Flow Cytometers
Flow Cytometers, No Longer the Domain of Only Immunologists
Advancements in flow cytometers revolve majorly around new approaches combining existing technologies with routine laboratory procedures, such as IFC that allows thousands of morphological and spatial properties to be measured for each individual cell.
Flow cytometers that made one of their first major contributions to biomedicine starting in the 1980s, during the global fight against HIV/AIDS, are so widely used nowadays for particle, tissue, and cellular research/analysis that they are almost taken for granted. Technologies have improved over the years and proved to be one of the most powerful asset in research and clinics. Patients’ clinical symptoms do not always follow the textbooks theories to direct doctors to a single genetic defect, and flow cytometry is key in unfolding this vital information. Thanks to flow cytometry, clinicians have been able to diagnose more than 300 genetic disorders and several different cancer cases. Clinical laboratorians are now able to analyze almost anything about generations of cells.
Use of more robust photodetectors and new laser emitters, LED lamps as emitters, and changes in analytical capacity (multidimensional analysis software) are a few recent improvements in flow cytometers. Major advancements in flow cytometers revolve around new approaches combining existing technologies with routine laboratory procedures, such as imaging, which makes the acquisition of images of cells possible in real time as they pass by the interrogation point. Flow cytometry is also being integrated with other technologies to provide richer data for understanding cellular makeup and function. In particular, it is being used in conjunction with next-generation sequencing applications such as RNA sequencing. The integration of cell-sorting technologies, such as index sorting, allows a finely detailed understanding of the cell receptor phenotype for each single cell in an RNA-sequencing application. Cytometers are easily integrated into high-throughput robotic systems, allowing scientists to use sophisticated flow cytometric barcoding technologies to enhance the speed of research.
The use of flow cytometry in clinical laboratory has also grown substantially due to the development of smaller, user-friendly, less expensive instruments, and increasing number of research applications such as in cytology, immunology, and microbiology, and neuroscience among other research fields. Besides, flow cytometry has a unique ability to investigate large cell populations at the single-cell level. This favors the adoption of flow cytometers for clinical application. Furthermore, flow cytometry is also gaining attraction in drug discovery and development, which helps transforming every stage of preclinical and clinical development, that guide new product evaluation, selection, and monitoring. Also in the field of medical diagnostics, flow-based technology is central to hematological testing.
One of the more challenging aspects of developing better drugs and improving upon the successes of IO is the need to find and analyze very rare cancer cell populations. Flow cytometry is ideal for this application, and has the ability to detect many rare events in a short period of time because of its fast electronics, which can achieve detection rates of up to 100,000 events per second. A million Indians are being diagnosed with cancer every year, and the burden is now expected to rise 70 percent over the next 18 years, according to Globocan. Many researchers also want to monitor multiple cell health parameters at the same time, such as viability, apoptosis, and proliferation. Having robust cell health assay combinations that use minimal sample preparation permits streamlined processing, and is compatible with high-throughput instrumentation. The growth in the flow cytometry market in the country is mainly driven by the combination of technological improvements, growing research activities in life science, and increasing clinical application of flow cytometry, such as diagnosis of HIV and cancer.
Indian Market Dynamics
The Indian flow cytometers market in 2017 is estimated at Rs. 211 crore, with reagents constituting 55 percent market share. Since prices were slashed by most vendors, the value of this segment dropped by about 5 percent over 2016.
Demand for analyzers was more or less stagnant at 100 units, whereas the market for cell sorters increased from 14 units in 2016 to 21 units in 2017. The analyzers, cell sorters, and reagents market continues to be dominated by BD India, followed by Beckman Coulter. Brands jostling for space in 2017 included Sysmex (Partec), Thermo Fisher, Merck (Millipore), MAC, and Bio-rad. Bio Legend had some success in 2017 in the reagents segment and Acea Biosciences (NovoCyte) in the analyzers segment.
The clinical segment continues to contribute 39 percent to the market, with HIV at 33 percent and research constituting the balance 28 percent. While the government corners 90 percent of the HIV segment sales, and 70 percent of the research segment, the clinical segment is popular with the private sector, which is responsible for 55 percent share of the market.
Orders were placed by NACO for CD4 (point of care), not strictly falling under the flow cytometers segment. Orders for 125 units of medium-throughput machines were placed with BD India; the approximate order value was Rs. 83.75 crore, and 75 units of low-throughput machines were placed with Alere Medical; the approximate order value was Rs 2.5 crore.
