Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

1. Intraoperative Multispectral Fluorescence Camera

1. A custom-made camera system was developed at developed at the Institute for Biological and Medical Imaging (IBMI, Technical University / Helmholtz Zentrum, Munich, Germany) in close in collaboration with SurgOptix (SurgOptix Inc, Redwood Shores, CA, USA). The setup of the intraoperative multispectral fluorescence camera system is shown in Figure 2.
2. Light passes through a system of optics and is separated into visible light, light at the emission (fluorescence) wavelength band and light at the excitation wavelength band. These bundles are detected by CCD-cameras (CCD) (Figure 2). Multi-spectral signals from all cameras are processed in order to correct for artifacts and yield true quantitative fluorochrome bio-distribution. Color and fluorescence signals can be displayed as separate images on external monitors or superimposed in one image.
3. The excitation and emission filters of the camera are set in the measurement herein to collect at 750 nm and 800 ±20 nm respectively.

2. Optical Contrast Agent Preparation

1. The fluorescent contrast agent indocyanine green (ICG, Pulsion AG, Munich, Germany) is prepared under sterile conditions. A concentration of 0.5 mg/mL is used. It is important to use sterile distilled water and not sodium chloride (NaCl), as the latter will cause the ICG to aggregate.
2. After preparation, the solution must be stored in a dark, cool place to avoid fast deterioration of the fluorescence intensity by bleaching. Note: from this point forward keep the ICG protected from light.

3. Intraoperative Imaging – Setup and Injection of ICG

1. The camera is positioned and initiated in the operating theatre prior to surgery to minimize interference with the standard surgical procedure, and connected to the high-definition screens (Figure 2B).
2. The camera is covered in standard sterile drapes (Carl Zeiss Vision BV, Sliedrecht, the Netherlands, OPMI Drape REF 306071) by the investigator in sterile OR clothing.
3. Note: care is taken to use only clear disinfecting agents, as colored disinfecting agents are often autofluorescent and might interfere with the imaging procedure. Moreover, sterile drapes and surgical markers can be autofluorescent. It is advised to test all materials used in the OR and the operating field for autofluorescence prior to surgery.
4. The fluorescent contrast agent indocyanine green (ICG) is prepared under sterile conditions. A concentration of 0.5 mg/mL is used. It is important to use sterile distilled water and not NaCl, as the latter will cause the ICG to aggregate. After preparation, the solution must be stored in a dark, cool place to avoid fast deterioration of the fluorescence intensity. Note: it is advisable to change gloves after preparing ICG, as even a small amount of spill of ICG may influence the image quality.
5. The imaging procedure starts after opening the abdomen. When the area of interest is exposed, the camera is maneuvered into the operating field. The zoom and focus are adjusted under sterile conditions. Lights in the operating theatre are switched off for better detection of the fluorescence signal.
6. 1.0 mL of 0.5 mg/ mL ICG is mixed with 1.0 mL of the standard blue dye (patent blue / bleu patenté, Guerbet, France) in one syringe. The surgeon injects the contrast agent in four quadrants around the primary tumor, preventing spill of the agent. Note: in case of spill of ICG on the surgical gloves, it is necessary to change gloves. Still images or real-time videos are acquired of the lymph flow and the appearance of the fluorescent sentinel lymph node (Figure 3). All images and videos are directly saved on the computer.
7. Apart from fluorescence, the SLN is also detected according to the standard protocol, either using a gamma probe or by visual inspection for a blue discoloration, or both. The radioactive tracer is generally administered one day prior to surgery, after which a lymphoscintigram is performed for detection of a radioactive SLN. This is part of the standard procedure and has no place in the fluorescence imaging protocol.
8. Real-time excision of the SLN is guided by both fluorescence and blue discoloration of lymph nodes. The real-time feature of the camera helps to detect for any remnant fluorescence or unexpected localizations of fluorescent nodes.
9. After excision of the SLN, ex vivo images are acquired of all excised lymph nodes for the presence of a fluorescent signal. The exposure time can be prolonged for higher resolution when capturing still images (Figure 4).
10. Histopathological examination of the lymph nodes reveals the presence or absence of tumor cells in the sentinel lymph node.

4. Representative Results

Fluorescent lymph nodes can be detected with a high signal-to-background ratio. Also, flow of ICG through lymphatic vessels can be monitored allowing real-time lymph node mapping. Results may be influenced by increasing depth of the node, possibly requiring different illumination procedures to achieve detection. Future application of specific tumor targeted fluorescent agents may provide intraoperative detection of a positive SLN with cancer cells using this technology.

Figure 1. The sentinel lymph node (SLN) theory. The SLN is the first draining lymph node(s) from the tumor.

Figure 2. Schematic picture of the multispectral fluorescence camera system (A). The basic setup of the camera system in the operating theatre (B).

Figure 3. Multispectral fluorescence imaging of a lymph node in vulvar cancer. Color image of a lymph node in vivo (A). Fluorescence image of the same lymph node, in vivo (B). Pseudocolor fluorescence image superimposed on the color image (C).

Figure 4. Multispectral fluorescence imaging of a lymph node ex vivo in cervical cancer. Color image of a lymph node ex vivo (A). Fluorescence image of the same lymph node, ex vivo (B). Pseudocolor fluorescence image superimposed on the color image (C).