BALf was obtained from patients with diffuse lung diseases. All patients gave informed consent to bronchoscopy and sampling of BALf and the study was approved by the Ethics Committee at the Medical University of Warsaw (No. KB/106/2004). Eighty three individuals in total were involved in the study, suffering from sarcoidosis (n = 22, mean age 42 yr., range 26–73 yr., 12 male/10 female), smoking-related interstitial lung diseases (sr-ILD) (n = 11, mean age 53 yr., range 27–77 yr., 8 males/3 females), eosinophilic disorders (n = 8, mean age 53 yr., range 30–69, 3 males/5 females), and lung fibrosis (n = 42, mean age 62 yr., range 30–80 yr., 27 males/15 females). Diagnosis of interstitial lung diseases was established according to ATS/ERS International Multidisciplinary Consensus Classification of the Idiopathic Interstitial Pneumonias . The following diseases were included in the group of eosinophilic disorders: chronic eosinophilic pneumonia (CEP), Churg Strauss Syndrome, and Wegener granulomatosis. The large group of lung fibrosis patients included patients with lung fibrosis in the course of hypersensitivity pneumonitis, with connective diseases, and with idiopathic pulmonary fibrosis (IPF).
At the time of collecting the BAL material none of the patients suffered from any known ongoing infection. BALf was collected according to the guidelines of Polish Respiratory Society , in line with the international guidelines , using 200 mL of saline, and total and differential cell counts were performed. The mean volume of recovery fluid was 100 mL. Cell smears were prepared immediately after the sample had been received at the laboratory, according to the guidelines . After filtration the fluid was centrifuged for 10 min at 400 × g, then the cell pellet was resuspended in PBS. Supernatants were collected in six 5-mL tubes and frozen at −20°C. Total cells were counted using a Bürker chamber whereas differential cell counting was performed on slides stained with the May-Grünwald-Giemsa method. Three hundred cells were used for differential cell counting. The proportion and number of alveolar macrophages (AM), lymphocytes (L), polymorphonuclear granulocytes (PMN) and eosinophils (Eo) were calculated. The smears of all samples were stained with haematoxylin-eosin and interference from any possible malignant cells was excluded. The presence of any abnormal constituents (cells, acellular material, microorganisms) was studied by using light microscopy (magnification × 1000).
BALf supernatants were lyophilized, weighted, and prepared for analysis. In brief, samples were heated in 2 M methanolic HCl at 80°C 18 hours. The liberated fatty acid methyl esters were extracted with heptane and the hydroxylated fatty acid methyl esters were purified by solid phase extraction as previously described . Prior to analysis, the hydroxy fatty acid methyl esters were transformed to trimethylsilyl ethers. Samples thus obtained were kept at 4°C until analysis by gas chromatography-tandem mass spectrometry (GC-MSMS) as described previously . Internal standard was added to quantify the data .
Apart from BALf supernatants we also analyzed BALf cell pellets in five samples from patients with different lung diseases.
The Kruskal-Wallis test was applied for data comparisons, since the data were non-normally distributed, and the Spearman rank test was applied for data correlations. Statistical significance was set at p < 0.05.