NIPT

Non-Invasive Prenatal Testing (NIPT) is carried out to confirm or exclude the presence of selected foetal chromosome disorders.

Odborné informácie

Referrals for TRISOMY test XY screening are given by specialists in gynaecology and obstetrics or clinical genetics.

 

History of NIPT throughout the world and in Slovakia:

1997discovery of free circulating foetal DNA,
1997non-invasive foetal sex determination using the detection of Y-chromosome sequences
1998quantitative analysis of free foetal DNA in the maternal circulatory system
1998non-invasive foetal RhD status determination
1999demonstration of the rapid elimination of free foetal DNA after birth
2000non-invasive DNA testing focused on monogenic inherited congenital disorders (achondroplasia)
2008pilot studies focusing on non-invasive foetal aneuploidy detection
2009first application and confirmation of the concept based on the potential of new generation sequencing (NGS) in detecting foetal trisomy 21
2010diversity of maternal and foetal DNA fragmentation characterized in detail
2010detailed foetal genome maps determined non-invasively
2011commercial NIPTs available for the first time
2012complete foetal genome sequence obtained non-invasively
2015the first foetal DNA test based on maternal blood is introduced in Slovakia to be used in clinical practice – TRISOMY test
2016TRISOMY test used more widely along with its the TRISOMY test + variant, which is designed to detect additional chromosome aberrations

Test principle

Our screening test is based on analysing the whole genome sequence of free foetal DNA isolated from the peripheral blood of a pregnant woman. The data obtained by low-coverage sequencing analysis 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 screening is to detect frequent foetal trisomy types in early stages of pregnancy to minimize the necessity of amniocentesis,* which poses certain miscarriage risks resulting from the amniotic fluid sampling procedure.

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

In practical terms, this means that every screen-positive TRISOMY test result must be subsequently confirmed by invasive sampling involving foetal genetic material (amniotic fluid or chorionic villi), which is subsequently genetically analysed (see references).

A screen-negative TRISOMY test result excludes the presence of foetal trisomy 21, 18 and 13. As a rule, a screen-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.

Referring physician

Referrals for TRISOMY test screening are given by specialists in gynaecology and obstetrics or clinical genetics.

Referrals for TRISOMY test XY screening are given by specialists in gynaecology and obstetrics or clinical genetics.

Referrals for TRISOMY test + screening are provided by specialists in medical genetics.

Sensitivity and specificity

NIPT screenings are generally characterized by high sensitivity and specificity. Foetal DNA tests provided by various laboratories usually vary in certain technical details. These details are reflected in various key NIPT parameters, particularly:

  • the rate of trisomic foetuses detected – DetectionRate (DR),
  • the rate of false positive results – False PositiveRate (FPR).

The sensitivity of NIPT screening with regard to trisomy 21 and 18 depends on specific laboratory parameters and the test types they use, typically ranging between 90 and 100% at a false positive rate of 0.1%.*

The sensitivity of NIPT screening with regard to trisomy 13 is lower (79 – 92%). Possible failure of NIPT screening may be caused by technical reasons or the fact that trisomy 13 pregnancies occur very rarely in the population. For these reasons, the false positive rate is estimated to be less than 0.1%.*

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)

TRISOMY test
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 validation study samples
nP – number of positive samples
CI – Confidence Interval
* 95 % CI not evaluated due to lack of samples

Number of samples with no results for NIPT: < 5%
Number of samples with no results for TRISOMY test: < 1%

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

A unique blood sample preparation procedure

The TRISOMY test laboratory procedure includes a unique process that ensures an increased proportion of foetal DNA compared to the initial amount. This is achieved thanks to the physical difference between foetal DNA and maternal DNA. Using this unique procedure for processing TRISOMY test samples, the laboratory typically manages to obtain an average foetal DNA proportion of 16% (the average proportion of foetal 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 foetal DNA than 7.5%, which significantly reduces the risk of test failure caused by a low content of foetal DNA fraction in the examined sample.

