William Ramsay
Authors: A. M. Hamilton
Journal: BMJ (2012)

<b>270</b> <h3><b>Objectives:</b></h3> Detection of sentinel lymph node (SLN) is important to evaluate tumor staging and therapeutic decision-making during surgery (<i>1</i>) (<i>2</i>) (<i>3</i>). In this experiment, our aim was to develop a new multi-modal probe for lymph node mapping by visual, fluorescence and SPECT/CT images. <h3><b>Methods:</b></h3> Binding affinities between various dyes such as naphthol blue black, patent blue VF, nitrazine yellow, reactive blue 4, and indocyanine green and mannosylated human serum ablumin (MSA) were tested. The binding fractions were determined by using Fujifilm LAS 3000 and analyzed by multi-gauge 3.0 after TLC. Mixture of 5 μL <sup>99m</sup>Tc-MSA (25.57 MBq) and MSA-conjugated with naphthol blue black (30 μL) was injected into the footpads of male BALB/c mice (5 weeks old, n=3) under anesthesia with 2% (v/v) isoflurane at 1 L/min oxygen flow. For comparison, 30 μL of naphthol blue black (3.3 mM) was injected into the opposite side. The visual, fluorescence and SPECT/CT images were taken at 10 min, 30 min, 1 h, and 2 h after injection. SPECT/CT images were analyzed with the DICOM browser in InVivoScope (IVS) program. <h3><b>Results:</b></h3> The required MSA for 100% binding of naphthol blue black, patent blue VF, reactive blue 4, and nitrazine yellow at 10 min were 2.5, 5, 10, and 20 mg, respectively, which represented that naphthol blue black showed the highest binding affinity among all. Binding efficiencies of all the dyes increased by time. After footpad injection of the MSA and naphthol blue black conjugate, accumulation at popliteal lymph node was investigated and quantified by fluorescence at 10 min. The accumulations were 4.11x10<sup>8</sup> and 4.03x10<sup>8</sup> for the conjugate and naphthol blue black, respectively. The conjugate showed the highest lymph node uptake 5.35x10<sup>8</sup> at 1 h. However, lymph node uptake of naphthol blue black continuously decreased almost to background level (2.35x10<sup>8</sup>) at 2 h. The visual imaging results were similar with fluorescence imaging studies. The conjugate showed lymph node uptake until 2 h. However, no naphthol blue black was found at the lymph node at 2 h. SPECT/CT images were obtained 10 min, 30 min, 1 h, and 2 h after footpad injection using male BALB/c mouse. <sup>99m</sup>Tc-MSA showed popliteal and sciatic lymph node uptakes (SUV 10.72 and 1.99) at 10 min and continually increased until 2 h (SUV 11.56 and 2.0) after injection. Visible image demonstrated that the conjugate remained at popliteal lymph node 2 h after footpad injection. Fluorescence signal was found from the dissected lymph nodes. <h3><b>Conclusion:</b></h3> We could prepare the conjugate of MSA and naphthol blue black successfully and confirmed by visual investigation, fluorescence imaging and SPECT/CT that the conjugate accumulated to lymph node until 2 h after footpad injection. Based on the results, we confirmed that the MSA and naphthol blue black conjugate is an excellent multimodal probe for lymph node mapping. <b>References</b>: <b>1.</b> Donald L. Morton, Duan-Ren Wen, Jan H. Wong, et al. technical details of intraoperative lymphatic mapping for early stage melanoma. <i>Arch Surg. </i>1992. <b>2.</b> Giuliano Mariani, Marco Gipponi, Luciano Moresco GV, et al. Radioguided sentinel lymph node biopsy in malignant cutaneous melanoma. <i>J Nucl Med. </i>2002. <b>3.</b> Jae Min Jeong, Mee Kyung Hong, Young Joo Kim, et al. Development of 99mTc-neomannosyl human serum albumin (99mTc-MSA) as a novel receptor binding agent for sentinel lymph node imaging. <i>Nuclear Medicine Communications. </i>2004.