A Sutureless Vascular Closure Device for Emergent Bovine Xenograft Implantation.

A novel vascular staple (C-staple) was developed that does not enter the vasculature lumen during anastomoses. The objective of this study was to demonstrate C-staple safety when used with a bovine xenograft and compare efficacy of the C-staple procedure with Anastoclip surgical clips or suturing when used with a bovine xenograft. Eight sheep had an acute comparison between suturing and C-staples using both common carotid arteries. Sixteen sheep had xenograft placement in the left carotid artery, eight with C-staples and eight with Anastoclips in a chronic study. Over 6 months, Doppler ultrasound interrogation of the common carotid arteries was performed. After 6 months, arteries were evaluated histopathologically. Cross-clamp and surgical times were longer in the C-staple group than the suture group, and xenograft implantation times were statistically longer with C-staples than with Anastoclips. After 6 months, C-staple biocompatibility was similar to Anastoclips. Patency and hemodynamics of the bovine xenograft were not statistically different between the two groups. C-staples performed as well as the Anastoclips except for implant times, likely due to delivery system differences. Histological findings and clinical outcomes were no different with the two devices. Further refinements of the C-staple delivery system are necessary before proceeding to clinical trials.

Preclinical characterization and validation of a dual-labeled trastuzumab-based imaging agent for diagnosing breast cancer.

OBJECTIVE: The combination of both nuclear and fluorescent reporters provides unique opportunities for noninvasive nuclear imaging with subsequent fluorescence image-guided resection and pathology. Our objective was to synthesize and optimize a dual-labeled trastuzumab-based imaging agent that can be used to validate an optical imaging agent with potential use in identifying tumor metastases in human epidermal growth factor receptor 2 (HER2) positive breast cancer patients. METHODS: [(111)In]-DTPA-trastuzumab-IRDye 800 was synthesized by a three-step procedure. Purity, stability, immunoreactivity, internalization and biodistribution were explored in HER2+ SKBR-3 cells. Biodistribution of [(111)In]-DTPA-trastuzumab-IRDye 800 was performed in a SKBR-3 xenograft model. RESULTS: [(111)In]-DTPA-trastuzumab-IRDye 800 demonstrated high purity by both chemical and fluorometric determinations. Both flow cytometry and the Lindmo assay demonstrated a high binding affinity of [(111)In]-DTPA-trastuzumab-IRDye 800 to HER2-overexpressing cells. The dual-labeled conjugate was stable in PBS, but not in serum after 24 h at 37 °C. Larger molecules (>150 kD) were seen after a 24 h-incubation in human serum. Biodistribution studies revealed tumor-specific accumulation of [(111)In]-DTPA-trastuzumab-IRDye 800 in SKBR-3 tumors, and tumor uptakes at 24 and 48 h were (12.42±1.72)% and (9.96±1.05)%, respectively, following intravenous administration. The tumor-to-muscle ratio was 9.13±1.68 at 24 h, and increased to 12.79±2.13 at 48 h. Liver and kidney showed marked uptake of the dual-labeled imaging agent. CONCLUSIONS: [(111)In]-DTPA-trastuzumab-IRDye 800 is an effective diagnostic biomarker that can be used to validate dual-labeled, molecularly targeted imaging agents and can allow these agents to be translated into clinical practice for identifying HER2+ lesions.

Seeing it through: translational validation of new medical imaging modalities.

Medical imaging is an invaluable tool for diagnosis, surgical guidance, and assessment of treatment efficacy. The Network for Translational Research (NTR) for Optical Imaging consists of four research groups working to "bridge the gap" between lab discovery and clinical use of fluorescence- and photoacoustic-based imaging devices used with imaging biomarkers. While the groups are using different modalities, all the groups face similar challenges when attempting to validate these systems for FDA approval and, ultimately, clinical use. Validation steps taken, as well as future needs, are described here. The group hopes to provide translational validation guidance for itself, as well as other researchers.

Near-infrared fluorescence imaging of lymphatics in head and neck lymphedema.

BACKGROUND: Lymphedema is a complication that may occur after surgical resection and radiation treatment in a number of cancer types and is especially debilitating in regions where treatment options are limited. Although upper and lower extremity lymphedema may be effectively treated with manual lymphatic drainage (MLD) therapies and devices that use compression to direct proximal flow of lymph fluids, head and neck lymphedema is more challenging. METHODS AND RESULTS: Herein, we describe the compassionate use of an investigatory technique of near-infrared (NIR) fluorescence imaging to understand the lymphatic anatomy and function, help direct MLD, and use 3-dimensional (3D) surface profilometry to monitor response to therapy in a patient with head and neck lymphedema after surgery and radiation treatment. CONCLUSION: NIR fluorescence imaging provides a mapping of functional lymph vessels for direction of efficient MLD therapy in the head and neck. Additional studies are needed to assess the efficacy of MLD therapy when directed by NIR fluorescence imaging.

Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging.

OBJECTIVE: To investigate the feasibility of assessing the efficacy of manual lymphatic drainage (MLD), a method for lymphedema (LE) management, by using near-infrared (NIR) fluorescence imaging. DESIGN: Exploratory pilot study. SETTING: Primary care unit. PARTICIPANTS: Subjects (N=10; age, 18-68y) with a diagnosis of grade I or II LE and 12 healthy control subjects (age, 22-59y). INTERVENTION: Indocyanine green (25 µg in 0.1 mL each) was injected intradermally in bilateral arms or legs of subjects. Diffused excitation light illuminated the limbs, and NIR fluorescence images were collected by using custom-built imaging systems. Subjects received MLD therapy, and imaging was performed pre- and posttherapy. MAIN OUTCOME MEASURES: Apparent lymph velocities and periods between lymphatic propulsion events were computed from fluorescence images. Data collected pre- and post-MLD were compared and evaluated for differences. RESULTS: By comparing pre-MLD lymphatic contractile function against post-MLD lymphatic function, results showed that average apparent lymph velocity increased in both the symptomatic (+23%) and asymptomatic (+25%) limbs of subjects with LE and control limbs (+28%) of healthy subjects. The average lymphatic propulsion period decreased in symptomatic (-9%) and asymptomatic (-20%) limbs of subjects with LE, as well as in control limbs (-23%). CONCLUSIONS: We showed that NIR fluorescence imaging could be used to quantify immediate improvement of lymphatic contractile function after MLD.

Assessment of free dye in solutions of dual-labeled antibody conjugates for in vivo molecular imaging.

PURPOSE: Recent preclinical and clinical studies show that dyes that excite and fluoresce in the near-infrared range may be used for tracking and detecting disease targets in vivo. A method for quantifying free dye molecules in antibody conjugate preparations is required for agent batch release and for translation into the clinic. PROCEDURES: Herein, we developed and validated a SDS-PAGE method to determine the percentage of free IRDye 800 CW in (DTPA)(n)-trastuzumab-(IRDye 800)(m) conjugate sample preparations in which high-performance liquid chromatography (HPLC) assessment of free dye was not possible. RESULTS: The SDS-PAGE assay was accurate and valid for free IRDye 800 CW amounts between 38 and 4 mol% of total dye. Gel sample preparation reagent affected the specificity of the assay, and lower and upper limits of quantitation and detection were determined. CONCLUSION: This method may be applicable to other near-infrared dye-conjugated antibody-based imaging agents in which HPLC assessment of purity is not feasible. This validated method for quality assurance will facilitate the translation of dual-labeled antibody conjugates for nuclear and optical imaging.

Subgingival delivery of oral debriding agents: a proof of concept.

OBJECTIVE: This study is a proof of concept to determine the efficacy of a custom-fabricated tray in placing antimicrobial and debriding agents in the periodontal pockets of persons with active gingival infections. Localized subgingival delivery of antimicrobial and antibiotic agents is routinely employed as adjunctive therapy for the treatment and management ofperiopathogens associated with periodontal disease. Because these delivery techniques often face time constraints and impose temporary restrictions on patient brushing and flossing, a custom-formed prescription dental tray can be used to deliver and maintain medications in periodontal pockets between office visits and without brushing or flossing restrictions. The ability of this tray to maintain sufficient concentrations of medication in the periodontal pockets to have a therapeutic effect is evaluated here with theoretical modeling and practical application. METHODS: Hydrogen peroxide is an oral debriding agent and oral wound cleanser with antimicrobial properties. The debriding effect of 1.7% hydrogen peroxide gel was tested in vitro on Streptococcus mutans biofilm using glass carriers for collection. Diffusion modeling tested the potential of the customized tray to place hydrogen peroxide gel into the sulcus in the presence of crevicular fluid flow. Changes in periodontal microflora with scanning electron microscopy analysis of in vivo paper point site sampling were analyzed before and after a thin ribbon of 1.7% hydrogen peroxide gel (approximately 0.7 gm) and a subtherapeutic dose (three drops) of Vibramycin (50 mg/5 ml) were placed via Perio Trays into periodontal pockets, ranging from 4-8 mm at daily prescribed intervals for two to five weeks. RESULTS: In vitro results indicate that 1.7% hydrogen peroxide gel breaks down the exopolysaccharide slime and cell walls ofS. mutans, and begins to debride the cells from glass carriers within 10 minutes. Diffusion modeling indicates that hydrogen peroxide can penetrate into the deeper pockets (9 mm), but also its concentration in these deep pockets will increase over wearing time in the absence of degradation by peroxidases and catalase. Site sampling data confirm diffusion modeling results, with evidence that medication delivered with the prescription tray reduced subgingival bacterial loads and enhanced healing of corresponding oral tissues. CONCLUSION: The prescription Perio Tray effectively placed medication in the gingival sulcus. Mathematical modeling indicated Perio Tray placement of hydrogen peroxide gel in periodontal pockets with depths up to 9 mm over 15 minutes treatment time was theoretically possible. Pathology reports reveal reductions in subgingival bacterial loads and improvements in pretreatment pocket depths of up to 8 mm after 1.7% hydrogen peroxide and Vibramycin Syrup were prescribed for use with the Perio Tray. The in vitro analysis indicating that hydrogen peroxide is the active and effective oral debriding agent needs to be confirmed with additional studies.

