Category Archives: Down Syndrome

Down Syndrome

Screening for Down syndrome: Beginning of a New Era?

DNADavid has written previously about the different types of screening tests for Down syndrome (trisomy 21). Although these tests have certainly gotten better over time, even the best among them is subject to less than perfect sensitivity (90%) and a false positive rate of between 2 and 5%. Given the prevalence of Down syndrome, this means that out of 16 women that screen positive for Down syndrome, only one will carry an affected infant. Therefore, 15 women are unnecessarily subject to potentially dangerous amniocentesis procedure. Recently, there have been reports of some molecular testing that may change the way we screen women for Down syndrome forever.

In 1997, researchers discovered that DNA from a fetus actually circulates in the blood of the mother during pregnancy. It goes away very rapidly after the baby is delivered. This amazing observation meant that it might be possible to diagnose certain diseases in the fetus by simply taking a sample of mom's blood. Since then, many studies have been done to try to develop a method to screen for Down syndrome using maternal circulating fetal DNA.

In 2008, two papers were published, from different laboratories, which described the detection of trisomy 21 using "massively parallel genomic sequencing" (MPGS). In this method, DNA fragments are isolated from a sample of mom's blood. These include a mix of mom's DNA and infant DNA. The ends of the DNA fragments get a small piece of adapter DNA attached to it which allows the fragments to hybridize to a surface coated with the complimentary adapter DNA sequence. All the fragments bound to the surface are then simultaneously amplified. After amplification, the DNA is sequenced. With the help of bioinformatics, the researchers are able to determine what chromosome the DNA fragment came from. The number of sequences that originate from a particular chromosome is counted and tabulated for each chromosome. If a fetus has an extra chromosome, then the percentage of chromosome 21 fragments is higher than expected.  Early studies suggested that this method might work with high sensitivity, but the studies were small.

Recently, a very large validation study was published that demonstrated that this method is not only sensitive, but also had a very low false positive rate. 

The study involved women at high risk of delivering an infant with Down syndrome from 27 prenatal diagnostic centers worldwide. The authors compared fetal karyotyping (the gold standard for diagnosing Down syndrome) to MPGS in 212 Down syndrome and 1484 matched normal pregnancies. Their research demonstrated that MPGS was able to detect Down syndrome with 98.6% sensitivity and only a 0.20% false positive rate. This represents a huge improvement over currently available methods.

Currently, MPGS is only available at specialized centers and is quite expensive. However, with the advent of studies such as this, the availability and price of MPGS is bound to come within reach. Hopefully, within the next 10 years, we will see the end of maternal serum screening for Down syndrome as we currently know it.

The Triple Test: time to say good bye

A recent post in the NACB blog titled "Jurassic Lab" invited readers to comment on which laboratory tests they thought should be considered obsolete.  I suggested that the Triple Test, a blood test used during the second trimester of pregnancy to screen for Down syndrome, should go the way of the dinosaur.

Labs that offer Down syndrome screening tests should seriously consider taking the Triple Test off of their menus. Why? There are a few reasons:

  1. It is not a great test. As a screening test for Down syndrome, the Triple test is only okay. If I were grading it, then I would give it a C. It detects about 70-75% of fetuses with Down syndrome and it has a false-positive rate of 5-8%.
  2. Better tests are available. I've already wrote about the different types of Down syndrome screening tests. Other tests, like the Quad and the Integrated tests perform much better than the Triple test. Given a choice, I imagine most mothers-to-be would prefer to have the best screening test available. It's abundantly clear that of all the tests used to screen for Down syndrome, the Integrated test is best. It has a detection rate of about 90-95% and (importantly) a false-positive rate that is lower than the Triple test. A low false-positive rate is important because it decreases the number of unnecessary invasive diagnostic procedures that are performed due to an abnormal result from a screening test.
  3. Lack of recommendation. Several national guidelines no longer recommend use of the Triple test and recommend better performing screening tests instead.

Clearly the Triple test is irrelevant. That doesn't mean that all the doctors that order Down syndrome screening tests have gotten the message. A 2009 survey of obstetricians in the US revealed that nearly a quarter of them (23%) continued to use the Triple test. That's lower than it used to be but still rather high for a test that shouldn't be ordered. Until laboratories that offer the Triple test take it off of their menus, it will continue to be (inappropriately) utilized.

Choosing a screening test for Down syndrome

Last month I wrote about the many different tests used for Down syndrome screening.  Today, there are 6 different screening tests that a woman can choose from should she opt for any testing at all.  Selecting one can't be an easy task for most women and, given the relatively short time that most physicians have to spend with patients today, many simply follow their doctor's advice and don't truly understand their options.

The choice of which Down syndrome screening test to have depends on the answers to a few questions:

  • When during pregnancy does the patient first seek obstetrical care for her pregnancy?

Some screening tests are performed in the first or the second trimester while other tests are performed in both first and second trimesters.  If a woman first seeks obstetrical care after the 14th week of pregnancy then only the second trimester tests can be offered.  That means either the Triple or the Quad tests.  If she presents earlier then all screening tests can be considered.

