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Advances in Noninvasive Prenatal Testing For Down Syndrome and other Trisomies
Author: David J. Moffa, PhD, BCLD Reviewer: Jenny Camele, MT (ASCP)

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Course Instructions
Please proceed through the course by clicking on the blue arrows or text links. Use the table of contents to monitor your progress. Your progress will be saved automatically as you proceed through the course, and you may later continue where you left off even if you use a different computer. You may encounter practice questions within the course, which are not graded or recorded.

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Course Info
This course carries the following continuing education credits:
G

P.A.C.E. Contact Hours: 1.50 hour(s) Course Number: 578-007-13 Florida Board of Clinical Laboratory Science CE - Molecular Pathology: 1.50 hour(s)

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Aneuploidy and Trisomy

Aneuploidy and Trisomies


Aneuploidy refers to an abnormal number of chromosomes and occurs during cell division as a result of improper chromosome separations between the cells. The condition can result in an extra or missing chromosome and is a common cause of genetic or birth disorders. Typically, chromosome abnormalities occur in 1 out of 160 live births. The most common abnormalities among these live births are the result of an extra chromosome in chromosomes 21, 18, and 13. Trisomy refers to the presence of three copies of a particular chromosome instead of the normal two copies. An extra copy in chromosome 21, 18 and 13 is called Down syndrome (trisomy 21), Edwards Syndrome (trisomy 18), and Patau Syndrome (trisomy 13), respectively.

Aneuploidy and Trisomy

Down Syndrome
Down syndrome (Trisomy 21) is the most common chromosomal abnormality in live births. It occurs in approximately 1 in 700 births. With increasing maternal age, this prevalence increases to about 1 in 400 (at age 35) and 1 in 100 (at age 40). The risk of having a baby with Down syndrome increases with maternal age, however, age cannot serve as the sole screening factor; 70% of babies with Down syndrome are born to women under 35, reflecting the increased number of pregnancies in that age group.

Aneuploidy and Trisomy

Edwards Syndrome
Edwards Syndrome (Trisomy 18 ) is the second most common trisomy. This condition occurs in about 1 in 5,500 live births, and the risk also increases with increasing maternal age. Approximately half of babies born with Trisomy 18 die within the first week of life and only five to ten percent survive to one year. Those individuals who survive have considerable intellectual disability.

Aneuploidy and Trisomy

Patau Syndrome
Patau syndrome is the third most common trisomy and occurs in approximately every 10,000 births. Infants typically die within the first month. Those that do survive have considerable intellectual disability, seizures, and fail to thrive.

Aneuploidy and Trisomy

Ungraded Practice Question


Which of the following are true statements? More than one answer is correct. Please select all correct answers

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c Trisomy is an aneuploidy in which there are three copies of a particular chromosome rather than the normal two copies. d e f g c Down syndrome is also known as Trisomy 21, as it occurs when an individual has an extra copy of chromosome 21. d e f g c Edwards syndrome is a more common chromosomal abnormality than Down syndrome. d e f g c Patau syndrome is trisomy 18. d e f g

Aneuploidy and Trisomy

Ungraded Practice Question


Which of the following are true statements? More than one answer is correct. Please select all correct answers c Trisomy is an aneuploidy in which there are three copies of a particular chromosome rather than the normal two copies. d e f g c Down syndrome is also known as Trisomy 21, as it occurs when an individual has an extra copy of chromosome 21. d e f g c Edwards syndrome is a more common chromosomal abnormality than Down syndrome. d e f g c Patau syndrome is trisomy 18. d e f g Feedback The true statements are: Trisomy is an aneuploidy in which there are three copies of a particular chromosome rather than the normal two copies and Down syndrome is also known as Trisomy 21, as it occurs when an individual has an extra copy of chromosome 21. Edwards syndrome (Trisomy 18) is not as common as Down syndrome. It occurs in about 1 in 5,500 live births. Down syndrome occurs in approximately 1 in 700 births. Patau syndrome is trisomy 13 as it occurs when an individual has an extra copy of chromosome 13.

Down Syndrome (Trisomy 21)

Down Syndrome (Trisomy 21):


In humans, when a sperm fertilizes an egg, an individual will inherit one chromosome pair from the mother's egg and one from the father's sperm resulting in 23 pairs of chromosomes or 46 in total for each individual. In Down syndrome, the affected individual will have a triplication of chromosome 21 (Trisomy 21). The condition can be the result of a developing egg or sperm cell that may divide incorrectly, causing an egg or sperm cell to have an extra chromosome number 21. When this cell joins with a normal egg or sperm cell, the resulting embryo has 47 chromosomes instead of 46. If this occurs, the affected individual will have three number 21 chromosomes instead of two. This type of error in cell division causes about 95% of the cases of Down syndrome. In about 3-4% of Down syndrome cases, a part of chromosome 21 breaks off during cell division and becomes attached to another chromosome in the egg or sperm cell. This can result in affected individuals having two normal copies of chromosome 21, plus extra chromosome 21material, which is attached to another chromosome. The resultant abnormality is called Translocation Down syndrome. It not clear precisely how extra genes from chromosome 21 can cause Down syndrome. Several investigators have suggested that the increased presence of specific genes may impair the interaction between various genes with some becoming more active while others are less active than normal. This alteration in the interaction among genes may lead to developmental changes to the body and may explain why some individuals with Down syndrome are more severely affected than others.

