An antiquated pregnancy test?

Many people are surprised at how many different types of "pregnancy tests" are available.  The use of quotations is deliberate because pregnancy tests don't detect pregnancy, they detect a hormone produced during pregnancy: human chorionic gonadotropin (hCG).  But, because hCG is usually only present during pregnancy, one can forgive the non-specific label.

Here's a table that illustrates the available hCG tests:

HCG test types

So hCG tests can be performed on urine or blood using a qualitative (yes/no) or a quantitative (concentration) test.  Because there is no clinical need to quantify hCG in urine, quantitative tests are usually not performed in urine.  Some laboratorians (yours truly included) do believe that quantitative urine hCG tests can be helpful in specific situations but that's a post for a different day.

In this post, I want to focus on the qualitative hCG test performed with a blood sample.  A recent paper suggested that this test should be considered antiquated (disclaimer: I was an author on that paper).  There are a few reasons why this might be true:

  1. The biggest value of qualitative hCG tests is that they can be performed at the point-of-care with test results available to the physician very quickly.  However, blood samples have to be centrifuged to separate the liquid part of the blood (serum) from the solid part (blood cells), and the centrifugation step is nearly always performed in the laboratory.  As such, qualitative hCG tests using a blood sample cannot be performed at the point-of-care.
  2. The vast majority of clinical laboratories can perform quantitative serum hCG tests.  Although these tests may take a bit longer to perform than qualitative tests, the time it takes to perform them is still rather fast (~20 minutes).
  3. Quantitative hCG tests can probably measure hCG at a concentration lower than the detection thresholds claimed by qualitative tests (~1 IU/L vs. 10-25 IU/L, respectively).  If that's the case, why not use the most sensitive hCG test for a blood sample?
  4. The only clinical use of a qualitative serum hCG test is to detect (or rule out) a possible pregnancy.  Therefore, it can be argued that the most analytically sensitive test available (i.e a quantitative test) should be utilized.

Given those reasons, I've been wondering if qualitative blood tests for hCG could be abandoned.  Why bother using them if quantitative tests are readily available and can detect hCG at lower concentrations than qualitative tests?  Seems like a no-brainer to me but, as a scientist, I need data to support my hypothesis.  This is something to investigate!

What the loss of a widely used fetal lung maturity test will mean to doctors and labs

The test most widely used to assess fetal lung maturity is going away.  Earlier this year I wrote about the different laboratory tests that doctors use to determine if a fetus' lungs were mature.  One of those tests, the surfactant-to-albumin ratio, is marketed and sold by Abbott Laboratories as the TDx FLM II test.  Last year, they announced that they were discontinuing the manufacture of that test.

The main reason this test will no longer be made is that the instrument that performs the test is several decades old.  The TDx line of instruments is aging and the technology it uses is becoming outdated.  The decision to stop supporting the TDx makes sense but it does leave labs that offer the FLM II assay in a bit of a quandary.  The Abbott test performs well and is rapid and simple to perform.  Doctors are familiar with it and know how to interpret the results.  The test is an excellent predictor of fetal lung maturity and the risk of respiratory distress syndrome as a function of both gestational age and TDx FLM II result is known.  Labs that currently offer the TDx FLM II test and that wish to continue offering a test for fetal lung maturity have to figure out what to do.  Doctors will also have to learn what the loss of this test will have on their clinical practice.

I recently published the results of a survey that provide some insight as to what might be expected. Here is a summary of those data:

  • Across the US, labs are performing fewer fetal lung maturity tests each year and the most widely used test is Abbott's TDx FLM II test.
  • Although 25% of physicians indicated that their use of fetal lung maturity tests is decreasing, 90% of them said that they could not provide their current level of care without access to a test of fetal lung maturity.
  • Without the TDx FLM II test, 65% of physicians would order the lecithin-to-sphingomyelin (L/S) ratio and 40% would order the lamellar body count (LBC).
  • Fewer than 20% of labs offer the L/S ratio and even fewer offer the LBC.
  • Laboratories could simply refer fetal lung maturity test requests to a lab that performs other lung maturity tests but doctors want results within 12 hours of ordering the test.  That expectation probably can't be realized if the test is not performed in the lab.

