Reflectance confocal microscopy for the diagnosis of eosinophilic esophagitis: a pilot study conducted on biopsy specimens

Abstract

Background: Diagnosis of eosinophilic esophagitis (EoE) currently requires endoscopic biopsy and histopathologic analysis of the biopsy specimens to count intraepithelial eosinophils. Reflectance confocal microscopy (RCM) is an endomicroscopy technology that is capable of obtaining high-resolution, optically sectioned images of esophageal mucosa without the administration of exogenous contrast.

Objective: In this study, we investigated the capability of a high-speed form of RCM, termed spectrally encoded confocal microscopy (SECM), to count intraepithelial esophageal eosinophils and characterize other microscopic findings of EoE.

Design: A total of 43 biopsy samples from 35 pediatric patients and 8 biopsy samples from 8 adult patients undergoing EGD for EoE were imaged by SECM immediately after their removal and then processed for routine histopathology. Two SECM readers, trained on adult cases, prospectively counted intraepithelial eosinophils and detected the presence of abscess, degranulation, and basal cell hyperplasia on SECM images from the pediatric patients. A pathologist blinded to the SECM data analyzed the same from corresponding slides.

Setting: The Gastrointestinal Unit, Massachusetts General Hospital.

Results: Eosinophils by SECM demonstrated a higher reflectance than the surrounding cells and other inflammatory cells. There was good correlation between SECM and histology maximum eosinophil counts/high-power field (R = 0.76, P < .0001). Intra- and interobserver correlations for SECM counts were very good (R = 0.93 and R = 0.92, respectively; P < .0001). For the commonly used eosinophil count cutoff of 15 per high-power field, the sensitivity and specificity of SECM for EoE were 100%. The sensitivity and specificity for abscess, degranulation, and basal cell hyperplasia were 100% and 82%, 91% and 60%, and 94% and 80%, respectively. Intra- and interobserver agreements for these microscopic features of EoE were very good (κ = 0.9/0.9, 0.84/1.0, 0.91/0.81, respectively).

Limitation: Ex vivo study.

Conclusions: This study demonstrates that RCM can be used to accurately count intraepithelial eosinophils and identify other microscopic abnormalities associated with EoE on freshly excised biopsy samples. These findings suggest that RCM may be developed into a tool for assessing eosinophilic infiltration in the esophagus in vivo.

Figure 1

Histopathologic and spectrally encoded confocal microscopy (SECM) images from esophageal biopsy of normal squamous mucosa from the mid esophagus of a 20-year-old female patient with a previous diagnosis of eosinophilic esophagitis and currently taking acid reflux medication. A, Histopathologic section (H&E, orig. mag. ×20) shows normal squamous mucosa with no evidence of eosinophils. B, Corresponding SECM image of normal squamous mucosa 80 μm below the surface shows transversely sectioned papillae (yellow arrowheads) at low magnification. C, High-magnification view of the SECM image demonstrates regions of higher reflectance surrounding the papillae. The thickness of this layer was measured to be 20 μm, consistent with the size of basal cells (white opposing arrows).

Figure 2

Images obtained from an 11-year-old male patient with a diagnosis of eosinophilic esophagitis who was on a regular diet. A, Videoendoscopy image demonstrates faint evidence of rings, white plaques, and a slightly diminished vascular pattern. B, Histopathology (H&E, orig. mag. ×20) demonstrates an abundance of eosinophils within the squamous epithelium (>25 eosinophils/high-power field) with evidence of degranulated eosinophils, abscess, and basal cell hyperplasia. Inset, corresponding to yellow dotted box in B shows an eosinophil with a bilobed nucleus. C, SECM, obtained at 50 μm from the surface, demonstrates a diffuse infiltration of highly scattering cells at low magnification. D, A higher magnification view of the yellow dotted box in C and inset shows that these cells are eosinophils with bilobed nuclei.

Figure 3

Spectrally encoded confocal microscopy (SECM) and histopathologic images of biopsy sample acquired from a 6-year-old male patient with eosinophilic esophagitis, demonstrating 1-to-1 cellular level matches. A, Digital histology (H&E, orig. mag. ×20) shows the papillae structure (red asterisks) and intraepithelial eosinophils. B, Expanded view of A shows intact eosinophils (red arrows) as well as eosinophilic cytoplasm fragments. C, D, Color transformation (red/green) of histology in A and B, respectively, allows the eosinophils (red arrows) to be seen more clearly. E, F, Corresponding registered SECM images demonstrate the papillae (green asterisks) and highly reflecting cells (green arrows) that are directly matched to the eosinophils seen by histology. Scale bars = 100 μm.

Figure 4

Representative orthogonally reformatted spectrally encoded confocal microscopy (SECM) images and scatterplot of histology versus SECM eosinophil counts (43 biopsy samples; reader 1). A, Cross-sectional SECM image of biopsy sample acquired from a 3-year-old male patient with no diagnostic abnormality identified by histopathology. B, Cross-sectional SECM image of biopsy sample acquired from a 13-year-old male patient with established eosinophilic esophagitis (EoE). The SECM image shows frequent highly scattering cells, consistent with eosinophils. C, Cross-sectional SECM image of biopsy acquired from a 6-year-old male patient with moderately active EoE. The SECM image demonstrates numerous eosinophils. D, The scatterplot of histology and SECM counts (reader 1) was rendered on the high-power field (550-μm diameter circular region) that contained the maximum eosinophil count. Scale bars = 50 μm.

Figure 5

Representative histology and spectrally encoded confocal microscopy (SECM) images of microscopic features of eosinophilic esophagitis. A, Histology image of eosinophilic abscess shows aggregates of eosinophils in the esophageal epithelium. B, Corresponding SECM image demonstrates a large number of closely spaced eosinophils. C, Histology image of eosinophil degranulation shows stellate eosinophils and extracellular eosinophilic granules. D, Corresponding SECM image demonstrates cells that have an irregular shape and poorly delineated cell boundaries. Extracellular highly scattering granular densities consistent with granules are also seen on the SECM image. E, Histology image of basal cell hyperplasia shows a thickened basal layer and elongated papillae. F, The corresponding SECM image demonstrates numerous eosinophils and a thickened highly reflecting layer surrounding the papillae of the lamina propria (compare with the layer surrounding the papillae seen in the normal esophagus in Figure 1C). The thickness of this layer was measured to be 70 μm (white opposing arrows). Scale bars = 100 μm.