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History and Basic Technique of Fluorescence Imaging for Hepatobiliary-Pancreatic Surgery
Abstract
Recently, fluorescence imaging using indocyanine green (ICG) has been used clinically to visualize the vascular/lymphatic anatomy and cancerous tissues in real time during surgery. Potentially, among the best indications for ICG fluorescence imaging are hepatobiliary and pancreatic diseases since not only the fluorescent property of ICG but also its biliary excretion property can be utilized for imaging. In fact, ICG fluorescence imaging is already being used in clinical settings to identify the anatomy of the bile duct during laparoscopic surgery as well as open surgery in cases of liver cancer. 5-aminolevulinic acid is another fluorescent probe that has been administered to humans for identification of malignant glioma, bladder cancer and epidermal tumor, although its application to hepatobiliary and pancreatic diseases has rarely been evaluated. Preclinically, numerous kinds of novel fluorescent probes are being developed to improve the sensitivity and specificity of ICG fluorescence imaging, making in vivo fluorescence imaging one of the most active research fields in the world.
Background
Microvascular damage is the main cause of delayed graft function (DGF) after kidney transplant. Assessing its extent may be helpful in predicting DGF to achieve better postoperative management, especially in terms of an immunosuppressive regimen. Our aim was to explore the capability of intraoperative indocyanine green (ICG) angiography to examine the microvasculature of the kidney.
Methods
We conducted a prospective cohort study on 37 kidney transplant recipients in a high-volume kidney transplant center. During surgery, after graft implant, an ICG angiography was performed through a high-definition Storz camera system (Karl Storz GmbH, Tuttlingen, Germany) with successive quantitative assessment of fluorescence using Icy bioimage analysis.
Results
All transplanted kidneys that showed immediate recovery of their function had a fluorescent intensity ≥49.953 with a mean of 96.930 ± 21. The fluorescence intensity for kidneys that showed a delayed recovery of their function never exceeded 55.648, and the mean was 37.718 ± 13. The difference between the 2 groups was statistically significant with a P value < .001. The only kidney that never recovered showed a fluorescence intensity consistently <25.220, the lowest detected.
Conclusions
This study demonstrates that intraoperative ICG angiography may be used to assess the microvasculature of the graft. A statistically significant difference in terms of fluorescent intensity can be highlighted between kidneys that immediately recover their function and those with delayed recovery. Further larger studies are needed to confirm the capability of the technique to predict DGF to optimize the transplanted patients’ management.
Intraoperative visualization of pancreatic tumors has the potential to improve radical resection rates. Intraoperative visualization of the common bile duct and bile duct anastomoses could be of added value. In this study, we explored the use of indocyanine green (ICG) for these applications and attempted to optimize injection timing and dose.
Eight patients undergoing a pancreaticoduodenectomy were injected intravenously with 5 or 10 mg ICG. During and after injection, the pancreas, tumor, common bile duct and surrounding organs were imaged in real time using the Mini-FLARE™ near-infrared (NIR) imaging system.
No clear tumor-to-pancreas contrast was observed, except for incidental contrast in 1 patient. The common bile duct was clearly visualized using NIR fluorescence, within 10 min after injection, with a maximal contrast between 30 and 90 min after injection. Patency of biliary anastomoses could be visualized due to biliary excretion of ICG.
No useful tumor demarcation could be visualized in pancreatic cancer patients after intravenous injection of ICG. However, the common bile duct and biliary anastomoses were clearly visualized during the observation period. Therefore, these imaging strategies could be beneficial during biliary surgery in cases where the surgical anatomy is aberrant or difficult to identify.
For hepatic tumors that cannot be identified on routine ultrasonography (US), we marked the target area using real-time virtual sonography (RVS) with indocyanine green (ICG)-ethanol (1:100) during surgery, and performed hepatic resection while observing the fluorescence. EXPERIMENT: An ICG-ethanol mixture locally injected into mouse liver was retained in the same area for more than 4 h. The same mixture locally injected into pig liver at a depth of 3 cm could be observed using an infrared camera.
An ICG-ethanol mixture (500 μl) was locally injected under RVS guidance into a metastatic hepatic tumor that was visible only on magnetic resonance imaging (MRI), and hepatic resection was performed while Photodynamic Eye (PDE) images were being observed. The metastatic lesion (3 mm in diameter in a pathological specimen) could be successfully resected.
This method was useful for resecting US-invisible hepatic tumors.
Accidental injury to the common bile duct is a rare but serious complication of laparoscopic cholecystectomy. Accurate visualization of the biliary ducts may prevent injury or allow its early detection. Conventional X-ray cholangiography is often used and can mitigate the severity of injury when correctly interpreted. However, it may be useful to have an imaging method that could provide real-time extrahepatic bile duct visualization without changing the field of view from the laparoscope. The purpose of the present study was to test a new near-infrared (NIR) fluorescent agent that is rapidly excreted via the biliary route in preclinical models to evaluate intraoperative real-time near infrared fluorescent cholangiography (NIRFC).
To investigate probe function and excretion, a lipophilic near-infrared fluorescent agent with hepatobiliary excretion was injected intravenously into one group of C57/BL6 control mice and four groups of C57/BL6 mice under the following experimentally induced conditions: (1) chronic biliary obstruction, (2) acute biliary obstruction (3) bile duct perforation, and (4) choledocholithiasis, respectively. The biliary system was imaged intravitally for 1 h with near-infrared fluorescence (NIRF) with an intraoperative small animal imaging system (excitation 649 nm, emission 675 nm).
The extrahepatic ducts and extraluminal bile were clearly visible due to the robust fluorescence of the excreted fluorochrome. Twenty-five minutes after intravenous injection, the target-to-background ratio peaked at 6.40 +/- 0.83 but signal was clearly visible for ~60 min. The agent facilitated rapid identification of biliary obstruction and bile duct perforation. Implanted beads simulating choledocholithiasis were promptly identifiable within the common bile duct lumen.
Near-infrared fluorescent agents with hepatobiliary excretion may be used intraoperatively to visualize extrahepatic biliary anatomy and physiology. Used in conjunction with laparoscopic imaging technologies, the use of this technique should enhance hepatobiliary surgery.
The cholecystic veins are thought to be an important metastatic route of gallbladder carcinoma to the liver. In the present study we evaluated the cholecystic venous drainage area, utilizing a novel method, indocyanine green (ICG) fluorescence angiography after superselective catheterization of the cholecystic artery, to detect and elucidate cholecystic venous flow.
Cannulation of the cholecystic artery was performed under laparotomy in nine patients who required a cholecystectomy. After ICG injection into the cholecystic artery, the cholecystic venous flow images were visualized with a near-infrared camera system and were analyzed according to site, shape, and time of fluorescence.
Fluorescence images of the cholecystic venous flow could be viewed as real-time images in all patients. We demonstrated that the route of the cholecystic venous flow could be classified into two patterns: type 1, in which the cholecystic veins flowed directly into the hepatic parenchyma adjacent to the gallbladder; and type 2, in which the veins flowed into sites separate from the gallbladder. In the type 1 pattern, fluorescence was observed in segment (S; defined according to Couinaud's nomenclature) 4a or S5 adjacent to the gallbladder in all cases. On the other hand, in the type-2 pattern, fluorescence was observed in S4a (6/9), S5 (8/9), S4b (2/9), S3 (2/9), S1 (1/9), S2 (1/9), and S8 (1/9) distant from the gallbladder. Overall, two-thirds of the cases showed fluorescence in segments other than S4a or S5.
Indocyanine green (ICG) fluorescence angiography is considered to be a useful method to detect and elucidate cholecystic venous flow in real time. This study showed that the cholecystic venous flow spread to the liver through two different pathways, one that flowed directly into the hepatic parenchyma adjacent to the gallbladder, while the other flowed into sites separate from the gallbladder. Taking these findings into consideration, we may therefore need to reconsider the preventive effects of a hepatic resection.
