November 1, 2018 (Vol. 38, No. 19)
For such a familiar technique, flow cytometry holds many surprises. It’s more compact, more maneuverable, and more available than ever. Consequently, flow cytometry is interjecting itself where it was once unknown or where it is still considered “nontraditional.” For example, flow cytometry is getting closer to clinicians, environmental field workers, and food safety inspectors. Flow cytometry is also finding new uses in familiar settings, such as the research laboratory and the biopharmaceutical production line.
Long capable of running phenotypic screens, flow cytometry is starting to apply its powers of discrimination to genetically modified cells. Flow cytometry is validating transfection, confirming whether desirable edits have been achieved, measuring the functional effects of gene editing, and enriching cell populations based on functional cell sorting.
Compatible with autosampling and high-throughput technology, sensitive to a rainbow of colors, capable of processing multiple inputs in parallel, and reconcilable with activities upstream and downstream (including PCR analysis and genome sequencing), flow cytometry is helping advance single-cell analysis. While the combination of flow cytometry and single-cell technology is still new, it is already defining new developmental states, identifying unculturable microbes, and revealing how cellular heterogeneity relates to health and disease.
These and other nontraditional applications are highlighted in this article, which reflects the views of three experts from leading providers of flow cytometry technology. Our experts emphasize what may be the most curious thing about flow cytometry: Although it forces cells to pass in single file, it also allows cell biology to spread out in many directions.
GEN: Flow cytometry continues to evolve from its origins as a research tool. Outside the laboratory, where is flow cytometry making significant contributions? Where is it going?
Dr. Guenther: Clinical uses of flow cytometry, which have exploded in recent decades, include validating hematopoietic stem cells pretransplantation and identifying irregularities in immune cell subsets that are present in cancer and immunodeficiency disorders. As instruments have become smaller and easier to maneuver, flow cytometers have also come to be used for near-patient disease diagnosis.
Additionally, scientists are adapting instruments for nontraditional flow cytometry applications, from biomanufacturing and bioprocessing monitoring (including water quality testing and agricultural and food safety certification) to veterinary medicine, oceanography, and ecological field research. Given their high-throughput capacity for detecting and quantifying analytes in solution, and given their multiplexing potential, flow cytometry systems will be quickly adapted to diverse research and industrial sectors.
Ms. Wright: Flow cytometry is an incredibly powerful tool for single-cell analysis because it lets labs quickly interrogate millions of cells. In recent years, the number of parameters—the “multiplexing” capacity of the technology—has increased dramatically. We can now analyze dozens of “markers,” both proteins and nucleic acids, with single-cell resolution and simultaneously derive valuable population statistics.
Looking forward, we see an increasing need to democratize flow cytometry. Today, it’s still considered a highly complex technique requiring an expert operator and a time-consuming experimental design process. As flow cytometry is increasingly used in new applications, particularly nontraditional immunology workflows, the technique must become less complex, and this will happen by making instruments smarter and preconfiguring reagents.
Ms. Talaga: Since its inception, flow cytometry has been important in clinical diagnosis. More recently, flow cytometry has been used to improve the analysis of small particles such as circulating exosomes, which promise to inform liquid biopsies. Now, flow cytometry is heading into single-cell analysis.
As flow cytometers become more capable—pushing the limits of color detection, for example—they allow us to collect vast amounts of information from small samples. Small vesicles have become important because of their biological significance, but instead of trying a number of other techniques, we can now turn to flow to simplify this area. We expect that in the near future, more scientists will either conduct analysis of single cells, in both biological and clinical samples, or verify processes by flow.
GEN: How is flow cytometry contributing to the “CRISPR craze” currently rocking the life sciences?
Dr. Guenther: CRISPR technology has a strong potential for the treatment of various diseases, either directly or indirectly. As a CRISPR tool, flow cytometry is important not only for validating the correct targeting performed by CRISPR, but also for measuring the functional effects of gene editing.
For example, flow cytometry can be used to screen cells for proper transfection, allowing for rapid high-throughput analysis of many different reactions. It can also be used to identify functional targets for CRISPR, especially in immune cells to monitor cytokine production and changes in surface markers. Additionally, the flow cytometer can easily be utilized as a screening platform for CRISPR, especially with the advancement of high-throughput autosampling capabilities.
Ms. Wright: As genome editing techniques become routine, demand is increasing for cell analysis tools. At the simplest level, if we modify the “programming” of a cell, we impact its function, so we need techniques to help us analyze what happens downstream. Have we disrupted, induced, or modified protein production? What is the efficiency of the modification within a target cell population? Have our changes affected other aspects of cellular function? Are there unexpected or off-target effects? Flow cytometry and functional cell sorting will be increasingly important techniques to help answer these questions and more as the field of genome editing evolves.
Ms. Talaga: In the past few years, we have seen an explosion of companies utilizing flow cytometry in the CRISPR process, doing everything from determining transfection rates to sorting cells. These activities, we have determined, are being facilitated by flow cytometry.
At Bio-Rad, we have incorporated flow cytometry into our CRISPR workflows. Besides monitoring transfection and enriching cell populations, we have been confirming edits and conducting downstream analyses. By pairing multiple tools and techniques, we have increased workflow efficiency. At the end of the day, scientists using CRISPR want to know that they have successfully edited their target cells before moving to downstream assays.
GEN: Now that flow cytometry is being adapted for immunology, will new applications emerge to advance immuno-therapy?
Dr. Guenther: As the immunotherapy field continues to expand, flow cytometry will play an important role in the development of varied immunotherapies. I see the method mostly involved in the quality control of the cell validation process during and after treatment, and in the monitoring of the therapeutic cells in the patient over time.
Many cellular subsets and functional markers of immune cells can be rapidly and efficiently analyzed by flow cytometry without the need of additional assays, so flow cytometry can also be applied to predict off-target effects. Finally, an exciting emerging area is the integration of flow cytometry into multi-omics for the application of precision medicine in immuno-oncology.
Ms. Wright: Traditional immunology was grounded in the principles of immune function, both innate and acquired. Immunotherapy is no different, and we’ll need to return to these principles to understand what a tumor does to evade the immune response and which aspects of the innate and adaptive immune systems can be harnessed to enable tumor elimination or avoidance altogether.
Some fascinating science is taking place in the immunotherapy field, particularly a focus on understanding the tumor microenvironment. This is putting a spotlight on flow cytometry as well as new technologies such as acoustically focused flow cytometry. These techniques are important in this research area because they can handle samples derived from tissues without complications such as clogging, and they enable efficient analysis of rare cell populations.
Ms. Talaga: Chronic viral infections and cancers have always tried to evade the immune system. Today, we are recognizing that the immune system cannot be studied as though it existed in isolation. Instead, its relationship to the microbiome must be recognized, as well as its (potential) vulnerability to combined therapies. We have been learning to reach across scientific disciplines and exchange both knowledge and techniques.
This combination of interdisciplinary work and knowledge sharing has led, in turn, to what we call the “emerging era of immunotherapies.” Scientists who incorporate flow cytometry into greater workflows—such as workflows encompassing PCR detection—are finding greater success. For example, PCR detection may be followed by electroporation and flow cytometry, allowing us to acquire new knowledge about, say, stem cells, and apply it into clinical work.