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* From the Asthma Research Group, Department of Medicine, McMaster University, Hamilton, Ontario, Canada.
Correspondence to: Malcolm R. Sears, MB, Asthma Research Group, Firestone Regional Institute for Respiratory Health, St. Joseph’s Healthcare, McMaster University, 50 Charlton Ave East, Hamilton, Ontario L8N 4A6, Canada; e-mail: searsm{at}mcmaster.ca
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Abstract |
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Materials and methods: In this cross-sectional study, we selected four groups of adult subjects by reported symptoms and diagnoses from among those previously randomly identified in a population study. Subjects were selected with no respiratory symptoms ever (normal group), or reporting a diagnosis of asthma (asthma group), or reporting recurrent wheezing not diagnosed as asthma (wheeze group), or reporting exposure to industrial irritants, but not asthma or wheezing (exposed group). Current respiratory symptoms, airway responsiveness to methacholine challenge, and sputum cell counts were determined. The study was completed by 107 subjects aged 20 to 44 years.
Results: There were no significant differences in FEV1 percent predicted, total cell count, and sputum eosinophil count among the four groups. Subjects with reported asthma had greater airway responsiveness as reflected in a lower bronchial reactivity (BR) index. There was a weak correlation between BR index and sputum eosinophils.
Conclusion: In a community setting, induced sputum eosinophil cell counts in subjects reporting asthma or wheezing were most often within the normal range and not sufficiently often abnormal to be useful in validating a diagnosis of asthma in epidemiologic studies.
Key Words: airway hyperresponsiveness • asthma • eosinophils • epidemiology • induced sputum
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main histologic characteristic of asthma is airway inflammation. New methods of examination of induced sputum have allowed repeatable, responsive, and valid noninvasive determination of the presence and nature of airway inflammation.2 Asthmatic patients with current symptoms often show an increase in some sputum cell counts, particularly eosinophils and metachromatic cells.3 If sputum cell analyses could be applied in epidemiologic studies, this might yield additional information on the nature of inflammatory processes associated with respiratory symptoms in the population, and help differentiate between nonasthmatic and asthmatic causes of reported symptoms. We hypothesized that subjects reporting asthma in epidemiologic surveys would show sputum eosinophilia that might be a useful marker of asthma in the community. However, the time involved in inducing sputum and analyzing the cell counts of the expectorated material is considerable. We therefore undertook a pilot study to determine whether measuring sputum differential cell counts, particularly of eosinophils, was a useful method of validating symptoms and diagnoses suggesting asthma in subjects who had recently participated in an epidemiologic study of respiratory disease. The null hypothesis was that there would be no difference in sputum eosinophil counts between subjects stating they had asthma and those who did not.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The four groups were as follows: subjects with no respiratory symptoms (normal group), subjects reporting a diagnosis of asthma (asthma group), subjects reporting recurrent wheezing not diagnosed as asthma (wheeze group), and subjects reporting exposure to industrial irritants but not asthma or wheezing (exposed group). The study was approved by the Research Committee of St. Joseph’s Hospital, and each subject gave written consent.
Current Symptoms
Although subject selection was based on previous responses to
the self-completed ECRHS questionnaire, the current status of symptoms
and diagnoses was further documented for the present study using the
same questions. The symptom score was based on the presence or absence
of five symptoms (wheeze, cough, shortness of breath, chest tightness,
sputum production) with a scale of zero (no symptoms) to 5 (all of the
above). Questions to identify the likely cause of recent symptoms were
included when applicable. Current treatments and occupational and
smoking histories were also checked.
Skin Tests
Allergy skin prick tests to 14 common inhalant allergen extracts
(Dermatophagoides pteronyssinus, Dermatophagoides
farinae, olive tree, birch tree, east-western tree mixture, common
ragweed, Kentucky grass, timothy grass, cat, cockroach, Cladosporium,
Aspergillus, Alternaria, and Penicillium) were available for each
subject from their previous participation in the ECRHS survey, or were
performed during this study. Any allergen wheal > 2 mm in size than
the negative control wheal was used to define atopy.
Airway Function Assessment
Subjects performed spirometry according to American Thoracic
Society standards5
on a spirometer (Koko; Pulmonary Data
Service Instruments; Louisville, KY) and then underwent a methacholine
challenge test6
to determine current airway
responsiveness. The test concluded with inhalation of salbutamol to
reverse any resulting airway constriction and to prepare for sputum
induction.
