Luminex assay of plasma IL-6, TNF-α, leptin, and adiponectin: Impact of anticoagulants, single-well analysis, and freeze–thaw stability
Lara Pattaroni, Elisabetta Venturelli, Daniele Morelli, Michela Bianchi, Giulia Guerra, Francesco Segrado, Sabina Sieri, Alessio Polymeropoulos, Claudia AgnoliBackground
Chronic inflammation from visceral obesity contributes to metabolic disorders and cancer through cytokine imbalance. Accurate cytokine measurement is crucial to understand these links. This study examined challenges in quantifying interleudin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), leptin, and adiponectin in historical biobank samples with limited volumes and varying plasma anticoagulants.
Methods
Luminex-MAGPIX platform was used to evaluate three analytical aspects. Single-well precision was assessed via intra- and inter-assay coefficients of variability (CVs). Plasma matrix effects were examined by pairwise comparison of analyte concentrations across citrate, heparin, and EDTA plasma from 10 volunteers. Cytokine stability was tested after one freeze-thaw cycle (analysis of variance).
Results
Single-well measurements showed good precision (CV <10% intra-assay, <15% inter-assay). Plasma matrix affected absolute concentrations. In particular, all analytes were lower in citrate than in EDTA and heparin, but relative relationships and subject-specific rankings remained preserved. Pearson correlation coefficients for IL-6, leptin, and adiponectin were high across matrices (r-range = 0.952–0.999), whereas correlations for TNF-α were lower (r-range = 0.897–0.972). Freeze–thaw stability was analyte- and matrix-dependent: leptin remained stable in heparin and EDTA but decreased 9.5% in citrate; adiponectin was stable in citrate but decreased in heparin (−9.0%) and EDTA (−11.8%). IL-6 and TNF-α were unaffected.
Conclusion
IL-6, TNF-α, leptin, and adiponectin were accurately quantified using single-well Luminex assays and showed reasonable tolerance to a single freeze–thaw cycle, offering a practical strategy for preserving valuable specimens. Although plasma anticoagulant affected absolute concentrations, the preservation of relative analyte rankings permits their use in most study designs but limit absolute value comparisons.