The traditional techniques of flow cytometry have been the stalwart of cell analysis for many years. The latest cutting-edge technologies, such as high-content analysis and super resolution microscopy, as well as other advances in flow cytometry, can lead to new discoveries while saving time and making the labs more efficient and productive. Classical flow cytometers are bulky sophisticated instruments, currently, more compact instruments no longer rely on extraneous water or air systems. The most recent ones, working with LEDs and/or light collection systems freed from voltage or gains, are even more self-contained and can easily fit a small bench.

The advancements such as mass cytometry technology (CyTOF) and imaging flow cytometry (IFC) have revolutionized the field of flow cytometry, boosting the number of measurable markers per single cell. This revolution also boosted the development of other single cell multi-parameter technologies such as spectral flow cytometry. Past few years have seen a rapid evolution of flow cytometry technology, moving toward more parameters with the development of instruments containing smaller and less expensive lasers and with the discovery of new dyes that extend the range of emissions that can be detected by those lasers. This has been exemplified by the emergence of violet diode lasers, associated quantum dot nanocrystals, and brilliant violet dyes that are excited by violet (and most recently by UV) lasers. These developments make flow cytometry in the range of 15 or so parameters more practical than ever. Flow cytometers of today must meet the data accuracy, high throughput and low serviceability requirements of customers, especially in the clinical laboratory. Some of the high end models have as many as six lasers and highly complex optical systems that must be properly aligned, calibrated and managed, especially during instrument operation.

Flow cytometry has emerged as a precise, rapid, and customizable platform for many state-of-the-art functional assays. The expanding researches are driving the need for flow cytometers in segments such as drug discovery. Monitoring immune cell activity, including phenotyping immune cell subsets, tracking cell proliferation, and measuring cytokine production can provide insights into the overall status of immune function in patients, particularly those undergoing immunosuppression after transplants, enduring cancer treatment, or suffering from autoimmune disease or other pathologies that affect the immune system. Imaging flow cytometry (IFC) has emerged as a useful and efficient tool for studying the signaling pathways in immunophenotypically defined subpopulations of immune cells.As the adoption of flow cytometers continues to grow, there is a strong focus on ease of use and intuitive data generation and interpretation.

Technological Advancements

Flow cytometry had been evolving slowly until imaging flow cytometry (IFC) became a resurgence of interest in the past few years. Imaging is indubitably indispensable for cell analysis. As cellular morphology analysis plays an important role in various biological studies and clinical diagnoses, such as cancer screening, conventional flow cytometry is much anticipated to incorporate imaging capabilities.

Microfluidics impedance cytometry is an emerging research tool for high throughput analysis of dielectric properties of cells and internal cellular components. This label-free method can be used in different biological assays including particle sizing and enumeration, cell phenotyping and disease diagnostics.

Advances in microfluidics and biomedical microelectromechanical systems (BioMEMS) are important in the development of impedance cytometers as it enables manipulation of small fluid volumes, and highly-sensitive measurement with the close proximity of micro-fabricated electrodes to single cells in micro-channels. Besides reducing reagent and sample volume consumption and expensive equipment, a key advantage over the traditional methods is that sample preparation and impedance detection modules can be readily integrated in a single device, lab-on-a-chip (LOC), for POC testing.

With the new technologies replacing the conventional techniques, the major vendors are focusing on improving the fluidics, optics, and electronics of the flow cytometers.

  • Increasing flow cytometer's spatial resolution provides high-speed comprehensive analysis and in-depth imagery of every individual cell. In addition, some erroneous results yielded in conventional flow cytometry can be eliminated by acquiring and analyzing the cell images to, for example, distinguish between cells, debris, and clusters of cells.
  • The optical subassembly in the flow cytometer that includes the light source, optics, filters, mirrors, mounts etc. is one of the most critical parts of the instrument. Depending on instrument complexity and customer requirements, a completely holistic optical system design is often required. This includes challenges such as mounting all the components in a small area, proper alignment, calibration and managing the light sources to avoid any unwanted beam path deviations.

