The PrenaTest® is developed and performed in Germany in accordance with German and European law. The lab process in accordance with the random massively parallel sequencing (rMPS) method is described below as it is performed in our diagnostic lab in Konstanz. You receive a clear result – not a risk assessment.
1. Extraction of cell-free DNA from the blood sample
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.
2. 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.
3. DNA sequencing (next generation sequencing)
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.
4. Bioinformatic data analysis with CE-marked PrenaTest® DAP.plus Software
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. left)
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 disorder. 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.
5. PrenaTest® results report
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 disorders with a high degree of reliability.
Calculation of the z-scores based on the chromosoms 21 test
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., 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., 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.