Blood Analysis Study 2

Blood samples were collected after an overnight fast into 10 mL EDTA and 10 mL SST tubes. EDTA tubes were subjected to centrifugation within 30 min of collection for analysis of cytokines.

EDTA plasma was analyzed by Crux Biolab (https://cruxbiolab.com.au/, accessed on 7 June 2017), using an immunoassay high sensitivity Luminex Panel for the following cytokines: IFNγ, IL-1β, IL-6, TNFα, IL-10, and IL-8. Calculated coefficients of variation (CVs) ranged from 9.6−19.3% for inter-assay variation and 0.8−7.3% for intra-assay variation. Limits for detection of the cytokines were as follows: 1.16−4770 pg/mL for IFNγ, 0.21−875 pg/mL for IL-1β, 0.74−3050 pg/mL for IL-6, 0.97−3960 pg/mL for TNFα, 0.20−810 pg/mL for IL-10, and 0.24−985 pg/mL for IL-8.

Serum was analyzed in-house on a Konelab 20XT auto-analyzer. Commercially available kits, reagents, and standards were obtained from Thermo Fisher Scientific Australia Pty. Ltd. and were used to analyze total cholesterol (kit code 981813), HDL-cholesterol (kit code 981823), triglycerides (kit code 981786), glucose hexokinase (kit code 981779), and C-reactive protein (kit code 981934). The first four tests were colorimetric assays, while CRP was an immunoturbidimetric assay. Samples were run in singular; however, for any unusually high or low results, they were analyzed again to confirm the reading. LDL levels were calculated using the Friedwald equation (Friedewald, Levy et al., 1972) [49].
