Computational protocol: Comparison of methods for the analysis of airway macrophage particulate load from induced sputum, a potential biomarker of air pollution exposure

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Protocol publication

[…] Sputum samples were kept on ice and sputum plugs were manually extracted and treated with 0.1 % Sputolysin (Merck Chemical Ltd, UK) for fifteen minutes to remove mucus. Phosphate Buffered Solution (Sigma-Aldrich, UK) was added and cells were filtered and centrifuged at 2200 rpm for ten minutes at 4 °C (Heraeus Megafuge 1.0R, ThermoFisher Scientific, USA). The pellet was re-suspended at 0.5×106 cells per ml and two × 100 μl of suspension was cytocentrifuged (Shandon Cytopsin 4, ThermoFisher Scientific) onto microscope slides at 450 rpm for 6 min to produce three cytospins per participant. Slides were fixed in methanol and stored until staining. One slide per participant was stained using Hemacolor Staining kit (Merck-Millipore, Germany) for ImageJ analysis. One slide was stained using Hemacolor Solution 2 (eosin) only (dipped for 9 s), so that only the cytoplasm was stained (a method previously developed for optimising Image SXM analysis []). One slide per participant was stained with Diff-Quik (Dade Behring, Deerfield, IL, USA) for differential cell counts : 400 cells were counted per participant, using a Leica DM IL light microscope at ×40 magnification. Cytospins with a leukocyte/squamous epithelial cell ratio of ≤5 were deemed inadequate and therefore excluded from the analysis []. [...] Cytospin slides for ImageJ analysis were photographed at ×60 magnification using Nikon Eclipse 80i digital microscope (Nikon Instruments Europe BV) with Nikon NIS-Elements BR software; 50 macrophages were captured per participant where possible (in cases where less than 50 macrophages were present on the cytospin a reduced number was used). Slides for Image SXM analysis were imaged at ×40 magnification using a Leica DM IL light microscope (Leica Microsystems UK Ltd) with a Nikon E990 digital camera (Nikon Inc, USA); where possible 50 microscope fields (with at least one macrophage per field) were captured per participant - all the macrophages captured in a field were analysed. In cases where less than 50 images from the whole cytospin contained a macrophage this reduced number of macrophages-containing images, and all macrophages within those images, were included in the analysis. Images for both methods were taken systematically using a predefined method to prevent duplication or biased image selection, as shown in Fig. .Fig. 1 […]

Pipeline specifications

Software tools ImageJ, Image SXM, NIS-Elements BR
Application Microscopic phenotype analysis
Organisms Homo sapiens