2.5. Duodenal Biopsy Analysis
atrophy
ATROPHY
The duodenal biopsies were analyzed for histological and ultrastructural changes in both study moments. Histologic analysis was performed using optical microscopy with hematoxylin–eosin staining observed by two pathologists, including one expert gastrointestinal pathologist. All samples were analyzed for the presence of mucosal atrophy according to the adapted Marsh–Oberhuber classification and average duodenal villi length in micrometers, with at least two villi being measured through the use of an automatic scale.For transmission electron microscopy, fragments from the duodenal biopsies were fixed in 3% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) overnight at 4 °C. The fixed fragments were rinsed and post-fixed in 1.0% osmium tetroxide for 1 h in the same buffer and dehydrated in graded ethanol passages. The fragments were embedded in a mixture of Epon and Araldite after two 15-min passages in propylene oxide. Thin sections, cut on a Reichert Ultracut II Ultramicrotome with a diamond knife, were stained on the grid with uranyl acetate and lead citrate and examined with a JEOL 1200EX electron microscope.All patient histologic and ultrastructural changes were compared with the duodenal samples of healthy controls.