What is Cell Free Fetal DNA Testing and How Does it Work?

Prenatal genetic testing has come a long way since it was first introduced in the 1960s. Particularly in the past seven years, prenatal genetic testing has been revolutionized by the introduction of cell-free fetal DNA testing. This simple blood test is now the most accurate way to screen for certain chromosome conditions in pregnancy without imposing any risk of a miscarriage. It can also tell the fetal sex with 99% certainty at as early as ten weeks. Surprisingly though, its uptake by OBGYN offices across the United States is hugely variable, leaving expectant parents confused about this new technology and if it’s right for them.

What is cell-free fetal DNA testing?

Cell-free fetal DNA testing is a blood test that analyzes fragments of fetal DNA in the maternal blood to screen for different chromosome conditions. About four days after fertilization the cells in the embryo begin to differentiate into two parts: the trophoblast (which will become the placenta) and the inner cell mass (which will become the baby). During pregnancy, cells from the placenta will naturally rupture, and fragments of the chromosome are released into the maternal bloodstream. Laboratories can identify these DNA pieces for genetic testing. A more accurate name for this would be cell-free placental DNA testing, but since the cells of the trophoblast originate for the same cells of the baby, the vast majority of the time placental DNA accurately represents fetal DNA.

How does cell-free fetal DNA testing work?

Most cell-free fetal DNA testing laboratories utilize a “counting method” to determine if a pregnancy is at increased or decreased risk for chromosomal conditions. The laboratory sequences fragments of placental DNA in the maternal bloodstream to determine which chromosome each fragment came from. The laboratory then compares the amount of material identified from each chromosome to a standardized expected amount. If more DNA from a particular chromosome is detected, there is an increased chance that the pregnancy has an extra copy of that chromosome.

What does cell-free fetal DNA screen for?

Most cell-free fetal DNA tests screen for conditions that occur when a baby has an extra chromosome: trisomy 21 (Down syndrome), trisomy 18, and trisomy 13, in addition to screening for fetal sex. These extra chromosome conditions become more common as a mother gets older but can happen at any age.

Some companies may also offer to screen for a few conditions that occur when there is a small missing piece of a chromosome (microdeletion syndromes) or conditions that occur when a baby has extra or missing sex chromosomes. Compared to the conditions listed above, there is much less data available to determine the accuracy of screening for these additional conditions. Screening for sex chromosome abnormalities or microdeletion syndromes with cell-free fetal DNA testing is not currently recommended by the American College of Obstetricians and Gynecologists (ACOG).

Can cell-free fetal DNA tell the gender?

Cell-free DNA can tell the sex of the baby with 99% certainty. Typically males have one X chromosome, and one Y chromosome and females have two X chromosomes. In determining the fetal sex, some cell-free fetal DNA laboratories only test for the presence or absence of the Y chromosomes, while others may check for the presence or absence of the X chromosome as well. Checking for both the X and Y chromosome can detect sex chromosome abnormalities, such as XXX or XXY. If the the mother has a sex chromosome abnormality, had an organ transplant or blood transfusion from a male, or if there is a miscarried twin in the pregnancy, results may be inaccurate.

When can cell-free fetal DNA testing be performed?

Unlike some other prenatal genetic blood tests that must be completed in specific windows of a pregnancy, cell-free fetal DNA testing becomes available at ten weeks gestation and remains available until delivery. Most laboratories require that there be a certain amount of fetal DNA present in the sample to run the test. This percentage is known as the fetal fraction. Cell-free fetal DNA can be detected in the mother’s bloodstream as early as five weeks, but most pregnant women will not have a high enough fetal fraction for testing until 9 or 10 weeks. Within hours of giving birth, there is no detectable cell-free fetal DNA in the mother’s blood, so DNA from past pregnancies would not compromise a test result.

What are the possible results of cell-free DNA testing?

In general, there are three possible results for a cell-free fetal DNA test.

  • Positive/High-risk: This means that the test has recognized concern for a condition it screened for, but does not say that the baby is necessarily affected. For example, if more fragments of chromosome 21 are identified than expected, it is likely that there is an extra copy of chromosome 21 present, which causes Down syndrome. Some companies simply put “positive” or “high-risk” for the condition of concern and others will try to calculate the chance that the result is actually correct using the information they have on the mother and specific values from the test. This is known as the positive predictive value. When the lab does not do this calculation for you, your doctor or genetic counselor can do a similar calculation.
  • Negative/Low-risk: This means that for all the chromosomes tested the expected amount of DNA was observed, so it is very unlikely that the baby would have any of those conditions. Cell-free fetal DNA testing is not a diagnostic test, so the results are not 100% certain, but a negative result typically means a <1 in 10,000 chance the baby has the condition.
  • Inconclusive: This type of result happens ~2% of the time and can occur for a variety of reasons. Most of the time it is because there was not enough of the fetal DNA in the sample for the lab to make a call. A low fetal fraction happens more frequently in mothers with a high body mass index. Recent studies showed a slightly increased risk for trisomy 18 or 13 following low fetal fractions. People who get an inconclusive result may decide to repeat the test, have a detailed ultrasound to look for any signs of chromosome conditions, or elect to have prenatal diagnostic testing.

