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    Cell-free DNA testing: Preferred for detection of fetal aneuploidy


    Dr. Lockwood, Editor in Chief, is Senior Vice President, USF Health, and Dean, Morsani College of Medicine, University of South Florida, Tampa. He can be reached at [email protected].


    I am not sure of how many genetic amniocenteses I have performed. My best guess is 5000 to 6000. I estimate that I’ve performed at least 1500 chorionic villus samplings (CVS). But like experience with mid-forceps and vaginal breech deliveries, broad experience with invasive prenatal diagnosis may soon be a thing of the past.

    A recent article suggests that the number of genetic amniocenteses and CVS procedures performed in this country may soon plummet. In The New England Journal of Medicine, Bianchi and colleagues reported that assessment of maternal plasma cell-free DNA (cfDNA) in low-risk women allowed for very high detection rates (ie, 100%) of fetal trisomy 21 and trisomy 18 with very low false-positive rates (0.3% and 0.4%, respectively).1 This study opens the door to replacement of relatively inaccurate maternal serum analyte and ultrasound-based fetal aneuploidy screening by cfDNA testing, although high-risk test results using cfDNA will still require confirmation by CVS or amniocentesis.

    What is cell-free DNA and how is it detected?

    Fetal cfDNA is released into the maternal circulation primarily from apoptotic placental cells, whereas maternal cfDNA is principally derived from hematopoietic cells. Fetal cfDNA tends to be found at lower levels and in shorter segments than maternal cfDNA. After 9 weeks’ gestation, the fetal fraction represents between 10% and 20% of total cfDNA in the maternal circulation and the fetal fraction increases throughout gestation, especially after 21 weeks.2

    There is no practical way to separate fetal from maternal DNA but by randomly amplifying all available cfDNA in a maternal plasma sample with polymerase chain reaction (PCR) and then utilizing a “next-generation” DNA sequencing method called massive parallel sequencing (MPS), the chromosomal origin of each DNA fragment can be obtained by comparing its sequence with known chromosome-specific human genome sequences. This MPS approach allows comparisons of the relative amount of each chromosome’s DNA. Thus, for fetal trisomies 21, 18, and 13, the quantity of the tripled chromosome’s DNA will be increased compared to disomic reference chromosomes. Although the absolute increase in a given trisomic chromosome’s DNA will be proportional to the fraction of fetal versus maternal cfDNA, relatively simple mathematical modeling can be used to determine thresholds strongly associated with aneuploidy.


    There are limitations to this approach. Because lower fetal fractions can affect fetal trisomy detection rates, sampling before 9 weeks’ gestation will be less accurate. The fetal fraction of cfDNA also decreases with increasing maternal weight, so obesity may also lead to higher rates of inconclusive results.2 Besides the impact of the fetal fraction of cfDNA, analysis of relative trisomic chromosomal DNA content can be influenced by amplification efficiency which is, in turn, dependent upon the relative amount of guanosine-cytosine (GC) base pairs in a given chromosome.3 Because the 21st and 18th chromosomes have abundant GC content, trisomies 21 and 18 have the highest detection rates. Alternatively, chromosomes 13 and X have lower GC content and detection of trisomy 13 and monosomy X is less efficient.3 However, new bio-mathematical approaches help adjust for variation in GC content and thus improve screening efficacy.


    Charles J. Lockwood, MD, MHCM
    Dr. Lockwood, Editor-in-Chief, is Dean of the Morsani College of Medicine and Senior Vice President of USF Health, University of South ...


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