Administration of 5-aminolaevulinic acid (ALA) leads to porphyrin accumulation in malignant and premalignant tissues, and ALA is used as a prodrug in photodynamic therapy (PDT). To understand the mechanism of porphyrin accumulation after the administration of ALA and to investigate whether ALA-induced protoporphyrin IX might be a suitable photosensitizer in Barrett's oesophagus and adenocarcinoma, we determined the activities of porphobilinogen deaminase (PBG-D) and ferrochelatase (FC) in various malignant and premalignant as well as in normal tissues of the human oesophagus. A PDT power index for ALA-induced porphyrin accumulation, the ratio of PBG-D to FC normalized for the normal squamous epithelium of the oesophagus, was calculated to evaluate intertissue variation in the ability to accumulate porphyrins. In malignant and premalignant tissue a twofold increased PBG-D activity and a marginally increased FC activity was seen compared with normal squamous epithelium. A significantly increased PDT power index in Barrett's epithelium and adenocarcinoma was found. Our results suggest that, after the administration of ALA, porphyrins will accumulate in a greater amount in Barrett's epithelium and adenocarcinoma of the oesophagus because of an imbalance between PBG-D and FC activities. The PDT power index here defined might be a useful indicative parameter for predicting the susceptibility of these tissues to ALA-PDT.
Recently, gastrointestinal cancer has also been identified as a target for sentinel node navigation surgery (SNNS). This study is the first to determine the feasibility of sentinel node (SN) mapping guided by indocyanine green (ICG) fluorescence imaging in gastrointestinal cancer.
Our series consisted of 22 patients with gastric cancer and 26 patients with colorectal cancer who had undergone standard surgical resection. ICG solution was injected intraoperatively into the subserosa around the tumor. Fluorescence imaging was obtained by a charge-coupled device (CCD) camera with a light-emitting diode with a wavelength of 760 nm as the light source and a cut filter to filter out light with wavelengths below 820 nm as the detector.
Immediately after the ICG injection, lymphatic vessels draining the tumor and round-shaped SNs were visualized by their bright fluorescence. Even SNs that were not green in color could be easily and clearly visualized by ICG fluorescence imaging. The SN detection rate and mean number of SNs were 90.9% and 3.6 +/- 4.5 (mean +/- SD), respectively, in patients with gastric cancer, and 88.5% and 2.6 +/- 2.4, respectively, in patients with colorectal cancer. Among the patients with gastric cancer, the accuracy and false-negative rates were 88.9 and 33.3%, respectively, in patients with T(1) stage cancer, and 70.0 and 60.0%, respectively, overall, in all the patients. Among the patients with colorectal cancer, the corresponding values were 100 and 0%, respectively, in patients with T(1) stage cancer, and 82.6 and 66.7%, respectively, overall, in all the patients.
Our preliminary results show that ICG fluorescence imaging allows easy, highly sensitive and real-time imaging-guided SN mapping in patients with gastric or colorectal cancer. SN mapping guided by ICG fluorescence imaging could be a promising tool deserving further clinical exploration.
In malignant hepatic neoplasm, anatomic resection could improve survival and limit complications from hepatectomy. Our purpose was to develop an intraoperative method for identifying segment and subsegment of the liver with high-sensitivity near-infrared fluorescence imaging.
The subjects were 35 patients with hepatic malignant liver disease who received hepatectomy in 2006. The segments of liver method of identification that used infrared observation camera system termed Photo Dynamic Eye-2 (PDE-2) with indocianine green (ICG) for the patient with malignant liver tumor (hepatocellular carcinoma: 13 cases; metastatic liver cancer: 18 cases; intrahepatic cholangio carcinoma: 4 cases) were performed before liver resection.
Although greenish stain of the liver surface after the injection of ICG via portal vein is not visible clearly without infrared observation camera system PDE-2, 1 minute after injection of ICG with fluorescent using infrared observation camera system PDE-2, demarcation of liver segment and subsegment was clearly detected. Ten minutes after injection of ICG with fluorescent using infrared observation camera system PDE-2, fluorescence of liver subsegment remained. Stained subsegment and segment of liver were identifiable in 33 (94.3%) of the 35 patients. There were no complications or side-effects related to the injection of patent blue dye.
We demonstrated here that near-infrared fluorescence imaging system is a novel and reliable intraoperative technique to identify hepatic segment and subsegment for anatomical hepatic resection.
Background:
Indocyanine green (ICG) fluorescent imaging has been used effectively to identify hepatocellular carcinoma (HCC) in intraoperative setting. However, whether extrahepatic metastatic lesions from HCC can also be detected by this imaging is unknown.
Methods:
This study was conducted on 17 patients with suspected extrahepatic HCC metastases in the lung (n = 3), adrenal gland (n = 1), lymph node (n = 7), peritoneum (n = 5) and both lymph node and peritoneum (n = 1). ICG was administered intravenously at a dose of 0.5 mg/kg prior to operation for liver function evaluation. Intraoperative ICG fluorescent imaging was performed with a near-infrared light camera system. The surgical specimens were also examined in all cases for the presence of ICG fluorescence.
Results:
Of 28 lesions for which ICG fluorescence was examined intraoperatively, 24 lesions exhibited fluorescence and were proved to be HCC metastases pathologically. Five of them were newly identified by ICG fluorescent imaging. The other four lesions included two HCC metastases and two benign tumors. Of 33 suspicious metastatic lesions extirpated, 26 lesions emitting fluorescence from the specimen were all metastatic HCC. The other 7 lesions consisted of 6 benign tumors and one HCC metastasis. Accordingly, the positive predictive value of in vivo and ex vivo ICG fluorescent imaging were both 100 %, while the negative predictive value of those methods were 50 and 86 %, respectively.
Conclusions:
Extrahepatic metastases from HCC exhibited ICG fluorescence when illuminated by near-infrared light, indicating their capability to transport ICG. This imaging can be a useful tool for intraoperative detection of metastasis in HCC patients.
Background & aims:
Although recent advances in preoperative imaging have enabled accurate estimation of the regional liver volume with venous occlusion, the extent of functional decrease in such regions remains unclear. In this study, the portal uptake function in postoperative veno-occlusive regions and non-veno-occlusive regions was evaluated by intraoperative fluorescent imaging after intravenous injection of indocyanine green (ICG).
Methods:
In 22 liver resection patients and 23 recipients and 18 donors of liver transplantation, fluorescent intensity on the remnant liver or the liver graft was evaluated in real time following intravenous injection of ICG (0.0025 mg per 1 ml of remnant liver volume).
Results:
Plateau ICG concentrations were significantly lower in the veno-occlusive regions (C(VO)) than in the non-veno-occlusive regions (C(Non)) in liver resection patients (median [range], 0.75 [0.29-2.0]μg/ml vs. 3.0 [0.46-6.4]μg/ml, p<0.001), donors (0.69 [0.29-1.9]μg/ml vs. 2.4 [0.46-6.4]μg/ml, p<0.001), and recipients (0.75 [0.34-1.8]μg/ml vs. 1.8 [0.54-6.4]μg/ml, p<0.001). Distributions of the C(VO)/C(Non) and the ratio of the hepatic uptake rate constant in the veno-occlusive regions to that in non-veno-occlusive regions were both around 40% (mean ± standard deviation, 0.36 ± 0.17 and 0.42 ± 0.16, respectively). When the functional remnant liver volume was calculated as a sum of non-veno-occlusive regions and veno-occlusive regions multiplied by C(VO)/C(Non), its ratio to the total liver volume was correlated with the improved postoperative/preoperative ratio of prothrombin time.
