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The influence of low-molecular-weight heparin (LMWH)
on DNA in the blood plasma of pregnant women
Dear doctor,
Newsletter March 2015
non-invasive prenatal tests (NIPT) to determine the most common fetal trisomies have permanently
changed prenatal diagnostics in the past three years. Hundreds of thousands of these tests have been
performed annually in that time. The level of cell-free fetal DNA (cffDNA level, fetal fraction) in the maternal blood plasma has been evaluated in this process to date as one of the most important criteria for
a successful NIPT analysis.
Quality from Germany
LifeCodexx AG, together with some of our medical partners, has now demonstrated for the first time since
the introduction of NIPT that the administration of medications has a crucial impact on the DNA in the
maternal blood plasma and thus on the quality of NIPT analyses.
info@lifecodexx.com
(0) 7531-9769460
Tel+49 (0) 7531-9769480
Fax+49 On the next few pages, you can
Chairman of the supervisory board:
Peter Pohl
Chairman: Dr. Michael Lutz
Freiburg im Breisgau District Court
Commercial registry 701989
read about the effects of low-molecular-weight heparin (LMWH) on the cell-free fetal DNA and
learn what practical steps you can take to minimize the influence of LMWH on the cffDNA level
in your patients.
We also report on a case which clearly shows the current limits of NIPT and we provide you with PrenaTest®
data from clinical practice. Do you have questions? Please contact us at any time.
With kind regards,
LifeCodexx AG, Konstanz, Germany
Dr. Michael Lutz
Chairman
The influence of low-molecular-weight heparin (LMWH) on
DNA in the blood plasma of pregnant women
cffDNA level is to date the most important quality criterion for NIPT
To date, the level of cell-free fetal DNA (cffDNA) in the blood of the pregnant woman has been considered to be the most
critical factor for a successful NIPT analysis. The cffDNA level is influenced by certain parameters such as the pregnant woman‘s
body weight [1, 2] or also by the presence of aneuploidy in the fetus [2 –5], for example. We have now shown for the first time
that the administration of low-molecular-weight heparin (for example: Mono-Embolex®, Clexane ® or Fragmin® P) significantly
influences the quality of the cffDNA tested and thus ultimately the accuracy of the NIPT result.
Investigation for the first time of the influence of LMWH in routine laboratory practice
Over a period of 3 months in 2014, 1614 PrenaTest ® analyses were performed during routine laboratory practice. The test results
were not able to be clearly interpreted in the case of 12 samples due to an increased GC level (that is, proportion of the DNA
bases guanine and cytosine of the aggregate of the bases – guanine, cytosine, adenine and thymine – in percent) of more than
44% in comparison to an average GC level of 42% in the majority of the samples analyzed. In accordance with the elevated GC
level, the z score for chromosome 18 was likewise elevated, while the z scores for chromosomes 13 and 21 turned out to be lower.
Thus the test results of those women who had received LMWH injections were not accurate and would lead to false-positive or
false-negative results if the GC level of a sample is not considered to be an important quality criterion.
After consulting the physicians of the patients concerned, we determined that nine of the twelve women were receiving prophylactic
LMWH. Medical reasons for the administration of LMWH were thrombosis prophylaxis, protein C deficiency, an increased risk of
pulmonary embolisms as well as recurrent miscarriages. Five of the patients underwent an additional blood draw shortly before
the next LMWH injection, that is, at a time when the LMWH level is the lowest. The PrenaTest® analysis was successfully performed thereafter in all five cases.
Conclusion
As the first worldwide to offer NIPT, we have been able to show since the introduction of NIPT in routine prenatal
diagnostic practice [6] that the administration of a drug can influence NIPT results.
The molecular cause of the effect of LMWH described is not yet fully explained; greater numbers of cases must be
gathered.
The GC level must be an important quality criterion in NIPT analytics. Not taking it into account can lead to false-positive
results for fetal trisomy 18 or false-negative results for fetal trisomies 13 and 21.
What you should do if your patient is receiving low-molecular-weight heparin (LMWH):
1. Ensure that blood is drawn for the PrenaTest® shortly before the next administration of LMWH.
The LMWH level in the patient‘s blood is the lowest at this point in time.
2. Note on the PrenaTest® request form that the patient is receiving LMWH.
2
1 Takoudes, T. & Hamar, B. Performance of non-invasive prenatal testing
when fetal cell-free DNA is absent. Ultrasound Obstet Gynecol. 2doi:
10.1002/uog.14715. [Epub ahead of print] (2014).
4 Canick, J.A., Palomaki, G.E. & Kloza, E.M. et al. The impact of maternal
plasma DNA fetal fraction on next generation sequencing tests for
common fetal aneuploidies. Prenat Diagn 2013; 33:667-674.
