Preliminary experience with a novel fluorescence lymphography using indocyanine green in patients with secondary lymphedema

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1 Preliminary experience with a novel fluorescence lymphography using indocyanine green in patients with secondary lymphedema Naoki Unno, MD, a,b Kazunori Inuzuka, MD, a,b Minoru Suzuki, MD, a,b Naoto Yamamoto, MD, a,b Daisuke Sagara, MD, a,b Motohiro Nishiyama, MD, a,b and Hiroyuki Konno, MD, b Shizuoka, Japan Background: Lymphoscintigraphy has largely been performed to diagnose lymphedema. It is, however a time-consuming and expensive technique, which has not been covered by Japanese medical insurance since the year In this report we introduce a new imaging technique of fluorescent lymphography to diagnose lymphedema. Methods: Fluorescence images of subcutaneous lymphatic drainage after subcutaneous injection of indocyanine green (ICG) at the foot were obtained using a newly developed near-infrared camera system. ICG fluorescent lymphography was performed in 12 patients with secondary lymphedema and 10 healthy volunteers. The 12 patients were diagnosed with secondary lymphedema according to the medical history and lymphoscintigram, of which 11 had a history of hysterectomy with extended lymph node dissection and local radiation therapy for uterine cancer. Lymphedema developed in one patient after femorotibial artery bypass for peripheral artery occlusive disease. Results: Four abnormal fluorescent patterns of the lymph drainage were observed in lymphedema: dermal backflow (an abnormal filling of the lymph capillaries), extended fluorescent signal at the dorsum and plantar region of the foot, dilated lymph channels with proximal obliteration, and diffuse glittering of fluorescent signals with scattered twinkling of the dye. Continuous lymph channels from the injection site of the foot to the groin were observed along the medial aspect of thigh in healthy subjects. Conclusion: ICG fluorescence lymphography is safe, simple, and minimally invasive. The device is portable and easy to use. The technique may be useful in clinical practice to identify presence of lymphatic disorder. (J Vasc Surg 2007;45: ) From the Division of Vascular Surgery, a Second Department of Surgery, b Hamamatsu University School of Medicine. Competition of interest: none. Reprint requests: Naoki Unno, MD, FACS, Division of Vascular Surgery, Second Department of Surgery, Hamamatsu University School of Medicine, Handayama, Hamamatsu, Shizuoka , Japan ( unno@hama-med.ac.jp) /$32.00 Copyright 2007 by The Society for Vascular Surgery. doi: /j.jvs Evaluation and diagnosis of the swollen extremity remains a challenge. The diagnosis of extremity swelling can frequently be established by a careful history and physical examination. Among the etiologies, lymphedema is one of the common diseases to be differentiated from other types of edema. Skin thickening and hyperkeratosis develop in the advanced form of lymphedema; however, an imaging modality is needed to establish the diagnosis because the clinical findings in the early stage of lymphedema may not be indistinguishable from other forms of edema. Among the imaging modalities, direct contrast lymphography accurately visualizes the lymphatics and contributes to the diagnosis of lymphedema 1-3 ; however, the tedious operative procedure to expose foot lymph as well as complications from the contrast agent have prevented widespread use of this procedure in clinical practice. 4,5 At present, lymphoscintigraphy is considered a major imaging modality for the diagnosis of patients with lymphedema and for evaluation of the swollen extremity. 6,7 It is, however, time consuming, expensive, and potentially teratogenetic during pregnancy. In particular, the cost of lymphoscintigraphy is not approved for reimbursement under the national medical insurance scheme in Japan. Although other diagnostic modalities such as magnetic resonance imaging, 8 computed tomography, 9 or ultrasound images show characteristic features related to the skin changes associated with lymphedema, 10 they can only suggest abnormalities of the lymphatics. The difficulty in diagnosing lymphedema prompted us to explore a new modality to diagnose lymphedema. In this article, we describe our preliminary experience with a novel imaging technique that uses fluorescence of indocyanine green (ICG) for the diagnosis of lymphedema of the lower extremity. PATIENTS AND METHODS Between March 