Down Syndrome (Trisomy 21)

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Down Syndrome, continued


Physical features that are often (but not always) associated with Down syndrome include: short stature, epicanthal folds, narrow palate, and transpalmar crease. Down syndrome is also associated with congenital heart defects, disorders of the immune and endocrine systems, decreased muscle tone, and varying degrees of cognitive delay (most being mild to moderate). Health symptoms and conditions will vary among individuals with Down syndrome, some having a number of the aforementioned conditions while others may have few if any health problems. It must be emphasized that most of the health problems associated with Down syndrome can be treated. Moreover, the life expectancy for individuals with Down syndrome is now approximately 60 years.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Screening Tests: Ultrasound


Typically, screening tests are used to identify pregnant women at risk for having a baby with Down syndrome. However, such tests do not yield a definitive diagnosis of the condition. At the present time, there are two commonly employed prenatal screening procedures: ultrasound and maternal serum screening tests. An ultrasound of the fetus can be done during pregnancy and may produce signs suggesting Down syndrome. Ultrasound signals may show an increased amount of skin behind the neck, a condition referred to as "nuchal translucency." There may be other signs, including decreased femur length, heart defects, and gastrointestinal defects. While the aforementioned ultrasound signals may tend to suggest Down syndrome, the definitive diagnosis cannot be based solely on the ultrasound findings. Ultrasound findings only suggest a slightly higher risk that a fetus may have Down syndrome. If there are positive ultrasound findings, the attending physician may recommend additional testing to confirm the diagnosis.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Screening Tests, continued: Maternal Serum Screening


Maternal serum screening tests may be performed during the first and second trimester of pregnancy. These serum tests involve the measurement of specific biomarkers in the maternal blood. These five biomarkers are typically used to assess the risk for Down syndrome: Maternal Serum Alpha-fetoprotein (MS-AFP) AFP is a fetal protein that is initially produced in the fetal yolk sac and liver; by the end of the first trimester of pregnancy, most if not all of the AFP is produced by the fetal liver. The concentration of AFP peaks in fetal serum at 10-13 weeks gestation. The fetal AFP normally diffuses across the placental barrier and into the maternal circulation so that MS-AFP rises throughout pregnancy to about 250 ng/mL at 32 weeks gestation. Lower MS-AFP values may be associated with increased risk for trisomy 21 (Down syndrome) or trisomy 18 (Edwards syndrome). Unconjugated estriol (uE3-estriol) Estriol is a female sex hormone that increases during pregnancy. It is produced by the placenta and passes into the maternal bloodstream during pregnancy. Its roles during pregnancy may be linked to the proper functioning of the uterus, softening of the cervix, and assistance in the lactation process. A biologically active form of estriol called uE3-estriol increases in the maternal circulation during pregnancy by the seventh to ninth weeks of gestation and continues to increase throughout pregnancy. uE3-estriol levels in maternal serum is typically decreased in the Down syndrome pregnancy. Human chorionic gonadotropin, total or free beta subunit (beta-hCG) HCG is a glycoprotein hormone produced by the placenta during pregnancy. It is present in blood and urine around 7-13 days following fertilization of the ovum. HCG has two subunit chains, alpha and beta. The beta subunit confers its specificity. A specific smaller part of the hormone, called free beta hCG may be used as a screening test during the first trimester of pregnancy. An increase in maternal serum free beta hCG (ie, greater concentration than in other pregnancies) may indicate an increased risk for Down syndrome. Total beta-hCG is tested as part of a triple or quad screen during the second trimester of pregnancy. An increase in total beta hCG in the maternal serum is also associated with increased risk of Down syndrome. Dimeric inhibin A (DIA) Inhibins are a family of glycoproteins mainly secreted by the ovaries and testicles. The beta subunits of the inhibins exist in two forms, the A and B forms. DIA is secreted by the ovaries and is designed to inhibit the production of the hormone FSH by the pituitary gland. The level of DIA is increased in the blood of mothers of fetuses with Down syndrome Pregnancy Associated Plasma Protein A (PAPP-A) PAPP-A is produced by the covering of the newly fertilized egg. It is thought to be involved in local proliferative processes such as wound healing and bone remodeling. Unexplained low levels of PAPP-A in the maternal serum during pregnancy may indicate increased chance for intrauterine growth restrictions, premature delivery, preeclampsia, and stillbirth. In the first trimester, low levels of this protein are seen in Down syndrome pregnancies. In the first trimester, serum screening is typically done in combination with an ultrasound to screen for nuchal translucency. Serum screening in the first trimester usually involves the measurement of two biomarkers in the maternal serum, free beta-hCG and PAPP-A. Combining the results of these two biomarkers with an ultrasound improves the screening process.

