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Airway Eosinophilia in Children with Severe Asthma

Predictive Values of Noninvasive Tests

Departments of Pediatric Respiratory Medicine and Histopathology, and Cell Biology and Clinical Studies Units, Imperial College of Science, Technology, and Medicine at the Royal Brompton Hospital and National Heart and Lung Institute, London, United Kingdom; and Department of Paediatric Cardiology and Pulmonology, Heinrich Heine University Düsseldorf, Düsseldorf, Germany

Correspondence and requests for reprints should be addressed to Prof. Andrew Bush, M.D., Department of Pediatric Respiratory Medicine, Imperial College of Science, Technology, and Medicine at the Royal Brompton Hospital and National Heart and Lung Institute, Sydney Street, London SW3 6NP, UK. E-mail: a.bush{at}rbht.nhs.uk

	ABSTRACT

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Rationale: Children with severe asthma experience persistent symptoms despite maximal conventional treatment. Fraction of exhaled nitric oxide (FE_NO) and sputum eosinophils are used as markers of airway inflammation to guide treatment with steroids, but no data are available on how reliable they are in predicting airway eosinophilia assessed bronchoscopically in these children.

Objectives: To determine how FE_NO and sputum eosinophils predict airway eosinophilia measured in both bronchoalveolar lavage (BAL) and endobronchial biopsy.

Methods: Twenty-seven children with moderate to severe persistent asthma attempted measurement of FE_NO and sputum eosinophils, followed by bronchoscopy, BAL, and endobronchial biopsy within 24 h.

Main Results: Significant correlations were found between eosinophils in sputum and both BAL eosinophils (n = 20, r = 0.45, p = 0.045) and FE_NO (n = 23, r = 0.42, p = 0.049). The relationship between FE_NO and BAL eosinophils was also significant with a stronger correlation (n = 24, r = 0.54, p = 0.006). The positive predictive value (PPV) for increased sputum eosinophil percentage (> 2.5%) to detect elevated eosinophils in BAL (> 1.19%) was 75%; the negative predictive value (NPV) was 63%. All patients with both increased sputum eosinophils and an elevated FE_NO value (> 23 ppb) had elevated eosinophils in BAL (PPV, 100%); the NPV of these two markers was 65%. Eight of nine patients without any sputum eosinophils had normal subepithelial eosinophil numbers (< 1.2%; NPV, 89%). However, the PPV of any sputum eosinophils for increased subepithelial eosinophilia was only 36.4%.

Conclusions: There was moderate agreement between both FE_NO and sputum eosinophils and BAL eosinophils. There was good NPV, but only poor PPV for these markers for mucosal eosinophilia.

Key Words: airway inflammation • bronchoalveolar lavage • endobronchial biopsy • exhaled nitric oxide • sputum induction

AT A GLANCE COMMENTARY TOP ABSTRACT AT A GLANCE COMMENTARY METHODS RESULTS DISCUSSION REFERENCES   Scientific Knowledge on the Subject Asthma is characterized by eosinophilia in sputum, in bronchoalveolar lavage, and in the bronchial wall, but the relationship between the three compartments is not known. What This Study Adds to the Field We show that, whereas bronchoalveolar lavage and sputum eosinophil counts are very similar, they do not relate to airway wall eosinophilia; it is unclear which is clinically important.

 
Children with difficult asthma experience persistent symptoms despite maximal conventional treatment. The management of these patients is complex, with little evidence to guide clinicians. Treatment decisions are usually made on the basis of reported symptoms and objective measures of lung function. However, recent research suggests that monitoring airway inflammation, in particular sputum eosinophils and fraction of exhaled nitric oxide (FE_NO), as a surrogate may also have a role in guiding steroid therapy (1–3). Such an approach is attractive for children with difficult asthma, in whom every attempt should be made to avoid the prescription of excessively high doses of inhaled or systemic corticosteroids.

