MATERNAL SERUM SCREENING

MATERNAL SERUM DOUBLE, TRIPLE, OR QUAD SCREENING SHOULD BE OFFERED TO WOMEN WHO ARE AT LOW RISK FOR ONTD, DS, AND T18

INTRODUCTION

Most families who have a child with an open neural tube defect (ONTD), Down syndrome (DS), or trisomy 18 (T18) have no prior family history of the same. Eighty percent of children with DS and 90 percent of children with ONTD are born to women less than 35 years of age. Yet, as of the mid 1980s, only women 35 years of age or older were offered prenatal diagnosis. Screening in low risk women in the United States began in 1985 with maternal serum alpha-fetoprotein (MSAFP) for the detection of ONTDs. In 1990, chorionic gonadotropin (CG) and unconjugated estriol (uE₃) were added to create a triple screen for DS detection. Recently, dimeric inhibin A (DIA) has been included, resulting in a quad screen, greatly reducing the false positive rate associated with such screening. Maternal serum screening is not diagnostic. Abnormal results require follow-up with targeted ultrasound examination and often a prenatal diagnostic procedure.

METHODOLOGY

The measurements of alpha-fetoprotein and chorionic gonadotropin use solid phase, two-site fluoroimmunometric methods. The assays use a direct sandwich technique, in which two monoclonal mouse antibodies are directed against two distinct antigenic determinants on the molecules. One antibody is solid phase and the second is europium-labeled. The estriol assay is a solid phase, time-resolved fluoroimmunoassay based on the competition between europium-labeled estriol and sample estriol for a limited number of binding sites on estriol-specific polyclonal antibodies. The measurement of inhibin-A is accomplished by the use of a pair of highly specific monoclonal antibodies in a two-site enzyme-linked immunosorbent assay (ELISA) format.

CLINICAL SIGNIFICANCE

The incidence of ONTDs varies depending on racial background and geographical location. It has decreased in recent years from approximately 1 in 1000 to 1 in 1700 due to fortification of the food supply with folic acid. The most common ONTDs include spina bifida (a developmental defect of the spine and overlying skin) and anencephaly (failure of proper development of the brain, skull, and overlying skin). Spina bifida usually results in some degree of paralysis of the lower limbs, difficulty with bowel and bladder control, and cerebral ventriculomegaly requiring shunt placement. Anencephaly is not associated with an extended lifespan; approximately 50 percent of the infants are stillborn with most of the remainder dying within hours or days of birth.

Down syndrome (DS) occurs in approximately 1 in 700 births regardless of race or geographical location. DS is caused by the presence of an extra 21st chromosome in every cell of the body. Persons with DS usually have moderate mental retardation and characteristic facial features. The risk for DS increases with maternal age, yet using a maternal age of 35 as a cutoff for offering diagnostic testing will only identify about 20% of affected pregnancies.

Triple screening using AFP, uE3, and hCG is currently the most frequently performed maternal serum screen for DS. It detects between 60-70 percent of DS-affected pregnancies. DIA can be used to increase DS detection by 7-10 percent over the triple screen detection rate or to lower the triple screen false positive rate by 50 percent. ARUP has chosen to decrease the DS false positive rate with the quad screen. Instead of 1 in 20 women screening positive for DS, this will be reduced to 1 in 40. Consequently, the quad screen will detect the same percentage of DS as the triple screen, but increase anxiety for only half as many patients. The quad screen will also lead to a 50 percent reduction in the number of unnecessary ultrasound examinations and amniocenteses performed, as well as a corresponding decrease in the number of fetal losses due to amniocentesis.

T18 is a chromosome abnormality caused by the presence of one extra 18th chromosome. Fifty percent of affected children are stillborn; five percent of infants who survive delivery will be alive at one year. Long-term survivors have severe mental retardation and are not ambulatory nor articulate. They often learn sign language.

LABORATORY DIAGNOSIS

Alpha-fetoprotein (AFP) is made by the fetal yolk sac and liver. Large quantities of AFP can be found in the fetal serum; it is excreted into the amniotic fluid through the fetal kidneys and lungs. Since ONTDs have no skin covering, large concentrations of fetal serum AFP gain access to the amniotic fluid. The AFP then passes from amniotic fluid to maternal blood where increased amounts are detected with maternal serum AFP (MSAFP) screening.

