First Trimester freeBeta / PAPP-A / Nuchal Translucency / Absent Nasal Bone Prenatal Screening for Down syndrome and Trisomy 18 syndrome

11-14 week Fetal Ultrasound Exam

 

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 (AFP) blood test was introduced to screen for open neural tube defects. In 1984, AFP was also shown to be a marker for Down syndrome fetuses in women under 35 years of age.

Over the years, screening test improvements have been introduced to enhance detection of more types of birth defects, to reduce the screen positive rate, and decrease the number of follow-up procedures performed.

In the late 1980's, new second trimester biochemical markers for Down syndrome (DS) and Trisomy 18 (T-18) were found (intact human chorionic gonadotropin [hCG], the freeBeta subunit of hCG, and estriol).

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).  (ACOG Bulletin, 1986).

Our researchers discovered that blood deteriorates over time, especially if subjected to temperature extremes. If, however, a blood dried on Screen Spot Plus paper, immediately stabilizes the proteins, eliminating hemolysis, analyte degradation and the need to centrifuge. Dried Blood  more accurately reflect the patients' true protein levels than liquid blood and offer better discrimination between affected and unaffected populations. Using dried blood significantly decreases screen positives to only half of liquid blood testing.  Dried blood is more effective than any other method to decrease screen positives while increasing detection 8-10%.

The timing of screening has been improved by the identification of first trimester markers for chromosomal and other abnormalities. In 1992, the measurement of the nuchal translucency (NT), (the fluid accumulation at the back of the fetal neck) at 11-13 weeks 6days, was shown to be a strong marker for Down syndrome, Trisomy 18 and other chromosomal abnormalities, birth defects, and perinatal risk. Around the same time the excellent biochemical markers 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. Absent or hypoplastic Nasal Bones (NB) was added as a very strong marker for screening chromosome and other anomalies in 2002.

Birth defects screening is a rapidly changing scientific field.  At present, however, many laboratories in the United States still provide the triple screen (AFP, hCG and estriol) or quad (triple plus InhibinA), tests that fall (10-24%) short of detection that can be accomplished with the standard second trimester AFP / freeBeta test (80% DS detection @ 2.9% screen positive rate)

First Trimester Chromosome and Anomaly Screening

This text outlines the USA National Fetal Medicine Foundation (FMF) NT Standard Screening protocol that has been implemented in the United States and over 70 countries, and offers the state-of-the-art first trimester screening for birth defects.

The highest detection rates and lowest screen positive rates for Down syndrome and Trisomy 18 are achieved by combining freeBeta / PAPP-A / NT / NB markers at 11-13 weeks 6 days.  First trimester screening involves a blood draw or finger stick to dry blood on filter paper and an ultrasonographic nuchal translucency (NT) measurement. The blood sample may be taken between 9 weeks and 13 weeks 6 days. The NT is measured between crown rump 45-84 mm (11 weeks 0 days and 13 weeks 6 days).  Although risks for Down syndrome and trisomy 18 can be generated with either the biochemical or the NT measurement alone, the detection rates are better when both are combined. The NT measurement must be taken by a Fetal Medicine Foundation NT certificate sonographer to ensure the uniformity and accuracy of the data being used for risk assessment NT measurements have to meet the USA National FMF -NT Standard and the American College of Obstetrics and Gynecology opinion before GeneCare can combine them with freeBeta / PAPP-A. GeneCare provides non-profit CME / CEU First Trimester Prenatal Screening and Ultrasound Diagnosis, Nuchal Translucency and Nasal Bone courses with hands-on training and continuing external quality control for those who are interested in incorporating this procedure into their practices.

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

Table 1: Performance of freeBeta/PAPP-A between 24-84mm CRL (9w and 13w6d) gestation.

Table 2: Performance of freeBeta/PAPP-A / NT between 45-84mm CRL (11w to 13w6d) gestation.

Comparison Criteria

The efficacy of a screening program is judged by several performance criteria:

Detection Rate

The detection rate is the percentage of affected fetuses identified as high risk by the test. Screening programs try to maximize this variable without raising the screen positive rate.

Without NT measurement, freeBeta / PAPP-A First Trimester Screening identifies 68% of Down syndrome cases, compared to triple screen's 60%. When NT measurement is added, detection rises to 91% (31% higher)!  This detection rate is unequaled by any other first or second trimester screening testing.

