Triple Marker Screen Test

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Triple marker screen test


The triple marker screen test (also called the maternal serum screening test or multiple marker test), is a blood test that is performed usually between the 14th and 18th week of pregnancy . This screening test measures the levels of three substances, alpha-fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated estriol (uE3) in the maternal blood. Each level is then divided by the median concentration of that substance for the given week of pregnancy to generate a multiple of the median value (MOM). These values, along with other maternal characteristics, such as maternal age, are analyzed by a computer program to indicate the probability that the fetus has Down syndrome . Down syndrome is a condition that includes mental retardation, skeletal abnormalities such as upslanted eyes and cleft palate, and organ abnormalities such as heart disease and intestinal obstruction. Approximately 80-95% of cases are caused by a nondisjunction of chromosome 21 in the developing gamete resulting in the presence of an additional chromosome 21.


Triple marker testing is a screening test that is used to identify the risk that a pregnant woman will give birth to an infant with Down syndrome. The test will also detect pregnancies at increased risk for Edward syndrome (trisomy 18) and Turner syndrome (monosomy X) and developmental defects associated with increased leakage of alpha fetoprotein from the fetus. The criterion used to define cutoff concentrations of the three markers is a risk for Down syndrome of one in 190. This is equal to the risk of miscarriage from amniocentesis . Women who screen "positive" (risk of 1:190 or higher) are recommended for amniocentesis. This procedure provides cells from the fetus that are cultured and analyzed to determine the number or chromosomes within each cell and detect structural chromosome abnormalities. This is the definitive method for diagnosing Down syndrome and other genetic conditions caused by an abnormal number of chromosomes (aneuploidy).


It is very important that the correct gestational age be determined by last menstrual period dating and recorded for the risk calculation. Errors in determining the age of the fetus lead to errors when interpreting the test results. Since an AFP test is only a screening tool, an abnormal test result is not necessarily indicative of a birth defect. Accurate gestational dating lowers the false-positive and false-negative rates associated with this screening test.

The nurse or phlebotomist collecting the blood sample for these tests should observe universal precautions for the prevention of transmission of bloodborne pathogens.


Prior to 1964, when the association between low levels of AFP and an increased risk for Down syndrome was reported risk assessment for chromosomal diseases was based upon maternal age. At age 35, the risk of carrying a Down syndrome pregnancy is approximately one in 270, and this was deemed sufficient to warrant amniocentesis. However, three of four Down syndrome pregnancies occur in women under 35 years old. When AFP testing was used along with maternal age, the rate of detection of Down syndrome increased to about 45%, but this level of sensitivity did not justify the screening of younger women because of the risk of miscarriage. The inclusion of uE3 and hCG testing has improved the detection rate to approximately 65-80% of cases for all age groups.


Acetylcholinesterase —A chemical found only inside neural tissue. Its presence in the amniotic fluid indicates an opening in the neural tube.

Amniotic fluid —Fluid within the uterine sac in which the fetus lives until birth.

Anencephaly —A severe and usually fatal brain abnormality caused by failure of the neural tube to close at its cranial end.

Embryo —The stage of human development prior to the second month of pregnancy.

Fetus —The stage in human development from the second month of pregnancy until birth.

Karyotyping —Chromosome analysis.

Neural tube —Tube that becomes the brain and spinal cord.

Oligohydramnios —Low amniotic fluid level.

Alpha fetoprotein

Alpha-fetoprotein (AFP) is a glycoprotein similar in size and structure to albumin. It is made principally by the fetal liver and is present at very low levels after birth. In several developmental defects the most prevalent of which is an open neural tube defect , spina bifida, the AFP leaks from fetal blood vessels into the amniotic fluid. The AFP crosses the placenta and can be measured reliably in the maternal circulation by week 14. Increased maternal serum AFP also occurs in the following conditons:

  • abdominal wall defects (omphalocele and gastroschisis)
  • anencephaly
  • Turner syndrome
  • trisomy 13
  • renal diseases (congenital nephrosis, polycystic kidneys , renal agenesis)
  • oligohydramnios (decreased amount of amniotic fluid)
  • more than a single fetus
  • maternal liver cancer and other malignancies

The cutoff for a positive screen is 2.5 MOM. A positive test should be repeated, and if positive the second time, should be followed by ultrasound. If ultrasound does not explain the high level (which may be caused by twins, anancephaly, or inaccurate dating), then amniocentesis is recommended. AFP and acetylcholinesterase levels in amniotic fluid along with high resolution ultrasound are used to predict the probability of an open neural tube defect. Decreased AFP levels, below 0.75 MOM, are seen in approximately 25% of Down syndrome pregnancies. AFP is measured by double antibody sandwich radioimmunoassay or enzyme immunoassays.

