Flow cytometry is an invaluable technique for the characterisation of highly heterogeneous cell populations.

Thanks to improvements in throughput, automation and multichannel detection capabilities, recent years have seen its use expand across a wide range of drug discovery applications. In this article, we look at novel developments in flow cytometer reagent and instrument design, and what these mean for drug discovery.

Flow cytometry is an optical cell counting and characterisation technique that enables large quantities of multiparametric information to be rapidly collected on highly heterogenous cell populations. Capable of analysing many thousands of cells in a matter of seconds, flow cytometry offers formidable statistical power for screening applications, while its ability to simultaneously measure dozens of parameters on an individual cell basis gives it considerable advantages over bulk analysis techniques that average out important phenotypic differences.

Improvements in speed, capacity and usability over the past decade have seen flow cytometry used for a growing number of drug discovery applications, including biomarker discovery, ligand binding assays, antibody screening as well as target identification and validation. In large part, the adoption of flow cytometry across the length and breadth of the drug discovery pipeline has been driven by the ease of which systems can be integrated with automation platforms, making high-throughput screening applications increasingly cost-effective while helping pharma innovate faster.

Despite these improvements, the need for pharmaceutical and biotechnology companies to do more with the same resources has put pressure on laboratories to derive additional value from their flow cytometry workflows. Fortunately, instrument and reagent vendors are meeting this need by developing solutions that make workflows more efficient and maximise the range of parameters that can be studied simultaneously.

Pushing the limits of multiparametric analysis

Flow cytometry measures how individual cells interact with light. The technique involves passing a thin stream of cells, often labelled using fluorescent dyes (known as fluorochromes), in front of one or more laser beams. Absorbed light is subsequently emitted by fluorescence at specific wavelengths and the intensity of this light is measured using sophisticated optics and electronics. Because cells with specific phenotypic features generate differing emission spectra, valuable cell characterisation information can be rapidly obtained.

Ongoing advances in instrument design have resulted in the commercial availability of flow cytometers with an extensive range of laser and detector options. Together with improvements in the range of fluorescent dyes available for cell staining, this has led to an expansion in the number of wavelengths or ‘channels’ that can be used to probe specific cell parameters. While these developments have enhanced the analytical power of multichannel flow cytometry as a screening technique, this also necessitates the careful selection of laser, fluorochrome and detector technologies to maximise the amount of useful information generated.

One of the most important factors affecting the quality of flow cytometry data is the choice of channels used to measure specific cell parameters. However, this task is complicated by the fact that some chromophores fluoresce at very similar wavelengths, with varying degrees of intensity. If a detector assigned to pick up signals from a fluorochrome of interest detects fluorescence emission from a second fluorochrome, the physical overlap of emission spectra (known as spectral spillover) can make data interpretation challenging. This issue is even more acute if the fluorochrome of interest is weakly-emitting or dim, making the effects of interference even more difficult to deconvolute. To address this issue, many instrument and reagent vendors are expanding and enhancing the range of detection channels available for flow cytometry experiments. One approach is to improve the quality of fluorochromes used for cell staining.

“The limited availability of high-quality dyes with high extinction co-efficients has been a major problem for laboratories, leading to sub-optimal resolution of positive and negative cell populations,” explains Dr Robert Balderas, Vice- President, Market Development for Biosciences at BD Biosciences. “Our polymer dye technology platform gives researchers access to a broad range of fluorochromes, many of which are 20 times brighter than traditional dyes. This not only facilitates the use of new laser lines, it adds additional options to existing ones too.”

Another strategy to enhance the quality of flow cytometry measurements, especially when using dim fluorochromes, is to improve instrument sensitivity. One way this can be achieved is by using advanced detection technologies.

“Some of the most exciting advances in flow cytometry design relate to improvements in detector technology,” notes Dr Nancy Li, Vice-President of Engineering at ACEA Biosciences Inc, part of Agilent Technologies. “Silicon photomultiplier (SiPM) technology, for example, offers high gain and high sensitivity for photodetection, which makes it possible to simultaneously measure a large number of fluorophores with excellent signal resolution. Our NovoCyte® Quanteon™ system is the first flow cytometer to adopt SiPMs for fluorescence channel detection.”

By increasing the number of viable channels that can be used for cell characterisation and improving instrument sensitivity, cell biology can be studied in much greater detail, potentially putting more translationally-relevant information in the hands of drug discovery scientists. These advances also allow the collection of more useful data from much smaller tissue samples. Not only is this more convenient for sample donors, it also allows laboratories to get more value from their existing workflows, helping to boost operational efficiency. As such, the latest advances in multichannel flow cytometry are poised to accelerate research and even improve success rates in a wide range of fields, including biomarker discovery and target identification.

Realising the full potential of automation

With throughput an important requirement for screening applications, manufacturers of flow cytometry platforms have devoted considerable resources to improving system capacity and speed in recent years. The commercial availability of instruments capable of processing large-scale micro-384 well plates at rates of tens of thousands of cells per second presents a wealth of opportunities for high-throughput screening applications.

Moreover, the steady adoption of automation into flow cytometry workflows has led to more efficient processes that allow laboratories to work with much smaller volumes, boosting efficiency and measurement precision. Despite this, poor instrument flexibility has meant that manual instrument set-up and switchover steps are typically still required – a time-consuming practice that prevents many organisations from fully realising the full efficiency benefits of automation.

