Repeatability of measures of inflammatory cell number in bronchial biopsies in atopic asthma.

Airway pathology is increasingly considered to be a major outcome in asthma research. The aim of this study was to examine the intra-observer, within-section and between-biopsy repeatability, together with the implications for statistical power of a computerized quantitative analysis of inflammatory cell numbers in the lamina propria in bronchial biopsy specimens from atopic asthmatic subjects. Thirty six atopic adults (aged 19-40 yrs) with mild to moderate asthma (baseline forced expiratory volume in one second (FEV1) > or =50% of predicted value, methacholine (PC20) range 0.02-18.2 mg x mL[-1]) at various levels of treatment (25 subjects on inhaled steroids) entered the study. Biopsies were taken from the (sub)segmental carinae of the right lower and middle lobe and from the main carina. Specimens were snap-frozen and immunohistochemical staining was performed on cryostat sections with monoclonal antibodies against: (secreted) eosinophil cationic protein (EG1, EG2), mast cell tryptase (AA1), CD45, CD22, CD4, CD8, CD25, and CD45RO. Using a computerized system, the number of positively stained cells in the lamina propria was counted. When considering all cell types together, satisfactory intraclass correlation coefficients (ICC) values were obtained for intra-observer, within-section and between-biopsy repeatability, being 0.90, 0.80 and 0.81, respectively. The analysis of repeatability for individual cell types revealed ICC values ranging 0.47-0.82 for intra-observer, 0.44-0.76 for within-section and 0.37-0.67 for between-biopsy repeatability. The results imply that a sample-size between eight and 25 subjects is needed to detect at least one doubling difference in cell number per 0.1 mm2 for a particular inflammatory cell type in a study, using a within-group design with alpha=0.05 and power of 0.80. A sample-size of 13-48 subjects per group is required to detect the same difference between the groups in a parallel design.

Relationship between the inflammatory infiltrate in bronchial biopsy specimens and clinical severity of asthma in patients treated with inhaled steroids.

BACKGROUND: Current guidelines on the management of asthma advocate the use of anti-inflammatory treatment in all but mild disease. They define disease control in terms of clinical criteria such as lung function and symptoms. However, the relationship between the clinical control of the disease and inflammation of the airways is not clear. A cross sectional study was therefore undertaken to investigate the relationship between airways inflammation and measures of clinical control and bronchial hyperresponsiveness in asthmatic patients treated with inhaled steroids. METHODS: Twenty six atopic adults (19-45 years) with mild to moderate asthma (baseline forced expiratory volume in one second (FEV1) > or = 50% predicted, concentration of histamine causing a 20% fall in FEV1 (PC20) 0.02-7.6 mg/ml) on regular treatment with inhaled steroids entered the study. Diary card recordings during the two weeks before a methacholine challenge test and bronchoscopic examination were used to determine peak flow variability, symptom scores, and use of beta 2 agonists. Biopsy specimens were taken by fibreoptic bronchoscopy from the carina of the right lower and middle lobes, and from the main carina. Immunohistochemical staining was performed on cryostat sections with monoclonal antibodies against: eosinophil cationic protein (EG1, EG2), mast cell tryptase (AA1), CD45, CD22, CD3, CD4, CD8, CD25, and CD45RO. The number of positively stained cells in the lamina propria was counted twice by using an interactive display system. RESULTS: There were no differences in cell numbers between the three sites from which biopsy specimens were taken. The PC20 for methacholine was inversely related to the average number of total leucocytes, EG1+, and EG2+ cells, mast cells, CD8+, and CD45RO+ cells in the lamina propria. These relationships were similar for each of the biopsy sites. Symptom scores, beta 2 agonist usage, FEV1, and peak flow variability were not related to any of the cell counts. CONCLUSIONS: Infiltration of inflammatory cells in the lamina propria of the airways seems to persist in asthmatic outpatients despite regular treatment with inhaled steroids. The number of infiltrating leucocytes such as mast cells, (activated) eosinophils, CD8+, and CD45RO+ cells in bronchial biopsy specimens from these patients appears to be reflected by airway hyperresponsiveness to methacholine, but not by symptoms or lung function. These findings may have implications for the adjustment of anti-inflammatory treatment of patients with asthma.