Of all the available tests that could potentially replace the TDx FLM II test, the LBC test is the most logical:

  • The instrumentation required to perform the test is an automated cell counter and these are widely available in clinical labs.
  • The test is performed rapidly and with high precision.
  • Several outcome-based studies have demonstrated the diagnostic utility of the LBC and it performs as well as the TDx FLM II test.

When I'm asked by laboratorians what they should do when the TDx FLM II test goes away I tell them that they should consider the LBC test.  Of all the other tests of fetal lung maturity, the LBC, for the reasons outlined above, is the most logical replacement test.  I also caution them that time is running out and that NOW is the time to communicate with their physicians about the need for the change and to get their input.

It's worth re-stating that perhaps, just maybe, we could consider stop performing fetal lung maturity tests altogether.  Time will tell.

    Predicting preterm birth

    Preterm baby Preterm birth is a serious problem. Defined as delivery before 37 completed weeks, infants born preterm are at high risk of complications such as respiratory distress syndrome, bleeding into the brain, poor neurological development, and cognitive impairment. Further, preterm birth is responsible for ~75% of death in newborns without genetic anomalies.

    Unfortunately, the vast majority of treatments provided to pregnant women with symptoms of preterm labor have done little to prevent preterm birth. About 13% of pregnancies end with preterm delivery and this percentage has increased steadily over the last few decades.

    Risk factors for preterm birth include mother's age, socioeconomic status, and being pregnant with more than one fetus. A woman who has had a previous preterm delivery is also at risk of delivering subsequent pregnancies early as well. Preterm birth is also more common among black women compared to other ethnic groups which appears to be unrelated to socioeconomic status or other factors.

    Symptoms of preterm labor are just like symptoms of term labor. However, early contractions occur frequently in pregnant women and don't always mean they are in actual preterm labor. In fact, most women with early symptoms go on to delivery term babies. This makes it difficult to diagnose true preterm labor when it occurs. A test that could accurately predict preterm labor would be helpful.

    Currently there is only one lab test approved for use to predict preterm labor. Fetal fibronectin (fFN) is a protein produced by the fetal membranes during pregnancy. Although it can be detected in cervicovaginal fluid early in pregnancy it usually becomes undetectable between weeks 25 and 35. After that, it becomes detectable again after 37 weeks. In women with preterm labor, fFN can be detected in cervicovaginal fluid during the time that it is usually not present. This may occur because of a break down of the fetal membranes or in response to inflammation.

    A recent study compared the ability of fFN and a interleukin-6 (IL-6), an inflammatory cytokine, to predict preterm birth. The tests were performed in 667 samples collected from 580 women with and without symptoms of preterm labor. About half of the women in the study were black.

    In both groups, a positive fFN result was significantly associated with preterm birth occurring within 14 days of testing. However, the actual likelihood of delivery early was still rather low. Like other studies, this one also showed that the strength of the fFN test is it's negative predictive value. That is, the absence of fFN is a much stronger indicator that preterm birth won't occur.

    IL-6 was also significantly associated with preterm birth but only in the black women. Like fFN, it also wasn't a strong predictor in this population but it was much better than fFN in either the black or non-black groups.

    There's still much to learn. We don't really understand the different processes (which are multifactorial) that lead to preterm delivery. Also, it's clear that the events that lead to preterm delivery are probably not the same between women of different ethnic backgrounds. As such, a single predictor of preterm delivery is probably not going to work well. Combination testing, such as fFN and IL-6 (or other, yet to be discovered markers) may yield more promising results than any one marker alone.

    Serum bile acids are used to diagnose a liver disease during pregnancy

    Intrahepatic cholestasis of pregnancy (ICP) is a long, rather complicated name, for a problem that affects about 1% of pregnant women in the United States. For reasons unknown, ICP is much more common in South America, particularly the country of Chile, where it is 10 times more frequent.

