NIPT: All you need to know about the non invasive prenatal screening test

Prenatal care consists of medical follow-up, which is essential and necessary to maintain the health of the pregnant woman and the baby, and its main objective is to early identify and treat diseases that may be harmful to the health of the mother and/or the child.

Conventional first trimester screening (ultrasound and serum biomarkers) allows the detection of the most frequent chromosomal changes in the fetus during pregnancy. 

This type of conventional screening is mainly intended for the detection of aneuploidies, such as:  

  • Chromosome 21 trisomy: Down Syndrome 
  • Chromosome 13 trisomy: Patau Syndrome 
  • Chromosome 18 trisomy: Edwards Syndrome 

Presenting a sensitivity of 85-90% for these syndromes, with a false positive rate of 5%. 

Technological advances in DNA analysis have enabled the development of non-invasive prenatal screening testing (NIPT) based on the study of fetal cell-free DNA in maternal blood, capable of studying different chromosomal conditions with greater sensitivity and specificity without generating risk for the mother and the baby.

In this scenario, the non-invasive prenatal test (NIPT) is the most recent development in prenatal screening, and has been increasingly offered in the clinical environment to detect not only the main fetal trisomies, but also an analysis of the whole fetal genome, in order to detect other fetal aneuploidies (changes in the number of chromosomes). NIPT is based on methods such as the next-generation sequencing (NGS) or other high-throughput analysis tools for fetal cell-free DNA in maternal blood serum. 

 

What is the NIPT and how is it performed? 

The NIPT is a non-invasive prenatal examination, used to screen the main chromosomal changes in the fetus, carried out from fetal cell-free DNA (cfDNA) in the maternal circulation, that is, cells from the fetus that are present in the mother’s blood.

 

  • The NIPT is performed by collecting peripheral blood:

 

cfDNA (cell free DNA) refers to DNA that exists as small fragments (less than 200 pb) in plasma or other body fluids, which are distinct from the DNA contained in the nucleus of an intact cell, and which are released from all organs during a series of cellular processes. The maternal plasma cfDNA contains both maternal and fetal sources of cfDNA. The source of fetal DNA is the trophoblast, while the predominant source of maternal DNA is the hematopoietic system. 

The NIPT for fetal aneuploidy uses new generation, high-throughput sequencing methods to quantify the proportional representation of each chromosome in the plasma cfDNA. The proportional representation of each chromosome in the plasma cfDNA reflects the chromosome size and the individual’s karyotype. While in a euploid pregnant woman (46,XX), a deviation from the expected chromosomal profile in the plasma cfDNA, due to the excess or deficient cfDNA fragments of a specific chromosome, suggests the presence of fetal monosomy or trisomy, respectively.  

 

Fetal fraction in NIPT result

A statistically significant variation in the count of cfDNA fragments for a given chromosome, commonly defined as a z score> 3, is a high-risk result.

The fetal fraction (FF) is the percentage of total maternal plasma cfDNA that is of fetoplacental origin. It is an assessment of both maternal and fetal cfDNA levels in maternal plasma.

  • Between the 10th and 20th weeks of gestation, the average FF is 10% to 15%. 

Thus, the fetal fraction is a function of biological factors (that is, the levels of maternal and fetal cfDNA in maternal plasma) and bioinformatics algorithms used to interpret the results of DNA sequencing, being essential in the quality control of the results of the non-invasive prenatal test (NIPT). 

It is important to note that, although the FF should be calculated routinely, there is still no consensus on whether it should be reported routinely in the report.

Among the factors that can influence the fetal fraction are: maternal weight, gestational age, placental problems, other biological and environmental factors, in addition to sample collection and storage under non-optimal conditions.

 

How to interpret the NIPT?

The NIPT consists of a prenatal screening for chromosomal changes, which does not exclude the possibility of other genetic diseases (such as monogenic diseases, for example). 

Since the NIPT is a screening test, the result must be evaluated by the requesting physician, within the clinical context and with other laboratory or imaging findings of the patient. 

