Reproducibility of Sputum Differential Cell Counts Is not Affected by Squamous Epithelial Cells

Abstract

Objective. Induced sputum is used to assess markers of inflammation in asthmatic individuals, and sputum cell differential counts provide an outcome to evaluate the presence, type, and degree of inflammation in the airways. Contamination of sputum slides with squamous epithelial cells (SECs) has been reported to adversely affect the reproducibility of sputum cell differential counts; however, this has not been studied in a controlled manner. Excluding sputum slides because of excessive squamous cell contamination can be problematic resulting in under-powering of studies. The aim of this study is to evaluate the effect of SEC contamination and cell dispersion on the reproducibility of differential counts of sputum cells prepared on glass slides. Study Design. A total of 33 sputum samples were induced from 11 subjects with mild asthma under baseline conditions and following an allergen inhalation challenge. Mucoid and salivary portions of each sample were divided and processed in parallel. To evaluate the effect of increasing the proportion of SEC and to evaluate the effect of increasing the number of leukocytes per high power field (HPF), four slides with varying leukocyte numbers and SEC percentages were prepared from each sample by combining and adjusting the volume of cell suspensions derived from mucous and saliva. The four slides were prepared to fall in the following categories: (A) 50 cells/HPF and <20% SEC; (B) 50 cells/HPF and >20% SEC; (C) 100 cells/HPF and <20% SEC; and (D) 100 cells/HPF and >20% SEC. All slides were blinded and counted twice by an experienced observer, and twice by an inexperienced observer. Results. The differential cell counts for eosinophils, macrophages, and neutrophils were highly reproducible under all conditions when enumerated by an experienced observer (ICC > 0.9), and furthermore, SEC contamination did not affect ICC when differential counts were enumerated by an inexperienced observer (ICC > 0.8). Conclusion. Our results demonstrate that slides containing SECs, up to 40% in this study, have reproducible differential cell counts.