Conclusions:
Portal uptake function in veno-occlusive regions is approximately 40% of that in non-veno-occlusive regions. Intraoperative ICG-fluorescent imaging enables real-time evaluation of the extent of the functional decrease in veno-occlusive regions, enhancing accurate estimation of the hepatic functional reserve for determining the surgical indications and procedures.
For safe laparoscopic cholecystectomy, surgeons must possess detailed knowledge of the anatomy of the bile duct and arterial system as seen through a laparoscope.
We developed an indocyanine green (ICG) reinjection technique for use in fluorescent angiography. Here, we evaluated the efficacy of the ICG reinjection technique in fluorescent angiography in discriminating the arterial system with the concomitant use of fluorescent cholangiography.
Twenty-eight patients were enrolled in the study. All patients underwent laparoscopic cholecystectomy without complication. After reinjection of ICG during surgery, fluorescence of the cystic artery was visualized in 25 patients (89%).
Fluorescent angiography using this ICG reinjection technique might enhance the safety of laparoscopic cholecystectomy.
Introduction: Laparoscopic hepatectomy has disadvantages in intraoperative diagnosis, because it offers limited visualization and palpability of the liver surface. Recently, we developed a novel fluorescent imaging technique using indocyanine green (ICG), which would enable identification of liver cancers during open hepatectomy. However, this technique has not yet been applied to laparoscopic hepatectomy.
Materials and Surgical Technique: A patient with a hepatocellular carcinoma (HCC) located in Couinaud's segment II was administered ICG (0.5 mg per kg body weight) intravenous injection 5 d before surgery, as a routine liver function test. The prototype fluorescent imaging system was composed of a xenon light source and a laparoscope with a charge-coupled device camera that could filter out light with wavelengths below 810 nm. Intraoperatively, fluorescent imaging of the HCC was performed by changing color images to fluorescent images with a foot switch. Then, the fluorescing tumor was clearly identified on the visceral surface of segment II during mobilization of the left liver for resection of segments II and III. On the cut surface of the specimen, the tumor showed uniform fluorescence and was microscopically diagnosed as a well-differentiated HCC.
Discussion: Laparoscopic fluorescent imaging using preoperative injection of ICG enabled real-time identification of HCC. This technique may be an easy and reliable tool to enhance the accuracy of intraoperative diagnosis during laparoscopic hepatectomy.
Intraoperative cholangiography (IOC) is especially helpful for the detection of anomalous biliary anatomy during laparoscopic cholecystectomy. Fluorescent cholangiography using an intravenously injected fluorophore and near-infrared (NIR) imaging provides similar anatomical detail to standard radiographic cholangiography without ionizing radiation, puncture of the biliary system, or additional operative time. This video shows a laparoscopic cholecystectomy performed under NIR cholangiographic guidance and highlights its ability to identify anomalous anatomy.
The attached video shows a laparoscopic cholecystectomy being performed on a 28-year-old female with a history of biliary colic and ultrasonographic evidence of cholelithiasis. This patient agreed to be part of a larger randomized study looking at near-infrared cholangiography and its ability to prevent common bile duct injuries (approved by the ethics review board of our institution and registered with clinicaltrials.gov Identifier# NCT01424215). This study uses the Pinpoint system (Novadaq, Ontario, Canada) for NIR imaging (Fig. 1). The Pinpoint mates a high definition white light laparoscopic view to the NIR cholangiography, providing an uninterrupted, augmented view of the anatomy. 1 cm(3) of indocyanine green was injected intravenously prior to the procedure.
As shown in the video, an anomalous duct was identified during dissection and development of the critical view of safety. Because of the possibility that this represented an aberrant right hepatic duct, the cystic duct was controlled and divided distal to the anomalous duct and the gall bladder excised from the fossa in the usual manner. The patient did well without sequelae at 1 week and 1 month follow-up.
Anomolous ductal anatomy of the biliary tree has been reported in up to 23 % of cases.1,2 The ability of IOC to elucidate biliary anatomy and thus prevent bile duct injury has led many to espouse routine cholangiography for all laparoscopic cholecystectomies.3,4 Near-infrared cholangiography (NIRC) is easy to perform, does not add steps to the operative procedure, and produces a similar anatomic roadmap of the hepatocystic triangle to that of standard IOC. Although the clinical significance of the anomalous duct identified in this video is unknown, this video highlights the excellent detail provided by NIRC. Recommendations regarding the routine use of this new technology await the results of an ongoing randomized control study.
Background and study aims:
Very recently, robotic single site cholecystectomy (RSSC) has been reported feasible and safe for selected cases. While an intra-operative cholangiography can be performed, data is scarce with respect to its use. Indocyanin green (ICG) has been shown to be a viable option to visualize biliary anatomy. Since the introduction of a new near infrared camera integrated to the da Vinci Si System (Intuitive Surgical, Sunnyvale, CA), the surgeon is able to assess the biliary anatomy by a non-invasive and non-ionizing method. This paper presents the first report of ICG imaging during a RSSC.
Patients and methods:
Twelve consecutive patients presenting symptomatic cholelithiasis were prospectively enrolled. They underwent RSSC approximately 45 minutes after intravenous administration of ICG (2.5 mg). The biliary anatomy was analyzed using a near infrared camera integrated to the robot before and after the robotic dissection.
Results:
Eight women and four men underwent the procedure. There was a port addition in one case and no peri-operative complications. Mean operative time was 85 minutes (range: 57-125). The cystic, common bile and common hepatic ducts were recognized by fluorescence imaging before the dissection in 91.7%, 50%, and 33.3% of patients, respectively. At least one structure was visualized in 100% of patients. After the completion of Calot's triangle dissection, the cystic, common bile, and common hepatic ducts were recognized in 100%, 83.3%, and 66.7% of cases respectively.
Conclusions:
RSSC using ICG for biliary tree fluorescence imaging can be performed safely. Fluorescent cholangiography enabled real-time identification of the extra-hepatic biliary anatomy using a near infrared camera integrated to the robot. Its routine clinical use merits further investigations.
The aim of this study was to improve fluorescence laparoscopy of pancreatic cancer in an orthotopic mouse model with the use of a light-emitting diode (LED) light source and optimal fluorophore combinations.
Human pancreatic cancer models were established with fluorescent FG-RFP, MiaPaca2-GFP, BxPC-3-RFP, and BxPC-3 cancer cells implanted in 6-week-old female athymic mice. Two weeks postimplantation, diagnostic laparoscopy was performed with a Stryker L9000 LED light source or a Stryker X8000 xenon light source 24 hours after tail-vein injection of CEA antibodies conjugated with Alexa 488 or Alexa 555. Cancer lesions were detected and localized under each light mode. Intravital images were also obtained with the OV-100 Olympus and Maestro CRI Small Animal Imaging Systems, serving as a positive control. Tumors were collected for histologic analysis.
Fluorescence laparoscopy with a 495-nm emission filter and an LED light source enabled real-time visualization of the fluorescence-labeled tumor deposits in the peritoneal cavity. The simultaneous use of different fluorophores (Alexa 488 and Alexa 555), conjugated to antibodies, brightened the fluorescence signal, enhancing detection of submillimeter lesions without compromising background illumination. Adjustments to the LED light source permitted simultaneous detection of tumor lesions of different fluorescent colors and surrounding structures with minimal autofluorescence.
Using an LED light source with adjustments to the red, blue, and green wavelengths, it is possible to simultaneously identify tumor metastases expressing fluorescent proteins of different wavelengths, which greatly enhanced the signal without compromising background illumination. Development of this fluorescence laparoscopy technology for clinical use can improve staging and resection of pancreatic cancer.
We identified the lymphatic drainage pathways from the pancreatic head guided by indocyanine green (ICG) fluorescence imaging to analyze optimal lymphadectomy for pancreatic cancer.
