Methods to examine sputum for indices of airway inflammation are evolving. We have examined the repeatability and the validity of an improved method to measure sputum cells and fluid-phase eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin (EDN), albumin, fibrinogen, tryptase, and interleukin-5 (IL-5). Sputum was induced with hypertonic saline twice within 6 d in 10 healthy subjects, 19 stable asthmatics, and 10 smokers with nonobstructive bronchitis. The method included the processing of freshly expectorated sputum separated from saliva, treatment with a fixed proportion of dithiothreitol 0.1% followed by Dulbecco's phosphate-buffered saline, making cytospins, and collecting the supernatant. The reproducibility of measurements, calculated by the intraclass correlation coefficient, was high for all indices measured with the exception of total cell counts and proportion of lymphocytes. Asthmatics, in comparison with healthy subjects and smokers with bronchitis, had a higher proportion of sputum eosinophils (median percent 5.2 versus 0.5 and 0.3), metachromatic cells (0.3 versus 0.07 and 0.08), ECP (1,040 micrograms/L versus 288 and 352), MBP (1,176 micrograms/L versus 304 and 160), and EDN (1,512 micrograms/L versus 448 and 272). Asthmatics differed from healthy subjects, but not from smokers with bronchitis, in the proportion of neutrophils (46.9% versus 24.1%), albumin (704 versus 288 micrograms/mL), and fibrinogen (2,080 versus 440 ng/mL). Smokers with bronchitis showed a trend for a higher neutrophil count and levels of albumin and fibrinogen than healthy subjects. The proportion of sputum eosinophils correlated positively with ECP, MBP, EDN, albumin and fibrinogen levels, and metachromatic cell counts correlated with tryptase. In asthmatics, IL-5 correlated with eosinophil counts. There was a significant negative correlation between sputum indices and expiratory flows and methacholine PC20. Thus, the methods of measuring cell and fluid phase markers in induced sputum used in this study are reproducible and valid. They can therefore be used to reliably measure these indices of airway inflammation.

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Inhalation of hypertonic saline to induce sputum may alter cells and fluid-phase markers in sputum. We have compared indices of inflammation in sputum produced spontaneously with sputum induced by an aerosol of hypertonic saline. Twenty-three asthmatics produced spontaneous followed by induced sputum on the same day. The sputum specimen was separated from saliva within 2 h, dispersed with dithiothreitol (DTT) and processed to obtain cytospins and supernatant. The statistical power to detect a 20% difference in sputum parameters was > 90%. Results are expressed as median and interquartile range [IQR]. Induced sputum had a higher proportion of viable cells (77.0 [19.0] versus 47.0 [38.0]%, p < 0.001), less squamous cell contamination (1.0 [1.2] versus 1.8 [34.0]%, p < 0.001) and better quality cytospins (score of 8.0 [4.0] versus 4.0 [2.0], p < 0.001). It also had lower fluid-phase levels of eosinophil cationic protein (ECP) (1,358 [1,102] versus 1,574 [2,479] microg/L) and fibrinogen (1,560.0 [3,130.0] versus 4,350.0 [5,970.0] ng/ml) but only the latter was significantly different (p = 0.02). Induced sputum was similar to spontaneous sputum in weight (200.0 [219.0] versus 270.0 [227.0] mg), total cell count (3.3 [4.1] versus 3.5 [4.5] x 10(6)/ml), proportion of nonsquamous cells, and levels of tryptase. The agreement between induced and spontaneous measurements was good, but fluid-phase levels were affected by the low viability of some spontaneous samples. We conclude that for the indices measured in asthmatic subjects, induced sputum separated from saliva is similar to lower respiratory secretions expectorated spontaneously and has the advantage of better cell viability.