Endobronchial biopsy and bronchoalveolar lavage (BAL) have traditionally been used for assessment of airway inflammation. However, bronchoscopy is not practical for most children or most centers. Even in specialist centers, bronchoscopic assessment of airway inflammation cannot be performed repeatedly. Longitudinal studies that have investigated the role of markers of eosinophilic airway inflammation to guide therapy have therefore used noninvasive methods, such as FE_NO and induced sputum. Previously, a number of research groups have demonstrated an association between FE_NO and airway eosinophils, measured bronchoscopically either using BAL or endobronchial biopsy, in children and adolescents with asthma (4–6). However, to our knowledge, there are no complete data in this age group investigating the relationship between noninvasive markers (induced sputum eosinophils, FE_NO) and eosinophils in either BAL or endobronchial biopsy. This is important because if the aim of treatment is to prevent bronchial inflammation, incorrect use of noninvasive markers could lead to the wrong doses of inhaled steroids being prescribed. The hypothesis of this study is that FE_NO and sputum eosinophils are closely related to BAL and mucosal eosinophilia.

The aim of this study was therefore to investigate the relationships between FE_NO and eosinophils in induced sputum, BAL, and the bronchial subepithelium in a group of children and adolescents with asthma undergoing bronchoscopy as part of their clinical evaluation. This involves assessment of lung function, FE_NO, and sputum cytology, followed by a bronchoscopy, BAL, and endobronchial biopsy. Most children follow our difficult asthma protocol, in which measurements are done after a course of systemic corticosteroids, usually prednisolone 40 mg daily, including noninvasive assessments of inflammation at the beginning of this course. The full details are discussed elsewhere (7), and given in more detail in the online supplement. We specifically aimed to determine how often FE_NO or sputum eosinophil values miscall BAL or endobronchial eosinophils. Some of the results of this study have been previously reported in the form of an abstract (8).

	METHODS

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Subjects
We recruited 27 children and adolescents with moderate to severe persistent asthma who underwent bronchoscopy, including endobronchial biopsy, as part of their clinical assessment (7, 9). Asthma was diagnosed according to American Thoracic Society (ATS) guidelines (10). Moderate to severe persistent asthma was characterized by "daily symptoms despite > 400 µg of inhaled budesonide (or equivalent)" (11). Patient characteristics are shown in Table 1.

FE_NO Measurement
FE_NO was measured using a chemiluminescence analyzer (NIOX; Aerocrine, Stockholm, Sweden) according to European Respiratory Society/ATS guidelines (12). The upper limit of normal for FE_NO was taken as 23.0 ppb (13).

Spirometry, Sputum Induction and Processing
Spirometry before and after bronchodilator and sputum induction and processing was performed as previously described (14). For subjects with a post-bronchodilator FEV₁ less than 65% predicted, sputum induction was performed with 0.9% instead of 3.5% saline. A normal sputum eosinophil percentage was defined as approximately 2.5% (14).

Bronchoscopy, BAL and Biopsy Processing
Bronchoscopy was performed as previously described (9). For the BAL, three aliquots of normal saline (1 ml/kg, maximum 40 ml) were instilled in the right middle lobe through the bronchoscope and the fluid retrieved by mechanical suction. Up to four endobronchial biopsies were taken from the subsegmental carinae of the right lower lobe using cupped forceps. Biopsies were initially fixed in formal saline.

BAL processing is described in the online supplement. A normal BAL eosinophil percentage was defined as less than 1.19% (15).

Endobronchial biopsies were processed into paraffin blocks. Five-micrometer sections were cut and stained with hematoxylin and eosin to assess morphology. Suitable sections (those with visible epithelium, reticular basement membrane, and subepithelium) were stained for eosinophils using EG2 antibodies as previously described (16). Positively stained cells were quantified using the validated stereologic technique of point counting as previously described (16), and expressed as volume density (%). For each subject, one section from between one to four biopsies was analyzed. A normal eosinophil volume density was defined as less than 1.2%, based on previous data from nonasthmatic control subjects (5, 16). All measurements were made independently and investigators were blinded to other results.

