March 15, 2016 (Vol. 36, No. 6)

The Seers of Flow Cytometry Predict Applications from Clinical Diagnostics to Drug Discovery

Although flow cytometry is a workhorse technology that has been around for more than 50 years, it still shows a lively gait. In fact, flow cytometry is about to break into a gallop, spurred by technological advances such as the integration of mass spectrometry and various forms of single-cell “omics.” Also, flow cytometry is enjoying wider use in clinical diagnostics and drug discovery.

Yet another development in flow cytometry is an increasingly rich color palette. The technology originated in the 1960s with just 2 colors. It now boasts a whopping 20 colors—and more are on the way.

It must be said, however, that flow cytometry, like any fast-advancing technology, will have its share of challenges. For example, flow cytometry will be characterized by increasing complexity. The technology will also have to find ways to enhance throughput and manage rapidly escalating data generation.

To help us anticipate how these and other challenges will be met, we asked flow cytometry experts to dust off their crystal balls and share their visions and expectations. Most agreed that challenges will be overcome and that flow cytometry’s future promises a colorful, almost dazzling array of improvements and applications.

Lower Costs, Smaller Footprints

Andrew L’Hullier, Ph.D., an R&D scientist at Enzo Life Sciences, says the trend toward making flow cytometry instrumentation smaller and more affordable will likely continue: “In recent years, there have been huge advances in the number of parameters that can be analyzed on a per-cell basis. On the one hand, lasers are smaller and cheaper than ever, and while a single-laser cytometer used to be a huge investment to be shared in a core lab, multilaser machines are now affordable on benchtop units.

“The drop in price has also lowered the bar for entry into the market, bringing in many more options. This, coupled with better technology for detecting fluorescence and more intelligent software that can analyze and compensate data for clean results, means many parameters can be analyzed with readily available and affordable cytometers. Further, other advances such as Q-dots have great promise to increase the number of colors that can be multiplexed.”

Not all advances will be as economical. “At the other end of the price spectrum, the advent of mass cytometry is allowing 40-plus parameters to be analyzed at a time on a per-cell basis,” notes Dr. L’Hullier. The investment, however, could lead to healthy returns—more information than ever before.

“Mass cytometry is still in its infancy, and has huge potential, not only for the number of parameters to be analyzed together, but also for what types of targets can be analyzed,” adds notes Dr. L’Hullier. “Mass spectroscopy techniques such as stable isotope labeling by amino acids in cell culture (SILAC) could make their way into mass cytometry in the future, giving an amazing wealth of information.”

Computational Challenges

Bob Smith-McCollum, head of marketing for flow cytometry at MilliporeSigma, agrees: “There continues to be an unending appetite for more colors, more channels, and faster event rates. But with these advances, customers still will require advances in software for sample acquisition, fluorescence compensation, and data analysis.”

Such issues are especially impactful for integrating flow cytometry with microscopy.  “Coupling these two technologies combines the resolution and detailed imagery provided by microscopy with the speed, sensitivity, and sample size of flow cytometry,” Smith-McCollum reports. “For many years, flow cytometry identified cell populations on scatter plots, but it couldn’t address the problem of incomplete information. That drove us to develop our Amnis® imaging flow cytometer systems.”

As such systems continue to develop, a challenge will be throughput and complexity. “Systems will need to be developed using fluidics technologies to advance event rates and sample throughput,” Smith-McCollum notes. “Also, as we continue to address where the target protein is located in cells, there will be a corresponding increase in data complexity.  This will necessitate advances in image analysis with much more powerful desktop computing capability.”

Connecting to Omics

Several of our experts agreed that flow cytometry is on a trajectory to integrate with multi-omics. The result will be a massive harvest of cellular information. “The ultimate goal in any flow cytometry application is to reliably increase the number of parameters that can be analyzed simultaneously,” says Dara Grantham Wright, senior vp/general manager, eBioscience, Affymetrix.

