Use of Induced Sputum Cell Counts to Investigate Airway Inflammation In

Thorax 1992;47:25-29 25 Use of induced sputum cell counts to investigate

airway inflammation in asthma Thorax: first published as 10.1136/thx.47.1.25 on 1 January 1992. Downloaded from

Isabelle Pin, Peter G Gibson, Roxanne Kolendowicz, Adele Girgis-Gabardo, Judah A Denburg, Frederick E Hargreave, Jerry Dolovich

Abstract production are characteristic features.' Direct Background Airway inflammation is examination of the inflammatory response considered to be important in asthma should help to improve understanding of the but is relatively inaccessible to study. pathogenesis and treatment of asthma. Less invasive methods of obtainling In patients with stable asthma bronchial sputum from patients unable to produce biopsy and bronchoalveolar lavage have been it spontaneously should provide a useful used to study airway inflammation2' but dis- investigational tool in asthma. comfort, inconvenience, and risks limit their Methods A method to induce sputum use. Examination of sputum is a less invasive with inhaled hypertonic saline was alternative,5 but sputum cannot always be modified for use in 17 asthmatic patients produced spontaneously. When sputum is not and 17 normal subjects who could not otherwise available induced samples may produce sputum spontaneously. The suc- allow secretions from the lower airways to be cess rate and safety of the method, the sampled. reproducibility of cell counts, and dif- Sputum induction by inhalation of hyper- ferences in cell counts between the tonic saline has been successfully used to asthmatic and normal groups were diagnose Pneumocystis carinii pulmonary examined. Hypertonic saline solution 3- infections in patients infected with HIV.' 5% was inhaled for up to 30 minutes We adapted this method for use in asthmatic after inhalation of salbutamol. Subjects subjects and examined (a) the success rate and were asked to expectorate sputum every safety of the method, (b) the reproducibility of five minutes. The quality of the sample cell counts, and (c) the differences in cell was scored on the volume of plugs and counts between normal and asthmatic the extent of salivary contamination. subjects. http://thorax.bmj.com/ Plugs from the lower respiratory tract were selected for a total cell count and for differential cell counts of eosinophils Methods and metachromatic cells (mast cells and SUBJECTS basophils) in direct smears. Seventeen normal subjects and 17 subjects Results Adequate samples from the with asthma were selected from among the staff lower respiratory tract were obtained in (adults) and asthmatic patients and their sib-

76% of first attempts. The mean fall in lings (adults or children) at the clinics of the on September 29, 2021 by guest. Protected copyright. the forced expiratory volume in one Firestone Regional Chest and Allergy Unit and second (FEV1) during inhalation of saline the Health Sciences Centre. All were non- was 5-3% and the maximum fall 20%. smokers or ex-smokers ofmore than five years. Eosinophil and metachromatic cell None had spontaneous sputum or symptoms of counts were reproducible (reliability a respiratory tract infection, or had been coefficient 0X8 and 0 7 respectively). When exposed to a seasonal allergen within the last compared with sputum from normal month. The normal subjects had no past or subjects sputum from asthmatic current symptoms of asthma, a forced expira- patients contained a significantly higher tory volume in one second (FEVI) >80% of proportion of eosinophils (mean 18-5% predicted values,9 a ratio of FEV, to vital (SE 3-8%) v 1-9% (0-6%)) and meta- capacity >70%, and normal airway respon- Asthma Research chromatic cells (0 50% (0-18%) v 0 039% siveness to methacholine (provocative concen- Group, Departments of Medicine and (0-014%)). In the asthmatic group the tration of methacholine causing a 20% fall in Pediatrics, Health differential eosinophil count correlated FEV, (PC20) >8 mg/ml)'0 (table 1). The asth- Sciences Centre, St with the baseline FEVI. matic subjects had a history of episodic dysp- Joseph's Hospi al and McMaster University, Conclusion Induced sputum is cap- noea with wheeze in the previous six months Hamilton, Ontario, able ofdetecting differences in cell counts and a PC20 methacholine <8 mg/ml (15 Canada L8N 3Z5 between normal and asthmatic subjects patients) or a spontaneous variability in peak I Pin merits further as a P G Gibson and development expiratory flow rate (PEF of >20% (two R Kolendowicz potential means of assessing airway patients). All were taking an inhaled 2 agonist A Girgis-Gabardo inflammation in asthma. when needed; 15 were treated with inhaled J A Denburg corticosteroid (daily dose 200-3000 and F E Hargreave Mg) J Dolovich one with prednisone 5 mg daily. Although a in the Reprint requests to: Airway inflammation is major factor asthma was stable in all subjects, control of the Dr Dolovich pathogenesis of asthma. Mast cell and eosino- condition was good" in only five subjects; the Accepted 21 May 1991 phil infiltration, epithelial damage, and mucus remaining 12 had more asthmatic symptoms 26 Pin, Gibson, Kolendowicz, Girgis-Gabardo, Denburg, Hargreave, Dolovich

