Methods to Increase the Percentage of Free Fetal DNA in Maternal Blood Samples and the
Implications for a Non-Invasive Prenatal Test
Ravinder Dhallan, M.D., Ph.D.1, Wei-Chun Au, Ph.D.1, Subhendra Mattagajasingh, Ph.D.1, Sarah
Emche, Ph.D.1, Philip Bayliss, M.D.2, Marian Damewood, M.D.3, Michael Cronin, Ph.D.1,
Victoria Chou, M.S.1, Michelle Mohr, M.S.1
SUMMARY
There has been significant growth and development over the last two decades in the field
of prenatal diagnosis. Yet, the need still exists for a non-invasive prenatal test that yields
diagnostic results. The use of free fetal DNA for detecting chromosomal abnormalities has
promise but has been limited by the seemingly low percentage of free fetal DNA in the maternal
circulation. Non-invasive prenatal diagnostic tests that are DNA-based would benefit from
higher percentages of free fetal DNA in the samples.
Our laboratory recently published a paper describing methods to increase the percentage
of free fetal DNA recovered from the maternal circulation. We demonstrated that inhibition of
maternal cell lysis, through the addition of formaldehyde to maternal blood samples and
implementation of careful sample processing protocols, resulted in a substantial increase in the
percentage of free fetal DNA recovered.
With an increased percentage of free fetal DNA in the maternal blood samples, the
sequence of fetal DNA can be discerned from maternal DNA using natural genetic markers, such
as single nucleotide polymorphisms. These natural genetic markers can be used to determine the
presence or absence of fetal chromosomal abnormalities. Therefore, methods for increasing the
percentage of free fetal DNA provide a solid foundation for the development of a non-invasive
prenatal diagnostic test.
1
Ravgen, Inc., Columbia, MD; 2Department of Maternal Fetal Medicine, Lancaster
General Women and Babies Hospital, Lancaster, PA; 3Department of Obstetrics and
Gynecology, York Hospital, York, PA.
THE NEED FOR A NON-INVASVIVE PRENATAL DIAGNOSTIC TEST
Prenatal diagnosis is useful for managing a pregnancy with an identified fetal
abnormality, and may allow for planning and coordinating of care during delivery and the
neonatal period.1 A variety of prenatal diagnostic tests are available but each test has limitations.
The two most commonly utilized non-invasive tests are ultrasound, which can be used as a
screening test for chromosomal abnormalities as well as a diagnostic test for certain structural
abnormalities of the fetus, and maternal serum marker testing.
Maternal serum marker tests are designed to measure the levels of specific markers in
the maternal blood. Serum marker testing does not yield direct diagnostic results, but rather leads
to the identification of a portion of the obstetrical patient population at risk for Trisomy 21 and
18, and open neural tube defects.2-4
The most efficient screening test in the second trimester is the “quad” screen, which
measures the levels of alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG),
unconjugated estriol (E3), and inhibin-A.5, 6 The “quad” screen is associated with false positive
rates in the range of 5-7%, and detection rates do not approach those achieved by invasive
prenatal diagnostic tests.6
Most recently, there has been interest in the use of nuchal translucency sonography for
first trimester screening.7,8 Nuchal translucency refers to the normal subcutaneous fluid-filled
space between the back of the fetal neck and the overlying skin. There seems to be a correlation
between increasing nuchal translucency measurements and the risk for Down syndrome, other
aneuploidies, and structural malformations.9
Malone and D’Alton pooled data from thirty studies involving the use of nuchal
translucency in an unselected general population and found the overall sensitivity for Down
Syndrome was 77% with a false positive rate of 6%.7 However, there was considerable
variability between these studies, with sensitivities varying from 29% to 100%, false positive
rates varying from 0.3% to 11.6%, and positive predictive values ranging from 1.6% to 50%.7
2
Nuchal translucency sonography appears to be useful as a first-trimester screen but further
improvements aimed at reducing the variability of the test are needed before it can recommended
as a screening test in routine obstetric practice.
The non-invasive tests discussed above have inadequate detection rates that are
accompanied by high false positive rates. Therefore, diagnostic tests including amniocentesis,
chorionic villus sampling (CVS) and percutaneous umbilical blood sampling remain the gold
standard for the definitive detection of fetal chromosomal abnormalities. The results from these
invasive diagnostic tests are highly reliable and give the patient specific information. However,
each carries a risk for pregnancy loss due to the procedure.10, 11 Many patients who are candidates
for these tests decline them due to the risk of pregnancy loss.
ALTERNATIVES TO EXISTING PRENATAL TESTS
An alternative to existing methods for prenatal diagnosis is to use fetal cells and fetal
DNA that exist in the maternal circulation.12-16 Fetal Trisomy 21 and 18 has been detected by
fluorescence in situ hybridization (FISH) using chromosome-specific probes on flow-sorted intact
fetal cells.17, 18 However, it has been estimated that there is only one intact fetal cell per cubic
centimeter of maternal blood, which makes the recovery of these cells labor intensive and very
difficult.19 While there has been progress in cell separation technology, the scarcity of intact fetal
nucleated cells has prevented the development of a reliable technique for prenatal diagnostics.