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In the second trimester during weeks 16-18, maternal serum assays for 3-4 levels of biomarkers are typically performed. The screening for these biomarkers has been established as a triple or quadruple (quad) screen, since the benefit of the screening lies in the combined use of the three to four biomarkers. The biomarkers used in the screening process may include MS-AFP, uE3-estriol, total beta HCG, and DIA. Increased serum levels of MS-hCG and DIA combined with decreased levels of UE3-estriol and MS-AFP suggests an increased risk of Down syndrome. Maternal age, family history, weight, race, and diabetic status are also used to determine a numeric risk for Down syndrome. It is important to understand that for women who have positive triple or quad screening results, only a very small number of them have babies who actually have a chromosomal abnormality.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Patient Information for Prenatal Maternal Serum Screening


Laboratories that perform the triple or quad screen usually require submission of specific patient information related to patient history and indications for performing the testing. Some of the following information may be requested on the patient request form:
G G G G G G G G

Estimated gestational age (weeks and days) Date on which the mother was the stated gestational age How gestational age was determined (eg, first day of last monthly period, estimated date of delivery, or ultrasound dating) Patient's weight, date of birth, and race Relevant patient history (eg, prior neural tube defects, Down syndrome, ultrasound anomalies) Was the patient receiving medication (eg, insulin) to control diabetes at the time of conception? Is there clinical evidence of multiple gestations (twins, etc)? Is this an initial maternal serum screening or was there a previous screening during this pregnancy?

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Laboratory Test Report for Prenatal Screening Panels


Test reports for prenatal screening panels typically include a calculated risk factor based on the normal levels for the test or tests. The average normal for the test is termed the population median and test results are compared to this normal and reported out as Multiples of the Median (MoM). The average value for a normal median for a singleton gestation is 1.0 MoM. Hence, any prenatal test that is below average or decreased would be considered less than 1.0 MoM, whereas, a test that is above average or increased would be greater than 1.0 MoM.* The laboratory typically establishes a MoM cutoff value for each test, which classifies each screen as either screen-positive or screen-negative. In addition, important considerations for determination of the risk factor for Down syndrome are the age of the fetus (gestational age), whether this is a singleton or multiple gestation, maternal age, and maternal race. All results are typically entered into a special software program that calculates the total risk. Results are reported as screen-negative or screen-positive and provide an estimate of the risk of chromosome abnormality. However, it is important to note that serum screening tests cannot diagnose Down syndrome. To accurately diagnose Down syndrome, a fetal chromosome analysis must be performed using an invasive procedure, such as chorionic villus sampling (CVS) or amniocentesis testing.

* Twin pregnancies would require a different screening cutoff level. For example, some laboratories use a screening cutoff between 3.5 and 5.0 MoM for twin pregnancies. HCG and DIA levels are approximately twice as high in twin pregnancies and uE3 level is approximately 1.7 times as high.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Prenatal Screening Test Panel Interpretation


A screen-negative result for prenatal screening indicates that the chance of the fetus having Down syndrome is low. Typically no follow-up testing is required unless there is an additional suspicion of a chromosome condition. A screen-negative result does not guarantee the absence of birth defects. In fact, other screening tests may be recommended, including additional ultrasound and/or non-invasive prenatal testing (NIPT) using a cell-free fetal DNA method.

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A screen-positive result indicates that there is a higher risk or chance of the fetus having Down syndrome and follow-up testing such as CVS or amniocentesis is necessary. It is important to note that a screen-positive result does not necessarily mean that the fetus has Down syndrome or other trisomy. Many women with screen-positive results have normal babies. In interpreting the test results, it is important to take into consideration that an underestimation of the gestational age can lead to a false-positive result or falsely high calculated risk. Race is an important consideration for correct interpretation of several prenatal screening tests and medians should be adjusted for tests. Both MS-AFP and beta HCG values are approximately 10 to 15% higher in Black women than in Caucasian women. DIA medians are approximately 8% lower in Black women than in Caucasian women. PAPP-A values are approximately 25% higher in Black women. Differences in test values for other races and ethnic groups (eg, Hispanic, Asian, Native American) also exist, although the differences tend to be small enough to not affect the risk calculations. However, values should be determined based on population tested, ie, if a specific race/ethnic group is the dominant population for the facility.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Diagnostic Tests
Diagnostic tests for Down syndrome (Trisomy 21) are considered invasive testing procedures. Note: The American College of Obstetricians and Gynecologists (ACOG) recommends that pregnant women of all ages have the option of bypassing the screening test and choosing instead to have a diagnostic test for Down syndrome. Diagnostic tests may be offered for several reasons, including: Will be age 35 or older at delivery Abnormal results obtained on screening tests or ultrasound Prior child or pregnancy with a birth defect

