Development of Label-Free Optical Methods for Real-Time Biochemical Evaluation of Pediatric Eosinophilic Esophagitis

Abstract

Eosinophilic esophagitis (EoE) is a chronic immunoinflammatory condition of the esophagus affecting ~1 in 700 people in the United States. Over the past 15 years the disease has increased 5-fold in prevalence and has an annual economic burden of approximately $1.3 billion. Given its chronic and progressive course, the intense eosinophilic inflammation seen in children with EoE can lead to esophageal fibrosis and dysphagia into adulthood if left untreated or mismanaged. Current diagnostic methods require repeated endoscopy with several random biopsies for histopathological confirmation of EoE, which is burdensome and can result in misdiagnosis and diagnostic delays. This doctoral research project aims to develop and optimize label-free optical methods for less-invasive, real-time, and quantitative evaluation of pediatric EoE. To this end, this work focused on applying in vivo Raman spectroscopy (RS), a non-invasive vibrational spectroscopy technique, during standard endoscopy to identify biochemical markers of EoE. In vivo RS revealed decreased carbohydrate, collagen, and lipid content and increased nucleic acid, non-collagen protein, and water content in patients with active EoE (aEoE). These biochemical markers provided 83-89% sensitivity and 83-86% specificity in differentiating aEoE patients from non-EoE controls and inactive EoE patients (treatment responders). To validate and identify the basis of the biochemical alterations observed in EoE, a range of methods were employed to analyze esophageal biopsies ex vivo, including stimulated Raman microscopy, second harmonic generation microscopy, two-photon excitation fluorescence microscopy, gold-standard histology, and histochemistry. Alterations in carbohydrate and nucleic acid content originated from the epithelium, whereas the protein and lipid alterations observed in vivo were caused by changes in subepithelial layers. Upon validating the in vivo RS approach, an optimized scanning endoscopic Raman fiber probe was developed to provide real-time non-contact biochemical mapping of the esophagus, to thoroughly assess the spatial heterogeneity of EoE toward clinical adoption. The scanning Raman probe was validated in esophageal phantoms and biological tissue, both ex vivo and in vivo, demonstrating accurate real-time biochemical mapping. Overall, this work lays the foundation for the clinical translation of Raman endoscopy to reduce the burden and delay in proper diagnosis of EoE.