It wasn’t that long ago that the concept of a relatively low-cost, benchtop flow cytometer was something of an oxymoron. The ability to quickly and simultaneously query multiple parameters on large numbers of individual cells was generally reserved for shared core facilities or well-equipped specialized laboratories. It required large, complex, expensive instrumentation typically operated by highly trained, often dedicated, specialists.
Today there are numerous instruments on the market that — depending on the your definition of “low-cost” and “benchtop” — fit the bill. These range from toaster-sized analyzers with limited capabilities and closed architecture all the way to multifunctional instruments that can hold their own against almost anything found in a core facility, including the ability to sterilely sort cells into different populations.
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With approximately 17 different companies each manufacturing one or more flow cytometers and sorters, the permutations can seem endless [1]. So, rather than calling out individual instruments and listing their features, this article will discuss the different factors a researcher should consider when purchasing a benchtop flow cytometer.
How many colors?
The first two questions someone is likely to ask about a flow cytometer are: How many lasers does it have? And, how many colors can it detect? The answers are typically closely linked, but they don’t have to be.
Almost all commercially available flow cytometers use one or more lasers to interrogate particles (cells) that flow past the beam at high speed. Fluorophores on or in the cells — either attached to antibodies or endogenous fluorescent proteins such as green fluorescent protein — are excited by the laser’s specific wavelength and emit their own characteristic spectrum that can be detected and interpreted by the instrument. Each particle also scatters the incoming laser beam in all directions, with the amount of forward scattered light (FSC) being an indication of size, and the side-scattered light (SSC) a measure of cellular “complexity” or granularity.
In most lower-priced flow cytometers, fluorescence is detected by a photomultiplier tube with a filter to restrict the bandwidth allowed to hit the detector. Because different fluorophores can be excited by the same wavelength but emit at different wavelengths, it’s not unusual to see, for example, a one-laser/three-color instrument. (If the latter also detected FSC and SSC, as is likely, it would be said to be a five-parameter instrument.)
“Each laser can support X many colors. Typically a red laser—the least expensive—can get three colors, and if you have a blue laser, you can measure up to six different colors,” explains Shervin Javadi, founder and CEO of Stratedigm. “For practical purposes, they’re additive.”
How many is enough?
Calling a flow cytometer “low-end” has a negative connotation, says Mike Olszowy, director of R&D for flow cytometry systems at Life Technologies. An instrument may have fewer lasers or detectors than competing instruments and/or a lower cost, but that doesn’t mean it’s lesser in terms of performance. Many of these still meet rigorous demands for resolution, speed and sensitivity.
Grant Howes, director of marketing for personal flow cytometry platforms at BD Biosciences, says more than 80% of flow cytometry analyses use four colors or fewer—making the two-laser, four-color BD Accuri™ C6 “capable of the vast majority of flow applications.”
But the standard C6, with both a blue and a red laser, isn’t a good match for the BD Horizon™ Brilliant Violet™ polymer dyes the company has been actively promoting for their ability to deliver a brighter signal without increasing background. (For that, a custom system with a 405-nm laser can be special-ordered.) So it’s important, he says, not only to look at how many lasers a system has, but to “make sure the lasers are compatible with the dyes you’re using.”
For instance, Miltenyi Biotec’s three-laser, 10-parameter MACSQuant® VYB (violet, yellow, blue) was designed around a 561-nm yellow laser that’s strong enough to excite a new generation of fluors, as well as "fruit dye" fluorescent proteins, notes product manager Julie Clark.
In addition to the number of lasers, “you probably want more colors so you can choose from a wider selection of different fluors, which allow you to avoid spectral crossover,” adds Olszowy.
All locked up?
Most flow cytometers have an open architecture, enabling the user to run a variety of analyses. This doesn’t mean they are open when it comes to configuration, though. A large proportion of lower-cost instruments are limited in the number of lasers or detectors they can accommodate. Some systems are completely closed: Users have no say over which lasers and filters the system ships with. Others allow a choice upon purchase, but the instrument cannot be reconfigured after it leaves the factory. Still other systems enable users to exchange lasers or filters as a service call, but virtually none have user-exchangeable lasers. In other words, if you think your needs may change moving forward, consider your options carefully.
