Blood and/or urine was collected from burn patients at admission, pre-operatively, and 5 days post-operatively for 4 weeks for serum hormone, protein, cytokine and urine hormone analysis. Blood was drawn in a serum-separator collection tube and centrifuged for 10 minutes at 1320 rpm; the serum was removed and stored at −70°C until assayed.
Serum hormones and acute phase proteins were determined using HPLC, nephelometry (BNII, Plasma Protein Analyzer Dade Behring, MD), and ELISA techniques. The Bio-Plex Human Cytokine 17-Plex panel was used with the Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA) to profile expression of seventeen inflammatory mediators interleukin [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), and tumor necrosis factor (TNF). The assay was performed according to the manufacturer’s instructions. Briefly, serum samples were thawed and then centrifuged at 4500 rpm for 3 minutes at 4°C. Serum samples were then incubated with microbeads labeled with specific antibodies to one of the aforementioned cytokines for 30 minutes. Following a wash step, the beads were incubated with the detection antibody cocktail, each antibody specific to a single cytokine. After another wash step, the beads were incubated with streptavidin-phycoerythrin for 10 minutes, washed, and the concentrations of each cytokine were determined using the array reader.3 (link), 18 (link)–20 (link)Urine creatinine, creatinine clearance, and cortisol were determined by standard laboratory techniques.
Serum hormones and acute phase proteins were determined using HPLC, nephelometry (BNII, Plasma Protein Analyzer Dade Behring, MD), and ELISA techniques. The Bio-Plex Human Cytokine 17-Plex panel was used with the Bio-Plex Suspension Array System (Bio-Rad, Hercules, CA) to profile expression of seventeen inflammatory mediators interleukin [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 beta (MIP-1β), and tumor necrosis factor (TNF). The assay was performed according to the manufacturer’s instructions. Briefly, serum samples were thawed and then centrifuged at 4500 rpm for 3 minutes at 4°C. Serum samples were then incubated with microbeads labeled with specific antibodies to one of the aforementioned cytokines for 30 minutes. Following a wash step, the beads were incubated with the detection antibody cocktail, each antibody specific to a single cytokine. After another wash step, the beads were incubated with streptavidin-phycoerythrin for 10 minutes, washed, and the concentrations of each cytokine were determined using the array reader.3 (link), 18 (link)–20 (link)Urine creatinine, creatinine clearance, and cortisol were determined by standard laboratory techniques.