“The number of parameters that can be interrogated is currently linked to the number of available dyes to simultaneously stain cellular markers, so broader availability of new dyes for multiplexed experiments is important,” Wright continues. “This dye challenge is affected by the number of excitation and light capture parameters available on an instrument configuration. The last challenge is with subsequent deconvolution of the data, so that various cellular attributes can be analyzed.”

Other challenges are more basic. “One research-related issue is the need for better methods for solid tissue disaggregation,” explains Wright. “Methods that could prepare solid tissue for flow cytometry are crucial for expanding the use of the technology to applications such as cancer studies.”

Further scientific progress increasingly relies on “multi-omic” approaches. Methods that can enable simultaneous cell, protein, and molecular analysis at a single-cell level are desired. Also, new platforms and test methods are required to make this a reality.

Wright asserts that Affymetrix has moved the field forward, for example, by commercializing single-cell analysis techniques such as Primeflow RNA, which “enables the simultaneous analysis of RNA and cellular protein in millions of cells using flow cytometry.”

Antibody Tools

One of the critical limiting factors in flow cytometry continues to be the availability of specific, highly validated antibodies for the detection of proteins of interest. This will also be the case for the future of flow cytometry, states Jody Bonnevier, Ph.D., manager, flow cytometry, R&D Systems, Bio-Techne. “As new research questions emerge,” she says, “new antibodies will need to be developed to help address these questions.”

Dr. Bonnevier maintains that although the development of additional dyes is necessary to detect more and more proteins simultaneously in multicolor flow, it’s just as important to develop new robust antibodies to label with those dyes. “R&D Systems continues to develop and manufacture 95% of the antibodies in our portfolio,” Dr. Bonnevier maintains. “By doing so, we maintain control over everything in the development process from immunogen design to immunization strategy, initial screening, and application testing.”

To enhance identification and specificity, development of new antibodies will be a priority.

“Antibodies need to be designed from the beginning to be flow cytometry reagents,” explains Dr. Bonnevier. “If we are after a difficult target (that is, low expression, part of a large complex of multiple proteins, located in an intracellular compartment), we can optimize the immunogen to be anything from a full-length biologically active protein to a short peptide aimed at an exposed epitope in a protein complex, or at a specific region that differentiates one family member from another closely related one.”

Important parameters in this pursuit include host species and immunization protocols to optimize antibody production. “Antibodies derived from specific hosts are screened at every step of the process for the intended applications,” informs Dr. Bonnevier. “And cellular models and in-house antibody testing need to be designed to stringently test the robustness in flow cytometry, before and after conjugation to a fluorochrome. Thus, antibodies will continue to be an important tool, even for future flow cytometry.”

Ramping Up Cell Numbers

Currently, most flow cytometry systems evaluate tens of thousands of cells. In future flow cytometry applications, that figure will likely grow, insists Gregory Kaduchak, Ph.D., engineering director, flow cytometry, Thermo Fisher Scientific. “As research continues to head toward both higher-throughput applications such as rare event detection as well as expanding into areas of research, the ability to detect a larger variety of cell types at increased rates in the many tens of millions will be required. This is forcing flow cytometry instrument developers to create innovative ways to run large samples with very large event numbers without increasing the time to result.”

When increased numbers of cells are utilized for more comprehensive analysis, it will become necessary to more efficiently extracting them from samples. “Sample preparation is the underappreciated part of analysis,” says Dr. Kaduchak. “The creation of innovative cell sample-preparation methods can be used to reduce workflows, de-bulk samples of unwanted cells, and provide more repeatable results.”

Furthermore, as cell throughput accelerates, sophisticated means of analyses must appear. Dr. Kaduchak anticipates that the analysis process could be automated so as to yield “something akin to a ‘red light’ and ‘green light’ type answer.” Currently, it takes a well-trained cytometrist to both design and perform multicolor experiments.