Table I Characteristics of normal and asthmatic subjects sample transferred to a Petri dish, where its macroscopic characteristics were recorded. Normal subjects Asthmatic subjects The quality of the sample was assessed by (n = 17) (n = 17) Thorax: first published as 10.1136/thx.47.1.25 on 1 January 1992. Downloaded from estimating the volume of lower respiratory No of females 9 11 tract secretions and the degree of salivary Mean (years) (range) 27 3 (14-58) 30 3 (11-69) Mean atopy score* 3-6 10-9 contamination. The volume was assessed by Mean (SE) FEV, (% pred) 108-9 (2 9) 88-9 (4-1) the size and number ofplugs; a cumulative size PC2, (geometric mean) (range) 41 7 (13-> 64) 0-21 (<0 03 6-1) of4-5 x 9 mm was estimated to be necessary to perform all investigations, including total and *Results of allergy prick tests expressed as sum of +, 1 + corresponding to weal diameter 1-2 mm greater than saline control, 2 + to 3-5 mm, 3 + to 6-9 mm, and 4 + to > 9 mm. differential counts. The quality of the sputum PC20 = provocation concentration of methacholine producing a 20% fall in FEV,. sample was scored by (a) visual inspection and inverted microscope examination (no plugs = 0, 4-5 x 9mm= 1,>4-5 x 9mm=2);(b) salivary contamination in total cell counts defined as the percentage of squamous cells than when at their best, an increased require- among nucleated cells (> 10% = 0, < 10% = ment for inhaled bronchodilator, or an FEV, 1, 0% = 2); (c) salivary contamination in <85% (but >40%) of their best recorded differential cell counts by assessing the pro- value. portion of squamous cells in the slides (too The study was approved by St Joseph's many squamous cells to permit a count = 0, Hospital research committee, and all subjects enough squamous cells to permit a count = 1, (parents in the case of children) gave informed no squamous cells = 2). A sample score of > 4 consent. was considered to be adequate, of 3 to be intermediate, and of < 2 to be inadequate. STUDY DESIGN Sputum analysis was performed as described Subjects' characteristics were documented by by Gibson et al,' except that Sputalysin (Cal- questionnaire, allergy prick tests with 12 com- biochem, San Diego, California) was used mon allergen extracts,'2 spirometry, and a instead of trypsin to suspend the plugs for total methacholine inhalation test (or diurnal varia- cell counts. Formal comparison of trypsin and tion in PEF). At a second visit, usually within Sputalysin suspensions in 18 sputum samples one week, sputum was induced between 0800 found good agreement between the total cell and 1300 hours. Twelve subjects (six rnormal counts (intraclass correlation coefficient 0-85). and six asthmatic) chosen at random were asked Differential cell counts of intact bronchial to have sputum induced on a second occasion epithelial cells and leucocytes were performed within one week at the same time of day. If on by counting 400 nucleated cells on each of two one of the two occasions an adequate sample slides fixed with methanol and stained with http://thorax.bmj.com/ was not obtained the induction was carried out May-Grunwald-Giemsa. Two further slides a third time within the same week. Cell counts were fixed with Carnoy's solution and stained were performed blind without knowledge of with 0 5% toluidine blue in 0 7 N hydrochloric the clinical characteristics of the patients. acid at pH 0 1, and 1500 nucleated cells were counted on to each to obtain a differential count INDUCTION OF SPUTUM of metachromatic cells. Inhaled bronchodilator was withheld for six

