Rita G. Laurence

Near-infrared fluorescent type II quantum dots for sentinel lymph node mapping

The use of near-infrared or infrared photons is a promising approach for biomedical imaging in living tissue. This technology often requires exogenous contrast agents with combinations of hydrodynamic diameter, absorption, quantum yield and stability that are not possible with conventional organic fluorophores. Here we show that the fluorescence emission of type II quantum dots can be tuned into the near infrared while preserving absorption cross-section, and that a polydentate phosphine coating renders them soluble, disperse and stable in serum. We then demonstrate that these quantum dots allow a major cancer surgery, sentinel lymph node mapping, to be performed in large animals under complete image guidance. Injection of only 400 pmol of near-infrared quantum dots permits sentinel lymph nodes 1 cm deep to be imaged easily in real time using excitation fluence rates of only 5 mW/cm2. Taken together, the chemical, optical and in vivo data presented in this study demonstrate the potential of near-infrared quantum dots for biomedical imaging.

Recovery and Viability of an Acute Myocardial Infarct After Transmyocardial Laser Revascularization

OBJECTIVES The short- and long-term effectiveness of transmyocardial laser revascularization was evaluated in the setting of an acute myocardial infarction. BACKGROUND Theoretically, transmyocardial laser revascularization allows direct perfusion of the ischemic area as ventricular blood flows through the channels to the myocardium. METHODS Infarcts were created by coronary occlusion in 30 sheep. Eighteen of these sheep were studied to assess short-term efficacy. The infarct was reperfused after 1 h by either removing the occlusion or by laser drilling using a high power carbon dioxide laser. The occlusions were left in place for the control group. To monitor regional recovery, percent systolic shortening was measured. To evaluate long-term effectiveness, 12 additional sheep underwent creation of an infarct. Six were treated with the laser, and six were untreated. The animals were restudied 30 days later. RESULTS In the short-term experiment, the control and reperfusion groups exhibited no recovery of regional contractility. The laser group demonstrated improvement throughout the recovery period. There was a significant difference in the area of necrosis within the same area at risk (reperfusion group 44 +/- 6% and control group 39 +/- 5% vs. laser group 6 +/- 2%). After 30 days, none of the control animals showed evidence of contraction in the infarct, whereas the laser-treated animals did. Histologic analysis of the laser-treated infarcts revealed patent channels surrounded by viable myocardium. The control-group infarcts were necrotic and scarred. CONCLUSIONS On the basis of both short- and long-term improved contractility, as well as diminished necrosis in the area at risk, these results indicate that transmyocardial laser revascularization may be an alternative method of treating ischemic heart disease.

Organic alternatives to quantum dots for intraoperative near-infrared fluorescent sentinel lymph node mapping.

Intraoperative near-infrared (NIR) fluorescence imaging provides the surgeon with real-time image guidance during cancer and other surgeries. We have previously reported the use of NIR fluorescent quantum dots (QDs) for sentinel lymph node (SLN) mapping. However, because of concerns over potential toxicity, organic alternatives to QDs will be required for initial clinical studies. We describe a family of 800 nm organic heptamethine indocyanine-based contrast agents for SLN mapping spanning a spectrum from 775 Da small molecules to 7 MDa nanocolloids. We provide a detailed characterization of the optical and physical properties of these contrast agents and discuss the advantages and disadvantages of each. We present robust methods for the covalent conjugation, purification, and characterization of proteins with tetra-sulfonated heptamethine indocyanines, including mass spectroscopic site mapping of highly substituted molecules. One contrast agent, NIR fluorescent human serum albumin (HSA800), emerged as the molecule with the best overall performance with respect to entry to lymphatics, flow to the SLN, retention in the SLN, fluorescence yield and reproducibility. This preclinical study, performed on large animals approaching the size of humans, should serve as a foundation for future clinical studies.