The lymphatic pathways in 20 patients undergoing pancreaticoduodenectomy were analyzed. We injected ICG into the parenchyma in the anterior (n = 10) or posterior surface (n = 10) of the pancreas head and observed the intraoperative lymphatic flows by ICG fluorescence imaging.
The seven main lymphatic drainage pathways were identified: (1) along the anterior or posterior pancreaticoduodenal arcade, (2) running obliquely down behind the superior mesenteric vein (SMV), (3) reaching the left side of the superior mesenteric artery (SMA), (4) running longitudinally upward between the SMV and SMA, (5) along the middle colic artery toward the transverse colon, (6) reaching the paraaortic (PA) region, and (7) reaching the hepatoduodenal ligament. The lymphatic pathway reaching the left side of the SMA was observed in 4 patients (20%), while that reaching the PA region in 17 patients (85%). The mean time to reach around the SMA was longer than that to reach the PA region.
We found that several lymphatic drainage routes were observed from the pancreatic head, suggesting that a lymphadectomy around the SMA might have a similar oncological impact as that of the PA region.
Anatomic hepatic segmentectomy1 plays an important role in the modern treatment of malignant disease. Segmentectomy maximizes postoperative hepatic functional reserve without compromising oncologic principles.2 This approach is particularly appropriate for a hepatocellular carcinoma segmental resection and may also be useful for other indications, such as a segmental resection for colorectal metastases and other hepatic tumors. The classic hepatic segment identification technique involves puncture of a segmental portal branch under the guidance of intraoperative ultrasonography. Vital blue dye is injected after temporarily clamping the hepatic artery to avoid dye washout from the segment.1 This staining technique is simple and useful for open surgery; however, it is much more difficult to reproduce these procedures laparoscopically and to confirm demarcation of the hepatic segment visually on the monitor. This problem may reduce the accuracy and frequency of anatomic segmentectomy using a laparoscopic blue dye approach.3 Recently, we have developed a laparoscopic fluorescent imaging system that allows visualization of indocyanine green (ICG) fluorescence to identify the biliary tract4 and liver cancers5 intraoperatively. Herein, we report our initial experience of applying this system to the visualization of hepatic segments during laparoscopic surgery, by injecting ICG dye into the segmental portal branch6 (positive staining) or by intravenously administering ICG dye after clamping the segmental portal pedicle (negative staining).
To demonstrate the usefulness of a fluorescence imager (photodynamic eye; PDE) for observation of lymph flow in lower rectal cancer (LRC).
Between October 2006 and January 2010, PDE observations were performed in 14 patients with LRC. After induction of general anesthesia, a total of 2mL of indocyanine green (ICG) (2.5mg/mL) was injected into the submucosal layer on the dentate line or the anal margin of the LRC.
Preoperative PDE observation was able to demonstrate several lymph flows running to the bilateral inguinal areas from the perianal area immediately after ICG injection in 13 of the patients (92.9%). Although these flows were pooled in the bilateral inguinal areas, there was no pooling of such lymph flows in the perianal area. Intraoperative PDE observation was able to demonstrate not only mesenteric lymph nodes in all patients but also bilateral lateral lymph nodes in 13 patients (92.9%). Although 6 patients had undergone sphincter-preserving surgery (SPS), no local recurrence was observed in such patients during the observation period.
PDE is able to visualize three sets of regional lymph nodes in patients with LRC, suggesting that it would be useful for determining the effectiveness of SPS for such patients.
This study evaluated the usefulness of photodynamic diagnosis (PDD) using oral 5-aminolevulinic acid (ALA) for the detection of peritoneal metastases in advanced gastric cancer.
First, the numbers of peritoneal metastatic nodules that were visible under conventional white light (WL) and ALA-induced fluorescence (ALA-F) were quantified in a mouse model of peritoneal metastasis to compare the tumor detection rate. Next, staging laparoscopy (SL) using ALA-PDD was performed in 13 advanced gastric cancer patients with serosa-invading tumors, and the detection sensitivity of ALA-PDD was compared to the observations using WL.
The tumor detection rate using ALA-F was significantly higher than the detection rate using WL (72% vs. 39%, respectively, P < 0.0001). Peritoneal metastases were detected in five patients using SL with ALA-PDD, and liver metastases were detected in one patient. These metastases were confirmed using histological examination. Three metastatic lesions that were invisible under WL were detected under ALA-F.
This study demonstrated that SL with ALA-PDD improved the detection sensitivity for peritoneal metastases. ALA-PDD may be an important technique for the preoperative staging of advanced gastric cancer, and ALA-PDD will provide useful information for the selection of therapeutic modality.
The ability of the unaided human eye to detect small cancer foci or accurate borders between cancer and normal tissue during surgery or endoscopy is limited. Fluorescent probes are useful for enhancing visualization of small tumors but are typically limited by either high background signal or the requirement for administration hours to days before use. We synthesized a rapidly activatable, cancer-selective fluorescence imaging probe, γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), with intramolecular spirocyclic caging for complete quenching. Activation occurs by rapid one-step cleavage of glutamate with γ-glutamyltranspeptidase (GGT), which is not expressed in normal tissue, but is overexpressed on the cell membrane of various cancer cells, thus leading to complete uncaging and dequenching of the fluorescence probe. In vitro activation of gGlu-HMRG was evident in 11 human ovarian cancer cell lines tested. In vivo in mouse models of disseminated human peritoneal ovarian cancer, activation of gGlu-HMRG occurred within 1 min of topically spraying the tumor, creating high signal contrast between the tumor and the background. The gGlu-HMRG probe is practical for clinical application during surgical or endoscopic procedures because of its rapid and strong activation upon contact with GGT on the surface of cancer cells.
Recently, a highly sensitive fluorescent imaging technique was developed for the real-time identification of hepatic tumors. The authors applied this procedure for the intraoperative detection of radiographically occult hepatic micrometastases from pancreatic cancer.
Forty-nine consecutive patients with pancreatic cancer who underwent surgical intervention were examined. Preoperative clinical images had not revealed any hepatic metastases. On the day before surgery, indocyanine green was injected intravenously. During the operation, the liver was observed with a near-infrared camera system, and abnormal fluorescent foci were examined by frozen-section histology. The patients with hepatic micrometastases were judged to have unresectable disease and underwent only palliative surgery followed by systemic chemotherapy using gemcitabine.
Abnormal hepatic fluorescence at least 1.5 mm in greatest dimension without any apparent tumor was observed in 13 patients. Among them, histologic examination confirmed micrometastases in 8 of 49 patients (16%). All patients with hepatic micrometastases had clinical T3 or T4 disease and high serum CA19-9 levels (P = .042). On follow-up computed tomography images that were obtained within 6 months after surgery, the patients with hepatic micrometastases manifested hepatic overt metastases (7 of 8 patients; 88%) more frequently than the patients without hepatic micrometastases (4 of 41 patients; 10%; P < .001). Regardless of histologic confirmation, the positive predictive value of abnormal fluorescence for the manifestation of hepatic relapse within 6 months was 77% (10 of 13 patients), and the negative predictive value was 97% (35 of 36 patients).
Indocyanine green-fluorescent imaging can detect hepatic micrometastases of pancreatic cancer during surgery. The hepatic micrometastases seem to have an adverse clinical impact identical to that of evident distant metastases.
The clear demarcation line is ideal for real-time surgical navigation imaging during hepatectomy.
The study population was comprised of 22 patients with moderate liver cirrhosis scheduled to undergo an anatomical liver resection for the treatment of hepatocellular carcinoma. This study set out to assess the clinical value of the concomitant intra-operative use of contrast-enhanced intra-operative ultrasound using Sonazoid™, and a fluorescence navigation system (PDE) with ICG, as a novel tool for patients undergoing an anatomical liver resection.
