Welcome to the news page. This page is updated regularly and provides the latest news and updates, including highlights from professional meetings, key research findings, society developments, and other information about noninvasive prenatal testing.
NIPT is neither negative nor positive, there is no result! Doctor, what is my problem?
While all non-invasive prenatal test providers are vocally about the high sensitivities and specificities of their tests, much less information is provided about the frequency tests fail to provide a result at all. Nevertheless, a significant proportion of women get to hear that NIPT does not provide a result. This percentage varies between 1% to over 10%! Some providers of the most commonly used tests do provide values for failure rates on their website. For VeriSeqv2 a 1.2% whereas NIFTY reports a 2.8% failure rate. Other common NIPT providers either only mention the possibility that there is no conclusive result (Panorama) or do not mention this possibility at all (Harmony, Clarigo, Veracity). We encourage those providers to also include this information to pregnant women and professionals. To rescue a ‘failed’ test, NIPT providers will often request a redraw. The redraw can rescue a significant number of tests which failed during a first draw. However, this rescue ability will be test dependent.
Why are tests ‘failing’? ‘Failing’ is put between parentheses on purpose. There are two types of failures. A true failure is a technical mistake which does not provide a test result. This can be administrative issues such as sample collection, labeling, and transportation or it can be technical issues including DNA extraction, amplification or sequencing. However, in most high-volume environments such technical artefacts are reduced to a minimum and make up only a small minority of the failures. More important are ‘failures’ where the laboratory test is perfectly executed. There is a biological reason for a test not to be able to discriminate between a positive or a negative test result. Rather than a failure, we call those ‘inconclusive results’. I will now zoom in into those reasons.
Probably the most important reason for a test to be inconclusive is a low fetal fraction. The placental DNA floating around in the maternal blood stream varies amongst pregnancies. Whereas the mean free-floating fetal DNA fraction (cffDNA) in maternal blood is about 10%, this percentage varies broadly. For many tests, a minimum fetal fraction of 4% is required to assure the test to be reliable. We now know that in about 2-4 % of all pregnancies, the cffDNA fraction is lower than 4%. It should be noted that fetal fraction determination varies amongst different NIPT and, hence, incidences of low fetal fraction may vary. In addition, the 4% rule is somewhat arbitrarily based on probability estimates that a test will still provide accurate results. Some test approaches can obtain reliable results with lower fetal fractions. Since the cfDNA fraction is directly proportional with the placental size and since the placenta grows during pregnancy, we see a gradual increase in cfDNA during pregnancy progression. Hence, a redraw a few weeks following a first inconclusive report, often provides a conclusive result. There are different reasons why the fetal fraction may be lower than average. Since all tissues of our body shed DNA in the blood stream, we see a negative correlation of the cfDNA fraction with increasing BMI. Women with high BMI have more tissue shedding DNA and the cffDNA fraction is reduced. Other reasons are often placenta related. Some placentas are smaller than others. When below the fifth percentile, the oby/gyn will call this intrauterine growth retardation. In such pregnancies, a lower amount of fetal DNA will be detected.
The second most frequent cause for an inconclusive result is that the probability for a trisomy is too high to be negative but too low to be positive. In this probability area the result is ambiguous. When a test value is within this ambiguous area, the test provider will report he cannot conclude about the presence or absence of trisomy 21 (or trisomy 13, 18, X or Y). The probability for a value to be in this ‘grey’ zone is test dependent and may vary amongst providers. Ambiguous test results can be the result of statistical variation (which is test dependent) or can also have a biological cause. As discussed in the previous newsletter, a trisomy may not be present in all cells of the placenta. If only a fraction of the placenta contains cells with trisomy 21 and the remaining cells are normal, this would result in a slightly elevated signal. This signal would be lower than predicted for a normal trisomy but higher than expected when normal. Such signals are often undetermined. Repeated sampling will not resolve the issue and the result will remain undetermined.
Finally, there are a flurry of other biological causes that can influence NIPT negatively. First, the presence of a maternal cancer would confound the fetal cfDNA analysis and may cause abnormal signals. This is because also cancer cells release DNA in the maternal bloodstream and the majority of cancers are characterized by chromosomal imbalances. The imbalances present in the circulating tumor DNA interfere with the circulating free fetal DNA anlaysis. Maternal cancers are predicted to occur about 1 every 2000 pregnancies. However, the signals are observed in about 1 every 10000 pregnancies. Maternal autoimmune diseases, such as lupus, can influence some test results. It appears that such autoimmune diseases could interfere with some tests. There is circumstantial evidence that other maternal medical conditions could, very occasionally, influence a NIPT.
In conclusion, the occurrence of a failed or inconclusive results is quite common. As Yuval writes: “Having received a ‘no result’ call both patient and physician are faced with a dilemma – should a repeat sample be drawn or directly opt for invasive testing? A redraw can often (>50%) lead to a positive result. For some causes of inconclusive results, the use of a test based on other principles may be an option to obtain a positive answer. Redraws may resolve some of the cases but may significantly increase turnaround time. The possibility of an inconclusive result should be a part of the pre‐test counseling. When assisting patients with the choice of an NIPT provider, the anticipated test failure rates should be described in no less detail than other often quoted metrics (such as sensitivity, specificity, and PPV). It should be made clear that NIPT test failures are associated with a decrease in actual sensitivity. Best advice may be to choose an NIPT platform that is associated with the lowest technical failure rate.”