Second trimester prenatal screening may include several blood tests, called multiple markers. These markers provide information about a woman's risk of having a baby with certain genetic conditions or birth defects. Screening is usually performed by taking a sample of the mother's blood between the 15th and 20th weeks of pregnancy (16th to 18th is ideal). The multiple markers include:

• hCG (human chorionic gonadotropin hormone): A hormone produced by the placenta 
• Estriol: A hormone produced by the placenta 
• Inhibin: A hormone produced by the placenta 
• AFP (alpha fetoprotein) or msAFP (maternal serum AFP): A blood test that measures the level of alpha-fetoprotein in the mothers' blood during pregnancy. AFP is a protein normally produced by the fetal liver, is present in the fluid surrounding the fetus (amniotic fluid) and crosses the placenta into the mother's blood. Abnormal levels of AFP may signal the following:
  • Open neural tube defects (ONTD), such as spina bifida
  • Down syndrome
  • Other chromosomal abnormalities or genetic conditions
  • Defects in the abdominal wall of the fetus (such as gastroschisis or omphalocele)
  • Twins - more than one fetus is making the protein
  • A miscalculated due date, as the levels vary throughout pregnancy

Multiple marker screening is not diagnostic. This means it is not 100 percent accurate and is only a screening test to determine who in the population should be offered additional testing for their pregnancy. There can be false-positive results, indicating a problem when the fetus is actually healthy; or false negative results, indicating a normal result when the fetus actually does have a health problem.

When a woman has both first and second trimester screening tests performed, the ability of the tests to detect an abnormality is greater than using just one screening independently. Most cases of Down syndrome can be detected when both first and second trimester screening are used. 

Abnormal test results of AFP and/or other markers may indicate the need for additional screening or testing. Usually, an ultrasound is performed to confirm the dates of the pregnancy, to assess the fetal spine for an ONTD, and to assess other body parts for defects. Additional screening, such as noninvasive prenatal testing (NIPT) can be considered to screen the pregnancy more accurately for aneuploidies (such as Down syndrome or trisomy 18) and sex chromosome differences. An amniocentesis may also be considered to perform diagnostic testing.