Following portal pedicle ligation for anatomical resection, 2 min after injection of ICG, the segments to be resected were detected as a negative-brightness area using PDE fluorescence. Sonazoid™ administration provides a parenchymal transectional line, as the margin of a loss of blood flow shows a hypo-enhanced image, and the resectional line of the parenchyma can be confirmed by CE-IOUS. Although the demarcation line of the liver surface after the portal pedicle ligation was apparent in 17 patients, the resection line using PDE was clearly detected in all 22 patients (p < 0.018).
The combined use of these methods is therefore considered to be useful and safe for surgeons, as an additional tool for performing a liver resection.
We hypothesized that two independent wavelengths of near-infrared (NIR) fluorescent light could be used to identify bile ducts and hepatic arteries simultaneously, and intraoperatively.
Three different combinations of 700 and 800 nm fluorescent contrast agents specific for bile ducts and arteries were injected into N = 10 35-kg female Yorkshire pigs intravenously. Combination 1 (C-1) was methylene blue (MB) for arterial imaging and indocyanine green (ICG) for bile duct imaging. Combination 2 (C-2) was ICG for arterial imaging and MB for bile duct imaging. Combination 3 (C-3) was a newly developed, zwitterionic NIR fluorophore ZW800-1 for arterial imaging and MB for bile duct imaging. Open and minimally invasive surgeries were imaged using the fluorescence-assisted resection and exploration (FLARE) surgical imaging system and minimally invasive FLARE (m-FLARE) imaging systems, respectively.
Although the desired bile duct and arterial anatomy could be imaged with contrast-to-background ratios (CBRs) ≥ 6 using all three combinations, each one differed significantly in terms of repetition and prolonged imaging. ICG injection resulted in high CBR of the liver and common bile duct (CBD) and prolonged imaging time (120 min) of the CBD (C-1). However, because ICG also resulted in high background of liver and CBD relative to arteries, ICG produced a lower arterial CBR (C-2) at some time points. C-3 provided the best overall performance, although C-2, which is clinically available, did enable effective laparoscopy.
We demonstrate that dual-channel NIR fluorescence imaging provides simultaneous, real-time, and high resolution identification of bile ducts and hepatic arteries during biliary tract surgery.
Survival after surgery and radiotherapy for the treatment of malignant gliomas is linked to the completeness of tumor removal. Therefore, methods that permit intraoperative identification of residual tumor tissue may be of benefit. In a preliminary investigation, we have studied the value of fluorescent porphyrins that accumulate in malignant tissue after administration of a precursor (5-aminolevulinic acid) for labeling of malignant gliomas in nine patients.
Three hours before the induction of anesthesia, 10 mg 5-aminolevulinic acid/kg body weight was administered orally. Intraoperatively, red porphyrin fluorescence was observed with a 455-nm long-pass filter after excitation with violet-blue (375-440 nm) xenon light and was verified by analysis of fluorescence spectra. Fluorescing and nonfluorescing samples taken from the tumor perimeters were examined histologically or used to study the photobleaching of porphyrins by excitation light and white light from the operating microscope. Plasma and erythrocyte porphyrin levels were determined by fluorescence photometry.
Normal brain tissue revealed no porphyrin fluorescence, whereas tumor tissue was distinguished by bright red fluorescence. For a total of 89 tissue biopsies, sensitivity was 85% and specificity was 100% for the detection of malignant tissue. For seven of nine patients, visible porphyrin fluorescence led to further resection of the tumor. Under operating light conditions, fluorescence decayed to 36% in 25 minutes for violet-blue light and in 87 minutes for white light. Plasma and erythrocyte porphyrin contents increased slightly, without exceeding normal levels.
Our observations suggest that 5-aminolevulinic acid-induced porphyrin fluorescence may label malignant gliomas safely and accurately enough to enhance the completeness of tumor removal.
Bile leakage is a common complication of hepatectomy, and is associated with an increase in sepsis and liver failure. There are no standard preventive methods against bile leakage after hepatic surgery. The aim of the present randomized clinical trial was to evaluate the application of indocyanine green (ICG) fluorescent cholangiography for preventing postoperative bile leakage.
102 patients who underwent hepatic resection without biliary reconstruction were divided into 2 groups. The control group (n = 50) underwent a leak test with ICG dye alone, and the experimental group underwent a leak test with ICG dye, followed by ICG fluorescent cholangiography using the Photodynamic Eye (PDE group, n = 52).
Among 42 patients with fluorescence in the PDE group, 25 patients had insufficient closure of bile ducts on the cut surface of the liver, which were closed by suture or ligation. There were 5 patients who developed postoperative bile leakage in the control group versus no bile leakage in the PDE group (10% vs 0%, P = .019).
ICG fluorescent cholangiography could detect insufficiently closed bile ducts that could not be identified by a standard bile leak test. ICG fluorescent cholangiography may have useful potential for prevention of bile leakage after hepatic resection.
Although the use of single-incision laparoscopic cholecystectomy (SILC) is spreading rapidly, this technique has disadvantages. It does not allow for sufficient surgical views to be obtained or for intraoperative radiographic cholangiography to be performed. Fluorescent cholangiography using a preoperative intravenous injection of indocyanine green (ICG) may be useful for identifying the biliary tract during both SILC and conventional laparoscopic cholecystectomy.
For seven patients undergoing SILC, 1 ml of ICG (2.5 mg) was administered by intravenous injection before the surgery. The prototype fluorescent imaging system consisted of a xenon light source and a 30° laparoscope (diameter, 10 mm) equipped with a charge-coupled device camera capable of filtering out light with wavelengths shorter than 810 nm. The laparoscope was introduced through an umbilical trocar. Fluorescent cholangiography then was performed by changing the color images to fluorescent images using a foot switch during dissection of the triangle of Calot.
Fluorescent cholangiography identified the confluence between the cystic duct and the common hepatic duct in all seven patients before and throughout the dissection of the triangle of Calot. The interval from the injection of ICG to the first obtained fluorescent cholangiography before dissection of the triangle of Calot ranged from 35 to 75 min.
Fluorescent cholangiography enabled real-time identification of the extrahepatic bile ducts during SILC without necessitating catheterization of the bile duct. Such properties of fluorescent cholangiography are expected to be helpful for ensuring the safety of SILC and expanding the indications for the procedure.
Bile leak remains a serious complication after hepatectomy. The conventional leak test by intrabiliary injection of normal saline solution is not sensitive. The authors present a new bile leak test using indocyanine green (ICG) fluorescence. After hepatic transection, ICG solution (.05 mg/mL) was intrabiliarily injected through a transcystic tube under distal common bile duct clamping, and fluorescent images were visualized using an infrared camera system. The ICG leak test was performed in 27 patients undergoing hepatectomy without biliary reconstruction. Bile leaks were intraoperatively found in 8 patients and fixed, resulting in no postoperative leaks. There was no adverse reaction to ICG. In contrast, postoperative bile leaks occurred in 2 of 32 patients who received the conventional leak test with normal saline solution between April 2007 and March 2008. The new bile leak test by ICG fluorography is useful to prevent postoperative bile leak.
Although intraoperative cholangiography has been recommended for avoiding bile duct injury during laparoscopic cholecystectomy, radiographic cholangiography is time consuming and may itself cause injury to the bile duct. Recently, a novel fluorescent cholangiography technique using the intravenous injection of indocyanine green (ICG) has been developed.
In 52 patients undergoing laparoscopic cholecystectomy, 2.5 mg ICG was injected intravenously 30 min before the patient entered the operating room or following intubation. A fluorescent imaging system, which consisted of a xenon light source and a laparoscope with a charge-coupled device camera that could filter out light wavelengths below 810 nm, was used. Fluorescent cholangiography was performed during dissection of Calot's triangle, and its ability to delineate biliary anatomy was compared with that of preoperative cholangiography.
