Cell-Free DNA Testing

What is cell-free DNA testing and how does it work?

All pregnant women have small pieces of DNA in their blood stream that comes from the baby. This fetal DNA circulates in the woman’s blood stream after it is released through the placenta starting early in the first trimester until the baby is born ¹.

Screening allows DNA fragments to be counted for each chromosome from the baby to determine if there are too many or too few. This is critical to know because scientists have determined that if the wrong amount of DNA fragments are present it could lead to birth defects.

For example, if a fetus has Down syndrome, then the amount of DNA for that chromosome will be more than compared to other chromosomes.

Prenatal cell-free DNA (cfDNA) screening is also known as noninvasive prenatal screening (NIPT) and is a relatively new method to screen for certain chromosomal abnormalities in a developing baby. NIPT involves taking a sample of blood from the mom’s vein rather than inserting a needle into the uterus like an amniocentesis.

Test results are generally available within seven to 10 days.

During a cfDNA screening, DNA from the mother and fetus are extracted from a maternal blood sample and screened for the increased chance for specific chromosome problems. The screening tests for conditions such as trisomy 21 better known as Down syndrome, trisomy 13 and trisomy 18.

It can also provide information about fetal sex and rhesus (Rh) blood type.

Down syndrome is the most common genetic condition in the U.S., affecting around 1 in every 700 babies.

To get a cfDNA screening, it is recommended for women who are at least 10 weeks pregnant and who also have access to genetic counseling, or other forms of counseling regarding options, risks, limits and benefits of first and second trimester screening.

Normally, because it screens only for trisomy 21, 18, and 13, NIPT covers only 80 to 90 percent of chromosome abnormalities that would be detected using amniocentesis.

However, some cfDNA screening tests might also screen for the increased chance for:

• Trisomy 16
• Trisomy 22
• Triploidy
• Sex chromosome aneuploidy
• Certain disorders caused by a chromosomal deletion (microdeletion syndrome), such as Prader-Willi syndrome
• Certain single-gene disorders associated with abnormalities of the skeleton or bones

Depending on the individual 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 various brand names for the test, including MaterniT21™, Panorama™, Harmony™, Prelude™, QNatal™, Innatal™, and InformaSeq™.

Cell-free DNA testing for Down syndrome

Knowing whether a child will be born with Down syndrome or not can have a profound impact on a family.

Women who are over 35 years old constitute an age associated with a higher risk of Down syndrome. Overall, the number of babies born with Down syndrome in the U.S. increased 30% between 1979 and 2003.

The medical costs of privately insured children from infant to 4 years old with Down syndrome are estimated to be around 12 times higher than those of children of the same age without the condition.

Accuracy of cell-free DNA Tests

Much has been written about the accuracy of cfDNA tests and if there is any controversy about the viability of this screening mechanism, it has to do with results and how they are reported and interpreted.

Keep in mind that cfDNA tests are screening tests and not diagnostic tests. This means that the test looks for the possibility that a condition might be present, but unlike a diagnostic test, it does not confirm the presence of the condition. False negatives and false positives do take place ².

It should be noted 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.

According to the genetic counseling website family-forecast.com, “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.”

In a 2014 study by researchers at University of California-San Francisco, 16,000 pregnant women were tested, and it was found that a cell-free DNA blood test carried out between 10-14 weeks gestation was more effective for diagnosing Down syndrome than standard screening methods.

The study also revealed the blood test was more effective for diagnosing two rarer chromosomal abnormalities - Edwards syndrome and Patau syndrome - than conventional techniques.

According to UCSF researchers, the cfDNA test has proved highly accurate in detecting Down syndrome in high-risk pregnant women, but its effectiveness among pregnant women at lower risk is unclear.

The study also concluded that “women who do opt for cell-free DNA testing should be informed that it is highly accurate for Down syndrome, but it focuses on a small number of chromosomal abnormalities and does not provide the comprehensive assessment available with other approaches.”

Risks and benefits of cell-free DNA testing

Before getting a NIPT test, you should be educated on the pros and cons of this type of test.

The Pros

NIPT screening poses no physical risks for you or your baby ³.

While NIPT screening might cause anxiety, it might help you avoid the need for more invasive tests, treatment or monitoring during your pregnancy.

When the NIPT test result is positive, then a baby is at increased risk of the particular trisomy identified. This can be a good first step to decide if a more accurate test should be performed to better determine if the baby is normal.

