Flow Cytometers Get Even More Powerful

Flow Cytometers Get Even More Powerful

Although being challenged by alternative platforms, for this decades-old analytical technique, the best, it seems, is yet to come.

Flow cytometer is a powerful tool that has evolved over the past few decades. It has long enabled basic and clinical research, with applications in fields as diverse as molecular biology, neuroscience, and plant and marine biology. Flow cytometers have now become essential instruments in biomedical research and routine clinical tests for disease diagnosis, prognosis, and treatment monitoring. Recently, its use in cancer immunology has grown impressively, because of the role it can play in extending the understanding of immune system and its response to cancer immunotherapies. 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 non-traditional flow cytometry applications, from bio-manufacturing and bioprocessing monitoring to veterinary medicine, oceanography, and ecological field research. Given their high-throughput capacity for detecting and quantifying analytics, and given their multiplexing potential, flow cytometry systems are being quickly adapted to diverse research and industrial sectors.

For such a familiar technique, flow cytometry holds many surprises. It is 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 non-traditional. Long capable of running phenotypic screens, flow cytometers are starting to apply their powers of discrimination to genetically modified cells. Flow cytometers are 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 auto-sampling 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 cytometers are 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. Although it forces cells to pass in single file, it also allows cell biology to spread out in many directions.

However, the increasing number of cellular parameters unveiled by genomic, proteomic, and metabolomic data platforms demand an augmented multiplexability. Also, the need for identification and quantification of relevant biomarkers at low levels requires outstanding analytical sensitivity and reliability. In addition, growing awareness of the advantages associated with miniaturization of analytical devices is pushing forward the progress in integrated and compact, microfluidic-based devices at the point-of-care (PoC). In this context, novel types of flow cytometers are emerging during the search to tackle these challenges.

Indian market

The Indian flow cytometers market in 2018 remained stable. Price points remained same as 2017 and the market saw an 8-10 percent increase in demand. It is estimated at Rs 229.52 crore with reagents constituting 55 percent share. Demand for analyzers is estimated at 110 units, and for cell sorters at 21 units. In 2018, an order for 200 systems was placed by NACO for CD-4 for POCT, with 125 systems on BD and 75 units on Alere.  BD India continues to dominate the flow cytometer segment.

New launches update

In March 2019, BD launched the BD FACSDuet automated flow cytometry system with CE-IVD certification. The new fully automated sample preparation instrument enables clinical laboratories to improve their efficiency by reducing errors and limiting the manual user interactions required to run assays on the BD FACSLyric clinical flow cytometer.

In January 2017, Beckman Coulter introduced the ClearLLab 10C System for the clinical flow cytometry lab. The new system includes the first 10-color CE-IVD panels of immunophenotyping reagents for both lymphoid and myeloid lineages. The tubes utilize DURA Innovations dry reagent technology for the panels, which requires no refrigeration. The ClearLLab 10C System incorporates the company’s new Kaluza C software to streamline and standardize clinical QC reporting to international guidelines.

Also, in January 2019, Luminex Corporation completed its previously announced acquisition of MilliporeSigma’s flow cytometry portfolio for USD 75 million in combined stock, asset, and inventory purchases. The acquisition is expected to contribute USD 40 million to USD 50 million in revenue to Luminex in 2019. This acquisition enables Luminex to enhance their existing offering of flow-based detection systems, while simultaneously expanding direct interactions with researchers conducting cellular analysis. Luminex’s newly acquired flow cytometry portfolio includes Amnis, the market-leading family of imaging flow cytometry products for cell-based analysis, as well as the Guava portfolio of products, which are economical, high-performance systems based on microcapillary technologies.

In December 2018, Sysmex Corporation launched the imaging flow cytometer MI-1000 and related software MI FISH Master as a system. This system utilizes imaging FCM technology to automate FISH testing for determining chromosomal abnormalities in circulating cells. The newly developed system comprising imaging flow cytometer MI-1000 and related software MI FISH Master captures the morphology and fluorescent images of large quantities of cells at high speed and at a high level of detail. These images are then analyzed automatically using imaging FCM technology to automatically detect chromosomal abnormalities contained in cells in the blood.