hours before the induction. FEV, and vital STATISTICAL ANALYSES on September 29, 2021 by guest. Protected copyright. capacity were measured before and 10 minutes Differential andtotal cell counts were calculated after salbutamol inhalation (two puffs, 200 ug) as arithmetic means with 95% confidence and then every five minutes during inhalation of intervals. PC20 results were log transformed for hypertonic saline solution. The hypertonic analysis. The relation between categorical data saline was nebulised with an ultrasonic nebul- (age, sex, diagnosis of asthma, and scores) was iser (Fisoneb, Fisons, Pickering, Ontario) on examined by the x2 test. The reproducibility of the maximum setting and was inhaled for five sputum cell counts was examined by analysis of minute periods for up to 30 minutes. Under variance with calculation of the coefficient of these conditions the mass median aerodynamic reliability, R (the ratio of the variance of cell diameter was 5 9 ,Mm and the output counts between subjects to the total variance in 0-6 /min.'3 The concentration of saline was cell counts (subjects and error variance)), increased at intervals of 10 minutes from 3% to higher values indicating higher reliability.'4 4% to 5%. If the FEV1 fell by > 10% from the For the study of reproducibility the two sam- post-bronchodilator value the concentration of ples with the highest scores were selected for saline was not increased. If the FEV1 fell by each subject. For the comparison of counts > 20% or if troublesome symptoms occurred between normal and asthmatic subjects and nebulisation was discontinued. Ten minutes their relation to FEVI, the ratio ofFEV, to vital after the start of nebulisation and every five capacity and PC20 the counts used were from minutes thereafter subjects were asked to rinse the first adequate sample collected (score >4) their mouth and throat carefully and to try to when there was more than one attempt or from cough sputum into a container. The nebulisa- adequate or intermediate samples when there tion was stopped after 30 minutes or earlier if a was only one attempt. Two tailed unpaired t sputum sample of good quality was obtained. tests were used to compare cell counts between groups. Correlations were examined by least SPUTUM ASSAYS square linear regression analysis. Significance The volume of sputum was recorded and the was accepted at the 95% level. Use ofinduced sputum cell counts to investigate airway inflammation in asthma 27

Results the normal subjects and 5-3% in the asthmatic SAFETY AND SUCCESS OF THE METHOD subjects. The nebulisation had to be stopped

The mean volume of nebulised hypertonic only once because the fall in FEV, exceeded Thorax: first published as 10.1136/thx.47.1.25 on 1 January 1992. Downloaded from saline was 12 ml per induction. Only one 20% (30%); the fall was quickly reversed with asthmatic subject developed a mild spontan- an inhaled fl2 agonist. eous cough during the process. The most Ofthe first 34 attempts at sputum inductions, common complaint was of a salty taste. The 26 produced adequate samples, one an inter- mean fall in FEV1 during nebulisation was 0 in mediate sample, and seven inadequate samples. A history of spontaneous sputum production (X2 = 7-65, p < 0-05) but not age, sex, or Figure 1 Identity plots 40 1 A the presence of asthma (x2 = 0-42, 1-5, 0-24 for eosinophils (top) and R = 0-8 respectively; df 2; NS) was associated with a metachromatic cells better quality of sample. (bottom): counts (as C%J percentages) from 30 - . Of the 12 adults with repeated studies, eight adequate sputum samples V had adequate samples on the first two occasions, collected on two days two had intermediate samples on the first within one week; R is the X- coefficient of reliability. 'a 20 - occasion but adequate samples on subsequent attempts, and one asthmatic subject had only Q one adequate sample out of two. One normal wLU0 subject had no adequate sample despite three 10 - attempts.

