SEE WHERE EETTINE FAT AND WE’LL TELL HOW FIX IT

Changes in the morphology of adipose tissue, characterized by changes in cell morphology such as enlargement of fat cells, are always associated with overweight and obesity. Cell size-related variables have been shown to be associated with common obesity-related diseases such as mild inflammation of adipose tissue, metabolic syndrome, and type 2 diabetes. Quantifying adipocyte morphology from images of histological specimens can be tedious. Here we present an easy way for the task using QuPath, a free and open source program with only built-in tools. Quantification of human adipose tissue samples according to the described protocol showed excellent correlation with ImageJ software with the Adipocyte Tools plugin combined with manual correction of error detection. Intraclass correlations between the two methods were good to excellent. The method described here can be applied to fairly large tissue sections and even to the analysis of entire slides.

KEY WORDS: Adipocyte volume, adipocyte volume, Cupat, Adipocyte tool, ImageJ, obesity
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  1. Introduction
    Obesity has become a major health problem worldwide [1,2]. The association between excess weight and comorbidities such as metabolic syndrome and type 2 diabetes is well established and appears to be related to adipose tissue dysfunction, often accompanied by mild inflammation [3–5]. The expansion of fat depots is accompanied by adipocyte hypertrophy, i.e., an increase in fat cell size (FCS), which represents adipose tissue dysfunction and general metabolic impairment [ 6 ]. For example, in obesity, FCS is positively correlated with the degree of macrophage infiltration, which is considered a marker of chronic inflammation [ 7 – 9 ]. Increased FCS in subcutaneous adipose tissue (SAT) reflects insulin resistance and fat accumulation in the liver [10–13]. Evaluation of the hyperplasia-hypertrophy-ratio and small adipocyte populations may also be important [14]. From this perspective, quantitative characterization of adipose tissue morphology, such as FCS and cell density, can be indirectly used to assess tissue dysfunction and inflammation. For example, FCS and cell density can be used to measure the effectiveness of therapeutic or surgical interventions for obesity. Therefore, the use of accurate, convenient, and reproducible methods to assess adipocyte size and number is encouraged.

Currently, FCS measurement methods are based on flow cytometry or direct histological analysis after collagenase treatment [6,15]. Histological analysis allows simultaneous assessment of tissue morphology, such as crown-like structures associated with dead or dying adipocytes, avoiding to some extent distortions due to cell rupture in cytometric methods [ 7 ]. The traditional method of evaluating histological samples is microscopic measurement, that is, the measurement of fat cells in photographs taken with a microscope camera [6]. However, affordable and accurate slide scanners have made it easy to digitize entire slides for further examination [16].

Evaluation of large adipocytes in histological specimens can be challenging. Commercial software for this purpose can be expensive and inconvenient to use [16]. However, there are open source versions such as ImageJ with numerous published scripts and plugins [17,18]. Adipocyte Tools is a widely used plugin and is reliable for micrograph quantification [16,19]. However, high-resolution whole-slide scans can be up to 2 GB in size and cannot be directly evaluated due to file size limitations. In contrast, QuPath, an innovative and increasingly popular open source software, can process larger images [ 20 ]. Internal plugins and scripts for measuring adipocytes have also been developed for QuPath [16,21].

Here we present a simple, intuitive and fast way to perform FCS measurements using QuPath with only built-in tools, no external plugins or scripts required. Measurements were compared to those obtained using the ImageJ + Adipocyte Tools plugin, and errors were manually corrected under conditions simulating conventional photomicrograph measurements. The described method can be extended to whole-slide measurements of adipose tissue sections containing at least 12,000 cells, as shown here.

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