Flow cytometric microsphere-based immunoassay: Analysis of secreted ctytokines in whole-blood samples from asthmatics

The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The Bow cytometer microsphere-based assay (FMBA) for cytokines consists of reagents and dedicated software, specific ally designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specif ic for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to ad just the stored curve periodically, and (iii) an internal standard to norma lize the detection step in each sample. Overall analytical performance, inc luding sensitivity, reproducibility, and dynamic range, was investigated fo r interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-gamma), a nd tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Res ults obtained with FMBA correlate well with commercial enzyme-linked immuno sorbent assay data (r > 0.98) for all cytokines assayed. This multiplex ass ay was applied to the determination of cytokine profiles in whole blood fro m atopic and nonatopic patients. Our results show that atopic subjects' blo od produces more IL-4 (P = 0.003) and less IFN-gamma (P = 0.04) than the bl ood of nonatopic subjects. However, atopic asthmatic subjects' blood produc es significantly more IFN-gamma than that of atopic nonasthmatic subjects ( P = 0.03), The results obtained indicate that the FMBA technology constitut es a powerful system for the quantitative, simultaneous determination of se creted cytokines in immune diseases.