Statistical Analyses
All analyses were performed using the Statistical Package of the Social Sciences (SPSS for Windows, release 12.0; SPSS, Inc., Chicago, IL). Correlations were measured using Spearman rank correlation. A p value less than 0.05 was considered statistically significant. Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values were calculated, and receiver operator characteristic (ROC) curves constructed according to Altman and Bland (17–19).

	RESULTS

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Details of the subjects are shown in Table 1. Seventeen children were approximately 12 yr old; eight of these children were male and nine were female. Nineteen patients were included in our difficult asthma protocol, receiving either a course of oral corticosteroids or a single intramuscular injection of triamcinolone 2 wk preceding the study (7). There were no significant adverse events during sputum induction and bronchoscopy. We found anatomic abnormalities in 3 of 27 (11%) patients: a short right main bronchus with an abnormal branching pattern in one patient and tracheomalacia in two patients. Six patients had positive BAL cultures (Staphylococcus aureus, n = 4; Haemophilus influenzae and Streptococcus pneumoniae, n = 1 each).

FE_NO could be measured in all subjects. The median (interquartile range [IQR]/absolute range [AP]) value was 20.3 (9.6–46.3/4.2–100.1) ppb. Eleven of 27 subjects had elevated values. Sputum induction was successful in 23 of 27 (85%) subjects. The median (IQR/AP) eosinophil percentage was 0.5% (0–2.5/0–23.25%). Detectable eosinophils were found in 12 subjects, of whom 5 had elevated values.

A satisfactory BAL cytospin was obtained in 24 of 27 (90%) subjects (median [IQR/AP] eosinophils: 1.0% [0.3–2.5/0–14.0%]). Ten subjects had BAL eosinophils above the normal range. Subepithelial eosinophils could be measured in 22 subjects. Five had values above the normal range.

There was a significant negative correlation between age and pre- and post-FEV₁ (r = –0.64, p = 0.001, and r = –0.61, p = 0.002, respectively) but not between age and FE_NO, sputum eosinophils, or BAL or biopsy eosinophils.

Pre- or post-bronchodilator FEV₁ alone did not correlate with any of the other parameters (FE_NO, or sputum, BAL, or biopsy eosinophils). However, bronchodilator reversibility correlated significantly with the percentage of sputum eosinophils (r = 0.473, p = 0.035), but not with FE_NO or BAL or biopsy eosinophils. There was no significant difference in pre- or post-bronchodilator FEV₁, FE_NO, or sputum, BAL, or biopsy eosinophils whether patients had been using rescue bronchodilator at least daily or less than daily.

Comparison between FE_NO and Sputum Eosinophils
There was 61% concordance (n = 14) between the two markers. Thirty percent of patients (n = 7) had a raised FE_NO but no elevation in sputum eosinophils; 9% (n = 2) had raised sputum eosinophils but normal FE_NO (Table E1 of the online supplement). There was a positive significant correlation between sputum eosinophils and FE_NO (n = 23, r = 0.42, p = 0.049; Figure 1).

View larger version (9K): [in this window] [in a new window]   Figure 1. Spearman's correlations (r) between sputum eosinophils and FENO. Open symbols indicate patients without systemic steroids. The upper limit of normal is shown by the dashed line.

View larger version (9K): [in this window] [in a new window]   Figure 2. Spearman's correlations (r) between biopsy and bronchoalveolar lavage (BAL) eosinophils. Open symbols indicate patients without systemic steroids. The upper limit of normal is shown by the dashed line.