Sentinel Lymph Node Mapping of the Gastrointestinal Tract by Using Invisible Light

BackgroundBecause many gastrointestinal (GI) tumors spread by way of lymphatics, histological assessment of the first draining lymph nodes has both prognostic and therapeutic significance. However, sentinel lymph node mapping of the GI tract by using available techniques is limited by unpredictable drainage patterns, high background signal, and the inability to image lymphatic tracers relative to surgical anatomy in real time. Our goal was to develop a method for patient-specific intraoperative sentinel lymph node mapping of the GI tract by using invisible near-infrared light.MethodsWe developed an intraoperative near-infrared fluorescence imaging system that simultaneously displays surgical anatomy and otherwise invisible near-infrared fluorescence images of the surgical field. Near-infrared fluorescent quantum dots were injected intraparenchymally into the stomach, small bowel, and colon, and draining lymphatic channels and sentinel lymph nodes were visualized. Dissection was performed under real-time image guidance.ResultsIn 10 adult pigs, we demonstrated that 200 pmol of quantum dots quickly and accurately map lymphatic drainage and sentinel lymph nodes. Injection into the mid jejunum and colon results in fluorescence of a single lymph node at the root of the bowel mesentery. Injection into the stomach resulted in identification of a retrogastric node. Histological analysis in all cases confirmed the presence of nodal tissue.ConclusionsWe report the use of invisible near-infrared light for intraoperative sentinel lymph node mapping of the GI tract. This technology overcomes the limitations of currently available methods, permits patient-specific imaging of lymphatic flow and sentinel nodes, and provides highly sensitive, real-time image-guided dissection.

Tissue-like phantoms for near-infrared fluorescence imaging system assessment and the training of surgeons

We demonstrate how to construct calibrated, stable, and inexpensive tissue-like phantoms for near-IR (NIR) fluorescence imaging applications. The bulk phantom material is composed of gelatin, intralipid, hemoglobin, and indocyanine green (ICG). Absorbance, scatter, background fluorescence, and texture can be tuned as desired. NIR fluorescent inclusions are comprised of ICG-labeled polystyrene divinylbenzene beads and Pam78-labeled hydroxyapatite crystals. The former mimic tumor masses of controllable size and contrast agent concentration, and the latter mimic microcalcifications in breast cancer. NIR-fluorescent inclusions can be positioned precisely in phantoms, with one or more regions having different optical properties, and their position can be verified independently using microcomputed tomography. We demonstrate how these phantoms can be used to calibrate and compare imaging systems, and to train surgeons to operate under NIR fluorescence image guidance.

Lymphatic drainage of the peritoneal space: a pattern dependent on bowel lymphatics.

BackgroundUnderstanding lymph drainage patterns of the peritoneum could assist in staging and treatment of gastrointestinal and ovarian malignancies. Sentinel lymph nodes (SLNs) have been identified for solid organs and the pleural space. Our purpose was to determine whether the peritoneal space has a predictable lymph node drainage pattern.MethodsRats received intraperitoneal injections of near-infrared (NIR) fluorescent tracers: namely, quantum dots (designed for retention in SLNs) or human serum albumin conjugated with IRDye800 (HSA800; designed for lymphatic flow beyond the SLN). A custom imaging system detected NIR fluorescence at 10 and 20 minutes and 1, 4, and 24 hours after injection. To determine the contribution of viscera to peritoneal lymphatic flow, additional cohorts received bowel resection before NIR tracer injection. Associations with appropriate controls were assessed with the χ2 test.ResultsQuantum dots drained to the celiac, superior mesenteric, and periportal lymph node groups. HSA800 drained to these same groups at early time points but continued flowing to the mediastinal lymph nodes via the thoracic duct. After bowel resection, both tracers were found in the thoracic, not abdominal, lymph node groups. Additionally, HSA800 was no longer found in the thoracic duct but in the anterior chest wall and diaphragmatic lymphatics.ConclusionsThe peritoneal space drains to the celiac, superior mesenteric, and periportal lymph node groups first. Lymph continues via the thoracic duct to the mediastinal lymph nodes. Bowel lymphatics are a key determinant of peritoneal lymph flow, because bowel resection shifts lymph flow directly to the intrathoracic lymph nodes via chest wall lymphatics.

