Induced Sputum Cell Counts in Healthy Adults

JOSE BELDA, RICHARD LEIGH, KRISHNAN PARAMESWARAN, PAUL M. O'BYRNE, MALCOLM R. SEARS, and FREDERICK E. HARGREAVE

Asthma Research Group, Department of Medicine, St. Joseph's Hospital, McMaster University, Hamilton, Ontario, Canada

	ABSTRACT

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Induced sputum cell counts provide a relatively noninvasive method to evaluate the presence, type, and degree of inflammation in the airways of the lungs. Their interpretation requires a knowledge of normal values from a healthy population. The objective was to examine the total and differential cell counts in induced sputum from a sample of healthy adults. A total of 118 healthy nonsmoking adults were studied. None had asthma or airflow obstruction (negative history, FEV₁  80% predicted, ratio of FEV₁ to vital capacity [FEV₁/VC]  80%, methacholine PC₂₀  16 mg/ml). Forty-six were atopic. Sputum induction produced an adequate sample in 96 subjects [53 males, mean age (range) 36 (18 to 60) yr]. The expectorate was processed within 2 h; sputum was selected, treated with dithiothreitol, filtered, and examined in a hemocytometer for total cell count and viability and on Wright-stained cytospins for a differential cell count. The mean, median (90th percentile) total cell count was 4.1, 2.4 (9.7) × 10⁶ cells/g and cell viability was 69.6, 72.0 (89.7)%. The proportions of eosinophils were 0.4, 0.0 (1.1)%, neutrophils 37.5, 36.7 (64.0)%, macrophages 58.8, 60.8 (86.1)%, lymphocytes 1.0, 0.5 (2.6)%, metachromatic cells 0.0, 0.0 (0.04)%, and bronchial epithelial cells 1.6, 0.3 (4.4)%, respectively. Female gender and atopy were associated with a significant elevation of eosinophils; mean difference between male/female was 0.3% (p = 0.043) and between atopic/nonatopic 0.4% (p = 0.024). This study has identified reference values for total and differential cell counts in induced sputum of healthy adults. Belda J, Leigh R, Parameswaran K, O'Byrne PM, Sears MR, Hargreave FE. Induced sputum cell counts in healthy adults.

	INTRODUCTION

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Airway inflammation is considered to be the primary cause of asthma and other airway diseases, and is associated with exacerbations (1) and airway remodeling (2). The measurement of airway inflammation has been facilitated by the examination of induced sputum for cell and fluid-phase constituents. The method is relatively noninvasive and is safe. The cell count results are repeatable, valid as illustrated by differences between diseases of different pathogenesis, and responsive to change induced by pro- and anti-inflammatory stimuli (3).

The interpretation of the results of induced sputum examination depends on a knowledge of normal values from a healthy population. So far these have only been published in small numbers of subjects in whom the characteristics were often not well defined.

In this study we aimed to determine reference values for total and differential cell counts in induced sputum from healthy nonsmoking adults.

	METHODS

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Subjects

A total of 118 healthy adult subjects were recruited from advertisements in the local newspapers and hospitals, from research staff, and from healthy subjects recruited from a randomly selected population in another epidemiologic study. They had no nasal or chest symptoms and no past history of asthma or other chronic respiratory diseases. No subject was a previous or current smoker. All had normal spirometry (FEV₁  80% predicted, ratio of FEV₁ to vital capacity [FEV₁/VC]  80%), and normal methacholine airway responsiveness (provocative concentration of methacholine causing a 20% reduction in FEV₁ [PC₂₀]  16 mg/ml). Forty-six subjects were atopic as indicated by one or more positive allergy skin tests to common allergen extracts. None had symptoms of a cold in the past month and none of the atopic subjects had been exposed to an allergen to which they were sensitized in the past week. The study was approved by the Research Committee of St. Joseph's Hospital and all subjects gave written informed consent.

Design

Subjects were seen in the research laboratory of the Firestone Regional Chest and Allergy Unit at St. Joseph's Hospital on one day. Subject characteristics were documented and spirometry and a methacholine inhalation test were performed. If these indicated that the subject was healthy, sputum induction and allergy skin tests were performed immediately thereafter.

