Background Airway inflammation is considered to be important in asthma but is relatively inaccessible to study. Less Airway inflammation is a major factor in the pathogenesis of asthma. Mast cell and eosinophil infiltration, epithelial damage, and mucus production are characteristic features.' Direct examination of the inflammatory response should help to improve understanding of the pathogenesis and treatment of asthma.In patients with stable asthma bronchial biopsy and bronchoalveolar lavage have been used to study airway inflammation2' but discomfort, inconvenience, and risks limit their use. Examination of sputum is a less invasive alternative,5 but sputum cannot always be produced spontaneously. When sputum is not otherwise available induced samples may allow secretions from the lower airways to be sampled.Sputum induction by inhalation of hypertonic saline has been successfully used to diagnose Pneumocystis carinii pulmonary infections in patients infected with HIV.'We adapted this method for use in asthmatic subjects and examined (a) the success rate and safety of the method, (b) the reproducibility of cell counts, and (c) the differences in cell counts between normal and asthmatic subjects. Methods SUBJECTSSeventeen normal subjects and 17 subjects with asthma were selected from among the staff (adults) and asthmatic patients and their siblings (adults or children) at the clinics of the Firestone Regional Chest and Allergy Unit and the Health Sciences Centre. All were nonsmokers or ex-smokers of more than five years. None had spontaneous sputum or symptoms of a respiratory tract infection, or had been exposed to a seasonal allergen within the last month. The normal subjects had no past or current symptoms of asthma, a forced expiratory volume in one second (FEVI) >80% of predicted values,9 a ratio of FEV, to vital capacity >70%, and normal airway responsiveness to methacholine (provocative concentration of methacholine causing a 20% fall in FEV, (PC20) >8 mg/ml)'0 (table 1). The asthmatic subjects had a history of episodic dyspnoea with wheeze in the previous six months and a PC20 methacholine <8 mg/ml (15 patients) or a spontaneous variability in peak expiratory flow rate (PEF of >20% (two patients). All were taking an inhaled 2 agonist when needed; 15 were treated with inhaled corticosteroid (daily dose 200-3000 Mg) and one with prednisone 5 mg daily. Although asthma was stable in all subjects, control of the condition was good" in only five subjects; the remaining 12 had more asthmatic symptoms

In recent studies, sputum smear cell counts were found to be reproducible and usefully applied to research in asthma and other airway conditions. However, cell definition on the smears is poor, and the procedure is tedious and has limited utility. The objective of this study is to improve the methods of sputum examination. The subjects used in this study were people with bronchitis or asthma from whom sputum could be obtained. By inverted microscopy, portions of fresh sputum were selected to exclude salivary contamination. These portions were exposed to different volumes of dithiothreitol for varied time intervals. We used the resulting cell suspensions to perform total cell counts and prepare cytospins for differential cell counts and immunohistochemical stains for GM-CSF, EG2, TNF alpha and IL-8. Cytospins were compared with smears for differential cell counts on the same sputum specimens. Excellent cell dispersion and definition in cytospins could be observed. The time required for differential cell counting on cytospins was reduced and cytospin counts were more reproducible than smears. Greater duration of treatment of sputum with dithiothreitol tended to increase total cell counts and significantly decreased EG2 staining but had no effect on differential cell counts or the cytokine cell components. Therefore the proposed method of sputum examination involving cell dispersion and use of cytospins overcomes a number of the disadvantages of the examination of smears.

Inhalation of hypertonic saline aerosol is a relatively noninvasive method to obtain sputum for examination of inflammatory processes in the airways. We investigated some technical factors which might influence the success of induction and sputum cell counts. In total, twenty six asthmatic and 13 healthy subjects, unable to raise sputum spontaneously, inhaled nebulized saline for three 7 min intervals. In three randomized, cross-over studies we repeated sputum induction on separate days with two ultrasonic nebulizers (De Vilbiss Ultraneb 99 and Fisoneb) and one jet nebulizer (Pari LL with Master Compressor) (Study 1, n = 15), with different saline concentrations (normal saline 0.9%; hypertonic saline 3% on 2 days; and hypertonic saline 3, 4 and 5%, sequentially) (Study 2, n = 14) and with pretreatment with either salbutamol or placebo (Study 3, n = 10). The latter two studies were double-blind. Sputum cells were dispersed with dithiothreitol, and the cell suspension was used to perform total cell counts and to prepare cytospins for differential cell counts. We compared success rate, cell counts, subject discomfort and percentage fall in forced expiratory volume in one second (FEV1) during the procedures. All sputum examinations were performed blind to the clinical procedures. The success rates and the cell counts of the specimens obtained with the two ultrasonic nebulizers were not different, whilst general discomfort was proportional to the saline output of the nebulizer. Induction of sputum by hypertonic saline was more successful than normal saline, but more disagreeable to the subjects. Induction with saline 3% on two days was only successful in 6 of 14 subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

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