With the capacity to study multiple parameters at a single-cell level, flow cytometry is a widely used and extremely popular technique. Providing detailed analysis of large populations of cells within a relatively short timeframe, its applications include immunophenotyping, evaluation of the homogeneity of isolated cellular sub-populations, and the diagnosis and monitoring of disease progression. As demands for increased plex and greater throughput continue to drive the evolution of flow cytometry systems, manufacturers are employing increasingly sophisticated methods to meet the needs of researchers.
“Following sample preparation, a flow cytometry assay is typically straightforward, less time-consuming, and less labor-intensive than other cellular multiplexing assays such as imaging,” explains Garret Guenther, Ph.D., global support manager for flow cytometry at ACEA Biosciences. “And because flow cytometry is capable of rapidly analyzing many thousands of individual cells, the technology provides unmatched statistical power when compared with nearly all other commonly used assays. However, to fully leverage the potential of the technique, several key areas require bigger and bolder flow cytometry breakthroughs.”
Advanced engineering
In Guenther’s opinion, these areas include more streamlined integration of single-cell analysis with multi-omics, higher throughput and faster time to results, and deeper incorporation of flow cytometry into cell biology and molecular biology workflows through easier to use instrumentation and software. “For this to happen, flow cytometry engineers must bring more advanced technologies and innovation to their instruments,” he says. “One recent trend is to replace traditional photo multiplier tubes with semi-conductor detectors. These have been implemented in several new-to-market flow cytometers, with ACEA Biosciences’ NovoCyte® Quanteon™ being a strong case in point. We have also prioritized continual updates to our powerful, intuitive flow cytometry software (NovoExpress®) with acquisition and analysis capabilities.”
Guenther notes that the Quanteon is unique in that it employs a series of solid-state, photon-level-sensitive detectors called silicon photomultipliers (siPM), which are ideal for dim signal and rare cell populations. “Through the incorporation of up to 25 independent siPM and by providing superior side scattering resolution that pushes the small particle detection limit down to the 100 nm range, the Quanteon offers exceptional sensitivity and a 7-log dynamic range,” he says. “Furthermore, when coupled with our NovoSampler® automatic loading systems, the Quanteon is applicable to high-throughput applications and a wide variety of sample formats, including single tubes, multi-tube racks, or microplates.”
Automating workflows
Another powerful flow cytometry system that is easily combined with automation to increase throughput is the Intellicyt® iQue Screener PLUS platform from Sartorius, which can acquire data, analyze it, and visualize the results for 96- and 384-well plates in as little as 5 minutes and 20 minutes, respectively. “We’ve combined a patented, rapid micro-volume sampling technology with flow cytometry-based detection ‘engines’ to provide a high-throughput suspension cell and bead screening system that requires only a few microliters of sample,” says Joseph M. Zock, North America regional business manager for bioanalytics. “The requirement to get more information from less sample is especially acute where limited primary cells are used. Add to this the need for increased sampling and studies as industry efforts in combinatorial therapies and personalized medicine continue to expand, and the importance of conserving precious sample material is greater than ever before.”
Zock considers that there is a growing trend toward high-throughput flow cytometry, driven by increased interest in immunology, immuno-oncology, and adoptive cell therapy within drug discovery. “SLAS recently published an issue of SLAS Discovery that was focused specifically on high-throughput flow cytometry,” he notes. “This included papers from our users within the therapeutic modalities of small molecule discovery (AstraZeneca), large molecule discovery (Takeda), and adoptive cell therapy (Kite Pharma). With screening productivity lacking in traditional flow cytometry, the pharmaceutical industry is demonstrating a noticeable move toward platforms such as ours.”
Large particle analysis
Addressing the problems inherent to large particle analysis and sorting, Union Biometrica offers a range of flow cytometry systems that have been optimized for large cells, cell clusters, small model organisms, hydrogel encapsulated particles, organoids and organotypic tissue fragments. Rock Pulak, director of life science technologies, clarifies that while the principles of flow cytometry are essentially the same, larger flow cells work at lower pressures than conventional flow cytometers to reduce shear forces and provide gentler sample handling. “Larger passage through the flow cell affects the fluidics, a factor that we have addressed thoroughly within our different instrument platforms,” he says. “Also, the sorting mechanism is very different than conventional flow sorting, involving a gentle air diversion mechanism.”
Adding that Union Biometrica currently offers three different instrument platforms, all of which have considerably larger flow cells than a conventional flow cytometer, Pulak notes that the company’s most recent flow cytometry system has the added capability of capturing a brightfield image of the objects that pass through the flow cell. “Our original instrument, the COPAS FP, is fitted with one of four possible sized flow cells, 250, 500, 1000, or 2000 micron,” he says, “while for researchers wishing to change from one sized flow cell to another we developed our user-friendly BioSorter for which all four sizes are available. Our COPAS VISION, launched earlier this year, expands on the strengths of our existing instruments to provide an extra dimension to large particle analysis.”
Image: COPAS/BioSorter instruments employ a gentle air sorting mechanism for large particle analysis and sorting. Image courtesy of Union Biometrica.
Future-proofing
With an extensive portfolio of flow cytometry systems designed to meet a range of differing research requirements, BD Biosciences launched their BD FACSymphony™ S6 cell sorter earlier this year, showcasing it at the International Society for Advancement of Cytometry. “This innovative flow cytometry system affords six-way and index sorting, and supports analysis of up to 30 parameters,” says Jennifer Lazar, global program and marketing manager for the BD FACSymphony S6 cell sorter. “It also includes a scalable architecture to facilitate future expansion up to 60 parameters and is the first instrument of its kind to offer high parameter cascade (HPC) detection arrays.”
Lazar explains that HPC detection arrays allow researchers to choose up to 20 parameters on a single laser line “With the BD FACSymphony S6 cell sorter, researchers can select up to 9 spatially separated lasers from more than 25 wavelength options,” she says. “They can also dictate the positions of the HPC detection arrays, a feature that provides exceptional flexibility.”
While trends toward high-throughput flow cytometry, the use of more sensitive detectors, and improved integration of flow cytometry systems with multi-omics information have already been highlighted, Lazar adds that future advancements within the technology are also likely to be seen within the informatics space. “By allowing researchers to combine large datasets that include cell characteristics such as surface phenotypes with intracellular protein markers, together with genomic and transcriptomic information, we will see the most detailed description yet at the individual cellular level,” she says. As the needs of the various cell culture communities continue to grow, flow cytometry systems will need to work hard to keep pace, but it seems that their future development is in safe hands.