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Sabbatinelli, G. Circulating Fetal Cells for NIPD. Encyclopedia. Available online: https://encyclopedia.pub/entry/17379 (accessed on 27 July 2024).
Sabbatinelli G. Circulating Fetal Cells for NIPD. Encyclopedia. Available at: https://encyclopedia.pub/entry/17379. Accessed July 27, 2024.
Sabbatinelli, Giulia. "Circulating Fetal Cells for NIPD" Encyclopedia, https://encyclopedia.pub/entry/17379 (accessed July 27, 2024).
Sabbatinelli, G. (2021, December 21). Circulating Fetal Cells for NIPD. In Encyclopedia. https://encyclopedia.pub/entry/17379
Sabbatinelli, Giulia. "Circulating Fetal Cells for NIPD." Encyclopedia. Web. 21 December, 2021.
Circulating Fetal Cells for NIPD
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This entry is adapted from the peer-reviewed paper 10.3390/diagnostics11122239

Prenatal diagnosis plays a crucial role in clinical genetics. Non-invasive prenatal diagnosis using fetal cells circulating in maternal peripheral blood has become the goal of prenatal diagnosis, to obtain complete fetal genetic information and avoid risks to mother and fetus. The development of high-efficiency separation technologies is necessary to obtain the scarce fetal cells from the maternal circulation. 

prenatal diagnosis circulating fetal cells NIPD

1. Introduction

During the early stages of fetal development, some cells migrate from the fetus to the maternal circulation and may represent an interesting target for non-invasive prenatal diagnosis (NIPD).
However, despite technological advances, an effective standardized test suitable for routine clinical practice is not yet available. An obstacle is the small number of fetal cells present in the maternal circulation and their extreme fragility, which can lead to loss during sample handling and the absence of a specific fetal marker.
Non-invasive prenatal testing based on circulating fetal cell-free DNA has been commercially available since 2011 and offers the opportunity to radically change prenatal screening. However, the introduction into actual clinical practice is challenging because of cost, differences in the scope of abnormalities detectable, and integration into existing testing [1][2]. Recently, the analysis of fetal cells from peripheral maternal blood has been shown to be more effective in helping to identify fetal aneuploidy, microdeletion syndromes, hemoglobinopathy, and blood groups than cffDNA, due to their intact fetal genome, free from maternal DNA contamination [3].

2. Fetal Cell Types and Strategies for Isolation and Enrichment

Herzenberg et al. were the first to demonstrate the enrichment of fetal leukocytes from maternal blood in 1979 using fluorescence-activated cell sorting (FACS) [4]; however, the most studied cell types are erythroblasts (nRBCs) and trophoblasts. A summary of the types of fetal cells identified and isolated from the maternal circulation and the most commonly applied markers are shown in Table 1.
Table 1. Fetal cell lines isolated from maternal peripheral blood and markers most commonly applied for validation of NIPD.
Fetal Cell Types in
Maternal Blood		 Markers Advantages Drawbacks
Lymphoid progenitors CD34 [5][6]
CD38 [5][6]		 In vitro proliferation [7] Long-term survival [5][6];
Not easy distinguishable from maternal cells [7]
Hematopoietic stem/progenitor cells CD34 [8][5][9][10][11][12][13] In vitro proliferation [7] Long-term survival [5];
Not easy distinguishable from maternal cells [7]
Mesenchymal stem cells Vimentin [10][14]
Fibronectin [10]
Vascular cell adhesion molecule [10][13]
CD14 [10][14]
CD45 [10][14]		 In vitro proliferation and differentiation [10][14][15];
Great therapeutical potential [10]		 Very low number [10][14];
Engraftment in maternal tissue soon after transplacental passage [14]
Leukocytes HLA antigens [4][13][16][17]
CD45 [13][18]		 Transplacental passage [19];
In vitro proliferation [19]		 Long persistence in maternal blood [5]
Nucleated red blood cells ζ and ε chain of embryonic Hb [20][21][22][23]
γ chain of HbF [22]
CD71 [24][25][26][27]
4B9 [28]
i-antigen [24]
CD147 [29]
Gly A [25]		 Short half-life [8];
Single nucleus [8];
Early appearance in maternal blood [8];
Surface and intracellular markers [20][22][23][24][25][27][28][29]		 Low number in maternal blood [8]
Extravillous cytotrophoblasts (EVTs) H315 [30]
GB17 [31]
GB25 [31]
Cytokeratins [32][33][34]
CD105 [34]
Human leukocyte G antigen [35][36]
CD141 [37]		 Specific intracellular markers [38];
Relatively distinctive cell morphology (size) [39]		 Passage in maternal blood is uncommon phenomenon in all pregnancies [38][39][40]; Placental origin (1% mosaicism) [41]

2.1. Trophoblast

Trophoblasts were the first cell type identified. Cytotrophoblast-derived cells are mononuclear, invade the uterine wall and spiral arteries, and spill into maternal blood.
One of the distinguishing features of trophoblastic cells is the expression of cytokeratins (CKs).
Sargent et al. and Johnson et al. used an antibody against trophoblast-specific H315 surface antigen, which later proved insufficient for adequate cell separation [30][32][33].
Cacheux et al. recovered trophoblasts from maternal blood using immunomagnetic leukocyte depletion and flow cytometry with the anti-trophoblast antibodies GB17 and GB25. However, Y chromosome identification by FISH showed a rate of only 4.25% [31]. Other studies report the use of human leukocyte G antigen to enrich circulating trophoblasts from the maternal circulation [23][24][28][36]. An additional isolation strategy was based on fetal trophoblast cell size [41].
The limitations currently associated with the use of these cells are their extremely low numbers in the mother’s blood. In addition, they are difficult to isolate because they become trapped in the lungs and are rapidly eliminated from the maternal circulation. Furthermore, the passage of trophoblastic cells does not appear to be a common phenomenon in all pregnancies [38][39][40].
The major disadvantage of using CKs and other intracellular antigens is the need to make membranes of fragile fetal cells permeable to antibody molecules.
In addition, trophoblastic cells, being of placental origin, have a 1% incidence of mosaicism, similar to that found in chorionic villus sampling (CVS) [41].

