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R. J. Fullard, L. E. Kehinde, K. S. Elder, M. K. Tran, N. M. Guyette; Magnetic Beads Produce Significant Artifacts in Luminex Tear Cytokine Assay. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3378.
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A Bio-Rad 27-Plex polystyrene bead cytokine assay was previously developed, validated and optimized for tears. A new magnetic bead version of the assay allows greater automation and removes filter plate factors that limit assay precision. This study applies the magnetic bead assay to tear cytokines.
Non-stimulated (NS) and stimulated tear samples were collected by micropipette from normal patients in sufficient quantity to run multiple aliquots on both the polystyrene and magnetic bead 27-Plex cytokine assay. An automated microplate washer was used, with filter plate and vacuum aspiration for the polystyrene bead assay, and plastic-based plate and magnetic plate holder for the magnetic bead assay. In a second study, 18 subjects collected 8 NS tear samples each over thirty days for polystyrene bead assay and 9 subjects collected 8 NS tear samples each over the same time-frame for magnetic bead assay. Samples were stored in assay buffer at -80°C prior to assay on a Luminex 200 system.
Mean cytokine intra-assay coefficient of variation (CV) for tear sample replicates with the magnetic versus polystyrene bead assay was 8.1% vs. 17.4% for duplicates, 10.6% vs. 16.2% for triplicates and 9.8% vs. 16.0% for quadruplicates. In study 2, tear component interference was observed with the magnetic bead assay, in particular for eotaxin, IL-9, IL-17, IL-5 and TNF-α, with 25-fold higher levels relative to the polystyrene bead assay and much less sample to sample variation. Magnetic bead interference varied by subject, some showing even higher levels for many cytokines, others consistently eliciting almost undetectable levels of many cytokines.
While Luminex magnetic bead tear cytokine assays show improved intra-assay CVs, tear components appear to be interfering to produce falsely high cytokine values and reduced discrimination between tear samples for most subjects, while preventing detection of many cytokines in others. Luminex cytokine assay results for tears using magnetic beads should be treated with caution. Further validation studies are needed.
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