The quality of lasers used in flow cytometers can directly affect instrument performance and product life. Superior quality lasers improve instrument resolution and sensitivity as well as reduce the overall cost of ownership. Laser quality can provide peace of mind that the data being generated is accurate and reliable.

Road Ahead

With the advent of big-data era, life scientists have started to grapple with massive volumes of data. Recent advances in high-throughput IFC allow continuous high-throughput capture of cell images. Various technologies have made the generation of multi-parametric imaging files highly feasible although efficient analysis and utilization of this huge amount of data still remains a challenge. Multimodality (i.e. transmission, scattering and fluorescence), functional flexibility (i.e. operation as conventional flow cytometry or IFC at users' choice), and compatibility with cell sorting are the three principal areas of development for IFC to gain wide acceptance as a workhorse for biomedical research and clinical application. Miniaturization of flow cytometers due to an increase in demand for point-of-care analysis is gaining traction. The next generation flow cytometers are expected to offer higher resolution and sensitivity, and expanded application space to areas such as stem cell research.

Flow cytometry has been instrumental in the discovery of rare cells and in understanding how they differ from more abundant cells. Rare cell analysis by flow cytometry is advancing through innovations in instrumentation and detection reagents, consisting mostly of fluorescently tagged antibodies to proteins expressed on cell surfaces. Adaptations of conventional cell based assays, such as FISH, augment more narrowly focused detection methods. With this type of co-development, the utility of flow cytometry for detecting rare cells and related events continues to improve even after instruments have achieved legacy status.

Flow cytometers market is pushing for more lasers and more channels to increase multiplexing capabilities. Dye development is following suit by providing more choices for upcoming and existing fluorescence channels. The aim is to use the whole spectra for excitation and emission, to achieve as many options as possible. In such a game of leapfrog, the winner is likely to be the end user.

Amardeep Gupta
Industry Speak
Accelerating Toward Clinical Flow Cytometry

It all started when Prof. Dr Wolfgang Ghde, 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. The new clinical flow cytometers will have the optical configuration up to 3 laser (Blue Red and Violet) and 14 colors. The clinical flow cytometer will have dedicated clinical software (6 colors) with CE-IVD and FDA (510 K) approval.

We are also looking for the opportunity to provide 21 CFR Part 11 compliant software. 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.

Amardeep Gupta
Business Manager – Flowcytometry,
Sysmex India Pvt. Ltd.

Amardeep Gupta
Second Opinion
Technology Trends

low cytometers had started as research tools which were laborious and time consuming. Advances in flow cytometers, their reagents, and analysis software have brought them to the clinical laboratory. Laborious and time consuming processes like alignment of lasers, cleaning protocol, sample streaming have become automated. Cell sorting, which required years of training, now requires less expertise due to automation. In fact, automation is approaching the stage where the user can press the appropriate process and walk away. This is ideal for commercial labs which do the same tests repeatedly.

Microfluidics have simplified the analysis and made it more efficient and faster. Integration of other methodologies has made flow cytometers even more useful e.g. image analysis, RNA fluorescent in-situ hybridization, and mass spectrometry. Small portable flow cytometers are also available now. Charge coupled device camera have replaced PMTs in some flow cytometers. These can provide precise location of markers within the cells.

State-of-the-art dyes are being developed, which are bright, stable, have narrow excitation and emission peaks, and are multicolor compatible. This will allow 20-30 colors to be analyzed on a routine basis. The aim is to develop single laser excitable dyes in the future. Kits are now available to conjugate antibody to the fluorochrome of the user's choice.

In research, flow-cytometers have moved from analysis of cells to analysis of intracellular contents and processes. Vast improvement in data analysis software has made it simpler and user friendly. The vast complex data generated can be stored in cloud based storage. Users can select display modes and even isolate specific cell subsets.

Thus the future is very bright for flow cytometers in clinical labs with simpler, faster analysis as well as in research where analysis of cells as well as intracellular contents and processes can be done with tailor made programs and dyes.

Dr Neena Verma
Chief Pathologist,
SRLLimited, Fortis Hospital
Noida


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