REPRODUCIBILITY OF SPUTUM CELL COUNTS 0- t. There was reasonably good reproducibility of 0 10 20 30 40 differential counts of eosinophils (R = 0-8), Eosinophils (%) day 1 metachromatic cells (R = 0-7) (figure 1), 0 36 - macrophages (R = 0-71), and polymorpho- B R - 0.7 nuclear cells (R = 0-73). The reproducibility of total cell counts (R = 0-25) was less satis- 0*30 - factory. co 0- 0-24 - COMPARISON OF CELL COUNTS BETWEEN C.T --ASTHMATIC AND NORMAL SUBJECTS 0 18 - There was no difference between asthmatic and .2 normal subjects in total or absolute cell counts. E http://thorax.bmj.com/ 0 0.12 - The analyses have therefore been expressed pc only as differential counts. The differential counts of eosinophils and metachromatic cells 0 06 - from asthmatic subjects were significantly higher than those from normal subjects (fig 2; 0 - *0 0 0 table 2) and the differential macrophage count ~' 6 was lower (p = 0-007, 0-02, and 0-002 respec- 0 006 012 018 024 0 30 0 36 tively). There was a trend for more neutrophils Metachromatic cells (%) dzay 1 in the sputum from asthmatic subjects, but the on September 29, 2021 by guest. Protected copyright. difference was not significant (p = 0-051). 50 - 2-7 Therewereno significant differences in total cell 0 0 counts, differential counts of intact bronchial F26 epithelial cells, or lymphocytes between the two groups. 40 - The relations between cell counts and clin- S 1-0 ical characteristics were examined. Mean

_ eosinophil and metachromatic cell counts did X not differ significantly between patients with 30 - 0-8 ao controlled and patients with uncontrolled -c S 0. 0 . asthma. There was an inverse correlation bet- 20 0 0 ween the count and both ._- 0 0-6 E eosinophil (%) FEV, C 2° (% predicted values) and the ratio of FEV, to - S 0 20 vital before bronchodilator on the u c, capacity day trthesputum wasws sampled (r = -0-71, S 0S E p < 0-001 and r = -0-57, p = 0-006 respectively). This relation was present for in 10 - FEV, - 0 2 the asthmatic group only (r = -0-61, p = 0-01) (fig 3). There was a weak correlation between the metachromatic cell count (%) and 0 PC20 when it was measurable (r = - 0-49, = Normal Asthmatic 'Normal Asthmatic p 0-03), but not when the asthmatic group alone was analysed. There was no relation Figure 2 Differential cell counts of eosinophils and metachromatic cells in nm ormal and between eosinophil or metachromatic cell asthmatic subjects. The solid bar represents the mean value for the counts in ti Open circles represent patients with controlled asthma, closed circles patients guth counts and either the allergy skin index or dose uncontrolled asthma. of inhaled corticosteroid. 28 Pin, Gibson, Kolendowicz, Girgis-Gabardo, Denburg, Hargreave, Dolovich

Table 2 Total and differential cell counts in induced sputum. Values are means (95% confidence intervals) Differential cell counts (% of total nucleated cells) Thorax: first published as 10.1136/thx.47.1.25 on 1 January 1992. Downloaded from Total cell count Bronchial Metachromatic Group ( x 1O6/ml) epithelial cell Neutrophil Macrophage Lymphocyte Eosinophil cell Normal (n = 14) 4-5 (2-3 to 6-8) 5-3 (1-8 to 8-8) 11-1 (5-1 to 17-0) 79-8 (73.5 to 86-2) 1-55 (0-7 to 2 4) 1-9 (0-6 to 3-2) 0 039 (0 009 to 0 068) Asthmatic (n = 15) 4-5 (2-0 to 7-1) 2-9 (1-8 to 4 0) 23-5 (12-2 to 34-7)* 56-7 (47.5 to 65-9)t 0-5 (0-2 to 0-9) 18-3 (10-2 to 26 4)t 0 50 (0 11 to 0 89)§ *p = 0-051. tP = 0002. tp = 0007. §p = 0-02.

