Objective: To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. Methods: Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. Results: An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. Conclusion: Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.

Doffini, A., Forcato, C., Mangano, C., Lattuada, D., Aversa, R., Maranta, C., et al. (2023). Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling. PRENATAL DIAGNOSIS, 43(1 (January 2023)), 14-27 [10.1002/pd.6275].

Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling

Doffini, Anna
;
Serafini, Marta;Biondi, Andrea;Perego, Sofia;Vergani, Patrizia;Ricciardi-Castagnoli, Paola;
2023

Abstract

Objective: To develop a multi-step workflow for the isolation of circulating extravillous trophoblasts (cEVTs) by describing the key steps enabling a semi-automated process, including a proprietary algorithm for fetal cell origin genetic confirmation and copy number variant (CNV) detection. Methods: Determination of the limit of detection (LoD) for submicroscopic CNV was performed by serial experiments with genomic DNA and single cells from Coriell cell line biobank with known imbalances of different sizes. A pregnancy population of 372 women was prospectively enrolled and blindly analyzed to evaluate the current workflow. Results: An LoD of 800 Kb was demonstrated with Coriell cell lines. This level of resolution was confirmed in the clinical cohort with the identification of a pathogenic CNV of 800 Kb, also detected by chromosomal microarray. The mean number of recovered cEVTs was 3.5 cells per sample with a significant reverse linear trend between gestational age and cEVT recovery rate and number of recovered cEVTs. In twin pregnanices, evaluation of zygosity, fetal sex and copy number profiling was performed in each individual cell. Conclusion: Our semi-automated methodology for the isolation and single-cell analysis of cEVTS supports the feasibility of a cell-based noninvasive prenatal test for fetal genomic profiling.
Articolo in rivista - Articolo scientifico
Fetal circulating cells, extravillous trophoblasts, aneuploidy, copy number variant, NIPT, prenatal diagnosis
English
28-nov-2022
2023
43
1 (January 2023)
14
27
none
Doffini, A., Forcato, C., Mangano, C., Lattuada, D., Aversa, R., Maranta, C., et al. (2023). Isolation of single circulating trophoblasts from maternal circulation for noninvasive fetal copy number variant profiling. PRENATAL DIAGNOSIS, 43(1 (January 2023)), 14-27 [10.1002/pd.6275].
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/398911
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