Applying our preparation, analysis and evaluation method characteristics to samples obtained from the pregnant women with healthy foetuses and the pregnant women with chromosome 21 trisomy foetuses used within our validation study, it is statistically possible to estimate the occurrence of false positive and false negative results depending on the portion of foetal DNA in maternal blood. Based on existing information, we know that the proportion of foetal DNA in maternal blood varies from subject to subject and the occurrence of samples with a low foetal fraction (< 4%) has been detected in less than 5% of pregnant women (the average proportion of foetal DNA is about 14.5 %).***

Comprising 164 analysed samples taken from pregnant women with normal foetuses and 39 pregnant women with trisomy 21 foetuses, our validation study suggests that the average proportion of foetal DNA in the samples obtained from pregnant women whose foetus suffered from trisomy 21 amounts to 16% (see the blue curve in Graph 1). Given this proportion of foetal DNA in the maternal blood system, 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 results) is lower than 1:9×1018.

Odborné informácie - graf

Thanks to our laboratory method, we did not observe proportions of foetal DNA lower than 7.5% in the set of samples under scrutiny. Enhanced by our own mathematical algorithm, the sensitivity of our test is so high that, even in the event of such low foetal DNA concentrations, TRISOMY test screening can practically detect all trisomy cases.

The probability of obtaining a non-informative result in a sample with the lowest proportion of foetal DNA observed in our study (7.5 %; see the 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 healthy foetuses (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 minimise the risk of obtaining a false negative or a false positive result caused by naturally occurring pregnancies with a low foetal fraction, the results of our analysis are evaluated with regards to a special zone that defines non-informative results – the so-called “grey zone”. In Graph 2, this zone is illustrated by grey shading. If an analysed sample falls within this zone, it is necessary to analyse it again. In this connection, another blood sample must be taken 14 days after the previous sampling.

Screening

***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 based on a custom algorithm

Obtained using whole-genome free foetal DNA sequencing analysis, data corresponding to individual NIPTs is evaluated using complex mathematical models. NIPT data sets vary from laboratory to laboratory and also depending on the procedure used. The objective of the evaluation is to calculate what is called the Z-score (Graph 4).

Analýza dát

Compared to the calculation methods used by our competitors, the advanced custom algorithm we use for TRISOMY test data evaluation allows us to achieve a higher average Z-score value with regard to the evaluated samples.

The higher median value of the Z-score in positive samples achieved using our advanced algorithm for TRISOMY test evaluation is automatically reflected in a more accurate distribution of negative and positive result values, which ultimately leads to a reduced grey zone (Area of Overlap) – an area that does not make it possible for us to decide clearly whether the test result is actually positive or negative.

Interference

Since TRISOMY test, TRISOMY test XY and TRISOMY test + are categorised under screening methods rather than diagnostic examinations, both false positive and false negative results may occur. A screen-positive result must therefore be diagnostically confirmed by any available diagnostic methods, i.e. by a genetic examination of the patient’s amniotic fluid.

A screen-negative result does not exclude the monitored 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 the presence of foreign DNA 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 either by technical or biological reasons. Such test failures are mainly caused by improperly obtained blood samples or improper transport conditions.

The most frequent biological reason for test failure is a relatively low proportion of foetal DNA in the obtained sample compared to the amount of maternal DNA. If the proportion of foetal DNA in the obtained sample is less than 4%, the reliability of the test drops significantly (Graph 5). According to the results presented by the studies published so far, the occurrence of samples with a low proportion of foetal DNA fraction ranges between 1.5-6.1%.

Results obtained by evaluating samples with foetal DNA proportions lower than 4% are not suitable for interpretation.

DNA grafy

Our analysis of 11,000 blood samples taken from pregnant women has shown that low foetal DNA fraction samples (<4%), which makes non-invasive prenatal testing complicated, represent almost 5% of all pregnant women.

****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)