An Experimental Study to Evaluate the Fluobeam 800 Imaging System for Fluorescence-Guided Lymphatic Imaging and Sentinel Node Biopsy

Abstract

Background. Visualization of the lymphatic system is challenging. Lymphatic imaging is a crucial diagnostic tool for benign and malignant lymphatic pathologies. Fluorescence-guided imaging allows selective lymphatic mapping and sentinel lymph node (SLN) identification. There are a few fluorescence systems, but some drawbacks remain due to technical and ergonomic aspects. The aim of this study was to evaluate the feasibility of the new Fluobeam 800 imaging system. Methods. After approval by the ethics committee, the system was evaluated for lymphography and SLN biopsy in an animal model. Five pigs each with 4 lymph node (LN) stations (n = 20 LN stations) were subjected to lymphatic imaging using indocyanine green (ICG). Additionally, the use of ICG was compared with ICG adsorbed to human serum albumin (ICG–HSA). Lymphatic vessels and SLN identification rates were measured. Results. After injection, a clear fluorescence signal of the lymphatic vessels was visualized leading to the LN station. Overall, ICG fluorescence imaging identified a mean of 2.0 lymphatic vessels and 1.1 (range = 1-2) SLN in 20 of 20 LN stations. Reverse lymphography was feasible. A clinical difference in resolution was not detected between use of ICG–HSA and ICG. Conclusion. This is the first study analyzing the feasibility of the Fluobeam 800 imaging system allowing transcutaneous real-time imaging. It enables detection of the SLN by fluorescence retention with increased detection depth and resolution. After fixation to the ceiling, the ergonomics advanced for simultaneous field navigation and dissection. The new system can be applied for lymphatic imaging for lympatico-reconstructive surgery and SLN biopsy.

Keywords

lymphatic imaging fluorescence lymph node solid tumors sentinel lymph node biopsy indocyanine green ICG lymphedema

Introduction

Lymphatic imaging has been established as an important diagnostic tool for benign and malignant diseases of the lymphatic system. Lymphatic imaging is required for lymphatico-reconstructive surgery in lymphedema patients^1-3 and for regional lymph node staging as the most important prognostic factor in patients with early tumor.^4,5 Lymphography and sentinel lymph node biopsy (SLNB) have been performed using various methods, including the radiocolloid and the blue dye method.^6,7 Additional techniques such as computed tomography with 3-dimensional reconstruction and magnetic resonance imaging have been described for lymphography and seem to have potential for clinical application.^8-10 However, drawbacks particularly with regard to handling, dimension, and spatial flexibility remain. Recently, invisible near-infrared (NIR) fluorescence guidance by indocyanine green (ICG) retention was shown to provide high sensitivity, good resolution, and real-time imaging for lymphatic imaging and SLNB.^11,12 Fluorescence imaging guided by ICG has further applications, for example, for late-fluorescence mammography¹³ and quantitative assessment of perfusion in perforator flaps.^14,15 During the past decade, only a few fluorescence imaging systems have been introduced and evaluated, including the FLARE system,¹⁶ the Photodynamic Eye (PDE),^17,18 the SPY System,¹⁹ and the IC-View System.^20,21 Fluorescence-guided lymphography has been validated using an intra-individual comparison to the radionuclide technique,²¹ but some drawbacks and controversies remain due to handling, technical and application aspects, ergonomics, and optimized definition of the available systems.²² In addition, the currently available systems have limitations of definition under room light conditions.

As a consequence, the aim of this study was to preclinically evaluate the feasibility of the new Fluobeam 800 system (Fluoptics, Grenoble, France) integrated into a suspension equipment in an animal model for lymphography and SLNB with regard to resolution, depth of penetration, ergonomics, and technical aspects in search for an optimized navigational tool.

Material and Methods

Study Design

The experimental study was designed to evaluate the feasibility of the Fluobeam 800 system for lymphatic imaging and lymph node (LN) identification using ICG in an animal model.

Ethics

The protocol of this study was approved by the Ethical Committee for Animal Research Val de Loire, University of Tours, France, for performing all experiments at the French National Institute for Agricultural Research (INRA), François Rabelais University Tours, Nouzilly, France. All animals received care in accordance with the European Convention on Experimental Animal Care.