A second alternative to existing prenatal tests is to analyze free fetal DNA in the maternal
circulation. Circulating fetal DNA has been used to determine the sex of the fetus through
detection of sequences present on the Y chromosome.15 In addition, several studies have
attempted to use free fetal DNA to screen for chromosomal abnormalities in the fetus.20-25
However, the use of free fetal DNA for detecting chromosomal abnormalities has been
limited by the seemingly low percentage of free fetal DNA in the maternal circulation. Lo et al.
reported a mean of 3.4% of free fetal DNA in maternal plasma in late first to mid-second
3
trimester and a mean of 6.2% free fetal DNA in late third trimester.15 Any method that can
increase the percentage of free fetal DNA in the sample would make it easier to distinguish fetal
DNA from maternal DNA.
RECENT ADVANCEMENTS IN THE FIELD OF FREE DNA
Our lab recently published studies describing methods to increase the percentage of free
fetal DNA recovered from the maternal circulation.26 We hypothesized that inhibiting cell lysis
during sample collection, shipping, handling, and processing would permit the recovery of a
larger percentage of free fetal DNA. By decreasing the amount of maternal cell lysis, and thus,
free maternal DNA, the percentage of free fetal DNA could be increased.
In the first phase of the study, we tested the effect of formaldehyde, a chemical known to
cross-link proteins and hinder cell lysis, on the percentage of free fetal DNA recovered from the
maternal circulation. Two samples of blood were collected from each of ten pregnant women:
one was treated with formaldehyde and the other was untreated. The mean percentage of free
fetal DNA in the untreated samples was 7.7%, while the mean percentage of free fetal DNA in
the formaldehyde-treated samples was 20.2% (P =0.02 for difference; Wilcoxon signed rank
test).26
The second phase of the study measured the percentage of free fetal DNA in 69
formaldehyde-treated samples of maternal blood obtained from a network of 27 clinical sites in
16 states.26 A median of 25% free fetal DNA was obtained for the 69 formaldehyde-treated
maternal blood samples. A detailed summary of the results from the second phase of the study is
provided in Table 1.26
4
Table 1. Summary of the percentages of free fetal DNA in samples from the second phase of the
study.
% Fetal DNA
Number of
Samples
% of total samples
(69)
3.1
4
6.2
7
12.5
17
25
22
50
8
Over 50%
11
5.8
10.1
24.6
31.9
11.6
15.9
The observed increase in the percentage of free fetal DNA likely resulted from a
combination of factors. First, formaldehyde stabilizes cell membranes, thereby preventing cell
lysis and the release of DNA.26 Prior to the venipuncture procedure, the amount of free maternal
DNA in the maternal circulation likely is low. However, the maternal cells may lyse during
sample collection, shipping, handling, and processing. The presence of formaldehyde helps to
protect the cells from lysis.
Second, the addition of formaldehyde may allow a larger recovery of free fetal DNA by
inhibiting enzymes that destroy DNA, such as DNases.26 Inhibition of DNA destroying enzymes
would permit a larger recovery of DNA already present in the sample, including free fetal DNA.
Also, the addition of formaldehyde may stabilize and preserve the structure of DNA, which may
increase the amount of DNA recovered.
Third, in conjunction with the addition of formaldehyde to the maternal samples, sample
processing protocols designed to minimize cell lysis likely contributed to increases in the
percentage of free fetal DNA.26 A centrifugation protocol was designed to minimize gravitational
forces imposed on the cells. In addition, all centrifugation steps were performed with the
acceleration and brake powers set to zero. Also, when removing the plasma sample, care was
taken to ensure the buffy-coat was not disturbed.
5
THE UTILITY FOR INCREASED PERCENTAGES OF FREE FETAL DNA
An increased percentage of free fetal DNA in the maternal blood samples makes it easier
to discern fetal DNA from maternal DNA using natural genetic markers, such as single nucleotide
polymorphisms (SNPs). For example, at certain SNP sites, the maternal genome will be
homozygous for a nucleotide, such as adenine (A), while the paternal genome is homozygous for
a different nucleotide, such as guanine (G), which means the fetal genome will be A/G at this
SNP site. The guanine represents a distinct fetal signal in the maternal blood sample. The
detection and quantitation of fetal DNA, in this case guanine, is more attainable with an increased
percentage of fetal DNA, and can be used to diagnose single gene disorders and chromosomal
abnormalities.
A ratio for the nucleotides at SNP sites that fit the pattern discussed above can be
quantitated and used to detect chromosomal disorders. When samples have a high percentage of
free fetal DNA, the difference between the expected ratio of the chromosomes for a healthy fetus
and an abnormal fetus is greater, which makes it easier to diagnose chromosomal abnormalities.
Thus, methods recently described for increasing the percentage of free fetal DNA provide a solid
foundation for the development of a non-invasive prenatal diagnostic test.
6
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