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Anselm Jorda,1 Sabine Eberl,1 Alina Nussbaumer-Pröll,1 Maysa Sarhan,2 Maria Weber,1 Lara Elisabeth Tegrovsky,1 Markus Wahrmann,2 Valentin al Jalali,1 Felix Bergmann,1,3 Lena Pracher,1 Amelie Leutzendorff,1 Matthias Farlik,4 Bernd Jilma,1 Markus Zeitlinger1 1Medical University of Vienna, Department of Clinical Pharmacology, Vienna, Austria; 2Medical University of Vienna, Department of Internal Medicine III, Division of Nephrology and Dialysis, Vienna, Austria; 3Medical University of Vienna, Department of Plastic, Reconstructive and Aesthetic Surgery, Vienna, Austria; 4Medical University of Vienna, Department of Dermatology, Vienna, AustriaCorrespondence: Markus Zeitlinger, Department of Clinical Pharmacology, Medical University of Vienna, Waehringer Guertel 18-20, Vienna, 1090, Austria, Tel +43 1 40400 29810, Email markus.zeitlinger@meduniwien.ac.atIntroduction: Lipopolysaccharide (LPS) stimulation of human whole blood ex vivo has been widely used to investigate human innate immune responses. However, there are uncertainties regarding the reproducibility and reliability of this assay.Methods: In this prospective, single-center study, cytokine responses (interleukin 8, interferon-α, interferon-γ, interleukin 10, interleukin 1-β, interleukin 6, and tumor necrosis factor-α) to ex vivo whole blood LPS stimulation were assessed in 12 healthy volunteers. Cytokine levels were measured at 0, 2, and 4 h using a multiplex immunoassay (Luminex ®). Stimulation was repeated after six weeks. We examined reproducibility across technical and biological replicates at baseline and between repeated experiments after 6 weeks based on the area under the curve (AUC) of the individual cytokines using Pearson’s correlation coefficient and the mean coefficient of variation.Results: The lowest mean coefficients of variation were observed for the technical replicates (5.4 to 9.2%), followed by the biological replicates (8.1 to 24.8%), and the repeated experiments after 6 weeks (17 to 31.2%). Between the baseline and 6-week AUCs, the following Pearson correlation coefficients R were observed: interleukin 10, 0.97; interferon-α, 0.84; interleukin 1-β, 0.83; interleukin 8, 0.79; interleukin 6, 0.73; interferon-γ, 0.73; and tumor necrosis factor-α, 0.63.Discussion: The level of agreement between the baseline and week-6 cytokine response to ex vivo LPS stimulation was high across the seven cytokines analyzed. While interleukin 10 exhibited the lowest level of variability over time, tumor necrosis factor-α showed the highest variability in repeated experiments, which should be considered in the design and interpretation of future studies.Keywords: LPS, lipopolysaccharide, inflammation, endotoxin |
