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

Abstract

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) ≥50% of predicted value, methacholine (PC20) range 0.02-18.2 mg·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 betweenbiopsy 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 mm 2 for a particular inflammatory cell type in a study, using a within-group design with α=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.

Do bronchial biopsies represent mast cell density in airways? A stereological study

“…This suggests that within a case, any given endobronchial biopsy from a (sub)carina is unlikely to reflect mast cell density across the airway wall generally in the vicinity of the biopsy site. Such a finding is not unexpected given previously observed intrasubject variations in the density of inflammatory cells within sections [37] and between sections [8,38] from the same biopsy site, and between biopsysized segments within an airway section [39].…”

Assessment of bronchial inflammation using an automated cell recognition system based on colour analysis

“…There are numerous advantages of immunohistochemistry over other techniques such as bronchioalveolar lavage, induced sputum or measurement of exhaled NO, to evaluate bronchial inflammation: good morphology, immunoreactivity for increasing number of antibodies, low cost, short procedure. However, light microscopy analysis requires particular attention including multiobserver analysis, blinded counting, and evaluation of both inter-and intraobserver reproducibility [25,26]. Computerized analysis can overcome part of these disadvantages provided that the automated recognition system is able to accurately discriminate a positive stain.…”

Rhinovirus infection in nonasthmatic subjects: effects on intrapulmonary airways

“…Six biopsy specimens were taken at (sub)segmental level. Three biopsies were immediately fixed in phosphate buffered saline (PBS) buffered formalin 10% (v/v) and three were embedded in ornithyl carbamyltransferase (OCT) medium (Miles Inc. Diagnostics Division, Elkhart, USA) and snap-frozen in isopentane cooled by iced carbon dioxide [9]. The formaline-fixed biopsies were embedded in paraffin and stored until further processing.…”

“…Fibreoptic bronchoscopy was performed according to a standardised and validated protocol [9]. Six biopsy specimens were taken at (sub)segmental level.…”

“…The sections were counterstained with Mayer9s haematoxyline (Klinipath, Duiven, the Netherlands). For negative controls, the primary antibody was omitted from this procedure [9].…”