G G G

Diagnostic tests, which use a sample of fetal cells, include one or both of the following procedures: Chorionic villus sampling (CVS): CVS is usually done between 10 and 12 weeks of pregnancy. It is considered an invasive procedure since the procedure involves the collection of chorionic villus cell samples from the placenta, either via a needle insertion into the abdominal wall or a catheter in the vagina. Samples are analyzed for fetal chromosome abnormalities. Amniocentesis: Amniocentesis is performed between gestational weeks 16 and 20 and is also classified as an invasive procedure. A thin needle is placed into the abdominal wall and a sample of the amniotic fluid is collected. The sample is analyzed for fetal chromosome abnormalities.

Both CVS and amniocentesis are highly accurate for diagnosing Down syndrome and/or ruling out the condition. However, because they are invasive procedures, there are risks, including miscarriage, preterm labor, infection, or injury to the fetus or mother.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Ungraded Practice Question


True or False: A definitive diagnosis of Down syndrome involves the identification of the presence of an extra number 21 chromosome : Select true or false j True k l m n j False k l m n

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

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Ungraded Practice Question


True or False: A definitive diagnosis of Down syndrome involves the identification of the presence of an extra number 21 chromosome : Select true or false j True k l m n j False k l m n Feedback Both CVS and amniocentesis diagnostic procedures involve the examination of fetal chromosomes and are considered definitive diagnostic testing for Down syndrome.

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Ungraded Practice Question


Unconjugated estriol (uE3-estriol) and dimeric inhibin A (DIA) are two of the tests that are part of the quadruple prenatal screening panel (quad screen), which is performed on maternal serum during the second trimester of pregnancy. Which of the following tests are also included on the quad screen? More than one answer is correct. Please select all correct answers c Maternal serum alpha-fetoprotein (MS-AFP) d e f g c Chorionic villus sampling (CVS) d e f g c Amniocentesis testing d e f g c Beta-HCG d e f g

Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies

Ungraded Practice Question


Unconjugated estriol (uE3-estriol) and dimeric inhibin A (DIA) are two of the tests that are part of the quadruple prenatal screening panel (quad screen), which is performed on maternal serum during the second trimester of pregnancy. Which of the following tests are also included on the quad screen? More than one answer is correct. Please select all correct answers c Maternal serum alpha-fetoprotein (MS-AFP) d e f g c Chorionic villus sampling (CVS) d e f g c Amniocentesis testing d e f g c Beta-HCG d e f g Feedback MS-AFP and beta HCG, uE3-estriol, and DIA, make up the quad screen. CVS and amniocentesis testing are diagnostic tests that identify chromosomal abnormalities.

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Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Noninvasive Prenatal Testing (NIPT) using Cell-free Fetal DNA (cfDNA)


A highly specific and sensitive NIPT method has recently become available as an alternative for prenatal screening tests that are currently in use. The NIPT method measures the level of circulating cell-free fetal DNA (cfDNA) present in maternal plasma to identify fetal aneuploidies, including Down syndrome (Trisomy 21), Edwards syndrome (Trisomy 18), and Patau syndrome (Trisomy 13). Fetal DNA represents approximately three to six percent of the total DNA in the maternal circulation. cfDNA typically can be detected in maternal plasma as early as the fifth postmenstrual week and almost always detected by the ninth postmenstrual week. Normally the percentage of fetal DNA increases with increasing gestational age. One of the major challenges with identifying cfDNA is separating fetal DNA from maternal DNA . At present, there are two major techniques for isolating fetal DNA from maternal DNA: Massively parallel signature sequencing (MPSS) and directed DNA analysis.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Massively Parallel Signature Sequencing (MPSS)


MPSS is a random, sequencing technique that analyzes millions of cfDNA fragments. MPSS analyzes the level of gene expression in a specimen by totaling the number of individual messenger-RNA (mRNA) molecules that are produced by each gene. The method sequences short segments of cfDNA from the mother and the fetus and then assigns them to specific chromosomes. After comparing the number of chromosome counts to a control value of other chromosomes, any excess of a particular chromosome (eg, 21) would suggest a trisomy. In the MPSS procedure, tagged polymerase chain reaction (PCR) products produced from complementary DNA (cDNA), which is the DNA synthesized from an mRNA, are amplified so that each corresponding mRNA molecule will yield about 100,000 PCR products with unique tags. The tags are used to attach the PCR products to microbeads. Several rounds of sequence determinations are performed and a sequence pattern or signature is identified from each microbead. The process is performed in parallel with about 1 million sequence signatures produced per overall assay. Each signature sequence is then analyzed, compared with all other signatures, and all identical signatures are counted. The level of expression of any single gene is then calculated. MPSS does requires analysis of very large numbers of DNA fragments per sample (about 25 million), which could potentially limit its clinical utility.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Massively Parallel Signature Sequencing (MPSS): Cloning of cDNA Fragments on Microbeads

