Inflammatory marker analysis
CLOT, BLOOD, SEPARATION, INFLAMMATION
As a convenience to participants, CRP assays were conducted by NATA accredited (Blood for the inflammatory marker (cytokine) analysis (5 ml) was collected at each time point into BD Vacutainer® SST™ serum separation tubes, under standard conditions at the time of collection of other study bloods. Blood was allowed to clot at room temperature for 30 to 45 min followed by centrifugation at 1000 × Inflammatory marker analysis was conducted using the Bio-Plex 200 multiplex immunoassay system (Bio-Rad). Bio-Plex 200 allows a robust quantification of multiple cytokines for multiple patients in a 96-well plate in 3–4 h. Sample preparation was conducted using an automated, magnetic-bead wash station to ensure highest reproducibility standards. A 37-plex Bio-Plex Pro™ human inflammation panel 1 (Bio-Rad, Cat. #171-AL001M) was used to analyse pro- and anti-inflammatory serum cytokine levels following the manufacture’s recommendations. Briefly, the Bio-Plex 200 was calibrated prior to use. Serum samples were thawed and diluted in standard diluent HB at 1:4 factor. Sera, standards, blanks, and controls were assayed in duplicate with Bio-Plex human inflammation panel beads in 96-well plates. Assay quantitation was performed using lot-specific normalised standards (#54298167) and controls (#64310917) for all samples and cytokines.