Flow Cytometry is generally understood to provide recognition of biomarkers (proteins, genes, small molecules) in individual cells on the basis of optical detection of stimulated emission from fluorescent tags bound to affinity products (antibodies, oligonucleotides, aptamers) that bind to the biomarkers. Indeed, Fluorescence Activated Cell Sorting, FACS, explicitly recognizes that fluorophores are innate to the method. The majority of flow cytometers are used for analysis; a minority are used for sorting intended to purify cell populations, and a subset of FACS applications takes advantage of sorting for cell population purification or so that the collected cells can be stripped and re-labeled for subsequent complementary assay.

"If at first the idea is not absurd, then there is no hope for it."
A. Einstein

Multiplex fluorescent assays in typical use are limited to about 4- or 5-plex due to overlap of the fluorescent signals, as shown below. There are reports, and intense development, of higher-order multiplex fluorescent assays (as many as 17 have been reported) through the use of time-delayed excitation and detection, use of quantum dots in combination with fluorophores and other means. Extension to more than, say, 20-plex seems to be unlikely, and the practitioners generally admit that more than 10-plex, possible on some commercial instruments, requires heroic experimental design to account and correct for the overlap.

Overlay of emission spectra of 8 fluorophores (shaded) and 8 quantum dots (lines). The substantial overlap into neighbor detection channels requires significant, time-consuming and error-prone compensation.

A new technology provides a transformative leap in the capability of quantitative massively multiplexed cell assay. Called Mass Cytometry, it is premised on the use of elements, or stable isotopes, as tags instead of fluorophores, with measurement of the tags using an Inductively Coupled Plasma Mass Spectrometer (ICP-MS). The advantage lies in the large number of available elements and stable isotopes (potentially greater than 100), the high resolution of the mass spectrometer between detection channels, and the large dynamic range (linearity) of detection of the ICP-MS (9 orders of magnitude for current commercial quadrupole-based systems for solution analysis).

ICP-MS spectra of 60 element isotopes. The high resolution of the detection channels and large dynamic range eliminate the need for compensation, enabling multiple parameter analysis with the ease of single channel assay.

These benefits, and others, result in the ability to perform multiplex assays of high order (as many as 100) without the need for mathematical correction of overlap, and with large dynamic range both for a given target biomarker and between different biomarkers (where, for instance, neighbor detection channels can clearly distinguish antigens that differ in concentration by up to 6 orders of magnitude, for solution asays).

Other benefits are high sensitivity (comparable to radioimmunoassay and chemiluminescent assay; of the order of attomoles/microlitre or as little as 100 copies of an antigen per cell); insensitivity to light or time (so that the experiments can be performed in a lighted laboratory, and can use reagents prepared or purchased long before --- unlike fluorophores) ; and stability of the immunolabeled samples so that the product can be stored and/or shipped for analysis (fluorophore activity rapidly decreases with time).

Because many thousands of distinguishable beads can be produced with the incorporation of multiple elements as a barcode, a massively multiplexed bead assay is enabled. Polystyrene beads are prepared to incorporate a specified amount of several elements, which are trapped inside the beads by chelation to prevent element leaching. The surface of the beads is functionalized with amino or carboxyl groups for convenient attachment of biomolecules (oligos, antibodies, etc). The elemental encoding of the bead can indicate the type of biomolecule that is attached, and hence the antigen or gene that it targets, or can correspond with a sample identifier. In most instances, binding of the target is flagged by a common reporter elemental tag that is sandwiched to the antigen or functionalized to the gene. Hence, for example, analysis at a rate of 1000 antisense-oligo-functionalized beads per second provides a 1000 gene assay that can be conducted with 30-fold redundancy in 30 seconds.

In addition, the new class of metalopolymer tags is also suitable for determination using conventional ICP-MS instruments in the instance that an average assay over a sample ensemble (i.e., solution assay) is desired. For example, where a tissue is sufficiently homogeneous, or the diagnostic allows for averaging over the biopsy, the sample may be stained with the metal-tagged antibodies and, following washing, may be acidified to lyse the cells and provide a homogeneous solution that can be analyzed according to long-standing standard ICP-MS protocols. The solution assay protocols still allow for massively multiplexed assay, with detection limits for each marker comparable to individual radio-immunoassay.