2.2. Erythroblast

Historically, nRBCs have been the most studied cells for NIPD. The first evidence of immature erythrocytes circulating in maternal blood dates back to 1957 in Kleinhauer’s studies [22]. The fnRBCs are the first hematopoietic cells to be produced during fetal development. Their passage through the maternal-placental interface is predominant over other fetal cell types, such as leukocytes and trophoblasts. They also have a short half-life (25–35 days), which does not allow their persistence in the maternal circulation [42][43]. In addition, they have a single nucleus with a complete genetic makeup and relatively specific intracellular and surface markers. Due to these characteristics, fnRBCs appear highly interesting targets for NIPD [8].

3. New Approaches

In fetal cell isolation, microfluidics allows the separation of cells based on size, deformability, and electrical and optical properties [44][45]. Antibodies with specific cellular markers can also be used [46]. Wang et al. were able to isolate 24 fnRBCs per milliliter of maternal blood in media using gelatin-coated microspheres, with anti-CD147 as a specific recognition molecule. Cells that bind to the microspheres are separated through a spiral microfluidic chip, based on the size difference between the microspheres binding fnRBCs and white blood cells. The release of fetal cells from the microspheres is achieved by enzymatic treatment. A fetal cell capture efficiency of approximately 81%, a purity of 83%, and a viability of cell release >80% were calculated using spiked samples [47]. The technology is very suggestive, and robust evidence is needed about its practical application when tested on a large series of patients.
Another fascinating technological innovation is the Cell RevealTM system, a silicon-based nanostructured microfluidic that uses immunoaffinity to capture trophoblasts and fnRBCs with specific antibodies. Huang et al. tested this technology, isolating 14–32 fnRBCs/4 mL and 1–44 EVT/4 mL of maternal blood. The fetal origin of the cells was confirmed by FISH on chip. The identified cells were retrieved by an automated cell harvester for molecular genetic analysis, such as comparative genomic array hybridization (aCGH) and next-generation sequencing (NGS) [48]. The cell capture rate was evaluated in spiking assays and estimated to be 88.1% [49].
In a recent study, Gur et al. presented a two-tiered microchip system that reduces sample preparation steps while implementing purity, using ETVs model cell lines. The system allows for direct processing of a whole blood sample and appears versatile enough to target a variety of antigens to achieve a high rate of cell recovery [50].
Microfluidic techniques are already used in many laboratories because of the considerable advantages they present, all of which are derived from the small volumes of liquid treated, high control of flow, less time needed to analyze a product, lower costs of reagents and waste products, greater control of concentrations and molecular interactions, and the possibility of performing parallel processes. Further development of such devices appears to be very promising.
However, limitations of approaches based on microfluidic systems remain, including the inability to directly process a standard tube of blood sample without an initial volume reduction step, the use of a single antibody for cell capture, and the retrieval of cells individually for downstream analysis [51]. In Table 2 a list of approaches for fetal cell isolation/enrichment is reported.
Table 2. A summary of technical approaches of isolation or enrichment of fetal cells in the maternal circulation.
Fetal Cell Type Isolation/Enrichment Technique Purity and Recovery Rates References
fnRBCs MACS N/A [20][27][52]
FACS N/A [53]
Density gradient centrifugation 82 ± 6.4% purity [37]
84 ± 4% recovery rate [37]
32.5 ± 11.6 recovered cells [54]		 [55][56][57][58][59]
High molecular filter method 46.3 ± 25.1 recovered cells [59]
Morphology-based micromanipulation Average 4.1 recovered cells [60]
Charge flow separation 0.503 ± 0.264 purity
11,409 ± 7.684 recovered cells		 [61]
Lateral magnetophoretic microseparator 87.8% purity
1–396 recovered cells		 [62]
Microfluidics 83% purity [63]
81% recovery rate [63]
88.17% recovery rate [64]		 [47][48][49]
Hyperaggregation N/A [65]
Trophoblasts MACS N/A [37]
FACS N/A [31]
Isolation by size of epithelial tumor/trophoblastic cells (ISET) 50.7% recovery rate [45]
48.1% recovery rate [46]		 [36][66][67][68]
Trophoblast retrieval and isolation from the cervix (TRIC) 99% purity [69]
106% ± 13 recovery rate [69]
675 (IQR, 399–1010) trophoblast yield [70]
97.9% purity [70]		 [69][70]
Microfluidics 1–32 EVT/2 mL [48]
Fetal cells * Avidin-biotin immunoaffinity column Enrichment up to 1000-fold [71]
Telomerase depletion assay N/A [72]
data Lectin-based method 7.8 ± 8.5 recovered cells [64]
Ikonoscope N/A [73]
N/A; not available. * not otherwise identified.

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Subjects: Genetics & Heredity
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Giulia Sabbatinelli
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