Fetal Cells (cbNIPT)
In prenatal care it might be necessary to get access to fetal DNA to test the developing fetus for genetic diseases. Conventional prenatal diagnostic methods such as chorionic villus sampling (CVS) or amniocentesis are stressful and invasive with a risk of spontaneous abortions. At the same time in the first trimester of pregnancy cells originating from the fetus can be found in the maternal blood circulation. Isolation of these fetal cells from a maternal blood sample is an attractive alternative to conventional procedures because it allows for low-risk, non-invasive prenatal testing (NIPT) while enabling isolation of the pure and intact fetal genome (Fig.1).
Fig. 1. A schematic representation (from Singh et al.) of the positioning of cell-based NIPT (cbNIPT) vs. conventional prenatal diagnostics methods and cell-free NIPT (cfNIPT). Being based on pure fetal cells, not contaminated by maternal genome, cbNIPT shows comparable strength of analysis as invasive procedure such as CVS. The method is a blood draw-based test with the risk and stress to the pregnant women being low as in the case of cfNIPT.
NIPT-based on the analysis of circulating cfNIPT is currently being introduced in many countries. However, its major challenge is low fetal genome purity because of an excess of maternal DNA in plasma as well as DNA fragmentation. This leads to difficulties for a reliable detection of atypical chromosomal aberrations in the fetalus genome. This obstacle can be solved by isolating pure and intact fetal cells from maternal blood and performing genetic analyses on whole genome-amplified fetal DNA.
Fetal cells circulating in the maternal blood are extremely rare: there are just 1 to 2 fetal cells per ml of blood. Thus recovering pure fetal cells from maternal blood in sufficient numbers for downstream genetic analysis is technically challenging. It requires an efficient upstream enrichment, detection with adequate cell-type specific markers and extremely specific, precise and gentle single cell isolation minimizing damage and loss of rare cells. The fetal cell isolation workflow based on the CellCelector combines rare cell detection and gentle recovery of individual single fetal cells from enriched blood samples providing both speed and precision (Fig 2).
Fig. 2. A typical workflow for isolation and genetic analysis of single fetal cells from maternal blood using the CellCelector single cell picker. CFC: circulating fetal cells; CMA: chromosome microarray analysis; NGS: next-generation sequencing.
Fig. 3. Example of a gallery of fetal cells detected using the CellCelector following the workflow described in Fig.2. Blue: DAPI; green: cocktail of fetal cell specific antibodies [images courtesy of Arcedi Biotech]
Fig. 4. Example of before and after picking images automatically saved by the CellCelector system. Single cell picking efficiency: 100% [images courtesy of Arcedi Biotech].
Proven sensitivity and accuracy of the novel cbNIPT method based on the CellCelector platform allowed our customer ARCEDI Biotech to introduce the test into prenatal care in the Central Region of Denmark in May 2018. Those women identified as ‘high-risk’ (>1:300) based on a combined first trimester screening now have the opportunity to choose between a cell-based non-invasive test along with a cell-free NIPT, or a conventional invasive testing. The new cbNIPT method provides zero no-calls, i.e. fetal cells are detected and isolated in all cases (Fig 5). In average 12.8 fetal cells are identified per sample. The figures below show a typical gallery of isolated fetal cells as well as examples of chromosomal aberrations detected utilizing this method. Presently the developed cbNIPT technique can detect aneuploidy, microduplication, unbalanced structural rearrangements and mosaic cases with a capacity to identify subchromosomal aberrations >10Mb (Fig.6). The ultimate goal would be offering the screening test to all pregnant women.
Fig. 5. Gallery of fetal cells (trophoblasts) enriched and detected from maternal blood in a male fetus with increased nuchal translucency (from Vestergaard et al.). The cells are stained with DAPI (Blue) and a cocktail of fetal cell specific antibodies (Green). After enrichment the CellCelector was used for detection and isolation of single fetal cells followed by WGA and aCGH analysis. The result confirming aneuploidy for chromosome 21 is shown in Fig.6 (case 1).
Fig. 6. Example of aCGH analysis on 5 cytogenetically abnormal cases. 1: trisomy 21; 2: trisomy 13 (mosaic); 3: trisomy 2 (mosaic); 4: partial trisomy 21 (12.4‐Mb duplication); 5: unbalanced translocation including 31‐Mb deletion on chromosome 4p and 30‐Mb duplication on chromosome 8p (Figure from Vestergaard et al.). All maternal blood samples were collected for cbNIPT prior to the invasive testing and analyzed blindly.
Toft, C.L.F. et al. Cell-based non-invasive prenatal testing for monogenic disorders: confirmation of unaffected fetuses following preimplantation genetic testing J Assist Reprod Genet 38, 1959–1970 (2021)
Jeppesen L.D., et al. Cell-based non-invasive prenatal diagnosis in a pregnancy at risk of cystic fibrosis Prenat Diagn. 2021 Jan;41(2):234-240
Hatt L, et al. Cell-based noninvasive prenatal testing (cbNIPT) detects pathogenic copy number variations Clin Case Rep. 2020
Vestergaard E.M. et al. - On the road to replacing invasive testing with cell‐based NIPT: Five clinical cases with aneuploidies, microduplication, unbalanced structural rearrangement, or mosaicism. Prenatal Diagnostics 37(11):1120-1124 (2017)
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