Methacholine Challenge
Methacholine challenge was performed according to the
standardized and validated protocol of Hargreave et al,6
preceded by 15 min of rest. Following baseline spirometry,
FEV1 was remeasured after inhalation of normal
saline solution, and after doubling concentrations of methacholine from
0.03 to 16.0 mg/mL in normal saline solution, delivered using a Wright
nebulizer (Aerosol Medical Limited; Colchester, Essex, United Kingdom)
(output, 0.13 mL/min) and inhaled by tidal breathing for 2 min with the
nose clipped. FEV1 was measured at 30 s and
90 s after each dose. If the FEV1 was lower
at 90 s than at 30 s, additional measurements were made at
180 s and every 2 min thereafter until the lowest
FEV1 was determined. Subsequent concentrations
were given at approximately 5-min intervals until the
FEV1 decreased 20% from the lowest post-saline
solution FEV1, or until the highest concentration
had been given. Once the FEV1 stopped falling
after the last inhalation, salbutamol was administered. The provocative
concentration of methacholine causing a 20% fall in
FEV1 (PC20) was calculated
by linear interpolation of the last two points. The dose-response slope
(bronchial reactivity [BR] index) was calculated by the method of
Burrows et al7
so that no data were censored.
Sputum Induction
Sputum was induced by the method developed and standardized at
this institution, as described by Pin et al8
and slightly
modified. An aerosol of hypertonic saline solution was inhaled in
increasing concentrations (3%, 4%, and 5%) generated by an
ultrasonic nebulizer (Fisoneb; Canadian Medical Products Ltd; Markham,
Ontario) with an output of 0.87 mL/min and particle size of 5.58 µm
aerodynamic mass median diameter.8
Each concentration was
inhaled for 7 min.
Sputum Analysis
Sputum examination was performed as described by Pizzichini et
al.2
The fresh expectorate was poured into a Petri dish,
and all portions that macroscopically looked more opaque or dense and
unlike saliva (selected portion) were placed in a 15-mL polystyrene
tube and weighed. This was treated with dithiothreitol (Sputalysin
10%; Calbiochem Corporation; San Diego, CA) freshly diluted 1:10 with
distilled water, in a volume equal to four times the weight of the
selected sputum. The mixture was vortexed for 15 s, gently
aspirated in and out of a pipette to ensure mixing, and placed on a
bench rocker (Dade Tube Rocker; Baxter Diagnostics Corporation; Miami,
FL) for 15 min. A further four volumes of Dulbecco’s
phosphate-buffered saline solution was added, and rocking was continued
for another 5 min. The suspension was filtered through a 48-µm nylon
mesh (BBSH; Thompson; Scarborough, Ontario) to remove cell debris and
remaining mucus. A total cell count was performed in a modified
Neubauer hemocytometer, and the cell viability was determined
simultaneously by the trypan blue exclusion criteria. The total number
of cells per milligram of processed sputum was calculated. Cytospin
centrifuges were prepared by placing 60 L of the cell suspension
adjusted to obtain 1.0 x 106/mL into a
cytocentrifuge (Shandon III; Shandon Southern Instruments; Sewickley,
PA) and spinning at 450 rotations per minute for 6 min. One cytospin
was dried and Wright stained, and a differential cell count was
performed on 400 nonsquamous cells.
Outcomes
The primary outcome was the percentage of eosinophils in sputum
in the different self-reporting symptom groups. The secondary outcome
was the level of airway hyperresponsiveness in the different groups and
its relationship with sputum eosinophil count and symptoms.
Analysis
Results were expressed as mean and SDs, except for data with
nonnormal distribution (sputum cell count), which were expressed as
median and interquartile range (IQR). Airway responsiveness was
expressed both as PC20 and as an index of
bronchial responsiveness calculated by a continuous index of the
decline of FEV1 during the methacholine test (BR
index).7
Despite usual transformations, the distribution of the sputum
eosinophil count could not be normalized. The mean normal sputum
eosinophil count has been reported as being
0.4 ± 0.9%.9
Most of the healthy subjects (normal and
exposed) of our population showed a sputum eosinophil count of < 1%
(Fig 1
). We have used 1% as an arbitrary cut point to express sputum
eosinophils as a binary variable 0 if < 1%, and 1 if 
1%. Blood
eosinophil count was also expressed as a binary variable, coding 0 if
< 0.2 x 109/L and 1 if
0.2 x 109/L. Symptoms of cough, wheezing,
dyspnea, chest tightness, and sputum production were recorded. The
current number of symptoms reported by the subjects was coded from 0
(no symptoms) to 5 (all five symptoms reported).
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8 mg/mL. Separate multiple
regression analyses of clinical and biological factors potentially
associated with airway responsiveness were performed. The four
subgroups were expressed as dummy variables using the normal group as
the reference group. Significance was accepted at a level of 95%. The
analysis was performed using statistical software (SPSS version 7.5;
SPSS; Chicago, IL). |
Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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8 mg/mL and
26 subjects with PC20 of < 8 mg/mL was not
significant (NS): 0.00 (0.4) vs 0.3 (1.3) [p = 0.08]. Likewise,
there was no difference in the median sputum eosinophil count between
nonatopic and atopic subjects: 0.00 (0.5) vs 0.3 (0.8) [p = 0.09]. As there was a substantial proportion of smokers in the asthma group and the wheeze group who may have a dominant neutrophilic inflammation, we compared sputum eosinophil counts excluding the smokers in each group. We again found no difference in the median sputum eosinophil count among nonsmokers in the four groups (p = 0.3).