Human Lymphatic Architecture and Dynamic Transport Imaged Using Near-infrared Fluorescence.

BACKGROUND: Although the importance of lymphatic function is well recognized, the lack of real-time imaging modalities limits our understanding of its role in many diseases. In a phase 0 exploratory study, we used dynamic, near-infrared (NIR) fluorescence imaging to assess the extremes of lymphatic architecture and transport in healthy human subjects and in subjects clinically diagnosed with unilateral lymphedema (LE), a disease that can be prevalent in cancer survivors. METHODS AND RESULTS: Active lymphatic propulsion was imaged after intradermal injections of 25 µg of indocyanine green (total maximum dose ≤400 µg) bilaterally in the arms or legs of control and subjects. Images show well-defined lymphatic structures with propulsive dye transport in limbs of healthy subjects. In LE subjects, we observed extravascular dye accumulation, networks of fluorescent lymphatic capillaries, and/or tortuous lymphatic vessels in all symptomatic and some asymptomatic limbs. Statistical models indicate that disease status and/or limb significantly affect parameters of apparent lymph propagation velocity and contractile frequency. CONCLUSIONS: These clinical research studies demonstrate the potential of NIR fluorescence imaging as a diagnostic measure of functional lymphatics and as a new tool in translational research studies to decipher the role of the lymphatic system in cancer and other diseases.

Single-dose intravenous toxicity study of IRDye 800CW in Sprague-Dawley rats.

OBJECTIVE: Fluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate. METHODS: Male and female Sprague-Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14 days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes. RESULTS: Based on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity. CONCLUSION: A dose of 20 mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW.

Direct evidence of lymphatic function improvement after advanced pneumatic compression device treatment of lymphedema.

Lymphedema affects up to 50% of all breast cancer survivors. Management with pneumatic compression devices (PCDs) is controversial, owing to the lack of methods to directly assess benefit. This pilot study employed an investigational, near-infrared (NIR) fluorescence imaging technique to evaluate lymphatic response to PCD therapy in normal control and breast cancer-related lymphedema (BCRL) subjects. Lymphatic propulsion rate, apparent lymph velocity, and lymphatic vessel recruitment were measured before, during, and after advanced PCD therapy. Lymphatic function improved in all control subjects and all asymptomatic arms of BCRL subjects. Lymphatic function improved in 4 of 6 BCRL affected arms, improvement defined as proximal movement of dye after therapy. NIR fluorescence lymphatic imaging may be useful to directly evaluate lymphatic response to therapy. These results suggest that PCDs can stimulate lymphatic function and may be an effective method to manage BCRL, warranting future clinical trials.

Near-Infrared Fluorescence Imaging in Humans with Indocyanine Green: A Review and Update.

Near-infrared (NIR) fluorescence imaging clinical studies have been reported in the literature with six different devices that employ various doses of indocyanine green (ICG) as a non-specific contrast agent. To date, clinical applications range from (i) angiography, intraoperative assessment of vessel patency, and tumor/metastasis delineation following intravenous administration of ICG, and (ii) imaging lymphatic architecture and function following subcutaneous and intradermal ICG administration. In the latter case, NIR fluorescence imaging may enable new discoveries associated with lymphatic function due to (i) a unique niche that is not met by any other conventional imaging technology and (ii) its exquisite sensitivity enabling high spatial and temporal resolution. Herein, we (i) review the basics of clinical NIR fluorescence imaging, (ii) survey the literature on clinical application of investigational devices using ICG fluorescent contrast, (iii) provide an update of non-invasive dynamic lymphatic imaging conducted with our FDPM device, and finally, (iv) comment on the future NIR fluorescence imaging for non-invasive and intraoperative use given recent demonstrations showing capabilities for imaging following microdose administration of contrast agent.