MSAFP concentrations are rising rapidly between 15-20 weeks gestation, usually 10-15 percent per week. A high AFP result means that the amount of AFP in the maternal blood is greater than that usually seen. It does not necessarily mean that a birth defect is present. Results are expressed in ng/mL and as “multiples of the median” (MoM), calculated as the AFP value in ng/mL divided by the median value based on gestational age of the fetus. Adjustments to MoM are made for maternal weight, race, number of fetuses, and insulin-requiring diabetes. The most common cause for an elevated AFP is an underestimated gestational age; 50 percent of twin pregnancies, 70-80 percent of pregnancies affected with spina bifida, and 95 percent of anencephalic pregnancies are also detected.

Chorionic gonadotropin (CG) is produced by the syncytiotrophoblast of the placenta after implantation, 5-8 days postconception. Maternal serum CG levels rise rapidly from about 5 IU/L at 8-11 days following conception, doubling every 2-3 days during the first six weeks of pregnancy. At 16-18 weeks, typical levels are 10,000-35,000 IU/L. Results are expressed in IU/L and as MoM, calculated as CG in IU/L divided by median value adjusted for gestation, maternal weight, and number of fetuses. Elevated CG levels at 16 weeks gestation can help predict the risk of fetal DS.

Estriol is the principal estrogen hormone in the blood during pregnancy. Estriol precursors are produced in the fetal adrenals and liver; estriol itself is synthesized in the placenta. Estriol exists in the maternal blood as a mixture of the unconjugated form together with a number of conjugates. Levels of total serum estriol can be affected by several factors (maternal renal system and administration of ampicillin), so uE3 is most often measured. Results are expressed in ng/mL, and as MoM, calculated as uE, divided by the median value adjusted for gestational age, maternal weight, and number of fetuses. Normally, levels of estriol increase during pregnancy. Low estriol levels are associated with DS and fetal distress.

Inhibin is secreted from the placenta and corpus luteum. Although dimeric inhibin comes in two types, A and B, only type A (DIA) is present in the maternal serum during pregnancy. Results are expressed in pg/mL and as MoM, calculated as DIA divided by the median value adjusted for gestational age, maternal weight, and number of fetuses. Unlike the other analytes, DIA levels remain relatively constant between 15-20 weeks gestation. Increased DIA levels are associated with an increased risk for DS.

Maternal serum AFP and uE3 values are usually 30 percent lower in DS pregnancies while maternal serum CG and DIA values are approximately two times higher in affected pregnancies. All four analytes are typically low in pregnancies affected with T18, with estriol being the strongest indicator. UE3 is the most effective analyte for the detection of T18. The triple and quad screens detect 60 percent of trisomy 18 with a 0.5 percent false positive rate.

Risk estimates are provided on all maternal serum screens. The risk for an ONTD is based on the AFP MoM, family history of ONTD, and insulin-requiring diabetes, while the risk for DS and T18 are based on maternal age, dating method, AFP MoM, CG MoM, uE3 MoM, and DIA MoM.

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APPROPRIATE USE OF TEST

Maternal serum double, triple, or quad screening should be offered to women who are at low risk for an ONTD, DS, and T18. If they have had a previously affected pregnancy, then they have an increased risk for recurrence and should consider undergoing a diagnostic test such as chorionic villus sampling or amniocentesis for a chromosome anomaly or targeted ultrasound and amniocentesis for an ONTD. If they refuse a diagnostic test and elect to undergo maternal serum screening, they need to be informed that not all cases of ONTDs, DS, or T18 will be detected. Furthermore, their history should be indicated on the test requisition to ensure an accurate calculation of risk.

Maternal serum screening is optimally performed at 16-18 weeks gestation, although risk estimates are given for tests performed anytime between 14.0-24.9 weeks gestation. When specifying the gestational age at the time of the screen, be sure to enter the completed weeks and days. Do not round the gestational age to the nearest week as ARUP uses day specific medians to calculate risks. It is better to use the estimated due date (EDC) than the gestational age because there are fewer clerical errors when EDC is used.