In September 2018, Agilent Technologies entered into an agreement to acquire ACEA Biosciences Inc. to expand its global revenue base, product portfolio, and strengthen its market share. In recent years, the ACEA xCELLigence RTCA technology has blazed a trail in immuno-oncology, pre-clinical drug discovery and development, as well as in basic academic research. The technology is complemented by the introduction in recent years of the NovoCyte and NovoCyte Quanteon portfolio of high performance benchtop flow cytometry systems. Complementary engineering and scientific expertise from both companies should also provide a rich pipeline of novel systems and products well into the future.

Moreover, in September 2018, ACEA Biosciences released the new 1.3.0 version of NovoExpress flow cytometry software, which supports the recently launched NovoCyte Quanteon flow cytometers and NovoSampler Q autosampler products, in addition to NovoCyte flow cytometer and NovoSampler Pro products. NovoExpress 1.3.0 is easy-to-use and intuitive software which supports the entire NovoCyte flow cytometer product portfolio and provides powerful and professional flow cytometry data analysis functionalities. These new data analysis functions enable simplified, high-throughput single-cell multiparametric data analysis at user’s fingertips.

In April 2018, Merck launched its new CellStream benchtop flow cytometry system – a compact, customizable flow cytometer that uses a camera for detection. Its unique optics system and design provide researchers with unparalleled sensitivity and flexibility when analyzing cells and submicron particles. The CellStream flow cytometry system expands the limits of sensitivity, allowing scientists to tailor their instrument to their needs in immunology, cancer research, and many other areas.

Global market

The global flow cytometry market was valued at USD 303.51 million in 2018 and is estimated to reach USD 490.11 million in 2024, witnessing a CAGR of 8.3 percent, according to Research and Markets. Rise in the use of flow cytometry in stem cell research, growing focus on immunology and immuno-oncology research, high incidence and prevalence of target diseases, availability of novel products, and the emergence and commercial application of new technologies in the field of flow cytometry are some of the key factors propelling the market growth. The significance of flow cytometry in clinical research is fueling the demand for flow cytometry techniques in R&D departments of the healthcare sector. The increasing incidences of HIV and cancer statistics show a significant unmet opportunity in cancer and AIDS diagnostics. There is a large void in diagnostics accessibility, particularly in the developing regions. Consequently, the increasing incidences of HIV AIDS and cancer can be considered a significant growth opportunity for the flow cytometry market.

The stem cell therapy segment is believed to have the largest market size and is expected to witness a CAGR of 8.4 percent. The key reasons for the large market share include the extensive use of flow cytometry for effective diagnosis and early detection of numerous diseases that have helped the market to gain immense share. There has been a growing market penetration in stem cell research, adoption of recombinant DNA technology for antibody production, and the evolution of tandem flow cytometry technologies that are expected to offer growth opportunities for the players operating in the flow cytometry market. Though flow cytometry has also found many applications in several stages of drug discovery, its routine widespread use for high throughput drug screening has been limited. This is unlike analogous technology, automated high content imaging (HCI), which, in terms of drug screening, is an established technology that is common in screening laboratories.

By technology, the cell-based technology segment accounted for the largest share of the market in 2018. However, the bead-based technology segment is expected to grow at the highest CAGR owing to the procedural advantages offered by this technology over other cell-based technologies, such as the capacity to detect multiple analytes (also known as multiplexing), high reproducibility, stability, and speed. On the basis of product and service, the reagents and consumables segment is expected to witness the highest growth in the coming years due to the development and commercialization of high-quality application-specific reagents and assays and continuous requirement of flow cytometry reagents by end users (due to the increasing number of flow cytometry-based research activities).