    What is it? It's a type of liver disease in which the flow of bile from the liver into the intestines is blocked. Normally, bile made in the liver is secreted and stored in the gall bladder where it eventually finds its way into the intestines where it aids in the digestion of dietary fats. For reasons that are poorly understood, this normal flow of bile may be interrupted during pregnancy leading to its elevation in the blood. Several studies support the idea that the reproductive hormones (mostly estrogen) plays an important role in the development of ICP.

    The most common symptom of ICP is intense itching (pruritis) during the third trimester. The majority of women with ICP describe the itchiness to be most severe in the palms of the hands and the soles of the feet but it can be felt on other areas of the body. Most women with ICP develop the symptoms after the 30th week of pregnancy although it can happen earlier in pregnancy.

    Apart from the itching, ICP is rather benign in women who develop it. The same cannot be said for her fetus, however. Although there is some debate about the risks of ICP to a fetus, the vast majority of reports describe several possible adverse fetal outcomes due to ICP. These include an increased risk of spontaneous preterm birth, an apgar score that is less than 7 at 5 minutes after birth, and increased rates of meconium staining of the amniotic fluid (a sign of fetal distress).

    An ICP diagnosis is really one of exclusion. All other possible causes of liver impairment should be excluded before diagnosing ICP. Although several laboratory tests may be used in an ICP investigation, the measurement of serum bile acids is considered to be the most sensitive test for ICP. Standard liver function tests such as ALT and AST are likely to also be elevated but are less sensitive and specific than bile acids. Bilirubin testing is of limited value.

    In normal pregnancy, the concentration of bile acids doesn't usually exceed 10 µmol/L. In ICP, concentrations of bile acids can get considerably higher, even up to 100 times greater than normal. Most evidence indicates that adverse fetal outcomes are less likely when the bile acid concentration is <40 µmol/L which allows more conservative management of the pregnancy and the ICP. Ursodeoxycholic acid (UDCA) is a naturally occurring, secondary bile acid that is often used to treat the disease. It appears to stimulate the excretion of the primary bile acids and decreases their delivery to the fetus. If the concentration of bile acids is high and the pregnancy is more than 37 weeks completed, delivery is usually the best course of action.

    Anti-müllerian hormone as a predictor of IVF outcome

    Human egg Investigators at Brown University have reported that the serum concentration of a hormone made by the ovaries can predict the success of in vitro fertilization (IVF). Specifically, the higher amounts of the hormone were correlated to a greater number of eggs retrieved and the probability of achieving pregnancy.

    The study measured blood concentrations of anti-müllerian hormone, or AMH. This is a rather interesting hormone. It plays an important role in both males and females but at different stages of human development.

    • In fetal development, AMH is synthesized by the testes of a male fetus and it functions to suppress the transformation of the müllerian ducts into the uterus and fallopian tubes. In the absence of AMH (i.e. a female fetus), the wolffian ducts fail to develop into ejaculatory ducts of the male reproductive system while the müllerian ducts complete their development.
    • AMH is synthesized by the female ovary but only after birth when it does not affect the development or maturation of the female reproductive tract. It increases slightly at puberty because of it functions to regulate the formation of ovarian follicles (eggs) by opposing the action of another hormone, follicle-stimulating hormone (FSH).

    Because of its role in follicle development, AMH can be useful as a marker of ovarian function. In particular it seems to be a good indicator of the growing pool of follicles in the ovary and so reflects the number of eggs in the ovary. That’s exactly what the scientists at Brown University reported.

    In 190 women undergoing IVF to achieve pregnancy, the concentration of AMH in a blood sample collected on the first day of ovarian stimulation with FSH was a strong predictor of favorable outcome. Women with the lowest concentrations of AMH had fewer eggs retrieved than those with higher concentrations. Also, the probability of achieving pregnancy was 2.5 times greater in the women with the higher AMH concentrations.

    What might these results mean from a practical perspective? The authors of the study comment that IVF patients could be better counseled and IVF management strategies modified by the AMH results. IVF is expensive. Women who seek to achieve a pregnancy by this route are highly motivated to obtain a positive outcome and so they may undergo several IVF cycles in an effort to become pregnant. Those with a low AMH concentration may opt for only a single IVF cycle or seek adoption or other routes of parenthood rather than invest thousands of dollars into what may be an unachievable biological goal.

