Understanding Test Results

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By Dr. Tara Sander Lee, Ph.D.

With any clinical laboratory test, there are inherent limitations. No genetic test or screen will always perform the way it should 100% of the time.  

There are two types of genetic tests, screening tests and diagnostic tests, and it is important to understand the distinction between the two.

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Prenatal Genetic Screening Tests

Screening tests are capable of predicting whether there is an elevated risk of the presence of a condition. By their nature, they are not diagnostic and do not confirm the presence of a condition. 

Standard prenatal genetic screening is often performed during the first and second trimester to calculate the risk of having a baby with a chromosomal disorder (change in baby’s DNA that can cause disease). Maternal age, serum analyte screening for biochemical markers (such as the triple screen or quad screen), and fetal nuchal translucency (NT) measurement are considered first-line non-genetic screening.1 However, these standard screening tests can not accurately predict the risk of the child having a genetic disorder.2  

An advanced method of non-invasive prenatal screening (NIPS; also known as NIPT) is used to reduce the need for invasive diagnostic genetic techniques such as amniocentesis.  NIPS uses cell-free fetal DNA (also known as cffDNA) found in the maternal circulation to screen for chromosomal disorders such as trisomy 21 (Down syndrome).3 DNA screening tests can be performed as early as the first trimester, often after 7 weeks gestation.

With any clinical laboratory test, especially NIPS, there are inherent limitations. Underlying conditions can limit NIPS performance and interfere with test results including placental mosaicism, maternal chromosomal abnormality, vanishing twin, organ transplant, etc. Incorrect reporting due to erroneous results, technical problems, and lab errors (i.e., false positives, false negatives, mixed specimens, mislabeling, etc.) is also a possibility. Multiple and past pregnancies may also interfere with the NIPS result.4 It’s also important to note that when NIPS is performed using the mother’s peripheral blood, the technique can only detect genetic changes in fragments of the baby’s DNA. These inherent limitations in DNA screening for genetic mutations will affect correct result reporting and interpretation.5 

It’s important to note that a negative NIPS test result does not ensure an unaffected pregnancy and a positive test result does not ensure an affected pregnancy. A false positive result occurs when a test result is reported as ‘positive’, yet the condition it is testing for is not actually present and should have been reported as ‘negative’.

NIPS tests are often marketed as highly accurate and many people are unaware of the high rate of false positives, especially among low-risk women (e.g., less than 35 years of age) that has been noted in both scientific and non-scientific literature.6 A comprehensive study across 21 different centers in the United States, which included 1,914 women (mean age, 29.6 years), observed much lower positive predictor values of 45.5% for trisomy 21 amongst low-risk women. This indicates that a significant proportion (over 50%) of “positive” test results for Down syndrome may not be truly positive when screening women mostly at low risk.7 Less frequent conditions have an even higher false positive rate. For this reason, the authors from this study highlight the “need for follow-up diagnostic testing to confirm true positive results before decisions are made about irrevocable clinical intervention.”8 They know that some parents might tragically terminate the life of their child based on an erroneous and incorrect NIPS lab result.

There have been several calls for federal action, advocating for stronger oversight to improve patient safety in the context of prenatal screenings.  House representatives also submitted a letter to the FDA regarding the high rate of false positives and increased oversight to all prenatal screens. Even the FDA has warned the public.9

 

Prenatal Genetic Diagnostic Tests

Diagnostic tests confirm the presence of a condition. Diagnostic tests are considered sufficiently accurate to make medical treatment decisions. While accurate, it should be noted that no test is 100% accurate all the time. 

Screening tests may be combined with diagnostic testing, usually between 10-18 weeks gestation, to increase the chance of correctly predicting a risk of a genetic disorder. Diagnostic DNA tests can be performed using fetal samples obtained via amniocentesis and chorionic villus sampling (CVS).  These tests are accurate, but the means to obtain fetal samples for DNA testing from the amniotic sac and placenta are invasive and carry their own risks for pregnancy loss.10 A patient with a positive NIPS screening test result is often referred for genetic counseling and should be offered invasive prenatal diagnosis for confirmation of test results. Parents may choose to have a diagnostic test or waive such testing if they are concerned with the possibility of pregnancy loss due to the test.  

Some platforms analyze cell-free fetal DNA fragments across the whole (or part) of the genome using next generation sequencing (NGS), targeted sequence analysis, and array-based techniques.  NGS platforms that screen fragments from the entire genome can be reliable, specific, and sensitive with a reported failure rate of 0.1% (inconclusive result) and false-positive rate of <0.1%.11  

A positive result from a diagnostic test along with anatomy scans via ultrasound may determine a treatment course of action before or after birth. 

 

Summary

Screening tests, which are often performed in the first or second trimester, will either rule out the presence of a condition with high accuracy or note an elevated risk of a condition. They do not confirm the presence of a condition and additional diagnostic testing is required if parents wish to confirm a condition.