Why can a cell-free fetal DNA test have a false positive?

There are several reasons why cell-free fetal DNA testing can give a false positive result, including a laboratory error or an unknown chromosomal or genetic condition in the mother, but the two most common are confined placental mosaicism and vanishing twin syndrome.

  • Confined placental mosaicism: This means that the chromosome condition of concern is in the placenta but not in the baby. Sometimes an embryo can begin with a trisomy condition, say Down syndrome, recognize the issues, and try to correct it but kicking out a chromosome. This is called trisomy rescue. The cells that have the extra chromosome will be sent to the trophoblast (placenta), and the “rescued” cells will be sent to the baby.
  • Vanishing twin: A vanishing twin pregnancy means that a pregnancy began as a twin pregnancy, but one twin miscarried very early on. The tissue of this twin will be absorbed into the other baby, placenta, and mother. Over 50% of pregnancies that miscarry have a chromosomal condition, so it is possible that tissue for a demised twin could influence the test result. This is also a reason why the fetal sex could be inaccurate. If the fetal sex reported on cell-free fetal DNA is male, but the ultrasound shows a female, it is possible that a demised twin was male and Y chromosome is still present in the pregnancy.

What is the controversy around cell-free fetal DNA testing?

When cell-free fetal DNA testing first became available it was marketed a non-invasive prenatal diagnosis. Diagnostic tests are those that are 100% accurate, and healthcare providers were excited to have a diagnostic testing option that did not have a risk of miscarriage. It did not take long before false positive cases were reported and doctors offices quickly learned that the test did not perform as advertised. This false promise left a mark on cell-free fetal DNA testing that for many healthcare providers remains today. While it still is more accurate than any other prenatal genetic blood test, it is not as accurate as it was made out to be. Also, the vast majority of the data on cell-free fetal DNA testing was collected from women who were already at increased risk to have pregnancies with chromosomal conditions, meaning there is limited (but growing) data that represents the average pregnant women.

How accurate is cell-free fetal DNA testing?

Cell-free fetal DNA testing is the most accurate way to screen for common chromosome conditions in a pregnancy, but it not considered a diagnostic test. False negatives and false positives do occur. Many cell-free fetal DNA testing companies report being >99% accurate, meaning that >99% of results accurately represent the pregnancy. However, it is important to remember that the vast majority of samples a company receives will be healthy pregnancies receiving low-risk results. Saying that the test is >99% accurate does not mean that there is a 99% chance your test result is accurate.

On average, 99.3% of pregnancies that have Down syndrome will receive a high-risk result on cell-free fetal DNA testing. This is called the detection rate or the sensitivity. The specificity, or the percentage of babies that do not have Down syndrome will receive a negative result, is 99.8%. This means that 0.2% of pregnancies without Down syndrome will receive a high-risk Down syndrome result (for a false positive). For trisomy 18 the sensitivity and specificity are 97.4% and 99.8% and for trisomy 13 are 91.6% and 99.9%.

The term positive predictive value means the chance that the baby is actually affected given a high-risk test result. This calculation accounts for the original risk that the baby had the condition. Because the chance to have a baby with a chromosome condition increases as women get older, a high-risk result on an older mother will have a higher positive predictive value than the same result in a younger mother.

Who can have cell-free fetal DNA testing?

The most current ACOG Practice Bulletin on prenatal genetic screening (#163) states that all women should be offered both screening and diagnostic testing for common chromosomal conditions, but does not go into detail as to who should be provided what test. Some doctors offices and genetic counselors give all pregnant patients the option of cell-free fetal DNA testing, while some prefer that their patients, particularly younger mothers, employ more traditional screening options. ACOG does not recommend using cell-free fetal DNA testing to screen for microdeletion syndromes or in pregnancies with multiples.

What are other names for cell-free fetal DNA testing?

Depending on your doctor’s office, cell-free fetal DNA testing can have many different names, including non-invasive prenatal screening (NIPS) or non-invasive prenatal testing (NIPT). Different laboratories that perform the testing have different brand names for the test, including MaterniT21™, Panorama™, Harmony™, Prelude™, QNatal™, Innatal™, and InformaSeq™.

How much does cell-free fetal DNA cost? Does insurance cover cell-free fetal DNA testing?

The cost and coverage of cell-free DNA testing will vary depending on if there is an increased risk for the child to have a chromosomal condition (such as the mother being over 35 years old at delivery), the laboratory performing the test, and your insurance company. To try to figure out an expected cost, ask your doctor or genetic counselor the name of the laboratory they most frequently use and reach out to a billing representative from the laboratory with your insurance card in hand. Many companies offer discounted prices when the test is not covered by insurance in the range of $100-$400 and may be able to further lower the price for those who meet financial aid requirements. Many doctors offices only work with one testing laboratory on a regular basis. If the cost is too much, consider reaching out to an independent genetic counselor to see if another laboratory might be a better financial option.

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