    The Triple Test: time to say good bye

    A recent post in the NACB blog titled "Jurassic Lab" invited readers to comment on which laboratory tests they thought should be considered obsolete.  I suggested that the Triple Test, a blood test used during the second trimester of pregnancy to screen for Down syndrome, should go the way of the dinosaur.

    Labs that offer Down syndrome screening tests should seriously consider taking the Triple Test off of their menus. Why? There are a few reasons:

    1. It is not a great test. As a screening test for Down syndrome, the Triple test is only okay. If I were grading it, then I would give it a C. It detects about 70-75% of fetuses with Down syndrome and it has a false-positive rate of 5-8%.
    2. Better tests are available. I've already wrote about the different types of Down syndrome screening tests. Other tests, like the Quad and the Integrated tests perform much better than the Triple test. Given a choice, I imagine most mothers-to-be would prefer to have the best screening test available. It's abundantly clear that of all the tests used to screen for Down syndrome, the Integrated test is best. It has a detection rate of about 90-95% and (importantly) a false-positive rate that is lower than the Triple test. A low false-positive rate is important because it decreases the number of unnecessary invasive diagnostic procedures that are performed due to an abnormal result from a screening test.
    3. Lack of recommendation. Several national guidelines no longer recommend use of the Triple test and recommend better performing screening tests instead.

    Clearly the Triple test is irrelevant. That doesn't mean that all the doctors that order Down syndrome screening tests have gotten the message. A 2009 survey of obstetricians in the US revealed that nearly a quarter of them (23%) continued to use the Triple test. That's lower than it used to be but still rather high for a test that shouldn't be ordered. Until laboratories that offer the Triple test take it off of their menus, it will continue to be (inappropriately) utilized.

    Laboratory testing in ectopic pregnancy investigation

    What’s the leading cause of maternal death in the first trimester? That would be an ectopic pregnancy, which occurs when the fertilized egg implants someplace other than the inside of the uterus. In the vast majority of ectopic pregnancies the zygote implants in the fallopian tube, an organ that, unlike the uterus, can’t expand to accommodate the growing embryo. If not diagnosed early, an ectopic pregnancy can cause the fallopian tube to rupture that can result in massive internal bleeding and death.

     Diagnosing an ectopic pregnancy is challenging. The typical symptoms of abdominal pain and vaginal bleeding are not present in all patients and can be due to reasons other than an ectopic pregnancy. Also, the risk factors of ectopic pregnancy are absent in about half of all women who have an ectopic pregnancy. Fortunately there are laboratory tests that assist in identifying an ectopic pregnancy.

     hCG testing is first used to confirm that the patient is, indeed, pregnant. Although a rapid, point-of-care urine test may be used to detect hCG, I think it is much safer to perform a serum hCG test because it can detect lower concentrations of hCG than a urine test can. If the test is negative, that is hCG is not present, then the patient isn’t pregnant at all. If hCG is detected then pregnancy is confirmed but, all by itself, a single hCG test cannot identify a normal from an ectopic pregnancy.

     To do that, other investigations are commonly put to use:

    1. Transvaginal ultrasound. Ultrasound imaging is used to visually observe if an embryo is in the uterus or the fallopian tube but it can also be inconclusive if no embryo is observed anywhere. If the ultrasound is inconclusive then the concentration of serum hCG can be useful. That’s because an intrauterine pregnancy should always be able to be seen by ultrasound when the serum hCG concentration is greater than 1500 to 2000 IU/L. If that threshold is reached and no embryo is seen in the uterus then the patient is likely to have an ectopic pregnancy. If that threshold is not present at the initial evaluation, then serial hCG testing is considered.
    2. Serial hCG testing. Collecting more than 1 blood sample over time for hCG testing is helpful because in a normal pregnancy serum concentrations of hCG increase by 53% or more every 2 days. An increase that is less than 53% is cause for concern. That’s because an abnormal increase in indicates an abnormal pregnancy. In an ectopic pregnancy, hCG can show a normal or abnormal increase so an abnormal increase by itself it isn’t diagnostic of an ectopic pregnancy.

