Autopsy and postmortem investigations

A complete autopsy was performed 72 hours after death. Vomit and gastric content were taken for analysis. To stop the residual proteolytic activity of pepsin, a portion of the gastric content was neutralised (to pH 7) with sodium hydroxide. All the biological samples were maintained at −20°C until the analysis. During autopsy, specimens from all organs were taken and fixed in formalin and embedded in paraffin. Histological sections were stained with H&E. In addition, immunohistochemical investigation of lung samples was performed using antitryptase antibody. We used 4 µm thick paraffin sections mounted on slides covered with 3-amminopropyl-triethoxysilane (Fluka, Buchs, Switzerland). Pre-treatment was necessary to facilitate antigen retrieval and to increase membrane permeability to antibodies: for fibrinogen, enzymatic digestion with Proteinase K in 20 mM Tris–HCl, pH 8.0 for 15 min (temperature 20°C); for tryptase, enzymatic digestion with proteolytic enzyme (Dako, Copenhagen, Denmark) for 5 min (temperature 20°C). The primary antibody was applied in a ratio of 1:1000 for tryptase and incubated for 120 min at 20°C. The detection system used was the LSAB+ kit (Dako), a refined avidin–biotin technique in which a biotinylated secondary antibody reacts with several peroxidase-conjugated streptavidin molecules. The positive reaction was visualised by 3,3-diaminobenzidine peroxidation, according to standard methods. The sections …