Variance in multiplex suspension array assays: intraplex method improves reliability

BACKGROUND: Flow cytometry based suspended microarray assays are susceptible to many sources of variance; multi-well replication and inter-instrument reproducibility is uncertain. METHOD AND RESULTS: An "intraplex" method was developed in order to minimize differences in sample readings between instruments. A full intraplex assay consists of a set m of microparticle set classifications assaying for the same analyte, with each of the m classifier sets having different sensitivity to analyte, and n classifier sets replicating each of the m levels of sensitivity, where m > 1 (generally m > 4 would be used). CONCLUSION: The intraplex method can compensate adequately for the sources of variance that have been identified in suspended microarray assays. It requires no changes to current equipment in use, and is a superior method of constructing precision assays. Additionally, Luminex(® )users may want to consider the evidence that shows that despite calibration to the same standard, two instruments may not give similar results for all concentrations of analytes.