FAQs - Section 3: How a Non-invasive Prenatal Test (NIPT) Works

A non-invasive prenatal test is an advanced prenatal screening test for certain common chromosomal aneuploidies or deletions. The most common clinically significant aneuploidies involve chromosomes 21, 18, 13, X and Y, and the most common clinically significant deletion involves chromosome 22q11.2. Aneuploidy of other chromosomes can occur, but is usually associated with an early miscarriage.

NIPT is an assessment for clinically significant aneuploidies involving the five chromosomes mentioned above and, if requested, a deletion of chromosome 22q11.2; other aneuploidies or deletions are not considered further in this section.

During pregnancy, a mother’s blood contains small fragments of DNA derived from the placenta. The placenta develops from the same fertilised egg as the fetus, and hence the genetic content of the placenta is usually the same as that of the fetus.

NIPT involves testing more than a million short fragments of these DNA fragments in maternal plasma. Some of these fragments come from the placenta, and most will be from the mother. To screen for aneuploidies, we count the number of fragments derived from each chromosome. In a mother with a chromosomally normal fetus, the proportion of fragments from each chromosome will be within a narrow normal range. But if the fetus has an excess or deficiency for a tested chromosome (or part of a chromosome), this proportion will change and can be detected.

Note that NIPT is an indirect assessment of fetal chromosome status using small amounts of placental DNA in maternal plasma. For this reason, NIPT is not a definitive diagnostic test. It is an excellent screening test, but clinically significant results should always be confirmed by a definitive diagnostic test, such as amniocentesis or chorionic villus sampling.

A variety of screening tests are used during pregnancy. Prior to conception, or early in the pregnancy, women are screened for medical conditions in the mother that may compromise the continuing health of the pregnancy. This includes tests of maternal infection, e.g. rubella, and abnormalities of haemoglobin, e.g. thalassaemia carrier status. NIPT does not replace these investigations as it is an examination of specific fetal chromosomes, not of maternal health.

In the third month of pregnancy, screening for structural and chromosome abnormalities in the fetus is done by maternal serum screening, plus fetal ultrasound. This is known as first trimester screening (FTS). FTS is reasonably sensitive, but it will miss a proportion of genetic abnormalities in the fetus. On the other hand, FTS has the advantage of being a general screen of structure and function, and can pick up abnormalities that may be missed by a more focused genetic test.

NIPT is a genetic test that screens for abnormalities in the number of particular chromosomes (21, 18, 13, X and Y) of the fetus, i.e. fetal aneuploidy, as well as screening for a 22q11.2 deletion. NIPT does not directly assess the structure or function of the fetus. NIPT is a much more accurate test for these specific abnormalities in chromosome number than other screening investigations, but it does not cover the same range of potential abnormalities.

Yes, there is an inverse relationship between maternal body weight and the concentration of fetal DNA (fetal fraction) in maternal plasma.

  • For a pregnant woman weighing 60 kg, there is a 99% chance that there will be sufficient fetal DNA for NIPT at 10 weeks’ gestation.
  • For a pregnant woman weighing 100 kg, the chance that there will be sufficient fetal DNA for NIPT drops to 90%.
  • For a pregnant woman weighing 150 kg, the chance that there will be sufficient fetal DNA for NIPT drops further to 50%.

This relationship between maternal weight and fetal fraction does not compromise the accuracy of Harmony. Irrespective of a woman’s weight, Harmony will provide a highly accurate screening test if there is sufficient fetal DNA. If there is insufficient fetal DNA, Harmony identifies this fact and does not provide a risk assessment.

Other aspects of the mother’s health are unlikely to interfere with NIPT. However, NIPT may not be accurate in a woman who has had organ transplantation or concurrent invasive cancer. The transplanted tissue or cancer can release DNA into maternal plasma, which interferes with the analysis underlying the test. In addition, mothers can rarely be mosaic for chromosomal aneuploidy themselves, and this can interfere with fetal chromosome analysis by NIPT.

NIPT examines DNA fragments from the placenta that are circulating in the mother’s blood. These DNA fragments, known as cell-free DNA, last only an hour or so, and are replaced continuously from the placenta throughout the pregnancy.

Once the baby has been delivered, the last remaining fragments of the placental DNA disappear from the mother’s circulation within a couple of hours. As a result, there are no remaining DNA fragments from the baby that might interfere with the Harmony test in the woman’s next pregnancy.

More than 15 years ago, it was recognised that DNA in the maternal plasma has contributions from both the mother and the fetus. Since then, numerous technical strategies have been developed to analyse the DNA and provide information regarding the risk of chromosomal abnormality in the fetus. Laboratories have developed different methods for detecting and measuring fetal DNA, as well as different computer programs to interpret these raw data.

