Flow Cytometry – Doing More With Less

Flow Cytometry – Doing More With Less

With recent advancements in flow cytometry technology, there are now possibilities to do more with less.

Flow cytometry was first developed for single-cell analysis in the late 1960s, and has been widely used as a powerful analysis tool for many diseases in the past two decades owing to its ability to count, characterize, and sort cells. The growing need for biological information at the single-cell level has driven the development of improved cytometry technologies. Flow cytometers have become essential instruments in biomedical research and routine clinical tests for disease diagnosis, prognosis, and treatment monitoring. 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.

Indian market

The Indian flow cytometers market in 2019 is estimated at Rs 232 crore. The market remained at the same level as 2018, by quantity, 110 units of analyzers and 21 units of cell sorters. Price points decreased marginally for analyzers, with about 20 percent installations on rentals. The cell sorters are offered in two categories, the high end and the basic models.

Reagents, as expected, picked some pace and showed an upward trend by about 5 percent over 2018, which explains the slight growth in the total market despite a slight slack in instrumentation revenue. The lull in instrumentation can largely be attributed to the low government spend, while the Indian elections were being held in seven phases to constitute the 17th Lok Sabha in May, 2019.

By application, research contributed 55 percent and clinical 45 percent to the market, with the latter at a 5 percent higher share than 2018.

BD India continues to dominate the flow cytometer segment. It is estimated that by volume the brand had a 100 percent share in the cell sorter segment in 2019 and around 60 percent share in the analyzer segment in 2019. Beckman Coulter had good success in the analyzer segment.

In June 2019, an order for 108 numbers of CD4 machines, under buyback, was placed by NACO for HIV monitoring on Sysmex. The estimated order value was Rs 92 crore with 1.08 million tests to be conducted every year, value translating into Rs 16.2 crore per annum. This is a rate agreement for six-year requirement for procurement of kits and reagents. The table top models are FDA/CE-approved for IVD use. The machine is capable of measuring absolute number of CD4 cells (absolute CD4 count) and CD4 percentage precisely in whole blood, with a throughput of at least 60 samples/day. The entire order was for 1 laser, 2 color instruments.

Flow cytometry on COVID-10 patients. The Journal of Infectious Diseases, Oxford Academic, has published an article on March 30, 2020, when the levels of peripheral lymphocyte subsets were measured by flow cytometry in 60 hospitalized COVID-19 patients before and after treatment, and their association with clinical characteristics and treatment efficacy analyzed. This was an aim to clarify the characteristics and clinical significance of peripheral lymphocyte subset alteration in COVID-19. The results indicated that total lymphocytes, CD4+ T cells, CD8+ T cells, B cells, and natural killer (NK) cells decreased in COVID-19 patients, and severe cases had a lower level than mild cases. The subsets showed a significant association with inflammatory status in COVID-19, especially CD8+ T cells and CD4+/CD8+ ratio. After treatment, 37 patients (67%) showed clinical response, with an increase in CD8+ T cells and B cells. No significant change in any subset was detected in nonresponsive cases. In multivariate analysis, post-treatment decrease in CD8+T cells and B cells, and increase in CD4+/CD8+ ratio, were indicated as independent predictors of poor efficacy. This led to the conclusion that peripheral lymphocyte subset alteration was associated with clinical characteristics and treatment efficacy of COVID-19. CD8+ T cells tended to be an independent predictor for COVID-19 severity and treatment efficacy.

The Indian industry is keen that in a similar manner, flow cytometric evaluation of the immune system on COVID-19 patients be done in India too. There are about 1300 installations of analyzers and cell sorters in India, excluding the ones at NACO, which are meant more for HIV. This becomes feasible as the ICMR has now relaxed its condition that sample processing need not be only done in BSL3 (Biosafety Level-3) laboratories, considering there are only about 13 or so labs in the country, but BSL2 laboratories could also be utilized.

