The Pregnancy Lab

More on noninvasive prenatal testing for fetal aneuploidy

We have written about nonivasive prenatal testing (NIPT) on this blog several times.  Because they are so new, the landscape around these tests is continually evolving.  The American College of Obstetricians and Gynecologists (ACOG) published guidelines on these tests in December of last year.  Just this week, the American College of Medical Genetics and Genomics (ACMG) released its policy statement on the same topic.  Note that the ACMG refers to these tests as "noninvasive prenatal screening" (NIPS) tests to emphasize that this is what they are: screening, not diagnostic tests.

The ACMG calls for caution before these tests become widely integrated into prenatal care due to the current lack of data obtained from prospective clinical trials.  While they acknowledge that NIPS tests have high sensitivity and specificity there are limitations to the technology and false-positive and false-negative results do occur.

A particular concern, and one that doesn't get as much attention as it should, is that most of the fetal DNA in the mother's blood sample originates from the placenta and not the fetus and it may not accurately reflect the fetal karyotype.  They emphasize (as have others), that abnormal NIPS test results must be confirmed by invasive diagnostic tests such as amniocentesis.

The policy statement also lists several limitations to NIPS tests.  Among them:

• They only detect aneuploidies (and some detect sex chromosome abnormalities).
• Certain chromosome abnormalities are not detected.
• The tests take longer to perform and result than more well-established tests.
• Data on the performance of the tests in twin and triplet pregnancies is not well established.

A recent paper published in the journal Obstetrics and Gynecology has a similar ring to it.  The authors make several interesting observations:

• First, they point out that well-established tests were developed in academic settings and came into use gradually and only after independent clinical studies generated data to support their use.  In contrast, NIPT (also developed in academic settings) was quickly licensed to commercial enterprises that have brought them to market without FDA review (as these are "lab-developed tests," FDA appoval is not required).
• From the analytical perspective, there are currently no guidelines regarding quality control and quality assurance for NIPT; a vital component of any lab test.
• The performance of NIPT in actual clinical practice settings (i.e. not a clinical study) is currently not well known or documented.  This is especially true for populations of women that have not been represented in the clinical studies (e.g. woman at low risk for having a fetus with an aneuploidy).
• The more well-established tests are able to detect fetal anomalies besides aneuploidy (e.g. open neural tube defects).

The authors also reflect on how NIPT should be incorporated into clinical care.  They agree with the ACOG recommendations that the tests should not be offered to low-risk women but they go a bit further and state that the most appropriate use of NIPT is as a second screening test used for those who have an abnormal result from convential, more well-established screening tests.  The latter point is something I have commented on before and I could not be in more complete agreement.

Should DNA-based tests for Down syndrome screening replace biochemical tests?

In a previous post I described the clinical performance of DNA-based screening tests for fetal aneuploidies like Down syndrome.  Overall, these tests have excellent detection rates (~99%) with very low false-positive rates (~0.2%).  In other words, these tests are about 99.0% sensitive and 99.8% specific.

With performance like that one might expect these to be considered diagnostic tests.  They are not! Although quite good, test results must not be interpreted as definitive evidence that a fetus does or does not have an aneuploidy.  Recent recommendations from the American College of Obstetricians and Gynecologists (ACOG) are quite clear on that issue.

In those same recommendations, ACOG also states that DNA-based screening tests may be performed only on women who are at increased risk of having a fetus with aneuloidy.  Among the indications listed for women considered to be at increase risk are:

• Maternal age 35 years or older at delivery
• Fetal ultrasound findings suggesting aneuploidy
• A previous aneuploid pregnancy
• Abnormal biochemical screening test results

The ACOG is right to avoid recommending that DNA-based screening tests are acceptable to use regardless of risk factors.  Unfortunately, many women who are not at increased risk are using these new tests as a primary screening test and that's not a good idea.

To understand why, considered a population of 100,000 pregnant women from the general population and assume that the prevalence of Down syndrome is 1 in 500 pregnancies.  That means that there would be 99,800 unaffected pregnancies and 200 pregnancies with Down syndrome.  The table below compares the results of the most commonly used biochemical screening test (the Quad test) to a DNA-based screening test.

