Shifting into Higher Gear

Shifting into Higher Gear

The designs of instruments and processing techniques are evolving to meet the needs of researchers and drug developers. The aim is to allow faster, more accurate experiments.

During the past 50 years, flow cytometry has established itself as the workhorse for high-throughput quantitative analysis of cells and other particles. Since its inception and its first use, the basic design of flow cytometers has remained almost unchanged, highlighting the robust design of the technology. The speed at which cells are analyzed, the high accuracy and resolution of the technology, and the low operating costs per sample have contributed to its success and widespread use. In recent years, there has been a widespread interest in development of flow cytometry tools. The original flow cytometry tools were only able to capture measurements of a single fluorophore. Today fluorescence-based flow cytometers can simultaneously extract measurements of up to 20 cellular markers. With the emergence of next-generation fluorochromes, thousands of new antibodies, a diverse array of fluorescent proteins, phospho-protein detection, and assays for RNA expression, flow cytometry is rapidly being adopted by almost every major discipline in the biological sciences. Improvements in software and hardware design have also made flow cytometry more user-friendly and enabled adoption beyond the traditional core of immunological researchers.

Flow cytometers were initially large and somewhat cumbersome instruments, but have evolved into table-top, user-friendly, multi-functional ones. Today’s flow cytometers are used in cell differentiation, chromosome analysis, cellular immunology, clinical hematology, cancer diagnosis, pharmacology, and toxicology, to name a few applications. They are further expected to undergo significant changes over the next 5 years as the technology establishes a growing base in biotechnology labs for applications like counting, sorting, biomarker detection, and protein engineering Significant changes owing to increasing research and commercialization in life science fields such as proteomics, genomics, pharmacogenomics, and stem cell research are creating big demand for flow cytometry.

Indian Market Dynamics

The Indian flow cytometers market in 2016 is estimated at 205 crore, with reagents constituting 60 percent market share. The analyzers and cell sorters market continues to be dominated by BD India and Beckman Coulter.

The government is moving toward centralized facilities, and for any instruments higher than average unit price of 25 lakh, tenders are not being finalized at regional level. Also, with the buying life cycle, especially for mid-end and high-end equipment normally being up to 12 months, some vendors abstain from participating.

Over the last couple of years, HIV spends gave way to cancer immunology where the private sector is more active. The government spent more funds for stem cell research and infectious diseases. Some buying was done by the private sector, for instance, Amrita Medical College, with funding by the government.

Vendor Update

In April 2017, BD (Becton, Dickinson and Company) received 510(k) clearance from the US Food and Drug Administration (FDA) for a flow cytometer system with a leucocount reagent assay used in residual white blood cell enumeration. When used together with the BD Leucocount kit and BD Trucount tubes, the new BD FACSVia system provides a simple solution for identifying and counting rWBCs in leucoreduced blood products. With an intuitive user interface, the fully automated BD FACSVia system improves overall lab efficiency and simplifies workflow.

In March 2017 Beckman Coulter Life Sciences announced the expansion of the multi-parameter capabilities of its patented CytoFLEX flow cytometer technology. Its latest model, the CytoFLEX LX, with up to six lasers and 21 fluorescent channels, delivers even more parameters with the same exceptional fluorescence sensitivity and superior nanoparticle detection demanded by high-end researchers.

Bio-Rad Laboratories, Inc., having acquired a high performance analytical flow cytometer platform from Propel Labs in February 2016, brought it to the Indian market. This enables advanced and novice users to perform basic and multi-parameter cytometry for a wide range of applications and chemistries.

Global Market

The global flow cytometry market is expected to reach USD 6.3 billion by 2020, up from USD 4.5 billion in 2016, reflecting a five-year compound annual growth rate (CAGR) of 9.1 percent, predicts BCC Research. The instruments segment, which accounted for the largest share at almost USD 2.3 billion in 2016, should increase at a five-year CAGR of 7.3 percent to reach almost USD 3 billion in 2020. Reagents as a segment would grow the fastest with a five-year CAGR of 11.9 percent.

Small-size high-throughput cytometers are expected to gain popularity over the coming years due to associated benefits such as ease in use and cost-effectiveness. Furthermore, improvement in fluorescent dyes and introduction of bench top cytometers are the other growth propellers. For instance, multicolor flow cytometry coupled with multiple lasers is the fastest growing application segment, which finds extensive applications in the field of R&D innovations in new drug development and is adopted by many contract research organizations.