Procedures

Subject characteristics were documented by questionnaire. Spirometry was performed according to American Thoracic Society specifications (6). Predicted values were taken from Crapo and coworkers (7). Baseline FEV₁ was expressed as the best of three reproducible values (with a maximal change of 5%). Methacholine inhalation tests were carried out by the method described by Juniper and colleagues (8) and the results expressed as PC₂₀ in noncumulative units. Allergy skin prick tests were performed (if not done during the preceding year) using a modified prick technique (9) with extracts of 12 common inhalant allergens and a negative (glycerol and buffer) and a positive (histamine acid phosphate 10 mg/ml) control. A positive test was indicated by a wheal  3 mm greater than the control. We defined atopy as one or more positive tests.

Sputum was induced by the method described by Pizzichini and coworkers (4) by inhaling increasing concentrations of hypertonic saline (3%, 4%, and 5%) each for 7 min, through a mouthpiece without a valve or nose clip. At the start and after each period of inhalation, FEV₁ was measured for safety. Subjects were asked to blow their nose, rinse their mouth, and swallow the water to minimize contamination from postnasal drip and saliva. Then they were instructed to cough sputum into a sterile container. The sputum was examined within 2 h as described by Pizzichini and coworkers (4). Briefly, all opaque or dense portions that looked different from saliva or were free of squamous cell contamination under the inverted microscope were selected from the expectorate, weighed and treated with 0.1% dithiothreitol (Sputalysin; Calbiochem Corp., San Diego, CA) and phosphate-buffered saline (PBS). The resulting suspension was filtered and a total nonsquamous cell count was performed in a hemocytometer and expressed as millions per gram of selected induced sputum. The proportion of salivary squamous cells was noted and cell viability was determined by the trypan blue exclusion method. From the remainder of the filtrate, two cytospins were made and stained with Wright's stain and a 400 differential nonsquamous cell count was performed. Another two cytospins were stained with toluidine blue and 1,500 cells were counted for metachromatic cells. The differential cell count, including metachromatic cells, was expressed as the absolute number of cells in millions per gram of sputum and as the percentage of the total nonsquamous and bronchial epithelial cells. The specimen was considered adequate if total and differential cell counts could be obtained; this required as little as 50 mg of selected material.

Statistical Analysis

In view of the importance of eosinophils in asthma pathophysiology, the study was planned to estimate the actual eosinophil count in the general adult population with a precision of ± 1%. This was done by assuming from previously published data (4) that the population mean percentage of eosinophils was approximately 0.6%. The criterion for significance, alpha, was set at 0.05 and the power at 80%, giving an estimated sample size of 88 subjects to which 20% was added (18 subjects) for expected attrition owing to unsuccessful sputum inductions.

Cell counts were expressed as arithmetic mean (standard deviation, SD) or median (interquartile range, IQR) depending on their normal distribution. The normal range was expressed by mean ± 2 SD, as well as the median and 10th and 90th percentiles.

The difference in the cell counts between gender and atopic groups was assessed by independent t tests. The effect of age and FEV₁ on sputum cell counts was assessed by linear regression. Significance was accepted at 95%.

	RESULTS

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Sputum induction was successful in 96 subjects (81% of the recruited subjects). The induced sputum samples had a mean weight (SD, range) of 291 mg (93, 179 to 447 mg) with cell viability of 70% (17, 23 to 96%). The selected sputum was mucoid and was minimally contaminated with saliva as indicated by a mean squamous cell count of 4% (SD 6%). It was of lower respiratory origin as indicated by the presence of macrophages and bronchial epithelial cells.

We examined the total and differential nonsquamous cell counts, the distribution of eosinophils and neutrophils as a percentage of the total inflammatory cells, and the upper acceptable levels of each cell (Tables 1, 2, and Figure 1). Induced sputum cell counts in healthy subjects are characterized by a predominance of macrophages and neutrophils and a paucity of eosinophils, lymphocytes, metachromatic cells, and bronchial epithelial cells.

View this table: [in this window] [in a new window]   TABLE 1 SPUTUM TOTAL AND ABSOLUTE DIFFERENTIAL CELL COUNTS (× 106 cells/g)

View this table: [in this window] [in a new window]   TABLE 2 SPUTUM DIFFERENTIAL CELL PERCENTAGES

View larger version (15K): [in this window] [in a new window]   Figure 1.   Histogram of eosinophils and neutrophils in absolute counts and percentages. X-axis labels represent the center of the interval.

