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Fully Automated Assessment of Inflammatory Cell Counts and Cytokine Expression in Bronchial Tissue

Departments of Medical Decision Making, Pulmonology, and Pathology, Leiden University Medical Center, Leiden; University Medical Center St. Radboud, Nijmegen; Erasmus Medical Center, Rotterdam; and Asthma Management Project University of Leiden (AMPUL) Study Group, Leiden, The Netherlands

Correspondence and requests for reprints should be addressed to J. K. Sont, Ph.D., Department of Medical Decision Making, J10-S Leiden University Medical Center, P.O. Box 9600, NL-2300 RC Leiden, The Netherlands. E-mail: j.k.sont{at}lumc.nl

Automated image analysis of bronchial tissue offers the opportunity to quantify stained area and staining intensity in a standardized way to obtain robust estimates of inflammatory cell counts and cytokine expression from multiple large areas of histopathologic sections. We compared fully automated digital image analysis with interactive digital cell counting and semiquantitative scoring of cytokine expression in terms of repeatability and agreement in bronchial biopsies in 52 patients with mild to moderate atopic asthma. Immunohistochemistry with antibodies against CD3, interleukin (IL)-4, IL-5, and interferon- protein was performed on frozen tissue sections, using 3-amino-9-ethylcarbazole as chromogen and hematoxylin as counterstaining. IL-4 and IL-5 messenger RNAs were localized by in situ hybridization without hematoxylin staining. Separation of 3-amino-9-ethylcarbazole and hematoxylin-stained pixels was achieved by linear combination of red- and blue-filtered gray-scale images. Using baseline biopsy specimens, fully automated CD3⁺ cell counts showed perfect repeatability (r = 1.0) and a strong linear relationship with the interactive procedure (r = 0.98). Automated densitometry showed perfect repeatability (1.0) and a moderate to strong relationship with semiquantitative scoring of protein and messenger RNA expression (r = 0.43–0.89). Relationships between automated and semiquantitative assessments of changes in cytokine expression during 2 years of follow-up were moderate to strong (r = 0.40–0.84). We conclude that fully automated cell counts and automated densitometric analyses in bronchial tissue of patients with asthma are unbiased and help to reduce variability in inflammatory outcomes.

Key Words: airway inflammation • asthma • biopsy (bronchial) • chronic obstructive pulmonary disease • cytokines • helper T cells, types 1 and 2

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