r = -001 bronchodilator. The mean fall in FEV1 was p = 0-01 15-3%. The method showed an increased proportion 50 - . of eosinophils and metachromatic cells in sputum from asthmatic subjects compared with normal subjects. This result is similar to 40 - those previously obtained with spontaneous sputum samples,5 bronchoalveolar lavage fluid,23"82' and bronchial biopsy specimens.4 co 30- The mean percentages ofeosinophils andmeta- ._ . chromatic cells were higher in induced sputum 0 than in bronchoalveolar lavage samples in 'a 20- 0 patients with mild stable asthma, in whom w eosinophils account for < 5% and metachromatic cells <0 5% of the cells.31920 They 10 - were lower than sputum counts during an * exacerbation of asthma.5 * 0 Despite the clear difference in mean differ- 0 - 0 0 . . r w - ential eosinophil and metachromatic cell I I 1 1 40 50 60 70 80 90 100 110 counts between normal and asthmatic subjects there was overlap between the two populations.

FEV, (% predicted) http://thorax.bmj.com/ Similar overlap was found with broncho- Figure 3 Plot of the differential counts ofeosinophils (as percentages) against FFEV, % alveolar and predicted measured before sputum collection in the asthmatic group. r is the correlo lavage samples23 bronchial biopsy coefficient; the line and p value correspond to thefitted regression equation. specimens.22 This may reflect normal variation, subclinical disease, inhibition by corticosteroid treatment,23126 or variability in sputum induction and cell counting. Lack ofreproducibility ofsputum cell counts has been recognised for some time.27 The

Discussion distribution of cells within the specimen may on September 29, 2021 by guest. Protected copyright. This is the first time that an attempt ha:s been not be homogeneous or the recognition of made to use induced sputum samp]les to different cell types may not be accurate, or both. examine the inflammatory response in assthma. The method of sputum examination in our The results show that such samples diiffered study was similar to that used in a previous between asthmatic and normal sulbjects. study on spontaneous sputum, in which the Further development and use of the tecbmique reproducibility of total and differential cell is indicated, especially as the only alterniatives counts was good.5 Microscopic selection of the are more invasive, with discomfort and risk. plugs for analysis is considered to be essential The mechanism whereby inhalation of an to ensure that only lower respiratory tract aerosol of hypertonic saline induces spuitum is secretions rather than saliva are selected for the not known. It may increase water flux inito the analysis.5 27 Reproducibility in our study, lumen of the airways and free or increaise the especially of total cell counts, was not as good volume of secretions so that they can be cexpec- possibly because induced sputum is less torated. An adequate or intermediate s-ample homogeneous than spontaneously produced was obtained from the lower respiratory tract in sputum. Further investigation of the methods 77% of first attempts and in 84% of s;econd and factors determining reproducibility are attempts. required. Inhalation ofhypertonic saline causes Xairway Our study was not designed to examine constriction in some asthmatic patients,'5*17 and relations between induced sputum cell counts to try to prevent this we gave an inhailed f2 and clinical and physiological characteristics of agonist before the procedures and increas;ed the asthma. Nevertheless, eosinophil counts concentration of hypertonic saline graidually correlated with the degree of airflow obstruc- while monitoring the FEV,. One asti imatic tion, as has been observed in bronchoalveolar patient (with a low ratio of FEVy tc vital lavage samples.2 Our observations suggest that capacity and PC20) had a fall in FEV1 of > 20%, induced sputum may have a role in the study of which reversed quickly with an irahaled asthma and, perhaps, other lung diseases. Use ofinduced sputum cell counts to investigate airway inflammation in asthma 29

We thank the subjects for participating and Fisons Canada for 13 Dolovich M, Chambers C, Newhouse M. Characterisation supplying the nebulisers. The study was supported by the of 3 systems used to deliver pentamidine (P) aerosol. Medical Research Council of Canada. PGG was supported by [Abstract.] Am Rev Respir Dis 1990;141:A153. the Canadian Lung Association and IP by Fondation Marc 14 Kramer MS, Feinstein AR. Clinical biostatistics. LIV. The

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