Global Market
The global flow cytometry market is estimated at USD 3410 million in 2017 and is anticipated to register a CAGR of 11 percent from 2017 to 2025 to reach USD 8100 million by 2025, according to Transparency Market Research. The continuous technological advancements in the field of flow cytometry, increasing incidence and prevalence of target diseases (such as HIV/AIDS and cancer), and growing adoption of flow cytometry in advanced research activities and clinical trials are some of the key factors driving the growth of the global market. However, high cost of flow cytometers and a dearth of skilled professionals and insufficient knowledge regarding the use of flow cytometers are restraining growth.
The reagents and consumables segment is expected to witness the highest growth in the coming years owing 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). In 2017, the instruments and accessories segment portrayed similar market shares; however, the accessories segment is likely to surpass the instruments segment in the coming years. The cell-based technology segment accounted for the largest share of the market in 2017; while, the bead-based technology segment is expected to grow at the highest CAGR attributing to the procedural advantages offered by this technology such as the capacity to detect multiple analytes (multiplexing), high reproducibility, stability, and speed.
Geographically, North America accounts for the largest share in the global flow cytometry market, followed by Europe. This mainly is attributed to research and advancement in technology, wide acceptance of the flow cytometry, rapid growth of the life sciences sector in developed countries such as the United States of America, and increase government initiatives in research studies for the prevalence of diseases. However, the Asia-Pacific market is projected to grow at the fastest rate and is likely to be a key revenue generator in the coming years. Increasing participation of China, India, Japan, and South Korea in flow cytometry-based research; expansion of research infrastructure in the region; and public–private finding aimed toward boosting advanced research practices are the key factors responsible for the growth of this market in the Asia-Pacific region.
Key players are continuously focusing on increasing their R&D activities to come up with new products and attract more customers by offering innovative and technologically advanced products. For instance, in January 2018, Cancer Genetics announced expansion of its immuno-oncology (IO) panel to a new advanced IO panel as complete IO, by using most all-inclusive flow-cytometry-based biomarker panels of the industry. In another instance, in October 2017, Sysmex introduced CyFlow antibodies for application in clinical flow cytometry.
In addition, rise in usage flow cytometry instruments offer ample business opportunities for the development of cutting-edge technologies that promote credibility and encourage product uptake. Rigorous investment in R&D activities for new product development and enhancement of existing product portfolio would help the key players to retain and enhance their share in the global market. Key companies operating in the global market include Becton, Dickinson and Company, Merck, Beckman Coulter, Thermo Fisher Scientific, Sysmex, Bio-Rad Laboratories, Luminex, Miltenyi Biotec, Agilent Technologies, biomérieux, EMD Millipore, Advanced Analytical Technologies, and Mindray.
Technological Advances
Flow cytometry continues to evolve at a fast pace and provide scientists with the ability to perform many highly-specialized assays simultaneously. With the development of greater throughput and sensitivity, the flow cytometer has become a unique tool for characterizing morphology and cell density changes during disorders. The evolution of optics and photonics is having a significant impact on cell analysis applications. Photonics developments being leveraged in flow cytometry include powerful new system design tools, small and reliable light sources, inexpensive and compact solid-state detectors, and bright, stable dyes. From better diagnostics to deeper understanding in cell biology, new photonics-enabled solutions are lighting the way.
Imaging flow cytometry. Recent advances in imaging technologies, electronics, and digital computing have enabled IFC. Equipped with 20◊, 40◊, or 60◊ objectives and up to two charge-coupled device cameras, IFC allows thousands of morphological and spatial properties to be measured for each individual cell. These include bright field, dark field, and up to ten fluorescent channels. The technology combines single-cell imaging capabilities of microscopy with the high-throughput capabilities of conventional flow cytometry. Recent advances in IFC are remarkably revolutionizing the single-cell analysis. Similar to its flow cytometry-based siblings, IFC is well-suited to image non-adherent or dissociated cells, key for many clinical applications such as analyses of bodily fluids like blood, whose structures can be distorted (smeared) by placement onto a slide.
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.
Hardware advances. Until recently, traditional flow instruments, because of their cost, size, and complexity, were almost exclusively located in centralized facilities and shared among users across departments or an entire institution. These factors limited the number and type of experiments that the average researcher could perform. However, recently flow cytometers have become smaller, more portable, reliable, cheaper, and easier to use, while retaining and often expanding their capabilities to keep up with scientific demands. Formerly built around bulky, power-hungry gas lasers, flow cytometer miniaturization is expected to continue. With many solid-state lasers now fitting comfortably in a shirt pocket, system designers are freed up from past constraints and can pack a lot of optical punch in a very small footprint. Size reduction of detectors is also playing a role, with the latest photomultiplier tubes 1/10th of their historical size, and even smaller silicon-based detector alternatives are starting to gain acceptance among users.