Comparison between FENO and sputum and BAL.
There was a significant relationship between sputum and BAL eosinophils (n = 20, r = 0.45, p = 0.045; Figure 3). There was also a positive relationship between FE_NO and BAL eosinophils and the correlation between both markers was even stronger (n = 24, r = 0.54, p = 0.006; Figure 3). We analyzed how well the presence or absence of elevated sputum eosinophil percentages or increased or normal FE_NO values predicted the presence or absence of eosinophils in BAL (Table 2). The PPVs were 75 and 64% and the NPVs were 63 and 77%, respectively. We also analyzed the predictive values for combining elevated sputum eosinophils and raised FE_NO values to predict increased BAL eosinophils. Because all children with both elevated FE_NO values and increased sputum eosinophil percentages had increased BAL eosinophils, the PPV was 100%. However, the NPV was 65% for this combination. Figure 3 also shows the ROC curves of FE_NO and sputum eosinophils for prediction of BAL eosinophilia at various cutoff points. The area under the curve (AUC) was 0.850 for FE_NO (p = 0.004) and 0.747 for sputum eosinophils (p = 0.063).

View larger version (35K): [in this window] [in a new window]   Figure 3. (A) Spearman's correlation between BAL eosinophils and FENO (for A–B, open symbols indicate patients without systemic steroids; the upper limit of normal is shown by the dashed line). (B) Spearman's correlation between BAL eosinophils and sputum eosinophils. (C) Receiver operator characteristics (ROC) curve for FENO for predicting BAL eosinophilia. (D) ROC curve for sputum eosinophils for predicting BAL eosinophilia.

View larger version (18K): [in this window] [in a new window]   Figure 4. (A) Spearman's correlation between subepithelial eosinophils and FENO (for A–B, open symbols indicate patients without systemic steroids; the upper limit of normal is shown by the dashed line). (B) Spearman's correlation between subepithelial eosinophils and sputum eosinophils. (C) ROC curve for FENO for predicting subepithelial eosinophilia. (D) ROC curve for sputum eosinophils for predicting subepithelial eosinophilia.

	DISCUSSION

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The subjects recruited to this study represent a highly selected subgroup of children and adolescents with problematic asthma, and the number of subjects studied is relatively small. It would have been valuable to try to correlate our findings with asthma severity or treatment, but the homogeneous severity of the group, and the difficulty of making comparisons between patients on prednisolone and triamcinalone, precluded us from performing this analysis. The findings cannot therefore be generalized to all patients with asthma. However, in practice, patients attending a tertiary center are those who will likely derive most benefit from (and have access to) regular monitoring of sputum eosinophils and FE_NO. The data are therefore of particular importance for clinicians working in specialist centers.

The majority of subjects underwent assessment of eosinophilic airway inflammation after treatment with high-dose systemic corticosteroids. This means that the relationships described here may have been altered by steroid therapy, and may not be relevant in steroid-naive patients with asthma, or those inhaling only a low dose of corticosteroids. Furthermore, the overall level of airway eosinophilia, for all methods used, was low and the range was narrow. In particular, over half the subjects had no detectable eosinophils present in their biopsy. Assessment of correlation between biopsy eosinophils and the other parameters is therefore difficult to interpret. However, the lack of any significant positive correlation between biopsy eosinophils and sputum eosinophils is in keeping with data from adult studies (20–22).

Of more practical relevance, and the major strength of this study, is that the data shed light on the relationships between eosinophilic airway inflammation assessed noninvasively and bronchoscopically. The combination of sputum eosinophils and FE_NO may help to identify, with confidence, patients with evidence of airway eosinophilia in BAL. However, it was not possible to predict the presence of biopsy eosinophilia—only its absence. The presence or absence of eosinophilia is of practical importance. Absence of eosinophils leads to consideration of corticosteroid reduction. Persistent eosinophilia with symptoms leads to intensification of corticosteroid therapy, or the addition of a steroid-sparing agent such as cyclosporine. It should be stressed that we do not know, nor does this study elucidate, which measure of eosinophilia is most useful in guiding therapy. However, it should be noted that this study was cross-sectional, and thus the significance of the bronchoscopic findings could not be assessed.

