Induced sputum in cystic fibrosis: within-week reproducibility of inflammatory markers
Introduction
The measurement of cell11ular and cytokine profiles in sputum as markers of inflammation offers a noninvasive method to determine lower airway inflammation in different disease processes [1], [2], [3]. Cystic fibrosis (CF) is characterized by lung inflammation resulting from chronic infection and the subsequent neutrophilic migration to the airways mediated by cytokines. Tumor necrosis factor-α (TNF-α), a proadhesion cytokine, allows for the subsequent diapedesis and migration of neutrophils into the damaged lung areas [4]. Furthermore, interleukin-8 (IL-8) activates neutrophils and sets up a chemotactic gradient, permitting their migration [5]. This chronic neutrophilic inflammation leads to an excess of free neutrophil elastase on the epithelial surface [6], which can injure epithelial cells and contribute to the loss of host defense systems [7]. In addition, the degeneration of the polymorphonuclear neutrophils causes the release of deoxyribonuclease from their nuclei, which in turn increases the viscosity of the CF sputum [8]. Due to the thick, tenacious nature of CF sputum, it is important to first establish the reproducibility of the actual measures in sputum to assess changes in cytokine and cellular markers. This analysis of induced sputum will further our understanding of the inflammatory process in CF and may thereby permit an evaluation of the response to therapies.
There have been many studies utilizing various dispersal agents and methods for specific biochemical and cellular evaluations of sputum from different patient populations [2], [9], [10], [11], [12], [13], [14], [15]. These have examined reproducibility of sputum fluid phase and cellular indices of airway inflammation in asthmatics [16], [17], [18], [19], [20], smokers [17], patients with COPD [21], and healthy subjects [17], [20], [22]. In patients with CF, sputum reproducibility studies have been rare. Ordoñez et al. [23] reported cytokine and cellular reproducibility in induced sputum of CF patients, dispersed with dithiothreitol, done on two study visits separated by 3 weeks of placebo treatment.
Dithiothreitol (DTT) is commonly used for the dispersal of sputum samples from asthma patients and shown to give reliable measurements of cellular and fluid-phase markers [12]. Cai et al. [13] showed that DTT alone was not as effective as DTT with the triple enzyme mixture [deoxyribonuclease (DNase), hyaluronidase, and β-galactosidase] in dispersing CF sputum perhaps because of the viscosity. Total cell counts (TCCs) and neutrophil counts were higher in the samples treated with DTT and enzyme mixture, but elastase immunoreactivity was lost. We used a modification of this method to assess the reproducibility of sputum samples taken within the same week for total and differential cell counts, IL-8, TNF-α, and neutrophil elastase. Within-week reproducibility was assessed to minimize the risk of changes due to the clinical condition and allowed for the assessment of biological variability in two separate induced sputum samples.
Section snippets
Subjects
Seventeen patients with a diagnosis of cystic fibrosis underwent sputum induction twice within the same week (3 days apart) with no change in patient management. All subjects were required to have been stable (no hospitalizations or courses of oral corticosteroids) for 2 months before entrance to the study. The study was approved by the Montreal Children's Hospital's Review Board and all subjects gave informed consent.
Sputum induction and processing
Before commencement of the sputum induction procedure, subjects were
Results
Of the 17 subjects (9 males, 8 females) [24.0 (5.0) years of age, 64.0 (18.0) FEV1 %predicted, median (quartile range)], there were 2 occasions where insufficient sample was obtained to allow for both chemical and cellular analysis. All patients tolerated the induction procedure; however, lung function was not measured during the procedure. One set of samples for %Neut and TNF-α analysis was discarded due to technical error. Data analysis was subsequently performed on 15 sample sets for TCC
Discussion
Our results show good reliability in our methodology for TCC, percentage of neutrophil counts, IL-8, and neutrophil elastase. Within-week reproducibility was chosen to avoid changes in clinical status, which can occur over several weeks. Prior studies have looked at cytokine and cellular analysis. The intraclass correlation coefficients are similar to those previously published in terms of cellular markers in other patient populations [1], [17], [18], [22]. The cell counts in our patients are
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