Clearly, the DNA-based test has several advantages over the Quad test.  Its positive predictive value is nearly 17 times greater than the Quad's and a positive DNA-based test result substantially increases the odds of having an affected fetus.  So why not use the DNA-based test as a primary screening test?  For the following reasons:

• No studies have been published that have evaluated the performance of DNA-based tests in women who are not at increased risk of having a fetus with an aneuploidy
• DNA-based tests are not widely available
• The time it takes to report results of DNA-based testing is about 3 times greater than it is with biochemical testing
• DNA-based tests are considerably more expensive than biochemical tests
• Relative lack of insurance coverage for DNA-based tests

Until these these limitations can be resolved, it makes more sense to use DNA-based testing as a secondary screening test.  In other words, it is only done after one of the risk factors described by ACOG (above) are met.  Doing so greatly improves the performance of both tests (see figure below).  A limitation of this approach is that the detection rate is that of the biochemical test which is not as high as it is with the DNA-based test.  Still, given the current limitations of DNA-based testing, this 2-step testing approach makes the most sense.

Molecular testing for Down syndrome: proceed with caution

Ann recently posted about massively parallel genomic sequencing using maternal blood as a
screening test for Down syndrome.  In it, she described a recently published, multi-center clinical study that validated this molecular test in just over 1,600 women.  The test detected 98.6% of the Down syndrome fetuses and had a false-positive rate of 0.2%.  Notably, this performance is considerably better than current biochemical marker Down syndrome screening tests.

However, there are some important limitations to this new test.

1. It’s expensive and available from only one company.  The Sequenom Center for Molecular Medicine, located in Grand Rapids, MI, developed the test and is currently the only place in the US that can do it.  It's marketed as the MaterniT21 test and costs $1,900 for the uninsured and ~$235 for those with insurance.  Biochemical screening tests cost much less.
2. After the blood sample arrives in the lab, test results are available in about 10 days.  Results for biochemical screening tests take about 1 day.
3. The test can only be performed in singleton pregnancies (not twins, triplets, etc).  Biochemical screening tests work best in singleton pregnancies, too, although risks can be estimated from a twin pregnancy.
4. The test fails to work in about 2% of cases, usually as a result of there not being enough fetal DNA detected in the mother's blood.  This is more likely to be the case in overweight women who have a higher blood volume which dilutes the amount of the fetal DNA.  The concentrations of the markers used in biochemical screening tests are also effected by maternal weight but there are effective ways to account for that so that the final result is not affected.
5. The test is only validated for the detection of Down syndrome and not other fetal aneuploidies such as trisomy 18 or trisomy 13.  The test does have the capability of detecting these disorders and, if they are detected, will be reported.  However, because the test has not been thoroughly investigated for detecting T18 or T13 a negative result doesn't rule out their presence.  Biochemical screening tests can detect these disorders although the detection rates are lower than they are for Down syndrome.
6. The test does not detect open neural tube defects (e.g. spinal bifida) that are usually screened for using biochemical screening tests.

I have no doubt that as the technology required to do massively parallel genomic sequencing becomes less expensive, tests like the MaterniT21 will become more affordable and mainstream.  Most of the limitations I described above will also be addressed with time and technological improvements.  As Ann indicated, this is the dawn of a new era in screening for fetal disorders.

Screening for Down syndrome: Beginning of a New Era?

David 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.

Screening tests for group B strep infection

The most common cause of life-threatening infections in newborns comes from a bacteria known as Streptococcus agalactiae (more commonly referred to as group B streptococcus or GBS).  This was stressed in a recent meta-analysis that reported that GBS infection remains an important, global cause of infant mortality.

The overall infection rate was 0.53 per 1,000 live births and, on average, about 10% of infected infants died.  Infants born in Africa were more likely to be infected (1.21 per 1,000) and die (22%) from the infection than infants born in the Americas or Europe (0.67-0.57 per 1,000 with 11 and 7% fatality rates).

It doesn't have to be this way because GBS infection is treatable with antibiotic therapy.  Indeed, in more developed countries, therapy is provided to women who carry the bacteria which prevents their baby becoming infected during delivery.  However, in poorer countries this is less likely to happen due to fewer resources.

