Plasma samples collected as described above, were aliquoted and stored at 280uC until analyses were done. A multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to manufacturer's instructions (Biosource Human Cytokine 30-plex Assay, Invitrogen). The following groups of cytokines were: inflammatory (GM-CSF, IL-1beta, IL-1RA, IL-6, IL-8, TNF-alpha); Th1/Th2 (IFN-gamma, IL-2, IL-2R, IL-4, IL-5, IL-10); Cytokine II (IFN-alpha, IL-7, IL-12p40/p70, IL-13, IL-15, IL-17); Chemokines (Eotaxin, IP-10, MCP-1, MIG, MIP-1alpha, MIP-1beta, RANTES); and Growth Factors (EGF, HGF, FGF-basic, G-CSF, VEGF). Briefly, 25 ul of plasma samples were diluted in 1:2 and incubated with antibody coupled beads for 2 h at RT uC. Complexes were washed twice with the use of a vacuum manifold before incubation with biotinylated detector antibody for 1 h at RT uC. Complexes were then washed twice followed by incubation with Streptavidin-phycoeryrhrin (RPE) for 30 min at RT uC. Complexes were washed thrice and incubated with wash buffer for another 3 min before detection in the Luminex 200 TM instrument. Results were acquired by the IS 2.3 software and the standard curves were plotted through a fiveparameter logistic curve setting.