Fluorescent cholangiography delineated the cystic duct in all 52 patients, and the cystic duct-common hepatic duct junction was visible before dissection of Calot's triangle in 50 patients. Fluorescent imaging also identified all accessory bile ducts that had been diagnosed before surgery in eight patients.
Fluorescent cholangiography enables real-time identification of biliary anatomy during dissection of Calot's triangle. This simple technique may become standard practice for avoiding bile duct injury during laparoscopic cholecystectomy, replacing radiographic cholangiography.
Histopathology is the gold standard in the diagnosis of liver diseases but may be complicated by sampling error and bleeding. Confocal laser endomicroscopy (CLE) is a novel imaging modality providing in vivo histology. Previous studies using CLE for liver microscopy suffered from limited imaging depth using blue laser light and fluorescein. The aim of the current study was to evaluate a novel near-infrared (NIR) light probe with indocyanine green (ICG) contrast for in vivo confocal imaging of the human liver during mini-laparoscopy.
The hand-held rigid laparoscopy probe (diameter 6.3mm) used a 780 nm diode laser. Subsurface images at different depths with < 1 microm lateral resolution were generated in real time by gently placing the sterile probe onto the liver in 22 patients under conscious analgo-sedation. Targeted biopsy was performed for histopathological correlation.
Maximum imaging depth was >350 microm. Typical aspects of normal liver architecture and liver diseases, such as nuclei, sinusoids, fibrous tissue, fatty inclusions, and bile ducts but not inflammation could be visualized at high resolution. The presence of steatosis and fibrosis was predicted correctly in 81% and 90%, respectively. No liver damage or severe adverse events occurred.
For the first time, ICG-augmented confocal mini-laparoscopy with a novel NIR probe allowed in vivo microscopy of human liver diseases, and with deeper imaging compared to the so far available blue laser systems. Such an increase of imaging depth could potentially also be used for submucosal imaging of the GI tract.
Preoperative imaging is widely used and extremely helpful in hepatobiliary surgery. However, transfer of preoperative data to a intraoperative situation is very difficult. Surgeons need intraoperative anatomical information using imaging data for safe and precise operation in the field of hepatobiliary surgery. We have developed a new system for mapping liver segments and cholangiograms using intraoperative indocyanine green (ICG) fluorescence under infrared light observation.
The imaging technique for mapping liver segments and cholangiogram based on ICG fluorescence used an infrared-based navigation system. Eighty one patients with liver tumors underwent hepatectomy from 2006, January to 2009, March. In liver surgery, 1 ml of ICG was injected via the portal vein under observation by the fluorescent imaging system. Fourteen patients were underwent laparoscopic cholecystectomy for chronic cholecystitis with gallstones. In laparoscopic cholecystectomy, 5 ml of ICG was administered intravenously just before operation and the bile duct was observed using the infrared-based navigation system.
This new technique successfully identified stained subsegments and segments of the liver in 73 of 81 patients (90.1%). Moreover, clear mapping of liver segments was obtained even against a background of liver cirrhosis. Fluorescent cholangiography clearly showed the common bile duct and cystic duct in 10 of 14 patients (71.4%). No adverse reactions to the ICG were encountered.
Application of this technique allows intraoperative identification of anatomical landmark in hepatobiliary surgery.
We evaluated the usefulness of intraoperative exploration of the biliary anatomy using fluorescence imaging with indocyanine green (ICG) in experimental and clinical cholecystectomies.
The experimental study was done using two 40-kg pigs and the clinical study was done in 12 patients for whom cholecystectomy was planned from January 2009 to June 2009. Initially we used a laparoscopic approach for the evaluation of fluorescence imaging of the biliary system in the two pigs. Then the clinical study was started on the basis of these experimental results. ICG (1.0 ml/body of 2.5 mg/ml ICG) was infused 1-2 h before surgery. With the subjects under general anesthesia we observed in real time the condition of the biliary tract under the guidance of fluorescence imaging employing an infrared camera or a prototype laparoscope. ICG was added intravenously to observe the location or flow condition of the cystic artery.
We obtained a clear view of the biliary tract and the location of the cystic duct in the two pigs. Local compression with a transparent hemispherical plastic device was effective for offering a clearer view. The biliary tract, except for the gallbladder, was clearly recognized in all clinical subjects. Local compression with a transparent hemispherical plastic device for open cholecystectomy and a flat plastic device for laparoscopy provided clearer visualization of the confluence between the cystic duct and common bile duct or common hepatic duct. The location of the cystic artery was revealed after division of the connective tissues, and the flow condition of the cystic artery was confirmed 7-10 s after intravenous re-infusion of ICG. There were no adverse events related to the intraoperative procedure or the ICG itself.
This method is safe and easy for the identification of the biliary anatomy, without requiring cannulation into the cystic duct, X-ray equipment, or the use of radioactive materials. Although fluorescence imaging is still at an early stage of application in comparison with ordinary intraoperative cholangiography, we expect that this method will become routine, offering a lower degree of invasiveness that will help avoid bile duct injury.
Accurate diagnosis of metastatic lymph nodes (LNs) is essential in choosing appropriate treatment for gastrointestinal carcinoma. Our aim was to evaluate the diagnostic power of 5-aminolevulinic acid (5-ALA) for LN metastasis in mouse rectal cancer. Colorectal cancer cell lines, isolated cells from normal LNs, and orthotopic mouse model incorporating enhanced green fluorescent protein-tagged and untagged human rectal cancer cells were studied after 5-ALA administration by using confocal microscopy, fluorescence stereomicroscopy, fluorescence lifetime imaging microscopy (FLIM), multichannel spectrophotometry and macroconfocal imaging system to precisely detect LN metastases. In vitro confocal microscopic analyses showed that all colorectal cancer cell lines tested were positive for 5-ALA-induced fluorescence, whereas isolated normal LN cells were negative. 5-ALA-induced protoporphyrin IX (PPIX) fluorescence, verified by FLIM and multichannel spectrophotometry, revealed LN metastases in mice-bearing human rectal cancer cells. Occult LN metastases, unrecognized on white-light imaging and simplified hematoxylin-eosin analyses, were readily detectable on 5-ALA-induced PPIX fluorescence imaging. In vivo macroconfocal images clearly revealed PPIX-fluorescence-positive cancer cells in draining lymph vessels and nodes. Together with specific speckled patterns of PPIX-fluorescence in metastatic lesions, the PPIX-fluorescence intensity ratio of metastatic and nonmetastatic lesions discriminated metastasis with 100% sensitivity and 100% specificity in excised whole LN samples. These results show that fluorescence diagnosis with 5-ALA is very accurate in the detection of LN micrometastases of mouse rectal cancer, suggesting that this feasible diagnostic approach is applicable to target sectioning of metastases of resected fresh whole node samples in pathology laboratories. (c) 2009 UICC.
Author Contributions:Study concept and design: Ishizawa and Kokudo. Acquisition of data: Ishizawa and Bandai. Analysis and interpretation of data: Ishizawa. Drafting of the manuscript: Ishizawa. Critical revision of the manuscript for important intellectual content: Bandai and Kokudo. Obtained funding: Ishizawa and Kokudo. Administrative, technical, and material support: Bandai. Study supervision: Bandai and Kokudo.
We have often encountered difficulties in identifying small liver cancers during surgery. Fluorescent imaging using indocyanine green (ICG) has the potential to detect liver cancers through the visualization of the disordered biliary excretion of ICG in cancer tissues and noncancerous liver tissues compressed by the tumor.
