Multiplexed analysis of clinical specimens apparatus and methods

A method of detecting multiple analytes in a sample, said method comprising:

• (a) exposing a pooled population of subsets of particles to a sample, the particles in each subset having (i) one or more characteristic classification parameters that distinguish the particles of one subset from those of another subset according to a predetermined discriminant function table, which includes fluorescence emission intensities, and (ii) a reactant specific for an analyte of interest;
• (b) adding at least one secondary reagent to the exposed pooled population, wherein the at least one secondary reagent comprises a solid particle label;
• (c) passing the exposed pooled population of subsets of particles through an examination zone;
• and
• (d) determining the identity and quantity of each analyte of interest, if present, in the sample by substantially contemporaneously:
• (i) detecting the at least one secondary reagent added to the population, (ii) collecting data relating to the one or more characteristic classification parameters, including the fluorescence emission intensities, (iii) collecting data relating to the presence or absence of a complex formed between the reactant and the analyte of interest specific to the reactant, (iv) classifying, without relying exclusively, if at all, on differences in particle size, each particle to its subset according to the predetermined discriminant function table, and (v) quantifying the amount of complex associated with each subset.