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The MPSS technique occurs in two major steps. In the first step, in vitro cloning of cDNA fragments on microbeads occurs. cDNA fragments are cloned onto microbeads using the Lynx Megaclone technology. Starting with one million mRNA molecules from a particular cell or tissue sample, Megaclone will produce one million beads, each containing 100,000 cloned copies of cDNA from each mRNA molecule. All molecules are covalently attached to the microbeads at their poly(A) ends, so the DpnII end is available for the sequencing reactions. The image on the right illustrates the in vitro cloning of cDNA fragments on microbeads. The procedure involves both microbead tagged vector library preparation and sample preparation. Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine,

http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Massively Parallel Signature Sequencing (MPSS): Sequencing Process and Analysis of Sequence Patterns

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In step two, the microbeads are loaded and the sequencing process is initiated. Typically, five rounds produce about 16-20 signature sequence products. Several rounds of sequence determinations are performed and a sequence pattern or signature is identified from each microbead. The process is performed in parallel with about 1 million sequence signatures produced per overall assay. Each signature sequence is then analyzed, and compared with all other signatures and all identical signatures are counted. The level of expression of any single gene is then calculated. The image on the right illustrates the sequencing process and analysis of sequence patterns. Source: National Center For Biotechnology Information (NCBI), National Institutes of Health, National Library of Medicine,

http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml. The MPSS technique is quite complex. If you would like a more in-depth explanation, please click on the "More Info" button below and access the resources that are provided.

More Info

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Directed DNA Analysis


Directed DNA analysis involves selective sequencing of relevant chromosomes. In directed DNA analysis, specific cfDNA fragments from maternal blood are used for selective sequencing. Digital analysis of selected regions (DANSR), a recently developed technique, uses directed DNA analysis and digitally analyzes sequencing results from targeted, selected genomic regions on particular chromosomes. This technique is touted as being potentially more efficient than MPSS because it uses fewer genetic fragments.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Commercially-Available cfDNA Prenatal Tests for Aneuploidy


Currently, there are three major commercially-available cfDNA NIPTs for detection of aneuploidy: MaterniT21Plus Test

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MaterniT21 PLUS test, developed and validated by Sequenom CMM, is a laboratory-developed test (LDT) that analyzes circulating cell-free DNA extracted from a maternal blood sample. This test is offered by Sequenom Center for Molecular Medicine and utilizes the MPSS technology to identify increased numbers of chromosomes. In the test, circulating cell-free DNA is purified from maternal plasma and the DNA is analyzed for chromosomal material and converted into a genomic DNA library for the determination of chromosome 21, 18, 13 and Y representation based on massively parallel DNA sequencing. The MaterniT21 Plus test has a reported performance characteristics of 99.1% sensitivity and 99.9% specificity for the detection of Down syndrome trisomy. Test results are reported as "negative" or "positive" for trisomies. Verifi Prenatal Test This test, offered by Verinata Health Inc., also utilizes MPSS technology. Verifi Prenatal Test uses a normalized chromosome value (NCV) calculation for each chromosome tested. This NCV calculation removes variation within and between sequencing to optimize the test precision. Test results are reported as no aneuploidy detected, aneuploidy suspected" for borderline cases, and aneuploidy detected for the respective chromosome tested. The test claims to have a >99.9% sensitivity and 99.8% specificity for detection of Down syndrome. Harmony Prenatal Test This test is offered by Ariosa Diagnostics and utilizes directed DNA analysis sequencing of specific cell-free DNA (cfDNA) fragments using digital analysis of selected regions (DANSR). It is a laboratory-developed test that analyzes cfDNA in maternal blood. As with other cfDNA tests, the Harmony prenatal test is best performed anytime after the tenthweek of pregnancy. Using the DANSR methodology, the Harmony Prenatal Test analyzes sequencing results of selected, targeted genomic regions on chromosome 21 (for Down syndrome assessment). The test results will yield an assessment of the risk for trisomy 21. Using a specific algorithm, the test results are reported out as an individualized risk for each trisomy. The test claims to have a >99% sensitivity and 99% specificity for Down syndrome detection. All of the aforementioned cfDNA tests are considered laboratory-developed tests and are not FDA-approved. Moreover, it must be stressed that at this time the cfDNA tests are not considered diagnostic and a low-risk test result does not suggest an unaffected pregnancy while a high-risk test result still requires an invasive confirmatory test. In addition, all cfDNA tests are typically intended for use in women 18 years or older with a singleton pregnancy at a minimum of 10 weeks gestation and who have been determined by their physician to be at risk for fetal trisomy. Such risks include advanced maternal age, positive results of prenatal screening tests (serum and/or NT ultrasound), the presence of ultrasound soft or hard markers, previous family history of genetic disorders, or a previous affected pregnancy for fetal aneuploidy. It is important to remember that at present, invasive prenatal diagnostic tests, such as CVS and amniocentesis, should be considered the most accurate and comprehensive way to assess fetal abnormalities.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


