Analysis of Sequential Aliquots of Hypertonic Saline Solution-Induced Sputum: Quantitative Culture

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.

Statistical Methods
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.