Geographically, North America held the largest share of the global flow cytometry market in 2018, followed by Europe. An increasing prevalence of diseases and the rising contribution from the US market are expected to help the market grow. In addition, there has also been a rise in the expansion of novel products that are anticipated to offer new growth opportunities in the coming years. However, the Asia-Pacific is expected to grow at the fastest rate during 2019–2024. The increasing participation of China, India, Japan, and South Korea in flow cytometry-based research; expansion of research infrastructure in the region; and public-private funding aimed towards boosting advance research practices are the key factors responsible for the growth of this market in the Asia-Pacific.

The flow cytometry market is considered highly consolidated due to the presence of very few players in the market. These players are holding a huge share of the market and are likely to remain in a similar competitive environment for the next few years. The rising importance of product development and the rise in R&D activities are projected to encourage the growth of the flow cytometry market. Becton, Dickinson and Co. is one of the leading players in the global flow cytometry. Its broad portfolio of flow cytometry products and well-established presence across the globe is the key factor accounting for its large share in this market. The company has adopted both organic and inorganic growth strategies such as product launches, agreements, partnerships, collaborations, and expansions in order to maintain its top position in the field of flow cytometry. The company is also focused on providing training and conducting workshops to increase the adoption of its products. Other major players in the market include Beckman Coulter, Thermo Fisher Scientific, Merck, Sysmex, Luminex Corporation, Miltenyi Biotec, Bio-Rad Laboratories, Sony Biotechnology, Agilent Technologies, bioMérieux, Enzo Life Sciences, Stratedigm, Cytonome/ST LLC, and Apogee Flow Systems.

Technological Advances

Most advances in flow cytometers have depended on co-creation, with scientists, research institutions, and companies working together. In recent years, researchers are paying increasing attention to the development of portable microfluidic diagnostic devices including microfluidic flow cytometers for PoC testing. Microfluidic flow cytometers, where microfluidics and flow cytometry work together to realize novel functionalities on the microchip, provides a powerful tool for measuring the multiple characteristics of biological samples. The development of a portable, low-cost, and compact flow cytometer can benefit the healthcare in underserved areas.

CRISPR Technology. In the past few years, the market has seen an explosion of companies utilizing flow cytometer in the CRISPR process, doing everything from determining transfection rates to sorting cells. These activities are being facilitated by flow cytometers. Manufacturers have incorporated flow cytometry into the CRISPR workflows. 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. By pairing multiple tools and techniques, new systems 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. Flow cytometers and functional cell sorting will be an increasingly important technology to help answer several questions as the field of genome editing evolves.

Integration with MS. Recently, a new analytical approach, called mass cytometry, which combines the precision of mass spectrometers with the power of flow cytometric analysis, has been developed. The application of both techniques in the field of cancer immunotherapy is very promising and a number of related applications are under development. If implemented, this may allow for the generation of large amounts of multidimensional data that are amenable to high-throughput analysis and this may offer unique opportunities and challenges for the field of biomarker development. The use of these powerful technologies will answer a multitude of questions with just one sample being analyzed and this will likely help to tailor cancer immunotherapies to each patient. However, at the moment the quantity and complexity of data obtained with this technology requires some analytical considerations.

Adding Spectroscopy to Flow Cytometers. Aided by recent innovations and advances in semiconductor detectors, telecom optics, and computation that enable sub-millisecond, full spectral measurements, spectral (or multi- or hyperspectral) flow cytometry has emerged in recent years and is now being adopted by a wider group of scientists. Spectral flow cytometers, which incorporate ultrafast optical spectroscopy, have simpler optical paths and fewer components than conventional flow cytometers, and they provide higher-quality results with fewer lasers. A major feature of spectral flow cytometry is, of course, spectral unmixing – that is, analysis of the spectral data to extract revealing information. Spectral unmixing provides accurate estimates of label intensity and resolve fluorochromes with significant spectral overlap. The algorithm used for this process replaces the compensation matrices of conventional flow cytometry and treats autofluorescence as an independent parameter. All of this adds up to less-complex, more-capable instrumentation as well as greater flexibility, as researchers gets much more choice in designing assays.