Which of the following statements are true regarding cell-free fetal DNA (cfDNA) testing? (choose all that apply) More than one answer is correct. Please select all correct answers c The cfDNA test measures the maternal DNA in plasma. d e f g c cfDNA can be detected in maternal plasma using the MPSS technique or by the Directed DNA analysis. d e f g c The MPSS technique analyzes millions of cfDNA fragments. d e f g c DANSR is used in the directed DNA analysis to digitally analyze selected regions. d e f g

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


Which of the following statements are true regarding cell-free fetal DNA (cfDNA) testing? (choose all that apply) More than one answer is correct. Please select all correct answers c The cfDNA test measures the maternal DNA in plasma. d e f g c cfDNA can be detected in maternal plasma using the MPSS technique or by the Directed DNA analysis. d e f g c The MPSS technique analyzes millions of cfDNA fragments. d e f g

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c DANSR is used in the directed DNA analysis to digitally analyze selected regions. d e f g Feedback These statements are true: cfDNA can be detected in maternal plasma using the MPSS technique or by the directed DNA analysis. The MPSS technique analyzes millions of cfDNA fragments. DANSR is used in the directed DNA analysis to digitally analyze selected regions. The only statement above that is not true is, "The cfDNA test measures the maternal DNA in plasma." The cfDNA test measures cell-free fetal DNA in maternal plasma.

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


Which of the following statements accurately describe the currently available cfDNA prenatal tests for fetal aneuploidy? More than one answer is correct. Please select all correct answers c The MaterniT21 Plus Test and the Verifi Prenatal Test use the MPSS technology. d e f g c The cfDNA tests are FDA-approved tests. d e f g c The Harmony Prenatal Test uses directed DNA analysis and the DANSR technique for digital analysis of selected regions. d e f g c A low risk test result using the Harmony prenatal test strongly suggests an unaffected pregnancy. d e f g

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


Which of the following statements accurately describe the currently available cfDNA prenatal tests for fetal aneuploidy? More than one answer is correct. Please select all correct answers c The MaterniT21 Plus Test and the Verifi Prenatal Test use the MPSS technology. d e f g c The cfDNA tests are FDA-approved tests. d e f g c The Harmony Prenatal Test uses directed DNA analysis and the DANSR technique for digital analysis of selected regions. d e f g c A low risk test result using the Harmony prenatal test strongly suggests an unaffected pregnancy. d e f g Feedback The MaterniT21 Plus Test and the Verifi Prenatal Test use the MPSS technology. The Harmony Prenatal Test uses directed DNA analysis and the DANSR technique for digital analysis of selected regions. None of the cfDNA tests are currently FDA-approved. They are considered laboratory developed tests. A low risk test result using the Harmony prenatal test or any of the other prenatal cfDNA tests does not suggest an unaffected pregnancy. Moreover, a high risk test result still requires a follow-up invasive confirmatory test.

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Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


The FIRST step in the massively parallel signature sequencing (MPSS) technique involves in vitro cloning of _________ on microbeads. Please select the single best answer j cDNA fragments k l m n j The sequencing products k l m n j Hybridized DNA products k l m n j Tagged sequencing products k l m n

Noninvasive Prenatal Testing (NIPT): Cell-free Fetal DNA (cfDNA)

Ungraded Practice Question


The FIRST step in the massively parallel signature sequencing (MPSS) technique involves in vitro cloning of _________ on microbeads. Please select the single best answer j cDNA fragments k l m n j The sequencing products k l m n j Hybridized DNA products k l m n j Tagged sequencing products k l m n Feedback The MPSS technique occurs in two major steps. In the first step, in vitro cloning of cDNA fragments on microbeads occurs. In step two, the microbeads are loaded and the sequencing process is initiated.

Updated Recommendations on NIPT For Fetal Aneuploidy

The American College of Obstetricians and Gynecologists (ACOG) Recommendations


In December 2012, ACOGs Committee on Genetics and the Society for Maternal-Fetal Publications Committee issued a recommendation that all pregnant women, regardless of age, should be offered blood-based screening or invasive diagnostic testing for the identification of most common fetal aneuploidies. In addition, the committees suggested that noninvasive prenatal testing (NIPT) that employs cell free fetal DNA (cfDNA) performed on maternal plasma offers tremendous potential as a screening tool for fetal aneuploidy. Moreover, the opinion clearly states that the NIPT screening test would be used most effectively in high-risk women and that the prior ACOG guidelines should apply relative to offering women prenatal assessment for aneuploidy, either by screening or invasive prenatal diagnosis. There are specific guidelines for offering the cfDNA testing as a primary screening tool for high-risk women. The cfDNA test should not be used for low-risk women. Guidelines on the indications for considering the use of cfDNA testing include the following:
G G G

