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Fetal Cell-Based NIPT by Droplet Digital PCR Demonstrated in Japanese Pilot Study

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NEW YORK – Researchers in Japan have shown that they can analyze genetic features of single circulating fetal cells in maternal blood without the need for isolating the cells or amplifying their genomes, potentially opening the door to fetal cell-based noninvasive prenatal diagnostic tests.

The technology, described in a paper in the Journal of Molecular Diagnostics last week, uses single-cell droplet digital PCR to analyze maternal blood cell populations that have been enriched for fetal cells by magnetic-activated cell sorting (MACS).

In their proof-of-concept paper, the scientists, led by Kenichiro Hata, chairman of maternal-fetal biology at the National Research Institute for Child Health and Development in Tokyo, demonstrated that they were able to detect SRY, a gene on the Y-chromosome, in single cells from blood samples of three pregnant women carrying a male fetus but not in samples from 10 women carrying a female fetus. All women were in their second or third trimester of pregnancy.

"In the future, by optimizing cell sorting and encapsulation as well as generating a more effective PCR environment in each droplet, this modified sc-ddPCR system may be a breakthrough analysis method that can be applied to various research realms, and possibly to clinical diagnostic testing," they wrote.

Most noninvasive prenatal DNA tests today analyze cell-free fetal DNA, which is mixed in with large amounts of maternal cell-free DNA in the mother's blood. However, it has long been known that a very small number of fetal or placental cells — an estimated fewer than 10 per milliliter — circulate in the blood of pregnant women. Being able to analyze their genetic material separately, without the background of maternal DNA, could potentially yield more accurate test results across the genome. However, it has proven extremely difficult to pick out and isolate cells of fetal origin from millions of maternal cells in each milliliter of blood.

"Cell-based NIPT would be a real game changer for prenatal diagnostics since it would enable the test to become diagnostic instead of a screening method," Lisa Jensen-Long, VP of marketing for the digital biology group at Bio-Rad Laboratories, said in an email. Jensen-Long was not involved with the study, but the droplet digital PCR technology used in the study is marketed by Bio-Rad.

Recently, researchers have made progress in getting to these fetal cells, though. In a study published in December, for example, a team from Baylor College of Medicine and Columbia University showed that they could detect and isolate trophoblasts, which originate from the placenta, in the blood of almost all of around 100 pregnant women, and sequence their DNA after whole-genome amplification. A Baylor spinout, Luna Genetics, now plans to develop clinical tests to analyze fetal cells from maternal blood for serious genetic conditions.

For their own study, the Japanese researchers bet on detecting genetic features in fetal cells without completely isolating them from the other blood cells, which usually requires the cells to be stained or fixed. Instead, they crudely enriched the fetal cells by MACS, collecting only those cells that don't express CD45 or CD14, which includes fetal cells.

They then analyzed DNA from individual cells, without amplifying it, using Bio-Rad's QX200 ddPCR system, which they had previously employed for a different type of single-cell analysis. Specifically, they used one reference probe, RPP30, and one Y-chromosome-specific probe for the SRY gene. One advantage of using that system, they wrote, is that it can analyze up to 3,000 individual cells per well.

One issue they encountered was that the crudely purified cell populations they analyzed contained some contaminants. Decreasing the amount of polymerase in the PCR reaction, they found, increased the signal they obtained from both probes together, thus increasing the sensitivity of their assay.

When they applied their test to cell fractions from 13 maternal peripheral blood samples, they found that it correctly called only the three samples from women with a male fetus as SRY-positive and all 10 samples from women with a female fetus as SRY-negative.

The hope is that other genes could be detected in fetal cells in a similar way by this approach. "Furthermore, not only copy numbers but also single-nucleotide alterations may be technically assessed at a single-cell level with this sc-ddPCR system," the researchers wrote.

While the Japanese study is showing proof of concept for fetal cell-based NIPT, "further work is required for optimization and confirmation with adequate controls along with a broader data set," Jensen-Long said. "It is also worth noting that fetal single-cell analysis would require the ability to assess the number of copies on a per-cell basis," she added, whereas the approach detailed in the study only determines whether or not cells possess a particular DNA target.

"The Bio-Rad QX200 system is ideally suited to perform single-cell analysis, and this is one of the publications that demonstrate how single cells can be counted using digital PCR technology," she said, adding that Bio-Rad itself is developing NGS and ddPCR solutions for the gene therapy single-cell market.

"The main limitation of this approach is that only one or a few genomic loci can be examined at one time," said Arthur Beaudet, CEO of Luna Genetics and the former chair of molecular and human genetics at Baylor College of Medicine, in an email. While it could be used to detect monogenic disorders in pregnancies with a known risk for such a condition, "it would not be suitable for genome-wide copy number analysis," he said.

He also pointed out that the samples the Japanese team analyzed were collected late in pregnancy. "It is not clear if there are sufficient fetal cells early in pregnancy to be successful with this approach," he said. In addition, he noted that the exact cell type analyzed using this method is not determined. 

In the meantime, others have pursued digital-PCR-based approaches for noninvasive prenatal testing. For example, BioCore of Korea in 2018 launched a test for fetal trisomy 21 that runs on the Bio-Rad QX200. However, that test analyzes cell-free DNA, not DNA from single cells.

Also, Bio-Rad said last year that it was seeing opportunities for its QXDx system, the clinical version of the QX200, in NIPT and early infant testing.