ICG had been intravenously injected for a routine liver function test in 37 patients with hepatocellular carcinoma (HCC) and 12 patients with metastasis of colorectal carcinoma (CRC) before liver resection. Surgical specimens were investigated using a near-infrared light camera system. Among the 49 subjects, the 26 patients examined during the latter period of the study (20 with HCC and 6 with metastasis) underwent ICG-fluorescent imaging of the liver surfaces before resection.
ICG-fluorescent imaging identified all of the microscopically confirmed HCCs (n = 63) and CRC metastases (n = 28) in surgical specimens. Among the 63 HCCs, 8 tumors (13%, including 5 early HCCs) were not evident grossly unless observed by ICG-fluorescent imaging. Five false-positive nodules (4 large regenerative nodules and 1 bile duct proliferation) were identified among the fluorescent lesions. Well-differentiated HCCs appeared as uniformly fluorescing lesions with higher lesion-to-liver contrast than that of moderately or poorly differentiated HCCs (162.6 [71.1-218.2] per pixel vs 67.7 [-6.3-211.2] per pixel, P < .001), while CRC metastases were delineated as rim-fluorescing lesions. Fluorescent microscopy confirmed that fluorescence originated in the cytoplasm and pseudoglands of HCC cells and in the noncancerous liver parenchyma surrounding metastases. ICG-fluorescent imaging before resection identified 21 of the 41 HCCs (51%) and all of the 16 metastases that were examined.
ICG-fluorescent imaging enables the highly sensitive identification of small and grossly unidentifiable liver cancers in real time, enhancing the accuracy of liver resection and operative staging.
The clear delineation between tumor and normal tissue is ideal for real-time surgical navigation imaging. We present a novel indocyanine green (ICG) fluorescence imaging technique to visualize hepatocellular carcinoma (HCC).
Ten patients with solitary HCC underwent hepatectomy between February and September 2007 at Osaka Medical Center for Cancer and Cardiovascular Diseases. ICG had been injected intravenously several days before surgery at a dose of 0.5 mg/kg body weight. After laparotomy, the liver was inspected with intraoperative ultrasonography (IOUS), and then with a near-infrared (NIR) fluorescence imaging system (PDE; Hamamatsu Photonics K.K. Hamamatsu, Japan).
All the 10 primary tumors showed bright fluorescent signals and could be completely removed with negative margins under the guide of PDE. In four cases (40.0%), new HCC nodules that were not detected by use of any preoperative examinations including IOUS were detected by PDE. These newly identified HCC nodules were very small in size and most of the tumors were well-differentiated HCCs.
This novel technique is simple and safe, and is therefore considered to be a promising tool for routine intraoperative imaging during a hepatic resection and further clinical exploration for HCC.
Near-infrared (NIR) fluorophores have several advantages over visible fluorophores, including improved tissue penetration and lower autofluorescence; however, only indocyanine green (ICG) is clinically approved. Its use in molecular imaging probes is limited because it loses its fluorescence after protein binding. This property can be harnessed to create an activatable NIR probe. After cell binding and internalization, ICG dissociates from the targeting antibody, thus activating fluorescence. ICG was conjugated to the antibodies daclizumab (Dac), trastuzumab (Tra), or panitumumab (Pan). The conjugates had almost no fluorescence in PBS but became fluorescent after SDS and 2-mercaptoethanol, with a quenching capacity of 10-fold for 1:1 conjugates and 40- to 50-fold for 1:5 conjugates. In vitro microscopy showed activation within the endolysosomes in target cells. In vivo imaging in mice showed that CD25-expressing tumors were specifically visualized with Dac-ICG. Furthermore, tumors overexpressing HER1 and HER2 were successfully characterized in vivo by using Pan-ICG(1:5) and Tra-ICG(1:5), respectively. Thus, we have developed an activatable NIR optical probe that "switches on" only in target cells. Because both the antibody and the fluorophore are Food and Drug Administration approved, the likelihood of clinical translation is improved.
One of the major complications encountered in hepatobiliary surgery is the incidence of bile duct and blood vessel injuries. It is sometimes difficult during surgery to evaluate the local anatomy corresponding to hepatic arteries and bile ducts. We investigated the potential utility of an infrared camera system as a tool for evaluating local anatomy during hepatobiliary surgery.
An infrared camera system was used to detect indocyanine green fluorescence in vitro. We also employed this system for the intraoperative fluorescence imaging of the arteries and biliary system in a pig. Further, we evaluated blood flow in the hepatic artery, portal vein, and liver parenchyma during a human liver transplant and we investigated local anatomy in patients undergoing cholecystectomy.
Fluorescence confirmed that indocyanine green was distributed in serum and bile. In the pig study, we confirmed the fluorescence of the biliary system for more than 1 h. In the liver transplant recipient, blood flow in the hepatic artery and portal vein was confirmed around the anastomosis. In most of the patients undergoing cholecystectomy, fluorescence was observed in the gallbladder, cystic and common bile ducts, and hepatic and cystic arteries.
Intraoperative fluorescence imaging in hepatobiliary surgery facilitates better understanding of the anatomy of arteries, the portal vein, and bile ducts.
Methods have been sought for the in vivo marking of tiny papillary tumors of the bladder and flat urothelial lesions such as dysplasia or carcinoma in situ, which can easily be missed during conventional endoscopy under white light. A new procedure is reported for the fluorescence detection of urothelial dysplasia and early bladder cancer. The method is based on intravesical application of 5-aminolevulinic acid (ALA). ALA if applied exogenously induces accumulation of protoporphyrin IX (PPIX) in the urothelium of the bladder. PPIX is an intensively red fluorescing agent. The mean ratio of fluorescence intensity between urothelial cancer and normal epithelium was found to be 17:1. Fluorescence excitation was achieved by violet light from a krypton ion laser (lambda = 406.7 nm) or from a xenon arc lamp with a bandpass filter system (lambda = 375-440 nm). Both light sources proved to be of equal suitability for fluorescence excitation. Fluorescence microscopy revealed that the PPIX fluorescence is strictly limited to the urothelium. It could not be detected from the submucosa or muscle of the bladder. Bladder wall biopsies were taken from 90 patients with suspicion of bladder cancer under fluorescence view. The fluorescence detection proved to be of high sensitivity (98%). No serious side effects which would preclude further clinical testing, especially no cutaneous photoreaction, were observed. Tumor-associated fluorescence induced by topical ALA application offers new perspectives in the diagnosis and treatment of bladder cancer. In case of suspicious or positive urine cytologic findings, ALA fluorescence cystoscopy may be useful for detecting the precise site of the malignancy. The procedure might be helpful in complete resection or coagulation of urothelial neoplasms. Due to this, diminishing recurrence rates are expected. However, this hypothesis has to be studied in prospective clinical trials.
Protoporphyrin accumulates in tissues after administration of delta-aminolevulinic acid, and can be used as a photosensitizer for photodynamic therapy. To determine the distribution of porphyrins in a large animal model after administration of this porphyrin precursor, delta-aminolevulinic acid was administered to anesthetized dogs (100 mg/kg body weight intravenously) and porphyrin concentrations were measured in tissues (liver, pancreas, prostate, bladder, muscle and skin), plasma and urine for 6-10 h. Porphyrins increased markedly (up to 50-fold) in plasma within 1 h, were still markedly increased at 8 h, and consisted mostly of coproporphyrin III and protoporphyrin. Tissue porphyrin concentrations increased more slowly, were highest in liver, pancreas and prostate 7-10 h after delta-aminolevulinic acid administration, and were predominantly protoporphyrin. Maximum porphyrin concentrations in liver were 3- and 4-fold higher than in pancreas and prostate, respectively. Urinary delta-aminolevulinic acid excretion increased and was greatest 2-4 h after dosing; urinary porphobilinogen and porphyrins increased more gradually and remained increased up to at least 8 h. Coproporphyrin III was the predominant porphyrin in urine at all times, but hepta-, hexa- and pentacarboxyl porphyrins increased proportionally after administration of delta-aminolevulinic acid. These results indicate that porphyrins accumulate in plasma as well as tissues and urine after administration of delta-aminolevulinic acid, and may contribute to tumor necrosis during photodynamic therapy.