Maternal age of 35 years or older at delivery Fetal ultrasonography findings indicating an increased risk of aneuploidy History of a prior pregnancy with a trisomy

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Positive test results for aneuploidy, including first trimester or integrated screen or quadruple screen

The opinion also suggests that an invasive prenatal diagnostic test, such as CVS or amniocentesis, should be considered the most accurate and comprehensive way to assess fetal abnormalities. Moreover, patients who have positive NIPT results should also receive invasive testing. The opinion emphasizes the use of pretest counseling, which should stress the following facts about the NIPT test:
G G G G

The test will only screen for common trisomies The test should not be used as a replacement for invasive tests A negative test does not ensure an unaffected pregnancy and Patients with positive results should be offered other testing.

In summary, ACOG issued the following conclusions on the use of cfDNA testing for fetal aneuploidy:
G G G

G G G G

The cfDNA test is expected to identify approximately 98% of cases of Down syndrome with a false-positive rate of less than 0.5% Patients at increased risk of aneuploidy can be offered the cfDNA test. The cfDNA test should only be an informed patient choice after pretest counseling occurs. Pretest counseling should indicate that the test will only screen for the common trisomies. The test should not be offered to low-risk women or women with multiple gestations. A family history should be obtained before the use of the test. A negative cfDNA test does not ensure an unaffected pregnancy. Patients with positive cfDNA tests should be referred for genetic counseling and offered invasive prenatal testing for confirmation of the test results. The cfDNA test does not replace the accuracy and diagnostic precision of prenatal testing with CVS or amniocentesis.

Updated Recommendations on NIPT For Fetal Aneuploidy

Recommendations By Other Organizations


In June 2012, the California Technology Assessment Forum (CTAF) issued an independent evaluation of the cfDNA technology used for the assessment of fetal aneuploidy for Down syndrome (Trisomy 21) in high-risk women. The assessment concluded that the cfDNA technology is promising and has a high sensitivity and specificity for evaluation of fetal aneuploidy in high-risk women. Later in October 2012, CTAF recommended the use of cell-free fetal DNA as a prenatal advanced screening test for fetal aneuploidy for Trisomy 21 and Trisomy 18 in high-risk women who meet all five CTAF criteria for safety and efficacy and improvement in health outcomes. In addition, The National Society of Genetic Counselors (NSGC) issued a position paper in February 2012 indicating support for the use of NIPT as an option for patients whose pregnancies are considered to be at an increased risk for certain chromosome abnormalities. NSGC emphasizes that NIPT only be offered if there is an informed patient consent, patient education, and counseling provided by a certified genetic counselor. In addition, patients with abnormal NIPT results should receive genetic counseling and be given the option of standard confirmatory diagnostic testing.

Updated Recommendations on NIPT For Fetal Aneuploidy

Conclusions
NIPT that employs cfDNA is not considered diagnostic at this time. Women having a positive NIPT result should be offered conventional invasive diagnostic testing such as CVS or amniocentesis for confirmation. With an abnormal ultrasound finding that suggests an increased risk of aneuploidy, clinicians may also recommend that a patient forego conventional diagnostic testing and use the NIPT to provide a second piece of evidence of the abnormality.

Updated Recommendations on NIPT For Fetal Aneuploidy

Ungraded Practice Question


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Which of the following policies reflect the guidelines and conclusions recently issued by The American College of Obstetricians and Gynecologists (ACOG) on the use of cell-free fetal DNA (cfDNA) testing for fetal aneuploidy? More than one answer is correct. Please select all correct answers c The test should be offered to all women, including those at low risk for fetal aneuploidy. d e f g c A family history should be obtained prior to the test. d e f g c The test should be performed only as an informed patient choice and after pretest counseling occurs. d e f g c The test can be considered as accurate as a diagnostic test such as CVS or amniocentesis. d e f g

Updated Recommendations on NIPT For Fetal Aneuploidy

Ungraded Practice Question


Which of the following policies reflect the guidelines and conclusions recently issued by The American College of Obstetricians and Gynecologists (ACOG) on the use of cell-free fetal DNA (cfDNA) testing for fetal aneuploidy? More than one answer is correct. Please select all correct answers c The test should be offered to all women, including those at low risk for fetal aneuploidy. d e f g c A family history should be obtained prior to the test. d e f g c The test should be performed only as an informed patient choice and after pretest counseling occurs. d e f g c The test can be considered as accurate as a diagnostic test such as CVS or amniocentesis. d e f g Feedback The cfDNA test is not for low risk women and it should not be considered as accurate as the CVS or amniocentesis test. It should be performed after a family history is obtained, as an informed patient choice, and after pretest counseling.