Positive results may reflect the presence of mosaicisms. Thus, the result can be confirmed by invasive tests as requested by the treating physician and the patient’s wish.

 

Who is the NIPT indicated for?

The NIPT has been considered an advance in prenatal care for screening chromosomal changes due to its clinical safety and ease of use. 

Prenatal screening for fetal chromosomal abnormalities is performed to identify women at a higher risk of having an affected fetus. It also allows informed decisions to be made about whether to proceed to the diagnostic test. 

The NIPT is indicated for pregnant women with at least 10 weeks of gestation (10 weeks + 0 days ), in the following situations: single or twin pregnancies; in vitro fertilization  (IVF); IVF with gamete donation and in cases of resorbed twins. 

 

What are the indications for performing the NIPT?
  • Advanced maternal age (over 35 years);
  • Parents with balanced Robertsonian translocation, mainly involving chromosome 21;
  • Abnormal ultrasound;
  • Suspected chromosomal syndrome;
  • Abnormal result in serum and biochemical screening;
  • Family history with higher risk for specific chromosomal aneuploidies;
  • Low-risk maternal anxiety.

 

What syndromes or diseases does NIPT screen for? 

The available NIPTs generally screen for all of the following chromosomal aneuploidies: 

  • Chromosome 21 trisomy:  Down Syndrome; 
  • Chromosome 18 trisomy: Edwards Syndrome;
  • Chromosome 13 trisomy: Patau Syndrome;
  • Chromosome X monosomy: Turner Syndrome (45,X);
  • Chromosome X trisomy: 47,XXX;
  • Klinefelter´s Syndrome: 47,XXY;
  • Jacob’s Syndrome; 47,XYY

They can also screen for aneuploidies on all other chromosomes.

Clinically significant fetal chromosomal changes usually involve gains or losses of genetic material. These can vary in size from small segments of chromosomes (called “microduplications” or “microdeletions”) to entire chromosomes (that is, aneuploidy).

Whole chromosomal imbalances, as well as copy number variation (CNVs), can also be detected using cfDNA, which includes CNVs from the whole genome and targeted microdeletion syndromes such as cri-du-chat syndrome (5p−), Digeorge syndrome (22q11.2), Prader-Willi and Angelman syndrome (15q−), Wolf-Hirschhorn syndrome (4p-), Jacobsen syndrome (11q−), Langer-Giedion syndrome (8q−) and 1p36. 

 

How does the SYNLAB NIPT work? 

The non-invasive prenatal screening tests offered by SYNLAB are performed using Next-Generation Sequencing Illumina®, paired-end (two readings are produced for each DNA fragment sequenced) of the whole genome using WGS (Whole Genome Sequencing) technology allowing to measure the amount of fetal cell-free DNA (cfDNA). Since fetal DNA is smaller than maternal cfDNA, chromosome counts in smaller DNA fragments improve sensitivity and specificity even in cases of low fetal fraction, with an overall detection rate of 99.1% (95% CI:  95-99.9%).

 

What are the types of non-invasive prenatal testing from SYNLAB?

SYNLAB offers the following NIPTs:

NeoBona test, which detects:

  • Down syndrome (chromosome 21 trisomy).
  • Edwards syndrome (chromosome 18 trisomy).
  • Patau syndrome (chromosome 13 trisomy).

      – Fetal sex (optional)

Through WGS Paired-end technology + percentage of fetal fraction. Indicated for single and twin pregnancies.

 NeoBona Advanced test, which detects: 

  • Down syndrome (chromosome 21 trisomy).
  • Edwards syndrome (chromosome 18 trisomy).
  • Patau syndrome (chromosome 13 trisomy).
  • Aneuploidies of sex chromosomes, X and Y. 
  • Fetal sex. 

Through WGS Paired-end technology + percentage of fetal fraction. Indicated for single pregnancies.