Human chorionic gonadotropin and unconjugated estriol

Human chorionic gonadotropin (hCG) and unconjugated estriol are hormones. Estriol is the major estrogen of pregnancy and is produced by the placenta from dihydroepiandosterone sulfate that is made in the fetal adrenal glands . Estriol levels rise steadily throughout pregnancy increasing about threefold from week 24 to full term. Human chorionic gonadogropin is also made by the placenta, and it supports the corpus luteum during gestation. The corpus luteum produces progesterone, which maintains the uterus during pregnancy. Chorionic gonadotropin peaks at about 10 weeks gestation and then falls to about 20-25% of peak levels for the remainder of pregnancy. During pregnancy, both hormones diffuse from the placental membranes into the maternal blood. Abnormal levels can be indicative of potential fetal distress and stillbirth. Like AFP, uE3 is lower than normal for the time of gestation. Conversely, hCG is increased above normal by about 25% for the time of gestation. Both hormones may be measured by radioimmunoassay or fluorescent or chemiluminescent enzyme immuno-assay.

When any one test exceeds the cutoff, testing should be repeated on a new sample and ultrasound should be performed in an attempt to explain the results and determine an accurate gestational age. If results are still positive and not explained by ultrasound, amniocentesis for chromosome karyotyping (chromosome counting and analysis) is recommended. When AFP, hCG, and uE3 are low for the gestational age, this may indicate trisomy 18. This condition is caused by an additional chromosome 18, and is associated with severe birth defects, mental retardation and death. The sensitivity for trisomy 18 is approximately 60-80% using cutoffs of 0.75 MOM for AFP; 0.60 MOM for uE3; and 0.55 MOM for hCG.


There is no specific physical preparation for this test. Fasting is not required.


After the blood sample is drawn, pressure should be applied to the puncture site until the bleeding stops to reduce bruising, and a bandage may be applied to the site. A warm pack may be applied to the site to relieve discomfort.


The complications associated with drawing blood are minimal, but may include bleeding from the puncture site, feeling faint or lightheaded after the blood is drawn, or blood accumulating under the puncture site (hematoma).


The various immunoassays for these analytes are associated with different normal ranges because the antibody specificity and assay detection limits are somewhat different. In order to allow for interlaboratory comparison of results, the results of analytes are expressed as multiples of the median value used by the laboratory. Normal ranges expressed in concentration (e.g. ng/mL) are dependent upon gestational age, but MOMs are age adjusted and do not change. These values are used to calculate risk. If the multiple of the median value is above2.0 MOM or 2.5 MOM (depending on the laboratory), the fetus is considered to be at a higher risk for a neural tube defect. The MOM value for amniotic fluid is then used to calculate the exact probability the fetus is affected (1:100, for example).

With respect to Down syndrome and trisomy 18, the MOM values are also used in the calculation of probability. The woman is considered to be "high risk" or "screen positive" for Down syndrome if the risk is greater than the standard risk for women who are 35 years old or older (one in 270). For trisomy 18, the cut-off is one in 150. In one study the triple marker screen test had a detection rate for Down syndrome of 67% and a false positive rate of 5%.

Health care team roles

The obstetrician orders the triple marker screen test, and explains its purpose and results to the patient. The nurse or phlebotomist collects the blood sample and transports it to the laboratory. Typically, a nurse calls the patient with her result. If abnormal, the pregnant patient is referred to a genetic counselor, who explains the test, the result, and diagnostic testing options.



Cunningham, Gary, et al. Williams Obstetrics. 20th ed. Stamford, CT: Appleton & Lange, 1997, 922-926.

Johnson, Robert, ed. Mayo Clinic Complete Book of Pregnancy and the Baby's First Year. NY: William Morrow and Company, 1997.


Canick, Jacob, et al. "Multiple Marker Screening for Fetal Down Syndrome." Contemporary OB/GYN (April 1992): 3-12.

Haddow, James, et al. "Reducing the Need for Amniocentesis in Women 35 Years of Age or Older with Serum Markers for Screening." New England Journal of Medicine 330 (16) (April 21, 1994): 1114-1118.


American Cancer Society. 1559 Clifton Rd. NE, Atlanta, GA 30329. <>.

March of Dimes Birth Defects Foundation, National Office. 1275 Mamaroneck Ave., White Plains, NY 10605. (888) MODIMES. <>.

National Cancer Institute. Building 31, Room 10A24, Bethesda, MD 20892. <>.

Rachael Brandt