Samples were run in triplicate and compared to a standard curve. In order to ensure that dithiothreitol did not degrade IL-8, we added 100 ng and 500 ng IL-8 for 90 min to each sample from three patients (ie, total of 15 samples) and generated standard curves. Dithiothreitol did not degrade IL-8. Urea concentration was measured at the clinical laboratory of the University of Washington Medical Center. Surfactant protein (SP)-A and SP-D were measured with a standard enzyme-linked immunosorbent assay as reported previously. Human SP-A was measured with a kit using two monoclonal antibodies provided by the Teijin Institute of Bio-Medicine (Hino, Japan). Recombinant SP-D was used as the standard for SP-D. The enzyme-linked immunosorbent assay was based on a sandwich method, using two monoclonal antibodies against human SP-D, 6B2 and 7C6, which were prepared against human SP-D purified from BAL fluids of patients with pulmonary alveolar proteinosis.
Quantitative microbiology was performed at Children’s Hospital and Regional Medical Center by methods previously de-scribed. The 1-mL aliquots removed for microbiologic cultures were plated within 2 h of the addition of dithiothreitol. canada-neighbor.com
Descriptive statistics and graphical displays were used to evaluate results by patient and sampling time. Analyses to examine the effect of sampling time on induced-sputum outcome measures were performed using generalized estimating equation methods. These methods allowed the fitting of linear regression models that accounted for the correlated nature of within-patient observations. The regression model for each outcome measure included indicator variables for the effects of sampling time, and a test that the time variables were jointly equal to zero was performed.