FreeBeta / PAPP-A Trisomy 18 screening NT measurement identifies 90% versus about 60% with the triple screen. Combined with NT measurement, the Trisomy 18 detection rate increases to 97%.

Screen Positive Rate

The Screen Positive Rate (SPR) 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 without decreasing detection.

First Trimester Screening (with or without NT Measurement) has a DS Screen Positive Rate (SPR) of less than 2-5%, compared to the triple screen's 7.6% more than a 50% reduction in screen 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 (either CVS at 11-12 weeks, early amniocentesis at 13-14 weeks, or  amniocentesis) required to identify an affected fetus. This variable is a function of both the detection rate and the screen positive rate and provides a critical 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 Prenatal Screening are aware that many women are referred for amniocentesis because of an increased screening risk, but most are found to have healthy babies. To be able to reduce the number of such women and diagnostic procedures without decreasing detection is the goal of screening, and Yield measures this ability. Approximately 1 in 80 (triple) and 1 in 75 (quad) women with an increased DS screen risk actually has a Down syndrome baby. First Trimester Screening improves the Yield to 1 in 25 (without NT measurement) or to 1 in 17 (with NT measurement), the highest of any prenatal screening test.

Additional Remarks

A 91% detection rate for Down syndrome and 97% for Trisomy 18 in women under 35 years of age is far more effective than any other known screening protocol and is available only in the First Trimester. Furthermore, these detection rates are achieved with the lowest SPR's and highest yields.

Open neural tube defects (ONTD's = open spine and/or skull defects) and open abdominal defects (OAD) cannot be biochemically screened for in the first trimester. Consequently, women who receive a First Trimester Screening report that does not show an increased chromosomal risk should have an AFP (alpha-fetoprotein) dried blood screening test at 13w 4d or later to obtain 98% detection of ONTDs.  Those few women who receive a First Trimester increased risk report for  chromosomal abnormalities can elect to have early amniocentesis and have an 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 placenta derived glycoprotein that consists of two subunits, alpha and beta. The alpha subunit is shared with other hormones such as thyrotropin (TSH), follitropin (FSH), and leutropin (LH). The beta subunit is specific for each hormone. Less than 1 percent of the beta subunit exists in the free form. Serum levels tend to be high in patients carrying a fetus affected with Down syndrome. The median concentration for freeBeta 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: its MoM is less than 1.2.  Furthermore, freeBeta is significantly reduced in the blood of women carrying Trisomy 18 fetuses, with a MoM of 0.18. Free Beta is the most specific and sensitive biochemical marker for Down syndrome and Trisomy 18.  The peer-reviewed medical literature has conclusively shown Intact or beta hCG does not detect Down syndrome or T-18 at 11-13w 6d and should not be used for First Trimester Screening.

PAPP-A

Pregnancy Associated Plasma Protein A (PAPP-A) is produced by the placental trophoblast. 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. These dramatic differences from normal 1.0 MoM provide excellent discrimination between affected and unaffected fetuses and raises detection while decreasing screen positives.

Nuchal Translucency

One of the single most important breakthroughs in birth defect screening is the correlation between nuchal translucency and  fetal anomalies and perinatal risk.  Nuchal translucency (NT) is an echo-free space between the skin and the soft tissue overlying the cervical spine that presents only during the first trimester of pregnancy. NT is increased in fetuses with chromosomal abnormalities, cardiac defects, certain genetic syndromes, and perinatal risks.

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. Over the next four years, eight additional publications reported from 1,368 cases of increased nuchal translucency wherein 53% (172 of 327) of fetuses with increased NTs had an abnormal karyotypes. The definition of increased NT thickness ranged from 2 to 6 mm in these studies and the incidence of chromosomal abnormalities ranged from 28 to 79%.  We no longer use fixed NT cutoffs, rather we combine the NT measurement with other risk factor to produce a delta NT before combining with freeBeta / PAPP-A.  NT MoM analysis, on the other hand, has been conclusively shown to lower detection and increase screen positives.

By far, the most important contributor to the study of NT has been the King's College researchers in London. Their data, now extending to well over 400,000 cases, shows 82% nuchal translucency Down syndrome detection, 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 good and bad interpretations as to why. The King's College group, the NICHD trials and many studies have proven 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 received special FMF training, NT certification, and external NT median and film monitoring. It is critical to note that all centers with initial low detection rates who subsequently received FMF - NT certification achieved greater than 80% detection rates, which validates the Fetal Medicine Foundation protocol.