“A lack of flexibility when changing between sample formats has proven to be a major bottleneck in flow cytometry workflows, particularly for multi-user research labs that use a lot of different plate formats,” explains Dr Xiaoti Guo, Global Commercialisation Product Manager at Bio-Rad Laboratories. “To run plated samples, for example, incumbent systems typically require users to switch to an external plate loader and manually attach the plate sampling tubing to the sample injection port. These steps can be cumbersome and often require extra attention.”

Some instrument manufacturers, such as Bio-Rad Laboratories, are addressing this issue by developing systems with integrated sample loaders that can accommodate all widely-used sample container formats. These systems reduce the need for manual changeover, freeing up scientists to work on other tasks. Additionally, the software solutions used to support these automated platforms are highly configurable, allowing laboratories to tailor their systems to their individual application needs. With these streamlined workflows, organisations engaged in drug discovery can benefit from more efficient processes, potentially accelerating successful outcomes.

Commercially-available flow cytometry systems

A broad range of flow cytometry systems is currently available on the market. A selection is highlighted here.

Bio-Rad Laboratories’ ZE5 Cell Analyzer (Figure 1) is designed with throughput, speed and flexibility in mind and can be configured with up to five lasers and 30 detectors. With this high parameter set-up, the system is capable of handling either high-colour panels or panels with minimal compensation; ZE5’s fast fluidics and powerful electronics enable high-speed analysis of particles at 100,000 events per second without compromising data quality.

Its innovative sample pump allows sampling from a volume as low as 2 microlitres, which greatly saves both samples and reagents. The sample loading platform accommodates 40- or 24-tube racks, 96-, 384- and deep-well plates, and its smart sample-handling features such as temperature control and vortexing capabilities are designed to maintain cell viability and prevent clogging. Furthermore, the system can be integrated into automated workflows and is ready to connect to laboratory information management systems and scheduling software to support new or existing automation solutions.

ACEA Biosciences’ NovoCyte® Quanteon™ is a four-laser benchtop flow cytometer that can be configured for up to 25 fluorescent channels to support the screening of large-scale panels. The system incorporates the most sensitive silicon photomultiplier detector technology on the market and offers a 7.2 log dynamic range, allowing both very dim and bright signals to be captured in the same analysis for the detection of rare events and low abundance phenotypic markers.

Combined with the NovoSampler Q™ autosampler, the system boasts rapid analysis speed and allows a 96-well plate to be processed in less than 20 minutes. Automated after-sampling rinse ensures minimal carryover between samples (<0.1%). The system is compatible with all of the most widely-used plate formats, from individual tube to 384-well plate, and can be integrated into automated workflows. The orbital shaker and barcode reader built in NovoSampler Q provide a complete walk-away solution when integrated with a laboratory automation system, while an Open Application Programme Interface allows for seamless operation with a scheduling software in a highly-automated format. Fully automated calibration can get NovoSampler Q up and running within five minutes following installation (Figure 2).

BD Biosciences offers a range of flow cytometers for research and clinical diagnostics applications. The BD LSRFortessa™ X-20 cell analyser (Figure 3), for example, is a compact system built for high performance multicolour analysis and can be configured with up to five lasers to facilitate the detection of as many as 20 parameters simultaneously.

Thanks to its fully-automated sample handling capabilities, the analyser can process a 96-well plate in under 15 minutes with less than 0.5% sample carryover. Its BD Accuri™ C6 Plus flow cytometer puts additional focus on usability and affordability, helping both novice and experienced users to benefit from flow cytometry. Incorporating two lasers, two light scatter detectors and four fluorescence detectors, the system also features a user interface that is designed to be intuitive for non-experts.

Beckman Coulter Life Sciences also offers a broad selection of systems for both routine and high-complexity flow cytometry applications. Its CytoFLEX Platform encompasses three compact instrument models designed to deliver optimal excitation and emission, minimise light loss and maximise sensitivity. The most advanced system in this range, the CytoFLEX LX, allows up to six lasers to be used, providing up to 21 fluorescence channels.

The Intellicyt® iQue Screener PLUS platform is an integrated instrument, software and reagent system that facilitates rapid, high-content, multiplexed analysis of cells and beads in suspension. Three optical configurations are available, based on either two or three lasers, which permit the use of up to 15 detection channels. The system is designed to deliver insights quickly, with a detection rate of up to 35,000 events per second and enhanced data visualisation tools that support rapid interpretation of results (Figure 4).

Conclusion

Improvements in flow cytometry reagents and instrumentation are helping to extend the analytical capabilities of this powerful technique. Recent advances in system flexibility, throughput and multichannel detection capacity are already helping scientists obtain more useful information from phenotypically diverse cell samples. As the insight that can be obtained from flow cytometry grows, its impact on existing and novel fields of drug discovery is set to expand. DDW

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This article originally featured in the DDW Spring 2019 Issue

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Helen Stewart-Miller is Director of PR Services and Dr Richard Massey is a science writing consultant at BioStrata, a life science specialist marketing agency. The company’s growing team in Cambridge (UK) and Boston (US) includes a significant number of people with deep scientific experience and knowledge. The agency offers a range of services from strategy, branding and message development through to content creation, creative design, digital marketing and public relations.