Screening for Down syndrome, Trisomy 18 syndrome, and
Open Neural Tube Defects in the First Trimester

Introduction
Screening a low-risk population to detect a group of patients at high risk for birth defects is a familiar concept to obstetricians and geneticists. In the late 1970's, maternal serum alpha-fetoprotein (MSAFP) was introduced as a screen for open neural tube defects (spina bifida). In 1984, MSAFP was also shown to be a valuable marker for fetuses with Down syndrome in women under 35 years of age.

Over the years, improvements have been introduced to enhance detection of birth defects or to reduce the number of follow-up procedures (amniocentesis or CVS) performed.

In the late 1980's, new second trimester biochemical markers (chemicals found in the mother's blood during pregnancy) for Down syndrome were found (intact human chorionic gonadotropin [hCG], the freeBeta subunit of hCG, estriol, etc.).

Improved statistical methods incorporating Bayesian and linear multivariate discriminate analysis were applied by GeneCare to the raw data, which dramatically improved detection levels over methods relying solely on multiples of the median (MoMs, a comparison of the woman's blood values to the normal range).

Our researchers discovered that a liquid blood specimen deteriorates over time, especially if subjected to temperature extremes. If, however, blood is spotted onto blot paper, the markers are immediately stabilized and dried onto the paper matrix, eliminating the effects of hemolysis, spontaneous analyte degradation and temperature fluctuation in transit. When analyzed, the blotted specimens more accurately reflect the patients' true biological state than whole blood samples and offer better discrimination between affected and unaffected populations. Dried blood significantly decreases the number of women who receive a screening report showing an increased risk of having a baby with a chromosome abnormality or other anomalies.

The timing of screening has also been improved by the identification of first trimester markers for chromosome abnormalities. In 1992, nuchal translucency, the measure of fluid accumulation at the back of the fetal neck, was shown to be a strong marker for chromosomal abnormalities, including Down syndrome and Trisomy 18. Around this time, excellent biochemical markers such as pregnancy-associated plasma protein-A (PAPP-A) and free beta-hCG (freeBeta) were shown to be the most specific and sensitive for Down syndrome and Trisomy 18 screening.

As the above discussion shows, birth defects screening is a rapidly changing scientific field. Undoubtedly, more improvements will be forthcoming. At present, however, many laboratories in the United States still provide the triple screen (AFP, hCG and estriol), a combination that falls substantially (21-24%) short of detection that can be accomplished with current second trimester technology. This text outlines a superior screening paradigm that has been implemented in most European countries and across the United States, and offers state-of-the-art first and second trimester screening for birth defects.

First Trimester Screening
The best detection of Down syndrome and Trisomy 18 occurs through a combination of biochemical and sonographic markers, which are assessed in the first trimester. First trimester screening involves a blood draw by finger stick onto filter paper and an ultrasonographic (by sonogram) nuchal translucency (NT) measurement. The blood sample may be taken between 8 weeks 4 days and 13 weeks 3 days of pregnancy. The NT is measured between 11 weeks 0 days and 13 weeks 6 days (crown rump 45-84mm). Although risks for Down syndrome and trisomy 18 can be generated with either the biochemical or the sonographic data alone, the detection rates are better when both are used. The NT measurement must be taken by a certified sonographer to ensure the uniformity and accuracy of the data being used for risk assessment.

The following two tables present the detection performance of freeBeta/PAPP-A by itself and freeBeta/PAPP-A in conjunction with NT measurement.

Table 1: Performance of freeBeta/PAPP-A between 8 weeks 4 days and 13 weeks 3 days gestation.

Table 2: Performance of freeBeta/PAPP-A + NT between 11 weeks 0 days and 13 weeks 6 days gestation.

* Initial positive rate, this rate decreases after ultrasound confirmation of gestational age
** Number of amniocenteses or CVS's necessary to identify one affected fetus

Comparison Critera

The quality of a screening program is judged by multiple variables:

Detection Rate

The detection rate is the percentage of affected fetuses that are identified as high risk by the test. Screening programs try to maximize this variable.

Without NT measurement, first trimester screening identifies 68% of Down syndrome cases, compared to the triple screen's 56-60%. When NT measurement is added, detection rises to 91% (31% higher)! This rate for Down syndrome is unequaled by any other known first or second trimester screening test.

Trisomy 18 screening in the first trimester without NT measurement identifies 90% versus 50-60% with the triple screen. With NT measurement, detection of Trisomy 18 increases to 97% in the first trimester.

