Analytical significance of encroachment in multiplexed bead-based flow cytometric assays

Multiplexed bead-based assays, using fluorescent dye-encoded beads, are finding widespread use in various profiling studies. The need to measure multiple quantitative responses simultaneously, the development of less expensive commercial flow systems, and the ease and cost effectiveness of manufacturing bead profiling kits of varied composition have all contributed to the popularity of this assay format. Maximizing the level of multiplexing in these assays requires tight spacing of fluorescent bead populations, and this leads to some degree of overlap or "encroachment" between populations. The degree to which encroachment affects analyte signal determinations depends upon both the extent of overlap and the relative analyte signals associated with the populations. In the work reported here, the impact of encroachment upon analyte signal for a subset of beads belonging to a multiplexed cytokine assay has been modeled and empirically evaluated.