The various methods have been assessed and validated to varying degrees. Not all types of NIPT are available in Australia, or are provided by an accredited laboratory in Australia. We recommend that doctors request NIPT only from an accredited provider who can provide documentation of the clinical accuracy of the test.

No.

The Harmony Prenatal Test was developed by Ariosa Diagnostics and has been used on more than 1,000,000 pregnancies worldwide. Sonic Genetics performs this test in Australia under licence. We follow the manufacturer’s approved protocols that are used in all laboratories providing the Harmony test worldwide, ensuring that we maintain the accuracy and quality that have given this test such a good international reputation.

Other tests available in Australia fall under two different categories:

  • The testing laboratory purchases a test kit from a manufacturer and adapts the protocol to establish its own assay. The manufacturer’s validation data cannot be used to verify the performance of the test, as the laboratory is not using the exact same method as the manufacturer.
  • Some Australian laboratories send NIPT samples overseas for testing in laboratories that are not accredited by Australian authorities.

Some laboratories use both strategies, doing part of the NIPT test in Australia and also sending the sample overseas for others tests, such chromosome deletion studies.

Sonic Genetics provides the Harmony test, including the test for 22q11.2 deletion, in our Australian laboratory. Our laboratory and staff are accredited by Australian regulatory authorities. We use the protocols that are specified by the manufacturer to ensure that the quality of all aspects of our test process is maintained.

No.

NIPTs are performed in Australia by one of two methods. One is unique to Harmony and the other is shared by other providers.

Before the risk assessment is performed, Harmony measures the fetal fraction in the sample, i.e. the proportion of the DNA in the blood sample that was derived from the placenta and fetus. If there is an insufficient quantity of fetal DNA, the risk assessment will not be performed.

At 10 weeks’ gestation, approximately 3% of samples will not have sufficient fetal DNA for analysis by Harmony. If there is insufficient fetal DNA, a NIPT cannot provide an accurate assessment of the risk of a fetal chromosome disorder. In a recent study in the US, blood samples were sent from non-pregnant women to laboratories that do not report fetal fraction; these laboratories failed to identify that there was no fetus and simply reported that there was a “normal female fetus”. The same samples were sent for the Harmony test, and they were successfully identified as not being suitable for a risk assessment.

Harmony also differs from other NIPTs in the method used. Harmony uses targeted testing to focus on the chromosomes of interest, and to differentiate fetal and maternal DNA fragments. Other NIPTs use a non-targeted approach, which requires the assessment of many more DNA fragments, most of which are not useful in providing the risk assessment.

Sonic Genetics regularly reviews the various implementations of NIPT and assesses their performance, scope and cost. Our recommended form of NIPT is the Harmony Prenatal Test, which has been developed by Ariosa Diagnostics in the US.

We recommend the Harmony Prenatal Test for the following reasons:

  • The Harmony Prenatal Test includes a precise measure of the amount of fetal DNA in the maternal circulation. This assures the quality of NIPT in two important ways. Firstly, this accurate measurement identifies samples in which there is insufficient fetal DNA to draw an accurate conclusion. This occurs in 3% of samples and is not necessarily identified by other methods of NIPT. This is particularly important in women with a relatively high body weight; increased maternal body weight is associated with a lower concentration of fetal DNA in maternal plasma. If a lack of fetal DNA is not recognised, the NIPT may yield a 'normal result' which simply reflects the mother’s DNA and says nothing about the fetus. The Harmony Prenatal Test confirms that there is sufficient fetal DNA to draw a conclusion before providing a test result.

    Secondly, the measurement of fetal DNA is incorporated into the risk assessment, allowing for a clearer delineation between normal and abnormal results, resulting in greater confidence for clinicians and patients. Other types of NIPT do not necessarily include a measurement of the amount of fetal DNA in their risk assessment.

  • The underlying principle of the Harmony Prenatal Test is to analyse a limited number of targeted DNA fragments. This, together with some technical aspects of test implementation, has resulted in a robust and highly reproducible assay. This is in contrast to the analysis of untargeted DNA fragments in some other forms of NIPT, necessitating analysis of 20 times more DNA fragments than is necessary with the Harmony Prenatal Test.
  • Most of the published experience with NIPT has involved small cohorts of women at relatively high risk of having an abnormal fetus. A publication in the New England Journal of Medicine (23 April 2015, 372:1589-97) documented the superb performance of the Harmony Prenatal Test in a prospective blinded study of more than 15,000 pregnant women of various ages and risk across multiple international sites.
  • The quality and stability of Harmony have been documented in more than 1,000,000 patient samples through the clinical laboratory of Ariosa Diagnostics in the USA and other Harmony laboratories worldwide. In contrast, other NIPT methods cannot necessarily provide the same assurance regarding consistency.