Initial Positive Rate

The initial positive rate (IPR) is the percentage of women reported to have an increased risk for Down syndrome and/or Trisomy 18. Screening programs try to minimize this variable.

First trimester screening (with or without NT Measurement) has an initial positive rate of less than 4%, compared to the triple screen's 5 to 7% (for Down syndrome and Trisomy 18) - about a 50% reduction in initial positives, which allows for fewer unneccessarily anxious women, reduced medical time and cost, and fewer diagnostic amniocenteses and CVS's.

Yield

Yield is the number of diagnostic procedures (including early amniocentesis at 12-14 weeks, CVS at 11-12 weeks, and amniocentesis) required to identify an affected fetus. This variable is a function of both the detection rate and the initial positive rate and provides a single measure of a program's performance. Screening programs try to decrease the number of diagnostic tests needed to identify the same number of affected fetuses.

Physicians who are familiar with maternal serum screening are aware that many women are referred for amniocentesis because of an increased risk, but most are found to have healthy babies. To be able to reduce the number of such women without decreasing detection is the goal of screening, and yield measures this ability. Approximately 1 in 80 women with an increased risk for Down syndrome via the triple screen actually has a baby with Down syndrome. First trimester screening improves this number to 1 in 25 (without NT measurement) or to 1 in 17 (with NT measurement).

Additional Remarks

A 91% detection rate for Down syndrome and 97% for Trisomy 18 in women under 35 years of age is vastly more effective than any other known screening paradigm and is available only in the first trimester. Furthermore, these detection rates are achieved with low IPRs and very high yields.

Open neural tube defects (ONTD's=open spine and/or skull defects) and open abdominal defects cannot be screened for in the first trimester. Consequently, women who recieve a first trimester screening report that does not show an increased chromosome risk should consider having an AFP (alpha-fetoprotein) dried blood screening test at 13.5-15 weeks to obtain the 98% detection rate of ONTDs. However, those women who receive an increased risk report for a chromosome abnormality can elect to have early amniocentesis and have a very accurate amniotic fluid acetylcholinesterase test to detect greater than 99% of ONTDs and most open abdominal defects.

First Trimester Markers

FreeBeta HCG

Human chorionic gonadotropin (hCG) is a protein made by the placenta that consists of two subunits, alpha and beta. hCG is present in two forms: intact hCG (consisting of both alpha and beta subunits) and free Beta hCG (the beta subunit of hCG alone). Serum levels of hCG tend to be high in patients carrying a fetus affected with Down syndrome. The median concentration for free Beta hCG in the blood of women carrying fetuses with Down syndrome, measured as a multiple of the median (MoM), is 1.9 (compared to 1.0 for unaffected fetuses). This large difference provides the basis for the discrimination between affected and unaffected fetuses. Intact hCG, on the other hand, does not discriminate as well: its median MoM is less than 1.3. Furthermore, free Beta is significantly reduced in the blood of women carrying Trisomy 18 fetuses, with a median MoM of 0.18. Free Beta is the most specific and sensitive marker for Down syndrome and Trisomy 18.

The main advantage of measuring free Beta hCG, instead of intact hCG, is a significantly higher detection rate for a given false positive rate. Free beta hCG is the only biochemical marker that is effective in both the first and second trimester. Screening for chromosomal anomalies is possible in the first trimester by using a combination of biochemical markers and ultrasound, with a better detection rate than second trimester biochemical screening.

PAPP-A

Pregnancy Associated Plasma Protein A (PAPP-A) is also produced by the placenta. PAPP-A is significantly reduced in the blood of women carrying fetuses with Down syndrome and Trisomy 18: the median MoMs are 0.44 and 0.32, respectively (compared to 1.0 for unaffected fetuses). These dramatic differences from normal provide excellent discrimination between affected and unaffected fetuses.

Nuchal Translucency

One of the single most important breakthroughs in chromosome abnormality screening was the ultrasonographic observation of a correlation between a nuchal translucency (accumulation of fluid at the back of the fetal neck) observed on ultrasound between 10 and 13 weeks and the incidence of Down syndrome and other abnormalities. NT is increased in fetuses with chromosome abnormalities, heart defects, and certain genetic syndromes.

