Ilab650 analyser

Blood samples were collected into Vacuette ® Z serum clot activator tubes (Greiner Bio-One) and left to stand at room temperature for 30 min to allow clotting before being transferred onto ice. Blood samples, for the preparation of plasma, were collected into fluoride oxalate tubes (Teklab) and put straight onto ice. At the end of the study day all samples were centrifuged at 1912 g, 4°C for 10 min. Plasma and serum aliquots were taken and frozen at -20°C for batch analysis at the end of the study.
Plasma glucose and serum total cholesterol, HDL-cholesterol, NEFA and TAG were measured on an ILab 650 analyser (Instrumentation Laboratory). Inter-assay CV were 2, 4, 9, 8 and 9 % respectively and intra-assay CV were ≤4, 9, 9, 7 and 8 %, respectively. Serum insulin concentrations were measured by RIA using a Human Insulin Specific RIA kit (Millipore). Interassay CV were 11 % and intra-assay CV were ≤9 %. Serum inflammatory markers, IL-6 and TNF-α, were analysed by the Clinical Immunology Service (University of Birmingham), using a bead-based multiplex assay (Bio-Plex Precision Pro Human Cytokine Assay kit; Bio-Rad). The plate was analysed on a Luminex-100 plate reader (Bio-Plex Systems; Bio-Rad Laboratories) using a low 'RP1' (low PMTphotomultiplier tube) target setting. Intra-assay CV were <10 %.