1. How does the IONA® test estimate the risk of a pregnancy being affected with a trisomy?
The IONA® test combines the prior (background) risk of a trisomy with a likelihood ratio to generate a final risk score. The likelihood ratio is derived from the relative amounts of chromosome 21, 18 and 13 in cell free DNA from a blood sample of a pregnant woman1.
2. Does the IONA® test provide information on fetal sex and how accurate is it?
Yes, but only if requested. In some regions fetal sex determination is not permitted. If specifically requested, the test is able to identify whether the fetus is genotypically male or female. The IONA® test’s fetal sex and trisomy analysis pipelines are independent; as such, while technical and clinical factors can in some occasional circumstances contribute to a sex determination failure, a failed fetal sex test has no implication for the accuracy of a trisomy test result.
The IONA® fetal sex test is 99% accurate. Discordances between phenotypic and genotypic fetal sex assessed by the IONA® cfDNA analysis may potentially arise with technical failure or very rare biological conditions2.
3. Why is IONA® described as a screening test rather than a diagnostic test?
As with all NIPT tests, the IONA® test analyses the chromosome ratios of cell-free DNA in the mother’s blood stream derived from the placenta. Various rare conditions associated with the placenta or with the mother’s genome can alter the chromosome ratios in the absence of a fetal trisomy; therefore, a high-risk result may not accurately represent trisomy in the fetus. For this reason, high risk results must be checked with an invasive diagnostic test such as amniocentesis and in the case of a low risk result it’s important to be aware that there is still a very small (but not zero) risk of trisomy. A low risk result does not exclude the presence of other genetic abnormalities in the fetus such as triploidy, trisomies involving other chromosomes, single gene disorders, microdeletions chromosomal rearrangements.
4. What is the IONA® test risk score?
The IONA® test risk score is a measure of the chance of a pregnancy being affected with a trisomy, given the background risk and the observed relative amounts of chromosome 21, 18 and 13. It is calculated by combining the background (prior) risk and a risk ratio derived from chromosome measurements, using Bayesian statistical methods. If the measured level of a chromosome is close to that expected in an unaffected pregnancy then this generates a low risk score for the corresponding trisomy; if the chromosome level is elevated this results in a high risk score.
The maximum risk score for each trisomy produced is capped to account for the impact of biological factors which could confound the test result. The caps are T21: 95%, T18: 75% and T13: 60%. These are the maximum risk estimates that can be displayed on the report. The minimum risk score generated is also capped at 1:1,000,000, to improve the readability of the report.
The risk score represents the risk of trisomy as judged by the relative level of chromosomes 21, 18 and 13 combined with the background risk, but as stated above there is always a very small possibility of a false negative due to biological factors (for example: confined placental mosaicism or a vanishing twin).
5. What is fetal fraction and how is it used in the IONA® test?
The fetal fraction is the proportion of cell-free DNA which is derived from the placenta.1 The IONA® test estimates fetal fraction using a combination of sex chromosome analysis and differential sequence representation. The IONA® test requires a minimal fetal fraction of ≥2% to generate a valid result. Where the fetal fraction is between 2 and 4%, a dynamic fetal fraction method is used to assess the validity of samples, if certain additional QC criteria are met. This feature allows IONA® to maximise the number of samples available for analysis.
6. Is maternal age considered?
By default, the IONA® test uses the prior risk derived from the published risks for trisomies 21, 13 and 18 at the patient’s age when providing a blood sample3.
7. Is it possible to customise the prior risk based on other data?
The IONA® test has the option to replace the default maternal age derived prior with the output from the First Trimester Combined Test (FTCT) as the prior to provide a more tailored risk score. Other commonly used tests such as the triple test, the quad test and the integrated test also generate a risk score but no studies have yet been performed to date to confirm that they are suitable for use with the IONA® test. Clinically validated first and second trimester screening tests are summarised in these reviews:
•Cochrane Database of Systematic Reviews 2015, Issue 11 First trimester serum tests for Down’s syndrome screening Alldred SK, Takwoingi Y, Guo B, Pennant M, Deeks JJ, Neilson JP, Alfirevic Z
•Cochrane Database of Systematic Reviews 2012, Issue 6 Second trimester serum tests for Down’s Syndrome screening. Alldred SK, Deeks JJ, Guo B, Neilson JP, Alfirevic Z
8. Can the IONA® test be incorporated into a contingent screening model?
Yes. The IONA® test is suitable as a first or second-line screening test. Healthcare Providers may first offer the First Trimester Combined Test and then offer the IONA® test to the women who are identified as high-risk.4
9. What is the cut-off for high-risk?
In the UK, the cut-off used by the NHS for a high or low-risk result is set to 1/150. Higher or lower cut-offs can be specified by the user to meet local requirements.