In 1990, Cullen reported the significant association of an abnormal collection of fluid behind the fetal neck in the first trimester of pregnancy and chromosomal abnormalities - particularly trisomies 21, 18, and 13. By far, the most important contributor to the study of NT thickness has been the King's College group in London. Their data, now extending to well over 100,000 cases, shows a sensitivity of abnormal nuchal translucencies for Down syndrome detection at over 80%, with comparable percentages for trisomies 13 and 18. Initially, other centers attempting to duplicate their work did not show as spectacular results, and there have been variable interpretations as to why. The King's College group suggests that the lower detection rates of others have been because personnel have not measured the NT with appropriate reliability at the correct gestational age. The King's College data has been confirmed by centers around the world after each center receives special training, certification, and continued NT median and film monitoring. It is important to note that centers with initially lower detection rates which subsequently received certification and underwent continuous monitoring have achieved greater than 80% detection rates, which validates the King's College protocol.

In order for the interpretive software to calculate an accurate risk for chromosome abnormalities, the measurement must be taken in a standardized way. For this reason, only measurements taken by sonographers who have received special certification from the Fetal Medicine Foundation, King's College, London in NT evaluation and continuing monitoring of medians and films are accepted for risk assessment. A partial list of NT certified healthcare providers who meet this high standard is presented below.

A United Kingdom Royal College of Obstetricians and Gynaecologists study group (1997) has recommended that "there is now sufficient evidence to consider that specific serum markers for Down syndrome at 9-13 weeks gestation (notably PAPP-A and free BhCG may be as effective as those serum markers in established use at 15-22 weeks gestation." Tsukerman and associates recently reported a scientific study that supports the RCOG's statement. They concluded, "On the basis of our experience in this pilot study, we strongly concur with this recommendation."

Cuckle and van Lith, (1999) recently concluded that "There is overwhelming evidence that screening for Down syndrome could be moved from the second to the first trimester of pregnancy.....The current study provides a robust parameter set based on the most extensive series of studies to date."

Moreover, two recent surveys of women's opinions found that most preferred first trimester screening. (Kornman et al., 1997) (Mononi et al., 1999). Mononi et al., reported all responding patients, 496 of 500, at 6-10 weeks gestation preferred early first trimester screening.

Increased NT in chromosomally abnormal fetuses usually resolves in the second trimester, making 11 weeks 0 days - 13 weeks 6 days gestation an important window for screening.

An important point to remember is that increased NT thickness per se does not constitute a fetal abnormality. Once chromosomal defects have been excluded, about 90% of pregnancies with fetal nuchal translucency below 4.5mm result in healthy live births. About 80% of pregnancies with NT of 4.5-6.4mm and 45% with NT of 6.5mm or more result in healthy live births.

If the combined free Beta/PAPP-A/NT Down syndrome and Trisomy 18 risks are not increased, the risk for these disorders is still increased if the NT is out of normative range (> 3.5mm). Additional testing by ultrasound, fetal echocardiography (a specialized sonogram of the heart to detect heart defects) or genetic testing are options that might be considered.

Nuchal Translucency Certified Healthcare Providers

A United Kingdom Royal College of Obstetricians and Gynaecologists study group (1997) has recommended that "there is now sufficient evidence to consider that specific serum markers for Down syndrome at 9-13 weeks gestation (notably PAPP-A and free BhCG may be as effective as those serum markers in established use at 15-22 weeks gestation." Tsukerman and associates recently reported a scientific study that supports the RCOG's statement. They concluded, "On the basis of our experience in this pilot study, we strongly concur with this recommendation."

Cuckle and van Lith, (1999) recently concluded that "There is overwhelming evidence that screening for Down syndrome could be moved from the second to the first trimester of pregnancy.....The current study provides a robust parameter set based on the most extensive series of studies to date."

Moreover, two recent surveys of women's opinions found that most preferred first trimester screening. (Kornman et al., 1997) (Mononi et al., 1999). Mononi et al., reported all responding patients, 496 of 500, at 6-10 weeks gestation preferred early first trimester screening.

Who Should Be Offered First Trimester Screening?
Maternal blood screening is designed for pregnant women who are at low risk for the disorders being screened. Therefore, screening programs described here are for women who are under 35 years of age at the time of delivery and who do not have a positive history of an ONTD or chromosome abnormality. The American College of Obstetricians and Gynecologists (1994) and the American College of Medical Genetics (1993) recommend women who are over 34 years or have a positive medical history should consider amniocentesis for prenatal chromosome testing not screening, in order to diagnose 99.9% of all chromosome abnormalities. Screening is not an adequate substitute because: 1) Screening only estimates a risk for 2 chromosomal abnormalities and does not give a diagnosis, 2) Screening will miss about 9 to 32% of Down syndrome pregnancies (depending on the type of screening) and the majority of the hundreds of other chromosomal abnormalities, 3) The parents ages and medical history more accurately determine the overall risk for chromosomal abnormalities than a limited screening risk, and 4) Screening will falsely reassure many patients who still have an increased chromosomal abnormalities risk due to age.

