Multiplexed analysis of clinical specimens apparatus and methods

A computer-readable medium, storing analyte-determining instructions for determining multiple analytes of interest in a sample, wherein a pooled population of subsets of particles is exposed to the 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, including fluorescence emission intensities, and (ii) a reactant specific for an analyte of interest, and wherein the exposed pooled population of subsets of particles is passed through an examination zone, said analyte-determining instructions being executable by a computer, said analyte-determining instructions comprising the step of:

• determining an identity and quantity of each analyte of interest, if present, in the sample by substantially contemporaneously
• (i) collecting data relating to the one or more characteristic classification parameters, including the fluorescence emission intensities,
• (ii) collecting data relating to a presence or absence of a complex formed between the reactant and an analyte of interest specific to the reactant,
• (iii) classifying, without relying exclusively, if at all, on differences in particle size, each particle according to its subset according to the predetermined discriminant function table, and
• (iv) quantifying an amount of complex associated with each subset.

A method of conducting a multiplexed assay to determine the identity and quantity in a sample of one or more differing analytes comprising:

• (a) passing through an examination zone a mixture comprising a pooled population of subsets of nearly identically sized particles, which population has been exposed to a sample, and the particles of each subset (i) exhibiting one or more characteristic classification parameters that distinguish the particles of one subset from those of another according to a predetermined discriminant function table, including fluorescence emission intensities, and (ii) having a reactant specific for an analyte of interest;
• (b) collecting, as the particles of the pooled population pass through the examination zone, data that relate to one or more characteristic classification parameters of each particle including the fluorescence emission intensities, and which relate to the presence or absence of a complex formed between the reactant and an analyte of interest specific to the reactant;
• and
• (c) substantially simultaneously with the collecting step (b) classifying, without relying exclusively, if at all, on differences in particle size, each particle according to its subset according to the predetermined discriminant function table and quantifying an amount of complex associated with each subset, to provide an identity and quantity of one or more differing analytes, if present, in the sample.