10. Some clinicians prefer to use the phrase high chance rather than high risk, what is the difference?
There is no clinical distinction between the terms ‘risk’ and ‘chance’. A risk is the chance of an event occurring, for example, a risk of Down’s syndrome of 1 in 100 means that if 100 women have this test result, we would expect that 1 of these women would have a baby with Down’s syndrome and that 99 would not. This is the same as a 1% chance that the baby has Down’s syndrome and a 99% chance that the baby does not.
11. What further action is recommended for a patient following a high risk result on the IONA® test?
All high risk results should be reviewed by the Healthcare Provider and confirmed by further testing.
12. Which invasive diagnostic test is suitable for follow up testing?
Both chorionic villus sampling (CVS) and amniocentesis have been recommended as suitable sampling techniques for obtaining samples for further testing but there is no clinical consensus about which is preferred. CVS has the benefit that it can be conducted earlier in the pregnancy, but it has a disadvantage compared to amniocentesis in that it tests a sample of the placenta rather than fetal cells.
13. Can the test report be given directly to pregnant women?
No. The IONA® test Screening Report should be provided to the requesting Healthcare Provider who will explain the results to the pregnant woman, enabling her to make informed choices about her pregnancy. This explanation may include providing the report to the patient.
14. Which blood tube should be used for sending samples?
We recommend a Streck Blood tube (CE marked cell-free DNA BCT Streck) as this keeps the blood stable for up to 14 days. Standard EDTA tubes are also suitable if the sample can be centrifuged to separate plasma within 8 hours of blood draw.
We can advise on sample packaging and shipping the samples either to our in-house laboratory services for analysis or to any other laboratory performing the IONA® test.
15. What is the turnaround time for the IONA® test?
The minimum, uninterrupted time to process 24 samples, from DNA extraction to report generation, is approximately 36 hours, including hands-on time.
The turnaround time will depend on each individual laboratory set-up. Results should be available within 3-5 working days after sample receipt in the laboratory. Results are shared via the MyNIPT® portal directly with the Healthcare Provider who requested the test.
16. Can the IONA® test be used on patients expecting twins?
Yes, the IONA® test is suitable for twin pregnancies. The IONA® Software algorithm assumes all monochorionic twins are identical and consequently the test performance is the same as for singleton pregnancies. In the case of dichorionic twins, or if the chorionicity is not known, the IONA® Software uses a specific proprietary algorithm which assumes that they are non-identical and increases the fetal fraction required for a valid result.
In monochorionic twins (monoamniotic and diamniotic), the detection rate is >99%. In dichorionic twins, scientific publications suggest that the detection rate is reduced from >99% to around 95%5. The test cannot tell which twin is high-risk. If a high-risk result is generated, selective invasive confirmatory testing would be required.
17. What could cause inaccurate test results?
The IONA® test is very accurate but as with all NIPT there are several confounding conditions and circumstances which can lead to a loss of specificity or sensitivity. These can be broadly summarised as:
1. Conditions arising during pregnancy
2. Pre-existing conditions in the mother
3. Medical interventions in the mother
4. Sampling or sampling handling errors and laboratory-related failure
17.1.1. My patient has shown twin demise (vanishing twin) on ultrasound, can I use the IONA® test?
The IONA® test is not validated for use when a demised twin has been identified. The validation data for twin pregnancies is based on the presence of two live fetuses in the uterus.
• Following demise of a fetus, there is a period in which DNA from the vanished twin can still be detected in the maternal circulation. We have not performed a demised twin study and there is little published data on this phenomenon but recent publications suggest that cell-free DNA from the demised twin can be detected for up to 8 weeks6, 15 weeks7, and 17 weeks8 after the demise, or even throughout the remaining pregnancy9.
• Circulating cell-free DNA from a demised twin which carried a trisomy could potentially cause a false positive (high risk) result if the surviving fetus were euploid and the level of DNA from the vanished twin was sufficient. Often a patient will be unaware that their pregnancy has involved a vanishing twin and the sample will be sent for analysis without this information on the test request form. In these cases, there is a slightly increased risk of a false positive result for the reasons outlined above.
• In the event of selective fetal reduction or termination the same issues could arise.
17.1.2 What issues can arise in the case of confined placental mosaicism (CPM)?
The main source of fetal associated DNA in the cell-free DNA of a pregnant woman is derived from the placenta10. Normally, the placenta and the fetus share the same genotype but in rare cases the placenta and the fetus differ due to a condition termed “confined placental mosaicism”11 . In these cases, the placenta could contain trisomic cells whilst the fetus was completely euploid – this could lead to a false positive result. Similarly, a euploid placenta and a trisomic fetus could result in a false negative result.
