Category Archives: Alpha-fetoprotein


Laboratory testing for premature rupture of membranes

This post is by a guest author, Douglas Stickle, Ph.D.  Dr. Stickle is a professor in the Department of Pathology at Thomas Jefferson University and the director of chemistry and point-of-care testing at Jefferson University Hospitals in Philadelphia, PA.

Rupture of membranes (ROM) is the term used to describe the breaking of the amniotic sac, as normally occurs before the onset of labor. If this happens earlier than the 37th week of pregnancy it is called preterm ROM (PROM). It’s a condition that can lead to a preterm birth, or, if very early, a preterm, premature birth.

Preterm baby 2When PROM happens, there is an increased risk of complications due to intrauterine infection, umbilical cord compression, and the neurodevelopmental disorders that are associated with a preterm delivery. Diagnosis of PROM is particularly important when the gestational age is incompatible with a viable birth, often considered to be a fetal age less than 24 weeks. In such cases, medical intervention is necessary to preserve the chances for a live birth.

Suspected cases of PROM are often investigated by laboratory analysis of fluid obtained from the vagina to detect properties or substances that should otherwise not be present unless the fluid contains amniotic fluid due to PROM. The simplest forms of testing are measurement of acidity (pH) of the fluid, or a test called "fern" testing. Fern testing refers to the fern-like appearance of amniotic fluid when it is dried on a glass slide. Both of these tests aren’t very accurate and so other tests have been developed to better identify patients with ruptured membranes.

These other tests are designed to detect molecules that are normally present in amniotic fluid but not vaginal fluid. For example, tests have been developed that detect alpha-fetoprotein (AFP) or insulin-like growth factor binding protein-1 (IGFBP-1). The presence or absence of these molecules in the specimen are determined by a lateral flow immunoassay. The assay works like commonly performed tests for human chorionic gonadotropin (hCG) (aka pregnancy tests).

These tests are highly sensitive to low concentrations of these molecules, which is both good and bad. It’s good because they can detect small amounts of the molecules and lead to a more accurate diagnosis. It’s bad because these two molecules are also present in maternal blood which means that if a sample is contaminated with blood, the certainty of a positive test to detect amniotic fluid is called into question.

From the doctor’s perspective, a practical advantage of the immunoassays is that their results are binary – the result is either positive or negative — whereas the pH test and the fern test are more subjective and difficult to interpret definitively. However, the AFP and IGFBP-1 tests may be subject to false-positive results as the gestational age of the fetus approaches term. This suggests that, at later stages of pregnancy, these biomarkers may signify imminence of delivery.

The gold standard, or best test, to diagnose rupture of membranes is a dye test, in which a colored fluid is injected into the amniotic fluid followed by direct observation to see if the dye subsequently appears in the vaginal pool fluid. Also, ultrasound imaging of the amniotic fluid volume may also assist in diagnosis of PROM, but in individual cases such imaging may be difficult to interpret. Given the low but finite risk of complications of the dye test, the AFP and IGFBP-1 tests are often preferred as first-line tests for preterm premature rupture of membranes.

Screening for neural tube defects

NeuronsA neural tube defect (NTD) is a birth defect of the spinal cord and/or brain.  The term is used to describe a group of disorders that occur very early in pregnancy and can be mild to severe or even fatal.

During the first 3 weeks of pregnancy, specific cells fuse to form a hollow tube (the neural tube) that forms the basis of what will become the spinal cord and brain.  A NTD occurs when that neural tube fails to close completely somewhere along its length.

The two most common NTDs are spina bifida and anencephaly.  Spina bifida is the most common.  There are different types of spina bifida and each has varying degrees of severity but it nearly always results in some nerve damage that can cause at least some paralysis of the legs.  Anencephaly is the most severe NTD and results in the lack of development of the brain and skull and is not compatible with life.  NTDs that are covered by skin are called “closed” defects while those that are not covered by skin are considered to be “open.”  Only open NTDs are detected by screening tests.

Alpha-fetoprotein (AFP) testing is used to screen for a NTD during the second trimester of pregnancy.  Ideally it takes place between 16 and 18 weeks of gestation but between 15 and 22 weeks is acceptable.  The concentration of AFP in fetal blood is 100,000 times greater than it is in maternal blood.  Some of the fetal AFP normally enters the maternal blood and so the AFP concentration in maternal blood will begin to increase.  A fetus with an open NTD will transfer more AFP into maternal blood than an unaffected fetus and so an unusually high AFP concentration in maternal blood can indicate that the fetus has an open NTD.

Because AFP concentrations normally increase during pregnancy (by about 15 percent each week), a statistic called the “multiple of the median” (MoM) is used to normalize the test result.  The MoM is a measure of how far an individual test result deviates from the median (middle) value of a large set of AFP results obtained from unaffected pregnancies.  For example, if the median AFP result at 16 weeks of gestation is 30 ng/mL and a pregnant woman’s AFP result at that same gestational age is 60 ng/mL, then her AFP MoM is equal to 60 divided by 30 (60/30) or 2.0.  In other words, her AFP result is 2 times higher than “normal.”

So how is the AFP MoM interpreted?  What is considered an abnormal result?  Although the AFP MoM cutoff varies by lab, the two most commonly used are 2.0 and 2.5.  Results above the cutoff are considered to be abnormal.  A cutoff of 2.0 will detect about 85 percent of open NTD and a cutoff of 2.5 will detect about 75 percent.  Most cases of anencephaly are detected with maternal serum AFP screening.  The figure below illustrates the distribution of AFP MoM results in women with unaffected fetuses, those with spina bifida, and fetuses with anencephaly.

Results to the right of the blue line (a cutoff of 2.5 MoM) would be interpreted as "abnormal" while an AFP MoM to the left of the line would be considered "normal."  Note that there is no single MoM cutoff that can completely separate unaffected from affected fetuses.  There will always be affected fetuses that screen normal and unaffected fetuses that screen abnormal.

Because this is a screening test, women with an abnormal result require additional testing to confirm if the fetus has a NTD.  More about these tests in future post.

Lastly, it’s important to keep in mind that most abnormal NTD screening tests are false-positives.  There are several reasons why AFP might be elevated in the absence of an open NTD such as: an abnormality in the fetal kidneys, a ventral wall defect (opening in the abdomen), the death of the fetus, a twin gestation, or, most commonly, underestimated gestational age.