NEW YORK (GenomeWeb) – Scientists at the University of California, Los Angeles and their collaborators are working toward a clinical noninvasive prenatal genetic test that relies on fetal cells isolated from the mother's blood or cervix.
In a paper published in ACS Nano today, the researchers, led by senior author Hsian-Rong Tseng, a professor of molecular and medical pharmacology at the Crump Institute for Molecular Imaging at UCLA, showed that they can obtain individual circulating trophoblasts from maternal blood using so-called NanoVelcro microchips and laser capture microdissection, and study them by array comparative genomic hybridization (arrayCGH) and short tandem repeat (STR) analysis.
A number of groups have been working on methods for extracting fetal cells noninvasively from maternal samples and analyzing them for genetic abnormalities. In theory, such tests could provide the same types of results as amniocentesis or chorionic villus sampling, both invasive procedures that are currently the gold standard for prenatal diagnostics.
The advantage over current NIPTs that analyze cell-free circulating DNA from the mother's blood, like those from Illumina, Natera, and Ariosa, is that the test would analyze pure fetal DNA without any maternal DNA mixed in and, therefore, likely be suitable for diagnostics, not just screening. "It would be of diagnostic power," Tseng said.
Art Beaudet, professor and chair of molecular and human genetics at Baylor College of Medicine, said there is great need in the prenatal field for a noninvasive test like this, which would "give you the same quality information you can get from amnio and CVS without doing an amnio or CVS."
However, he said that while some experts, including himself, believe noninvasive fetal cell-based tests will be the answer, others think that with new computational and technical strategies, the same information could be obtained from cell-free DNA.
For their new study, the UCLA researchers captured circulating trophoblasts from the blood of six women with normal pregnancies between eight and 14 weeks of gestation, and nine pregnant women between 15 and 23 weeks of gestation with genetic aberrations in their fetuses, including trisomy 21, trisomy 18, trisomy 13, and tetrasomy X.
They detected between two and six trophoblasts in 2 milliliters of blood from the normal pregnancies and a higher number — ranging from 10 to 25 trophoblasts per 2 milliliters of blood — from the abnormal ones. Those numbers, Beaudet said, are the highest reported so far for this type of assay.
To isolate the trophoblasts, red blood cells were first removed from the blood by gradient centrifugation. The remaining peripheral blood mononuclear cells were added to a NanoVelcro microchip, a patented nanostructured chip carrying a capture agent to trap EpCAM-expressing trophoblast. The fetal cells were then stained with four different agents in order to distinguish them from blood cells and cellular debris. Finally, the researchers used laser microdissection to obtain pure trophoblasts.
For the genetic analysis, they pooled three trophoblasts, performed whole-genome amplification of the DNA, and subjected the genetic material to array CGH or an STR assay.
For the normal pregnancies, array CGH was able to confirm that all fetuses were male, and at least half of the trophoblast STR signatures could be matched to either maternal or paternal STRs. In addition, array CGH detected all chromosomal aberrations in the abnormal pregnancies.
Following this pilot project, the team is now working on improving the reproducibility of the test, which is currently only successful in about 50 to 75 percent of samples, Tseng said, mainly because it is so difficult to fish the few circulating fetal cells out of a background of billions of maternal blood cells.
Another hurdle, he said, is to amplify genomic DNA from single or a few fetal cells evenly.
One possibility to improve the test would be to start with a larger volume of blood, Tseng said, but that would increase the sample processing time. Instead, his team recently switched to using Pap smears, which contain more fetal cells than blood samples, and has been getting "very exciting data" in an ongoing study, he said, obtaining about 300 to 600 cells per sample.
Last year, another team, led by researchers at Wayne State University School of Medicine, published a study in which they retrieved trophoblasts from Pap smears, using magnetic nanoparticles with an affinity reagent, and profiled their DNA for STRs and single nucleotide variants. PerkinElmer has exclusively licensed the method, and a company founded by the researchers, Advanced Reproductive Testing, is working on tests to identify women at risk of placental disorders.
Critics have said that it is unclear whether the Pap smear procedure might increase the risk of miscarriage, but Tseng said it is at least less invasive than amniocentesis or chorionic villus sampling. A study his team is currently conducting, in collaboration with the Cedars-Sinai Medical Center in Los Angeles, the department of obstetrics and gynecology at UCLA, and Cathay General Hospital in Taiwan, is analyzing Pap smears taken from women scheduled to undergo elected abortions, so an increased miscarriage risk is not a concern.
Pap smear samples, he said, could be regarded as a kind of "stepping stone" until methods sufficiently improve to collect fetal cells from blood samples.
In addition to enhancing fetal cell isolation, the researchers are also working on increasing throughput, using high-speed imaging and machine learning to locate and recognize fetal cells, and automated dissection methods to retrieve them. As of now, they can process 10 to 20 samples per day, Tseng said.
Turnaround time is currently about 48 hours, from sample processing to analysis by array CGH, which he said is faster than many cell-free NIPTs.
UCLA has licensed the NanoVelcro technology for fetal cell isolation to FetoLumina Technologies, a spinout founded by Tseng and his colleague Tom Lee that plans to offer the test as a clinical service within a year. The company is currently working with undisclosed partners in the US, China, and Taiwan to develop the test, Tseng said.
The cost of the test will likely be high at the beginning, he said, and drop over time with technology improvements.
Another startup Tseng cofounded, CytoLumina Technologies, focuses on developing the NanoVelcro technology for isolating circulating tumor cells, which uses different cellular markers but is otherwise similar to retrieving trophoblasts.
The major difference between the two applications, Tseng said, is that in prenatal diagnostics, clinicians already know how to interpret and utilize the genetic information, whereas in oncology, that is not always the case, especially when it comes to genome-wide data.
A potential competitor for FetoLumina is Baylor Genetics, he said, which has been developing its own noninvasive fetal cell test.
Last year, the Baylor team, led by Beaudet, published two articles in Prenatal Diagnosis — one in collaboration with RareCyte, the other with Arcedi Biotech in Denmark — in which they showed that they could use arrayCGH and whole-genome sequencing to detect chromosomal abnormalities in individual trophoblasts isolated from maternal blood.
"We're in a similar situation," Beaudet said. "We've shown it can work but we haven't launched a test."
Similar to the UCLA group, the main problem has been to retrieve sufficient fetal cells from maternal blood samples consistently, he said, and it is difficult to predict when Baylor Genetics will be ready to launch the assay as a clinical test.