Market consolidation is one trend that would continue over the next 5 years as players strengthen or retain their position. This development has produced stronger, vertically integrated competitors that are better able to compete as sole-source vendors to end-users. Its competitive nature prompts key players to have continuous product development and forge partnership alliances to aid market penetration. Manufacturing plantsare being set up in untapped regions of developing countries. Competitiveness is expected to intensify with the increase in product extensions, technological innovations, and new applications of the products and services provided.

Technology Trends

The past decades have marked a number of key technological advances in flow cytometry, all of which increase the number of parameters that can be measured simultaneously from single particles. For conventional flow cytometry, the most notable advance has been the extension of the classical design with additional and more powerful lasers. Together with advances in fluorophore design, 18-parameter flow cytometry is now routinely used; 30-parameter flow cytometers have recently become commercially available and 50-parameter flow cytometry is projected to be available soon. This will allow the user to perform analyses comparable to those using another recent innovation in the field, mass cytometry, which is more widely known as cytometry by time-of-flight (CyTOF). Further technological advances have focused on satisfying the increasing need for polychromatic approaches to flow cytometry.

Spectral flow cytometer. The arrival of the spectral cytometer opens a new era in flow cytometry by allowing detection of the whole spectrum of each particle, from ultraviolet to infrared. It gives a great deal of information and details on the fluorescence emission. In conventional flow cytometry, light is classified and filtered for every detector leading to overlapping of different fluorochromes of the spectra. However, the spectral cytometer is able to simultaneously discriminate between the spectrums without them overlapping. It is capable of using up to 32 different parameters. Spectral flow cytometry distinguishes the shapes of emission spectra along a large range of continuous wave lengths. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter, which was a challenge during data analysis in conventional flow cytometers. Thus, spectral flow cytometry is expected to discriminate fluorochromes with similar emission peaks and provide multi-parametric analysis without compensation requirements.

CyTOF. Cytometry by time-of-flight (CyTOF) is a new method for detecting antibodies bound to cells. Even with careful panel design, loss of resolution occurred from auto fluorescence and spectral spillover in classical flow cytometers that relied on fluorescent tags. The greater the number of fluorochromes used, the larger these problems become magnified. Now, the CyTOF overcomes this limitation of flow cytometry. In CyTOF system, antibodies are labelled with metal-conjugated probes instead of fluorophores, and a time-of-flight detector is used to quantify the signal, avoiding the problem of spectral overlap associated with classical fluorophore-based flow cytometry. Use of traditional labeling techniques with minimal change to current protocols, easy panel design, minimal to no compensation need, and no autofluorescent are some of the advantages offered by this new technology.

IFC. Imaging flow cytometry (IFC) combines the single-cell imaging capabilities of microscopy with the high-throughput capabilities for conventional flow cytometry. Recent advances in imaging flow cytometry are remarkably revolutionizing single-cell analysis. It enables potentially powerful, multiplexed single-cell analysis. Where conventional flow cytometry has been found wanting, image cytometry has filled in the gaps to provide both quantitative power of flow cytometry and fluorescence localization power of imaging in a single platform. It combines flow cytometry with a high-resolution multispectral imaging system, acquiring several images per cell, at current rates of 5000 cells per second. Hundreds of image-based features can subsequently be extracted, giving additional information regarding morphology, co-localization, and cell signaling. Imaging mass cytometry uses mass cytometry to reconstruct tissue images. Imaging software is subsequently used to combine all spot information and extract information at the single cell level.

Optofluidic microflow cytometers. They combine the field of micro-optics and microfluidics bringing a host of new advantages to conventional cytometers. Optofluidic microflow cytometers integrated with optics provide significant enhancements for flow cytometry. Low costs, higher sensitivity, free optical alignment and smooth interaction interfaces are the obvious advantages. Although conventional flow cytometers have gained profound success in cell sorting and analysis, they are bulky, demand large amounts of expensive reagents with complicated processing steps, are complicated in manipulation, and require high maintenance costs. These limitations restrict their use in POC diagnostics, in situ pathogen monitoring and other application where portability, handling small volume samples, low operation costs and ease of operation are essential. Owing to the recent development of lab-on-chip (LOC) technology, microfabrication and micromachining techniques, the miniaturization of a flow cytometer can be achieved. Optofluidic microflow cytometers offer significantly lower costs and size reductions, as well as low reagent requirements and portability advantages over a bench-top flow cytometer.

