The Luminex xMAP™ serum assays were performed in 96-well microplate format. Analytes were chosen based on knowledge of pathophysiology of malignancy and markers reported in the literature. Multiplex bead-based immunoassays for cytokines were purchased from Invitrogen. Other assays for proteins were developed in the Luminex Core Facility of University of Pittsburgh Hillman Cancer Center (Pittsburgh, PA, USA) according to the protocol by Luminex Corporation. The analytes were: Eotaxin-1, interferon-alpha (IFN-α), interferon-gamma (IFN-γ), interleukin (IL) 10, 12 p40, 13, 15, 17, 1α, 2, 4, 5, 6, 7, 8, interferon γ-inducible protein 10 (IP-10), monocyte chemotactic protein α (MCP-1), monocyte-induced γ interferon (MIG), macrophage inflammatory protein 1-α (MIP-1 α), macrophage inflammatory protein 1-β (MIP-1β), regulated upon activation, normally T-expressed and presumably secreted (RANTES), tumor necrosis factor alpha (TNF-α), tumor necrosis factor receptor I (TNF-RI), tumor necrosis factor receptor II (TNF-RII), death receptor 5 (DR5), epithelial growth factor (EGF), basic fibroblast growth factor (bFGF), granulocyte colony stimulating factor (G-CSF), granulocyte monocyte colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF).
Internal (spiked) control analytes were included for validation and standard curves. For intra-assay precision, CVs were calculated from six replicates of a control serum run on a single plate. For inter-assay precision, CVs were calculated from at least five separate experimental runs of the same control serum. The intra-assay variability was 3.5–7%. Inter-assay variability was 11–15%. Correlation with appropriate ELISA was 89–98% and recovery from serum was 80–110%. Table 2 provides a complete list of the 32 factors originally assayed via the xMAP procedure, 19 of which met the inclusion criteria for statistical analysis (see below).