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Comparative Study
. 2015 Nov 5:15:137.
doi: 10.1186/s12890-015-0135-7.

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

Hannah Jary  1 , Jamie Rylance  2   3 , Latifa Patel  4 , Stephen B Gordon  5   6 , Kevin Mortimer  7   8
Affiliations
Comparative Study

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

Hannah Jary et al. BMC Pulm Med. .

Abstract

Background: Air pollution is associated with a high burden or morbidity and mortality, but exposure cannot be quantified rapidly or cheaply. The particulate burden of macrophages from induced sputum may provide a biomarker. We compare the feasibility of two methods for digital quantification of airway macrophage particulate load.

Methods: Induced sputum samples were processed and analysed using ImageJ and Image SXM software packages. We compare each package by resources and time required.

Results: 13 adequate samples were obtained from 21 patients. Median particulate load was 0.38 μm(2) (ImageJ) and 4.0 % of the total cellular area of macrophages (Image SXM), with no correlation between results obtained using the two methods (correlation coefficient = -0.42, p = 0.256). Image SXM took longer than ImageJ (median 26 vs 54 mins per participant, p = 0.008) and was less accurate based on visual assessment of the output images. ImageJ's method is subjective and requires well-trained staff.

Conclusion: Induced sputum has limited application as a screening tool due to the resources required. Limitations of both methods compared here were found: the heterogeneity of induced sputum appearances makes automated image analysis challenging. Further work should refine methodologies and assess inter- and intra-observer reliability, if these methods are to be developed for investigating the relationship of particulate and inflammatory response in the macrophage.

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Figures

Fig. 1
Fig. 1
Systematic digital image acquisition. The pathway used to acquire digital images of cytospin ‘spots’ is shown
Fig. 2
Fig. 2
Image SXM and ImageJ methodology. Image SXM (a, b & c); digital images of the cytospins (a) were manually edited to remove all non-macrophage cells and debris (b). Image SXM then calculated the area of cytoplasm [27] and particulate matter (red), mapped out in the output image (c). ImageJ (d, e & f): for each macropghage, the threshold level was adjusted manually until the black areas of particulate matter seen in the original image (a) turned red (b). The particulate matter within the cytoplasm was then selected by freehand (c)
Fig. 3
Fig. 3
Participants and samples. The flow chart shows the number of consented and recruited patients, and how many samples were obtained and included in the final analysis
Fig. 4
Fig. 4
An example of inaccurte Image SXM analysis. Comparing the original image (a) to the output image (b), the total cellular area [27] of the airway macrophage on the left has been overestimated, and the partcilate matter (red) of the airway macrophage on the right has been overestimated
Fig. 5
Fig. 5
Airway macrophage heterogeneity. The morphology of the airway macrophages (shown with red arrows) was varied within the same sample (a) and between different participant samples (a & b). The particulate load also varied between macrophages in the same sample (a)
See this image and copyright information in PMC

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