Real-time intra-operative near-infrared fluorescence identification of the extrahepatic bile ducts using clinically available contrast agents

BACKGROUND Iatrogenic bile duct injuries are serious complications with patient morbidity. We hypothesized that the invisible near-infrared (NIR) fluorescence properties of methylene blue (MB) and indocyanine green (ICG) could be exploited for real-time, intraoperative imaging of the extrahepatic bile ducts during open and laparoscopic surgeries. METHODS In all, 2.0 mg/kg of MB and 0.05 mg/kg of ICG were injected intravenously into 35-kg female Yorkshire pigs and the extrahepatic bile ducts were imaged over time using either the Fluorescence-Assisted Resection and Exploration (FLARE) image-guided surgery system (open surgery) or a custom NIR fluorescence laparoscopy system. Surgical anatomy was confirmed using x-ray cholangiography. The contrast-to-background ratio (CBR), contrast-to-liver ratio (CLR), and chemical concentrations in the cystic duct (CD) and common bile duct (CBD) were measured, and the performance of each agent was quantified. RESULTS Using NIR fluorescence of MB, the CD and CBD could be identified with good sensitivity (CBR and CLR > or =4), during both open and laparoscopic surgeries, from 10 to 120 min postinjection. Functional impairment of the ducts, including constriction and injury were immediately identifiable. Using NIR fluorescence of ICG, extrahepatic bile ducts did not become visible until 90 min postinjection because of strong residual liver retention; however, between 90 and 240 min, ICG provided exquisitely high sensitivity for both CD and CBD, with CBR > or =8 and CLR > or =4. CONCLUSION We demonstrate that 2 clinically available NIR fluorophores, MB fluorescing at 700 nm and ICG fluorescing at 800 nm, provide sensitive, prolonged identification of the extrahepatic bile ducts and assessment of their functional status.

Intraoperative near-infrared fluorescence imaging in perforator flap reconstruction: current research and early clinical experience.

Despite recent advances in perforator flap reconstruction, there can be significant variability in vessel size and location. Although preoperative evaluation may provide valuable information, real-time intraoperative methods have the potential to provide the greatest benefit. Our laboratory has developed the Fluorescence-Assisted Resection and Exploration (FLARE) near-infrared (NIR) fluorescence imaging system for intraoperative visualization of details of the underlying vasculature. The FLARE system uses indocyanine green, a safe and reliable NIR fluorophore already FDA-approved for other indications. The system has been optimized in large-animal models for the identification of perforator size, location, and perfusion and has also been translated to the clinic for use during breast reconstruction after mastectomy. In this article, we review our preclinical and clinical data, as well as literature describing the use of similar NIR fluorescence imaging systems in plastic and reconstructive surgery.

Real-time intraoperative assessment of the extrahepatic bile ducts in rats and pigs using invisible near-infrared fluorescent light

BACKGROUND Currently, only x-ray fluoroscopy is available for visualization of the extrahepatic bile ducts intraoperatively. We hypothesized that with an appropriate fluorophore and imaging system, invisible near-infrared (NIR) light could be used for image-guided procedures on the extrahepatic bile ducts. METHODS We quantified the performance of three 800 nm NIR fluorophores, differing primarily in their degree of hydrophilicity, for real-time imaging of the extrahepatic bile ducts in rats and pigs: IR-786, indocyanine green (ICG), and the carboxylic form of IRDyetrade mark 800CW (CW800-CA). The signal-to-background ratio (SBR) of the common bile duct relative to liver and pancreas was measured as a function of the dose of contrast agent, injection site, and kinetics using an intraoperative NIR fluorescence imaging system described previously. Bile samples were examined by high performance liquid chromatography tandem mass spectrometry (HPLC/MS) to determine the chemical form of fluorophores in bile. RESULTS Non-sulfonated (IR-786) and di-sulfonated (ICG) NIR fluorophores had poor efficiency and kinetics of excretion into bile. Tetra-sulfonated CW800-CA, however, provided sensitive, specific, and real-time visualization of the extrahepatic bile ducts after a single low-dose given either intraportally or intravenously via systemic vein. A SBR >/=2 provided sensitive assessment of extrahepatic bile duct anatomy and function for over 30 min post-injection, including the detection of millimeter-sized, radiolucent inclusions in pigs. CW800-CA remained intact chemically after secretion into bile. CONCLUSION The combination of invisible NIR light and an IV injection of CW800-CA provides prolonged, real-time visualization of the extrahepatic bile duct, without ionizing radiation and without changing the look of the operative field.

Real-Time Simultaneous Near-Infrared Fluorescence Imaging of Bile Duct and Arterial Anatomy

BACKGROUND 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.