Enhanced Imaging of the Esophagus: Confocal Laser Endomicroscopy

9.1

Introduction

Barrett’s esophagus (BE) is associated with the risk of development of esophageal adenocarcinoma. Current surveillance according to the Seattle protocol includes white light endoscopy (WLE) with the collection of random four-quadrant biopsy specimens over every 1–2 cm of the columnar-lined esophagus. The aim of such surveillance is detection of neoplasia, ideally at an (endoscopically) curable stage. This state-of-the-art approach is labor intensive and prone to sampling error since only a minor part of the mucosal surface is undergoing microscopic analysis ex vivo. Despite high definition (HD) and virtual or spraying surface contrast enhancement to augment WLE, its ability to reliably detect premalignant lesions remains suboptimal. This is partly based on the fact that high-grade intraepithelial neoplasia (IN) intramucosal cancer in BE is found in a patchy pattern side by side with lower grades of dysplasia and metaplasia.

Endomicroscopy is a technique in which the mucosa is magnified by a confocal scanner. When combined with WLE this technique can be used to highlight areas that are suspicious for dysplastic epithelium. A fluorescent agent is injected intravenously prior to image acquisition and images are viewed at real time during endoscopy. This tool is best used when examining small areas of the esophageal mucosa. Endomicroscopy is fundamentally different from light microscopy: the use of the confocal technique allows microscopy even underneath the tissue surface in intact tissue without the need to physically shine light through thin tissue sections. Such optical sectioning permits magnification to about 1000-fold and reveals subtle structural details of the mucosa on a (sub-)cellular level. Since its first description over 10 years ago, many trials have covered confocal laser endomicroscopy (CLE) in BE. This indicates a clinical need to optimize detection of Barrett’s associated IN and reflects the low confidence of many gastroenterologists in untargeted quadrant biopsies to incidentally pick up preneoplastic lesions. Volumetric laser endomicroscopy (VLE) shows overlap in naming and indication but relies on optical coherence tomography, a technically different cross-sectional imaging technique that is covered in Chapter 8 : “Enhanced Imaging of the Esophagus: Optical Coherence Tomography.”

9.2

Confocal Endomicroscopy Devices

Two CLE systems are currently used in clinical practice, an endoscope-based system (eCLE; Pentax, Tokyo, Japan) and a probe-based system (pCLE; Mauna Kea Technologies, Paris, France) . Both are point techniques that achieve very high resolution of a small mucosal area.

In eCLE, that is not marketed at this point (early 2015) but still available in many endoscopy suites, a miniaturized confocal scanning device is integrated into the distal tip of a standard resolution endoscope. The tip of the scanner protrudes slightly and is visible in the endoscopic image at the seven o’clock position so that it can be placed onto the point of interest under endoscopic guidance. The free working channel can be used for labeling, targeted biopsies, or other interventions. The scanning mechanism relies on a single optical fiber working as a pinhole, mounted into a resonant magnetic tuning fork that scans the area at high resolution at two different speeds. Lateral resolution is 1024×1024 pixels (~0.7 µm) at a frame rate of approximately 0.6 s ⁻¹ or 512×1024 pixels at 1.2 s ⁻¹ . The depth of imaging can be actuated by the user at 7 µm steps from surface to about 200 µm.

In probe-based CLE (pCLE), the confocal probe is fitted through the working channel of any gastrointestinal endoscope. This carries with it the advantage of using it with different types of endoscopes, including state-of-the-art HD scopes. The probes are available with different diameters that even allow use within the bile duct or through an FNA needle during endoscopic ultrasonography (which are outside the focus of this review). The probes use a fiber bundle for laser light propagation and collection of fluorescence. The imaging plane is fixed for the different probe types and can be somewhat adapted by exerting different extent of pressure with the probe. Most trials have used the Gastroflex UHD CLE probe. Some authors use a short transparent cap on the endoscope to facilitate stabilization of the probe tip on a region of interest. Resolution of pCLE is lower than with eCLE, but image acquisition faster, providing microscopic video sequences at real time.

In both systems, the excitation wavelength is 488 nm (blue light), and emitted light is captured in the green range. Imaging relies on the application of a fluorescent agent. In most trials, fluorescein is injected intravenously at 2.5–5 mL of a 10% solution. Fluorescent contrast is available for tissue imaging after few seconds. Fluorescein is partly bound by plasma proteins and also extravasates into the tissue. As a result, the tissue structure becomes visible almost immediately with good resolution of the mucosal structures and the capillaries of the lamina propria. Nuclei are not discernible, but structural information of the mucosa is usually sufficient as a basis for a therapeutic decision (see later). Fluorescein is safe, and adverse events are rare . While some early trials have also used topical acriflavine (which visualizes nuclei) , this is largely abandoned due to a theoretical risk of harboring mutagenic effects of the nuclear staining. Cresyl violet results in an indirect visualization of nuclei but has only been evaluated in small trials .

In CLE, the resultant image on the screen is parallel to the tissue surface, ie, perpendicular to sectioning in conventional histopathology. Interpretation of the microscopic images is usually performed online in order to make use of the advantage of having a microscopic tissue analysis available during the endoscopic session. This requires a thorough knowledge of the mucosal histopathology by the endoscopist. For starting CLE in the endoscopy unit, it might be beneficial to ask a histopathologist into the room for the first CLE sessions, however, this is not mandatory. Usually, the endoscopist bases his microscopic diagnosis on a two-step decision: the first being the differentiation of normal versus abnormal (pattern recognition) and the second being appreciation of the fine suspicious alterations in abnormal tissue (detail description). This will be explained later for Barrett’s esophagus and associated neoplasia in more detail.