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). We compared the measurements of human ATM using flow cytometry, immunohistochemistry (IHC) and RT-PCR of ATM markers. METHODS: We evaluated a new software program (AMCounter) to help measure ATM using IHC and compared this to flow cytometry and RT-PCR. RESULTS: IHC had good intra-individual reproducibility for total (CD68), pro-inflammatory (CD14) and anti-inflammatory (CD206) ATM. The AMCounter improved inter-reader agreement and was more time efficient. Flow cytometry had acceptable intra-individual reproducibility for the percent of CD68(+) cells that were CD14(+) or CD206(+), but not for ATM/g tissue. ATM/g 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 ATM’s per 100 adipocytes. Of interest, were also statistically significant correlations between RT-PCR CD68 and IHC CD68, CD14 and adipose flow cytometry measures of CD68(+), CD68(+)/CD14(+) and CD68(+)/CD206(+) ATM’s per g tissue. CONCLUSIONS: The AMCounter software helps reproducibly and efficiency measures of IHC ATM’s. Flow cytometry, immunohistochemistry and RT-PCR measures of adipose inflammation provide somewhat different information.