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Coeliac disease is characterised by inflammation of small intestinal mucosa accompanied by abnormal villous architecture. It is now accepted that some patients with positive coeliac serology tests may have minor mucosal lesions that may not be apparent on routine histopathological analysis. The aim of the study was to perform detailed examination of enterocyte morphology and cytoskeletal structures using a high content analysis technology. Duodenal biopsies from 14 untreated and 10 treated coeliac patients and from 20 non-coeliac controls were examined. Tissue sections from six patients (study group subjects) before and after the development of gluten-sensitive enteropathy were also investigated. Immunohistochemical studies were performed on paraffin-embedded sections using an anti-α-tubulin antibody. Significant differences in enterocyte morphology and intracellular cytoskeletal structures were demonstrated in patients with proven coeliac disease and in the study group subjects. These changes were present in study group biopsies before evidence of enteropathy, as assessed by routine microscopy. This is the first study to demonstrate detailed characteristics of enterocyte morphology in coeliac patients using a high content analysis approach. The use of this technology allows a quantitative analysis of enterocyte intracellular structures from routine biopsy material and permits detection of subtle changes that precede the characteristic histological lesion.