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Flow Cytometers

Using technology to combat cancer

As flow cytometry improved in capability and precision, its applicability to immunologists and cancer researchers grew repidly. However, the best is yet to come!

Flow cytometry has long enabled basic and clinical research, with applications in fields as diverse as molecular biology, neuroscience, and plant and marine biology. Recently, its use in cancer immunology has grown impressively because of the role it can play in extending our understanding of the immune system and its response to cancer immunotherapies. As the capabilities and precision of the analytical platforms continue to improve, so too will their beneficial effect on immunology and cancer research communities. For a decades-old analytical technique, the best, it seems, is yet to come.

Immuno-oncology, or cancer immunotherapy, is a novel approach that is transforming the cancer therapy landscape and revolutionizing the standard of care for patients, particularly for more challenging advanced and metastatic cancers. The rapid advancement of immuno-oncology has resulted in the demand for sensitive, accurate, and reproducible analysis technologies like flow cytometry.

Flow cytometry has long been the favored analysis platform in immunology and other cellular and molecular research fields. Commercial development and marketing of user-friendly benchtop instruments has resulted in the progression of flow cytometric analysis from a specialized, expensive research tool to a significant clinical methodology for routine diagnosis and prognosis of many diseases, including immunological and hematological disorders. Thus, the adoption of flow cytometry by immuno-oncology in pre-clinical and clinical studies was a natural evolution.

Flow cytometry is a high-throughput technology that can simultaneously analyze multiple cellular markers (surface and/or intracellular) at a single-cell level. This powerful technology can be used to accurately identify, quantify, and monitor cellular phenotypes, signaling pathways, and functional responses revealing deep insights into the complex milieu of the immune system and tumor microenvironment. Multi-parameter conventional flow cytometry can analyze up to 30 markers, while smaller, more focused panels of markers have traditionally been preferred in clinical settings to facilitate straightforward analyses.

The progression of flow cytometry in immuno-oncology and the clinical implications of the findings have highlighted the importance of best practices and standardized proc edures to generate reliable and reproducible data. The minimum information about a flow cytometry experiment (MIFlow-Cyt) standard developed by the International Society for the Advancement of Cytometry and the guidelines for the use of flow cytometry and cell sorting in immunological studies are common frameworks for research reporting in flow cytometry and immunology communities. Committees and working groups, such as the International Clinical Cytometry Society, European Society for Clinical Cell Analysis, and Australasian Cytometry Society, are dedicated to establishing similar guidelines and suitable standards in clinical flow cytometry.

Novel cytometry platforms, such as mass cytometry, genomic cytometry (single-cell RNA sequencing), and spectral cytometry, have been developed to more deeply profile immune cell populations. Conventional flow cytometry measures cellular marker expression using fluorochrome-conjugated antibodies. The limitation on the number of markers traditionally analyzed in clinical flow cytometry is due primarily to the spectral overlap of fluorescence emission signals; panel design and data analysis become progressively more labor-intensive and complex as the number of markers and fluorochromes increases.

Mass cytometry is a valuable high-dimensional analysis platform that is not constrained by fluorescence emission overlap (antibodies are conjugated to metal reporters) and can be used to analyze over 40 markers. However, to date, it has not been widely adopted in clinical settings for real-time analysis due to slower throughput and limited availability of commercial reagents. Single-cell RNA sequencing significantly increases the dimensionality of possible markers used for analysis and allows for unbiased single-cell transcriptome profiling, though the low throughput and high reagent and sequencing costs are limitations for its use in clinical settings.

Spectral cytometry offers high-dimensional analysis potential, comparable to mass cytometry, while retaining the real-time analysis and high-resolution properties, as well as the reasonable cost of conventional flow cytometry. While fluorescence emission overlap remains a consideration, capturing the full unique spectral signature of each fluorochrome lessens the issue. In conjunction with the release of many new and improved fluorochromes, spectral cytometry is quickly emerging as the most promising new technology in clinical flow cytometry.

With the combination of technological advancements and maintained efforts toward high standards of rigor and reproducibility, the future is exciting for immuno-oncology and flow cytometry. The number of tumors potentially amenable to immunotherapeutic intervention is growing and the field continues to make remarkable advances.

Flow cytometry data analysis is built upon the principle of gating, a key step necessary to visualize relevant correlations in multi-parameter, multi-population data. However, gating is often seen as something of a dark art that needs to be completed manually by those with specialist expertise to avoid errors and inconsistencies. In reality, with appropriate rules in place, gating simply becomes a laborious task – an ideal candidate for automated data-handling processes. This is especially true when the number of markers grows; the number of 2-D scatter plots generated increases exponentially. As a result, automated gating algorithms have been developed that function by grouping comparable populations together.

Automated gating is based on the mathematical modelling of the fluorescence intensity distribution of particle populations. As well as drastically reducing analysis time, automated gating addresses the challenge of subjectivity in manual methods, and could even lead to the discovery of novel, biologically relevant populations that had not previously been considered.

Not all automated approaches solve all challenges though, for example, random variation in automated clustering algorithms can lead to inconsistent results, such that comparing results from automated gating with each other, as well as with traditional manual gating results, can be difficult.

Although a range of software platforms exist that enable automated gating, there is a lack of tools available that enable data sharing. There is, therefore, a need for a solution that allows gated and analyzed data to be exported from one platform and imported into another, allowing users to reproduce analyses from raw files and facilitate demonstrable reproducibility.

New level of workflow efficiency is required

Shruti Iyer
Assistant Product Manager – Flow Cytometry,
Sysmex India Pvt. Ltd.