Animals

Five healthy, domestic female pigs with a mean body weight of 65 kg were applied for the study, resulting in 20 regional LN stations (each 2 inguinal and 2 superficial cervical/axillary). The pigs were fasted overnight with unlimited water access. For anesthesia, premedication was started with an intramuscular injection of 2 mg/kg BW xylazine (Rompun 2%, Bayer-Schering AG, Leverkusen, Germany) with 20 mg/kg BW ketamine (Imalgene 1000, Merial SAS, Villeurbanne, France). The pigs received 2 peripheral vein cannulas for induction and maintenance of the general anesthesia and for fluids (Physiologic serum of NaCl 0.9%, CEVA Santé animale, Libourne, France). The pigs were orally intubated with a cuffed endotracheal tube (Rüsch, Waiblingen, Germany) and anesthesia maintained with closed circuit of isoflurane 3% and oxygen and ventilated with a veterinary anesthesia ventilator (Model 2000, Hallowell EMC, Pittsfield, MA) in volume-controlled mode (100% oxygen). The experiments were performed as nonlethal experiments with postoperative humane care of 2 mg/kg BW Flunixin Meglumin (Finadyne, Intervet, Beaucouzé, France) for 4 days and antibiotics as amoxicillin 7 mg/kg BW (Clamoxyl suspension, Pfizer, Paris, France) for 3 days after wound closure with continuous stitching (Prolene, Ethicon, Norderstedt, Germany).

The dermal anatomy of the pig is relatively comparable to the human anatomy. In some areas, it is thinner and thus provides increased detection by transcutaneous transmission. The female pig has a widespread network of inter-areolar lymphatics. There is rare evidence, but the molecular anatomy is expected to be comparable.

Lymphography and Sentinel Lymph Node Biopsy

The 5 consecutive pigs were all subject to a cranial (cervico/axillary LN) or caudal (inguinal LN) periareolar injection of ICG versus ICG adsorbed to human serum albumin (ICG–HSA) with increasing doses in the first step of the dose finding study (1.0, 0.5, 0.1 mg ICG/ICG–HSA). The pigs were fixed in a supine position to allow safe approach to all LN stations. Each injection was performed with 1 mL solution.

ICG solutions were made from stock solution (2.5 mg/mL) using either 5% glucose solution or 20% human serum albumin. The injections were carried out parallel on the left and right side to compare ICG (0.5 mg/mL in 5% glucose solution) with ICG–HSA (0.5 mg/mL in 20% human serum albumin).

To prevent interference, the injections were made at the most cranial and caudal areolars in both rows with respect to intersectional lymphatic vessels in the pig.

Planar Fluorescence Imaging

The planar fluorescence imaging system Fluobeam 800 was applied, which has been built up with a camera and an integrated NIR light source with a maximum absorption between 750 nm and 800 nm and a maximum emission between 780 nm and 850 nm. The excitation was provided by a class 1 expanded laser source at 780 nm. The irradiance on the imaging field was 100 mW/cm². The fluorescence signal was collected by a CCD through a high pass filter with a high transmittance for wavelength >830 nm. The field of view was 10 cm × 7.5 cm. The detection limit of the system at 10 images/second on a 10-µL droplet on a dark background was 0.5 pmol for ICG/HSA and 8 pmol for ICG in gluconated water. The mobile camera system (Figure 1) was integrated into a suspension attachment fixed to the ceiling with sterile handholds to allow “above-field” navigation and simultaneous dissection. After subareolar injection into the nearest nipple areola complex (NAC) to the investigated LN station, the dye uptake to the lymphatic vessels and the drainage to the LN station was visualized and recorded on hard disk in real time with the camera fixed in the OR light.

Figure 1.

The Fluobeam 800 system: (A) Isolated system before (by courtesy of Fluoptics); (B) After integration in the suspension equipment (*).

Operation Room Light Source

To allow fluorescence navigation under operation room (OR) light condition, a modified LED light source (Maquet SAS, Ardon, France) was applied with a filter (low pass filter that cuts radiation under 800 nm). The OR light involved the following technical features: illumination max., 104 300 lux; radiance, 382 W/m²; energetic radiant, 3.66 W/m² lux; color temperature, 3642 K; color rendering index, 93.72; diameter d10, 260 mm; diameter d50, 139 mm.

Outcome Measures

Technical and clinical feasibility of real-time lymphography, lymph node visualization, and detection rate were analyzed for each LN station. The number of lymph vessels after injection was recorded.

A clinical comparison between ICG and ICG–HSA was performed with regard to transit time, dye retention, and resolution of the image.

The suspension of the camera to the OR ceiling was analyzed with regard to ergonomic aspects.

