Non- invasive prenatal diagnosis using cell-free fetal DNA (cffDNA) in maternal plasma

Invasive prenatal diagnosis for chromosome anomalies is being offered to all
women with an elevated risk. If chosen by the pregnant woman and her partner
it is perfomed with chorionic villus sampling or amniocentesis. These tests
entail a small but unavoidable risk of iatrogenic pregnancy loss of ca 0,5 %.
In the Netherlands invasive prenatal diagnosis is performed 8000 times on an
annual base. Non-invasive diagnosis of monogenic conditions inherited from the
father has been possible since 1997 and has been used amongst others by
ourselves in fetal sexing for medical reasons and fetal Rh D blood group
typing. Since 2011 the introduction of massive parallel sequencing has made it
possible to quantify cell free fetal DNA in maternal plasma and link it to its
chromosomal origin. This has opened the possibility for fetal Down syndrome
detection in maternal plasma. The test is now being offered in patient care in
a number of centers abroad. The published sensitivity for trisomy 21 is high
(100 % with a CI 95.9-100). the percentage false positive results is 1 %,
substantially lower than the currently used combination test (5 %). In the
Netherlands a multidisciplinary consortium called NITRO (www.nipdnederland.nl)
is active. Amongst others geneticists, gynaecologists, laboratory specialists
in clinical genetics and ethicists of all academic centres participate as well
as the National Institute of Public Health (RIVM). The aim of the consortium
is to introduce NIPD in the Netherlands. A first step is that each
participating centre gets expertise in performing the test with the locally
available equipment and platforms, and gives testimony of its ability to do so
according to the standards.

Developing a non-invasive test for detection of trisomy 21,13 orr 18 in the
unborn fetus
Developing a standardised protocol, inclusive quality criteria for the use of
massive parallel sequencing of celfree fetal DNA in maternal plasma
Optimalising throughput, turnaround time and costs

In order to achieve the study objectives we require plasma and DNA fractions of
women with and without a foetus with trisomy 21,13 or 18 in utero. For this
purpose we will collect blood from women who report for invasive prenatal
diagnosis and who request foetal karyotyping. These women have a higher than
average risk for a foetus with one of these trisomies. Blood will be sampled
before the invasive procedure. After blood sampling the material will be
transported asap to the labs of the Department Medical Genetics UMCU. Cells
will be separated from plasma according to already validated procedures.
Plasma will be stored. From samples where the foetus appears to have a trisomy
according to the analysis of chorionic villi or amniotic fluid, cffDNA will be
extracted. The same will be done for a number of samples where the foetus has a
normal karyotype. Fetal DNA analysis will be done using the platforms
available in the laboratories of the Department of Medical Genetics.

Risks and burden associated with venapunction. One venapunction will be done,
preceding the invasive procedure. No extra hospital or lab visits will be
required. The amount of blood taken (20 ml) is comparable to the amount taken
for normal diagnostic procedures during pregnancy. Participation in the project
does not reveal additional information, and the patient will not be informed of
the results of the blood test.
The patient will be requested to send us a follow up form at the end of
pregnancy. She will be given a standard form and prestamped envelope for this
purpose This is already a standard procedure, part of our normal care
evaluation process. Finally she will be asked if she agrees us asking
additonal information on the outcome of pregnancy from her caregiver, if
necessary to evaluate the effect of pregnancy complications on the amount of
ffDNA in maternal plasma in case of outliners. Risks and burden are hence
minimal.