Profiling Immune Mediators in Serum and CSF

We used multiplexed bead assay techniques for establishing the profiles of 39 immune mediator proteins that included cytokines, chemokines, and growth factors in serum and CSF. The selected panel was the most comprehensive available. Assay reagents and plates were obtained from well-validated commercial sources (Millipore®) [42 (link)]. The procedures followed recommendations and well-established protocols for evaluation of serum [42 (link), 43 (link)] and the CSF [44 (link), 45 (link)]. Only the first freeze-thaw aliquots were used for assay measurements. To achieve uniformity in the longitudinal assessment, assays for samples collected at different timepoints from the same individual were run simultaneously. Masked samples were measured in duplicates and blank values subtracted from all readings. Measurements and data analysis of all assays were performed with the Luminex-200® system in combination with Luminex manager software (Bioplex manager 5.0, Bio-Rad, Hercules, CA). We used standard operating procedures to guarantee the consistency, reproducibility, and reliability of the assays. Samples that exhibited unexpected or unacceptable variance (i.e., evidence of bead clumping, coefficients of variation greater than 20%, or unusual distributions of values) were re-tested.