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.

Clinicians have been interested in the macroscopic and protocol (fig 1), based on that described by Pin et al 7 using a relatively low output ultrasonic nebuliser (output 0.9 ml/ microscopic appearance of sputum in asthma since the last half of the 19th century when Charcot-Leyden crystals, min, particle size 5.6 m), results in successful sputum induction in 76% of normal and asthmatic subjects who Curschmann's spirals, and their association with sputum eosinophilia was first recognised in patients with asthma. 1 cannot produce sputum spontaneously. Cell counts and biochemical content of induced and spontaneous sputum Forty years ago Morrow Brown suggested that the microscopic examination of sputum might be clinically useful are similar with the exception of fibrinogen which is present in higher concentrations in spontaneous sputum. 11 With by showing that the presence of eosinophils in a crude Leishman stained sputum smear identified patients whose salbutamol premedication and careful monitoring of forced expiratory volume in one second (FEV 1 ) during sputum wheeze was responsive to corticosteroids.2 Recently, with the recognition that even mild asthma is associated with induction in mild asthma significant bronchoconstriction rarely occurs, 7 but it is more common in patients with evidence of airway mucosal inflammation in bronchial biopsy specimens and bronchoalveolar lavage fluid, 3 4 there more severe or uncontrolled asthma. 12 In a recent study a third of sputum inductions in patients with asthma has been renewed interest in the use of sputum to assess airway inflammation non-invasively.exacerbations who were overusing inhaled 2 agonists were complicated by a >10% fall in FEV 1 which emphasises the This review describes the development over the last eight years of new and reliable techniques to assess airway need to perform the inductions carefully. 12 We and other investigators have induced sputum in asthma using similar inflammation using sputum differential cell counts and measurement of molecular markers of inflammation in concentrations of hypertonic saline delivered by ultrasonic nebulisers with a higher output and, whilst there might be the sputum fluid phase. We review studies where these measurements have been made in normal and diseased a higher success rate, this is at the expense of increased adverse effects including mild bronchoconstriction. [13][14][15] subjects and assess their validity, repeatability, and responsiveness. Finally we describe current, and speculate A recent preliminary report has suggested that the cellular and biochemical content of sputum induced by a high on future, applications of sputum measurements of airway inflammation in asthma in both research and clinical setoutput ultrasonic nebuliser changes with sequential inhalatings. Developments in methodologyEarly attempts to provide reliable sputum differential cell counts used smears of spontaneously produced sputum stained with May-Grunwald-Giemsa. 5 Additional staining with toluidine blue was required for accurate meta...

We have investigated the time-course of symptoms, forced expiratory volume in one second (FEV1), and the airway inflammatory changes in sputum selected from saliva and blood of 10 patients with severe exacerbation of asthma betwen presentation and after 1, 2, 3, 7, and 21 days of treatment. The sputum was induced by a modified standard protocol, and we examined its safety. The severe exacerbation of asthma was defined by the presence of nocturnal symptoms disturbing sleep and/or the need for inhaled short acting beta2-agonist > or = 8 puffs/d and an FEV1 after bronchodilator < 60% of predicted. The treatment consisted of additional prednisone 30 mg daily for 5 d followed by reduction to zero by day 10. Abnormal findings [median (interquartile range)] in spontaneous and induced sputum included low viability of cells [52.0 (34.0)%]; eosinophilia [20.0 (16.4)%]; many free eosinophil granules; and increased levels of fluid-phase ECP [1960 (9204) microg/L], fibrinogen [6045 (10720) microg/L], and IL-5 [160 (212) pg/ml]. Peripheral blood eosinophils [10.4 (7.6)%] and ECP levels [34.0 (35.0) microg/L] were increased. After treatment, symptoms, FEV1, blood eosinophilia, and serum ECP improved in the first 24 h. Sputum eosinophils and ECP did not improve until 48 h and fibrinogen not until 7 d. The improvement in sputum eosinophils and ECP levels was correlated with improvement of FEV1 and in fluid-phase IL-5. Thirty sputum inductions were performed safely in the majority with inhaled isotonic or 3% saline (23.3% or 63.3%, respectively) over a short duration (mean 8.4 min). The patients who had a fall in FEV1 of > or = 10% (10 occasions) after induction differed from those with a fall of < 10% only in the amount of inhaled beta2-agonist used by the patients in the preceding 24 h [8.0 (5.0) versus 4.0 (3.0) puffs/d, p = 0.01]. The results suggest that spontaneous or induced sputum can be used safely to follow the kinetics of effects of antiinflammatory treatment in a severe exacerbation of asthma. The clinical and blood indices improve before those in sputum, raising the possibility that examination of sputum is a better guide in these patients to follow the effects of treatment.