Providing therapy to every pregnant women is not practical because not all women are colonized with GBS and so a key preventative strategy is to identify those women who do carry the bacteria.  The most sensitive test is culture performed on samples collected from the vagina and rectum.  The Centers for Disease Control and Prevention (CDC) published guidelines in 2010 that called for the routine GBS screening in all pregnant women at 35 to 37 weeks of gestation.  Testing needs to happen close to delivery (normally at ~40 weeks) because women can be colonized with GBS at anytime.  That is, a negative test result obtained earlier in pregnancy wouldn't rule-out the possibility that colonization then occured sometime after testing.  Women with a positive culture are treated with antibiotics during labor to prevent the transmission of GBS to their infant.

Although culture is considered the gold standard test for GBS screening, it is not perfect because some infants born to culture-negative women still get infected with GBS.  Also, culture techniques give results in 1–3 days, a time frame that may not be useful should an expectant mother go into labor prior to having the culture test performed.  For these women, DNA-based tests can be used.

These tests detect the presence of GBS using a DNA amplification technique like PCR and give results in a few hours rather than days.  Currently, these types of tests are not as sensitive as culture (i.e. they can give false-negative results) and so they aren't recommended for routine screening of women who are not in labor.  Their sensitivity is improved by using an enriched sample (one where the bacteria are allowed some time to multiply in a growth media), the use of this type of sample is impractical for women in labor when results are needed quickly.

Until an effective vaccine to prevent GBS infection is available, laboratory testing will remain an essential tool for identifying and preventing GBS.

Determining a baby's gender in the lab

It's rare that I come across a pregnant woman who doesn't already know the gender of the baby she is carrying.  Ultrasound assessment of a fetus is routinely performed during pregnancy to identify fetal anomalies and, very often, gender identification is offered.  After the 13th week of pregnancy, ultrasound is quite accurate in determining a baby's sex.  There are circumstances, however, where knowing a fetus is a boy or a girl is important.  For example, male fetuses born to women who are carriers of X-linked genetic disorders are at risk of inheriting the disorder.  In such instances, knowledge of a fetus' gender before 13 weeks can be advantageous.

Today, the Journal of the American Medica Association (JAMA) published a systematic review and meta-analysis (a systematic method of evaluating statistical data based on results of several independent studies of the same problem) that sought to determine the performance of DNA tests performed on maternal blood to determine fetal sex.  As a reminder, DNA from a fetus circulates in maternal blood.  This cell-free fetal DNA can be detected and amplified and is the basis of other emerging diagnostic tests.

This report evaluated the results from 57 different studies that tried to determine fetal gender using a test performed on a sample of mother's blood or urine.  The 57 reports contained results from 6,541 women; 54% who delivered boys and 46% who delivered girls.  Because this was a meta-analysis, several different testing techniques were used in the included studies but all of them looked for the presence or absence Y chromosome-specific molecular markers.  If those markers were detected, the fetus predicted to be a male.  If they were not detected, the fetus was predicted to be a female.

Collectively, the tests performed very well.  They correctly identified 95.4% of the male fetuses (test sensitivity) and 98.6% of the female fetuses (test specificity).  The tests did not work well if maternal urine was used as the test sample which is not surprising because cell-free fetal DNA is very difficult to detect in urine.  The report also noted that real-time quantitative PCR (RTQ-PCR) methods had better performance than conventional PCR techniques.

As might be expected, test performance improved along with the gestational age of the fetus.  At less than 7 weeks test sensitivity (see above for definition) was 74.5% but increased to 95% after 7 weeks at was 99.6% at more than 20 weeks.  That makes sense because there is more fetal DNA circulating in the mom's blood as the pregnancy progresses and the fetus increases in size.

Because of the potential clinical use of the test to identify male fetuses of mother's with X-linked genetic diseases, the authors caution that these types of sex-predicting tests should be optimized to achieve high sensitivity (detection of male fetuses) even if it compromises the specificity (detection of female fetuses).  They also cautioned that any tests developed and offered by laboratories should thorough validate that a negative (i.e. female fetus) test result reflects the presence of a female fetus and not simply the inability to detect any fetal DNA (which would also give a negative (female) result).

