Non-invasive prenatal tests (NIPT) are carried out to confirm or exclude the occurrence of three chromosomes in a fetus, that is to say chromosomes 21 (Down syndrome), 13 (Patau syndrome), and 18 (Edwards syndrome).

History of NIPT throughout the world and in Slovakia:

1997discovery of free circulating fetal DNA,
1997non-invasive fetal sex determination using the detection of Y-chromosome sequences,
1998quantitative analysis of free fetal DNA in the maternal circulatory system,
1998non-invasive fetal RhD status determination the,
1999rapid elimination of free fetal DNA after birth demonstrated,
2000non-invasive DNA testing focused on monogenic inherited congenital disorders (achondroplasia),
2008pilot studies focusing on non-invasive fetal aneuploidy detection,
2009first application and confirmation of the concept based on the potential of new generation sequencing (NGS) in detecting fetal trisomy 21,
2010diversity of maternal and fetal DNA fragmentation characterized in detail,
2010detailed fetal genome maps determined non-invasively,
2011commercial NIPT available for the first time,
2012complete fetal genome sequence obtained non-invasively,
2015the first fetal DNA test based on maternal blood is introduced to Slovakia and used in clinical practice – TRISOMY test.

The Principle

Our screening test is based on analyzing the whole genome sequence of free fetal DNA isolated from the peripheral blood of a pregnant woman. The data obtained by the sequenator analysis with low coverage is evaluated using a custom calculation algorithm and verified in accordance with the procedure published by Brianchi et al. (2012). *


* Bianchi DW et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012 May; 119 (5):890 – 901.

Clinical Significance

The objective of the high sensitivity and specificity TRISOMY test is to detect frequent fetal trisomy types in early stages of pregnancy to minimize the necessity of amniocentesis,* which carries certain miscarriage risks resulting from amniotic fluid sampling.

Despite its very high sensitivity and specificity in detecting the monitored trisomy types, TRISOMY test is a screening method, not a diagnostic examination.

In practical terms, every positive TRISOMY test result must be subsequently confirmed by invasive sampling of fetal genetic material (amniotic fluid or chorionic villi) and subsequent genetic examinations of the sample obtained (see references).

A negative TRISOMY test result excludes the presence of trisomy 21, 18 and 13 in the fetus. Generally, a negative test result does not need to be validated by an invasively obtained sample (see references).

* Bianchi DW et al. Genome-wide fetal aneuploidy detection by maternal plasma DNA sequencing. Obstet Gynecol. 2012 May; 119 (5):890 – 901.

Requesting Physician

The test can be indicated by a specialized gynecologist, an obstetrician or a clinical geneticist.

Sensitivity and Specificity

NIPT is generally characterized by high sensitivity and specificity. Fetal DNA tests provided by various laboratories usually vary in certain technical details. These details bring about different NIPT key parameters, particularly:

  • the rate of detected trisomic fetuses – Detection Rate (DR)
  • the rate of false positive results – False Positive Rate (FPR)

The sensitivity of NIPT with regard to trisomies 21 and 18 depends on specific laboratories and test types carried out, typically ranging between 94.4 and 100 % at a false positive rate of 0 – 2.05 %.

The sensitivity of NIPT with regard to trisomy 13 is lower (79 – 92 %). The potential failure of NIPT can be caused by technical reasons or very rare occurrences of trisomy 13 pregnancies in our population. The false positive rate expected is between 1 and 2 %.

NIPT – Detection of Chromosomal
Aneuploidy – Reliability

TrialDown syndrome
DR (n (%))FPR (n (%)
Chiu et al., 201186/86 (100)3/146 (2,1)
Ehrich et al., 201139/39 (100)1/410 (0,24)
Palomaki et al., 2011209/212 (98,6)3/1471 (0,20)
Bianchi et al., 201289/90 (98,9)0/410 (0,00)
Sparks et al., 201236/36 (100)1/123 (0,81)
Ashoor et al., 201250/50 (100)0/297 (0,00)
Norton et al., 201281/81 (100)1/2888 (0,03)
Total590/594 (99,3)9/5745 (0,16)

Sensitivity and Specificity

n = 203Sensitivity95 % CISpecificity95 % CInP
T21> 99 %90,97-100 %> 99 %97,78-100 %39
T18> 99 %*> 99 %*5
T13> 99 %*> 99 %*2

n – total number of samples in the validation study
nP – number of positive samples
CI – Confidence Interval
* 95 % CI not evaluated due to lack of samples

Number of samples with no result (NIPT): < 5 %.
Number of samples with no result (TRISOMY): < 1 %.

The sensitivity and specificity of the TRISOMY test is fully comparable with other NIPT methods. The result of the analysis is based on a validation study and supported by an analysis of reference samples with a known karyotype.

All trisomy 21, 18 and 13 samples included in the study were successfully detected by the TRISOMY test.