1 and September 7, 2006, 16 extremities in 12 individuals (11 women, 1 man) with secondary lymphedema underwent ICG lymphography at our department. The subjects were years old (range, 38 to 86 years). Eight patients had unilateral involvement, and four had bilateral involvement. Ten healthy volunteers (7 men, 3 women; years old) without any leg symptoms were also examined as a control study. Each patient with secondary lymphedema had previously undergone a physical examination, lymphoscintigraphy, and a duplex ultrasound study of the venous system of the lower extremities to check for the venous insufficiency. A medical history had also been obtained from each patient. The 11 women had a history of hysterectomy with extended lymph node dissection and local radiation therapy

2 JOURNAL OF VASCULAR SURGERY Volume 45, Number 5 Unno et al 1017 Fig 1. A, The device used in this study. The image can be observed at the monitor of the laptop computer, and the images were recorded on the hard disk. B, Acquisition of images of fluorescent lymphography at the bedside. The instrument is equipped with a charge-coupled device camera and light-emitting diode, and is easy to use. for uterine cancer, and leg edema had manifested after surgery. The male patient complained of leg edema after femorotibial artery bypass for peripheral artery occlusive disease. All patients were diagnosed as having secondary lymphedema by medical history or lymphoscintigram. Among them, seven patients were previously diagnosed by other physicians according to medical history and lymphoscintigram. Five patients in this study underwent lymphoscintigram to confirm the lymphatic disorder. None of the healthy volunteers underwent lymphoscintigram under the study protocol. All patients showed leg edema at the time of investigation. The 11 patients with hysterectomy and radiation underwent only compression therapy after manifestation of leg swelling. Seven of the 11 women were at clinical stage II, and the remaining four patients were at stage III. 11 The male patient had been treated with compression and massage therapy after bypass surgery for peripheral arterial disease and was classed as stage I. Fluorescence lymphography. With approval of the ethical committee and written informed consent, fluorescence micrography was performed. A 27-gauge needle was used to subcutaneously inject 0.2 ml of indocyanine green (ICG; Diagnogreen 0.5%; Daiichi Pharmaceutical, Tokyo, Japan) at the dorsum of the foot. The patient was asked to stand immediately after the injection. After that, no standard exercises were prescribed as in lymphoscintigram. Fluorescence images of the subcutaneous lymphatic drainage were obtained using a newly developed nearinfrared camera system (PDE; Hamamatsu Photonics K.K. Fig 2. A and B, Normal fluorescent lymphography in healthy volunteers. Panoramic image shows continuous lymph channels from the injection site at the foot to the groin along the medial aspect of thigh.

3 1018 Unno et al JOURNAL OF VASCULAR SURGERY May 2007 Fig 3. Characteristic pattern of fluorescent lymphography in patients with secondary lymphedema. A, Dermal backflow. B, Extended fluorescent images at the dorsum and plantar region of the foot. C, Dilated lymph channels with proximal obliteration. D, Diffuse glittering of fluorescent signals with scattered twinkling of the dye. Hamamatsu, Japan) that activates ICG with emitted light at a wavelength of 760 nm and filters out light with a wavelength below 820 nm. The light source was a lightemitting diode (LED), and the detector was a chargecoupled device (CCD) camera. The instrument is portable and handy to use. The fluorescence images were continuously observed on the monitor of a laptop computer (LaVie G, Type T; NEC Co., Tokyo, Japan; Fig 1, A). The video images of the lymphatic drainage were converted digitally into AVI or MPEG2 formatted data and recorded on the hard disk of the computer. The movie files were later processed to still panoramic pictures that showed the whole limb by using Mofix image processing software (Emaki Inc, Aizuwakamatsu, Japan). Image interpretation. A physician who did not know the clinical diagnosis of the patients performed the ICG fluorescence lymphography and interpreted the findings of the real-time video images of subcutaneous lymphatic drainage for the primary physician who was aware of the patients past medical history and clinical diagnosis. The video images were later converted to panoramic images