Information for professionals

Non-Invasive Prenatal Testing (NIPT)
Method & clinical application
Information for professionals
Quality from Germany
Non-Invasive Prenatal Testing (NIPT )
The test method of the PraenaTest ® is based on the analysis of cell-free DNA (cfDNA) in the pregnant woman's blood.
The cfDNA is present in fragments and freely circulates in the maternal blood. In addition to maternal cfDNA, it also
contains approx. 2 – 40% (on average approx. 10%) cell-free fetal DNA (cffDNA). The cffDNA essentially comes from
the cytotrophoblast, that is, from placental cells of the growing embryo and is thus of extra-embryonic origin (Fig. 1). It
develops through apoptosis and necrosis of the trophoblast cells and is continually excreted into the pregnant woman's
bloodstream. The lifespan of the DNA fragments is about two hours. Within a few hours after birth of the child, the
cffDNA is no longer detectable in the mother‘s blood. With the PraenaTest ®, the cffDNA in the maternal blood is used
to determine whether there is a chromosomal imbalance for a particular chromosome to thus determine a corresponding
chromosomal disorder in the unborn child. Prenatal tests based on this method are known as non-invasive prenatal
testing (NIPT).
DNA sequencing (next generation sequencing)
Fig. 1: Schematic diagram of the transfer of the cffDNA in the
maternal bloodstream
The objective is to determine whether the quantity of
sequences for the respectively investigated chromosome
exceeds the normal range found in the case of a euploid,
i.e. normal, chromosome set. The 15 million sequences
generated are initially sorted according to defined quality
criteria using a human reference genome. About 6 million sequences can be used from this for the subsequent
analysis. These sequences are distinguished by the fact
that they are found just once in each case in a particular
region of the human genome and correspond 100% with
the reference genome. The amount of associated sequences per chromosome is then determined (Fig. 2).
To be able to sequence the DNA, the DNA fragments
which are present in small quantities are first set up in
a genomic library and amplified. Then the DNA is decoded with the most modern analytical equipment and
using the random massively parallel sequencing (rMPS)
method. About 15 million sequences are generated per
sample.
Placenta
cffDNA
Maternal DNA
X
Y
15 million
X
Y
sequences
DNA sequences
X
Y
Bioinformatic data analysis with
CE-marked PraenaTest ® DAP.plus software
Assuming that the plasma sample of a pregnant woman contains 100 chromosome-21-specific fragments, about 10 of
the fragments would be of fetal origin and 90 fragments
would be of maternal origin in the case of a euploid fetus.
In a comparable plasma sample from a pregnant woman
with a fetus with trisomy 21, 15 out of 105 chromosome-21specific fragments would be from the fetus and 90 from
the mother. The PraenaTest ® method reliably detects this
difference of 1:1.05.
X
Y
6
million
sequences
X
X
X
X
X
X
X
Y
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
Quantification
Fig. 2: Bioinformatic data analysis with PraenaTest ® DAP.plus software
PraenaTest®-analysis in the diagnostic laboratory of LifeCodexx AG
Taking the percentage of associated sequences per chromosome based on the total quantity of the 6 million associated
sequences into account, the so-called z-score is then calculated. The z-score is a statistical value which is used to determine
whether the sample tested has a chromosomal aneuploidy. It is calculated for the chromosome tested in each case from
a sample. The threshold values of the z-scores which are used to differentiate a positive from a negative test result differ
for trisomies 21, 18 and 13 due to biological and analytical factors. To determine gonosomal aneuploidy (Turner, triple X,
Klinefelter and XYY syndrome), additional evaluation criteria are used and thus the z-score alone is not significant (Fig. 3).
Extraction of cell-free DNA from the blood sample
PraenaTest ® results report
First the blood plasma is isolated from the maternal blood. Then the cell-free DNA is extracted from the blood plasma.
This comes from the mother and unborn child and is not separated from each other.
As a final step, the result is professionally interpreted and documented in the results report. Making a diagnosis as
well as the associated human genetic counseling (in Germany according to section 10 German Genetic Diagnostics Act
(GenDG)) must be carried out by a physician. Based on the results report as well as in the context of all other relevant
clinical findings, the responsible physician may therefore either rule out or confirm the presence of one of the investigated
chromosomal aneuploidies with a high degree of reliability.
Measuring the level of cell-free fetal DNA
A precondition for a successful analysis is a level of fetal DNA in the mixture with the maternal DNA of at least 4%
(minimum of 8% in twin pregnancies). First the percentage of cell-free fetal DNA is calculated from the total quantity. This
is done using a proprietary assay (QuantYfeX®). If the proportion of cffDNA is too low, it is recommended that a repeat
blood sample be taken later in the pregnancy.