A Unique Blood Sampling Procedure

The TRISOMY test laboratory processes include a unique procedure that provided an increased proportion of fetal DNAcompared to the initial state. This is ensured by the physical diversity of fetal DNA and maternal DNA. Thanks to this procedure, the laboratory can obtain an average proportion of fetal DNA in the amount of 16 % in the TRISOMY test samples (the average proportion of fetal DNA in the published studies ranges from 10 to 13 %). More importantly, no set of samples processed using this special procedure has achieved a lower proportion of fetal DNA than 7.5 %, which significantly reduces the risk of test failure caused by a low content of fetal DNA fraction in the examined sample.

Applying our preparation, analysis and evaluation method characteristics on the basis of pregnant women with a healthy fetus and pregnant women with a detected trisomy of chromosome 21 used within our validation study, it is statistically possible to estimate false positive and false negative results depending on the portion of fetal DNA in maternal blood. Following the information that is available, we know that the portion of fetal DNA in maternal blood is subjective and the occurrence of samples with low fetal fraction (< 4 %) has been detected in less than 5 % of pregnant women (an average proportion of fetal DNA is about 14.5 % – Hudecova et al., 2014). **

Comprising 164 analyzed samples taken from pregnant women with a normal fetus and 39 pregnant women with detected trisomy 21, our validation study suggests that an average proportion of fetal DNA in the samples obtained from pregnant women whose fetus suffered trisomy 21 amounted to 16 % (the blue curve in Graph 1). If the proportion of fetal DNA in maternal circulation gives such values, the probability of obtaining a non-informative result is ~ 1:3 x 1013 and the probability of obtaining a false negative result (i.e. wrong) is lower than 1:9×1018.


The probability of obtaining a non-informative result in a sample with the lowest proportion of fetal DNA in our study with 7.5 % (red curve) is ~ 1:15, and the probability of obtaining a false negative (i.e. wrong) result is less than 1:740.

The analysis of samples from pregnant women with a healthy fetus (blue curve) suggests that the probability of obtaining a non-informative result is ~ 1:161 and the probability of obtaining a false negative (i.e. wrong) result is less than 1:30,000.

In order to minimize the risk of obtaining a false negative or false positive result caused by pregnancies with low fetal fraction which naturally occur in our environment, the results of the analysis are evaluated using a special zone that defines non-informative results – the so-called “grey zone”. In our graph (Graph 2), this zone is represented by grey shading. If the sample analyzed falls within this zone, it is necessary to analyze it repeatedly. In this regard, an additional blood sample is required within a two-week span.


** Hudecova I, Sahota D, Heung MM, Jin Y, Lee WS, Leung TY, Lo YM, Chiu RW.Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies.PLoS One. 2014 Feb 28;9(2):e88484. doi: 10.1371/journal.pone.0088484

Data Evaluation Using a Custom Algorithm

Obtained through whole-genome sequence analysis of free fetal DNA, the data corresponding to individual NIPTs is evaluated using demanding mathematical models; the data varies depending on the specific laboratory and procedure used. The objective of the evaluation is to determine the so-called Z-score (Graph 4).


The improved custom algorithm we use for TRISOMY test data evaluation allows us to achieve a higher average Z-score value in the evaluated samples compared to the calculation methods used by our competitors.

The higher average value of the Z-score in positive samples achieved using our improved algorithm for TRISOMY test evaluation is automatically reflected in a more accurate distribution of negative and positive result values, which results in a reduction of the so-called grey zone (Area of Overlap), where it is not possible to decide clearly whether the test result is actually positive or negative.


Since TRISOMY test is a screening method rather than a diagnostic examination, both false positive and false negative results may occur. A positive result of the analysis must be diagnostically confirmed by diagnostic methods that are currently available, i.e. subsequent genetic amniotic fluid examination.

A negative result does not exclude the tested trisomy types in absolute terms. There may be inaccuracies that are caused by naturally occurring phenomena, such as placenta mosaicism, vanishing twin syndrome, point mutations, gene inactivation, or other types of genetic or epigenetic mechanisms, or presence of foreign DNAs in maternal blood.

In a certain small percentage of cases, it is not possible to obtain a clear NIPT result. Such tests are considered unsuccessful.*** The failure may be caused by either by technical or biological reasons. An improper blood sample or unsuitable transport conditions are the main technical causes of such test failures.

The most frequent biological reason of test failures is a reduced proportion of fetal DNA in the obtained sample compared to the content of maternal DNA. If the proportion of fetal DNA in the obtained samples is less than 4 %, the reliability of the test drops significantly (Graph 5). According to the results presented by the studies published so far, it is possible to expect samples with a reduced proportion of fetal DNA fraction within the range of 1.5-6.1 %.

The results obtained by the evaluation of samples with fetal DNA content lower than 4 % are not suitable for interpretation.


*** M. M. Gil, M. S. Quezada, R. Revello, R. Akolekar, and K. H. Nicolaides, „Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis,“ Ultrasound Obstet. Gynecol. 45 (3), 249-266 (2015)