Global market

The global flow cytometry market is projected to reach USD 6.4 billion by 2025 from USD 4.0 billion in 2019, at a CAGR of 8.3 percent, estimates MarketsandMarkets. The major factors driving the growth of the flow cytometry market are the technological advancements in flow cytometers, increasing adoption of flow cytometry techniques in research activities and clinical trials, the growing focus on immunology and immuno-oncology research, increasing incorporation of AI platforms in flow cytometry workflows and advancements in flow cytometry software, high incidence and prevalence of HIV-AIDS and cancer, and availability of novel application-specific flow cytometry products.

The cell-based technology segment held the largest share of the overall flow cytometry market in 2019. The large share of this segment is mainly attributed to the increasing utilization of this technology in research and development activities of novel therapeutic drugs and advantage of analyzing characteristics and properties of targeted cells with ease. However, bead-based technology is expected to experience a rapid adoption during the forecast period of 2019–2025. This technology efficiently discriminates between uniformly sized particles, based on their intrinsic properties, and possesses high stability and reproducibility as compared to the cell-based technology. Moreover, high speed and less time for consumption further supports the rapid adoption of this technology.

The reagents segment accounted for the largest share of the overall flow cytometry market in 2019. Factors like the development and commercialization of high-quality application-specific reagents and assays and the continuous requirement of flow cytometry reagents by end users (due to the increasing number of flow cytometry-based research activities) are expected to drive the growth of the reagents and consumables market in the coming years.

The pharmaceuticals and biotechnology companies segment accounted for the largest share of the overall market in 2019, and is also expected to dominate the flow cytometry market over the next 5 years, based on end user. The growth of this segment is mainly attributed to the increasing chronic cases, which encourages the development of new drugs and rise in R&D expenditure by the companies. However, during the forecast period, the diagnostic laboratories segment is expected to register the fastest growth in the flow cytometry market. Some of the major factors contributing to the growth of this segment are the rising prevalence of chronic and infectious diseases, growing awareness regarding various chronic diseases, rising willingness to get accurate diagnosis, and growing number of laboratories adopting flow cytometers as the advanced diagnostic technology.

The flow cytometry market is dominated by North America in 2019. The favorable business environment, continuously growing number of research activities, the rising number of drug discovery initiatives by US-based pharmaceutical and biotechnology companies, and the strong presence of key players are factors propelling the growth of the North American flow cytometry industry. However, countries like China and India are expected to grow with a higher CAGR in coming years. The factors driving the growth of the flow cytometry market in these countries are increasing incidence of chronic diseases, technological advancements, growing initiatives toward research activities, and presence of streamlined regulations for IVD products.

The major players operating in the flow cytometry global market are Becton Dickinson and Company, Beckman Coulter, Inc., Thermo Fisher Scientific, Merck, Sysmex, Luminex Corporation, Miltenyi Biotec GmbH, Bio-Rad Laboratories, Sony Biotechnology, Agilent Technologies, bioMérieux, Enzo Life Sciences, Stratedigm, Cytonome/ST LLC, Cytek Biosciences, and Apogee Flow Systems Ltd.

Vendor update

In April 2020, flow cytometry startup BennuBio closed on USD 5 million of a USD 7 million Series B financing round, which it plans to use to expand commercial efforts, accelerate development of new features for its initial product, and develop additional complementary products. The funding round was led by new investor Co-Win Ventures, with additional investment by existing investors Tramway Partners, Cottonwood Technology Fund, and Sun Mountain Capital. Xin Huang, managing partner at Co-Win Ventures, will also join BennuBio’s board of directors.

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 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.

Technology trends

Over the last five decades, flow cytometry has developed rapidly in terms of the number of its applications and the quantity and dimensionality of the data it generates. There have been significant advances in all three V’s of flow cytometry data – velocity (throughput/speed of data acquisition), volume (data content), and variety (sample types and signal-acquisition technology).

