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

Abstract

Background and aims: Current methods to diagnose malignant biliary strictures are of low sensitivity. Confocal endomicroscopy is a new approach that may improve the diagnosis of indeterminate biliary strictures. The purpose of this study was to evaluate indeterminate biliary strictures using probe-based confocal laser endomicroscopy and to understand the histologic basis for the confocal images.

Methods: 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.

Results: Earlier published criteria were used to evaluate possible malignancy in the confocal images obtained in the 14 patients. None of the individual criteria were found to be specific enough for malignancy, but a normal-appearing reticular pattern without other putative markers of malignancy was observed in all normal patients. Multiphoton reconstructions of intact rat bile ducts revealed that the reticular pattern seen in normal tissue was in the same focal plane but was smaller than blood vessels. Special stains identified the smaller structures in this network as lymphatics.

Conclusions: Our limited series suggests that a negative confocal imaging study of the biliary tree can be used to rule out carcinoma, but there are frequent false positives using individual earlier published criteria. An abnormal reticular network, which may reflect changes in lymphatics, was never seen in benign strictures. Better correlation with known histologic structures may lead to improved accuracy of diagnoses.

FIGURE 1

Reticular pattern (arrows) in a normal bile duct. This pattern was observed by confocal imaging using the Cholangio-Flex confocal miniprobe.

FIGURE 2

An example of irregular epithelium. The epithelium has lost its normal structure of regular, adjacent cells. This pattern was observed by confocal imaging using the CholangioFlex confocal miniprobe.

FIGURE 3

Normal bile duct epithelium observed by confocal imaging using the CholangioFlex confocal miniprobe.

FIGURE 4

Normal bile duct observed by confocal imaging using the GastroFlex UHD high-definition confocal miniprobe. Note that blood vessels (arrows) can be identified as white (fluorescein-filled) structures that contain red blood cells, which exclude fluorescein. Dark reticular structures (arrowheads) are seen in the same focal plane.

FIGURE 5

Serial optical sections of rat common bile duct examined by multiphoton microscopy. The duct was examined in a freshly isolated, unfixed, and unstained duct. The image is pseudocolored to reveal emission signals detected at 320 to 380 nm (blue), 410 to 490 nm (green), and 510 to 650 nm (red). A, Surface section reveals confluent epithelia with interspersed pits (green). B, Section collected approximately 25 μm beneath the luminal surface reveals glands (green) that extend from the pits, surrounded by connective tissue (blue). C, Section collected approximately 50 μm beneath the luminal surface reveals termination of the glands (green), surrounded by connective tissue (blue). At this depth a vascular network (red) is now visible within the stroma.

FIGURE 6

Histologic sections of rat common bile duct. A, Hematoxylin and eosin-stained section along the length of the duct reveals epithelia lining the lumen with occasional pits that extend into glands surrounded by connective tissue. Vascular structures can be seen at the periphery of the connective tissue. Pancreatic acini surround the duct. B, D2-40 immunostaining of lymphatics (arrows) identifies multiple small structures in the periphery of the connective tissue, at nearly the same depth from the luminal surface as blood vessels.