Analysis of Sequential Aliquots of Hypertonic Saline Solution-Induced Sputum: SI
Using a modification of the protocol described by Fahy et al, modified by the Cystic Fibrosis Foundation Therapeutics Development Network, subjects inhaled nebulized sterile hypertonic saline (3%) solution for 20 min from an Ultra-Neb 99 ultrasonic nebulizer (DeVilbiss; Somerset, PA). This nebulizer generates particles of a mean mass median diameter of 4.5 μm and has an output of 2.4 mL/min. After expelling saliva, subjects were encouraged to cough, and sputum was collected at 4-min intervals.
Samples were processed as previously described. All specimens were processed within 30 min of collection. The samples were weighed, and the volume was calculated from the weight. The whole sample, including plugs, was diluted 1:8 0.1% dithio-threitol (Sputolysin; Behring Diagnostics; Somerville, NJ) in phosphate-buffered saline solution. The sample was then mixed gently by vortex mixer and placed in a shaking water bath at 37°C for 15 min. Periodically, the samples were removed from the water bath for further brief gentle mixing with a plastic transfer pipette to ensure visible liquefaction.
Following liquefaction with dithiothreitol, 1 mL was removed for quantitative culture. The liquefied sputum sample was used to determine the total cell count, and cytospin samples were prepared and stained (Hema 3; Fisher Scientific; Santa Clara, CA). The remainder of liquefied sputum was centrifuged at 2,000 revolutions per minute for 5 min. The supernatants were aspirated and frozen at — 70°C for later analysis. Slides were batched, and a blinded observer counted 500 nonsquamous epithelial cells and cell differentials per sputum preparation. Interleukin (IL)-8 concentrations were measured using a quantitative “sandwich” enzyme immunoassay technique (Quantikine; R&D Systems; Minneapolis, MN).