Improvements in percutaneous catheter interventions and new technical demands in the practice of coronary surgery have increased the need for an accurate and easy-to-use imaging modality for validating the quality of bypass grafts in the operating room. This report examines the initial clinical use of fluorescent cardiac imaging, a technology that uses indocyanine green (ICG) with a portable imaging device to visualize coronary anatomy and grafts intraoperatively.
The modality was evaluated at two institutions in 20 patients undergoing non-emergent CABG or MIDCAB with respect to safety, feasibility of use, and image quality. Images were generated and acquired with a portable laser diode/infrared camera device after injection of 0.5 ml of ICG (0.5-5.0 mg/ml) either intravenously, via the antegrade cardioplegia cannula, or via the cardiopulmonary bypass circuit.
There were no ICG- or imaging device-related complications. The technology was easy-to-use during conventional CABG as well as MIDCAB and adequately demonstrated coronary anatomy, filling of the grafts, and graft patency in all but two patients. In one patient, the use of the modality resulted in the intraoperative recognition and revision of a non-functioning graft.
This technology is user-friendly in the operating room, appears to be safe, provides good-quality images of coronary anatomy and grafts, and holds promise as an intraoperative graft validation tool for conventional and minimally invasive CABG.
Sentinel lymph node biopsy using a vital dye is a convenient and safe method to assess lymph node status in breast cancer. However, intensive training is necessary to obtain a satisfactory detection rate and to avoid false-negative results. This paper presents a novel method using indocyanine green fluorescence imaging to detect sentinel lymph nodes.
Fluorescence images were obtained using a charge coupled device camera with a cut filter as the detector, and light emitting diodes at 760 nm as the light source. When indocyanine green was injected around the areola, subcutaneous lymphatic channels draining from the areola to the axilla were visible by fluorescence within a few minutes. The sentinel lymph node was then dissected by fluorescence navigation.
Sentinel lymph node biopsy using the present method was performed on eighteen patients. Subcutaneous lymphatics were detectable by fluorescence in all patients, and sentinel nodes were successfully identified in 17 of 18 cases (detection rate:94%). It was possible to detect the lymphatic channels and nodes receiving indocyanine green with higher sensitivity by the fluorescence signal than by the green color.
Sentinel node biopsy guided by indocyanine green fluorescence imaging is a promising technique for further clinical exploration.
The expression of gamma-glutamyltransferase (GGT), a cell surface enzyme involved in cellular glutathione homeostasis, is often significantly increased in human tumors, and its role in tumor progression, invasion and drug resistance has been repeatedly suggested. As GGT participates in the metabolism of cellular glutathione, its activity has been mostly regarded as a factor in reconsitution of cellular antioxidant/antitoxic defences. On this basis, an involvement of GGT expression in resistance of cancer cells to cytotoxic drugs (in particular, cisplatin and other electrophilic agents) has been envisaged. Mechanistic aspects of GGT involvement in antitumor pharmacology deserve however further investigations. Recent evidence points to a more complex role of GGT in modulation of redox equilibria, with effects acting both intracellularly and in the extracellular microenvironment. Indications exist that the protective effects of GGT may be independent of intracellular glutathione, and derive rather from processes taking place at extracellular level and involving reactions of electrophilic drugs with thiol metabolites originating from GGT-mediated cleavage of extracellular glutathione. Although expression of GGT cannot be regarded as a general mechanism of resistance, the involvement of this enzyme in modulation of redox metabolism is expected to have impact in cellular response to several cytotoxic agents. The present commentary is a survey of data concerning the role of GGT in tumor cell biology and the mechanisms of its potential involvement in tumor drug resistance.
The authors prospectively compared a new technique of surgical microscope-based indocyanine green (ICG) videoangiography with intraoperative or postoperative digital subtraction (DS) angiography.
The technique was performed during 187 surgical procedures in which 124 aneurysms in 114 patients were clipped. Using a newly developed setup, the ICG technique has been integrated into an operating microscope (Carl Zeiss Co., Oberkochen, Germany). A microscope-integrated light source containing infrared excitation light illuminates the operating field. The dye is injected intravenously into the patient, and intravascular fluorescence from within the blood vessels is imaged using a video camera attached to the microscope. The patency of parent, branching, and perforating arteries and documentation of clip occlusion of the aneurysm as shown by ICG videoangiography were compared with intraoperative or postoperative findings on DS angiography. The results of ICG videoangiography corresponded with intra- or postoperative DS angiography in 90% of cases. The ICG technique missed mild but hemodynamically irrelevant stenosis that was evident on DS angiography in 7.3% of cases. The ICG technique missed angiographically relevant findings in three cases (one hemodynamically relevant stenosis and two residual aneurysm necks [2.7% of cases]). In two cases the missed findings were clinically and surgically inconsequential; in the third case, a 4-mm residual neck may require a second procedure. Indocyanine green videoangiography provided significant information for the surgeon in 9% of cases, most of which led to clip correction.
Microscope-based ICG videoangiography is simple and provides real-time information about the patency of vessels of all sizes and about the aneurysm sac. This technique may be useful during routine aneurysm surgery as an independent form of angiography or as an adjunct to intra- or postoperative DS angiography.
In living-donor liver transplantation (LDLT), hepatic arterial thrombosis and portal venous thrombosis are critical problems that can result in graft loss. Only intraoperative Doppler ultrasound (IDUS) is able to evaluate blood flow in the reconstructed vessels. The aim of this study was to evaluate the utility of a newly developed fluorescence imaging technique using indocyanine green (ICG) for visualizing reconstructed vessels.
In three patients who had undergone LDLT, IDUS was performed after reconstruction of the portal vein and hepatic artery. Fluorescence images were then recorded, using a SPY system (Novadeq Technologies), which employs ICG as a fluorescent imaging medium activated by light. The ICG (3.75 mg) was injected intravenously, then, 10 s later, the images were recorded for 30 s (first photographic recording). Two minutes later, the same procedure was repeated (second photographic recording), and 40 min later, images were obtained without injection of ICG (third photographic recording).
After portal venous reconstruction, IDUS demonstrated a nonphasic and continuous waveform, with a mean velocity of 52.1 cm/s and a mean portal blood flow volume of 69.5 ml/s per kg. After hepatic arterial reconstruction, a pulsatile waveform with a mean peak systolic velocity of 52.4 cm/s and a mean resistance index of 0.76 was obtained. The first photographic recording clearly visualized the blood flow in the reconstructed hepatic artery, without kinking or stenosis, in all three patients. The second photographic recording visualized the flow in the portal vein without stenosis, kinking, or stagnation. The third photographic recording demonstrated the excretion of ICG into bile, thus confirming bile production by the grafts.
Fluorescence imaging can clearly visualize the reconstructed hepatic artery and portal vein and demonstrate the production of bile by a transplanted liver graft. A combination of IDUS and the new system can guarantee the patency of the reconstructed vessels.
Lymphedema is known to be caused by many pathologic conditions; however, its diagnostic and therapeutic strategies remain to be unestablished. In this study, we investigated the usefulness of a novel lymphographic method based on fluorometric sensing using indocyanine green (ICG) dye for imaging lymphatic vessels using rat models. The real-time imaging system enabled visualization of superficial lymphatic vessels with a diameter of 0.1 mm in 33 frames/second. In addition, morphologic changes in lymphatic vessels in a radiation-induced lymphedema model were detected even at the latent stage. These results suggest that this imaging technique is acceptable as an evaluation method for the lymphatic system.