by image processing software by other physicians who had no information about the patients. These images were retrospectively interpreted by yet another physician. RESULTS All fluorescent lymphography was successfully performed without any complications. Visual interpretation of the image patterns demonstrated a distinct normal pattern and several abnormal patterns. In healthy volunteers, the normal fluorescence lymphographic pattern of the superficial lymphatic system of the lower extremities shows continuous lymph channels from the injection site on the foot to the groin along the medial aspect of thigh (Fig 2). All 20 extremities from the healthy volunteer group showed a similar normal pattern. As shown in Fig 3 (A to D), four abnormal patterns of lymph drainage were seen in patients with secondary lymphedema: (A) fluorescent dermal backflow, which may be an abnormal filling of the lymph capillaries; (B) extended fluorescent images at the dorsum and plantar region of the foot, (C) dilated lymph channels with proximal obliteration, and (D) diffuse glittering of fluorescent signals with scattered twinkling of the dye like the Milky Way. In patients with secondary lymphedema, 15 of 16 swollen legs showed dermal backflow that is regarded as lymphatic flow obliteration. Extensive spreading of the fluorescent dye beyond the dorsum of the foot was also found in all swollen legs. Proximal obliteration of lymph vessels with dilated lymph vessels was found in eight of 16 legs. Six of 16 swollen legs showed diffuse glittering of fluorescent signals with scattered twinkling of the dye like the Milky Way. These image patterns were reproducibly interpreted by the three different physicians. The lymphoscintigram showed typical findings of lymphedema in all 12 patients with secondary lymphedema, such as slow removal of the tracer from the injection

4 JOURNAL OF VASCULAR SURGERY Volume 45, Number 5 Unno et al 1019 Fig 4. A, A 70-year-old woman with right lower extremity lymphedema who underwent hysterectomy, bilateral iliac node dissection, and pelvic radiotherapy for uterine cancer 30 years earlier (clinical stage III). B, Lymphoscintigram shows extensive dermal pattern in the right leg. C, Indocyanine green fluorescence lymphogram shows diffuse dermal backflow, extended fluorescent images at the dorsum and plantar region of the right foot, and Milky Way sign. site, dermal backflow, and fewer visualized lymph nodes. Panoramic images covering the whole leg showed a combination of abnormal findings of fluorescent staining and facilitated understanding of the disease (Fig 4 and 5). The image demonstrated the distribution of abnormal lymphatics extending throughout the whole leg, and the combination of dermal backflow, extended fluorescent signals at the dorsum of foot, or Milky Way sign were observed. DISCUSSION Isotope lymphoscintigraphy has been widely used to diagnose lymph stasis and resultant lymphedema. Because the technique is minimally invasive and can be performed without discomfort, lymphoscintigraphy has replaced direct contrast lymphography. The lymphoscintigram is an expensive and time-consuming procedure, however, and the technique has only been performed on selected patients in clinical practice. Without a lymphoscintigram, physicians must establish the diagnosis of lymphedema purely from clinical criteria; therefore, a new diagnostic modality to examine patients with swollen extremities is needed in clinical practice that is effective and can be performed without discomfort to patients. In particular, the technique should be safe. To achieve these goals, we focused on ICG fluorescence lymphography. Fluorescence lymphography was first performed in human skin by Bollinger et al 12 after injection of fluorescein isothiocyanate (FITC)-dextran. 12 By videomicroscopy, fluorescence microlymphography visualized the superficial dermal network of lymphatic capillaries. The technique was applied to patients with primary lymphedema and identified the larger spreading of the injected fluorescent dye in the superficial lymphatic capillary network. 13 All of the previous methods of fluorescence lymphography have used FITC-dextran to visualize microlymph vessels. Although fluorescence microlymphography is useful to understand changes in lymphatic capillaries and the intravital anatomy of cutaneous lymphatic microvessels in a localized area, 14 it is not suitable for screening lymphedema and has failed to enter clinical practice to date.