In order for the interpretive software to calculate an accurate risk for chromosomal abnormalities, the NT measurement must be standardized way. For this reason, only measurements taken by sonographers who meet the US National FMF - NT Standard for NT measurement and external audit are accepted for risk assessment by GeneCare.

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."

Cuckle and van Lith, (1999) 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, all six surveys of women's opinions found that almost all women preferred first trimester screening. (Kornman et al., 1997, Mononi et al., 1999), not second trimester screening.

Who Should be Offered First Trimester Prenatal Screening?
First Trimester screening is designed for pregnant women who are at low risk for the disorders being screened. Therefore, the  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 chromosomal 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 chromosomal abnormalities. Screening is not a substitute for amniocentesis, because: 1) It does not give a diagnosis,  2) It estimates a risk for only 2 chromosomal abnormalities, 3) It will miss many of the hundreds of other types of chromosomal abnormalities, 4) It may falsely reassure some patients who still have an increased chromosomal abnormality risk due to age.  If you have decided to have an amniocentesis, this test is not necessary.

1st and 2nd Trimester Prenatal screening should not be offered 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 most 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 screening risks are limited to 2 chromosomal abnormalities, mother's age and medical history risks more accurately determine each  high-risk patient's risk of having a baby with a chromosomal 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 Prenatal 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 chromosomal 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 chromosomal abnormality risk not addressed. Genetic counseling is strongly suggested for all high risk patients to help them understand why screening is not an equal substitute for diagnostic amniocentesis chromosome analysis.

Twins

First Trimester freeBeta / PAPP-A / NT Screening should be offered to low risk women who are carrying twins to detect 80% of DS pregnancies. Nuchal translucency screening is thought to be equally sensitive in both singleton and multiple pregnancies.  Adding biochemistry reduces the Screen Positive rate from 12.4% to 7.2%.

Second Trimester Screening
Patients who elect to have Second Trimester Prenatal Screening 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).

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 test results (as from a biochemical assay) from affected and unaffected fetuses overlap. 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 the normative 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.

Screening tests should be reported as "within the normative range" or "increased risk" because screening provides a patient specific risk, not a diagnosis. The terms "normal" or "negative" and "abnormal" or "positive" suggest the screening result is a diagnosis. These terms may over reassure some patients or cause undue concern to others.

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 some 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 screen positive rates are inproved by obtaining pregnancy outcomes. Physicians and patients should be aware that only labs with outcome data can correctly quote detection and screen positive rates.

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.
• 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 pregnancies.
• 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 fetal ultrasound exam is recommended for patients with an increased risk screen for gestational age, multiple pregnancy and fetal anomalies. GeneCare offers fetal ultrasounds by collaborating with other experienced specialists. Ultrasound can detect most but not all ONTDs, some (but not most) cases of Down syndrome and few other chromosomal abnormalities. Many journal articles have documented that the fetal chromosomal abnormality risk for patients after a normal targeted fetal ultrasound will still exceed the possible risk of miscarriage associated with amniocentesis (less than 1 in 200 chances). Therefore, the patients may be offered amniocentesis.

CVS or Early Amniocentesis

Women with increased risk for a chromosomal abnormality because of first trimester screening or ultrasonographic findings are considered candidates for CVS or early amniocentesis (13-14 weeks). CVS has been associated with an increased risk for terminal transverse limb deficiencies, and oromandibular hypogenesis 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 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 procedures are offered through GeneCare's network of consultants.

One advantage of early amniocentesis is that amniotic fluid can be assayed for amniotic fluid AFP (AFAFP) and acetylcholinesterase (AChE), tests for ONTDs. AFAFP has identified all detectable open spine and skull defects between 8w4d and 22w3d. AchE determination is performed on all increased AFAFP specimens and has identified all detectable open spine and skull defects between 8w4d and 22w3d. For women electing CVS, AFP/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 or targeting fetal ultrasound screening in the second trimester. This approach will achieve 91% detection of these 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. The first trimester blood sample and NT measurement do not have to be obtained concurrently. However, GeneCare must know upon receipt of the biochemical specimen that an NT measurement is pending or it will generate a report based on biochemistry alone.

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.

First Trimester References - Available upon request.

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