Category Archives: AFP

AFP

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.

AFP and NTD
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.

Why so many Down syndrome screening tests?


Throughout pregnancy, women have to make lots of decisions.  Two of those are “Do I want to have a screening test to see if my baby has an increased risk of Down syndrome?” If the answer to that is yes then the logical next question would be “What tests can I choose from?”  That is not an easy question for most women to answer without some guidance from their doctors and it’s not easy for many doctors to help provide that guidance.  Why?  Simply put, there are many tests from which to choose.

Let’s be clear on an important concept up front.  Screening tests are not diagnostic tests.  A test that screens for Down syndrome doesn’t identify if a baby has Down syndrome; it identifies babies that are at increased risk of having Down syndrome.  Women with abnormal or positive screening test results can undergo additional tests that can be used to confirm if their baby does or does not have Down syndrome but the screening tests cannot do that. 

All Down syndrome screening tests require a blood sample from the mother.  Biochemical markers in the blood are measured in the laboratory and the results used to calculate the risk that the baby has Down syndrome.  Those biochemical markers include:

  • Alpha-fetoprotein (AFP)
  • Human chorionic gonadotropin (hCG)
  • Unconjugated estriol (uE3)
  • Dimeric inhibin A (DIA)
  • Pregnancy-associated plasma protein A (PAPP-A)

Some screening tests also include the measurement of nuchal translucency (NT) that is obtained by an ultrasound scan of the fetus.  The NT is the width of the space between the spine and skin at the fetus’ neck.

All together, these 5 biochemical markers and 1 ultrasound marker can be used in various combinations to create the different Down syndrome screening tests.  There are 6 to choose from:

Table

Some, like the Triple (3 markers) and the Quad (4 markers) tests are performed on a blood sample collected during the 2nd trimester and don’t require the NT ultrasound measurement.  The Combined test (so called because it “combines” the biochemical and ultrasound tests) is performed only in the 1st trimester.  All the other tests use two different blood samples (one collected from the mother in the 1st and the other in the 2nd trimester) and may or may not also include the NT measurement.  No wonder this is confusing!

Why are there so many different tests?  Two reasons: first because Down syndrome screening tests evolved over many decades (and continues to evolve) and second because the medical community is often slow to change its habits.

In the not so distant past, one “test” was used to determine Down syndrome risk: the age of the mother.  We actually still use the mother’s age in determining Down syndrome risk.  The risk of having a baby with Down syndrome increases as the age of the mother increases.  We now use this age-based risk as a starting point; a risk that is the modified by the results of the screening test.

In 1988, the Triple test was introduced as a way to adjust the age-based risk using the measured concentrations of AFP, hCG, and uE3 in the mother’s blood.  A few years later, the Triple test turned it into the Quad test when it was discovered that the addition of DIA to the Triple test improved the Down syndrome detection rate.  However, the Triple test didn’t disappear and labs simply continued to offer it as well as the Quad test.

The Combined test appeared in 1999 and had a Down syndrome detection rate that was similar to that of the Quad test.  Unlike the Quad test, however, the Combined test provided women a way to get Down syndrome screening test results many weeks sooner.  So, it was added to the menu along with the Triple and the Quad tests.

It wasn’t long before someone thought to “integrate” the Combined test with the Quad test and thus was born the Integrated test.  And, for those women without access to the specialized equipment needed to perform the ultrasound NT measurement, the Serum Integrated test required only the mother’s blood samples to be tested.  While both of these tests provide the greatest Down syndrome detection rates, the need for the second blood sample collected in the 2nd trimester means that early risk-assessment is not possible.

And finally, the Sequential test developed as a modification of the Integrated test.  While the Integrated test delivers its results in the 2nd trimester after all testing has been completed, the Sequential test offers women results in the 1st trimester only if the risk of Down syndrome is very high.  In the absence of a high risk, results are provided only after the test is completed in the 2nd trimester.

Because it’s a test with a lot of history and experience behind it and because it’s well known to the doctors that order Down syndrome screening tests, the Quad test still leads the pack in terms of test usage.  The Triple test is slowly falling out of favor (as it should) and the Combined, Integrated, and Sequential tests are gaining more traction, albeit slowly.  As I said earlier, old habits are slow to change.