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(Chest. 2004;126:1811-1814.)
© 2004 American College of Chest Physicians

The Influence of Age on Induced Sputum Differential Cell Counts in Normal Subjects*

Rebecca A. Thomas, MB; Ruth H. Green, MD; Chris E. Brightling, PhD; Surinder S. Birring, MD; Debbie Parker, BSc (Hons); Andrew J. Wardlaw, MD and Ian D. Pavord, DM

* From the Institute for Lung Health, Department of Respiratory Medicine, Glenfield Hospital, University of Leicester NHS Trust, Leicester, UK.

Correspondence to: Ian Pavord, DM, Department of Respiratory Medicine, Glenfield Hospital, Groby Rd, Leicester LE3 9QP, UK; e-mail: ian.pavord{at}uhl-tr.nhs.uk


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Sputum induction is increasingly used as a research technique and as a clinical tool. In order to evaluate abnormal results, normal ranges need to be fully developed. Although a number of studies have described normal ranges, none have investigated the effect of the age of the subject on these results. This study was undertaken to assess whether there are age-related differences in sputum cell differential cell counts in a population of normal, healthy volunteers.

Study design and participants: Induced sputum samples were obtained from 66 healthy, nonsmoking subjects (24 men) with a mean age of 44 years (age range, 18 to 74 years). Differential cell counts were related to age.

Results: Sputum neutrophil counts were found to correlate significantly with the age of the volunteers (r = 0.58; p < 0.001). Macrophage counts showed a proportionate, inverse correlation with increasing age (p < 0.01), but no correlation was seen for any other cell type. On subanalysis according to age range, the mean neutrophil differential increased from 26.9% (SD, 19.8%) [17 patients] in the group of patients who were 0 to 29 years of age to 68.5% (SD, 20.6%) [11 patients] in the group of patients who were > 60 years of age.

Conclusion: In our healthy volunteer population, the induced sputum differential neutrophil count increased significantly with age. These findings highlight the need for age matching in controlled studies.

Key Words: age • induced sputum differential cell counts


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
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The first reports on the microscopic appearance of sputum and its use in a clinical setting were made almost 50 years ago.1 Over the last decade, the technique has evolved into a simple, safe, valid, and repeatable noninvasive method for measuring airway inflammation in the clinic and in a research setting.

Initial reports identified the patterns of inflammatory cells seen in various respiratory diseases,234 with small subject cohorts used as the healthy population. As the technique became more standardized and validated,56 several authors described normal ranges in larger populations of healthy adults.78 Belda et al7 collected induced sputum samples from a total of 118 healthy nonsmoking adults, with an age range of 18 to 60 years (mean age, 36 years), whereas in the second study,8 induced sputum was obtained from 114 healthy volunteers with a mean age of 38 years. A subanalysis for gender differences was performed, but the effect of subject age was not reported. We set out to investigate the effects of age on sputum differential cell counts in a healthy adult population.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study Population
Induced sputum differential cell counts were obtained from 66 healthy adults with an age range of 18 to 74 years (mean, 44 years; SD, 16 years). All were current nonsmokers with a < 5 pack-year history, and 24 were men. No patients reported symptoms of respiratory disease, and all had FEV1 values > 70% predicted, FEV1/FVC ratios of > 80%, and normal airway responsiveness (ie, provocative concentration of methacholine producing a 20% decrease in FEV1, ≥ 16 mg/mL).

Study Design
Healthy control subjects were recruited into the study over a 6-year period. Details of the clinical history were obtained, and subjects were excluded if they had experienced any respiratory symptoms or had a history of respiratory disease. All subjects scored 0 on a 100-mm global respiratory symptom visual analog scale.9 Spirometry was performed with a spirometer (Compact spirometer; Vitalograph; Buckinghamshire, UK). The methacholine challenge was performed using the tidal breathing method10 with doubling doses of methacholine (0.03 to 16 mg/mL) administered with a Wright nebulizer.

Sputum was induced and processed as has been previously described.5 Briefly, 3%, 4%, and 5% saline solutions were inhaled in sequence, each for 5 min, via a low-output (0.9 mL/min; median particle size, 5 µm) ultrasonic nebulizer (Medix; Harlow, UK), 10 min after inhaling 200 µg salbutamol. Induced sputum was collected and processed within 2 h of expectoration. Sputum plugs were separated and mixed with 0.1% dithiothreitol. After processing and preparation of a cytospin, differential cell counts were obtained after staining with Romanowsky stain.

Statistical Analysis
All analyses were performed using a statistical software package (SPSS-pc, version 10.1; SPSS; Chicago, IL). All differential cell counts followed a normal distribution, so they are expressed as the mean (SD), and correlation was performed using the Pearson correlation coefficient.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The mean sputum differential cell count with SD for the total population and subdivided by age is shown in Table 1 . There was a significant, positive correlation between increasing age and sputum neutrophil count in the total population (r = 0.58; p < 0.001) [Fig 1 ] and when analyzed separately by gender (women: r = 0.57; p = 0.001; men: r = 0.60; p < 0.01). The increase in sputum neutrophil count was particularly evident in the group of subjects aged 50 to 59 years, and it did not appear to increase beyond this age (Table 1). There was a proportionate decrease in macrophages with age (r = –0.58; p < 0.01), but no correlation was seen for any other cell type.