5 1020 Unno et al JOURNAL OF VASCULAR SURGERY May 2007 Fig 5. A, A 69-year-old woman with left lower extremity lymphedema who underwent hysterectomy, bilateral iliac dissection and pelvic radiotherapy for uterine cancer 12 years earlier (clinical stage II). B, Lymphoscintigram shows markedly delayed transport with dermal pattern in the left leg with diminished number of lymph nodes in the left inguinal region. C, Indocyanine green fluorescence lymphogram shows diffuse dermal backflow in the left leg. In this study, we introduced a novel system of fluorescence lymphography for diagnosing lymphedema. With our technique, real-time imaging of lymphatic drainage of the whole leg was obtained without patient discomfort. The instrument to acquire images is equipped with an LED and CCD camera and is very portable and handy to use. During the examination, the patients can simultaneously observe their video images together with the physician on a monitor of a laptop computer. ICG is a very safe compound that has been very widely used in a variety of clinical situations, such as examination of hepatic function, cardiac output, and retinal angiography. It absorbs light in the near-infrared range, with a maximum at 805 nm. The excitation wavelength of ICG that produces the maximum fluorescence is 765 nm. ICG fluorescences with a maximum at 840 nm in plasma. 15 The fluorescence of ICG in the near-infrared wavelength can deeply penetrate living tissue and is advantageous for obtaining visual information. The use of ICG fluorescence has previously been reported for diagnosing burn depth, 16 evaluation of skin-flap viability, 17 and intraoperative evaluation of bypass graft patency. 18 A previous study reported that fluorescence was observed from ICG solution embedded 1 cm deep in material with optical properties compatible with human tissue. 19 In subcutaneous tissue, most of ICG binds to albumin, which is drained to the lymphatic vessels. These properties of ICG have been used to detect sentinel nodes by fluorescence navigation in breast cancer patients. 19,20 We focused on ICG as a method for visualizing lymph vessels. To our knowledge, this is the first report of ICG fluorescent lymphography of lymphedema. In this study, injected dye to the dorsum of the foot drained to the groin along the medial aspect of the thigh within 30 minutes in healthy volunteers. In patients with lymphedema, however, several characteristic images such as dermal back flow, obstructed fluorescence images with dilatation and tortuosity, and Milky Way sign were obtained. In particular, dermal backflow was always observed in patients with secondary lymphedema, indicating that this technique may be a better screening test to identify the presence or absence of lymphatic disorder. Further study is needed to determine whether fluorescence images with dilatation and tortuosity represent proximal obstructive lymphatics with distal dilation. If this is confirmed, the sign may facilitate patient selection for surgical procedures such as lymph-venous anastomosis. The other abnormal pattern Milky Way sign, identified by diffuse glittering of fluorescent signals with scattered twinkling of the dye, may reflect the extended destruction of lymphatic channels. If this is true, the patients with the Milky Way sign may be better treated not with microsurgical lymphatic reconstruction but with compression and massage therapy.

6 JOURNAL OF VASCULAR SURGERY Volume 45, Number 5 Unno et al 1021 In this preliminary study, qualitative interpretation of the ICG fluorescence lymphography agreed well with actual clinical diagnoses of secondary lymphedema determined by physical findings, medical history, and lymphoscintigram. In lymphoscintigraphy, qualitative interpretation of images has resulted in excellent sensitivity (92 %) and specificity (100 %) for the diagnosis of lymphedema. 21 Further, larger-scale studies are needed to determine the sensitivity and specificity of ICG fluorescence lymphography in diagnosing lymphedema among patients with swollen legs. However, we consider that this technique could replace lymphoscintigraphy for screening lymphedema. Because ICG is already used in other clinical tests with low cost, we believe that once the usefulness of the ICG fluorescence lymphography for the identification of lymphatic disorder is confirmed, the Japanese government may reimburse for ICG fluorescence lymphography. In lymphoscintigraphy, quantitative analysis of lymph transport was performed by obtaining dynamic images or measuring the time it took for the injected dye to reach the groin. 22 Although the method was time-consuming and difficult to reproduce, careful observation using the transport index allowed quantitative analysis of lymph transport, which could facilitate not only patient selection but also follow-up observation in lymph vessel reconstruction In this preliminary study, we have only performed qualitative analysis. Quantitative analysis of lymph transport using ICG fluorescence may also be possible with careful evaluation of dynamic images of fluorescence. CONCLUSION This novel technique of ICG fluorescence lymphography is safe and minimally invasive. The device is portable and easy to use, and real-time fluorescent images can be obtained. Panoramic images of the whole leg showed several characteristic patterns of lymph drainage in patients with secondary lymphedema. These findings suggested that ICG fluorescence lymphography may be useful in daily practice to differentiate lymphedema from edemas resulting from other causes. AUTHOR CONTRIBUTIONS Conception and design: NU Analysis and interpretation: NU, KI, NY, HK Data collection: NU, NU, KI, NY, DS, MS Writing the article: NU Critical revision of the article: NU, KI, NY, HK Final approval of the article: NU Statistical analysis: NU Obtained funding: Not applicable Overall responsibility: NU REFERENCES 1. Kinmonth JB. Lymphangiography in man: a method of outlining lymphatic trunks at operation. Clin Sci 1952;11: Kinmonth JB, Taylor GW, Tracy GD, Marsh JD. Primary lymphoedema. 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