The two biggest trends in flow cytometry are high content data and the merging of technologies from separate disciplines. For example, the last 5 years or so have seen the emergence of mass cytometry, which merges the disciplines of flow cytometry and mass spectrometry. In its latest iteration, an image cytometry module has been incorporated to generate unprecedented amounts of content (number of measured parameters) from relatively small amounts of patient tissue. Spectral flow cytometry has also established itself as an important emerging technology. Indeed, mass cytometry can now measure up to 50 features on a single cell simultaneously, using antibodies tagged with rare earth metals, and imaging flow cytometry allows for 1000’s of morphological features and multiple fluorescence markers to be analyzed per cell.

Flow cytometry, therefore, has inarguable potential as a clinical tool for disease diagnosis, prognosis, and therapeutic monitoring. However, some challenges remain in translating the full promise of flow cytometry into clinical practice. Here, some of the current clinical applications of flow cytometry will be discussed, as well as some of the compelling new applications being researched.

Cancer detection, classification, and clinical management. Flow cytometers were originally developed to provide a rapid screening method for cellular aneuploidy (abnormal number of chromosomes) and to measure cell-cycle distribution, both of which are important for tumor prognosis and treatment. However, it has since been realized that for many cancers, especially in the early stages, the accompanying changes in DNA content are not detectable. Despite this initial setback, flow cytometer is now routinely used in basic research and clinical practice, particularly in oncology. Immunophenotyping has become one of the most common flow cytometery techniques and is used to characterize cell subpopulations based on the presence of different surface antigens, which can be detected with flow cytometery using fluorescently labeled antibodies.

For example, leukemias and lymphomas express a specific set of cell surface markers, depending on their stage and differentiation pathway. Therefore, using immunophenotyping, flow cytometery is frequently applied to the clinical diagnosis and sub-classification of these cancers. Flow cytometery is also used to identify and sort hematopoietic stem cells (stem cells that can differentiate into blood cells) from the peripheral blood, following intensive chemotherapy for blood cancers. These stem cells can then be used to repopulate patients’ depleted bone marrow.

Other oncology applications include minimal residual disease (MRD) detection and the detection of apoptosis (cell death) for determining the efficacy of cancer treatments. Flow cytometery can detect very low levels of disease (as few as 1 malignant cell among 10,000 normal cells), which can be important in the clinical management of cancer.

Similarly, flow cytometery of liquid biopsies could be used to detect circulating tumor cells in the bloodstream. These cells are extremely rare, and with its high sensitivity, flow cytometery is perfectly poised to make a significant impact in this area. This approach has potential for the clinical detection of early-stage cancer as well as the detection of circulating metastatic or drug-resistant cancer cells.

Immunology applications. In addition to the numerous oncology applications of flow cytometery, it is also a highly versatile tool for clinical immunology. Prior to an organ transplant, flow cytometery can be used to crossmatch the patient’s serum with donor lymphocytes to detect antibodies that could result in organ rejection. Postoperatively, the analysis of various cell markers on the peripheral blood lymphocytes can indicate early transplant rejection, detect bone marrow toxicity arising from immunosuppressive therapies, and help differentiate infections from organ rejection. For blood transfusions, flow cytometery can be used to detect contamination of blood with residual white blood cells, which can have adverse effects such as pulmonary edema.

Researchers are using flow cytometery to diagnose primary immunodeficiency disorders with the use of immunophenotyping and functional assays. These disorders are caused by genetic mutations that result in defects in the immune system, such as X-linked (Bruton’s) agammaglobulinemia and X-linked hyper-IgM syndrome. Over 300 of these disorders have been identified thus far, and the causative mutations lower immune defense against the attack of infections.

In addition to oncology and immunology applications, flow cytometers are also used to diagnose a variety of rare hematologic disorders as well as autoimmune/autoinflammatory disorders like spondylarthritis (arthritis of the spine). Another area of research that is likely to give rise to increasing clinical applications in the future is that of platelet activity, which is important in many clinical conditions.