A reliable predictor of benefit from corticosteroid treatment in patients with chronic airflow limitation is needed. In a single-blind, sequential crossover trial of placebo and prednisone (30 mg/day) treatment, with each given for 2 wk, we investigated whether an increased proportion of sputum eosinophils (>= 3%) predicts a beneficial effect of prednisone in smokers with severe obstructive bronchitis. Patients were seen before and after each treatment. Clinical measurements were made blind to the laboratory findings and vice-versa. Eighteen of 20 patients completed the study. Eight had sputum eosinophilia and similar clinical and physiologic characteristics to those of 10 patients without a finding of sputum eosinophilia. Only in patients with sputum eosinophilia did prednisone, as compared with placebo, produce a statistically significant and clinically important mean effect on effort dyspnea of 0.8 (95% confidence interval [CI]: 0.3 to 1.2), p = 0.008, and in quality of life of 1.96 (95% CI: 0.5 to 3.3), p = 0.01, associated with a small improvement in FEV1 of 0.11 L (95% CI: - 0.04 to 0.23 L), p = 0.05. In these patients, prednisone also produced a significant decline in the median sputum eosinophil percentage, from 9.7% to 0.5% (p = 0.002), eosinophil cationic protein (ECP), from 6, 000 microgram/L to 1,140 microgram/L (p < 0.001), and fibrinogen, from 25. 3 mg/L to 5.4 mg/L (p < 0.001). These findings indicate that in smokers with severe airflow limitation, sputum eosinophilia predicts a beneficial effect of prednisone treatment. Improvement in FEV1, after prednisone treatment in this population, is small, and may not be appreciated in clinical practice.

Sputum examination is being used increasingly as a noninvasive method to assess airway inflammation. Expectorated sputum has variable contamination with saliva. Methods of processing have included the selection of portions of the sample considered to be representative of pulmonary origin versus use of the whole specimen, which is confounded by varying volumes of saliva. We compared cell profiles and eosinophilic cationic protein (ECP) concentration in sputum selected from the expectorate and in the usually discarded residual portion to determine to what degree salivary contamination is minimized and if the results are representative of lower respiratory secretions. Sputum was induced with hypertonic saline in six healthy and nine asthmatic subjects. All portions considered to be of pure lower respiratory tract origin were selected from the residual. The selected and residual portions were treated with dithiothreitol, total cell counts and cell viability were obtained, cytospins were made for differential cell counts and supernatant was collected for ECP assay. Selected portions of the specimens, in comparison with the residual portion showed: little squamous cell contamination (median 1.2 vs 70%; p < 0.001); higher total cell counts.mL-1 (5.1 vs 0.5 x 10(6) cells.mL-1; p < 0.001); higher number of viable nonsquamous cells per sample (1.9 vs 0.6 x 10(6) cells; p < 0.001); higher slide quality score (7 vs 4; p < 0.001); and higher levels of ECP (768 vs 136 micrograms.L-1; p < 0.001). There were no differences in the differential cell counts of eosinophils (1.3 vs 3.8%), neutrophils (44 vs 32%), and lymphocytes (0.6 vs 0.6%). While the proportion of macrophages was lower (36 vs 54%; p < 0.05), the absolute number (41 vs 19 x 10(4) cells; p < 0.05) was higher in the selected portion. In summary, selection of all portions of induced sputum from the expectorate minimized the confounding influence of saliva. Loss of nonsquamous cells in the residual portion was variable but usually less than one third of those in the selected portion. With one exception, this loss had little influence on the differential counts of inflammatory cells. Similar observations apply to eosinophilic cationic protein levels. We conclude that, in healthy subjects and treated asthmatics, inflammatory markers in the selected portion of the expectorate can be used to represent those in the lower respiratory tract in general.

The role of inhaled corticosteroids in the management of chronic obstructive pulmonary disease (COPD) remains controversial. The purpose of this study was to evaluate whether sputum eosinophilia (defined as eosinophils o3%) predicts clinical benefit from inhaled corticosteroid treatment in patients with smoking-related clinically stable moderate-to-severe COPD.Forty consecutive patients with effort dyspnoea (mean age 67 yrs; 52 pack-yr smoking history; post-bronchodilator forced expiratory volume in one second (FEV1) ,60% predicted, consistent with moderate-to-severe smoking-related chronic airflow limitation) were enrolled. Subjects were treated with inhaled placebo followed by inhaled budesonide (Pulmicort Turbuhaler1 1,600 mg? day -1 ), each given for 4 weeks. While the treatment was single-blind (subject level), sputum cell counts before and after treatment interventions were double-blind, thus removing bias. Outcome variables included spirometry, quality-of-life assessment and 6-min walk test.Sputum eosinophilia was present in 38% of subjects. In these, budesonide treatment normalised the eosinophil counts and, in comparison to placebo treatment, resulted in clinically significant improvement in the dyspnoea domain of the disease-specific chronic respiratory questionnaire (0.8 versus 0.3) and a small but statistically significant improvement in postbronchodilator spirometry (FEV1 100 mL versus 0 mL; p,0.05).In conclusion, sputum eosinophilia predicts short-term clinical benefit from high-dose inhaled corticosteroid treatment in patients with stable moderate-to-severe chronic obstructive pulmonary disease.

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.

The study highlights the variability in estimates of depression prevalence in COPD. It could be explained by methodological differences across the included studies. This suggests that a standardization is critical to improve precision of the estimates.