, which could alter how well lymphatic channels or sentinel nodes are visualized during surgery. That concern is biologically plausible, too. Prior imaging literature has long noted that both ICG and methylene blue are concentration-sensitive fluorophores, and quenching behavior is a known issue in fluorescence-guided imaging systems. (pubmed.ncbi.nlm.nih.gov)

There’s also a parallel conversation underway about access and cost. A recent JAVMA report on 10% fluorescein sodium for intraoperative sentinel lymph node mapping in dogs, presented as “Expanding access to cancer care,” positions fluorescein as a practical, lower-cost option for canine mapping. In that preliminary study, 6 client-owned dogs with cutaneous or subcutaneous tumors received intradermal peritumoral injections of 10% fluorescein sodium diluted 1:120 with saline. Lymphatic tracts were visualized in 5 of 6 dogs, sentinel nodes were identified in all 6, mean time to tract fluorescence was 6.7 minutes, and mean time to sentinel node fluorescence was 8.0 minutes. No adverse events were reported. Histopathology found metastatic mast cell disease in 4 of 6 sentinel nodes, with additional disease in 1 of 2 second-tier nodes. The authors concluded that fluorescein was safe, feasible, and accessible, with potential to support wider adoption of sentinel node mapping in general practice. The Veterinary Cancer Society featured that work in its 2026 conference program, suggesting the idea is already drawing attention from the oncology community. Fluorescein isn’t new to veterinary lymphatic mapping, either: a 2006 Veterinary and Comparative Oncology study reported that intradermal fluorescein and patent blue violet identified sentinel nodes in 98% of injection sites in healthy dogs. Taken together, the field seems to be moving in two directions at once, toward more sophisticated fluorescence imaging and toward simpler, cheaper tracers that may broaden access. (vetcancersociety.org)

Expert reaction specific to the new Animals paper was limited in public sources, but the surrounding literature points to the same operational message: fluorescence imaging is highly dependent on dye handling, concentration, timing, optics, and background signal. Reviews in surgical imaging emphasize that ICG and methylene blue are not interchangeable and can behave differently depending on the application and imaging platform. Inference: for veterinary surgeons, the most important takeaway may be less about abandoning methylene blue and more about validating local protocols before mixing or co-administering tracers in routine cases. That point is worth separating from methylene blue’s broader clinical utility. In other areas of animal health, the dye still performs as a useful therapeutic agent: a separate 2025 Animals study in Nile tilapia found that methylene blue incorporated into feed prevented mortality during acute nitrite intoxication, lowered circulating methemoglobin, and was associated with less severe gill pathology than control feed. In other words, the issue here is not that methylene blue is inherently problematic, but that its optical behavior may conflict with fluorescence-based mapping when paired with ICG. (pmc.ncbi.nlm.nih.gov)

Why it matters: For veterinary professionals, this is a workflow story as much as a research story. If methylene blue reduces ICG fluorescence, teams may need to rethink whether they should inject the dyes together, separate them in time, lower concentrations, or use a different tracer strategy altogether. That has implications for surgical planning, equipment purchasing, training, pathology submission, and discussions with pet parents about the goals and limits of sentinel node mapping. It also matters for interpretation: a “negative” fluorescent field may reflect tracer interference, not true absence of drainage. At the same time, the fluorescein data suggest there may be practical alternatives for clinics that want real-time mapping without the cost or workflow demands of near-infrared systems. (pubmed.ncbi.nlm.nih.gov)

What to watch: The next step is likely in vivo validation in dogs and cats, ideally comparing mixed versus sequential tracer administration, standardized dosing, and platform-specific performance, while also testing whether lower-cost options like fluorescein can deliver acceptable accuracy in real-world oncology practice. It will also be useful to see whether future work can define when methylene blue remains best used as a visual adjunct, when it should be separated from ICG-based imaging, and how tracer choice should vary by tumor type, anatomy, and practice setting. (vetcancersociety.org)