Cytokine measurements
TUMOUR NECROSIS
The plasma was analysed for 27-cytokines (Bio-Plex Pro Human Cytokine 27-plex Assay) in single replicate using Luminex xMap Technology on a Bio-Plex 200 system (Bio-Rad Laboratories, California, USA). This assay quantifies IL-1β, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8/CXCL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic fibroblast growth factor (b-FGF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-ɣ), eotaxin/CCL11, IP-10/CXCL10, macrophage chemoattractant protein/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, (RANTES)/CCL5, tumour necrosis factor (TNF), platelet-derived growth factor (PDGF) and vascular endothelial growth factor.The assay uses detection antibodies for the biomarker of interest, conjugated with a fluorescent indicator. The acquired fluorescent intensity is used to calculate the concentration (pg/mL) of each cytokine using a standard curve. The standard dilution series curves for b-FGF, IL-10, IL-15 and IL-17 were distorted, and the results were not credible. These results were therefore discarded, and not included in the analyses. Interassay coefficient of variations (CV) was <15% for all analytes (mean 5.13%), and most intra-assay CVs were <11% (mean 5.10%). Many of our samples were out of range (OOR) in respect to the manufacturer’s tolerance. To obtain continuous datasets, we established a new global standard curve that was used to calculate the concentration of all cytokines with >10% of the values OOR. For cytokines with <10% of values OOR, we replaced the missing values using the lowest measured concentration for that cytokine.