Isolation and characterization of fetal nucleated red blood cells from maternal blood as a target for single cell sequencing‐based non‐invasive genetic testing

Autor: Noriko Ito, Kazuhiro Tsukamoto, Kosuke Taniguchi, Ken Takahashi, Aikou Okamoto, Hiroaki Aoki, Yuka Otera‐Takahashi, Michihiro Kitagawa, Hiroko Ogata‐Kawata, Hideaki Morita, Kenichiro Hata, Kazuhiko Nakabayashi
Jazyk: angličtina
Rok vydání: 2021
Předmět:
Zdroj: Reproductive Medicine and Biology, Vol 20, Iss 3, Pp 352-360 (2021)
Druh dokumentu: article
ISSN: 1447-0578
1445-5781
DOI: 10.1002/rmb2.12392
Popis: Abstract Purpose Although non‐invasive prenatal testing (NIPT) based on cell‐free DNA (cfDNA) in maternal plasma has been prevailing worldwide, low levels of fetal DNA fraction may lead to false‐negative results. Since fetal cells in maternal blood provide a pure source of fetal genomic DNA, we aimed to establish a workflow to isolate and sequence fetal nucleated red blood cells (fNRBCs) individually as a target for NIPT. Methods Using male‐bearing pregnancy cases, we isolated fNRBCs individually from maternal blood by FACS, and obtained their genomic sequence data through PCR screening with a Y‐chromosome marker and whole‐genome amplification (WGA)‐based whole‐genome sequencing. Results The PCR and WGA efficiencies of fNRBC candidates were consistently lower than those of control cells. Sequencing data analyses revealed that although the majority of the fNRBC candidates were confirmed to be of fetal origin, many of the WGA‐based genomic libraries from fNRBCs were considered to have been amplified from a portion of genomic DNA. Conclusions We established a workflow to isolate and sequence fNRBCs individually. However, our results demonstrated that, to make cell‐based NIPT targeting fNRBCs feasible, cell isolation procedures need to be further refined such that the nuclei of fNRBCs are kept intact.
Databáze: Directory of Open Access Journals
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