We use cookies to understand how you use our site and to improve your experience. This includes personalizing content and advertising. To learn more, click here. By continuing to use our site, you accept our use of cookies. Cookie Policy.

Features Partner Sites Information LinkXpress
Sign In
Advertise with Us
Eurofins Technologies

BIO-RAD LABORATORIES

Provides full range of instrumentation, reagent kits, software and quality control systems to clinical laboratories. ... read more Featured Products: More products

Download Mobile App




Events

ATTENTION: Due to the COVID-19 PANDEMIC, many events are being rescheduled for a later date, converted into virtual venues, or altogether cancelled. Please check with the event organizer or website prior to planning for any forthcoming event.
06 Jun 2020 - 09 Jun 2020
Virtual Venue
08 Jun 2020 - 11 Jun 2020
Virtual Venue

Fetal Cell-Based NIPT Demonstrated by Droplet Digital PCR

By LabMedica International staff writers
Posted on 27 Feb 2020
Print article
Image: MACS Technology is a fast and gentle method for the isolation of viable and functionally active cells by magnetic labeling (Photo courtesy of University of Konstanz).
Image: MACS Technology is a fast and gentle method for the isolation of viable and functionally active cells by magnetic labeling (Photo courtesy of University of Konstanz).
Noninvasive testing techniques are often used for fetal diagnosis of genetic abnormalities, but are limited by certain characteristics, including non-informative results. Thus, novel methods of noninvasive definitive diagnosis of fetal genetic abnormalities are needed.

Most noninvasive prenatal diagnostic tests (NIPT) on DNA today analyze cell-free fetal DNA, which is mixed in with large amounts of maternal cell-free DNA in the mother's blood. However, it has long been known that a very small number of fetal or placental cells, estimated at fewer than 10/mL, circulate in the blood of pregnant women.

Scientists at the National Research Institute for Child Health and Development (Tokyo, Japan) recruited 32 pregnant women without any obstetrical complications or abnormalities and malformations as determined by fetal ultrasonography at 20 weeks of gestation. Peripheral blood was obtained from each pregnant woman and non-nucleated cells were removed.

The washed nucleated blood cells were mixed with 10 μL each of CD45 microbeads and CD14 microbeads (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) and incubated for 15 minutes at 4 °C. The CD45–CD14– cell fraction was collected, white blood cells and large cells were removed to align the cell size, and cells were washed twice. The cells sorted by magnetic-activated cell sorting (MACS) were resuspended, processed and the stained cells were observed using the Axio Imager 2 fluorescence microscope system (Carl Zeiss Microscopy, Jena, Germany).

Single-cell–based droplet digital polymerase chain reaction (sc-ddPCR) was performed and after using the QX200 Droplet Generator, the PCR was subsequently analyzed with the signal of each droplet using the QX200 Droplet Reader (Bio-Rad, Hercules, CA, USA). DNA was extracted from each of the cell suspensions or umbilical cord blood samples. The team performed genetic confirmation of circulating male fetal cells in the CD45–CD14– cell fraction. The team used one reference probe, RPP30, and one Y-chromosome-specific probe for the SRY gene. One advantage of using that system is that it can analyze up to 3,000 individual cells per well.

The team’s modification of the sc-ddPCR system enabled the assessment of rare circulating fetal cells in the peripheral blood samples of pregnant women carrying male fetuses. However, the observation of more RPP30+SRY+ droplets in each sample was expected because two to six circulating fetal cells had been contained in 1 mL of maternal blood. When they applied their test to cell fractions from 13 maternal peripheral blood samples, they found that it correctly called only the three samples from women with a male fetus as SRY-positive and all 10 samples from women with a female fetus as SRY-negative.

The authors concluded that they had demonstrated that the modified sc-ddPCR system was able to effectively assess the genomic DNA of each targeted cell in a crudely sorted biological sample. This system is unprecedented in that the DNA of numerous individual live cells can be simply analyzed with high sensitivity and specificity without any whole-genome amplification, cell-fixation, and cell-staining steps. Moreover, this study serves as a proof of concept for noninvasive prenatal definitive diagnosis with extremely rare circulating fetal cells using our modified sc-ddPCR system. The study was published in the February 2020 issue of the Journal of Molecular Diagnostics.

Related Links:
National Research Institute for Child Health and Development
Miltenyi Biotech GmbH
Carl Zeiss Microscopy
Bio-Rad



Print article

Channels

Microbiology

view channel
Image: Stenotrophomonas maltophilia colonies on sheep blood agar. Cultivation 48 hours in an aerobic atmosphere, 37 °C (Photo courtesy of microbiologyinpictures).

Global Spread of the Multi-Resistant Pathogen Stenotrophomonas Maltophilia Revealed

Stenotrophomonas maltophilia strains occur in several natural and human associated ecosystems. The bacterium was long regarded as relatively unproblematic, but is now considered to be one of the most feared... Read more
Copyright © 2000-2020 Globetech Media. All rights reserved.