PoC Microfluidic Flow Cytometry. In recent years, research is focused on developing microfluidic flow cytometers with the motivation of creating smaller, less expensive, simpler, and more autonomous alternatives to conventional flow cytometers. These devices could ideally be highly portable, easy to operate without extensive user training, and utilized for research purposes and/or PoC diagnostics, especially in limited resource facilities or locations requiring on-site analyses. Microflow cytometers that combine microfluidics and miniaturized detection systems are a promising solution for PoC diagnosis. The recent innovations in particle-focusing and detection strategies are used to fulfill the criteria of high-throughput analysis, automation, and portability, while not sacrificing performance. The ongoing contribution of microfluidics demonstrates that it is a viable technology to advance the current state of flow cytometry and develop automated, easy to operate, and cost-effective flow cytometers.

Large Particle Analysis. Addressing the problems inherent to large particle analysis and sorting, manufacturers now offer a range of flow cytometers that have been optimized for large cells, cell clusters, small model organisms, hydrogel-encapsulated particles, organoids, and organotypic tissue fragments. 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 manufacturers have addressed thoroughly within the different instrument platforms. Also, the sorting mechanism is very different than conventional flow sorting, involving a gentle air diversion mechanism.

Challenges Faced while Using Flow Cytometers

Flow cytometers have been categorized as relative and quasi-quantitative, that is readouts from unknown samples cannot be compared to a calibration standard with a known quantity of the test analyte. What remains challenging is to tackle the lack of reference material suitable for measurements by flow cytometers. For assessing its accuracy, there are only substitute solutions as of now. Scientists need to timely adapt assay development to different project needs, which are exploratory in nature. But within these scopes they do not need to produce and document all the evidence with the stringency required to complete bioanalytical method validation. Achieving this is generally costly and time-intensive.

Besides analytical limitations specific to flow cytometers, analyzing datasets that are generated from phenotyped samples can be another source of variability. No universally applicable approach is in place that is to be followed. Some may argue it, therefore, resembles more of an art, as the data analyst can take a lot of freedom to place gates for identifying target cells. Certainly, the analysts’ educational background and subject matter expertise plays a role more than ever before, since marker analysis is no longer limited to standard immune populations. Most multicolor panels are now customized to also characterize very specific, very rare subsets. All these factors require ongoing training for qualified analysts to be aware of the underlying biology, to know how to include appropriate controls and how to apply proper gating strategies for consistent results across experiments.

Road ahead

Recently, the World Health Organization (WHO) incorporated flow cytometers in its model list of essentials in vitro diagnostics. This underlines this platform’s relevance for primary patient care, and is expected to see more recognition beyond its status as a sophisticated research tool. Though being known for more than 50 years, fluorescence-based flow cytometers regained momentum in the current new era of immunotherapies. But not just immunology-focused researchers have turned to this technology. The frontier of flow cytometer applications is expanding. While the benefits offered by flow cytometers are well known throughout the scientific community, adoption has lagged due to its cost-prohibitive, workflow-intensive nature. Manufacturers are now on a mission to make the advanced instrumentation that was once available to very few scientists accessible to more researchers.

It is always daring to envision what the future holds, to predict which tools will become important or not. Although being challenged by alternative platforms, it is still believed that the market will not witness flow cytometry becoming obsolete, and phased out from applied research. A lot will depend on how the technology can advance to become more quantitative. Ultimately, the more accurately measurements by flow cytometers can be performed, the more valuable its results can be for the users relying on them, may it be a clinician, a drug developer, or a food-safety technician. Going forward, it will be essential that datasets can be considered valid, that is fully reproducible, irrespective of by who, where, and on which benchtop flow cytometer these were generated. For this decades-old analytical technique, the best, it seems, is yet to come.

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