| Autor: |
Doffini, Anna, Forcato, Claudio, Mangano, Chiara, Lattuada, Debora, Aversa, Roberta, Maranta, Chiara, Giovannone, Emilia D., Buson, Genny, Bolognesi, Chiara, Maiocchi, Rebecca, Dori, Martina, Jamal, Liyana, Ahmad, Raidah B., Yeo, George S. H., Yeo, Tai Wai, Saragozza, Silvia, Silipigni, Rosamaria, Serafini, Marta, Biondi, Andrea, Perego, Sofia |
| Zdroj: |
Prenatal Diagnosis; Jan2023, Vol. 43 Issue 1, p14-27, 14p |
| Abstrakt: |
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. Key points: What's already known about this topic?Fetal circulating extravillous trophoblasts (cEVTs) have been isolated from maternal blood for both aneuploidy and copy number variant (CNV) classification.Previous reported methodologies require mostly manual low throughput workflows and identify putative fetal cells based on immunophenotype. What does this study add?We present a novel semi‐automated workflow for the isolation of circulating fetal trophoblasts and single‐cell genomic profiling of submicroscopic variants.With this methodology, the feasibility of fetal genetic confirmation of each individual cell, a limit of detection (LoD) for CNVs down to 800Kb and the assessment of zygosity, fetal sex and genomic profiling in the same cell in twin pregnancies was demonstrated. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
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