Lymphatic imaging in humans with near-infrared fluorescence.

While the lymphatic system is increasingly associated with diseases of prevalence, study of these diseases is difficult owing to the paucity of imaging techniques with the sensitivity and temporal resolution to discriminate lymphatic function. Herein, we review the known, pertinent features of the human lymphatic system in health and disease and set the context for a number of emerging studies that use near-infrared fluorescence imaging to non-invasively assess tumor draining lymphatic basins in cancer patients, intraoperatively guide resection of first draining lymph nodes, and to interrogate the difference between normal and aberrant lymphatic structure and function.

New horizons for imaging lymphatic function.

In this review, we provide a comprehensive summary of noninvasive imaging modalities used clinically for the diagnosis of lymphatic diseases, new imaging agents for assessing lymphatic architecture and cancer status of lymph nodes, and emerging near-infrared (NIR) fluorescent optical imaging technologies and agents for functional lymphatic imaging. Given the promise of NIR optical imaging, we provide example results of functional lymphatic imaging in mice, swine, and humans, showing the ability of this technology to quantify lymph velocity and frequencies of propulsion resulting from the contractility of lymphatic structures.

Imaging of lymph flow in breast cancer patients after microdose administration of a near-infrared fluorophore: feasibility study.

PURPOSE: To prospectively demonstrate the feasibility of using indocyanine green, a near-infrared (NIR) fluorophore at the minimum dose needed for noninvasive optical imaging of lymph nodes (LNs) in breast cancer patients undergoing sentinel lymph node mapping (SLNM). MATERIALS AND METHODS: Informed consent was obtained from 24 women (age range, 30-85 years) who received intradermal subcutaneous injections of 0.31-100 microg indocyanine green in the breast in this IRB-approved, HIPAA-compliant, dose escalation study to find the minimum microdose for imaging. The breast, axilla, and sternum were illuminated with NIR light and the fluorescence generated in the tissue was collected with an NIR-sensitive intensified charged-coupled device. Lymphoscintigraphy was also performed. Resected LNs were evaluated for the presence of radioactivity, blue dye accumulation, and fluorescence. The associations between the resected LNs that were fluorescent and (a) the time elapsed between NIR fluorophore administration and resection and (b) the dosage of NIR fluorophores were tested with the Spearman rank and Pearson product moment correlation tests, respectively. RESULTS: Lymph imaging consistently failed with indocyanine green microdosages between 0.31 and 0.77 microg. When indocyanine green dosages were 10 microg or higher, lymph drainage pathways from the injection site to LNs were imaged in eight of nine women; lymph propulsion was observed in seven of those eight. When propulsion in the breast and axilla regions was present, the mean apparent velocities ranged from 0.08 to 0.32 cm/sec, the time elapsed between "packets" of propelled fluid varied from 14 to 92 seconds. In patients who received 10 microg of indocyanine green or more, a weak negative correlation between the fluorescence status of resected LNs and the time between NIR fluorophore administration and LN resection was found. No statistical association was found between the fluorescence status of resected LNs and the dose of NIR fluorophore. CONCLUSION: NIR fluorescence imaging of lymph function and LNs is feasible in humans at microdoses that would be needed for future molecular imaging of cancer-positive LNs.

The immuno cross-reactivity of gutta percha points.

OBJECTIVE: The purpose of this study is to test the postulated immuno cross-reactivity between proteins derived from raw gutta percha (RGP), gutta percha point (GPP) and natural rubber latex (NRL). METHODS: Antigenicity and cross-reactivity of proteins were determined by the FITkit (FITBiotech, Finland) and ELISA inhibition assays. RESULTS: Antigenicity of proteins derived from RGP or GPP was not demonstrated. Except for NRL glove extracts, neither extracts from RGP or GPP were reactive in ELISA inhibition assay. SIGNIFICANCE: There is no immunologic cross-reactivity in vitro between proteins derived from RGP or GPP, and from NRL gloves. Thus, therapeutic use of GPP is unlikely to initiate adverse immuno-reactivity in individuals previously sensitized to NRL proteins.