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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)
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.
Course Info
This course carries the following continuing education credits:
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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)
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.
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.
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.
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
Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies
Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies
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
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
* 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
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
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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
Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies
Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies
Current Screening and Diagnostic Tests for Down Syndrome and other Trisomies
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.
Massively Parallel Signature Sequencing (MPSS): Sequencing Process and Analysis of Sequence Patterns
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
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.
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.
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
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:
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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:
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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.
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.
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
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,
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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