References

References
Abnormalities of chromosome number (Aneuploidy). Available at: http://www.utmb.edu/pedi_ed/CORE/MedicalGenetics/page_11.htm. Accessed May 1, 2013. Aetna Clinical Coverage Policy, Serum Marker Screening for Down Syndrome, Policy #0464, reviewed 2/4/12. Available at: http://www.aetna.com/cpb/medical/data/400_499/0464.html. Accessed May 1, 2013. AFP test information. American Pregnancy Organization. Available at: http://americanpregnancy.org/prenataltesting/afp.html. Accessed May 1, 2013. American College of Obstetricians and Gynecologists. ACOG Committee Opinion No. 545, December 2012. Available at: http://www.acog.org/Resources_And_Publications/Committee_Opinions/Committee_on_Genetics/Noninvasive_Prenatal_Testing_for_Fetal_Aneuploidy. Accessed May 1, 2013. Aneuploidy: NCBI, US Library of Medicine. Available at: http://www.ncbi.nlm.nih.gov/books/NBK21870/. Accessed May 1, 2013. CAP Commission on Laboratory Accreditation- Laboratory Accreditation Program. Chemistry and Toxicology Checklist. Northfield, IL: College of American Pathologists: September 2012. Cigna Coverage Policy. Down syndrome screening. Policy #0211, reviewed 7/15/12. Available at: http://www.cigna.com/assets/docs/health-careprofessionals/coverage_positions/mm_0211_coveragepositioncriteria_down_syndrome_screening.pdf. Accessed May 1, 2013. Definition of MSAFP (maternal serum alpha-fetoprotein. Available at: http://www.medterms.com/script/main/art.asp?articlekey=4446. Accessed May 1,

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2013. Down syndrome: Genetics home reference. US National Library of Medicine, NIH. Pub. 1/17/13. Available at: http://ghr.nlm.nih.gov/condition/downsyndrome. Accessed May 1, 2013. Down syndrome genetic testing basics, Aetna Intelihealth, updated 6/28/11. Available at: http://www.intelihealth.com/IH/ihtIH/WSIHW000/32193/35641.html. Accessed May 1, 2013. Down syndrome. Mayo Clinic web site. Available at: http://www.mayoclinic.com/health/down-syndrome/DS00182. Accessed May 1, 2013. Down Syndrome: The ARC, 2013 update. Available at: http://www.thearc.org/page.aspx?pid=2546. Accessed May 1, 2013. Down Syndrome: The March of Dimes web site Information. Available at:http://www.marchofdimes.com/baby/birthdefects_downsyndrome.html? gclid=CIyNoJCP-rQCFQHonAodSnEAXg. Accessed May 1, 2013. Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing. California Technology Assessment Forum. [Draft]. Available athttp://www.ctaf.org/sites/default/files/u39/121017_draft_prenatal.pdf. Accessed May 1, 2013. MPSS technical information. National Center for Biotechnology Information, NCBI. Available at: http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechMPSS.shtml. Accessed May 1, 2013. Morris JK, Mutton DE, Alberman E. Revised estimates of the maternal age specific live birth prevalence of Down's syndrome. J Med Screen.2002;9(1):26. Newberger DS. Down syndrome: Prenatal risk assessment and diagnosis. Available at: http://www.aafp.org/afp/2000/0815/p825.html. Accessed May 1, 2013. Resta RG. Changing demographics of advanced maternal age (AMA) and the impact on the predicted incidence of Down syndrome in the United States: Implications for prenatal screening and genetic counseling. Am J Med Genet A. Feb 15 2005;133A(1):3136. Rodeck CH, Whittle MJ. Fetal Medicine: Basic Science and Clinical Practice. Philadelphia: Churchill Livingstone; 2009. Sietske NH, Perlstein D. Downs syndrome. Available at: http://www.medicinenet.com/down_syndrome/article.htm. Accessed May 1, 2013. Test ID: MAFP. Mayo Clinic Website. Available at: http://www.mayomedicallaboratories.com/test-catalog/Clinical+and+Interpretive/81169. Accessed May 1, 2013. The Harmony Prenatal Test. Test information. Aiosa Diagnostics. Available at: http://www.ariosadx.com/about-the-science/. Accessed May 1, 2013. Torres TT, Metta M, Ottenwlder B, Schltterer C. Gene expression profiling by massively parallel sequencing. Genome Res. 2007 Nov 21 PMID: 18032722. Triple screen testing. AACC Lab tests online. Available at: http://labtestsonline.org/understanding/analytes/triple-screen/tab/test. Accessed May 1, 2013. Tufts Health Plan provider policy. Genetic testing of maternal serum. Policy #2210630. Available at: http://www.tuftshealthplan.com/providers/pdf/mng/genetic_testing_maternit.pdf. Accessed May 1, 2013. Verifi Prenatal Test. Test information. Verinata Health. Available at: http://www.verinata.com/providers/provider-overview/. Accessed May 1, 2013.

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Resources
Please review the resources below, then click on the navigation arrows to continue this course. Additional Resources Adobe Acrobat PDF file

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