Results

No complications occurred related to the injection of the dye or handling with the camera system.

Comparison Between ICG and ICG–HSA

Six LN stations were used for dose finding reasons. ICG with ICG–HSA were compared by simultaneous bilateral injections using increasing doses (1.0, 0.5, 0.1 mg). ICG–HSA had a stronger fluorescent signal and moved faster through the lymphatic vessels compared to ICG alone (Figure 2). LN identification after incision was easier using ICG–HSA. The resolution of 0.1 mg ICG–HSA was comparable to 0.5 and 1.0 mg. Subsequent investigations were performed using 0.1 mg ICG–HSA.

Figure 2.

ICG versus ICG -HSA. Left: ICG in 20% human serum albumin solution (HSA). Right: ICG in 5% glucose solution.

Lymph Node Navigation

The average total operation time (time from injection to excision of the lymph node) was 8.5 minutes (range = 4 to 25 minutes), and the average time from incision to extraction of the lymph node(s) was 3.5 minutes for identification ranging between 1 and 27 minutes.

Under sterile precautions, the camera was adjusted to focus the lymph vessels and LN stations, and the camera detected the local enrichment due to dye retention in real time (Figure 3A). After incision of the skin above the local fluorescence retention (Figure 3B), the LN showed a sharp configuration in situ, clearly demarcating the afferent lymphatic vessels of the LN (Figure 3C and D). All 20 LN stations were subject to an injection of ICG, lymphography, and dissection of the SLN. In the axillary region, the lymph nodes were generally buried deeper in the tissue and more difficult to excise (Figure 4). Overall, ICG fluorescence imaging identified a mean of 2.0 lymphatic vessels and 1.1 (range = 1-2) LNs in 20 of 20 LN stations (detection rate = 100%). The mean transcutaneous detection depth in the pig was 2.5 cm. To evaluate the retention time, all pigs were scanned on postoperative days 5 and 20 showing a clear persistent emitting signal in the lymphatic vessels.

Figure 3.

Planar fluorescence image after injection of the dye providing lymphatic imaging with guidance to the SLN: Local enrichment after fluorescence dye retention (A), after incision of the skin with visualization of afferent vessels and the SLN as clear spots (B), after dissection into a depth between 1.5 and 2 cm and identification of 2 LNs with clear, high definition (C), and after excision, the afferent lymphatic vessels are still clearly visualized (D).

Figure 4.

The axillary approach: Lymph nodes were localized approximately 2 to 4 cm below the cutaneous level. Marking the end of the lymphatic vessel and a local retention of fluorescent dye enabled dissection of deeply buried lymph nodes.

Lymphography

For reverse lymphography, 0.1 mg ICG–HSA was injected into 2 web paces of each hind leg in several depots. This was followed by taking a fluorescence real-time movie showing a clear signal due to dye retention with the main 3 to 4 lymphatic vessels (Figure 5). Lymphatic vessels were clearly visible transcutaneously, and all lymphatic vessels ended at the position where lymph nodes had been excised inguinally before.

Figure 5.

Lymphography: After web space injection, a clear configuration of the vessels was detected.

Discussion

Fluorescence-guided navigation for lymphatic imaging has been shown to be a reliable, reproducible, and safe technique.^23-25 It has been applied for lymphatico-venular bypass surgery and free vascularized lymph node transfer,^17,26 for SLN biopsy in varying solid tumors,^20,27 and in reconstructive surgery.²⁸

In this experimental study, the Fluobeam 800 imaging system was successfully evaluated and showed feasibility for lymphography and SLN biopsy. It demonstrated a high transcutaneous resolution and detection depth of a minimum of 2.5 cm, providing accurate detection of the lymphatic vessels and the LN. Simultaneous LN navigation and dissection was improved by the suspension equipment.

In comparison to currently available fluorescence imaging systems, the Fluobeam 800 differs with regard to resolution, application under room light conditions, and ergonomic aspects. While the FLARE imaging system (Center for Molecular Imaging, Boston, MA) and the Spy-System (Novadaq, Toronto, Canada) are fixed to a portable cart with an articulating arm, the PDE (Hamamatsu, Japan, and Pulsion, Munich, Germany) and IC-View system (Pulsion, Munich, Germany) are handheld probes.^18,29-31 Due to the relatively small size of the Fluobeam 800 camera, a portable handheld system is also available but was not evaluated in this study. The appropriate ergonomics are up to the individual requirements of the health care professional, and thus a handheld probe allows increases flexibility, but a major disadvantage is that simultaneous navigation and dissection is restricted to a 2-surgeon approach.^18,32,33 A combination of a portable probe that can be fixed to suspension equipment for sterile navigation reflects optimized ergonomics.

