Simple SummaryThe increasing relevance given to sentinel lymph node assessment in veterinary medicine has led to the necessity of evaluating different mapping techniques and their combination. This study was performed in dogs with mast cell tumors after preoperative assessment identification of draining nodes using lymphoscintigraphy. The aim was to assess whether the addition of methylene blue dye to intraoperative detection of radioactivity in lymph nodes increased the surgical identification of such nodes. Dogs included totaled 103, for a total of 116 mast cell tumors and 196 biopsied lymph nodes. Adding the removal of blue-stained lymph nodes to radioactive positive nodes, the detection of metastatic lymph nodes increased from 90% to 95%. A low percentage of nodes detected in the single lymphocenter were both unstained and non-radioactive. Interestingly, all lymph nodes considered overtly metastatic at histopathology were radioactive. Only a few self-limiting side effects were recorded. The results of this study reported an advantage of the combination of intraoperative gamma probe and methylene blue for guiding the dissection of sentinel lymph nodes in dogs with mast cell tumors.AbstractMethylene Blue (MB) is combined with radiopharmaceutical for intraoperative sentinel lymph node (SLN) mapping, but its role during SLN extirpation has not been investigated yet in veterinary medicine. The aim of this study was to assess whether MB increased surgical detection of SLN beyond the use of intraoperative gamma-probe (IGP) alone in clinically node-negative dogs with mast cell tumors (MCTs) following the detection of sentinel lymphocentrums (SLCs) via preoperative planar lymphoscintigraphy. Dogs enrolled underwent MCT excision and SLC exploration guided by both MB and IGP. Data recorded for each SLN were staining (blue/non-blue), radioactivity (hot/non-hot), and histopathological status (HN0-1 vs. HN2-3). A total of 103 dogs bearing 80 cutaneous, 35 subcutaneous, and 1 mucocutaneous MCTs were included; 140 SLCs were explored, for a total of 196 SLNs removed. Associating MB with IGP raised the SLNs detection rate from 90% to 95%. A total of 44% of SLNs were metastatic: 86% were blue/hot, 7% were only blue, 5% were only hot, and 2% were non-blue/non-hot. All HN3 SLNs were hot. Combining MB with IGP can increase the rate of SLN detection in dogs with MCTs; nonetheless, all lymph nodes identified during dissection should be removed, as they might be unstained but metastatic.