Ongoing research continues to refine NIPT testing to make it more accurate and to try and be more inclusive so that other genetic conditions can be offered through this kind of screening.

The Cons

It does not test for other genetic conditions like missing and duplicated pieces of chromosomes or chromosome abnormalities other than trisomy 21, 18 and 13.

It cannot detect genetic conditions like cystic fibrosis, beta-thalassemia or sickle cell anemia. Those conditions can only be identified by amniocentesis. cfDNA also does not screen for spina bifida or birth defects.

NIPT is a screening test, not a diagnostic test, which means that sometimes the results are inaccurate ⁴. It cannot tell you with 100% certainty whether or not a fetus will be affected by a given disorder.

By its very nature, a screening test does not tell with 100% certainty whether or not a fetus will be affected by a given disorder.

Other first trimester screenings may offer a wider range of results. There are many other noninvasive screening tests that have been offered to all pregnant women for many years.

A first trimester screen is done at 11-13 weeks of gestation and includes a measurement of fluid behind the neck of the fetus (nuchal translucency) by ultrasound and a blood test of the mother. The quad screen is a blood test (AFP) done at 15 to 20 weeks.

These tests screen for birth defects, additional chromosome abnormalities and pregnancies at risk for complications like preeclampsia and poor fetal growth.

Because prenatal cell-free DNA screening doesn't screen for all chromosomal or genetic conditions, a negative test result will not insure an unaffected pregnancy.

There can be false positive and false negative results with NIPT so any abnormal result should be followed-up by other diagnostic tests like an amniocentesis.

In addition, while the detection rates for Down syndrome are high, the test is less accurate and effective for trisomy 18 and 13. When birth defects are revealed in an ultrasound or when a woman is at a high risk of a chromosome abnormality, she should be offered more accurate tests such as CVS or amniocentesis.

There are cases when the NIPT test cannot provide a result because the amount of fetal DNA is too low. In these cases, other screening tests should be considered.

False positive results can occur from placental chromosome abnormalities, cancer cells, chromosome abnormalities of the mother, twins or triplets, use of donor egg, and miscarriage of one twin.

One study reported in the New England Journal of Medicine reported that “the positive predictive value of cfDNA was found to be less than 50% in low risk patients – that is, more than half of the results were false positives.”

Some testing companies may 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. But there is much less data available to determine the accuracy of screening for these additional conditions.

As such, screening for sex chromosome abnormalities or microdeletion syndromes with cfDNA testing is not currently recommended by the American College of Obstetricians and Gynecologists.

Does insurance cover cell-free DNA tests?

When genetic testing is recommended by a patient’s doctor, many health plans will cover the cost of the test.

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, the laboratory performing the test, and your insurance company.

Different providers will have different policies regarding coverage, so it may be best to talk with your provider first. Also be sure to ask about genetic counseling coverage as well.

Many testing companies may offer discounts if a test is not covered by insurance. Be sure to ask if a discount is available if you need to pay for the entire test out of pocket. There may also be lower costs or further discounts if you have limited income and meet financial aid requirements.

It is okay to shop the price of the test around to many providers if you think you can get a better deal. Most doctors only use one testing laboratory, but this does not mean you need to be subject to that limitation.

There are also cases when a patient may deliberately choose to pay for a test on their own because it could affect their insurance coverage if positive test results are reported. There are privacy protection laws in place, but genetic discrimination can still take place. Consider this issue as you move forward.

Who should consider cell-free DNA screening?

NIPT testing should be considered by pregnant women who want to assess the risk of a developing baby having certain chromosome disorders.

It is currently offered only to woman at increased risk for trisomy 21 (Down syndrome), 18 and 13, which includes the following:

• Women 35 years of age and older who do not want amniocentesis or CVS
• Ultrasound findings consistent with a trisomy
• Previous baby with a trisomy
• Abnormal first trimester or quad screen test result

Because it can produce false positives, the NIPT test is not recommended in the circumstances:

• Low-risk women (under age 35, normal ultrasound)
• Earlier than 10 weeks of gestation
• Twin or triplet pregnancies
• Women over 250 pounds
• To determine sex of the baby
• Fetal birth defect seen on ultrasound
• Pregnant via a donor egg
• Taking certain blood thinners

If you are obese, your doctor will be able to discuss other screening and diagnostic test possibilities with you.