We also compared cell counts by gender, atopy, FEV₁, slow VC, and FEV₁% (Table 3). Absolute and percentage eosinophil counts were higher in females [absolute counts: mean difference (95% confidence interval) 0.003 × 10⁶ cells/g (0.000 to 0.015) (p = 0.049); percentage counts: 0.274% (0.001 to 0.651) (p = 0.043)] and in atopic subjects [absolute counts: 0.006 × 10⁶ cells/g (0.001 to 0.020) (p = 0.044); percentage counts: 0.436% (0.059 to 0.813) (p = 0.023)]. Atopy was not more common in female subjects, and these differences remained significant when adjusting atopy by gender. No other cell types showed significant differences between gender or atopy and no cell type showed a significant relationship with spirometric indices.

View this table: [in this window] [in a new window]   TABLE 3 SPIROMETRY AND SPUTUM CELL PERCENTAGES BY GENDER AND ATOPY*

	DISCUSSION

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The results of this study identify a normal range of cell counts in induced sputum for nonsmoking healthy adults. They show that the majority of the cells are neutrophils and macrophages, whereas eosinophils, lymphocytes, and bronchial epithelial cells are scarce and metachromatic cells (basophils/mast cells) are almost absent. They also show a small effect of gender and atopy on eosinophil counts. This knowledge of the normal range, and the factors that can modify this, are needed for the accurate interpretation of sputum cell results.

This is the first report to examine the normal range of sputum cell counts in healthy adults. Although many studies have included healthy subjects within their study sample (3, 4, 10- 26), defining the normal range was not their objective, the sample sizes were small ranging between six and 20 (14, 15), and the definition of the healthy subject varied and was incomplete. For these reasons, the reports did not allow the normal range from a healthy population to be described with enough precision. In the present study the sputum induction, for convenience, followed immediately after a methacholine inhalation test. We considered that this was unlikely to influence the results, a view that has been supported by Spanevello and coworkers (27) who found no effect on cell counts 1 h after methacholine inhalation. The results reported here are close to what we recorded previously in 10 subjects (4), with the exception of the total cell count which is higher.

The generalization of the normal values reported here should take into account the environment within which the study was conducted. Hamilton is an industrial midsize city and atmospheric pollutants may increase lung macrophages (28) or neutrophils (29, 30). Furthermore, the method of processing sputum may have an influence on both the total and differential counts. We have examined sputum selected from the expectorate of sputum plus saliva, whereas others examined the whole expectorate. The total cell count will be higher when it is expressed per gram of selected sputum, but it will be reduced by the process of selection and filtering the suspension of dithiothreitol and PBS-treated sputum before counting. The differential cell count should not be altered by whether sputum or expectorate is examined, although there is one report (23) that eosinophils are higher and neutrophils are lower in selected sputum. We believe the latter result was probably caused by salivary squamous cell contamination in the expectorate which can interfere with the accuracy of counting of these cells (31).

We have shown a significant positive influence of atopy and female gender on the eosinophil counts. However, this influence is small as indicated by the difference between atopic and nonatopic subjects of 0.44% and between female and male subjects of 0.27%. The influence of atopy on eosinophils is recognized (32) and eosinophils have receptors that are highly responsive to some hormones (33, 34). Other studies have also identified an influence of atopy and gender on blood eosinophilia. Mensinga and coworkers (35) described higher blood eosinophil counts in subjects with positive allergy skin tests. However, in contrast to our results and those of others (37), they observed higher eosinophil counts in males. A possible explanation for the latter is that their random sample from the general population included symptomatic and smoking subjects, which are confounding factors.

In summary, we have determined the cell profile of induced sputum for the general adult population with enough accuracy to provide a normal range for similar populations using similar methods of induced sputum examination. Sputum eosinophil counts are slightly higher in atopic subjects and females.

	Footnotes

Correspondence and requests for reprints should be addressed to Dr. F. E. Hargreave, Firestone Regional Chest and Allergy Unit, St. Joseph's Hospital, 50 Charlton Avenue East, Hamilton, ON, L8N 4A6 Canada. E-mail: hargreav{at}fhs.mcmaster.ca

(Received in original form March 18, 1999 and in revised form July 21, 1999).

Acknowledgments: The authors thank the subjects for volunteering for this study, Dr. G. S. Berlyne and Dr. Catherine Lemière for the recruitment of subjects, Denise O'Shaughnessy, Corinne Walker, and Megan O'Connor for help with the clinical procedures, and Ann Efthimiadis, Sharon Weston, Susan Carruthers, and Tracy Rerecich for performing the cell counts.

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