Flow cytometry has become an essential tool for separation and characterization of single cells in heterogeneous populations. Immune cell and stem cell classification for research and clinical applications are examples, among a wide array of applications. View a brief primer on Flow Cytometry.

Drawbacks of the technique traditionally included relatively low-throughput workflows, arising from limited multiplexing and sample processing speeds. Technical limitations affecting accuracy, as well as complex operation, have also been difficulties in the past.

State-of-the-art flow cytometry systems are harnessing multiplexed detection and automatable workflows, significantly enhancing the scale and speed of analysis.

Advanced focusing technologies are boosting the strength and accuracy of cellular characterization – with overall implications for the fields of immunology, oncology, cell therapy, and other areas of clinical importance.

Flow cytometry protocol

At the basic level, flow cytometry can detect the volume, cell-size, and structural complexity of cells, typically by capturing side and forward scattering light, or fluorescence, projected by a laser light source on a flow cell. The presence of specific DNA, RNA, protein, antibodies, etc. requires use of fluorophore conjugated antibodies and lasers of specified wavelengths.

The sample flow cell is typically attached to sensitive detectors to capture the intensity of emitted light on fast time scales. These signals are then used to deduce the state of the cells or internal components relative to blanks or control cells.

Traditional workflows are complex, sometimes prone to variability in sample prep -- and some markers are cytotoxic, further challenging accuracy. Overall, the analysis time can be lengthy, especially with large cell populations and mixed phenotypes.

New flow cytometry technologies

Three main aspects of technology development are together having significant impact of the power and performance of modern flow cytometry instrumentation.

Multiplex flow cytometry

The advent of multicolor flow cytometry has led to an evolution in the workflows of the traditional technique.

The number of targets which can now be measured in a given sample or experiment has increased and is now limited only by the number of light sources and detectors available on a given instrument.

The use of multiple laser light sources can be coupled with fluorophores with distinct absorption profiles – expanding the number of antibodies and targets available for measurement.

Multiple lasers can also be used in separate channels or in tandem to measure differing absorption profiles within the same sample or cell.

The use of multiple detectors enables the measurement of emitted light covering a broad spectrum of fluorophore signals – again individually, in combination, or in sequence.

The net benefit of these advancements is the ability to now measure, for instance, cellular effects of experimental stimuli, the origin or fate of cellular features over time, or the coexistence of multiple immune markers.

Multiplexed detection conitnues to evolve with a large array of adaptable configurations now available.

Flow cytometry processing speed and automation

High-throughput processing is another technology feature driving the evolution of flow cytometry instrumentation.

Traditional instruments have been limited by, not only the number and arrangement or light sources, detectors, and flow cells, but by the inability to interface with automation.

Even high-end, multifunctional instruments, while powerful, have traditionally been restricted by the operator and sample interface, as well as data analysis and management at the tailend.

Upgrades to the sample interface and fluidics handling systems has now enabled more streamlined coupling of automation modules.

Plate robots, plate hotels, incubators, and reagent handling products can together serve to significantly increase the rate of sample preparation.

The ability to process 96- or 384-well plates (or more) has clear implications on processing rates for sample intensive applications.

Control software to manage all of these features, with the ability to interface directly with laboratory information management systems (LIMSs), creates a central command interface – decreasing complexity of operation and increasing workflow efficiency.

In total, these advanced features should now support large-scale, high-throughput applications such as biomarker discovery and phenotypic screening – workflows which were unachievable with previous flow cytometer technologies.

Advanced flow cytometry focusing and accuracy

A third enhancement for modern flow cytometer performance involves increased accuracy arising from new focusing technologies.

Acoustic focusing involves the use of ultrasound waves to funnel cells into and through the flow cell and detection point. The advantages of this approach over the more typical liquid flow cell alignment includes higher throughput processing of more dilute samples or populations of specific cells, such as stem cells, in heterogenous samples.

Although in the experimental stage, combined ultrasound (US) backscatter and photoacoustic (PA) (combined acoustic and laser irradiated) approaches will theoretically enable single cell in vitro label-free multiparametric analysis based on two difference physical interactions. Photoacoustic signals contain information about the cellular components such as lipids, mitochrondria, DNA, etc. Ultrasound backscatter collects information on cell shape, size, and biomechanical properties.

Together, these powerful techniques may promise to significantly increase the accuracy and scope of flow cytometry applications.

Summary

Technology development is moving quickly to keep pace with modern research and clinical approaches towards immunotherapy and cell therapy in a variety of disease scenarios.

It will be fascinating watch as these new technologies continue to expand and flow into the marketplace.