Labinsky H, Panipinto PM, Ly KA, et al. - Cell composition and soluble protein production was calculated by flow cytometry and multiplex ELISA in synovium obtained from OA individuals who underwent knee replacement surgery (n = 35) in order to create a novel, multi-parameter approach to phenotype synovial responses in knee OA. Testing disaggregation conditions exhibited that aggressive digestion enhanced synovial cell yield and mesenchymal staining through flow cytometry, however, it negatively affected CD4+ T cell and CD56+ natural killer (NK) cell staining. Less aggressive digestion conserved these markers and showed highly variable T cell infiltration (n = 32). Mesenchymal subpopulations related to various non-mesenchymal populations, including macrophages and T cells were recognized by the correlation analysis. IL-6 measured by flow cytometry related robustly to IL-6 released by ex vivo culture of synovial tissue and was highest in mesenchymal cells co-expressing CD90 and CD34. IL-6, IL-8, complement factor D (CFD), and IL-10 release positively associated with tissue cellularity. Moreover, raised CD8+ T cell numbers also related to retinol-binding protein 4 (RBP4). At last, combining flow cytometry and multiplex data recognized patient clusters with various types of inflammatory responses. Thus, a novel approach to investigate OA synovium, recognizing patient-specific inflammatory clusters was used. Moreover, this study debates that phenotyping synovial inflammation could give new insights into OA patient heterogeneity and biomarker development.