In this study, a low pass filter for cutting radiation under 800 nm was used in a commercially available OR light with regular room light. In addition, the FLARE system and the PDE were successfully applied under room light conditions, but xenon and halogen bulbs had to be switched off.^33,34 In contrast, room and OR light had to be switched off during ICG navigation using the IC-View and the SPY System due to interference with NIR light.^19,22 The optical resolution for lymphography and LN detection during navigation was excellent in this study (Figures 2, 3, and 5). In comparison of the Fluobeam 800 with reports on other systems, there is shortage of validated measures, which reflect the subjective parameter of resolution, which in general has been described as “good,” “accurate,” or “high.”^16,19,26,35 The transcutaneous detection depth of 2.5 cm for the Fluobeam 800 was above-average, whereas the other systems range between 1.5 and 3 cm.^16,19,26,35 In comparison to the validated cameras, the FLARE system allows optional generation of color-coded NIR merge imaging for improved navigation,¹⁶ which has been applied in different studies, but with limitation due to complexity of the system.^36-38 All systems provided a low power density of the emitted laser energy, so no tissue warming was seen and no further need for safety goggles was required.

Nevertheless, all systems lack “white” or “fluorescence” balance to prevent “over-fluorescence” and to increase the detective capacity after repetitive injections. The main technical features are summarized in Table 1.

Table 1.

Comparison of Technical Items of Available Fluorescence Systems.^a

	Fluobeam 800	SPY	PDE	FLARE System
Integration	Suspension equipment and portable	Trolley	Handheld	Trolley
	Laser class M	Portable	Portable	Portable
		Laser class IIIB	LED class 1M	LED class 1M
Operation room light compatibility	Yes	No	No	Yes
Field of view	7.5 × 10 cm	7.4 × 10.1 cm	8.6 × 11.6 cm to 9 × 13 cm	2.2 × 1.7 cm to 15 × 11.3 cm
Number of pixels	316 000	300 000	300 000	308 000
Working distance	20 cm	26-34 cm	15-25 cm	45 cm
Resolution	4 cycles/mm	3.17 cycles/mm	2.83 cycles/mm	?
Visible image	No	Black and white	No	Yes, and merge
Zoom	No	No	No	Yes

IC-View no longer available.

In this study, slight differences were seen in dye retention and lymphatic transition time between ICG and ICG–HSA in the pigs, with limited evidence due to the small sample size. In contrast, Hutteman et al demonstrated no significant difference with regard to fluorescence brightness and identification rate.³⁸ In addition, lymphatic transport in humans includes a longer transit time compared to pigs due to anatomical differences. Moreover, the mean transit time in this study was 0.5 cm/s, whereas the transit time for axillary staging in humans was shown to have a mean of 3 to 5 cm/min for ICG.³⁹ Varying transit and drainage times have to be considered between the superficial dermal and the deep subfascial lymphatic vessel network. Consequently, intradermal and subdermal injections seem to be necessary to reflect the complete drainage pattern. However, a subareolar injection is widely recommended for breast cancer patients in the recent literature.^40,41 The retention time of ICG in the lymphatic system was reevaluated on days 5 and 14 after the procedure and showed persistent fluorescence with slightly decreasing levels. A possible reason for prolonged retention may be an unspecific binding of the dye to lymphatic vessels, but the mechanism has to be evaluated in further studies.

With reference to the available systems, the costs can be calculated with approximately $13 per case (ICG ~ $40/ampulla; isolated procedure costs [$140; ~20 min at $7]; equipment ~$40 000, use for 10 years at 300 procedures in a medium volume center).

Conclusion

Integration of the Fluobeam 800 system into a suspension equipment improved the ergonomics and allowed both sterile navigation and surgical dissection by the OR team. The camera has been optimized in terms of resolution and an increased depth of detection up to 2.5 cm. This study showed that the system is feasible for lymphography, which justifies the application of the system in clinical trials. It can be compared to the available systems with regard to the major technical features, but a limitation of this study remains that the imaging system was not directly compared with any other system, which has to be pursued in further studies. A handheld camera of the system is available due to the relatively small size.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: BG Trauma Centre Ludwigshafen, Germany, Maquet SAS, Ardon, France.

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