A Straightforward Method for Adipocyte Size and Count Analysis Using Open-source Software QuPath

Figures

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  Figure 1.. Comparison of the ability of QuPath and ImageJ plugin Adipocyte Tools to detect the adipocytes in images of H&E stained 5 µm adipose tissue sections. Section quality and concomitant success of correctly rendering the cell area was graded on a visual scale of good-average-poor. See text for details. Scale bar 200 µm.

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  Figure 2.. Comparison of alternate pixel classifiers in illustrative cases of poor quality (H&E) histological samples. For size measurement, rendering the seemingly empty adipocyte area correctly is crucial and spillover of staining results in difficulties of the QuPath’s pixel classifier to appropriately detect the cell area (b-d). Training the classifier to recognize weakly stained spillover may help, but sometimes it results in inclusion of weakly stained sections of cell membranes, resulting in detection of falsely conjoined cells (c), or even reversal of detections (d). Scale bar 200 µm.

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  Figure 3.. ImageJ plugin Adipocyte Tools rendered fat cell detections (a) can be manually corrected (b) if necessary, effectively resulting in delineation of cells equivalent to full manual detection. With the QuPath protocol, there are similar options. Scale bar 200 µm.

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  Figure 4.. Adipocyte Tools with manual correction (MC) and QuPath based methods show good intermethod consistency. The QuPath based method measured on average 13.7 µm2 smaller area, 0.8 µm smaller diameter, and 7.9 higher density (cells/10E6 µm2). A-C: Scatter-plots with linear regression for mean area, mean diameter and mean density. D-F: Respective Bland-Altman plots with middle dashed-line set at mean difference level.

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  Figure 5.. The QuPath based method is applicable for analysis of large adipocyte areas as well. A-B: Illustration of QuPath-based method finding the adipocytes in a larger area (7.1x10E6 µm2). Scale bar 500 µm. C-D: Analysing the whole slide requires more effort but can be achieved in pieces on an average powered workstation (total area 62x10E6 µm2). Red boxes (10E6 µm2) demonstrate areas used for basic analyses with ImageJ + Adipocyte Tools plugin. Scale bar 1,000 µm.