Confocal endomicroscopic examination of malignant biliary strictures and histologic correlation with lymphatics.

Fourteen patients with indeterminate biliary strictures underwent endoscopic retrograde cholangiopancreatography with examination of their common bile duct with fluorescein-aided probe-based confocal laser endomicroscopy. Standard brushings and biopsies were performed. In parallel, rat bile ducts were examined either with conventional staining and light microscopy or with multiphoton microscopy.

MATERIALS AND METHODS Patients

Fourteen consecutive patients referred to the Gastrointestinal Procedure

Center at our institution for ERCP to rule out biliary malignancy were recruited to participate in this study, which was approved by the Yale University Human Investigations Committee. Written informed consent was obtained before study participation. Inclusion criteria included age above 18 years and the ability to provide informed consent. Exclusion criteria included having a known allergy or a prior adverse reaction to fluorescent contrast agents or chromoendoscopy stains; below the age of 18 years or mentally or legally incapacitated or otherwise unable to give informed consent; are pregnant or breastfeeding; coagulopathy; and impaired renal function.

Confocal Imaging

A confocal miniprobe (Cellvizio, Mauna Kea Technologies, Paris, France) was used to examine the biliary mucosa in all cases. All 14 patients undergoing ERCP received up to 10 mL of 0.5% to 10% intravenous fluorescein approximately 2 to 3 minutes before imaging. Biliary strictures and bile ducts were imaged using either a CholangioFlex confocal miniprobe in most patients or a GastroFlex ultra-high definition (UHD) high-definition confocal miniprobe. The CholangioFlex confocal miniprobe is compatible with a 1.2 mm operating channel, has a lateral resolution of 3.5 μm, a depth of focus of 40 to 70 μm, and a field of view of 325 μm. The GastroFlex UHD confocal miniprobe was used in 1 patient. It is compatible with a 2.8 mm working channel, has a lateral resolution of 1 μm, a depth of focus of 55 to 65 μm, and field of view of 240 μm. The CholangioFlex miniprobe was inserted into the common bile duct (CBD) through a Spyglass (Boston Scientific, Natick, MA) catheter or Olympus swing tip catheter. The GastroFlex UHD miniprobe was inserted into the CBD using a freehand technique. The bile duct was cannulated with a standard ERCP catheter which was removed and a 0.035-inch guide wire left in place. The UHD probe was then inserted adjacent to the guidewire.

Histology

Standard biopsy forceps and brushings were used to obtain cytology and tissue from the biliary tree during ERCP when clinically indicated. Biopsy specimens were collected in 10% buffered formalin (Val Tech Diagnostics, PA), were routinely processed, paraffin embedded, and sectioned at 3 μm. They were stained with hematoxylin and eosin (H&E) and examined using standard bright field (white light) microscopy.

Multiphoton Studies of Bile Duct Histology

Fresh, unfixed-rat bile ducts were examined with a multiphoton microscope to correlate confocal images with the corresponding histologic structure. Studies were approved by the Yale Animal Care and Use Committee. Adult male Sprague Dawley rats were anesthetized, subject to laparotomy to expose the inferior vena cava and CBD, and then injected with 1% fluorescein (1 mL/kg) in the inferior vena cava. Three minutes later, the CBD was removed and transferred to a dish containing cold saline and cut open lengthwise to expose the luminal surface, then the animal was euthanized. The duct was then placed in an incubation chamber fitted on the stage of a Zeiss LSM 510 NLO confocal/multiphoton microscope (Thornwood, NY). A mode-locked, femtosecond-pulsed Mai Tai laser (Spectra Physics, Mountain View, CA) tuned to 735 nm was used as a light source, and custom-built nondescanned (external) detectors were used to collect emission images at 320 to 380 nm, 410 to 490 nm, and 510 to 650 nm as described earlier. 15 Immunohistology of Bile Ducts In correlative separate studies of rat bile ducts, whole CBDs were collected in 10% buffered formalin, and then processed, paraffin embedded, and sectioned at 3 μm intervals. Sections were pretreated with 0.01 mol/L citrate buffer, and then endogenous peroxidase activity was blocked by 3% H 2 O 2 in absolute methanol. Each section was preincubated in rabbit serum for 10 minutes, then incubated with lymphatic tissue-specific mouse monoclonal antibody D2-40 (DAKO #M3619; Carpinteria, CA) diluted 1:500 for 60 minutes. Subsequently, sections were rinsed in phosphate-buffered saline, diaminobenzidine was used as a chromogen, and sections were counterstained with H&E and examined using standard bright field (white light) microscopy.