Comparison of Methods for Analyzing Human Adipose Tissue Macrophage Content

Objective: The relationship between inflammation, obesity, and adverse metabolic conditions is associated with adipose tissue macrophages (ATM). This study compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC), and real-time polymerase chain reaction (RT-PCR) of ATM markers. Methods: A new software program (AMCounter) was evaluated to help measure ATM using IHC, and this was compared to flow cytometry and RT-PCR. Results: IHC had good intraindividual reproducibility for total (CD68), proinflammatory (CD14), and anti-inflammatory (CD206) ATM. The AMCounter improved interreader agreement and was more time efficient. Flow cytometry had acceptable intraindividual reproducibility for the percentage of CD68⁺ cells that were CD14⁺ or CD206⁺, but not for ATMs per gram of tissue. ATMs per gram of tissue was much greater using IHC than flow cytometry. The flow cytometry and IHC measures of ATM from the same biopsies were not correlated. There were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and CD206 ATMs per 100 adipocytes. Also of interest were statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14, and adipose flow cytometry measures of CD68⁺, CD68⁺/CD14⁺, and CD68⁺/CD206⁺ ATMs per gram of tissue. Conclusions: The AMCounter software helps provide reproducible and efficient measures of IHC ATMs. Flow cytometry, IHC, and RT-PCR measures of adipose inflammation provide somewhat different information.