Diagnostic tests, which are often performed in the second trimester, are accurate at confirming the presence of a condition, although no test is 100% accurate. Some diagnostic tests, such as amniocentesis, come with their own risks to baby and parents may choose to have these tests or decline further testing.

 

Sources:

    1. Rink, B.D. and M.E. Norton, Screening for fetal aneuploidy. Semin Perinatol, 2016. 40(1): p. 35-43.
    2. For example, when predicting the risk of having a child with Down syndrome, there is a high false-positive rate of incorrect reporting (a negative result is reported as positive) ranging from 1-14% and incredibly low positive predictor values (PPV, the proportion of positive test results that are true positives) of 4.2%. (Bianchi, D.W. et al., DNA sequencing versus standard prenatal aneuploidy screening.  N Engl J Med 370:9, 2014.) 
    3. Scientists can detect cell-free fetal DNA from a mother’s blood sample as early as 4 weeks and 5 days after fertilization. (G. S. Dawe et al., Cell migration from baby to mother.  Cell Adhesion & Migration 1:19-27, 2007.) Cell-free fetal DNA is consistently detected from seven weeks (ibid), remains level between 10 and 21 weeks, (Wapner, R.J and Dugoff, L.  Prenatal diagnosis of congenital disorders, in Creasy and Resnik’s Maternal-Fetal Medicine: Principles and Practice 8th Edition, R., Resnik, Lockwood, C.J., Moore, T.R., Greene, M.F., Copel, J.A., and Silver, R.M., Editor. 2019, Elsevier: Philadelphia, PA. p. 506.) steadily increases after 24 weeks, peaks at birth, and then declines postpartum. (H. Ariga et al., Kinetics of fetal cellular and cellā€free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis.  Transfusion 41:1524-30, 2001). Once the cell-free DNA sample is collected, NIPS uses advanced molecular techniques to determine a child’s genetic susceptibility to a genetic disorder. (ACOG Committee on Genetics, Committee Opinion No. 640: Cell-Free DNA Screening For Fetal Aneuploidy. Obstet Gynecol. 126(3): p. e31-7, 2015) The most common genetic for screening include the targets for trisomy 13, 18, 21, 22q11 deletion syndrome (i.e., DiGeorge syndrome) and some x-linked disorders.
    4. Some studies have shown that cell-free fetal DNA is rapidly cleared from the maternal blood, with 100% clearance within 1-2 days postpartum (A. Kolialexi et al., Rapid Clearance of Fetal Cells from Maternal Circulation After Delivery.  Ann N Y Acad Sci 1022, 113-8, 2004), (Y. M. D. Lo et al., Rapid Clearance of Fetal DNA from Maternal Plasma.  Am. J. Hum. Genet. 64:218–224, 1999), suggesting that fetal DNA from past pregnancies should not interfere with current tests.  However, other studies have found the persistence of fetal DNA for decades in the mother. (D. W. Bianchi et al., Male progenitor cells persist in maternal blood for as long as 27 years postpartum.  Proc Natl Acad Sci USA.  93:705-708, 1996), (Invernizzi P. et al., Presence of fetal DNA in maternal plasma decades after pregnancy.  Human Genetics, 110(6): 587-591, 2002.)
    5. One widely utilized NIPT screening test on the market has a positive predictive value (PPV) of 81%, meaning that there is a significant chance that a positive test result is NOT a true positive. (Norton ME et al., Cell-free DNA Analysis for Noninvasive Examination of Trisomy, New England Journal of Medicine 372, 1589, 2015; doi: 10.1056/NEJMoa1407349) But even this reported PPV value is deceiving, because PPV is based on test sensitivity, specificity, and the prevalence of the condition in the population being tested. Because the prevalence of Down syndrome increases with maternal age, PPVs will be higher in patients of advanced maternal age (>35 years old) and will likely increase when other aneuploidy risk factors are known (e.g., ultrasound abnormalities) (National Society of Genetic Counselors, NIPT/Cell free DNA screening predictive value calculator.  Available at: https://www.perinatalquality.org/Vendors/NSGC/NIPT/)
    6. When They Warn of Rare Disorders, These Prenatal Tests Are Usually Wrong The New York Times Jan 1, 2022 https://www.nytimes.com/2022/01/01/upshot/pregnancy-birth-genetic-testing.html 
    7. Bianchi, D.W. et al., DNA sequencing versus standard prenatal aneuploidy screening.  N Engl J Med 370:9, 2014.   
    8. Ibid
    9. https://www.fda.gov/news-events/press-announcements/fda-warns-risks-associated-non-invasive-prenatal-screening-tests#:~:text=Today%2C%20the%20U.S.%20Food%20and,invasive%20prenatal%20tests%20(NIPT)
    10. Rink, B.D. and M.E. Norton, Screening for fetal aneuploidy. Semin Perinatol 40(1): p. 35-43, 2016
    11. Illumina Verifi Prenatal Test: https://www.illumina.com/clinical/reproductive-genetic-health/nipt/sendout-testing-for-labs.html