It appears from the current study that the relationship between sputum and either BAL or biopsy is less in children than in adults with asthma (20–22). The percentage of sputum eosinophils may be due to the smaller pediatric sample sizes, which may be less representative for the complete airways. However, it is clear from the current study and from previous studies involving adults (20–22) that the different techniques described sample different airway compartments. The relationship between eosinophils in sputum and BAL is consistently stronger than that between sputum and biopsy. A possible explanation for this is the fact that both sputum and BAL sample inflammatory cells from a much larger area of the airways than is possible with biopsy. Another explanation may be that eosinophils traverse from the peripheral blood into the airway lumen far distal from the major airways that are sampled by biopsy, and thus are not seen in the proximal airways.

The data from the current study cannot be used to determine which is the most appropriate method for measuring eosinophilic airway inflammation in children and adolescents with difficult asthma. We do not know whether intraluminal or intramural eosinophils are more important in causing or monitoring this disease, and this study cannot answer that question. It is possible that clearing infiltrating eosinophils from within the airway wall, as assessed by biopsy, might even require lower steroid doses or a shorter treatment period than that used in this study, as we had fewer patients with biopsy eosinophilia than patients with BAL eosinophilia in our study. In clinical practice, noninvasive methods are more acceptable for monitoring of inflammation over time, with invasive methods more suited to one-off investigations of airway pathology in children with loss of symptom control despite high doses of inhaled and/or systemic steroids. The data from the current study help to highlight the similarities and differences between the methods described. However, if, as seems likely, targeting asthma therapy to reduce airway eosinophilia is desirable (1), then we need better markers than FE_NO and induced sputum if this is to be optimally achieved. For example, sputum cytokine pattern, perhaps in combination with FE_NO and sputum eosinophils, may be shown to be useful in future. Until then, children with difficult asthma who are on high doses of steroids may benefit from endobronchial biopsy to prevent overtreatment. However, it must be stressed that serial endobronchial biopsy is unlikely ever to be acceptable. Furthermore, the significance of proximal airway mucosal eosinophilia has been called into question by a recent study demonstrating that patients with asthma in apparent complete remission had ongoing airway wall eosinophilia to a similar degree to that of symptomatic patients with asthma (23).

In summary, these data show that, in a group of highly selected children on high-dose inhaled and systemic steroids, FE_NO and induced sputum do not completely reflect but are a pointer to airway eosinophilia as measured by BAL or endobronchial biopsy. Furthermore, BAL and endobronchial biopsy themselves seem to measure inflammation in different compartments. However, the study shows a relationship between FE_NO, sputum eosinophils and BAL eosinophils. A combination of elevated sputum eosinophils and elevated FE_NO levels may be used as a predictor for BAL eosinophilia. An absence of sputum eosinophils or a normal FE_NO level may help to exclude mucosal eosinophilia measured in endobronchial biopsy. However, better noninvasive markers are needed to guide antiinflammatory therapy, at least in this difficult group of patients, if the goal of treatment is to reduce airway wall eosinophilia, rather than merely treat symptoms.

	FOOTNOTES

 
Supported by the European Respiratory Society (C.L.), the National Asthma Campaign, UK (D.N.R.P.), the Society of Pediatric Pneumology (German, Austrian and Swiss; A.Z.), and the Royal Brompton and Harefield Charitable Fund B0437 (P.L.H.).

This article has an online supplement, which is accessible from the issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200603-352OC on September 14, 2006

Conflict of Interest Statement: C.L does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. F.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.G.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.L.H. is an inventor of international patent PCT GB 2005/001966 "Protease inhibitors and their therapeutic applications" owned by Imperial College Innovations. She has no other current relationships with a commercial entity. N.M.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.T.H. has received research grants in 2005 to 2006 of $400,000 from GlaxoSmithKline, $300,000 from Oxagen, $200,000 from Novartis, and $100,000 from Millenium. D.N.R.P. has received $1,600 in 2005 from AstraZeneca for speaking at meetings and attending an advisory board meeting. A.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 10, 2006; accepted in final form September 14, 2006

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This Article Abstract Full Text (PDF) Online Supplement All Versions of this Article: 200603-352OCv1 174/12/1286    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Lex, C. Articles by Bush, A. Search for Related Content PubMed PubMed Citation Articles by Lex, C. Articles by Bush, A.