Nuchal translucency (NT) is an ultrasound measurement of space under the skin behind the fetal neck and is performed only during the first trimester.  An increased NT is strongly associated with Down syndrome and other chromosomal abnormalities.  It's a component of the Combined test, the Integrated test, and the Sequential test.

Chorionic villus sampling (CVS) is the removal of a small piece of placental tissue in order to obtain fetal cells in order to obtain the fetal karyotype.  The karyotype will identify the number of chromosomes the fetus has inherited and is usually only performed after an abnormal Down syndrome screening test.  This procedure is performed in the first trimester because amniocentesis can't be performed this early in pregnancy.

If CVS is not available then options include the Integrated, the Triple, or the Quad test.  If the NT is not available then the choice of screening tests is limited to the Serum Integrated test, the Triple test, or the Quad test (the same is true if both NT and CVS are not available).

  • When does the mother want the results of the screening test?

If the woman wants the earliest assessment of the risk of carrying a fetus with Down syndrome then she should opt for the Combined test.  The test is performed only in the first trimester and results are available sooner than with any of the other test options.  However, the test doesn't perform as well as some of the other tests.  On average, the Combined test detects about 85% of all Down syndrome fetuses at a 5% false-positive rate.

If she's willing to wait until the second trimester for the test results then the best screening test is the Integrated test.  This test gives the highest Down syndrome detection rate (about 95%).  The test can also be performed without an NT (the Serum Integrated test) and will provide the same detection rate with a slightly higher false-positive rate.

If the patient desires early risk assessment but is willing to wait for test results if needed, then the Sequential test may be appropriate.  The Sequential test reports results in the first trimester only if the risk of having a Down syndrome fetus is very high.  If the risk is not high, then results are not reported until the second trimester test is completed.

The table below summarizes the performance of the 6 different Down syndrome screening tests.  The "detection rate" (DR) is the percentage of Down syndrome pregnancies that are correctly identified by the test and the "false positive rate" (FP) is the percentage of unaffected pregnancies identified as abnormal.

Performance of maternal serum screening tests

Selecting a test for Down syndrome screening doesn't have to be complicated.  Patient's should be informed about the pros and cons of each test (and screening tests in general).  It's often said that information is power and, when it comes to Down syndrome screening, women should be empowered to choose the test that is right for them.

      A perfect Down syndrome screening test?

      In a January post, I wrote about a possible new Down syndrome screening test that detected 100% of affected fetuses and had a very low false-positive rate.  Recently a different group of scientists in Cyprus reported they had developed a perfect test to detect Down syndrome: one that correctly detects all Down sydrome fetuses but had zero false positive results.

      While the earlier report utilized a time-consuming and expensive technique called massively parallel sequencing, this new study focused on a process called DNA methylation.  Methyl groups are chemical structures that are naturally attached to regions of DNA.  They function to turn genes on or off.  Because DNA from fetuses have different methylation patterns compared to their mothers', fetal DNA can be distinguished from mom DNA in a blood sample taken from the mother.

      After enrichment of the methylated fetal DNA from the mother's blood, quantitative PCR was used to amplify specific methylated regions of chromosome 21.  Fetuses with Down syndrome were identified because they have three, rather than two, copies of chromosome 21, and the increased copies were readily detected by the test.  Out of 40 women who were 11 to 14 weeks pregnant when tested, the method was 100% accurate: all of the 14 Down syndrome and all of the 26 unaffected fetuses were correctly identified.

      In contrast to a sequencing approach, the techniques used in this study are rather easy and fast to perform and don't require expensive equipment or software.  It is also a lot cheaper.

      This is a promising test but there's still a lot more to be done before it makes the leap into clinical use.  The Cyprus researchers are planning to do larger-scale clinical trials with many more women.  Let's hope that the results from those trials are as exciting as this early report.

      Why so many Down syndrome screening tests?

      Throughout pregnancy, women have to make lots of decisions.  Two of those are “Do I want to have a screening test to see if my baby has an increased risk of Down syndrome?” If the answer to that is yes then the logical next question would be “What tests can I choose from?”  That is not an easy question for most women to answer without some guidance from their doctors and it’s not easy for many doctors to help provide that guidance.  Why?  Simply put, there are many tests from which to choose.

      Let’s be clear on an important concept up front.  Screening tests are not diagnostic tests.  A test that screens for Down syndrome doesn’t identify if a baby has Down syndrome; it identifies babies that are at increased risk of having Down syndrome.  Women with abnormal or positive screening test results can undergo additional tests that can be used to confirm if their baby does or does not have Down syndrome but the screening tests cannot do that. 

