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

We studied pathologically and ultrastructurally five patients suspected of having a pandemic S-OIV virus who developed ARF requiring ventilatory support. Nasal swabs for RT-PCR for H1N1 were collected from all patients. The OLBs indicated by the clinicians were carried out after receiving consent from the families. These patients had a severe evolution of the virus and more information about the physiopathology of the disease was required in order to provide adequate treatment. If no improvement of the respiratory status was seen in the patients with ARF after $5 days (defined as no decrease of the Lung Injury Score) an OLB was indicated. 15 Lung tissue sections (4 mm thick), prepared from 10% formalin-fixed, routinely processed, paraffin-embedded blocks, were stained with hematoxylin-eosin. The following methods of histochemical staining were carried out: Grocott's methenamine silver stain, Brown-Brenn, and Ziehl-Neelsen. The following pathological changes were analyzed: a) necrotizing bronchiolitis, b) alveolar collapse, c) dilatation of the airspaces, d) hyaline membrane, e) fibroplasia, f) squamous metaplasia, g) multinucleated cells, h) alveolar hemorrhage, i) acute inflammatory exudates, j) atypical pneumocytes. Pathological changes were graded, using two sections, 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. This semiquantitative analysis is currently routinely used in most studies of the department of pathology of the University of Sã o Paulo Medical School. 16, 17 For immunohistochemistry, the avidin-biotin-peroxidase complex and streptavidin-biotin enzyme complex immunostaining methods were used with antibodies against: lymphocytes CD4 (clone: MO834, dilution 1:1000), CD8 (clone: M7103, dilution 120), CD20 (clone: M755, dilution 140), macrophages-histiocytes CD68 (clone: M814, dilution 130), mouse monoclonal antibodies from DAKO, Carpinteria, CA, USA; S100 (clone: Z311, dilution 11000) rabbit polyclonal antibodies from DAKO; CD1a (clone: MCA1657, dilution 1: 200) mouse monoclonal antibodies from Serotec, Oxford, UK; natural killer, NK (clone: MS136P, dilution 11000) mouse monoclonal antibodies from Neomarkers, Fremont, CA, USA; interleukin 4 (IL-4) (dilution 140), IL-10 (dilution 140) goat polyclonal antibodies from R&D Systems, Minneapolis, MN, USA; IFNc (clone: MAB285, dilution 130), mouse monoclonal antibodies from R&D Systems; tumor necrosis factor alpha (TNFa) (clone: AF210NA, dilution 140) all mouse monoclonal antibodies from R&D Systems; inducible nitric oxide synthase (iNOS) (dilution 1500) polyclonal rabbit from Calbiochem, La Jolla, CA, USA.