     These two tests are usually used together in an algorithm that looks something like this:



    Ectopic algorithm


    The take home message here is that, as helpful as lab tests are, the diagnosis of ectopic pregnancy cannot be accomplished by lab tests alone. All clinical data must be evaluated in order to arrive at the final diagnosis.


    A new way to detect gestational diabetes mellitus

    Diabetes definition At first glance, screening pregnant women for gestational diabetes mellitus (GDM) seems like it should be straightforward.  After all, the tests are designed to identify pregnant woman with high concentrations of glucose (sugar) in their blood and laboratory tests that measure glucose are accurate and precise.  So what’s the problem?

    For one, experts don’t agree on how best to screen pregnant women for GDM.  While nearly everyone agrees that both mom and baby can have adverse outcomes if GDM goes undetected and untreated, there is lack of consensus on the best way of identifying GDM.

    Consider how it has been done for several years here in the United States using either a 1 or 2 step process.  In the 2-step approach, a screening test is done first followed by a diagnostic test if the screening test is abnormal.  To do the screening test, blood glucose is measured 1 hour after the non-fasting patient drinks a 50-gram dose of glucose.  A glucose result that is greater than 140 mg/dL is usually used as the cutoff although a lower cutoff of 130 mg/dL is also used (again, no consensus).  A woman that has an abnormal screening test (i.e. glucose concentration greater than the cutoff) will go on to have the diagnostic test.  In the 1-step approach the screening test is skipped completely and only the diagnostic test is performed.

    The test used to diagnose GDM is the oral glucose tolerance test (OGTT).  The OGTT requires women to be fasting and then drink either a 75- or a 100-gram dose of glucose.  Blood samples are collected every hour for 2 or 3 hours if using the 75- or 100-gram dose, respectively.  The test is considered positive, and GDM confirmed, if 2 or more of the glucose results are above designated cutoffs (which differ depending upon the glucose dose given).

    Now, new criteria have recently been advocated.

    The International Association of Diabetes in Pregnancy Study Groups (IADPSG) has made recommendations for glucose tolerance testing in pregnancy based on the results of the Hyperglycemia and Adverse Outcomes (HAPO) study.  That study clearly demonstrated that the risks of adverse maternal and fetal outcomes continually increase as maternal glucose concentrations increased.  Importantly, the relationship between glucose concentration and risks were continuous.  That is, there were no obvious glucose cutoffs above which risks increased.  The new recommendations from the IADPSG address this issue.

    The IADPSG advocates for the use of the 75-gram OGTT in pregnant women between 24 and 28 weeks gestation.  The test is performed following an overnight fast of at least 8 hours and blood is collected at 1 and 2 hours after the glucose load.  A diagnosis of GDM is made when any of the following glucose results are met:

    • Fasting: greater or equal to 92 mg/dL
    • 1 hour: greater or equal to 180 mg/dL
    • 2 hour: greater or equal to 153 mg/dL

    A couple of questions are called for here: 

    1. Why were those cutoff selected?  These are the glucose concentrations above which the adverse risks of hyperglycemia were 1.75-fold higher than for women whose glucose results were lower.  Other thresholds were considered but higher cutoffs missed lots of women with adverse pregnancy outcomes and lower cutoffs identified 25% of women as having GDM.
    2. What is the impact will this test have on the prevalence of GDM?   It will definitely increase.  Currently, about 7% of pregnant women are diagnosed with GDM in the US each year.  Using the IADPSG approach that will more than double to about 18% of pregnant women.

    Although the American Diabetes Association adopted the IAPDSG criteria and recommends that approach to identifying women with GDM, it does recognize that there is the potential for harm.  For example, more interventions such as earlier delivery and increased C-section rates are likely to occur due to the increase in the prevalence of GDM.  Also, an increased number of women being diagnosed with GDM will be accompanied by a rise in health care costs.  Despite those considerations, the ADA supports the new criteria in light of the increased rates of obesity and diabetes throughout the US and the world.