If you decide to have the NIPT test done, an ultrasound exam will be done at the same time to confirm viability, the number of fetuses and due date. The fluid behind the fetal neck (nuchal translucency) and the position of the heart in the fetal chest (cardiac axis) can also be measured as additional screening for birth defects.

NIPT screening does not assess risk of fetal birth defects such as spina bifida or other birth defects. Women undergoing NIPT screening should have an ultrasound later in the pregnancy (18 to 22 weeks) to rule these conditions out.

When to ask your healthcare practitioner about having a cell free DNA test performed

Cell-free DNA testing is a good screening test for high-risk women, but it is not recommended for low risk patients.

Screening tests will have different performance based on background risk, and in low risk patients the abnormality rates are low, meaning that a positive test is usually more likely to be a false positive.

If you are considering a cfDNA test, it’s smart to meet with a genetic counselor first. A counselor will be able to provide information on the following points:

• cfDNA tests are screening tests, not diagnostic tests.
• Abnormal results must be confirmed with diagnostic testing such as CVS or amniocentesis before an irreversible action, such as terminating a pregnancy, are taken
• Although cfDNA tests have greater sensitivity and specificity than traditional screening, false positives and false negatives still occur.

How early can you do cfDNA testing?

You can undergo cfDNA testing as early as the 10th week of your pregnancy ⁵. Check with your doctor who will accurately advise you based on your specific circumstances.

There must be a certain amount of fetal DNA present in a sample to run the test. This amount 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.

What to expect when getting a cell-free DNA test

A cfDNA test is a relatively simple procedure. A blood sample is drawn in a doctor’s office and is sent to a lab to interpret results.

One of the biggest benefits is that drawing blood does not impose any kind of risk on a miscarriage ⁶.

In scientific terms, most cell-free fetal DNA testing laboratories use 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.

This screening can also provide information about fetal sex and rhesus (Rh) blood type.

Once the results come back, your doctor will likely pair them with the results of your first-trimester ultrasound or nuchal translucency screening to determine if further testing is needed. If it’s positive, your doctor may recommend following up with amniocentesis or CVS to confirm the result and check for other problems cfDNA test can’t detect.

How long do cfDNA test results take to receive?

Typically, results take about 7 to 10 days, although in some instances results could take as long as two weeks.

It depends on what testing company is used and the amount of their backlog.

Can cfDNA testing tell me the gender of my child?

Yes, with up to 99% accuracy.

Most of the time, males have one X chromosome, and one Y chromosome. Females have two X chromosomes. In determining the sex of the fetus, some 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. If 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.

If you get abnormal cfDNA test results

A simple draw of blood makes getting a cfDNA test easy. What may be difficult is waiting for and getting the results.

For the most part, there are three possible results for a cfDNA 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. This does not mean that the fetus has Down syndrome, it simply means there is a higher risk of having it.

Some medical professionals will try to calculate the chance that the result is actually correct by generating what is known as Positive Predictive Value. Using information known about the mother and specific values from the test, a percentage number will be generated to help interpret the results in a standardized way.

If a lab does not do this calculation, then 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.

cfDNA testing is not a diagnostic test, so the results are not 100% certain, but a negative result typically means a there is only about a 1 in 10,000 chance the baby has the condition.

Inconclusive: This type of result happens about 2% of the time and can occur for a variety of reasons. Most of the time, it is because a mother has a high body mass index or there are low fetal fractions. In these cases, the test can be repeated, an ultrasound can be performed, or a mother can elect to have prenatal diagnostic testing.

For many parents, knowing as much as possible as soon as possible is better. Finding out about potential conditions allows parents to prepare for a special needs baby, including making plans to deliver at a facility that can better address the baby’s specialized health needs at birth.

What to do if your tests results show a risk of birth defects

If test results are positive, one of the best things to do is to discuss your options with your doctor and a genetic counselor.

There’s a high likelihood that more tests will be recommended, giving you a fuller understanding of the health of your baby. Further testing using the sample from amniocentesis or CVS can be used to confirm a diagnosis ⁷.

The baby may indeed be healthy and normal, but confirmation is critical to making choices that lie ahead. This can include preparing for the needs of a newborn who may have birth defects, or it can lead to a discussion about the termination of a pregnancy if the condition is confirmed.

You also need to understand that chromosomal abnormalities can't be corrected.

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