Combined first trimester screening (maternal serum screening with fetal ultrasound) and NIPT provide complementary information about the fetus. There is overlap in the range of conditions that they detect, and each screening method can identify abnormalities that will be missed by the other. For this reason, some clinicians recommend combined first trimester screening and, subject to the result, NIPT. Others recommend detailed ultrasound and NIPT for all patients.

Pending the publication of national clinical practice guidelines, the choice and sequence of screening tests used in early pregnancy is a matter of clinical judgement. Sonic Genetics recommends that non-specialist practitioners providing antenatal screening align their practice with their preferred specialist obstetric service.

Harmony represents a choice that an informed woman may select to provide her with additional information regarding her pregnancy. There are three ways in which Harmony can be included in the management of pregnancy.

  • If a woman has conventional first trimester screening and is identified as being at increased risk of having an aneuploid fetus, NIPT is a safe alternative to invasive chromosome testing by amniocentesis or CVS. In this situation, a normal NIPT result is almost certainly normal and invasive testing is not indicated. (If the risk estimated from first trimester screening is very high, e.g. greater than one in 10, it may still be appropriate to recommend invasive testing; discussion with a specialist is recommended). However, if the NIPT result is high risk, it is very likely that the fetus does have aneuploidy; diagnostic invasive testing is recommended for definitive confirmation. This approach provides efficient use of NIPT (not all women will have the test), but it is limited by the overall accuracy of first trimester screening. In other words, this strategy will fail to identify some abnormal pregnancies because of the limitations of first trimester screening.
  • A second strategy is to offer all pregnant women both conventional first trimester screening and NIPT. The combination of tests has a very high accuracy for detecting fetal aneuploidy, as well as providing a more general screen of fetal structure and biochemical function. This approach is more expensive overall, as a woman is being offered NIPT irrespective of the result of conventional first trimester screening.
  • The third strategy is called 'contingent screening', and is hybrid of the first two strategies. The principles are that a woman has conventional first-trimester screening. If she is at high risk of fetal aneuploidy, she is offered invasive genetic testing by amniocentesis. If she is at moderate risk of fetal aneuploidy, she is offered NIPT. If she is at low risk of fetal aneuploidy, further testing is not medically warranted. The definitions of what constitutes high, moderate and low risk of aneuploidy remain matters of debate and analysis. These parameters are not dictated by Sonic Genetics. We recommend that non-specialist practitioners align their practice with their preferred specialist obstetric service.

Harmony is not indicated if the fetus is known to have major congenital malformations. Such malformations could be caused by a variety of chromosome abnormalities or be non-chromosomal in origin. The Harmony test provides an assessment for selected abnormalities and, in this setting, the underlying abnormality may be missed by Harmony. The more appropriate investigation may be fetal chromosome studies obtained by CVS or amniocentesis or a more detailed scan.

Harmony has not been validated:

  • in pregnancies with three (or more) fetuses.
  • in the presence of a demised twin on ultrasound at the time of collection.
  • at a gestation of fewer than 10 completed weeks.
  • in women with an organ or marrow transplant.
  • in mothers aged less than 18 years; however, we will issue a result for mothers aged 16–17 years, noting the lack of validation data in this age group.

Harmony can be used in:

  • IVF pregnancies using egg or sperm from the couple or donor.
  • IVF pregnancies where a surrogate is used.
  • twin pregnancies.

NIPT is not for everyone

Many women view NIPT as an empowering investigation that gives them assurance in their pregnancies. However, for others, the possibility that NIPT might reveal a major problem for the developing baby means that they hold back in feeling and sharing the joy of being pregnant until the test result is back. Instead of liberating these women to enjoy their early pregnancy, they feel that they must place any sense of involvement in the pregnancy 'on hold' until the test result is back.

If the NIPT result is indeed abnormal, this raises the question of what to do next. Would the patient consider a more invasive investigation, such as amniocentesis, to confirm the NIPT result? And if the amniocentesis is abnormal, would she continue the pregnancy or consider a termination of pregnancy?

These are not trivial issues, and the associated decisions are not lightly made. For that reason, NIPT represents an important option for a woman to consider, but it is not an obligation. It would be very important that a pregnant woman consider the pros and cons of NIPT, discussing them with her partner and her doctor, before deciding whether to proceed with the investigation.