Fetal Sex Determination Using Cell-Free Fetal Dna (cffDNA) in Maternal Blood

Objective. The detection of paternally inherited sequences in maternal plasma, such as the SRY gene for fetal sexing or RHD for fetal blood group genotyping, is becoming part of daily routine in diagnostic laboratories. Due to the low percentage of fetal DNA, it is crucial to ensure sample stability and the efficiency of DNA extraction. We evaluated blood stability at 4°C for at least 24 hours and automated DNA extraction, for fetal sex determination in maternal plasma.Methods. A total of 158 blood samples were collected, using EDTA-K tubes, from women in their 1st trimester of pregnancy. Samples were kept at 4°C for at least 24 hours before processing. An automated DNA extraction was evaluated, and its efficiency was compared with a standard manual procedure. The SRY marker was used to quantify cfDNA by real-time PCR.Results. Although lower cfDNA amounts were obtained by automated DNA extraction (mean 107,35 GE/mL versus 259,43 GE/mL), the SRY sequence was successfully detected in all 108 samples from pregnancies with male fetuses.Conclusion. We successfully evaluated the suitability of standard blood tubes for the collection of maternal blood and assessed samples to be suitable for analysis at least 24 hours later. This would allow shipping to a central reference laboratory almost from anywhere in Europe.

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Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation

Prenatal Diagnosis ◽

10.1002/pd.2582 ◽

2010 ◽

Vol 30 (10) ◽

pp. 918-923 ◽

Cited By ~ 30

Author(s):

Ranjit Akolekar ◽

Daniel H. Farkas ◽

Anna L. VanAgtmael ◽

Allan T. Bombard ◽

Kypros H. Nicolaides

Keyword(s):

Sex Determination ◽

Fetal Sex ◽

Fetal Dna ◽

Cell Free Fetal Dna

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Cell-Free RNA Is a Reliable Fetoplacental Marker in Noninvasive Fetal Sex Determination

Clinical Chemistry ◽

10.1373/clinchem.2015.244962 ◽

2015 ◽

Vol 61 (12) ◽

pp. 1515-1523 ◽

Cited By ~ 7

Author(s):

Elke Mersy ◽

Brigitte H W Faas ◽

Sabine Spierts ◽

Leonie M H Houben ◽

Merryn V E Macville ◽

...

Keyword(s):

Nucleic Acids ◽

Sex Determination ◽

Prenatal Testing ◽

Plasma Samples ◽

Rt Pcr ◽

Fetal Sex ◽

Fetal Dna ◽

Cell Free Fetal Dna ◽

One Step ◽

Reliable Marker

Abstract BACKGROUND Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate. METHODS We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes. RESULTS The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples. CONCLUSIONS Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination.

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