MAD reference
0,0069
Example: Calculation of the z-score based on the chromosome 21 test
z (Chr21) sample =
Relative frequency
In a sample with a euploid genome, approximately 76,000 sequences can be allocated to chromosome 21. The percentage
of these sequences as compared to the total quantity of 6 million sequences is 1.27% (Fig. 3, sample 1). If there is fetal trisomy 21
in a sample tested, that is, an additional copy of chromosome 21, there are about 79,000 sequences which belong to chromosome 21. The percentage in this case is 1.32% (Fig. 3, sample 2). To calculate the z-score for a particular chromosome,
the percentage is compared with the respective percentage of a reference collective (that is, a set of samples with a euploid
chromosome set). If it deviates to a certain degree from the median of the reference collective, fetal trisomy 21 can be determined with a high degree of certainty for the sample in question. z-scores < 3 are typically found in pregnancies without fetal
trisomy 21. The distribution of the z-scores of such pregnancies corresponds to the Gaussian normal distribution. z-scores ≥ 3
indicate the presence of fetal trisomy 21.
Percentage (Chr21)
Median
reference
1,26
Max.
deviation
from median
1,28
1,5
z-score
www.lifecodexx.com
Sample 1
1,27
euploid trisomy 21
1,27 – 1,26
0,0069
Fig. 3: z-score calculation of
chromosome 21
2
Y
X
A sample calculation to illustrate the
proportions of DNA
Maternal
blood
Y
X
0
PraenaTest ® – Get Clarity. Reliable. Rapid. Safe.
1,5
% (Chr21) sample – Median(Chr21) reference
MAD(Chr21) reference
z-score
<3
Sample 2
1,32
1,32 – 1,26
0,0069
8,7
3
8,7
3
Europe‘s first non-invasive prenatal test (NIPT)
with a choice of 3 test options for individual medical issues
Fast
Safe
Clear result –
no risk assessment
4 – 6 working days
with express service
Sensitivity 99%, false-positive rate 0.1%
(for trisomies 21, 18 and 13)
Determination of the
cffDNA level after
sample receipt
8 – 10 working days with
standard service
The only NIPT with CE-marked
software according to the In-Vitro
Diagnostic Directive 98/79/EC.
Can be used even in the case of twin pregnancies and
after fertility treatment
As the only NIPT in the European Union, the PraenaTest ® was
also independently tested for investigation in the case of twin
pregnancies. It can be used unrestrictedly following all forms
of assisted reproductive technology therapies and also following egg donation.
Paid for by many health insurance plans today in individual cases
More than 20 private and statutory health insurance plans in
Germany and Switzerland are already covering the costs of the
PraenaTest ®, depending on the patient‘s individual situation.
In addition, in 2015, the Federal Joint Committee (G-BA) will
include the PraenaTest ® „on approval“ in the standard care of
the German national health insurance (GKV)*. An important
precondition for this is the use of the PraenaTest® software as
a CE-marked medical device.
* Trial regulation according to section 137e para. 7 Code
of Social Law [SGB] V
For patients at increased risk of fetal aneuploidy
National and international associations recommend the use of
NIPT in pregnant women with an increased risk of fetal aneuploidy as a primary diagnostic procedure in combination with
an ultrasound, for example in the case of increased age or
twin pregnancy, as well as a follow-up test following a positive
first-trimester screening (FTS). The PraenaTest ® is also suitable
for high-risk pregnancies which are already beyond the time
frame for first-trimester screening.
Only the original PraenaTest ® is exclusively developed and
performed in Germany
Rely on certified and independently monitored quality according to German and European legislation, combined with the
highest degree of scientific expertise. We support you in your
everyday practice. Contact our scientific experts who perform
the PraenaTest ® every day and who are continually developing
it further.
If you have any questions, please call us
Telephone +49 (0) 7531 9769460
[email protected]
LifeCodexx AG Jakob-Stadler-Platz 7, 78467 Konstanz, Germany
Telephone +49 (0) 7531-9769460, Fax +49 (0) 7531-9769480
www.lifecodexx.com
© LifeCodexx AG, PraenaTest ®/PrenaTest ® are registered trademarks of LifeCodexx AG, Germany
Reliable
WM-1103-EN-001 / November 2014
From the blood of pregnant women at increased risk for fetal aneuploidy, the PraenaTest ® determines trisomy 21 alone, trisomies 21, 18 and 13 or additionally gonosomal aneuploidy (Turner, triple X, Klinefelter and XYY syndrome) as desired, starting
at week 9 + 0 since LMP. If desired, the sex of the child can also be determined.