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  		Table 1.. Differential Cell Counts and Total Cell Count for All Subjects and Subgrouped by Age*

 


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  		Figure 1.. Relationship between sputum neutrophil differential and age. {blacksquare} = male patients; {square} = female patients; straight line = mean regression.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the sputum neutrophil differential cell count increased with age in normal healthy adults. The effect was seen in both male and female subpopulations, and was most evident in subject > 50 years of age. A similar trend for sputum neutrophil counts to increase with age has been reported in patients with asthma.11 Our findings suggest that this effect is not disease specific, although further studies assessing the effect of age on sputum neutrophil counts in other disease categories are required to be sure on this point. The effect of age on sputum cell counts has important implications for the interpretation of sputum differential cell counts in older subjects and of research findings in an older population

Two studies78 have previously estimated normal ranges for sputum differential cell counts in large healthy populations. Both studies examined > 100 subjects, but neither reported a significant age effect. However, both studies included relatively few subjects > 50 years old, so such an age effect could have been missed, particularly as our data suggested that the age effect is principally evident in subjects who are > 50 years of age. Our study also was limited by a small number of subjects in the older age range, but the effect seen seems large and therefore likely to be real. However, larger studies are required to address this important question more fully.

Why does subject age affect the sputum differential cell count in this way? The effect could relate to the difficulty in recruiting a true healthy population at this age, although we think that our criteria are reliable and will be widely accepted. Nevertheless, it is possible that subtle, subclinical disease is present in some of our patient population, perhaps as a result of longstanding environmental exposure. There may also be age-related changes in the immune response in the lung, since BAL percentages of CD4+ lymphocytes and the CD4+/CD8+ ratio increase in the fifth decade,12 particularly in women. Conceivably, this may lead to an amplification of the immune response to environmental and other triggers. However, the age effect seen in our study was not confined to women, suggesting that this mechanism is unlikely to be of fundamental importance in increasing neutrophil counts. Another possibility is that the effect of age on sputum neutrophil count was due to age-related differences in neutrophil viability after expectoration. Further studies are required to investigate this possibility. It is important to emphasize that any such effect is likely to be relevant to other studies since we used standard methods to induce and process the sputum.5

Our results have important implications for the interpretation of research findings. Previous studies have reported increased sputum neutrophil differential cell counts in patients with chronic obstructive airways disease13 and idiopathic chronic cough.14 However, the control populations were not well-matched for age, suggesting that these observations may represent an artifact. Our findings highlight the importance of careful age matching in future comparative studies.


   Footnotes
 
Dr. Thomas was supported by The Wellcome Trust and the University of Leicester NHS Trust. The study was supported by grants from Astra Zeneca, the National Asthma Campaign, and The British Lung Foundation.

Received for publication March 17, 2004. Accepted for publication July 15, 2004.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Brown, HM (1958) Treatment of chronic asthma with prednisolone. Lancet ,1245-1247
  2. Fahy, JW, Lim, J, Wong, H, et al Cellular and biochemical analysis of induced sputum from asthmatic and from healthy subjects. Am Rev Respir Dis 1993;147,1126-1131[ISI][Medline]
  3. Gibson, PG, Girgis-Gabardo, A, Morris, MM, et al Cellular characteristics of sputum from patients with asthma and chronic bronchitis. Thorax 1989;44,693-699[Abstract]
  4. Hargreave, FE, Popov, T, Kidney, J, et al Sputum measurements to assess airway inflammation in asthma. Allergy 1993;48,81-83[Medline]
  5. Pavord, ID, Pizzichini, MMM, Pizzichini, E, et al The use of induced sputum to investigate airway inflammation. Thorax 1997;52,498-450[ISI][Medline]
  6. Pizzichini, E, Pizzichini, MMM, Ethimiadis, A, et al Indices of airway inflammation in induced sputum: reproducibility and validity of cell and fluid-phase measurements. Am J Respir Crit Care Med 1996;154,308-317[Abstract]
  7. Belda, J, Leigh, R, Parameswaran, K, et al Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000;161,475-478[Abstract/Free Full Text]
  8. Spanevello, A, Confalonieri, M, Sulotto, F, et al Induced sputum cellularity. Am J Respir Crit Care Med 2000;162,1172-1174[Abstract/Free Full Text]
  9. Brightling, CE, Monterio, W, Green, RH, et al Induced sputum and other outcome measures in chronic obstructive pulmonary disease: safety and repeatability. Respir Med 2001;95,999-1002[CrossRef][ISI][Medline]
  10. Juniper, EF, Cockcroft, DW, Hargreave, FE Histamine and methacholine inhalation tests: a laboratory tidal breathing protocol 2nd ed. 1994 Astra Draco AB. Lund, Sweden:
  11. Woodruff, PG, Khashayar, R, Lazarus, SC, et al Relationship between airway inflammation, hyperresponsiveness and obstruction in asthma. J Allergy Clin Immunol 2001;108,753-758[CrossRef][ISI][Medline]
  12. Mund, E, Christensson, B, Larsson, K, et al Sex dependent differences in physiological ageing in the immune system of lower airways in healthy non-smoking volunteers: study of lymphocyte subsets in bronchoalveolar lavage fluid and blood. Thorax 2001;56,450-455[Abstract/Free Full Text]
  13. Keatings, VM, Collins, PD, Scott, DM, et al Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153,530-534[Abstract]
  14. Jatakanon, A, Lalloo, UG, Lim, S, et al Increased neutrophils and cytokines, TNF-alpha and IL-8, in induced sputum of non-asthmatic patients with chronic dry cough. Thorax 1999;54,234-237[Abstract/Free Full Text]



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This Article
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