Multicolor flow. As understanding of the complexity of the immune system grows, better multiplexing (measuring multiple cell parameters simultaneously) is required. In contrast to the CD4 assay used to monitor AIDS therapy, now multiple lasers are used for many immunology applications, with each laser exciting several fluorescent labels in different spectral bands simultaneously. However, current multiplexing approaches (maximum 20 parameters) have hit a brick wall of sorts enabling researchers to explore different directions to break the logjam. For instance, a company is developing a new technology for multiplexing that preserves the same workflows of conventional flow cytometry – including the option to sort – but with an expanded array of available detection channels, and where the need for compensation is reduced or eliminated. Manufacturers are mapping the practical limits of this approach to deliver a very high-channel-count analyzer (30+) with the same footprint and hardware complexity of a mid-range machine.
Detection of nanoparticles. Some fields of research are demanding performance that pushes detection technology to the limit. Scientists are recognizing that very small, nanometer-sized bioparticles (extracellular vesicles or EVs) can reveal important information about cancer and other disease states. Since light signals (both scattering and fluorescence) from particles drop rapidly with particle size, flow instruments designed to detect micron-sized cells have struggled to extend their reach to the measurement of EVs. Most current commercial units hit the sensitivity floor at or above 200 nm, and only a few have pushed it to 100 nm. The detection sensitivity of flow cytometers needs a boost to adequately characterize EVs. Recent researchers have demonstrated detection of EVs below 100 nm, and of viruses below 50 nm, by making improvements such as more powerful lasers, tighter focusing, and longer integration times.
The Road Ahead
Flow cytometers have versatile applications in basic research, clinical practice, and clinical trials. Every phase of flow cytometry is moving forward as a result of advanced technology. There are newer fluorochromes, better protein reagents, and improvement in automated data analyses. Newer technologies have allowed researchers to take a simple concept and multiply what they can do with it. Despite this promise, IFC is currently primarily used in research rather than clinical practice. Data analysis is the primary hurdle – it is often prone to variation, manual tuning, and interpretation. User-friendly, robust, and standardized workflows that can facilitate machine learning, especially deep learning, will accelerate the paradigm shift from low- to high-content analysis in IFC. Furthermore, cloud computing can overcome the computational infrastructure hurdles. These developments are key for practical IFC applications to reach the clinic.
Although conventional flow cytometry is considered high throughput as it analyzes up to 100,000 cells per second, it is considered to be low in information content because typically only a single feature (integrated intensity) is measured per fluorescence marker. The future trend is to increase the number of parameters that can be simultaneously measured by developing instruments with more lasers and detectors in combination with new fluorochromes that can be used in concert with one another. Mass cytometry can measure in excess of 40 markers simultaneously using antibodies tagged with rare earth metals. This platform significantly increases the number of parameters measured beyond what is currently achievable with conventional flow cytometry and will drive the adoption of machine learning techniques when analyzing such multidimensional data.
Industry Speak
Technology Trends in Flow Cytometry
Amardeep Gupta
Business Manager – Flow Cytometry,
Sysmex India Pvt Ltd
It all started when Prof. Dr Wolfgang GÖhde, launched the world’s first fluorescence flow cytometer in 1968. The original name of this technique was pulse cytophotometry, and he named it as ICP-11 (Immuno Cytophotometer). 10 years later in 1978, at the Conference of the American Engineering Foundation in Pensacola, Florida, the name was changed to flow cytometry. Since its inception, this technique has started developing interest in many scientists thereafter. Advances in this field have been tremendous, and even now there are many developments day by day.
Initially, flow cytometers were used as experimental devices in research institutes only. However, the uses and application of this instrument gained great momentum, and other industries have started to explore this technique too, and now it has become a major market. Today’s flow cytometers sport multiple lasers and fluorescence detectors, which aid users in labeling multiple antibodies and in identifying precisely a target population by its phenotype.
Apart from research segment, flow cytometry is now being applied in clinical, industrial, essential healthcare, agriculture, aquaculture, microbiology, etc. and the list goes on. People have now understood the importance of this technique, and now improvising in its existing technical aspects. Advances in laser sources, sample preparation automation, detection sensitivity, digital-signal processing speed, rare-event analysis, and cell-sorting performance, coupled with a broad portfolio of reagents with a varying spectrum of dyes and fluorochromes, provide the tools necessary to answer questions in immune monitoring, immunophenotyping, stem-cell analysis, cell-signaling, and signal transduction.