While there are some direct-to-consumer labs that offer this type of DNA-based testing to determine fetal sex, the test is marketed as one of convenience to satisfy a mother's curiosity regarding her baby's gender.  Currently, I am unaware of any clinical laboratories in the United States that offer such testing for diagnostic purposes although the tests are available for that reason in the European Union.  I predict that this type of test will become more widely available in clinical labs over the next few years.  There is much promise to be realized from tests based on circulating cell-free DNA!

Noninvasive antenatal testing using cell free fetal DNA

Today's post is by a guest author, Joshua Cook, M.D.  Dr. Cook is a pathology resident at the University of Utah.

Fetal cells, largely of placental origin, circulate freely in the mother’s bloodstream during gestation.  In some cases fetal cells are detectible in maternal blood for years post delivery.  Cell free fetal DNA (cffDNA) is also present in the peripheral blood of pregnant women, though it disappears rapidly at delivery.  cffDNA appears early in the first trimester and tends to increase in concentration as gestation progresses.  A number of studies over the last decade have shown this naked fetal DNA provides a noninvasive method for rendering prenatal diagnoses without risk to the fetus or mother.

Consider hemolytic disease of the newborn (HDN).  About 10% of pregnancies in Caucasian populations involve a rhesus Rh(D)- negative mother carrying an Rh(D)-positive fetus.  Such a mother is at risk of becoming sensitized, or isoimmunized, to Rh(D)-positive red blood cells following exposure to fetal blood. 

Small physiologic fetal-maternal hemorrhages commonly occur at delivery or during miscarriage.  Exposure of the mother’s immune system to fetal red blood cells may result in the generation of maternal antibodies to fetal antigens (sensitization or isoimmunization).  These antibodies may then cross the placenta in subsequent pregnancies and harm a future baby.  In pregnancies with Rh(D) incompatibility [Rh(D)-negative mom with Rh(D)-positive fetus] the mother has historically had about a 10% risk of becoming sensitized. 

Knowledge of the fetal RHD genotype in Rh(D) negative women greatly simplifies antenatal management.  Modern medical practice requires that immunoprophylaxis (anti-D IgG immunoglobulin; aka RhoGAM) routinely be given to all non-sensitized RhD-negative woman in pregnancy to prevent isoimmunization.  This had led to a dramatic decrease in HDN since the 1960’s.  When administered, the anti-Rh(D) antibodies circulate in the mother’s bloodstream and prevent her immune system from reacting to Rh(D)-positive fetal blood.  The standard of care is to provide anti-Rh(D) administration at 26 to 28 weeks of gestation, and after suspected antepartum fetal-maternal hemorrhage (i.e. amniocentesis, chorionic villous sampling, pregnancy loss, ectopic pregnancy).  Anti-D IgG is also given with within 72 hours of delivery if the baby is found to be Rh(D)-positive.

Because the Rh(D) status of the fetus is usually not known prior to birth, about 40% of immunoprophylaxis is given unnecessarily to Rh(D)-negative mothers who are carrying Rh(D)-negative fetuses.  cffDNA testing restricts immuneprophylaxis to the 60% of pregnancies with a true Rh(D)-positive fetus.  Fetal RHD genotyping using maternal plasma is about 95% accurate at 11 -13 weeks gestation and is nearly 99.8% accurate by 26 weeks (when the first dose of immuneprophlyaxis is administered).  Accuracy improves throughout gestation because the amount of cffDNA in maternal peripheral blood increases with gestational age. 

cffDNA testing is currently used in Europe to identify high risk pregnancies [i.e. Rh(D)-positive fetus  in a previously sensitized mother] which benefit from intensive antenatal monitoring.  cffDNA RHD genotyping is used to determine the risk of chorionic villous sampling (CVS) in sensitized women.  CVS is a technique used in the prenatal diagnosis of fetal aneuploidies (such as Down syndrome) and other inherited genetic disorders; however it is relatively contraindicated in isoimmunized women since procedure related fetal-maternal hemorrhage exacerbates pre-existing sensitization.  If the fetus lacks Rh(D) then boosting the maternal amnestic response is not an issue.

Cost and outcome studies for noninvasive cffDNA prenatal screening are in progress, but obstetricians may soon have a powerful new tool in their armamentarium.

 

 

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.

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. 

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.