1st and 2nd Trimester blood screening should not be provided to high-risk patients (women over 34 years of age at EDD or with a positive medical history) as a substitute for amniocentesis  chromosomal analysis because:

• Screening provides a risk for two out of the hundreds of types of chromosome abnormalities, not a diagnosis, whereas amniocentesis can diagnose 99.99% of the hundreds of types of fetal chromosome abnormalities.
• Screening will miss about 1 out of 10 (10%) Down syndrome pregnancies for women under 34 years of age. Screening will miss the majority of other chromosomal abnormalities.
• A within normal screening result only suggests a lower risk. Only a diagnostic amniocentesis can determine whether a fetus has a chromosome abnormality.
• Because the two screening risks are limited to a few chromosome abnormalities, mother's age and medical history risks more accurately determine each  high-risk patient's risk of having a baby with a chromosome abnormality.
• Screening will falsely reassure many women who are carrying an affected fetus. (see position statements from ACMG 1993, ACOG 1994).

GeneCare recognizes that some high-risk women will elect to have maternal blood screening instead of diagnostic amniocentesis. Although screening will provide a risk assessment for two disorders, these patients should be counseled that screening will not adequately address the total chromosomal risk they face and may give them a false sense of security. The fetal chromosome risk of a 35 year old woman includes 1.  Down syndrome (only 40% of the total screening risk) and 2.  hundreds of other chromosome abnormalities (60% of the total risk). A within normal range Down syndrome report will only reduce a percentage of the Down syndrome risk, which leaves most of the fetal chromosome abnormality risk not addressed. Genetic counseling is strongly suggested for all high risk patients to help them understand why screening should not be considered an equal substitute for diagnostic amniocentesis chromosome analysis.

First trimester biochemical screening may be offered to low risk women who are carrying twins or triplets, although screening is not as sensitive as in singleton pregnancies. Nuchal translucency screening is thought to be equally sensitive in both singleton and multiple pregnancies.

Screening Methods
Patients who elect to have the screening test after nondirective genetic counseling should be scheduled for blood draw between 13w4d and 22w3d. AFP/Free beta testing may ideally be scheduled at 14w with known dates and 15w with uncertain dates (based on our published data).

In order to achieve the detection rates discussed in this text, blood samples must be fixed onto the dried blood paper attached to the requisition as soon as possible after leaving the patient's body. Blood can be obtained by using a sterile lancet. Alternatively, a sample of blood may be drawn into a red top tube (no additives) and then immediately spotted onto the dried blood paper after the draw. GeneCare uses a simple device (DIFF-SAFE) which is inserted into the blood tube to dispense 5 drops of blood onto the paper.

Screening and Diagnostic Procedures

Screening versus Diagnosis

In order to distinguish between affected and unaffected fetuses, one or more tests are needed to tell them apart. If, after applying a test, no overlap in the distribution of results of the affected and unaffected populations is seen, the method is diagnostic. This is the case with a chromosome test for Down syndrome: discrimination between affected and unaffected should be greater than 99%.

Screening tests differ from diagnostic tests in that the distributions of results (as from a biochemical assay) from affected and unaffected fetuses overlap. Some affected fetuses are not detected by the screening tool, while some unaffected fetuses are falsely identified as being at greater risk for an abnormality. The more the distributions overlap, the poorer the discrimination between the two groups. Screening tests are limited to detecting certain birth defects and do not guarantee a perfect baby. A result within normal range does not necessarily mean there are no abnormalities present, and an increased risk result does not guarantee that abnormalities are present. An increased risk screen places a woman at increased risk for abnormalities and warrants diagnostic testing by amniocentesis or CVS.

