In the armamentarium of the healthcare community, flow cytometry has commonly been regarded as a tool best applied in research laboratories rather than in clinical laboratories. A workhorse technology that offers tremendous flexibility and power for detecting cellular abnormalities and identifying a wide range of pathogens, flow cytometry has nevertheless been mostly restricted to studies which contribute understandings that often find application through other diagnostic methods.

But in recent years, routine clinical applications of flow cytometry have begun to increase in number and complexity, driven largely by advances in technology. In small steps originating with a number of different instrument manufacturers and reagent suppliers, clinical flow cytometry is benefitting from the availability of better reagents, increased attention to the automation of pre-analytical processing steps, more colors to represent data points, more powerful lasers, better detectors, and extremely high-end informatics processing to deal with all of the data that the new instruments can generate.

Advances in flow cytometry technologies have been significant, leading to greater automation, higher performance, and an increased range of capabilities for clinical applications. The technology has exploded over the past 5-10 years, with a lot of new market entrants offering instrumentation, and a lot of new discoveries in reagents, fluorophores, and dyes. In the past, flow cytometry was so complex that it was associated with a lot of ease-of-use issues. But now it has become much easier to use. The latest generations of instruments are finally catching up with technologies that have been available in other fields for many years, including the adoption of user-friendly software. There are a lot of new developments in the flow cytometry equipment that will be coming out in the near future.

Shifting Paradigms

The first cytometers were systems capable of merely three or four parameters, using a single laser and four detectors, and were the size of a small car. Nowadays, flow cytometers including cell sorters can analyze more than 30 parameters, and new technology in bench-top analyzers can deliver exponentially better performance in a footprint less than 45 cm2.

This paradigm shift of higher performance in a small, less expensive instrument is driven by investigators who want to capture the power of flow cytometric analysis. The democratization of flow cytometry is enabled by key advances in technology. Prominent concepts in other scientific fields such as the telecommunications industry are being leveraged to allow the subsystems to be miniaturized while at the same time providing even better performance. These compact high-performance systems not only deliver better performance than historically expensive systems, but they are also easy to set up, operate, and maintain, enabling a greater number of laboratory scientists to leverage the power of flow cytometry.

Advancing Technology

Several important developments in flow cytometry technology are in use nowadays. The use of more robust photodetectors and new laser emitters, the use of LED lamps as emitters, and changes in analytical capacity (multi-dimensional analysis software) are examples of recent improvements. Although these represent important advances, a major overhaul in flow cytometry consists of new approaches combining existing technologies with routine laboratory procedures, such as imaging.

Imaging through cytometry. This makes possible the acquisition of images of cells in real time as they pass by the interrogation point. At this point, the presence of target markers is assessed, and the subcellular localization of these markers can be imaged. Although imaging during cytometry has long been proposed, it is not yet widely used by research laboratories, probably due to the low image resolution. However, over the last 10 years, technology has advanced to the point where it is now possible to resolve some of the issues with this approach.

Mass cytometry. Another technology that is already a reality but not yet widespread is flow cytometry accompanied by mass spectrometry by time-of-flight. Relatively recently, the mass cytometry approach has been characterized by coupling with a fluid-flow cytometry system, which allows the separation and organization of cells in suspension with the resolution of mass spectrometry. Using antibodies labeled with non-fluorescent heavy metals, cells are separated one-by-one and injected into an ionization chamber, where the metals bound to antibodies are identified by their TOF characteristics. This is not yet a widely used technology, but some researchers believe it will be possible to simultaneously assay up to 100 markers in each cell by mass cytometry.

Seeing the light. New systems use unique laser designs that are focused onto a flow cell with integrated optics. These systems can ensure both maximum excitation of the dyes not only on cells, but also maximum collection of the emitted light for integration and measurement. When designing a compact flow cytometer, the use of fiber optics to carry light is an efficient way of transmission, providing flexibility in laying out system components. Another recent development is a key concept borrowed from the telecommunications industry, the wavelength division multiplexor (WDM), which is used for light detection and measurement. These detectors are highly sensitive semiconductor devices used to measure each parameter.

New high-multiplexing capabilities of flow cytometry make it possible to analyze multiple biological targets in parallel without appreciably impacting sample analysis time. Improvements in software have also contributed to simpler test processes, including post-analysis color compensation to the test parameters. In the past decade, there have been a lot of improvements in the reagents available for clinical applications. Getting standardized reagents has been a big improvement. While flow cytometry testing has already benefitted from significant improvements in specimen processing and standardized instrument calibration, further improvements in automation are also on the horizon. There will be more automatic processing to take specimens all the way through a standardized process. A good improvement in the near term will be the achievement of greater standardization across laboratories.

Way Forward

Pushing the norms of conventional flow cytometry, the cytometry systems could enable complex research into high-content applications in cell biology, as well as a deeper understanding of the advantages gained from the emerging nanoparticle frontier. Because market advances are making flow cytometry simpler, smaller, and more affordable, more and more usage of it would be observed. Over the next 5-15 years, more assays and novel ways to utilize flow cytometry would be devised.

In the not-too-distant future, aspects of flow cytometry could be fused with molecular biology, which could become a very exciting area in terms of determining specific types of cancers and how they can be treated. While applications of clinical flow cytometry are increasing, it is uncertain whether the technology would ever rival the major players that already dominate laboratory medicine. In the meantime, flow cytometry is continuing to evolve in new directions with even greater utility for clinical testing.


 

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