Extended Prenatal Panel, which detects:

  • Down syndrome (chromosome 21 trisomy).
  • Edwards syndrome (chromosome 18 trisomy).
  • Patau syndrome (chromosome 13 trisomy).
  • Aneuploidies of sex chromosomes, X and Y. 
  • Fetal sex. 
  • DiGeorge syndrome. 
  • Angelman syndrome.
  • Prader-Willi syndrome.
  • Wolf Hirschhorn syndrome
  • Cri-du-chat syndrome.
  • 1p36 deletion. 

Through WGS Paired-end technology + percentage of fetal fraction. Indicated for single pregnancies.

Extended Prenatal Panel + All Chromosomes, which detects:

  • Down syndrome (chromosome 21 trisomy).
  • Edwards syndrome (chromosome 18 trisomy).
  • Patau syndrome (chromosome 13 trisomy). 
  • Aneuploidies of sex chromosomes, X and Y. 
  • Fetal sex. 
  • DiGeorge syndrome. 
  • Angelman syndrome.
  • Prader-Willi syndrome.
  • Wolf Hirschhorn syndrome
  • Cri-du-chat syndrome.
  • 1p36 deletion. 
  • Aneuploidies in all chromosomes. 

Through WGS Paired-end technology + percentage of fetal fraction. Indicated for single pregnancies.

 

How are SYNLAB non-invasive prenatal tests done?

To perform the examination, only a small sample of maternal peripheral blood is needed, in a specific tube using a kit provided by SYNLAB, from the 10th gestational week (10 weeks + 0 days). Before that, the fetal fraction is very low, and there is a greater chance of failure of the test; however, there is no upper limit on gestational age (FF increases according to gestational age).

 

The examination consists of the analysis of fragments of fetal cell-free DNA by sequencing, with subsequent quantification of the DNA fractions of the examined chromosomes in relation to a control pattern, using a bioinformatics algorithm to release the result.

 

Results consistent with cfDNA-based aneuploidies consist of screening and should always be confirmed by a diagnostic technique, such as fetal karyotype or fetal DNA analysis (amniocentesis or chorionic villus biopsy), before any other medical intervention. In such cases, it is indicated that the patient receives adequate genetic counseling.

 

What is the difference between neoBona and other non-invasive prenatal screening tests?

NeoBona® is the first screening test based on paired-end cfDNA that uses an innovative computer algorithm and provides double verification of chromosome count data, generating the T-SCORE (single trisomy score calculation) that integrates several parameters to provide reliable results even in cases of very low fetal fractions. This allows results to be obtained in the vast majority of cases (recollection rate is about 1.5%).  The TSCORE takes into account the chromosome count, the fetal fraction, the determination of the fragment size distribution and the depth of the sequencing, thus quantifying the probability of fetal trisomy.

 

In addition, neoBona’s paired-end WGS sequencing technology allows a deeper and more comprehensive analysis of cfDNA when compared to conventional single-end WGS technology, generating more efficient sequence counts, increasing analysis accuracy.

 

If any risk is detected for the assessed syndromes, a free confirmatory test is offered to the patient, in which the analysis is performed by sending material obtained by an invasive procedure (amniocentesis or cordocentesis).

 

The sensitivity and specificity rates for neoBona are:

  • For Down Syndrome (T21): it is 100% (95% CI: 94.3-100%) and 99.96% (95% CI: 99.9-100%). 
  • For Edwards Syndrome (T18): it is 97.1% (95% CI: 84.7- 99.9%) and 100% (95% CI: 99.9-100%)
  • For Patau Syndrome (T13): it is 100% (95% CI: 75.3-100%) and 99.98% (95% CI: 99.9-100%), respectively.

The maximum specificity of neoBona allows to reduce the number of false positives to practically zero (<0.1%), avoiding a high number of unnecessary invasive procedures, while its sensitivity is greater than 99%, which means that, in practice, it is very similar to a diagnostic test, which detects 100% of occurrences. 