“The need for such expertise effectively keeps flow cytometry from the greater scientific community,” complains Dr. Kaduchak. “If a system can remove these barriers and allow for the democratization of flow cytometry by automating such tasks, then more researchers could incorporate flow cytometry into their tool box to extend and expand their research. This of course would require the creation of many new standards, but the payoff would be significant.”

Clinical Realm

Flow cytometry has led to many advances in immunology, hematology, and pathology.  It is invaluable in clinical applications as well as in research. “Flow cytometry first entered clinical labs as a result of the HIV epidemic,” recalls Jeannine Holden, M.D., director, scientific affairs, flow cytometry, Beckman Coulter. “Even before we had discovered the virus, it was clear that decreasing CD4+ T cells were an indication of worsening disease.”

Challenges affecting flow cytometry in the realm of R&D also extend to the clinical use of flow cytometry with additional concerns such as reimbursement. According to Dr. Holden, there are three “holy grails” in flow cytometry that challenge both research and clinical cytometrists. She summarizes them as follows: sample preparation, antibody cocktailing, and data analysis.

“All three are time-consuming and predominantly manual, and consequently they represent potential sources of error that may obscure important findings,” Dr. Holden observes. “They also drive labor costs, an increasingly important issue for clinical labs facing reimbursement pressure. Vendors will need to find solutions that address workflow in clinical laboratories, ideally by targeting these three areas in a coherent and complementary manner.”

Drug Discovery and Beyond

It has been historically difficult for standard flow cytometry to expand into areas such as pharmaceutical drug discovery. But that will change in the future, suggests Jason Whalley, Ph.D., director of cell biology at Bio-Rad. He remains optimistic even though  progress has been uneven.

“Flow cytometry has seen an expansion in the products offered in the low- to mid-range instrument specifications,” he explains. “However, there has been little innovation in the high-specification instrument range.

“High-specification instruments offer the expansion of flow analysis due to their detection and analytical capabilities. Improving the ease of use, screening capabilities, and data analysis of these high-end instruments will increase the adoption within pharmaceutical and biotechnology companies, especially as drug discovery is moving to a phenotypic screening approach.”

Joseph M. Zock, senior director, product management, IntelliCyt, weighs in on the issue: “Reasons for this bottleneck include a lack of sample throughput, need for experts to make adjustments, large sample/reagent requirements per assay, and a lack of plate-level analytics including the deconvolution of highly multiplexed cell-based data.”

According to Dr. Zock, this bottleneck hasn’t prevented progress in the industry: “Work meant to eliminate this bottleneck helped our company evolve the flow cytometer into a detection engine for drug discovery. We combined the instrument with a rapid, microvolume sampling technology and a revolutionary software experience. The resulting system, the iQue® Screener, can automatically process multiple microplates in minutes (384-well plate in under 15 minutes) while using a fraction of the well volume of standard flow cytometers; analyze an entire plate of data at a time; and provide ‘plate to well to cell’ data visualizations including profiling each well against multiple, user-defined criteria.

“These improvements in speed, miniaturization, usability, content, and insight are paving the way for ‘evolved flow’ to take a critical role in areas like antibody discovery, immuno-oncology, and adoptive cell therapy.”

Further, flow cytometry may play an important role in the evolution of personalized medicine. “The future of medicine will be to personalize by understanding a patient’s unique physiology and applying a combinatorial therapeutic approach,” Dr. Zock suggests. “This will require an enormous number of correlative studies. Flow cytometry will need to be even faster and easier!”

Flow cytometry platforms are evolving to contribute to drug discovery efforts. For example, the iQue Screener PLUS, developed by IntelliCyt, integrates microvolume sampling technology and software-assisted automation, analysis, and visualization tools, in an effort to streamline screening workflows. According to IntelliCyt, the instrument enables rapid, high-content, multiplexed analysis of cells and beads in suspension in 96-, 384-, and 1,536-well plates. The company adds that plates can be processed rapidly (less than 5 minutes for 96 wells) and that assays can be miniaturized to conserve samples and reduce reagent use.

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