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Flow Cytometers

The best, it seems, is yet to come

While the value of performing multi-parametric analysis at a single-cell level is widely recognized, there are still those who regard flow cytometry as a complex technique that is best left to the experts.

Flow cytometry is a technique that is continually evolving. Improvements to instrumentation, reagents, and software have all been key in enabling researchers to generate increasingly detailed profiles of individual cells. Recently, its use in cancer immunology has grown impressively, because of the role it can play in extending experts understanding of the immune system and its response to cancer immunotherapies. However, while the value of performing multi-parametric analysis at a single-cell level is widely recognized, there are still those who regard flow cytometry as a complex technique that is best left to the experts. For a decades-old analytical technique, the best, it seems, is yet to come.

Recent advances
Flow cytometry and cell sorting are similar techniques with markedly different goals. Both are used to characterize individual cells within a heterogeneous population, but while flow cytometry represents the end of the line for the cells undergoing analysis, cell sorting often provides cells with a new beginning. Some of the best practices for ensuring the success of these two established techniques and some modern advances that underpin a growing number of flow cytometry and cell sorting applications include:

Smaller, more powerful instrumentation. A striking difference between early flow cytometry and modern forms of the technique is the size of the instrumentation. One of the main reasons flow cytometry has been able to reach into new and developing fields is that benchtop flow cytometers can be positioned in a small lab space rather than requiring a dedicated facility. Another important differential is the increased number of lasers, filters, and detectors that have contributed to larger panel sizes. Modern instrumentation also encompasses spectral cytometers that allow for detection of more readouts from a single laser.

Best practices for flow cytometry and cell sorting success. With both flow cytometry and cell sorting requiring single cell suspensions, several shared best practices are essential. Although cells will not be recovered at the end of a flow cytometry experiment, cell health and viability must still be maintained. Cell death can switch on numerous cellular pathways with the capacity to alter cellular behaviors and impact the target of interest, and it also releases a plethora of biochemicals that can influence surrounding cells. Steps for ensuring cell viability include lowering the temperature to decrease metabolic activity; adding a protein source to buffers to stabilize cells; and reducing any physical or shear stresses resulting from pipetting or centrifugation. Additional measures involve passing cells through a nylon mesh filter and including EDTA in buffers to eliminate clumps; optimizing cell numbers for the experimental system, with 1-10 x106 cells/mL being a good starting point;

Journeying toward upward trajectory

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

Flow cytometry 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 are 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 frequently used in basic research, clinical practice, and trials. It is used in cell counting, cell sorting, determining cell characteristics and function, detecting microorganisms, biomarker detection, protein engineering detection and diagnosis of health disorders etc.

According to the study conducted by at National Institute of Health in 2019, explains FCM for the diagnosis of pediatric acute leukemia has high reliability with an accuracy of greater than 98 percent.

The global FCM market size is projected to grow from an estimated USD 4.0 billion in 2019 to USD 6.4 billion by 2025, at a CAGR of 8.3 percent. Market growth is largely driven by factors such as technological advancements in cytometers, the increasing adoption of FCM techniques in research activities and clinical trials, growing focus on immunology and immuno-oncology research, increasing incorporation of AI platforms and advancements in software, high incidence and prevalence of HIV-AIDS and cancer, and the increasing availability of novel application-specific flow cytometry products. It can also be used in wide range of different applications as in microbiology, biotechnology, AgroSciences, Plant breeding, aquaculture, and for quality control in pharmaceutical industry as well as in food and beverage industries.

The market is categorized into reagents and consumables, instruments, services, software, and accessories. Wherein the first two is expected to dominate the market. Factors such as the development and commercialization of high-quality application-specific reagents and assays and the continuous requirement of FCM reagents by end users are expected to drive the growth in the coming years.
Acquisitions and strategic collaborations, new product launches, and advanced technologies are driving the global flow cytometry market.

Sysmex is committed to advancements in flow cytometry, building upon our hematology knowledge and strengthened by our acquisition of Partec GmbH, a pioneer in flow cytometry. Our comprehensive line of flow cytometry products includes analyzers, consumables, and reagents.

and using viability dyes to distinguish live and dead cells. Time may also be a factor if cells are required to undergo extended treatments or incubations.

Modern technologies opening up new possibilities. Many innovative developments have helped to improve the scope and reliability of flow cytometry in recent years. Fluorescent labels within the UV and IR range, along with novel tandem dyes, have increased panel sizes for greater multiplexing capabilities, while the majority of antibody manufacturers have invested considerable effort to improve the validation of their reagents. Many of conjugated RabMAb antibodies have been validated for specificity against knockout cell lines, including application-specific validation using flow cytometry, whereas REAlease releasable antibody technology provides easy removal of antibodies after cell labeling. Depending on the downstream application, it can be important to remove bound antibodies from cells. For example, this might be required if cells are to be activated, if certain epitopes need to be made accessible again, or if cells are to be transplanted into patients or animal models.

One area seeing the greatest disruption is that of cell sorting instrumentation, with manufacturers promising faster, gentler, scalable, sterile sorting to support a growing range of applications. Flow cytometers and cell isolation products are typically very bulky and require a lot of specialized equipment, meaning many users have to book time at a core facility to carry out their experiments. Recent cell sorter has a footprint of less than 2 cubic feet, allowing it to be placed anywhere in the lab—even in a hood. Not only does the small footprint provide researchers with greater flexibility, but because sorting takes place within a disposable microfluidic cartridge, sample-to-sample contamination and biohazard aerosols are eliminated.

Supporting new applications. Common applications of flow cytometry include immunophenotyping, cell cycle analysis, the study of apoptosis, and biomarker discovery and diagnostics, yet the technology is branching into many other research areas as well. For instance, flow cytometry is now used to analyze other regulated cell death pathways such as autophagy and necrosis, and it has recently found itself at the center of research for COVID-19 where it has been used to determine immune responses, recovery, and antibody levels through cellular and bead assays. The multiparameter data acquired, at speed, has certainly helped with research into the basis of the disease and will be an important tool for determining vaccine efficiency.

Going forward
The current advancements in medical research, as well as those in the diagnostic capabilities provided by flow cytometric technology, will provoke a shift in the flow cytometric business landscape. Instead of the technology being used only for research, the market can be revolutionized by less complex tools that rely on cloud computing and AI to assist medical personnel in making diagnostic and therapeutic decisions for patients.

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