Noninvasive Screening for Cytogenetic Disorders (Fetal Aneuploidy Including Microdeletions)

Trophoblast cells

Trophoblasts were the first fetal cell type to be detected in the maternal circulation but present challenges to their use for noninvasive prenatal diagnosis. First, few highly specific antibodies are available for isolation and enrichment. Second, these multinucleated cells are not easily amenable to standard cytogenetic techniques, such as FISH, but require molecular techniques such as chromosomal microarray. Third, because they are placental in origin, there is a 1% incidence of confined placental mosaicism similar to that seen with chorionic villus sampling. Despite these difficulties, contemporary studies continue to show their potential value.

Paterlini-Brechot’s group reported the genetic diagnosis of 63 fetuses at risk for either cystic fibrosis (CF) or spinal muscular atrophy (SMA) using circulating trophoblast cells. Recovery of trophoblast cells was performed by the initial isolation of epithelial tumour/trophoblastic cells (ISET) by size using a calibrated filter. Cells were subsequently laser dissected off the filter and genotyped using maternal and paternal short tandem repeats (STRs) to confirm those that were trophoblast. Of the cells greater than 15 μM in size selected by filtering, half were confirmed as fetal. These cells then underwent specific diagnostic testing. Approximately, 1.5 cells per millilitre of maternal blood were present, and approximately 5 to 10 cells were analysed per case. All fetuses affected by CF or SMA were correctly diagnosed and confirmed by chorionic villus sampling (CVS).

Further evidence of the potential value of trophoblast recovery was presented by Hatt and colleagues using specific markers for cells most likely belonging to the endovascular subgroup of extravillous trophoblast (EVTs). Expression of the endothelial/vascular marker of these originally ectodermal cells is the result of adaptation to their vascular environment. Fetal cells were isolated from maternal blood (gestational age, 11–13weeks) using magnetic cell sorting enrichment (with a novel antibody combination for the endothelial/vascular markers CD105 and CD141). Trophoblast cells were demarcated using a cocktail of cytokeratin antibodies. In 85% of the samples, it was possible to obtain X and Y signals by FISH for gender determination with a 91% specificity. Concordance to fetal sex of 100% was possible if three or more fetal cells could be found in a sample.

Recently, Beaudet and his group developed methods for the detection of chromosomal and subchromosomal abnormalities in fetal trophoblast cells in maternal circulation using array comparative genomic hybridisation (CGH), next-generation sequencing (NGS), or both. They first separated nucleated cells from maternal blood collected at 10 to 16 weeks’ gestation by density fractionation and then immunostained them to identify cytokeratin-positive and CD45-negative trophoblasts. Array CGH, NGS, or both was used for analysis after whole-genome amplification and genotyping and identified normal fetuses and fetuses affected with Klinefelter syndrome; trisomies 21, 18 and 13; and chromosome 15 deletion syndrome.

The presence of trophoblast cells in cervical mucus has also been reported, first by Shettles in 1971 using quinacrine mustard fluorescent staining for identification. The cells were readily identified by their morphology and unique immunohistochemistry using specific antibodies. However, since then, identification of fetal cells in cervical mucus has been inconsistent with detection rates between 50% and 60%.

More recently, Bolnick and colleagues published results on 56 women between 5 and 20 weeks of gestation in whom cervical specimens were collected using a cytobrush and human leukocyte antigen G trophoblast-specific antibodies to identify cells. The average number of cells recovered per patient was 746 ± 59 across the gestational ages. There was minimal maternal cell contamination, and 95% to 100% of the cells isolated expressed confirmatory fetal-specific parameters. Nonetheless, to date, technical challenges have prevented this technology from developing into a clinical tool. Although some groups are still exploring this approach, none has consistently demonstrated success.

Fetal nucleated red blood cells

Fetal nucleated red blood cells have been detected in the maternal circulation and have a relative short half-life, making them specific to the current pregnancy. This has led to their evaluation as a target for prenatal diagnosis. The advantages of this cell are that they can be identified through a marker profile which is characteristic for erythroid precursor cells and which varies from other blood cell subpopulations. For example, fNRBCs express the transferrin receptor (CD71) on their cell surfaces and do not express CD45 as do maternal leukocytes.

Stringent isolation criteria are required to isolate and identify the fetal cells which are present at a concentration of 1 in 10 ⁵ to 10 ⁷ maternal nucleated cells. After initial staining with cell-specific markers, enrichment of fetal cells most often uses immunomagnetic or flow cytometric cell separation techniques, either alone or in combination. Other approaches attempted include separation by centrifugation using a Ficoll gradient, filtration on a chip, lateral displacement and magnetophoresis, lectin-binding, dielectrophoresis, micromanipulation, laser microdissection and pressure catapulting. This relatively large assortment of approaches shows the lack of consensus on the single best approach for isolation.

Confirmation of the fetal origin of the cells is required after enrichment because maternal immature nucleated RBCs are present and can express markers similar to those on fetal cells such as the transferrin receptors. Immune labelling with embryonic or fetal globin antibodies followed by selected cell dissection or capture is presently the most frequently used approach.

Initially, cytogenetic techniques such as FISH were used for cytogenetic evaluation but proved difficult because of the cellular disruption that occurred during separation and isolation. More recently, molecular approaches which only require a small amount of fetal DNA have proven more efficient.

The National Institute of Child Health and Human Development Fetal Cell Isolation Study (NIFTY) is the largest to date to evaluate fNRBC-based techniques for noninvasive prenatal diagnosis. Results showed that the sensitivity and specificity of the cell-based methods were not satisfactory for aneuploidy detection. In the NIFTY trial, the authors used various potentially unique NRBC cell surface identifiers and both magnetic-activated cell sorting (MACS) and fluorescent-activated (FACS) approaches. The results of this trial, which included 2744 samples, showed an aneuploidy detection rate by FISH of trisomies 13, 18 and 21 and the sex chromosomes of 74.4% with an FPR of 0.6% to 4.1%. This study demonstrated the limitations of NRBC identification, separation and analysis using approaches available at the time. Isolation of fetal nucleated cells for prenatal diagnosis continues to be a costly, labour-intensive and time-consuming process and will remain so until reliable automated approaches become more readily available.