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.
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