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Leveraging Microfluidics Experience, Formulatrix Set to Launch First Digital PCR System

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This story has been updated from a previous version to correct information about the QuantStudio 3D consumables and to add pricing information.

Formulatrix, a Boston-area company known primarily for its protein crystallization and liquid-handling products, is gearing up to launch its first PCR product — an inexpensive, plate-based digital PCR platform — at the end of this month.

The product, called the Constellation digital PCR system, uses 96-well plates with each well containing 498 partitions, enabling users to conduct digital PCR with a relatively small number of reaction volumes but very high sample throughput, or a much larger number of reaction volumes at lower throughput, depending on the application.

The company believes that the platform's sweet spot will fall somewhere in between the types of applications enabled by droplet-based digital PCR systems and standard quantitative PCR, with copy number variation assessment topping the list of ideal applications, Kabir Yamana, a product manager at Formulatrix, told PCR Insider.

"It seems like one of the big issues with digital PCR in general is that it's been a lot more work than qPCR to set up the reaction, and so hopefully ours will be a solution to that, enough that maybe people doing normal qPCR would want to switch," Yamana said. "What we are going for is to bring digital PCR data to the qPCR people."

Established in 2002 primarily to commercialize automated protein crystallization products, Waltham-based Formulatrix eventually expanded into automated liquid handling, leveraging a proprietary microfluidics diaphragm pumping technology.

The Constellation digital PCR system is the company's first foray outside of those markets, Yamana said.

"It's not using the exact same microfluidic technology, but we used some of our other microfluidic experience," Yamana said. "And we have imaging experience from the [protein] crystallization [business], so that's where all the [digital PCR] instrument technology came from."

First previewed at the American Society of Human Genetics meeting in Boston in October, the Constellation platform consists of a specially designed microfluidic plate and a benchtop analysis instrument. The consumable plate contains 96 microwells, each containing 496 individual partitions. Once the sample and PCR reaction mixture are pipetted into the input wells and the plate is sealed, the user places the plate into the benchtop instrument's priming chamber, which automatically loads the individual partitions with reaction mixture. A roller then compresses the bottom seal and blocks off the connecting channels, isolating individual copies of target DNA in the partitions.

The microplate can then be thermocycled using any commercial flat-block thermal cycler, thus amplifying target DNA molecules. The partitions containing target DNA fluoresce and the microplate can then be imaged using the Constellation instrument, which has built-in software to count the number of positive reactions.

According to Yamana, a user could investigate a single sample per well, which would allow them to look at 96 samples per plate with a low density of individual PCR reaction volumes. Or, they could use multiple wells for individual samples, thus increasing the density of individual reaction volumes to as many as 47,616 per plate.

"It really depends on what you want to get out of it," Yamana said. "For the really high-throughput people who don't need as much precision, you could do just a single sample per well, so 96 different samples. But if you had some really high copy number you were trying to detect, something like five or six, and you were worried that you wouldn't get the precision based on the statistics of only having 496 wells, then you could do replicates to improve your precision there. It's flexible in that way."

Initially the company envisioned customers running 96 individual samples at a high throughput for situations where copy number varied between zero, one, or two — "like zyogsity," Yamana said. "That was the original application that we started with, but of course you can use it for many other things."

Architecturally speaking, the Constellation system is most akin to Fluidigm's BioMark HD platform or EP1 system and associated integrated fluidic circuit chips. However, according to Yamana, Formulatrix expects to be competitive on price and throughput. "You can run a lot of our plates relatively quickly, and the system cost is not very high," Yamana said. "I think the BioMark is pretty expensive and out of the range of most people doing qPCR."

Formulatrix, he said, is targeting a cost of about $50,000 for the Constellation instrument and about $75 per 96-well microplate, with quantity discounts on the microplate.

In an email to PCR Insider, a Fluidigm spokesperson said that Fluidigm's BioMark HD platform cost depends on configuration but usually is in the range of $220,000 to $250,000. However, he noted that the system can perform quantitative real-time PCR, gene expression, single-cell gene expression, SNP genotyping, protein expression, copy number variation, and sample quantification for sequencing in addition to digital PCR.