Maternal Serum Screening

Anything that can be done to reduce the overlap between affected and unaffected fetuses will enhance detection and improve the quality of the screening test. GeneCare uses several innovative approaches to reduce the overlap between distributions and thus enhance detection of maternal serum screening:

• Certified filter paper (903) specimen collection fixes the patient's chemicals, eliminating sample degradation and guaranteeing that the patient's sample is representative of her true biological state.
• Patented assays with the highest documented specificity and sensitivity accurately measure the freeBeta and PAPP-A concentrations on dried filter paper.
• Unlike large general laboratories, no default data is ever used. If patient information (such as weight, family history, etc.) is not provided, it is obtained from the referring physician prior to analysis.
• Unlike some other laboratories, positive family history is considered in assessing the patient's prior risk for Down syndrome and Trisomy 18.
• Once the patient's prior risk is determined by multivariate discriminate analysis of her clinical information and her conditional risk is determined by discriminate analysis of her biochemical and sonographic information, Bayesian analysis integrates these two risks to generate a true patient-specific risk for Down syndrome and Trisomy 18.
• The detection rate and initial positive rate are increased by obtaining pregnancy outcomes. Physicians and patients should be aware that only labs with outcome data can quote detection and initial positive rates.
• GeneCare combines the patient's prior risk, determined by analysis of her clinical information, and her biochemical and ultrasound information to generate a true patient-specific risk for Down syndrome and Trisomy 18.

Summary of First Trimester Screening Parameters 

• FreeBeta/PAPP-A detects 68% of Down syndrome, 90% of Trisomy 18, some other chromosomal abnormalities and some multiple pregnancies (twins, triplets, etc.).
• FreeBeta/PAPP-A and nuchal translucency detects 91% of Down syndrome and 97% for Trisomy 18 and some other chromosome abnormalities.
• No screening protocol identifies all disorders being screened, nor does it detect other physical or mental defects not being screened.
• Multiple gestation screening is not as sensitive as singleton screening with biochemical analysis, while NT screening is unaffected by multiple fetuses.
• Report results rely on the accuracy of the clinical information submitted.
• Patient-specific risks are determined by sophisticated statistical distributions (not simply MoMs) and can be accurately recalculated as needed only by GeneCare.
• First trimester screening is not a substitute for second trimester AFP screening for ONTD's. It provides earlier testing for chromosome abnormalities.

Targeted Fetal Ultrasound
A targeted ultrasound exam is recommended for patients with an increased risk screen to evaluate gestational age and to assess multiple pregnancy and fetal anomalies (birth defects or markers associated with a chromosome abnormality). GeneCare offers the highest resolution fetal ultrasounds by collaborating with other qualified specialists. Ultrasound can detect most but not all ONTDs, some (but not most) cases of Down syndrome and few other chromosome abnormalities. Many journal articles have documented that the fetal chromosome abnormality risk for patients after a normal targeted fetal ultrasound will still exceed the possible risk of miscarriage associated with CVS or amniocentesis (less than 1 in 300 chances). Therefore, patients with an increased risk for Down syndrome or Trisomy 18 should be offered amniocentesis or CVS.

CVS or Early Amniocentesis in the First Trimester
Women with increased risk for a chromosomal abnormality because of first trimester screening or ultrasonographic findings are considered candidates for CVS or early (first trimester) amniocentesis. CVS has been associated with a slightly increased risk for terminal transverse limb deficiencies when performed before 10 weeks gestation. CVS is also associated with a small increased risk of miscarriage. In the USA, early amniocentesis has been shown to be very safe when performed by experienced physicians using continuous ultrasound guidance. The choice of procedure is usually made on the basis of local availability and experience. Both are offered through GeneCare's network of consultants.

One advantage of early amniocentesis over CVS is that amniotic fluid can be assayed for amniotic fluid AFP (AFAFP) and acetylcholinesterase (AChE), tests which are diagnostic for open neural tube defects (ONTDs). AFAFP has identified all detectable open spine and skull defects between 8 weeks 4 days and 22 weeks 3 days of pregnancy. AchE determination is performed on all increased AFAFP specimens and has identified all detectable open spine and skull defects between 8 weeks 4 days and 22 weeks 3 days. For women electing CVS, maternal serum MSAFP/freeBeta screening can be performed in the second trimester to detect 98% of ONTDs.

Optimal Screening Protocol
Maximum screening efficiency for Down syndrome, Trisomy 18 and ONTDs is achieved through first trimester biochemical (PAPP-A/freeBeta) and sonographic (nuchal translucency [NT]) screening, followed by AFP/freeBeta screening in the second trimester. This approach will achieve 91% detection of the two chromosomal abnormalities and 98% detection of ONTDs. Alternative screening protocols (first trimester biochemical screening alone, NT measurement alone or second trimester biochemical screening) cannot achieve these detection levels.

Timing of Tests
As with any screening test, there is a time range to offer the first and second trimester screening tests. Gestational age is best estimated by ultrasound when available, followed by the date of onset of the last menstrual period and finally by physical exam. The times to perform the various tests are indicated below.

 Reference:Gen Care Medical Genetics center