    False Negative Pregnancy Tests

    A false negative pregnancy test means that the test tells the patient they are not pregnant when they really are. Why does this occur? There are four well known reasons pregnancy tests can give false negative results.

    1) The most common reason is testing too early after fertilization occurs. The pregnancy hormone, hCG, is not produced until implantation occurs and it takes several days for the hCG concentrations to get high enough in blood and then urine to give a positive signal. Most devices give positive results around the day of a woman's expected period, but this can vary widely.

    2) Another reason for false negative results is dilute urine. If a woman drinks a lot of fluids, the concentration of hCG in the urine will be more dilute. For this reason many doctors recommend that testing be performed on the first urine of the morning because this tends to be the most concentrated (because you probably have not drank anything all night long).

    3) A very rare cause of false negative results occurs when very, very, high concentrations of hCG are present. This is called the high-dose hook effect. The hCG assays works by forming a so-called "sandwich" with two different antibodies as the "bread" and the hCG molecule as the "meat." The hook effect occurs when the hCG concentration is so high that it saturates both antibodies and there are so many molecules that the antibodies don't actually form a sandwich. This is rare because women don't normally produce enough hCG to saturate both antibodies. The hook effect should be of concern in a hospital setting, but most women should not be concerned about a hook effect with their urine. A hook effect can be confirmed if testing shows a positive result after sample dilution.

    4) Finally, the other reason for false negatives was only recently described and is referred to as the "variant hook effect." This is much more common than the hook effect. As pregnancy progresses, there are actually different variant forms of hCG that begin to appear in the urine. After about 5 weeks of pregnancy (i.e. 3 weeks after the expected period) concentrations of hCG beta core fragment are higher than all other forms of hCG. This is perfectly normal. Unfortunately, the concentration of hCG beta core fragment can saturate one of the antibodies used in the assay in certain pregnancy kits, and the other antibody doesn't recognize the beta core fragment. As a result, no sandwich forms and the test is read as a negative. The farther in pregnancy a woman is, the more likely that this false negative will occur. Similar to the hook effect, the variant hook effect can be confirmed if testing shows a positive result after diluting the sample.

    Posted by Ann M. Gronowski, PhD 5/8/11

    False-positive results from qualitative serum pregnancy tests

    OK.  I know I've written a lot about false-positive hCG tests already, both in February and earlier this month. Bear with me as I still have more to say on this issue.

    I spoke on the topic of hCG testing at a clinical lab science conference a couple of weeks ago and got this question from someone in the audience:

    "I was told that one way to investigate a possible false-positive, quantitative hCG result obtained from a serum sample was to test the sample using a qualitative hCG test because interfering antibodies don't affect those tests; is that true?"

    (If you are unfamiliar with the concept of interfering antibodies and want to learn more, read this.)

    My response to this question was "No, that is not true."  I'm uncertain why one would think that qualitative serum hCG tests were somehow immune to any influence from interfering antibodies but that wasn't the first time I had fielded that same question.  Clearly, this is not an isolated misconception.

    To support my response that qualitative tests can also produce erroneous results due to interfering antibodies I referred to a case report we described a short while ago.  You can find that report here.  The case describes a 46-year-old woman who was not sexually active yet whose blood sample produced a positive result using a qualitative serum pregnancy test.  Because the positive result was unexpected, her doctor asked us to measure the concentration of hCG in the same serum sample.  That result was less than 2 IU/L (normal is less than or equal to 5).

    When we repeated the qualitative test after treating the serum sample with a blocking agent that removes interfering antibodies, the result was interpreted as negative.  A urine sample obtained from the patient also produced a negative qualitative hCG test result.  The logical conclusion was that an interfering antibody produced a false-positive result with the qualitative test.

    I don't know how often this scenario might occur but it's clear that labs shouldn't rely on the notion that a qualitative hCG test is immune to any influence from interfering antibodies.