Airway Inflammation and Cellular Stress in Noneosinophilic Atopic Asthma: Measurement of Soluble Mediators

Oct-30-2014

Commercial immunoassays were used to measure sputum supernatant IL-8, GM-CSF, MPO, and ECP levels, as previously described. All measurements were performed twice for the same sample, and the results were averaged. The mean intrasample coefficient of variation was < 4%. in detail
All assays were validated according to European Respiratory Society guidelines. Spiking recoveries of ECP, MPO, IL-8, and GM-CSF levels were assessed by adding a known quantity of purified mediator to different aliquots of sputum samples. Considering that recovery might differ between individuals due to differences in factors in the sputum that interfere with detection, sputum from a range of subjects with asthma and COPD was used (NEA, 5 patients; EA, 5 patients; COPD, 5 patients). The expected concentration of the spiked mediator, assuming 100% recovery, was in the mid-part of the standard curve. The spiking recovery of each mediator was calculated as the percentage of the expected value, which was the sum of the spiked purified mediator and the mediator concentration in the original sputum sample. The median recovery values were 88.3% (range, 71.2 to 98.9%) for ECP, 82.1% (range, 47.8 to 94.6%) for MPO, 84.5% (range, 73.4 to 95.3%) for IL-8, and 82.8% (range, 67.4 to 92.7) for GM-CSF.
Statistical Analysis
Before between two group comparisons were made, differences among three groups were tested using the Kruskal-Wallis test for nonnormally distributed variables and the analysis of variance test for normally distributed variables. Differences between two groups were tested using the Mann-Whitney test for nonnormally distributed variables and the t test for normally distributed variables. Normality was tested by the Shapiro-Wilk test. A statistical software package (StatsDirect; Camcode; Cambridge, UK) was used. A p value of < 0.05 was considered to be significant.