A chromatic explosion: the development and future of multiparameter flow cytometry

Abstract

Multiparameter flow cytometry has matured tremendously since the 1990s, giving rise to a technology that allows us to study the immune system in unprecedented detail. In this article, we review the development of hardware, reagents, and data analysis tools for multiparameter flow cytometry and discuss future advances in the field. Finally, we highlight new applications that use this technology to reveal previously unappreciated aspects of cell biology and immunity.

Figure 1

Bulk measurements include irrelevant cell populations that introduce noise, and reduce the power to detect cells of interest. If a population of cells, defined by the phenotype A⁻ B⁺ C⁺ D⁻, correlates strongly with disease or immunity, then assays with fewer than four markers include irrelevant populations that introduce noise into the assay. Note that the measurement of too many may also reduce power, unless the additional markers are excluded from analysis.

Figure 2

The development of multiparameter flow cytometry. The width of each curve represents the time lag between research discovery and commercialization, while the colour gradient indicates the degree to which each technology has been broadly adopted. For example, advances in hardware (electronics, detectors and lasers) largely occurred in the late 1990s, leading to 20-parameter instrumentation. Instrumentation first developed in 2001 is now widely available; therefore, the colour gradient of this curve is mostly dark. Advances in reagents lagged somewhat behind, and the period between research discovery and commercialization was much longer than for instrumentation. Notably, a number of monoclonal antibody–fluorochrome conjugates are not yet commercially available. Data analysis techniques in the larger flow community remain nascent and represent the area where the greatest need for development remains.

Figure 3

A selection of fluorochromes commonly used in flow cytometry. For each fluorochrome, the optimal laser source for excitation is indicated by coloured dark lines (violet = 405 nm, blue = 488 nm, green = 532 nm and red = 633 nm). Some fluorochromes, such as quantum dots, are suboptimally excited by a number of other laser lines, which are represented by the dimmer lines. Each fluorochrome’s range of emission is indicated by the grey/black bars, with the darkest region corresponding to the wavelengths of strongest emission. APC, allophycocyanin, FITC, fluorescein isothiocyanate; PE, phycoerythrin; QD, quantum dots.