      All Down syndrome screening tests require a blood sample from the mother.  Biochemical markers in the blood are measured in the laboratory and the results used to calculate the risk that the baby has Down syndrome.  Those biochemical markers include:

      • Alpha-fetoprotein (AFP)
      • Human chorionic gonadotropin (hCG)
      • Unconjugated estriol (uE3)
      • Dimeric inhibin A (DIA)
      • Pregnancy-associated plasma protein A (PAPP-A)

      Some screening tests also include the measurement of nuchal translucency (NT) that is obtained by an ultrasound scan of the fetus.  The NT is the width of the space between the spine and skin at the fetus’ neck.

      All together, these 5 biochemical markers and 1 ultrasound marker can be used in various combinations to create the different Down syndrome screening tests.  There are 6 to choose from:


      Some, like the Triple (3 markers) and the Quad (4 markers) tests are performed on a blood sample collected during the 2nd trimester and don’t require the NT ultrasound measurement.  The Combined test (so called because it “combines” the biochemical and ultrasound tests) is performed only in the 1st trimester.  All the other tests use two different blood samples (one collected from the mother in the 1st and the other in the 2nd trimester) and may or may not also include the NT measurement.  No wonder this is confusing!

      Why are there so many different tests?  Two reasons: first because Down syndrome screening tests evolved over many decades (and continues to evolve) and second because the medical community is often slow to change its habits.

      In the not so distant past, one “test” was used to determine Down syndrome risk: the age of the mother.  We actually still use the mother’s age in determining Down syndrome risk.  The risk of having a baby with Down syndrome increases as the age of the mother increases.  We now use this age-based risk as a starting point; a risk that is the modified by the results of the screening test.

      In 1988, the Triple test was introduced as a way to adjust the age-based risk using the measured concentrations of AFP, hCG, and uE3 in the mother’s blood.  A few years later, the Triple test turned it into the Quad test when it was discovered that the addition of DIA to the Triple test improved the Down syndrome detection rate.  However, the Triple test didn’t disappear and labs simply continued to offer it as well as the Quad test.

      The Combined test appeared in 1999 and had a Down syndrome detection rate that was similar to that of the Quad test.  Unlike the Quad test, however, the Combined test provided women a way to get Down syndrome screening test results many weeks sooner.  So, it was added to the menu along with the Triple and the Quad tests.

      It wasn’t long before someone thought to “integrate” the Combined test with the Quad test and thus was born the Integrated test.  And, for those women without access to the specialized equipment needed to perform the ultrasound NT measurement, the Serum Integrated test required only the mother’s blood samples to be tested.  While both of these tests provide the greatest Down syndrome detection rates, the need for the second blood sample collected in the 2nd trimester means that early risk-assessment is not possible.

      And finally, the Sequential test developed as a modification of the Integrated test.  While the Integrated test delivers its results in the 2nd trimester after all testing has been completed, the Sequential test offers women results in the 1st trimester only if the risk of Down syndrome is very high.  In the absence of a high risk, results are provided only after the test is completed in the 2nd trimester.

      Because it’s a test with a lot of history and experience behind it and because it’s well known to the doctors that order Down syndrome screening tests, the Quad test still leads the pack in terms of test usage.  The Triple test is slowly falling out of favor (as it should) and the Combined, Integrated, and Sequential tests are gaining more traction, albeit slowly.  As I said earlier, old habits are slow to change.

      New blood test for Down syndrome screening

      This is impressive!  A study reported yesterday in the British Medical Journal describes a new blood test that could vastly decrease the number of invasive procedures that are currently used to confirm if a fetus has Down syndrome.

      Current blood tests used for Down syndrome screening can identify up to 90-95% of Down syndrome pregnancies but about 5% of normal fetuses are incorrectly identified as having Down syndrome.  That means that lots of women have follow-up tests, like amniocentesis, to definitively determine if their baby is affected.  That's a problem because amniocentesis is an invasive procedure that can lead to the loss of the pregnancy.  Current estimates put the risk of fetal loss due to amniocentesis at about 1 out of 300. 

      914335_85510317 This new blood test measures the amount of DNA from chromosome 21 that is present in the mother's blood.  Down syndrome is caused by having an extra copy of chromosome 21.  We know that a pregnant woman's blood contains small amounts of her baby's DNA but that amount is very
      low compared to the amount of her own DNA in her blood.  Using a technique called massively parallel sequencing, this test looks for increased amounts of pieces of chromosome 21 in the mother's blood.  In a Down syndrome pregnancy, the mother's blood will have more pieces of chromosome 21 because the fetus has an extra copy of that chromosome.

      The study used blood collected from 753 women who were at high-risk of having a baby with Down syndrome.  86 of these pregnancies were carrying a fetus with Down syndrome and the test was able to correctly identify 100% of them.  While that alone is impressive, even more exciting is that only 2.1% of the unaffected fetuses were incorrectly identified as having Down syndrome.  Stated another way, in 98% of the cases Down syndrome could be ruled-out sparing the need for invasive follow-up testing.  Like some of the existing blood tests used to screen for Down syndrome, this new test can be performed in the first trimester of pregnancy.

      Although the test is not yet available, there is a company that is working on making it available soon.