Acoustic focusing. Modern flow cytometers handle thousands of events per second. However, when increasing the flow rate, one has to lower the overall resolution. Scientists have solved this issue with acoustic-assist focusing. They use sound energy to usher the particles into the center of the sheath fluid, where hydrodynamic focusing takes place. This allows high resolution at a high flow rate. In addition, this technology also enables the possibility of running multiple streams at the same time in one flow cytometer, thus tremendously speeding up the acquisition time. Hydrodynamic fundamental to flow cytometry enablesmake better judgments on how to fine tune the machine and, become better at running a flow cytometer.

Future Outlook

Flow cytometry has become a powerful tool for biological research, capable of generating volumes of valuable data when employed correctly or mountains of misleading data if potential pitfalls are not avoided. Although labs are equipped with better tools than ever before to do flow cytometry, it can still be a complex technique. Analysis of flow cytometry data is considered to be one of the most challenging and time-consuming steps in flow cytometry experiments, primarily due to the absence of an efficient automatic analysis approach to analyze the high dimensional data generated by advanced flow cytometers. In the context of high-throughput screening, flow cytometry has traditionally been slow, low-throughput and not amenable to automation. Thus, there is high demand for bioinformatics tools for automatic analysis of flow cytometry data.

A push has been underway to package flow cytometry in more compact, affordable and intrinsically more reliable units. Fueled by the need for point of care diagnostic applications, a significant effort has been made to miniaturize flow cytometry. However, despite recent advances, current micro flow cytometers remain less versatile and much slower than their large-scale counterparts. Various fixes are under development. Improvements in the optical design (reduction of stray light, increased collection of scattered light, reduction in electronic and system noise sources), increase in the available optical power from solid-state lasers, and signal-processing techniques are all being deployed in the push for detecting the smallest possible particles quickly and reliably.

For a long time, flow cytometers had to rely on massive, electricity-guzzling, water- or forced-air-cooled gas lasers, such as argon-ion and helium-cadmium. With the arrival of solid-state alternatives, the footprint – both physical and carbon – of flow instruments has been significantly reduced. The latest laser products to hit the market are as small as a pack of mints enabling transformative instrument designs in terms of alignment stability and overall size.

One of the biggest challenges of flow cytometry lies in acquiring, storing, and processing a massive number of cell images. There are many software packages and tools for use in high-throughput image analysis but the pipelines of these tools are for offline image analysis. Computational requirements are unprecedented. In order to combine cell sorting with imaging flow cytometry to fully realize its tremendous potential, real-time image construction and analysis is required. Hence, the ability to produce, measure, and analyze cell images, and sort cells in a real-time manner will be the next major milestone.

Second Opinion
A Dynamic Area of Diagnostics

Flow cytometry is a technology that simultaneously measures and then analyzes multiple physical characteristics of single particles, usually cells, as they flow in a high-pace fluid stream through a beam of laser light. Flow cytometers are automated instruments that quantitate properties of single cells, one cell at a time. Light scattering at different angles can distinguish differences in size and internal complexity whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. Three main systems make up the flow cytometer instrument: The Fluidics – the purpose of which is to transport the particles in a stream of fluid to the laser beam where they are interrogated; the Optics which is made up of lasers which illuminate the particles; and the Electronics which convert the light signals to electronic pulses that a computer can process. The information obtained is both quantitative and qualitative. A variety of specimens can be analyzed including whole blood, bone marrow, serous cavity fluids, FNAC aspirated material, CSF, urine, and solid tissues. Most flow cytometry is analytical – after the information is obtained as it passes through the cytometer, the sample is discarded. Some flow cytometry is preparative – living cells are sorted into separate containers based on the properties of each cell.

The common clinical uses of flow cytometry are: Immunology – histocompatibility cross matching, transplant rejection, HLA B27 detection, and immunodeficiency diseases; Oncology – DNA content, S phase of tumors, and measurement of proliferation markers; and Hematology – CD34 cell enumeration, leukemia and lymphoma phenotyping, autoimmune, and alloimmune disorders, anti-platelet antibodies, immune complexes, and FMH quantification, PNH, leucocyte adhesion deficiency, identification of lymphocyte sub populations, and immunohematology.

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable to the development of smaller, user-friendly, less expensive instruments, and a continuous increase in the number of its clinical applications.

Dr Meenakshi Sharma,
Consultant Hematopathologist,
Manipal Hospital, Jaipur

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