Fluidigm also offers two types of integrated fluidic circuit chips — Digital Array IFCs and Quantitative Real-time Digital PCR IFCs. These chips cost between $200 and $1,000 each, depending on how many are ordered at a time and what reagents are involved, he said.

Another platform similar to the Constellation is the QuantStudio 3D, a chip-based, endpoint digital PCR platform from Thermo Fisher brand Life Technologies. In a bid to drive more widespread adoption of digital PCR, Life Tech launched the platform in late 2012 with an approximately $30,000 price tag. As of the publication of this article, the QuanStudio 3D has an online list price of $39,000 to $44,000, which, according to a Thermo Fisher spokesperson, includes the instrument plus a choice of kits with varying amounts of consumables, chips, and auxiliary hardware. Meantime, the system's QuantStudio 20K Digital PCR chips cost about $100 for a package of 12, or around $8.50 per chip, the spokesperson said.

Again, Yamana said he believes the Constellation will compete on price (in terms of cost per sample analyzed) and especially ease of workflow.

"[Life Tech's] workflow is a little bit more difficult," he said. "Each chip does a single sample, and it's actually … a good amount of work to set up each chip. It does have 20,000 partitions per chip, but it's very much for low throughput … you wouldn't want to do 96 samples with that."

On the other side of the digital PCR spectrum are droplet-based platforms from companies' such as Bio-Rad and RainDance Technologies. The power of these systems lies primarily in the enormous number of individual reaction volumes they can create, making them ideal for applications such as rare allele detection and copy number variation — but only when there is a variation of more than zero, one, or two gene copies, Yamana said.

"What we are going for with this product is not as much to compete with Bio-Rad and RainDance," he said. "There are definitely applications — trying to detect really rare targets — where more partitions is better. But in cases where you're talking about precision, like if you're trying to measure the concentration of a particular target, you pretty quickly run up to a limit on how precise your upstream steps are."

For example, he said, "let's say you want to measure the concentration of a certain sample of DNA. Typically when people are pipetting you get a [coefficient of variation (CV)] of around 5 percent. Basically that's the precision when people are pipetting. And that basically is the lower limit on your precision that you can measure … because of these upstream pipetting steps."

For an instrument like Bio-Rad's QX200, "that statistical, sort of optimal precision is about 0.5 percent, which is awesome, but you're never going to use that 0.5 percent, because none of your other processes are that precise."

He said that using appropriate internal controls and stringently optimizing the assay will help take care of these issues, but "there are very few sort of copy number variation situations where you need under 0.5 percent. According to our internal research, the majority of people doing qPCR are doing things that are not as stringent."

Essentially, he summarized, most everyone currently does qPCR, and digital PCR is right now a niche application where, "if you have this really rare target, you really can't get your data any other way. But there are a lot of cases where you can get data with qPCR, and you might want to get even better data with digital PCR, but you're not willing to sort of sacrifice the throughput and ease of use and cost that it would take to switch to, say, the RainDance system."

Besides copy number variation, Formulatrix believes that the Constellation will be useful for many of the other applications for which digital PCR has become generally useful. These include library quantification upstream of sequencing and methylation analysis.

"We haven't really narrowed it down to a few applications, but we're looking," he said. "I think gene expression is another one when people want to detect smaller changes in expression. We haven't really been focusing on a specific application. We've been asking people to come to us and bring us their applications."

Formulatrix is currently running samples in house from external collaborators, but it is not planning to put beta systems of Constellation out in the field. Instead, the company hopes to commercially launch the platform at the end of this month. "Generally what we've done with other products is commercially launch them, and the people who buy it early get the option to return it or, if we make substantial improvements, get an upgrade … so they're not penalized for being early adopters."

And, Yamana said, the company is already working on developing different plate architectures to give potential customers more flexibility in the way they run their digital PCR experiments. "We're going to be increasing our density as a continuous project," he said. "We might have a higher number of wells in the future. And we're considering other formats, so if people want to do 24 wells or 16 wells — maybe with a higher number of partitions but not as high a throughput."

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