Classification and properties of 64 multiplexed microsphere sets

We describe a practical method for the analysis of multiple analytes in a single sample. The vehicle for each separate measurement consists of a set of microspheres identifiable by characteristic fluorophores embedded in the particles. The use of robust, bench-top flow cytometers (flow microfluorimeters) for the analysis of the multiple sets of microspheres is facilitated by hardware and software, which acquire the data from the cytometer, classify the microspheres according to sets, and collate measurement information from each microsphere set in real time. This measurement system can analyze up to 64 analytes in a single sample. The advantages of multiplexed assays using flow cytometry include robust measurements, because each microsphere set is measured repeatedly. The advantage of the assay's is consistent with simultaneous measurement of many parameters as well as the speed with which the flow microfluorimeter (cytometer) makes measurements (many hundreds per second). Here, we describe the properties of the microspheres, the calibration of the cytometer, and the influence of the properties of the microspheres on the sensitivity of measurements.