Other systems do offer an open configuration. Stratedigm, for example, offers a single platform, allowing users to start out with a single laser and two colors for $68,000. “Later, if you want to upgrade and add a second or third or fourth laser, or increase the colors up to 18, you can, as a field upgrade,” says Javadi. “You’re able to build on top of your existing investment.”
Several vendors also offer plate loaders and stackers (hotels), barcode readers, samplers and other accessories that can either be added at the factory or after the fact, as your needs evolve.
How much?
At a list price of $49,000, the Accuri C6 is in the lower-middle range of benchtop instruments, which start at $14,000 for the closed-architecture Muse® Cell Analyzer from EMD Millipore. Most instruments cost $75,000 to $100,000 for a two-laser, four- to six-color instrument, says Olszowy, whose two-laser, six-color Life Technologies Attune® lists for $99,000 in the United States. Large, extremely capable analyzers that also happen to sit on a bench, though, may cost several times that.
Purchasing decisions aren’t based solely on cost, of course, and different instruments boast different appeals. Some of these, such as ease of use, are subjective—is it easier to use an instrument you’re familiar with, one based on a touch-screen, one that is preconfigured or one whose parameters can be user-controlled?
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Other variables are more objective. For instance, some cytometers are very small—like the 8” x 10” Muse, while others take up considerable bench space.
Similarly, instruments may be fully self-contained, with integrated touch-screen user interfaces; others require an external computer or display screen. The fluid and waste containers, too, are sometimes hidden inside the housing of the unit, attached to the side or fitted atop the housing or kept on a “fluidics cart” typically stored beneath the bench.
The MACSQuant line features an on-board magnetic-bead separation column. Using it to look for a rare cell type in a large sample, for example, cuts down the time necessary to read the sample as well as the size of the resulting file, says Clark. “Instead of looking at millions of data points, you can look at thousands.”
Several instruments can provide an absolute cell count. Attune’s acoustic focusing technology, for example, uses direct-drive pumps to impel the cells forward, “which lets you measure volume, and therefore concentration, of the cells without the addition of counting beads,” explains Olszowy.
That acoustic focusing technology also lets almost 10 times more volume be moved through at a given time compared to conventional instruments — about 1 ml/min — meaning dilute samples can run without first being concentrated. And it also allows the cells to be slowed down, “spending more time in front of the laser, which means better illumination of fluorochromes, collection of more photons, and therefore a reduction in variability on the sample,” he adds.
Sort of flow cytometry
Benchtop cell sorters are also available.
Some are fairly modest. Bio-Rad Laboratories' new S3™ Cell Sorter, introduced in June, has two lasers, four colors and retails for about $150,000. It boasts on-board fluidics, a sensitivity comparable to other instruments in the lower-end market, and can sort 30,000 cells per second, comparable even to some higher-end sorters.
Sony Biotechnology’s SH800 Cell Sorter has at its heart a plastic, disposable microfluidics chip ($25 to $40). “Plug it in like a DVD. Then throw it away. The next user can put in a brand new chip that hasn’t seen any other samples, so there is minimal or no cross-contamination from one end user to the next,” points out product manager Deena Soni. Prices start “in the low $200[,000]s, depending on the configuration, hookups, etc.”
Also entering the sorting market is Miltenyi Biotec, whose MACSQuant Tyto, a three-laser, eight-color, disposable, microchip cartridge-based cell sorter, currently is in beta testing, Clark says.
So how to choose? First, decide what you need now and what you’re likely to need in the future. Then, do your legwork, see which systems meet those needs and get quotes. Ask current users for their opinions, perhaps by posting on the Purdue Cytometry Discussion List. Finally, says Javadi, get a demo instrument in your lab and run your own samples and standards. Because ultimately it doesn't matter how many other researchers have had a positive experience with a given instrument; what matters is how it works for you.
Reference
[1] Picot, J, et al., “Flow cytometry: retrospective, fundamentals and recent instrumentation,” Cytotechnology, 64:109-130, 2012. [PubMed]
Image: BD Biosciences' BD Accuri C6 with CSampler option.