The path ahead

The latest developments in flow cytometery are allowing ever more refined gating of target subpopulations, and thousands of parameters can now potentially be measured per cell to generate unique fingerprints. However, the current approach in advanced flow cytometery is typically to capture all the data first and then manually (and subjectively) select only a few, specific features for further analysis. Thus, much information of interest may be lost, preventing powerful comparisons of cell subpopulations or disease phenotypes that may never otherwise be visible to the researcher. Therefore, there is a pressing need for objective, automated, and robust data analysis tools, and, to gain widespread use, user-friendly software interfaces. Arguably, for this reason, advanced flow cytometery remains primarily a research rather than a clinical tool. Experts suggest that it may be possible to overcome this data analysis hurdle by applying machine learning approaches, coupled with further standardization of flow cytometery workflows. The most exciting applications of high content data revolve around the use of machine learning, in particular, deep learning, to extract relevant meaning from large data sets. Machine learning, coupled with big data, has the potential for driving diagnosis and treatment options tailored to the patient’s disease in a timely manner. In addition, researchers still need to figure out how to design a workflow that convincingly validates diagnostic results, especially if the diagnosis employs the power of machine learning. Such developments are necessary before the rich information content of advanced flow cytometery technology can be fully applied in the clinic.

In terms of other future advances in the field, there are exciting and unique applications of sorters in fields like cell therapy and regenerative medicine. Also, creating key applications of imaging cell sorters in pharmaceutical fields may accelerate global drug discovery. Disease heterogeneity makes it hard to validate findings. Perhaps the use of flow cytometry with sorting capability can help such validation, where events-of-interest collected by flow cytometry can be validated with other downstream assays. Finally, with multiple layers of data (types) incorporated altogether, there are now possibilities to do more with less, i.e., label-free sample measurement, which could lead to more direct, faster, and smarter diagnoses. Rare events (e.g., metastatic cancer cells) may soon be detected better than before.

Second Opinion

Flow cytometry in molecular medicine

Dr Pravin D Potdar
Faculty and Professor of Genomics and Stem Cell Biology,
Dr. A.P.J. Abdul Kalam Educational & Research Centre

The increasing prevalence of chronic diseases has greatly boosted the demand for flow cytometry technology in diagnosis and therapies of various diseases. There is a definite need for rapid, accurate, and sensitive diagnostic technology to precisely diagnose the disease at the gene level. Flow cytometry is a technology utilized to investigate a single-cell population from a heterogeneous population of cells, according to their different cell-surface molecules, size, and volume. The availability of specific monoclonal antibodies to their surface receptor molecules widely broadened the spectrum of clinical applications of this technique for the diagnosis and therapies of various hematological and genetic disorders. In addition, extensive development in bioinformatics and statistical analysis allows us to manipulate various parameters with several different combinations to offer a definite conclusion. Introduction of advanced methods, such as high-throughput multifunctional analysis, rapid detection, higher resolution, and improved cell sorting, enhances the efficiency in characterizing and identifying novel drugs for definite therapy.

The most common application performed on the flow cytometer is the immunophenotyping of cells types, present in blood or body fluid. It is the most important diagnostic tool in diagnosing of lymphomas and leukemia. It is also used to know the specific stem cell population in donor blood cells for bone marrow transplantation in leukemia patients. Flow cytometry is also useful in defining four distinct phases of the cell cycle, along with determining cell cycle replication states and cell aneuploidy associated with chromosomal abnormalities. Flow cytometry can be useful in distinguishing cell apoptosis and necrosis on the basis of differences in morphological, biochemical, and molecular changes occurring in the dying cells. The important signal transduction mechanism of the intracellular flux of calcium into the cell, which responds to the stimuli, can be monitored by flow cytometry.

