Variance in multiplex suspension array assays: microsphere size variation impact

Size variation is demonstrated by transmission electron microscopy. Size variations of microspheres are shown to occur in stepwise increments. A strong correspondence between microsphere size distribution and distribution of fluorescent events from assays is shown. An estimate is made of contribution of microsphere size variation to assay variance.A probable significant cause of variance in suspended microsphere assay results is variation in microsphere diameter. This can potentially be addressed by changes in the manufacturing process. Provision to users of mean size, median size, skew, the number of standard deviations that half the size range represents (sigma multiple), and standard deviation is recommended. Establishing a higher sigma multiple for microsphere production is likely to deliver a significant improvement in precision of raw instrument readings. Further research is recommended on the molecular architecture of microsphere coatings.A suspended microarray assay system uses small particles such as microrods or microspheres that contain some method for identifying a set, often termed a classifier. Classifiers are often 2 (or 3 in the cased of the new Luminex 3-D system) fluorophores dedicated to the task of identifying a particle set, but may be transponders or some other method. An assay used to detect an analyte is bound to the surface of a set of identically classified particles, which are generally in the size range 3–15 microns. These particles are added to a liquid containing the analyte. (In systems such as "smart dust", the assay may be distributed in the field to detect analytes and read differently.) The final step in the assay activates a reporter fluorophore that provides a signal. In systems using fluorophores for classification, the reporter fluorophore is distinct, and will have a significant frequency difference from the classification fluorophores. The particles are run through a flow cytometer, which is generally optimized for the specif