17.2 Pre-existing medical conditions in the mother
The IONA® test measures the ratio of chromosome 21 and other chromosomes to the total amount of cell free DNA in the circulation. The test is designed to expect maternal cell free DNA with the karyotype 46,XX and any existing conditions that could perturb this ratio would exclude the woman from taking the IONA® test. Existing conditions include both inherited and acquired chromosomal imbalances
17.2.1 Can the IONA® test be performed if one or other of the parents has a translocation?
• The test is suitable for couples with Robertsonian Translocations involving chromosomes 21 or 13 who are at elevated risk of a Down Syndrome or Patau Syndrome affected pregnancy respectively.
• The chromosomal complement of cells with trisomy 21 or trisomy 13 incorporating a Robertsonian Translocation is lacking one copy of the p arm of chromosome 21 or 13. These genomic regions do not contribute a significant part of the data used by the IONA® algorithm, so their absence has minimal impact on the IONA® test itself. Although the IONA® test is able to detect Down’s syndrome in pregnancies from parents who carry a Robertsonian Translocation the test is not able to determine the presence or absence of such a translocation in the fetus.
• If it is known that the mother or father has a relevant Robertsonian Translocation, this should be noted on the Test Request and Patient consent form with as much detail as possible.
•Other balanced or non-balanced translocations may lead to gametes with chromosomal rearrangements which could result in problems with the pregnancy. The IONA® test is not designed to detect these conditions.
17.2.2 Can the IONA® test be used on pregnant women who have cancer?
No, malignant tumours shed cell-free DNA fragments which could interfere with the IONA® test potentially giving an erroneous result so if a pregnant woman is known to have cancer the IONA® test is not suitable12.
17.2.3 Are there rare inherited genetic conditions which can preclude the use of the IONA® test?
The IONA® test is not suitable in cases where the mother carries a genetic condition that changes the level of chromosomes 21, 18 or 13 in her cell-free DNA fraction. Such rare conditions would include mosaicism or chimaerism involving a trisomic cell line as well as partial duplications or chromosomes 21, 18 and 13 in the germline.
17.2.4 Is the test suitable for women with Turner syndrome (monosomy X)?
No. The method used by the IONA® test to calculate the fetal fraction is not compatible with mothers who have mosaic or complete Turner syndrome.
If a sample is received and generates a result indicative of monosomy X in the mother then a repeat sample is not recommended and the Healthcare Provider will be informed.
17.3 Medical interventions in the mother
Medical treatments that could perturb the chromosomal ratio of cell free DNA in circulation would exclude the woman from having an IONA® test.
Cell free DNA is short lived in the circulation13 so it is only treatments which introduce cell lines which can continue to produce cell free DNA over time which are of concern. The introduction of cell free DNA per se is not an exclusion criterion.
17.3.1 Can the IONA® test be used on pregnant women who have had a blood transfusion?
A blood transfusion could potentially interfere with the IONA® result if DNA derived from the transfusion was present in the mother’s blood stream. DNA in blood is associated with the leucocyte fraction. For this reason, caution is required for women who have had a non-leucocyte depleted blood transfusion within the last 12 months. Transfusions of red blood cells or plasma will not interfere with the IONA® test.
17.3.2 Is the IONA® test suitable for women who have had an organ transplant?
No. An organ used in a transplant would not have the same genotype as the recipient and if DNA from the transplant was present in the cell free DNA fraction, it could cause an erroneous result. If the transplant was from an individual with Down’s syndrome the level of cell free chromosome 21 DNA could be elevated. If the organ was from a male donor, Y DNA could be introduced into the circulation leading to a mis-estimation of the fetal fraction.
17.3.3 Can the IONA® test be used on women who have had immunotherapy?
Immunotherapy can refer to a range of treatments designed to boost immune response during cancer therapy, the use of monoclonal antibody therapy or the use of polyclonal antibodies such as IVIG treatment during IVF therapy. In all cases the question of whether use of the therapy should exclude treatment with IONA® depends on whether the treatment introduces cells into the body which can potentially generate cell free DNA and thereby affect the background chromosome ratios in the circulation. IVIG treatments for example contain antibodies but not cells so would not be an exclusion criterion.
17.3.4 Can the IONA® test be used on women who have had stem cell therapy?
No, stem-cell therapy is the use of cultured stem cells to treat or prevent a disease or condition – the cells are often derived from the patient’s bone marrow but could also come from other sources such as the umbilical cord. Given the potential for the stem cells to have a different karyotype to the mother the IONA® test is not recommended for women who have undergone heterologous stem cell therapy.