Flow cytometry (FCM) is a technique used to detect and measure characteristics of a population of cells or particles. A sample containing particles or cells labelled with fluorescent markers is mixed with fluid and compressed into the flow cytometer. The maximum number of cells can be quickly examined, and data gathered are processed by a computer. FCM is used in cell counting, cell sorting, determining cell characteristics and function, detecting micro-organisms, biomarker detection, protein engineering detection, and diagnosis of health disorders.

The flow cytometry market globally is expected to register a CAGR of >12 percent. Some of the key growth drivers include increase in global incidence and prevalence of HIV/AIDS and cancer; continuous adoption of flow cytometry techniques in research and academia; undertaking public-private initiatives in the fields of immunology and immuno-oncology research and technological advancements; steep rise in the usage of AI platforms in flow cytometry workflows; and expensive instruments and the evolution of the tandem FCM technology.

Covid-19 pandemic has also positively impacted this market and played a vital role in exponentially increasing demand of the FCM technology, as it is used in several Covid-19 research studies to investigate how the immune system reacts to SARS-CoV-2.

With the increasing growth of the FCM technology, the specialized flow cytometry laboratories today are facing several challenges. The number of requests and the complexity of the analyses are increasing, while at the same time, the requirements for documentation are growing, and achieving the required traceability of reagents and processes is a complicated feat. To add to this, the number of skilled staff and resources is declining.

The need for the hour is to take the flow cytometric analysis to a new level of workflow efficiency and deliver the results with absolute confidence.

Sysmex’s this vision to automate the process of sample preparation to reporting the measurement result has come true with the launch of its new clinical flow cytometry automation system. This is an important milestone for Sysmex India, which we see as a logical step, and a perfect complement to our hematology diagnostic solutions.

The global flow cytometry market size is expected to reach a value of USD 11.5 billion by 2027, according to Grand View Research, Inc. The market is expected to expand at a CAGR of 8.9 percent from 2021 to 2027. The high prevalence of chronic diseases, the introduction of technologically advanced flow cytometry solutions, and increasing R&D investments in the pharmaceutical industry are expected to propel market growth in coming years.

In 2021, the instruments segment dominated the market for flow cytometry and accounted for the largest revenue share of 35.4 percent, and is expected to maintain its dominance over the next 6 years. This high share is attributed to new advancements in technology and the introduction of novel cytometers by key players. The high price of these instruments is also contributing toward the high revenue generation in this segment. The reagents and consumables segment also held a significant revenue share in 2021 in the market for flow cytometry owing to their increased adoption in diagnostics and research.

In 2021, by revenue, the cell-based segment dominated the flow cytometry market, accounting for 76.6 percent. Increasing demand for early diagnosis and growing awareness, pertaining to associated benefits of cell-based assays, are factors contributing to its highest share. Furthermore, advancements in technologies of cell-based assays, such as innovation in instruments, labels, affinity reagents, software, and algorithms, are anticipated to drive adoption in the coming years.

The research segment dominated the market in 2021, with a 49.6-percent market share, and is expected to maintain its dominance over the next six years. This high share can be attributed to increasing R&D activities, pertaining to cancer and infectious diseases, including Covid-19. In addition, increasing R&D investments in the pharmaceutical and biotechnology industry are also expected to create a conducive environment for market growth.

Technological advancements, resulting in enhanced accuracy, portability, and cost-effectiveness, are expected to present this market with future growth opportunities. Small-sized high-throughput cytometers are anticipated to gain popularity in the near future due to their associated benefits, such as ease-of-use and cost-effectiveness. Furthermore, improvements in fluorescent dyes and the introduction of bench-top cytometers are factors expected to drive market growth. Rapid advancements in multicolor flow cytometry, which has extensive applications in new drug development, have led to easy cellular analysis by simultaneous evaluation of several parameters. These devices are extensively adopted by many contract research organizations.

The restraining factors that challenge the growth of the flow cytometry market are the availability of alternate and cheaper techniques, high costs associated with flow cytometry reagents and instruments, budget restrictions for research purposes, and expensive installation and maintenance costs. High costs associated and the scarcity of labor and skilled technicians in the labs and clinics also hamper the flow cytometry market growth. Additionally, the lack of awareness among the end-users and less accessibility of technical expertise is further restraining growth.

Regionally, North America is the biggest market in terms of market share globally, followed by Europe. Factors driving the flow cytometry market in North America are increasing the application of flow cytometry techniques in cancer research, increasing adoption of low-cost and compact flow cytometers in clinical diagnostics and research laboratories, and the rising quality of infrastructure for laboratory and clinical research. The US is estimated to account for the leading shares in the North American market due to many clinical labs for research and major pharmaceuticals and a high occurrence of cancer rates and other chronic conditions.

Major players operating the global flow cytometry market are Becton, Dickinson, and Company, Beckman Coulter, Inc., Thermo Fisher Scientific, Inc., Merck KGaA, Sysmex Partec GmbH, Luminex Corporation, Miltenyi Biotec GmbH, Bio-Rad Laboratories, Inc., Sony Biotechnology, Life Technologies Corporation Inc., EMD Millipore Corporation, Miltenyi Biotec, Agilent Technologies, Inc. and Affymetix Inc.

Although there are other analytical techniques in common use for disease diagnosis, the robust, precise, and versatile nature of flow cytometry means that it is unlikely to become obsolete any time soon. The increasing sophistication and accuracy of flow cytometers, the use of new fluorescent dyes and alternatives, and the introduction of new flow cytometry techniques all make the technique future-proof.

As well as this, the development of more sophisticated software and the application of machine learning to the technique means that the potential for this analytical technique is vast. Flow cytometry remains at the forefront of disease diagnosis and research, providing data that helps clinicians provide better outcomes for patients.

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