A high MSAFP result can often be explained by an underestimated gestational age, multiple gestation pregnancy, or occasionally a birth defect such as a ONTD or ventral wall defect on a targeted ultrasound examination. If a MSAFP result is greater than or equal to 2.5 and less than 3.0 MoM, the AFP can be repeated. If the result is 3.0 MoMs or higher, a targeted ultrasound should be scheduled as soon as possible. If a targeted ultrasound, with optimal views of the fetal posterior fossa and spine by an experienced sonographer, reveals no anomalies, the risk for an ONTD can be reduced by 95 percent. The patient should still be offered the option of proceeding with amniocentesis to measure amniotic fluid alpha-fetoprotein (AFAFP) and acetylcholinesterase (ACHE) to decrease the risk for spina bifida by 99 percent. Most patients feel comfortable forgoing amniocentesis if a targeted ultrasound examination is normal. Occasionally maternal habitus, fetal position, or decreased amniotic fluid volume may preclude optimal ultrasonographic views necessitating amniocentesis to reduce the risk for a ONTD after an elevated MSAFP result.

Some increased MSAFP values will remain unexplained even after a normal targeted ultrasound or amniocentesis. As the MSAFP value increases above 3.0 MoM, there is corresponding increase in the risk for a poor pregnancy outcome including: prematurity, a small-for-gestational-age infant, and stillbirth. Women with CG or DIA MoMs greater than 2.0 are at increased risk for preeclampsia and small-for-gestational-age infants. These pregnancies should be followed more closely throughout the remainder of gestation to ensure an optimal outcome.

Recalculations for incorrect pregnancy dates should be requested only if ultrasound examination indicates a due date that is more than 10 days different than estimated by the patient’s last menstrual period. Ultrasound measurements are accurate to within ± 10 days during the second trimester of pregnancy. Be careful when requesting a recalculation for a pregnancy at increased risk for DS, as a shorter femur length may falsely indicate that the pregnancy is less advanced than it is. The recalculation could inappropriately reduce the DS risk. Biparietal diameter should be used when dating such a pregnancy. T18 fetuses typically measure 7 to 14 days less than expected by menstrual dates during the second trimester. Again, one must exercise caution when requesting a recalculation in such circumstances.

Never repeat a maternal serum screen that indicates an increased risk for DS unless the first screen was drawn before 14 weeks gestation. This practice has been shown to decrease maternal serum screening sensitivity for DS.

Whether to order the double, triple, or quad test depends on the purpose. All three tests have the same ONTD detection rate of ~85 percent. The double screen is the most economical. For the detection of at least 60 percent of T18 and DS, the triple or quad screen must be considered. For the highest detection rate of DS and T18, in combination with the lowest false positive rate, the quad screen is the best choice.

See Table 1 below for the sensitivity and false positive rates of using maternal age in combination with maternal serum double, triple, and quad screening.

Table 1

Screening Test	% DS Detection	% T18 Detection	% False Positive	DS Cutoff
Double	50	30	5-7	1/270
Triple	60-70	60	5-7	1/190
Quad	60-70	60	2-3	1/150

Figure 1: Maternal Serum Screening: High AFP

Figure 2: Maternal Serum Screening: No Increased Risks



Figure 3: Maternal Serum Screening: Down Syndrome



Figure 4: Maternal Serum Screening: Trisomy 18

Test Highlights
0080269	Alpha Fetoprotein, hCG, Estriol, Inhibin (Maternal Screen)	AFP MS4

Methodology:	Fluoroimmunometric/Enzyme-Linked Immunosorbent Assay (ELISA)
Performed:	Tue
Reported:	Within 2 days
Specimen Required:	Collect : One 7 mL plain red. Transport: 3 mL serum, frozen. Remarks: Patient must be between 14-25 weeks gestation. The following information is required and must accompany the sample for test interpretation: 1) Patient’s date of birth, 2) Current weight, 3) Due date, 4) The method used for determining the due date (US, LMP), 5) Date of last menstrual period, 6) The number of fetuses present (if known), 7) The patient’s race*, 8) If the patient requires insulin to control diabetes, 9) If there is a known family history of neural tube defects, and 10) The physician’s name and phone number. Avoid repeated freeze/thaw cycles. Unacceptable Conditions: Plasma, severely hemolyzed, or lipemic samples. Stability: Refrigerated: 24 hours; Frozen: 1 year
Reference Interval:	By report (Intervals are based upon weeks of gestation)
CPT Code(s):	82677 Estriol; 86336 Inhibin A; 82105 AFP; 84702 hCG
*Note	When mothers are of mixed race, please ask the physician to list the race as OTHER and then provide both of the maternal races on the request slip. For example, an expectant woman whose mother is Caucasian and whose father is African American should be listed as: OTHER (Caucasian and African American). Remember, the race needed is the mother’s race, not the race of the fetus.
Related Tests	0080427 Alpha Fetoprotein (Amniotic Fluid) with Reflex to Acetylcholinesterase AFP AF 0080108 Alpha Fetoprotein, hCG, Estriol (Maternal Serum) AFP MS3 0080426 Alpha Fetoprotein, hCG (Maternal Serum) AFP MS