17.4 Sampling and sample handling errors and laboratory-related failure.
Inaccurate test results may occur in rare circumstances beyond our control, which include but are not limited to: Sampling and sample handling errors (including courier/shipping delay, contamination or degradation of a sample) and laboratory-related failures.
18. Is the test suitable for a pregnant mother with a high Body Mass Index (BMI)?
Yes. Women with an elevated BMI tend to have a lower ratio of placental DNA to maternal DNA in their blood.14 This low fetal fraction is the result of more maternal DNA being released from apoptosis/necrosis of stromal vascular cells and adipose tissue15,16 and also due to an increased blood volume. This results in a dilution of the cell-free placental DNA in the mother’s plasma. Nonetheless, if the fetal fraction in the sample is above the threshold required by the IONA® test, the sample will be deemed valid and the accuracy of the result is not affected. The lower levels of fetal fraction found in women with an elevated BMI mean that there is a slightly increased rate of test failure in this population.
19. Is the IONA® test suitable for pregnancies resulting from IVF?
Yes. In an IVF pregnancy it is likely that the age of a donor egg cell differs from the age of the mother. The consent form and the IONA® software are designed to ensure that the correct prior risk (i.e. the age of the donor egg) is used to calculate the result.
20. Will heparin treatment interfere with the result of the IONA® test?
There are some reports in the literature that low molecular weight heparin can interfere with NIPT performance17. Heparin has been directly tested and shown to not impact the performance of the IONA® test. It is not clear whether heparin has an indirect effect by altering the level of cell-free DNA in circulation, but in any event, as the data available indicates that the accuracy of the test is not compromised, heparin treatment is not an exclusion criterion for the IONA® test.
21. What are the clinical implications of a failed test?
•The blood sample supplied for the IONA® test normally provides sufficient plasma to allow for a repeat test without the need for a repeat sample. If a repeat sample is requested this could be due to:
1. Rare laboratory technical issues that prevented the test from operating properly,
2. Sample failed incoming quality checks (e.g. hemolysis or labelling errors).
3. Very low fetal fraction that was below the minimum requirement of the IONA® test.
4. A result very near the cut-off which was flagged by the IONA® test for repeat analysis.
•In the case of technical issues and failed quality checks there are no clinical implications and the patient should be encouraged to provide a repeat sample.
•A low fetal fraction is associated with early gestational age and with high BMI14 and often a satisfactory yield will be obtained on a repeat sample with a clear-cut result18. Low fetal fraction is also associated with trisomy 18 and trisomy 1319
•There is no clear consensus amongst the clinical societies whether a test which has failed for low fetal fraction indicates a repeat NIPT or an invasive test. The decision whether to proceed directly to invasive testing following a low fetal fraction result or whether to repeat the test will depend upon the individual circumstances of the pregnant woman and the judgement of her clinician.
•If the test result is very near the cut-off it is flagged by the IONA® software as a quality control (QC) check. If the second test (from the first sample) is also flagged for the same reason, then this indicates that there is something potentially unusual about the sample such as confined placental mosaicism. Such an intermediate result will be communicated directly to the healthcare professional with a recommendation for further investigation. Repeat testing is not recommended.
•Samples from women with complete or partial Turners Syndrome fail to generate a result in the IONA® test. A repeat sample is not recommended, and the Healthcare Provider will be informed.
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2. Discordant sex between fetal screening and postnatal phenotype requires evaluation. Byers HM, Neufeld-Kaiser W, Chang EY, Tsuchiya K, Oehler ES, Adam MP. J Perinatol. 2019;39(1):28-33. doi:10.1038/s41372-018-0278-5
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5. Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis Ultrasound Obstet
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15. Review: Cell-free fetal DNA in the maternal circulation as an indication of placental health and disease
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17.The association between anticoagulation therapy and, maternal characteristics and a failed cfDNA test due to a low fetal fraction. Burns W, Koelper N, Barberio A, Deagostino-Kelly M, Mennuti M, Sammel MD, Dugoff L. Prenat Diagn, 2017;37, 1125-1129
18. Palomaki and Kloza, Prenatal cell-free DNA screening test failures: a systematic review of failure rates, risks of Down syndrome, and impact of repeat testing. Palomaki GE, Kloza EM. Genet Med. 2018 Nov;20(11):1312-1323
19. Screening for trisomies by cell-free DNA testing of maternal blood: consequences of a failed result. Revello R, Sarno L, Ispas A, Akolekar R, Nicolaides KH. 2016 Ultrasound Obstet Gynecol 47 698-705.
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