As now Partec GmbH (the pioneers of flow cytometers), have joined their hands with Sysmex Corporation, Japan. Worldwide, there has been a tremendous boost to the clinical segment of flow cytometry. Partec (now Sysmex) being in the market since last 47 years, now along with the pioneers in clinical field, Sysmex would be soon launching a dedicated clinical flow cytometer for all the clinical application along with the reagents kit. There have been some great innovations expected in the coming years when we see the association of flow cytometer with the best of hematology analyzers, creating a haemat-flow technology. Already imaging flow cytometry has created a boom in the industry, and people are eying for new technologies and advances.
Second Opinion
A Powerful Tool in Research and Diagnostics
Dr Rajarshi Kar
Assistant Professor, Department of Biochemistry,
University College of Medical Sciences
The advent of flow cytometry has revolutionized the field of diagnostics and research. The differential count of white blood cells can be done in seconds and with great accuracy. Flow cytometry can analyze thousands of cells individually in a very short time and can provide enormous amount of data. With the inclusion of multiple lasers and more than 12 detectors, expression of multiple proteins can be evaluated on a very small amount of the same sample. The addition of a sorter has increased the power of this technique manifold.
Any cell of interest can be separated and collected from a mixture of cells in viable form and can be further used for downstream applications. For example, stem cells can be isolated and enriched and further cultured in vitro and used for therapeutic applications. I have been working on ovarian cancer for the last 14 years and used flow cytometry to estimate apoptosis in primary cultures. It helped me to evaluate the response of primary culture of ovarian cancer to various chemotherapeutic agents. The newer models of flow cytometers have software modules to evaluate apoptosis, cell count, and expression studies using commercially available kits.
The inclusion of propidium iodide based evaluation of apoptosis module will make apoptosis studies cheaper for researchers in third world countries. Another major problem faced in flow cytometry is evaluation of cells that get clumped during preparation of cell suspension. Robust techniques to differentiate clumped cells from polyploidy will help researchers new to flow cytometry to effectively analyze their samples. Flow cytometer is a powerful tool in the hands of an astute researcher and is a must-have instrument in institutes seriously pursuing research.
Second Opinion
Wide Applications of Flow Cytometers
Dr Anuradha Sekaran
Chief of Pathology,
Asian Institute of Gastroenterology
Flow cytometry is used to analyze the physical and chemical characteristics of fluorescently labeled cells in a fluid as it passes through a laser beam and emits light of varying wavelengths. There are continuous advancements in molecular diagnostics, monoclonal antibodies, lasers, and software. Moreover growing demand for understanding of immunologic regulating systemic diseases and increase in adoption rate by healthcare facilities are some major factors driving the growth of the market. However, lack of availability of technical expertise, huge investments in flow cytometry instruments are hindering the growth of the market.
Based on the technology segment, cell-based flow cytometry leads the market globally as they are widely used in the study of tumor stem cells and in the study of disease mechanism. Among applications, the demand for clinical diagnosis is increasing owing to growing demand for disease diagnostic tools. On the basis of product and services the market has been segmented into reagents and consumables, Flow cytometry instruments, software and services. Flow cytometry instruments segment has been further classified in to cell analyzers and cell sorter.
Current technology applications include, multicolor flow cytometry – up to 18 independent color parametric can be analyzed; multiflex broad based assays – enable the measurement of variety of soluble or intracellular proteins; stem cells and side population cells – stem cells and early progenitor cells in hematopoietic tissue can be identified; cell tracking/labeling and sorting; cell signaling analysis to measure phosphorylation status of enzymes in cell signaling pathways; and imaging flow cytometry – flow cytometry combined with digital microscopy.
Utility of flow cytometry in diagnosis, diagnosis and classification of hemato-lymphoid malignancies; diagnosis of primary immunodeficiency disorders; diagnosis of PNH; tissue typing for HLA B 27; enumeration of stem cells in peripheral blood; and identification of circulating malignant cells.
Application of flow cytometry in therapeutics, monitoring of immune status in HIV patients; monitoring of immunosuppressive therapy; T-cell cross matching for transplantation; monitoring for post–transplantation rejection episodes; and detection of minimal residual disease in cancer.
Limitations of flow cytometry, lack of expertise in sample processing and data interpretation; poor standardization across institutions; cells must be viable for analysis; and caution should be exercised while interpreting flow cytometry in the absence of histopathological and clinical correlation.
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