Flow cytometer – Applications in immunophenotyping

Dr Rohit Joshi
Senior Scientist, Division of Biotechnology,
CSIR-Institute of Himalayan Bioresource Technology

Flow cytometry is used in investigation of whole cells and cellular components such as organelles, nuclei, DNA, RNA, chromosomes, cytokines, hormones, calcium flux, protein content, cell proliferation, and cell cycle. In biomedical area, it is used in cell-cycle analysis, gene expression, vaccine analysis, phagocytosis, immunophenotyping of blood cells, rapid and quantitative measurement of apoptotic cells, cell viability, alteration in the plasma membrane, DNA fragmentation from permeabilized cells, and intracellular cytokine detection besides monitoring the progression of hematological diseases such as leukemia and AIDS. It is also used to assess the reaction of basophils to allergens for the detection of allergic response in the skin test. Moreover, polychromatic flow cytometry is used in preclinical tumor immunology and in cancer immunotherapy such as Ag binding, expression of activation and inhibitory markers, cytokine production, cytotoxicity, and proliferation. Cytokine flow cytometry is used to quantify the percentage of antigen-specific T-cells and determine their phenotypic characteristics, for assessing the activity of natural killer cells, monocytes, and dendritic cells. Flow cytometry with T and B cell markers is employed to enumerate depleting T and B lymphocytes and detection of HLA alloantibody after solid-organ transplantation. Similarly, Raman flow cytometer directly probes intracellular molecules and large heterogeneous populations of single live cells in a label-free manner, and is free from cellular toxicity, photobleaching, interference with biological functions, and nonspecific binding. Further, in vivo flow cytometry allows non-invasive real-time monitoring and detection of circulating tumor cells. Recently, smartphone detectors with built-in and compact cameras have created new possibilities for inexpensive point-of-care diagnostics and healthcare delivery that enables on-site, rapid, reproducible, accurate, inexpensive, and sensitive imaging and sensing of biologically relevant targets of interest.

Technology trends in flowcytometry – Combination flowcytometry

Dr Neena Verma
Deputy Lab Head,
SRL Limited, Fortis Hospital

Flowcytometry (FCM) is an excellent technology to detect a large number of parameters on a single cell, examining large number of cells, in a short time, on a miniscule quantity of sample. However, its widespread use has been precluded by drawbacks like labor-intensive data analysis, operator-dependent interpretation of results, and bulky equipment.

To overcome these drawbacks, FCM is being combined with other technologies to get the best of both, in one equipment.

The biggest game changer will be the use of artificial intelligence (AI) in FCM. AI seems to be the ideal solution to save time, labor, and give objective results. FCM captures vast amount of data but when analyzed manually, only a few features are selected for further analysis, losing other useful data. AI can give meaning to the vast data acquired by FCM.

Image flowcytometry (IFC) combines the imaging data of immune-fluorescence with the high-throughput quantitative data of FCM, and helps in localization of the marker within the cell. Introduction of deep neural networks in AI can revolutionize IFC. The large number of single-cell images, obtained through IFC, can be used to train the networks to rapidly identify cells, and diagnose common disorders/identify rare cells and bring it out from research to clinical diagnostics.

Imaging cell-sorter flowcytometry can accelerate drug discovery in pharmaceutical field as only events of interest are collected (instead of getting lost in the vast data) to be analyzed further.

Mass cytometry combines FCM with mass spectrometry. It uses elemental metal isotopes, conjugated to monoclonal antibodies, to evaluate around 50 parameters simultaneously on individual cells with minimal overlap between channels.

Combination of flowcytometry with immunomagnetic separation detects microbial contaminants in food samples. This may be extended to clinical samples to give rapid results overcoming the shortcomings of traditional cultures.

Spectral analyzers are the next generation of multicolor flow cytometers, with unparalleled sensitivity and detection range. These are extremely useful for separating labels with narrow-emission spectra.

Flowcytometry-on-a-chip would be the dream come true owing to its low manufacturing cost, drastic reduction in the size of the flow cytometer, and bringing flowcytometry to the bedside for point-of-care diagnosis.

With rapid amalgamation of various other technologies, FCM has the potential to be the best clinical tool for diagnosis and monitoring of diseases.

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