 

In conventional screening the sensitivity for reaching 90%, while the specificity is 95% (with a 5% false positive index), in which out of every 100 healthy fetuses 5 are erroneously classified as positive, and are submitted to invasive procedures that endanger the mother and the fetus.

 

What is the difference between NIPT and invasive tests and its risks for the pregnancy?

NIPT is a screening test, while the diagnostic test requires an invasive procedure, which can be performed between 11 and 14 weeks of gestation by chorionic villus biopsy (CVS) of the placental tissue. 

 

Alternatively, after the 15th week of gestation, the sample can be obtained by amniocentesis. Both procedures have a small risk of causing miscarriage. The degree of risk is commonly reported as 0.5-1%, although recent studies have suggested that the actual risk related to the procedure may be much less.

 

Another issue is the rates of false positives and false negatives: 

The NIPT false positive rate for Down syndrome, for example, is generally 0.1% (which means that the cfDNA test is positive for such a change, but the fetus is later determined to be unaffected). In a pooled study, the cumulative false positive rate was less than 0.4%.

 

Most false positives result from the presence of an increase in chromosome 21-specific DNA, the origin of which does not reflect the chromosomal composition of the fetus in the current pregnancy. Possible origins of this increased DNA include confined placental mosaicism (presence of two or more karyotically different cell lines present in the placenta and absent in the fetus), an absorbed twin, maternal mosaicism and other maternal medical conditions (presence of bone marrow or tissue transplantation, for example).

 

Although it is difficult to determine the true rate of false-negative results, these results are more common when the fetal fraction is low and/or when placental mosaicism is present. 

 

Overall, there are four main reasons for low fetal fraction

  • Early gestational age: the fetal fraction is much smaller before the 10th week of pregnancy. In the 10th week, the fetal fraction is around 13%; 
  • Maternal obesity: the higher the maternal BMI the lower the fetal fraction, possibly due to the dilution effects of the greater maternal plasma volume; 
  • Fetal aneuploidy: fetal fraction is lower in pregnancies with chromosomes 13 and 18 trisomies, and chromosome X monosomy, for example: 
  • Problems with sample quality: the degradation of white blood cells in the maternal blood increases the fraction of maternal cfDNA and dilutes the fraction of fetal cfDNA. To avoid this complication, samples must be collected and stored properly to avoid DNA degradation.

 

False-negative rates for the most commonly targeted aneuploidies are not sufficient to guarantee a “diagnostic test” status. In this case, the diagnostic test used is the fetal karyotype test.

The fetal karyotype test can be performed from a chorionic villus biopsy (CVS) or by amniocentesis. All of these collection procedures for performing the karyotype are invasive; therefore, they represent a small risk for the pregnancy, and amniocentesis is still considered the gold standard for invasive prenatal examinations. 

The conditions that contraindicate the use of NIPT are, in general, cases of fetal malformations, screening for less common aneuploidies, nuchal translucency equal to or greater than 3.5 mm, suspected fetal triploidy and congenital infections, due to the very high risk level of genetic alteration, in which the direct indication of invasive fetal karyotype is justified.

Discordant results between NIPT and invasive fetal karyotype may occur due to biological processes, such as aneuploidy confined to the placenta, an absorbed twin, maternal aneuploidy or maternal cancer. 

It is worth mentioning that NIPT is a major advance in the screening of prenatal aneuploidy, but it is a screening test, and not a diagnostic test, in which it represents a risk for fetal chromosomal changes, and its confirmation must be performed by chorionic villi sampling or amniocentesis. In addition, genetic screening is optional and is at the discretion of each individual patient, together with their physician.

Furthermore, the interpretation of NIPT must always be performed by the requesting physician based on the patient’s clinical and family history and in conjunction with other laboratory findings.

 

About the SYNLAB Group

The SYNLAB Group is a leader in providing medical diagnostic services in Europe, providing a full range of clinical laboratory analysis services to patients, healthcare professionals, clinics and the pharmaceutical industry. Resulting from the Labco and Synlab merger, the new SYNLAB Group is the undisputed European leader in medical laboratory services.

 

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