NEW YORK – Releasing an updated model of its flow cytometry system last month, Cytek Biosciences plans to offer researchers the ability to multiplex 40 fluorescent biomarkers on a cell in a blood sample for research and clinical purposes.
While the Fremont, California-based firm believes that the updated Aurora platform, which uses five lasers and 64 detectors to analyze fluorochromes in a single tube, will minimize human error and costs for spectral analysis, some experts are hesitant to use the platform in cell-based research.
Flow cytometry aims to count the number, size, granularity, and other properties of cells in a heterogeneous population. Standard flow cytometry lasers excite certain fluorescent markers (fluorochromes, antibodies, or stains) on a cell as it passes through the beam. Detectors in the instrument record and quantify the relative amount of light emitted by fluorescent markers in the cell, which the tool presents to researchers through a histogram.
However, flow cytometer users can run into a myriad of technical issues, such as dealing with limited sample volumes and lacking enough lasers to excite a target amount of fluorochromes. Each laser also can only excite a certain number of fluorochromes on a cell before inducing spectral overlap.
"On an 18-detector instrument, you're only probably testing about 12 to 14 colors … before the dyes stop playing well together," David Adams, director of the University of Michigan Flow Cytometry Core, explained. "For example, a violet laser has 16 detectors [associated with it], while a red laser uses eight detectors because there's only so much useful spectrum for fluorescent detection."
According to Cytek CEO Wenbing Jiang, the updated Aurora platform uses five optical lasers (ultraviolet, violet, blue, yellow-green, and red) to excite 40 fluorochromes on cellular antibodies, which are then recorded by 64 detectors.
"This allows us to use many fluorochromes of dyes on the market that our competitors cannot use," Jiang said. "We've also developed our own unique, special dyes internally that match [commercially available] dyes, plus our internal dyes developed specifically for the Aurora platform."
Jiang said that several of Cytek's users apply the Aurora for immunological research purposes. For example, he noted that some research groups have used the currently available four-laser Aurora platform to identify mutated cells in blood samples from leukemia patients.
With standard flow cytometry panels, a patient's blood sample must be separated into multiple tubes to identify biomarkers linked to different types of leukemia, Jiang said, noting that Aurora only needs a single tube of blood to identify the fluorescent antibodies. While researchers still need to run controls prior to running a multicolor tube to measure the different emission spectra recorded by Aurora, Jiang said that they can save the controls in the software and reuse them with the same panel in future tests.
"After chemotherapy, no one really has that many bone marrow samples available for testing and splitting into several different tubes," Jiang said. "But because you don't need to split blood samples into several tubes with Aurora, you can have more cells per tube, which leads to more specific results."
Jiang also argued that the updated Aurora system can analyze up to 30,000 to 40,000 cells per second while maintaining a "competitive sensitivity." Flow cytometry platforms on the market currently offer anywhere from 20,000 to 100,000 cells per second, but not always at the sample level of multiplexing.
While Cytek has only demonstrated the capability of the updated platform internally, the firm is partnering with undisclosed cancer centers across the US, Europe, and China to validate the system in future studies.
"While some cancer centers have been using [Fluidigm's] CyTOF system, we believe that with our technology, they can convert their existing panels into a flow cytometry platform," Jiang said. "The problem with Fluidigm's mass cytometry is that it's expensive, cells are destroyed, and it's difficult to prepare samples."
According to Steven Porcelli, scientific director at the Albert Einstein College of Medicine Flow Cytometry Core, flow cytometry is often used by researchers who perform basic immunology research in a variety of different diseases, including primary immune system diseases (rheumatoid arthritis or lupus) and cancer. Porcelli highlighted that flow cytometry has also rapidly become an important tool for cancer immunotherapy development.
Porcelli's team at Einstein has applied the earlier version of the Aurora platform to study lymphocytes that infiltrate tumors in animals grafted with cancer to better understand the details of individual immune responses.
"Cytek's tool collected light coming out over a wide range of wavelengths for each cell and for each laser that we've used to excite the cell," Porcelli explained. "Instead of giving you a high or low value for a tracer, it allows you to distinguish many different tracers from each other because you create a kind of fingerprint of the wavelengths being emitted."
Porcelli also said that Aurora allows researchers to label more biomarkers on an individual cell and distinguish them from each other. If a machine allows a user to test 20 biomarkers, he argued that researchers could easily figure out how to push the technology to its current limits and reach up to more than 30 colors.
"This might not sound like a lot, but when going from 13 to 20 or 30 colors, you're talking about a vast expansion in terms of the complexity of the dataset," Porcelli explained. "If you have the right kind of analytical tools, then the info derived can lead to strong conclusions for downstream [applications]."
An academic flow cytometry expert at a US core facility who wished to remain anonymous due to a conflict of interest involving other vendors in the space, however, has certain misgivings about Aurora's capabilities. The source, who has attended several Cytek workshops at vendor shows and independently tested the earlier version of Cytek on liquid samples, has chosen not to use the technology at the flow cytometry core.
