A New Wave Of Innovation In Flow Cytometers

There has been an explosion in technological advances, which has seen an expansion in the number of commercially available fluorescent dyes tagged to antibodies, and instrumentation capable of detecting over 18 colors simultaneously.

Flow cytometry has often been regarded as an immunologist’s playground, and accordingly, flow cytometers are most often found in or are associated with immunology research labs. But with the capacity to study multiple parameters at a single-cell level, flow cytometers are now being widely used and have become a powerful tool for biological research and clinical diagnostics, and its applications have been essential to innumerable advances in cell biology and immunology, as well as for understanding diseases such as immunodeficiency and cancer. Many aspects of cell function, physiology, or components of cells can be measured using cytometric techniques, and therefore, researchers from a wide range of disciplines can find themselves in the driver’s seat of a cytometer. Just as long as a fluorescent probe, such as an antibody or dye is available to target a cell or component of interest, the types of analyses that can be performed on cytometers are virtually limitless.

Over the last decade there has been an explosion in technological advances, which has seen an expansion in the number of commercially available fluorescent dyes tagged to antibodies, and instrumentation capable of detecting over 18 colors simultaneously. Applying high dimensionality (18+ colors) to precious or limited samples is now in high demand and is driving innovation in the field. Conventional flow cytometer designs sought to meet the multicolor requirements by adding more lasers and detectors, which significantly increases the cost of ownership, maintenance, and usage. However, more recently, innovations in optical detection have seen a new wave of instruments enter the market. One such new-generation flow cytometer is a spectral analyzer that represents a new level of capability for complex multicolor flow cytometry. This technology is the new kid on the block and an apparent game-changer in the multicolor arena.

With the advent of monoclonal antibodies and an increasing number of fluorescent dyes, together with continuing improvements in the computerized hardware and associated software, the development of reliable techniques for performing polychromatic flow cytometry analysis has been possible. As demands for increased plex and greater throughput continue to drive the evolution of flow cytometry systems, manufacturers are employing increasingly sophisticated methods to meet the needs of researchers. At present, there are over 50 vendors in the flow cytometry business, selling flow cytometers ranging from high-end of up-to 21- parameter systems to just one or two-color point-of-care systems.

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 a 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).

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 20x, 40x, or 60x 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.

Way forward
While trends toward high-throughput flow cytometry, the use of more sensitive detectors, and improved integration of flow cytometry systems with multi-omics information are taking place, future advancements within the technology are likely to be seen within the informatics space. By allowing researchers to combine large datasets that include cell characteristics such as surface phenotypes with intracellular protein markers, together with genomic and transcriptomic information, one will see the most detailed description yet at the individual cellular level. As the needs of the various cell culture communities continue to grow, flow cytometry systems will need to work hard to keep pace, but it seems that their future development is in safe hands.

Apart from the 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 are improvising its existing technical aspects. There are continuous advancements in molecular diagnostics, monoclonal antibodies, lasers, and software. Moreover, growing demand for understanding 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 and huge investments in flow cytometry instruments may hinder growth.