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Mar 1, 2014 (Vol. 34, No. 5)

Flow Cytometry Gets Particular

  • Flow cytometry has reached a stage in its evolution where fundamental technical improvements are paying huge rewards.

    “We can now go a lot farther, and delve more deeply into fundamental scientific problems,” says Jeannine Holden, M.D., director of scientific affairs for flow cytometry at Beckman Coulter Life Sciences. “We’re reaching the point where signal to noise is not about instruments, but about the biology.”

    Multilaser, multidetector systems, combined with improved chemistries and fluorophores, have emerged in rapid succession, enabling scientists to answer scientific questions that were previously unapproachable.

    Flow is now capable of analyzing and sorting microparticles, which was previously impractical due to instrument variability and noise. Microparticles are heterogeneous, submicrometer, extracellular vesicles that express antigens specific to their cells of origin. Illnesses as diverse as sepsis, preeclampsia, and heart disease are associated with elevated microparticle populations.

    “We’ve known that microparticles were significant, but we previously lacked the tools to study them,” Dr. Holden adds. Beckman Coulter’s AstriosEQ cell sorter, for example, identifies and sorts microparticles.

    Flow cytometry has traditionally used cell surface markers to identify and sort cells, but with a few exceptions observing intracellular events, or intra-extracellular interactions, involved cumbersome workflows. Recent advances now allow the simultaneous examination of surface and intracellular events, and in some cases their interactions.

    Several vendors offer kits for achieving this. Beckman Coulter’s PerFix reagent kit reduces prep and handling time by about 50%, according to the company. A variation on this theme, PerFix Expose, covers phosphorylated targets, which opens up possibilities for studying cell signaling.

    “The ability to study surface and intracellular markers simultaneously, in many thousands of cells responding differently to experimental stimuli, in one sample, has been a breakthrough,” Dr. Holden explains. After sorting, researchers may recover selected populations for further study. “Flow doesn’t provide the detail of microscopy, but it hits a sweet spot for maximizing information from large numbers of cells.”

    Vendors of flow cytometry reagents have done well to standardize kits to eliminate dispensing-related variability. Global diagnostics companies, for example, are mindful that assays be consistent across the hall or across the globe.

    One way to eliminate reagent dispensing issues entirely is through wells or tubes that contain reagents in dry form out of the box. The sample becomes, in effect, the reconstitution buffer. Through its July 2013 acquisition of ReaMetrix, Beckman Coulter became a leading purveyor of stable, dry, ready-to-use reagents in familiar container formats. “The rigor that’s been the standard in clinical work is entering research as well,” Dr. Holden comments. “The multiparameter aspect of flow no longer represents a multiparameter means of introducing variability.”

  • Mass Cytometry

    Complex cell analysis has significantly raised the ante for developers of classical flow cytometry instrumentation. Users are demanding more lasers and detectors, to measure more colors and parameters from a single sample. At the extreme end, companies such as DVS Sciences (acquired by Fluidigm at the end of January), offer an instrument platform, CyTOF, that combines cytometry with time-of-flight mass spectrometry.

    Both CyTOF and flow cytometry quantify multiple events simultaneously using labeled antibodies. But instead of fluorescent tags, CyTOF combines antibodies with heavy metal isotopes. “With conventional flow cytometry, spectral spillover limits how many probes you can combine,” says Tad George, Ph.D., the company’s senior director of scientific applications.

    In CyTOF, labeled cells pass through a nebulizer, and the aerosol is blown into a torch at 7500K. Detection occurs through ion optics—first a quadrupole, which focuses high-molecular-weight ions and rejects species of low molecular weight, then a time-of-flight device, which quantitates species by molecular weight.

    “The advantage here is that a mass spectrometer can resolve to one atomic mass unit, so there is no overlap,” Dr. George notes.

    DVS currently sells 34 probes, but the theoretical number of channels, 120, will make flow cytometry experts jealous.

    Critics will note that CyTOF is expensive and demands a high skill level—the same knock on conventional mass spectrometry 30 years ago. Dr. George admits as much, but explains that CyTOF is a tool for big science—for discovery—and not for routine cell characterization.

  • Broader Spectra, Narrower Channels

    Click Image To Enlarge +
    Sony Biotechnology’s SP6800 can help investigators avoid the limitations of filters. At the same time, it can offer investigators more options in their fluorochrome pairings.

    As European marketing manager at Sony Biotechnology, Mark Dessing represents a side of Sony with which most consumers are unfamiliar. Dessing notes that on the analysis side (versus sorting), flow cytometry is moving toward higher content analysis and greater user-friendliness: “Sorting has been around for a long time, and the underlying technology has not changed much. But advances in software and electronics have enabled high-speed sorting and the ability to observe more cell populations simultaneously.”

    Dessing believes that cell sorting will eventually adopt some form of microfluidics, which promises to deliver compactness and biosafety. These possibilities have encouraged Sony to invest in microfluidics, even though the technology has been criticized for its long switching times. These result from pressure changes or micromechanical movements, which occur much more slowly than the length of a droplet, about 10 microseconds.

    “That means cells must be spaced far apart,” Dessing remarks, which is problematic when sorting millions of cells. Another issue is that cells do not move as predictably through microchannels. “If you don’t know where the cell is, you don’t know what to do with it.”

    Sony’s system overcomes some of the issues with microfluidics by analyzing on a chip but sorting through a conventional streaming air system.

    Another intriguing Sony innovation is the SP6800 Spectral Analyzer, which collects and analyzes the entire spectrum from a fluorescence event. Whole-spectrum analysis has been used in confocal microscopy for years, but in flow systems the analysis must occur within the four microseconds the cell whizzes by. Full-spectrum measures more colors simultaneously, and can deconvolute those that overlap.

    “Conventional optical filters provide good data, but they miss half or more of the light,” Dessing explains. Spectrum analysis uses the entire spectrum for every cell, which thanks to modern computing allows spectral unmixing. New algorithgms allow calculation of spectral overlap almost instantaneously.”

    At the moment, spectrum analysis works only in analysis mode, but Dessing expects applications in cell sorting down the line.

  • Democracy Now!

    Cell sorters have followed a development trajectory similar to those of many lab instruments. According to Mike Olszowy, Ph.D., flow cytometry R&D director for biology at Life Technologies, instruments sport automation features that make them easier to set up and run, often on a more robust platform, in a much smaller footprint, and at lower cost.

    “This has led to the democratization of flow cytometry that is occurring more rapidly on the sorting side compared with cytometry analyzers,” Dr. Olszowy says. Flow cytometers are emerging from core facilities supervised by experts, and winding up on more benchtops for individual lab workers and small groups.

    “Instruments that are simpler, easier to operate, and affordable are increasingly becoming more popular, particularly in diverse fields outside of immunology,” asserts Melissa Ma, product manager at BioRad’s gene expression division.

    While high-end flow systems are growing as well, instrument personalization has become a target for both labs and instrument vendors. “More researchers are searching for systems they can use in their own lab, that do not require years of experience to run,” Ma adds. Democratization must occur to create opportunities for researchers with diverse expertise levels. The challenge for companies is to design those instruments.”

    “Democratization and spatial diminution are evident throughout laboratory equipment markets—as evidenced by HPLC, mass spectrometry, and other types of equipment—but the evolution toward smaller footprints and price tags is different for every instrument type.

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