Dataset Information

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

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.

SUBMITTER: Doffini A

PROVIDER: S-EPMC10107339 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Publications

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

Doffini Anna A Forcato Claudio C Mangano Chiara C Lattuada Debora D Aversa Roberta R Maranta Chiara C Giovannone Emilia D ED Buson Genny G Bolognesi Chiara C Maiocchi Rebecca R Dori Martina M Jamal Liyana L Ahmad Raidah B RB Yeo George S H GSH Yeo Tai Wai TW Saragozza Silvia S Silipigni Rosamaria R Serafini Marta M Biondi Andrea A Perego Sofia S Vergani Patrizia P Ferrazzi Enrico E Ricciardi-Castagnoli Paola P Musci Thomas J TJ Grati Francesca Romana FR

Prenatal diagnosis 20221208 1

<h4>Objective</h4>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.<h4>Methods</h4>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 imbal ...[more]

PMID: 36443901

Similar Datasets

Project description:Noninvasive prenatal testing (NIPT) based on cell-free DNA in maternal circulation has been accepted worldwide by the clinical community since 2011 but limitations, such as maternal malignancy and fetoplacental mosaicism, preclude its full replacement of invasive prenatal diagnosis. We present a novel silicon-based nanostructured microfluidics platform named as "Cell Reveal™" to demonstrate the feasibility of capturing circulating fetal nucleated red blood cells (fnRBC) and extravillous cytotrophoblasts (EVT) for cell-based noninvasive prenatal diagnosis (cbNIPD).The "Cell Reveal™" system is a silicon-based, nanostructured microfluidics using immunoaffinity to capture the trophoblasts and the nucleated RBC (nRBC) with specific antibodies. The automated computer analysis software was used to identify the targeted cells through additional immunostaining of the corresponding antigens. The identified cells were retrieved for whole genome amplification for subsequent investigations by micromanipulation in one microchip, and left in situ for subsequent fluorescence in situ hybridization (FISH) in another microchip. When validation, bloods from pregnant women (n = 24) at gestational age 11-13+6 weeks were enrolled. When verification, bloods from pregnant women (n = 5) receiving chorionic villus sampling or amniocentesis at gestation age 11+4-21 weeks with an aneuploid or euploid fetus were enrolled, followed by genetic analyses using FISH, short tandem repeat (STR) analyses, array comparative genomic hybridization, and next generation sequencing, in which the laboratory is blind to the fetal genetic complement.The numbers of captured targeted cells were 1-44 nRBC/2 ml and 1-32 EVT/2 ml in the validation group. The genetic investigations performed in the verification group confirmed the captured cells to be fetal origin. In every 8 ml of the maternal blood being blindly tested, both fnRBC and EVT were always captured. The numbers of captured fetal cells were 14-22 fnRBC/4 ml and 1-44 EVT/4 ml of maternal blood.This report is one of the first few to verify the capture of fnRBC in addition to EVT. The scalability of our automated system made us one step closer toward the goal of in vitro diagnostics.

Dataset Information

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

Objective

Methods

Results

Conclusion

Publications

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

Similar Datasets

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