MEDICARE COVERAGE OF LABORATORY TESTING Please remember when ordering laboratory tests that are billed to Medicare/Medicaid, or other federally-funded programs, the following requirements apply: 1. Only tests that are medically necessary for the diagnosis or treatment of the patient should be ordered. Medicare does not pay for screening tests, except for certain specif­ically approved procedures, and may not pay for non-FDA approved tests or for those tests considered experimental. 2. If there is reason to believe that Medicare will not pay for a test, the patient should be informed. The patient should then sign an Advance Beneficiary Notice (ABN) to indi­cate that he or she is responsible for the cost of the test if Medicare denies payment. 3. Effective January 1, 1998, the ordering physician must provide an ICD-9 diagnosis code or narrative description, if required by the local fiscal intermediary or carrier. 4. Organ- or disease-related panels should be billed only when all components of the panel are medically necessary. 5. Both ARUP- and client-customized panels should be billed to Medicare only when every component of the customized panel is medically necessary. 6. Medicare National Limitation Amounts for CPT codes are available through CMS or its intermediaries. Medicaid reimbursement will be equal or less than the amount of Medicare reimbursement. The CPT codes for the tests profiled in this test bulletin are in the "Test Highlights" section. In addition to this source, however, ARUP strongly recommends that clients reconfirm CPT code information with their local intermediary or carrier. Also, if you have further questions regarding the appropriate use of any test, please contact ARUP's Client Information Services Department. The regulations described above are only guidelines. Additional procedures may be required by your local intermediary or carrier.

For Technical Information, Contact: Cynthia Gin, B.S., MT(ASCP) Technical Supervisor, Special Chemistry Lab (801) 583-2787 x 2255 (800) 242-2787 x 2255 For Scientific & Clinical Information, Contact: Edward Ashwood, M.D. Medical Director, Special Chemistry Lab (801) 583-2787 x2105 (800) 242-2787 x2105 Chris Miller, M.S., CGC Genetic Counselor, Molecular Genetics Lab (801) 583-2787 x2946 (800) 242-2787 x2946 References º Aquilina J, et al. Second-trimester maternal serum inhibin A concentration as an early marker for preeclampsia. Am J Obstet Gynecol 1999 Jul; 181 (1):131-6 º Ashwood ER. Evaluating health and maturation of the unborn: the role of the clinical laboratory. Clin Chem 1992; 38:1523-1529. º Ashwood ER. The clinical chemistry of pregnancy. Burtis CA and Ashwood ER, eds. Tietz textbook of clinical chemistry, 3rd ed. 1999; Philadelphia: W.B. Saunders Co., 1860-1861, 1736-1775. º Goel V, et al. Psychological outcomes following maternal serum screening: a cohort study. CMAJ 1998:159:651-6. º Haddow JE, et al. Prenatal screening for Down syndrome with use of maternal serum markers. N Engl J Med 1992; 327:588-593. º Haddow JE, et al. Second trimester screening for Down syndrome using maternal serum dimeric inhibin A. J Med Screen 1998; 5:115-119. º Lambert-Messerlian GM, et al. Second-trimester maternal serum progesterone levels in Turner syndrome with and without hydrops and in trisomy 18. Prenat Diagn 1999 May; 19(5):476-9. º Palomaki GE, et al. Risk-based prenatal screening for trisomy 18 using alpha-fetoprotein, unconjugated estriol, and human chorionic gonadotropin. Prenat Diagn 1995; 15:713-23. º Phillips OP, et al. Maternal serum screening for fetal Down syndrome in women less than 35 years of age using alpha-fetoprotein, hCG, and unconjugated estriol: a prospective 2-year study. Obstet Gynecol 1992; 80:353-358. º Spencer K, et al. Maternal serum activin A and inhibin A in trisomy 18 pregnancies at 10-14 weeks. Prenat Diagn 2001 Jul; 21(7):571-574. ARUP Laboratories 500 Chipeta Way Salt Lake City, UT 84108 (800) 242-2787 (801) 583-2787 Reorder Number: 1039-02 www.aruplab.com May 2002