Benjamin L Wright1,2, Alfred D Doyle3, Kelly P Shim3,4, Rish K Pai5, Suzanne M Barshow3,4,6, Jennifer L Horsley-Silva7, Huijun Luo3, Matthew A Rank3,4, Elizabeth A Jacobsen3,8, David A Katzka9, Hirohito Kita3,8, Evan S Dellon10. 1. Division of Allergy, Asthma, and Clinical Immunology, Department of Medicine, Mayo Clinic, 13400 E Shea Blvd., Scottsdale, AZ, 85259, USA. wright.benjamin@mayo.edu. 2. Division of Pulmonology, Department of Pediatrics, Phoenix Children's Hospital, 1919 E Thomas Rd, Phoenix, AZ, 85016, USA. wright.benjamin@mayo.edu. 3. Division of Allergy, Asthma, and Clinical Immunology, Department of Medicine, Mayo Clinic, 13400 E Shea Blvd., Scottsdale, AZ, 85259, USA. 4. Division of Pulmonology, Department of Pediatrics, Phoenix Children's Hospital, 1919 E Thomas Rd, Phoenix, AZ, 85016, USA. 5. Department of Laboratory Medicine and Pathology, Mayo Clinic, 13400 E Shea Blvd., Scottsdale, AZ, 85259, USA. 6. Division of Pediatric Allergy, Immunology & Pulmonary Medicine, Duke University Medical Center, DUMC 2644, Durham, NC, 27710, USA. 7. Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, 13400 E Shea Blvd., Scottsdale, AZ, 85259, USA. 8. Department of Immunology, Mayo Clinic, 13400 E Shea Blvd., Scottsdale, AZ, 85259, USA. 9. Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, 200 1st St. SW, Rochester, MN, 55905, USA. 10. Division of Gastroenterology and Hepatology, Department of Medicine, University of North Carolina School of Medicine, 4140 Bioinformatics Building, Campus Box 7080, Chapel Hill, NC, 27599, USA.
Abstract
BACKGROUND: Diagnosis of eosinophilic esophagitis (EoE) requires manual quantification of tissue eosinophils. Eosinophil peroxidase (EPX) is an eosinophil-specific, cytoplasmic granule protein released during degranulation. AIMS: The objective of this study was to evaluate image analysis of EPX immunohistochemistry as an automated method for histologic diagnosis of EoE. METHODS: We performed a secondary analysis of prospectively collected esophageal biopsies obtained from adult subjects with EoE and controls. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated for peak eosinophils per high power field (eos/hpf). The same slides were de-stained and re-stained to detect EPX for direct comparison. Slides were digitized, and EPX staining area/mm2 was quantified using image analysis. Paired samples were compared for changes in EPX staining in treatment responders and non-responders. RESULTS: Thirty-eight EoE cases and 49 controls were analyzed. Among EoE subjects, matched post-treatment biopsies were available for 21 responders and 10 non-responders. Baseline EPX/mm2 was significantly increased in EoE subjects and decreased in treatment responders. EPX quantification correlated strongly with eos/hpf (r = 0.84, p < 0.0001) and identified EoE subjects with high diagnostic accuracy (AUC 0.95, p < 0.0001). The optimal diagnostic EPX-positive pixel/area threshold was 17,379 EPX/mm2. Several controls (5/49) with < 15 eos/hpf on H&E staining exceeded this cutoff. CONCLUSIONS: EPX/mm2 correlates strongly with eos/hpf, accurately identifies subjects with EoE, and decreases in treatment responders. Automated quantification of intact eosinophils and their degranulation products may enhance pathologic assessment. Future studies are needed to correlate EPX/mm2 with symptoms, endoscopic findings, and esophageal distensibility.
BACKGROUND: Diagnosis of eosinophilic esophagitis (EoE) requires manual quantification of tissue eosinophils. Eosinophil peroxidase (EPX) is an eosinophil-specific, cytoplasmic granule protein released during degranulation. AIMS: The objective of this study was to evaluate image analysis of EPX immunohistochemistry as an automated method for histologic diagnosis of EoE. METHODS: We performed a secondary analysis of prospectively collected esophageal biopsies obtained from adult subjects with EoE and controls. Tissue sections were stained with hematoxylin and eosin (H&E) and evaluated for peak eosinophils per high power field (eos/hpf). The same slides were de-stained and re-stained to detect EPX for direct comparison. Slides were digitized, and EPX staining area/mm2 was quantified using image analysis. Paired samples were compared for changes in EPX staining in treatment responders and non-responders. RESULTS: Thirty-eight EoE cases and 49 controls were analyzed. Among EoE subjects, matched post-treatment biopsies were available for 21 responders and 10 non-responders. Baseline EPX/mm2 was significantly increased in EoE subjects and decreased in treatment responders. EPX quantification correlated strongly with eos/hpf (r = 0.84, p < 0.0001) and identified EoE subjects with high diagnostic accuracy (AUC 0.95, p < 0.0001). The optimal diagnostic EPX-positive pixel/area threshold was 17,379 EPX/mm2. Several controls (5/49) with < 15 eos/hpf on H&E staining exceeded this cutoff. CONCLUSIONS: EPX/mm2 correlates strongly with eos/hpf, accurately identifies subjects with EoE, and decreases in treatment responders. Automated quantification of intact eosinophils and their degranulation products may enhance pathologic assessment. Future studies are needed to correlate EPX/mm2 with symptoms, endoscopic findings, and esophageal distensibility.
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