Pathological and ultrastructural analysis of surgical lung biopsies in patients with swine-origin influenza type A/H1N1 and acute respiratory failure

Capelozzi, Vera Luiza; Parra, Edwin Roger; Ximenes, Manoel; Bammann, Ricardo Helbert; Barbas, Carmen Silvia Valente; Duarte, Marid Irmd Seixas

Immunohistochemical reactions were carried out in accordance with the manufacturer's instruction. Diaminobenzidine was used as the color substrate, and Meyer's hematoxylin was used for counterstaining. Cell immunophenotypes and immune expression of cells using the different methods of immunohistochemical staining were identified and graded according to a five-point semiquantitative intensity-based scoring system as: 0 = negative, 1 = positive in 1-25%, 2 = positive in 26-50%, 3 = positive in 51-75%, and 4 = positive in 76-100% of examined tissue. 18 Small blocks (1 mm 3 ) of lungs were fixed in 2% glutaraldehyde/2% paraformaldehyde in cacodylate buffer overnight, then fixed in 1% osmium tetroxide, dehydrated, and embedded in araldite. Ultrathin sections obtained from selected areas were double-stained and examined in a Philips TECNAI 10 electron microscope at 80 kV. For each electron microscopy image (15/case), the following structural changes were analyzed: a) cytoplasmic swelling, b) degenerative changes, c) sloughing of necrotizing alveolar epithelial cell type I (AECI) and II (AECII), d) denudation of the epithelial basement membrane, e) hyaline membranes, f) alveolar septal collapse, g) viral particles such as tubuloreticular structures (TRS) and cylindrical confronting cisternae (CCC), h) multinucleated AECII. Ultrastructural findings were graded according to a five-point semiquantitative severity-based scoring system as: 0 = normal lung parenchyma, 1 = changes in 1-25%, 2 = changes in 26-50%, 3 = changes in 51-75%, and 4 = changes in 76-100% of examined tissue. 16, 17