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Study Demonstrates Utility of Akonni TruTip Tech to Enrich Fetal DNA for NIPD

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This article has been updated from a previous version to include information provided by a Novartis spokesperson.

Scientists from Akonni Biosystems and Novartis Vaccines and Diagnostics have published research demonstrating that Akonni's TruTip sample prep technology is better than established methods at enriching cell-free fetal DNA from maternal DNA samples for subsequent downstream detection and analysis.

Based on these results — and because the TruTip method is more amenable to automation and working with larger sample volumes — the researchers contend that the method has the potential to significantly improve non-invasive prenatal diagnostic testing, regardless of the downstream detection method used.

As such, Akonni is currently collaborating with a number of undisclosed companies — though not Novartis, which has discontinued its NIPD program — to evaluate TruTip as a sample prep method for NIPD tests based on sequencing, PCR, and other molecular detection techniques, Rebecca Holmberg, director of application development at Akonni and lead author on the study, told PCR Insider.

Interest in NIPD is growing due to its ability to help diagnose potential health risks such as miscarriage and fetal deformations in a more non-invasive manner — requiring only a simple maternal blood draw — than traditional prenatal diagnostic methods such as amniocentesis and chorionic villus sampling.

And although numerous companies and research groups are developing a variety of molecular detection approaches for NIPD, sample prep remains a challenge, Holmberg said.

"The field of NIPD is really growing, and there have been a lot of really exciting advances on the detection side, but there has really been a gap on the sample prep side," Holmberg said. "And these types of samples are really challenging because of the nature of the sample — you have both maternal DNA and fetal DNA present, and you need to distinguish one from the other. Also, a really small amount of fetal DNA is present, so you need to process quite a large volume in order to get enough to analyze on the back end."

Holmberg also noted that, currently, nucleic acid extraction and purification using Qiagen spin column technology is the gold standard for recovery of fetal DNA from maternal plasma. This method, while generally reliable, is difficult to automate and can be confounded by larger sample sizes, both of which could be problematic as the field of NIPD grows.

"There will be more and more samples that need to be processed," Holmberg said. "Companies and research facilities are trying to get ahead of the game and process [samples with] high throughput. Currently, Qiagen is a manual process, and it has its challenges when you try to automate it."

According to Akonni, however, the TruTip technology may be more amenable to such applications. As the company described in a paper published last month in PLoS One, TruTip consists of "a porous rigid silica monolith inserted into a pipette tip that connects to standard laboratory pipettors or automated liquid handling systems. The binding matrix has greater porosity than a typical silica filter, which effectively lowers the backpressure, allowing bidirectional flow and processing of viscous samples such as plasma or blood without the high force of a vacuum manifold or centrifuge."

Furthermore, the sample can be passed across the binding matrix multiple times, increasing the probability of binding and enabling the processing of sample volumes larger than the volume of the pipette tip itself, the researchers noted.

In addition, in the case of fetal DNA extraction, the TruTip method has an extra step that decreases the recovery of DNA fragments larger than 600 base pairs from the sample, thus effectively enriching for smaller molecular weight DNA — in other words, the type of fragmented cell-free circulating fetal DNA often found in maternal blood samples.

In their study, the researchers compared the ability of TruTip with that of Qiagen's Circulating Nucleic Acids Kit to recover fetal DNA from maternal plasma. First, the researchers tested the ability of both technologies to process spiked samples using fragmented DNA to simulate cell-free DNA components and thus assess absolute recovery.

Holmberg and colleagues found that both the TruTip and Qiagen methods were able to efficiently isolate DNA over a range of fetal DNA in a background of female DNA similar to the concentrations found in early gestation. They also observed that TruTip resulted in a higher percentage of fetal DNA than the Qiagen kit in all but two of the 50 samples tested in this first experiment.

Next, the scientists compared the performance of the methods using 19 blinded clinical samples with previously unknown amounts of fetal and maternal DNA, and using whole blood samples that had been incubated up to 48 hours prior to plasma processing, which has been shown to result in increases in maternal DNA due to lysis of maternal leukocytes in the sample.

Here, too, the TruTip method consistently isolated a higher percentage of fetal DNA. Finally, the researchers showed that the extraction product eluents were amenable to both downstream qPCR and droplet digital PCR detection, demonstrating that the TruTip method could be used as a sample prep method in front of various downstream detection methods.

Holmberg said that the group has also demonstrated that the TruTip method works for enriching cell-free fetal DNA from maternal DNA for downstream detection using next-generation sequencing, although the group did not publish data from these experiments in the PLoS One paper.

"It's really widely applicable to whatever downstream detection method you want to use," Holmberg said.

The field of NIPD is relatively new to Akonni, which developed TruTip as a sample prep technology to be used with its own nucleic acid detection platforms, such as its TruDrop gel droplet-based PCR microarray biochip (PCR Insider 3/15/2012, 9/30/2010, and 7/29/2010).

"But in the process of developing the technology, we've found that it's really applicable to any back-end detection technology," Holmberg said. "So it's kind of spun off from there into its own entity. We've seen a need and had people calling about the TruTip technology and asking us if we had a solution to this application [of NIPD]."

One of those early collaborators was Novartis Vaccines and Diagnostics, which up until 2012 had been working with Fluidigm and possibly other technology vendors to develop PCR- or digital PCR-based non-invasive prenatal diagnostics.

However, in May 2012 Fluidigm disclosed that this partnership had dissolved (PCR Insider, 5/10/2012). A Novartis spokesperson this week told PCR Insider that the company is no longer active in the area of NIPD.

Holmberg said that Akonni is "agnostic" right now in terms of pairing the TruTip technology with various NIPD analysis methods, and said that the company is working with "a lot of different collaborators" in this area.

"Sequencing is a main platform that people are pursuing," she said. "Everyone has their own tweak on how they are doing their sequencing for NIPD."

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