The expert said that Aurora's method to process and analyze collected information differs from traditional flow cytometry methods. Arguing that Cytek offers two to three algorithms that researchers can use to process the collected data differently, the source is uneasy about the potential ambiguity and biased results the tool might produce.
However, Jiang said in an email that the source might be confusing the firm with another manufacturer of spectral cytometers, which offers different unmixing algorithms to unmix the data. In contrast, Jiang said that Cytek only uses one spectral unmixing algorithm — called "ordinary least squares" — to unmix the spectral data.
The source also claimed that when Cytek launches longitudinal studies with large groups of samples with different institutions, the firm applies very specific controls and dyes to validate the instrument's results. The source said that certain academic groups have consequently reported that they cannot compare data produced by Aurora with older data produced by standard flow cytometers.
If researchers want to begin a new study using Aurora, the anonymous source argued that they can therefore only compare data with existing data produced with the updated flow cytometry system.
Jiang acknowledged that because Aurora can support parameters compared to conventional systems, panels developed on Aurora "leveraging the tool's more advanced capability may not perform" on conventional systems. However, he argued that the same controls used for a conventional cytometer can be used on Aurora, and that the firm has several references who have had no trouble comparing data using the same sample on the Aurora versus a conventional cytometer.
Cytek has several well-known and up-and-coming competitors in the flow cytometry market, and both the anonymous source and University of Michigan's Adams noted that these platforms each have their own niche advantages.
Becton Dickinson sells its line of FACS research cell analyzers, which Adams said are "agile instruments" that will work with a variety of samples regardless of the group's experimental goal. BD's website notes that researchers can use the tools for both clinical and research applications.
Danaher's Beckman Coulter offers its three-laser CytoFlex platform, which can look at up to 13 fluorochromes per run. Adams said the tool provides "very clean signals" and offer high sensitivity for research applications.
Sony sells its SP Spectral Analyzer multi-laser series of instruments, which Adams said are easy to operate and can run a similar number of fluorochromes as Aurora without using a complicated compensation matrix. The firm's site notes that its ID-7000 spectral analyzer can be configured with up to seven lasers and 188 detectors, allowing researchers to experiment with 44 colors.
"Once Cytek's Aurora system came out, Sony announced a 44-color spectral analyzer," Adams said. "It's good to see that they're pushing the technology so that Cytek and Sony can compete."
However, Jiang argued that Cytek's system is a powerful and cost-effective tool because researchers can run several fluorochromes in a single tube, instead of requiring multiple diluted liquid samples.
"When researchers use multiple tubes to characterize cells by pipetting samples into them and trying to ensure the same percentage of samples in each, this is both expensive and adds the potential for human error," Jiang said.
Jiang also emphasized that most systems capable of running higher complexity panels start at $500,000 and can get close to $1 million, in addition to requiring specialized reagents. In contrast, he claimed that Cytek's spectral analyzer "costs a fraction of that price," despite declining to provide a specific price for the updated system. He believes that anticipated cost savings will not only stem from Aurora's hardware, but also in "time savings for operators, reagent costs, and by removing human error."
While no issues inherently exist by increasing the number of lasers and detectors on a spectral analyzer, Adams said that additional capabilities can encumber a user with developing the appropriate experimental design in a study. Applying complicated versions of a system on cell populations can therefore potentially lead to the risk of using the machine improperly and producing bad data.
"That's why in small labs, you tend to see low to mid-level instruments, but shared resource labs tend to have high-capability instruments, not only because they're more expensive, but there's a higher level of expertise needed to guide users through higher-parameter experiments," Adam said.
The anonymous source, however, believes that extensively training an individual to use Aurora properly would minimize the risk of making mistakes with the instrument. The expert argued that a larger group classically educated on standard flow cytometry instead might increase the risk of producing unnoticed errors using Cytek's system.
Einstein's Porcelli said that his lab does not plan to solely use the current Aurora system as its only research tool, but noted the team will spend time understanding how to use Aurora effectively and maximize its full utility before upgrading to the updated 40-color version.
"When you purchase a new [flow cytometry] tool, you need to figure out its maintenance cost, then ask what supportive services you need with it to actually work," Porcelli said. "If you know you're getting vast amounts of data, but you don't have analysts who can help you process it, you might as well not have the tech."
Porcelli also emphasized that his group requires time to assess new technology and that it moves a bit slower than commercial users until "we're pretty sure that the [flow cytometry platform] will be worth the investments because they are considerable for us."
The anonymous source, on the other hand, said that it has chosen not to incorporate Aurora in the lab for research use. While believing that Cytek's advanced algorithms and technology may theoretically work, the source will wait to see what happens when researchers attempt to validate the updated Aurora platform in peer-reviewed studies.