Prenascan

Information about the examination method and scope of examination

How the test works

PRENASCAN belongs to a group of blood-based genetic non-invasive prenatal tests (NIPT) that, between the 10th and 26th week of pregnancy, examine fetal DNA freely circulating in the mother's blood (cffDNA). NIPT is mostly focused on losses (deletions) or gains (duplications) of gene blocks and changes in the number (aneuploidy) of paired organizational units of genes – chromosomes. Trisomy refers to an extra, third chromosome, while monosomy, on the other hand, refers to the loss of one chromosome from a pair.

The main source of cffDNA is the placenta connecting the mother to the fetus and ensuring its nutrition and metabolic exchange. The placenta and the fetus have the same genetic basis - they originate from the fertilized egg. Placental cells constantly divide and die, releasing fragments of fetal DNA into the mother's bloodstream, which mix with freely circulating fragments of the mother's DNA.

Fetal fraction

Fetal fraction (FF) is the proportion of free fetal DNA to the total content of free DNA in the mother's blood. The usual lower limit of FF at which a result can still be issued is FF 4%.
However, the PRENASCAN methodology allows results to be issued even at lower FF values. Low FF may be a sign of lower placental activity and a risk factor for the further course of pregnancy (for example, the risk of lower birth weight).

Test methodology

Automated procedure: reliable result

Sample mix-up is not a risk with the automated line for the PRENASCAN test.

The PRENASCAN test uses CE-IVD and IVDR certified VeriSeq NIPT v2 Solution technology (Illumina Inc., USA).

The CE-IVD and IVDR certification of the method meets the high EU requirements for the quality and safety of laboratory diagnostic tests used for the detection, diagnosis, and monitoring of various diseases.

Scope of examination

The PRENASCAN test is based on the analysis of whole-genome data, not just selected regions.

The PRENASCAN test is validated for:

Aneuploidies (numerical abnormalities) of all fetal chromosomes, including rare ones. The most serious aneuploidies include:
- changes in the number of non-sex chromosomes:
  - Trisomy of chromosome 21 (Down syndrome), Trisomy of chromosome 18 (Edwards syndrome), Trisomy of chromosome 13 (Patau syndrome), Trisomies of chromosomes 9, 16, and 22 increasing the risk of spontaneous abortion.
- changes in the number of sex chromosomes X and Y:
  - Turner syndrome (monosomy X), Klinefelter syndrome (trisomy XXY), Triple X syndrome (XXX), and Jacob's syndrome (XYY).
- Rare chromosomal abnormalities (aneuploidies of all other chromosomes), which account for a significant portion of all serious prenatal findings.
Detection of partial deletions and duplications of at least 7Mbp in size for all autosomes.

We further analyze the results of the PRENASCAN test with our own bioinformatic analysis, which allows, depending on the nature of the specific sample (FF level, etc.), to analyze even small partial deletions and duplications (also microdeletions and microduplications) with a size of less than 7 Mbp, including, for example, DiGeorge syndrome and others (see Table 3).

Rare chromosomal aneuploidies and partial deletions and duplications are often limited to the placenta and may be a risk factor for the further course of pregnancy. They are confirmed in the fetus in less than half of the cases. In some cases, the found chromosomal abnormality is of maternal origin.

The test does not analyze defined regions. A list of these regions can be found in the VeriSeq NIPT Solution v2 Excluded Regions File (illumina.com).

	T21 (Down syndrome)	T18 (Edwards syndrome)	T13 (Patau syndrome)
Sensitivity	>99.9%	>99.9%	>99.9%
Specificity	99.90%	99.90%	99.90%

Table 1: Measured sensitivity and specificity values of the VeriSeq NIPT Solution v2 technology used by PRENASCAN for trisomies of chromosomes 21, 18, and 13. The sensitivity of the test indicates the proportion of all pregnancies with a fetus affected by trisomy that were detected by PRENASCAN (more than 99% of all). Specificity is the proportion of pregnancies with a newborn without trisomy for which the PRENASCAN result was negative. From the publication DOI: 10.1093/clinchem/hvab067.

	Chromosomal aneuploidies and accompanying findings	Rare aneuploidies of non-sex chromosomes	Partial deletions and duplications of chromosomes (≥7Mb)
Sensitivity	95.5%	96.4%	74.1%
Specificity	99.3%	99.8%	99.8%

Table 2: Measured sensitivity and specificity values of the VeriSeq NIPT Solution v2 technology used by PRENASCAN for other chromosomal aneuploidies and partial deletions and duplications of chromosomes. From the publication DOI: 10.1093/clinchem/hvab067

Example of syndromes caused by partial deletions/duplications

18q deletion Syndrome	10q deletion Syndrome	Jacobsen Syndrome
14q11-q22 deletion Syndrome	2q33.1 deletion Syndrome	15q26 overgrowth Syndrome
5q21.1-q31.2 deletion Syndrome	Wolf-Hirschhornův syndrome (4p deletion)	8p23.1 duplication/deletion Syndrome
Monosomy 9p Syndrome	Cri du Chat (5p deletion)Syndrome	22q11 (incl. DiGeorge syndrome)
Langer-Giedion Syndrome (LGS)	Cat-eye Syndrome (CES)	11q11-q13.3 duplication Syndrome

Table 3: Random selection of syndromes that can potentially be detected by the PRENASCAN test.

Fetal sex

PRENASCAN detects fetal sex based on the presence of DNA fragments specific to the male Y chromosome.

