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Prenatal diagnosis

Microdroplets for safe and rapid prenatal diagnoses

Although the methods used to carry out amniocentesis are quite sophisticated, there is still a 0.5 percent risk of miscarriage following the intervention. Therefore, an EU-funded project called AngeLab is developing a rapid test that only requires a blood sample of the mother rather than amniotic fluid. The test yields information on the genetic health of the foetus within only a few hours. As part of the project, researchers from the Hahn-Schickard site in Freiburg have developed an innovative droplet PCR system which enables the non-invasive analysis of foetal DNA from maternal blood. Droplet PCR involves the fractionation of a DNA sample into several thousand tiny droplets, and subsequent PCR in each individual droplet. The DNA can thus be analysed rapidly and without any risk to the child.

Amniocentesis is a medical procedure during which a thin hollow needle is inserted through the abdominal wall of a pregnant woman into the amniotic sac with the foetus in order to extract amniotic fluid. The procedure is carried out under constant ultrasound guidance. Amniocentesis can nevertheless lead to complications, for example through the loss of amniotic fluid or the incomplete closure of the foetal membranes which can trigger contractions resulting in miscarriage in about 0.5 percent of cases. Amongst other things, the amniotic fluid contains foetal cells, which can be cultivated and analysed for potential genetic defects. This procedure usually takes several weeks before the results are available and it goes without saying that the intervention itself as well as the subsequent waiting period are very stressful for the women involved.

Dr. Nadine Borst and her group of researchers at Hahn-Schickard in Freiburg have developed an innovative droplet PCR for application in prenatal diagnostic testing. © Hahn-Schickard

The EU-funded AngeLab (A New GEnetic LABoratory for non-invasive prenatal diagnosis) project was launched in 2012 to improve this situation. AngeLab brings together 15 partners from all over Europe with the aim of developing safe and efficient non-invasive prenatal tests that do not interfere with the mother’s body, and so prevent damage to the unborn child. The large-scale project also involves scientists from the Hahn-Schickard research institute in Freiburg. AngeLab seeks to develop in vitro methods that enable the diagnosis of genetic diseases in foetuses from the mother’s blood, i.e. without the need of amniotic fluid. The blood of a pregnant woman contains both maternal as well as foetal DNA. The cell-free foetal DNA fragments can be isolated using special methods.

DNA analyses: quickly and safely from maternal blood

In order to address the ambitious research objective, AngeLab was divided into three parts. The developed systems are modular and can be combined with each other depending on the application for which they are used. In the first and second parts, the European project partners focused on the development of methods with which foetal DNA can be extracted from the mother’s blood and subsequently be analysed. This work led to two lab-on-a-chip systems, one that uses magnetophoresis to automatically extract foetal DNA, and another one that uses immunoprecipitation. The researchers from Freiburg were involved in the third part of the project, which focused on the development of an effective digital PCR procedure using the DNA samples extracted with the methods developed in the first two parts to detect cystic fibrosis or determine the foetal rhesus factor within a few hours.

The Hahn-Schickard researchers, now led by Dr. Nadine Borst, have successfully designed an innovative digital droplet PCR method, with which it is possible to fractionate a DNA sample into thousands of tiny microdroplets. This has the advantage that rare components can be detected and quantified more easily than using other PCR methods. In November 2016, the AngeLab project was awarded the European Union’s Innovation Award.

Disk creates thousands of microdroplets

The Freiburg researchers' droplet PCR involves a rotating plastic disk called LabDisk. The microstructures on the disk form an inlet chamber that is connected by a fine channel with a larger reaction chamber, where DNA amplification and readout take place. As the term implies, droplet PCR relies on droplets that need to be created before the DNA samples can be analysed. To generate these droplets, the disk is first loaded with fluorinated oil. Afterwards the aqueous reaction mixture, which includes the DNA sample, is introduced to the inlet chamber. Through centrifugation around 7.500 droplets from that ideally contain only one DNA molecule. PCR is performed in each individual droplet using the DNA molecule inside as a template. “We use specific primers and probes for amplification and signalling,” said the biochemist. “A microarray scanner is used to read the disks. Positive and negative droplets can be identified from the intensity of the fluorescent signal.” Software programmes are used for analysis. Some manual steps are still necessary, but the researchers are working to achieve a fully automated process.

LabDisk (on the left-hand side of the image) and DropChip (on the right-hand side): microfluidic cartridges used for non-invasive prenatal diagnoses. © Hahn-Schickard, Photo: Bernd Müller

“The DNA molecules are distributed randomly during the droplet generation process. Depending on the number of DNA molecules that are present in the sample, some droplets might contain more than just one DNA molecule,” said Borst. “However, as long as positive and negative droplets are present, statistical methods can be used to calculate the absolute count of DNA molecules. The determination of the absolute count without the need for other reference samples is a big advantage of digital droplet PCR over other PCR techniques.”

In addition to the LabDisk, the researchers have also developed a further microfluidic cartridge called DropChip. The plastic chip has the shape of a microscope slide, and can therefore be used for analysis with standard laboratory devices. “With each DropChip, we can process two samples in parallel. The LabDisk can process up to eight samples or additional operations could be integrated,” said Borst. The entire process from the generation of the droplets to PCR readout takes around two hours and thirty minutes.”

Droplets are generated by way of a tear-off mechanism

The idea of using droplets stems originally from Dr. Friedrich Schuler who invented the method called “centrifugal step emulsification” at the Hahn-Schickard research institute a few years ago. An inlet chamber is connected to a reaction chamber by a microfluidic channel. A backward facing step, known as terrace, is located at the junction of the channel and the reaction chamber. The disk is loaded with oil and the aqueous reaction mixture is introduced to the inlet. Centrifugation forces the mixture to flow through the channel and droplets tear-off at the backward facing step. “The process is very reproducible, resulting in thousands of droplets of the same size,” explains the Freiburg researcher. “The break-up mechanism involving step geometries has been known for quite some time. However, our method is the only one that relies solely on the centrifugal force created by the rotating disk. The method, which involves only a short centrifugation time, is therefore very robust and easy to use.”

Development of other rapid tests

EU funding will end in June 2017. For the three project parts, samples of around 400 pregnant women were collected to verify the results of the new tests. Two companies, NIPD Genetics (CY) and DNA-Data (SP), are involved in the project and want to commercialise the systems. “After completion of the project, we hope that the devices will be turned into marketable commodities,” said Borst. “However, before the rapid test can be put on the market, we will have to seek marketing authorisation according to the relevant guidelines.”

The researchers at the Hahn-Schickard research institute in Freiburg are already working on other projects that might benefit from the results obtained by AngeLab. “The technology is available, and we now want to develop it further, for example for use in isothermal amplification methods that are used in the diagnosis of HIV or for the detection of resistant hospital pathogens,” added Borst. In addition, the researchers are working on the development of devices with which the complete analysis process can be performed fully automatically.

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