2 Ashoor, G., Syngelaki, A., Poon, L.C., Rezende, J.C. & Nicolaides, K.H. Fetal
fraction in maternal plasma cell-free DNA at 11 – 13 weeks‘ gestation:
relation to maternal and fetal characteristics. Ultrasound Obstet Gynecol.
41(1), 26-32 (2013).
5 Palomaki, G. et al. Circulating cell free (ccf) DNA testing: are some test
failures informative? Prenat Diagn. , doi: 10.1002/pd.4541. [Epub ahead
of print] (2014).
3 Palomaki, G.E. et al. DNA sequencing of maternal plasma to detect Down
syndrome: an international clinical validation study. Genet. Med. 13,
913–920 (2011).
7 Hofmann, W. et al. NIPT: Welche Unterschiede zwischen den Tests gibt
es tatsächlich? Frauenarzt 55 (2014); Nr. 11; 1063-1065.
www.lifecodexx.com
6 Zur Publikation eingereicht.
8 Richtlinien zur pränatalen Diagnostik von Krankheiten und Krankheitsdispositionen, Deutsches Ärzteblatt 100, Heft 9 (28.02.2003), S. A583.
Case Report
Mosaics occur more frequently in the extraembryonic placental tissue (trophoblast/chorion) than
in the embryonic tissue, that is, the set of chromosomes of the placenta is not always identical
to the set of chromosomes of the fetus. Following chorionic villus sampling (CVS), this leads to
fetoplacental discrepancies in approximately 1 to 2% of cases. These mosaics limited to the
placenta can also lead to discrepant results (referred to as „false positive“ and „false negative“)
in the case of NIPT as well, because the cell-free „fetal“ DNA tested actually comes from the placenta. A corresponding example of a possible false-negative NIPT case presentation results from
a complete discrepancy of the cytotrophoblast and the mesodermal chorion. CVS was performed
on a 36-year old pregnant woman in gestational week 11+0. In addition to maternal age, the indication was a significantly abnormal ultrasound with generalized dermal edema, abdominal wall
defect and several soft markers. The result of the chromosomal analysis from the CVS short-term
culture (cytotrophoblast) was unremarkably female: 46,X X. This finding did not match the abnormal
ultrasound findings. The chromosomal analysis from the CVS long-term culture (mesodermal chorionic
cells), on the other hand, revealed an abnormal female finding with trisomy 18: 47,X X +18. This
finding also explains the abnormal ultrasound findings. In week 13 + 4 since LMP, the patient had
a blood sample drawn for a scientific PrenaTest ® analysis. The result was unremarkable with a
cffDNA level of 17.78% for chromosome 18 (z score 0.5).
PD Dr. rer. nat. Markus Stumm,
human geneticist
Center for Prenatal Diagnostics
and Human Genetics,
Kudamm 199, Berlin
Conclusion
The PrenaTest® analysis led to the same result as CVS short-term culture. This means that the
cffDNA predominantly comes from cells of the cytotrophoblast. This has also been able to be
confirmed in other case studies. This results in the important finding that, despite the high degree of accuracy of NIPT analyses, the diagnostic reliability of a short-term CVS culture can be
achieved at a maximum.
PrenaTest® evolving needs 2014
PrenaTest® data from clinical practice (08/2012 – 12/2014)
Abnormal results*:
As a supplement to our letter to the editor in
Results reported: 17,527
Trisomy 13: 19/16,310
FRAUENARZT 55 (2014), no. 11 [7], we would like
Unremarkable results: 97.8%
Trisomy 18: 61/16,310
to sum up the evolving needs:
Detection rate: 98.7%
Trisomy 21: 295/17,527
•Four out of ten pregnant patients decided on
False-positive rate: 0.13%
Turner syndrome: 5/2105
the PrenaTest® option 3 (determination of
Failure rate: 0.6%
Klinefelter syndrome: 5/2105
Twin pregnancies: 3.75%
T21/18/13, gonosomal aneuploidy as well as
47XYY syndrome: 3/2105
sex determination).
Triple X syndrome: 5/2105
• Likewise, an average of four out of ten patients
selected the express service to shorten the
waiting time to 4 – 6 working days.
* 08/2012 to 01/2013: T21 determination
Reprints of the letter to the editor can be reques02/2013 to 12/2014: T21/18/13 determination
ted via fax using the form on the back of this page.
07/2014 to 12/2014: Determination of gonosomal aneuploidy
PrenaTest ® – Get Clarity. Reliable. Rapid. Safe.
3
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I am interested in your letter to the editor: NIPT: Welche Unterschiede zwischen den Tests gibt es tatsächlich
(NIPT: What are the actual differences between the tests?) (Reprint from FRAUENARZT, Vol. 55, November 2014).
Please send me a copy.
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WM-1111-EN-001 / March 2015
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Quality from Germany