Note on the examination:

Whether you wish to report the fetal sex or not, the PRENASCAN test will analyze changes in the number (aneuploidies) of sex chromosomes X and Y for karyotypes XO (Turner syndrome), XXX (Triple X syndrome), XXY (Klinefelter syndrome), and XYY (Jacobs syndrome). The increased risk of these syndromes will always be published in the PRENASCAN report.

Concordance of fetal sex classification using the method employed by the PRENASCAN test with clinical reference
Percentage agreement	100% XX	100% XY	90.5% XO
Percentage agreement	100% XXX	100% XXY	91.7% XYY

Table 4: Concordance of the VeriSeq NIPT v2 method results, used by PRENASCAN, with the clinical assessment of the newborn. XX – girl, XY – boy, XXX – Triple X syndrome, XXY - Klinefelter syndrome, XO – Turner syndrome, and XYY – Jacobs syndrome.

Sex in twins

Sex determination: in twins, the analysis of sex chromosomes is limited to determining the presence of the Y chromosome. If fragments specific to the Y chromosome are detected, PRENASCAN cannot distinguish whether both twins are male (brothers) or if it is a brother and a sister.
Scope of examination: the patient can choose with the PRENASCAN test whether to perform only the basic analysis focused on the risks for trisomies of chromosomes 21, 18, 13, or an analysis of all non-sex chromosomes and their partial chromosomal aneuploidies (deletions and duplications of parts of chromosomes). In twins, it is not possible to perform an analysis of sex chromosome aneuploidies (e.g., Turner syndrome with karyotype X0).
Although the algorithm settings for twin pregnancies are more complex than in singleton pregnancies, the accuracy of PRENASCAN in twins is comparable to the accuracy in singleton pregnancies. We recommend evaluating NIPT results in twins together with other methods of prenatal care, especially ultrasound examination.

	T21 (Down syndrome)	T18 (Edwards syndrome)	T13 (Patau syndrome)
Sensitivity	>99.9%	>99.9%	NA
Specificity	>99.7%	>99.7%	>99.7%

Table 5: The test methodology used by our PRENASCAN has repeatedly demonstrated very good test performance in a number of laboratories in Europe and worldwide, including in multiple pregnancies. Data from the table according to DOI [https://doi.org/10.1055/s-0043-1770066.ISSN](https://doi.org/10.1055/s-0043-1770066.ISSN) 0974-2727

Vanishing twin syndrome

If, after the fertilization of two embryos, one of them disappears in the early stages of pregnancy and only one fetus continues to develop, we speak of Vanishing Twin Syndrome. The disappearance of one embryo is documented by ultrasound examination and does not necessarily involve any complications in the further course of the pregnancy. DNA fragments of the vanished twin may remain in the mother's bloodstream for some time and may cause a false positive NIPT result or incorrect sex determination. In such a case, the examination is only possible if the disappearance of the second embryo occurred by the 8th week of gestation and the blood sample was taken at least 8 weeks thereafter. Even then, there remains up to a 5% risk of false positivity. A question about Vanishing Twin Syndrome is included in the PRENASCAN request form.

examination.genetic-difference-between-placenta-and-fetus

Genetic differences between the placenta and the fetus and their impact on the NIPT result

Although the genetic basis of the fetus and placenta is the same, in approximately 2% of pregnancies, differences in the genetic makeup of the fetus and placenta arise in the first weeks of pregnancy, which we refer to as mosaicism. Because cffDNA originates from the placenta, changes limited only to parts of the placenta (placental mosaicism) can lead to false positive results, and conversely, a disorder limited only to fetal cells (fetal mosaicism) can lead to false negative NIPT results. Therefore, we recommend that every positive PRENASCAN result be verified by direct examination of fetal tissue obtained by amniocentesis. A negative PRENASCAN result should be supplemented by an ultrasound examination in the 20th week of pregnancy.

Chimerism

Maternal chimerism is a condition where a woman has two or more genetically distinct cell lines in her body. This phenomenon, like fetoplacental mosaicism (see above), can affect the results of non-invasive prenatal testing (NIPT) (distortion of sex, false positive and negative results).

Notice on the general limitations of NIPT tests

In addition to vanishing twin syndrome, fetoplacental mosaicism, and chimerism, other factors such as the use of heparin and its analogues, the proportion of fetal fraction, and others can distort the NIPT test result. For comprehensive information on the examination, see PRENASCAN Informed Consent. The PRENASCAN test is a screening test, therefore its conclusions must be consulted with a specialist taking into account the results of other examinations. A positive result from the PRENASCAN test must always be confirmed by a diagnostic test. A negative PRENASCAN result should be supplemented by an ultrasound examination in the 20th week of pregnancy.

Importance of including NIPT in comprehensive care for the pregnant patient

The results of genetic, biochemical, and ultrasound examinations complement each other, and their combination provides the highest possible level of safety for patients and their fetus.
The highest reliability is ensured by comprehensive programs for the care of the pregnant patient.

Fetal heart action

Ultrasound transvaginal examination

Biochemical examination in the first trimester

Examination of PAPP-A, free β hCG, PIGF (recommended sampling date is from 11+0 weeks at any laboratory including GENNET)

First trimester screening

including NT (nuchal translucency) measurement, calculation of the risk of pregnancy complications – preeclampsia, growth restriction, premature birth, detailed assessment of fetal morphology (sampling in the 12th-13th week of pregnancy)

PRENASCAN test

Sampling takes place between the first trimester and the 22nd week

Detailed fetal morphology

Ultrasound examination in the 20th-22nd week of pregnancy

There are also other comprehensive programs for the care of pregnant patients, such as BabyVision+, where a transvaginal ultrasound examination and PRENSACAN are performed as early as the 10th week.