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

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

doi:10.1002/rmb2.12392

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

Reprod Med Biol. 2021 Jun 14;20(3):352-360. doi: 10.1002/rmb2.12392. eCollection 2021 Jul.

Authors

Affiliations

¹ Department of Maternal-Fetal Biology National Center for Child Health and Development Tokyo Japan.
² Department of Pharmacotherapeutics, Course of Medical and Dental Sciences Nagasaki University Graduate School of Biomedical Sciences Nagasaki Japan.
³ Department of Obstetrics and Gynecology The Jikei University School of Medicine Tokyo Japan.
⁴ Aoki Obstetrics and Gynecology Clinic Tokyo Japan.
⁵ Sanno Birth Center Tokyo Japan.
⁶ Department of Allergy and Clinical Immunology National Center for Child Health and Development Tokyo Japan.

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.

Keywords: fetal nucleated red blood cell (fNRBC); fluorescence‐activated cell sorting (FACS); non‐invasive prenatal testing (NIPT); single cell; whole genome amplification (WGA).