Correlations among airway responsiveness, smoking habits and sputum and blood eosinophils, atopy, and symptoms are shown as Spearman’s correlation coefficients in Table 2 . Airway responsiveness was weakly but significantly correlated with both sputum eosinophils and atopy, while smoking habits were weakly but significantly correlated with both FEV1 and symptoms. Blood and sputum eosinophil counts were also correlated.
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our study, subject selection was from a community population self-reporting asthma or respiratory symptoms by questionnaire. In this setting, subjects did not require demonstration of airway hyperresponsiveness or FEV1 reversibility to be classified as having self-reported asthma. Indeed, 16 of the 31 self-reported asthmatic subjects (52%) had a PC20 of > 8 mg/mL, and only 11 subjects (35%) had a PC20 of < 4 mg/mL. It is therefore possible that a proportion of subjects classified as asthmatics by questionnaire did not have current asthma. Moreover, Turner et al10 found almost half the adult subjects with mildly uncontrolled asthma recruited for a drug trial did not have increased sputum eosinophils. Considering that not all asthmatic subjects with symptoms show increased eosinophil counts even when recruited in an asthma clinic, and that the subjects classified as asthmatics by questionnaire had mild asthma or did not have current asthma with increased airway responsiveness, it is not surprising that our asthmatic group did not have a significant increase in their sputum eosinophil count.
Gibson et al11 did find a significant increase in sputum eosinophils in an epidemiologic study of children, especially in those with airway hyperresponsiveness. We likewise found a higher sputum eosinophil count in subjects with airway hyperresponsiveness (PC20 < 8 mg/mL), but this difference did not achieve statistical significance and most eosinophil counts were within the normal range. Gibson et al11 undertook their study to determine whether childhood asthma was associated with airway inflammation, not to determine the usefulness of sputum as an epidemiologic tool. In their study,11 despite selecting those with symptoms within the last 2 weeks, 30% of symptomatic asthmatic children had no eosinophils in the sputum. The number with eosinophils within the normal range is not reported. Hence, in their study as well as in ours, sputum eosinphilia was not highly sensitive to the diagnosis of asthma even with recent symptoms, although somewhat more sensitive if airway responsiveness was present.
The use of inhaled corticosteroid by almost one third of our subjects reporting asthma may have reduced sputum eosinophil counts in this group, but the relatively low use of inhaled corticosteroid also reflects the mild nature of the disease reported in this study. In contrast to the ease with which an inhaled ß-agonist can be withheld in an epidemiologic study to allow measurement of baseline spirometry or airway responsiveness, inhaled corticosteroid withdrawal is not practicable given that the treatment effect may last several weeks and there are risks of exacerbating asthma by such withdrawal. This is a further reason for not advocating sputum cell measurements routinely in epidemiologic studies.
The relationship between the level of airway responsiveness and the magnitude of the airway inflammation is variable. Although some studies11 12 found a relationship between airway responsiveness and airway inflammation represented by the percentage of mast cells and eosinophils, the correlation between airway responsiveness and airway inflammation is not straightforward.13 14 15 16 17 18 We found a weak correlation between sputum eosinophil count and airway responsiveness, but sputum eosinophilia explained little of the variation in airway responsiveness. Airway responsiveness probably reflects many factors, one of which is airway inflammation. Cellular analysis of induced sputum appears to provide less positive information than measurement of airway responsiveness in epidemiologic studies. We did not find a significant correlation between respiratory symptoms and sputum eosinophils or airway responsiveness, but symptoms were correlated to smoking habits.
In summary, the spectrum of sputum eosinophil counts in adult subjects self-reporting asthma or wheezing in an epidemiologic survey did not allow the use of this test for confirmation of a diagnosis of asthma, whereas the degree of airway responsiveness was increased in subjects reporting asthma. Hence, this pilot study suggests that the analysis of differential cell counts in induced sputum is not appropriate in epidemiologic studies to validate reported diagnoses or symptoms suggesting asthma. Toelle et al19 have suggested that current symptoms (wheezing within the last 12 months) together with documented increased airway responsiveness is a useful definition for current asthma in epidemiologic studies. While this definition will exclude some with mild asthma, sputum cell counts do not add to the precision of the diagnosis.
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Footnotes |
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Support was provide by a grant from the Father Sean O’Sullivan Research Centre, St. Joseph’s Hospital, Hamilton.
Dr. Lemière was the recipient of a fellowship from Merck Canada.
Received for publication October 24, 2000. Accepted for publication February 27, 2001.
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This article has been cited by other articles:
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  L.-P. Boulet, V. Boulet, and J. Milot How Should We Quantify Asthma Control?: A Proposal Chest, December 1, 2002; 122(6): 2217 - 2223. [Abstract] [Full Text] [PDF]
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