Laboratory procedure
The laboratory analyses of the BAL samples shipped by veterinarians from all over Denmark, including cytospin preparations and differential cell counts, were performed by the diagnostic laboratory services at the Large Animal Teaching Hospital in Denmark. The samples were shipped on ice, stored in EDTA tubes, and received by the laboratory within 24 hours. A two-hundred µl aliquot of the BAL EDTA sample was instilled into the funnel of a cytospin (StatSpin® Cytofuge, USA) and centrifuged at 93 g for seven minutes. The cytospin samples were fixed in methanol (ChemSolute®, Th. Geyer, Denmark) and stained with May-Grünwald-Giemsa (Merck, Germany). Slides were examined under light microscopy at 400 x magnification using the five-field counting technique (Fernandez et al., 2013) by experienced observers.
Four-hundred cells were counted and classified as percentages of alveolar macrophages, lymphocytes, neutrophils, mast cells or eosinophils.