Category Archives: False Result

False Result

Conventional aneuploidy screening remains “most appropriate” choice for general population


OpinionThe American Congress of Obstetricians and Gynecologists (ACOG) have updated their guidance on cell-free DNA (cfDNA) screening tests for fetal aneuploidy. In it, they state that any patient (i.e. women at high-risk OR low-risk for having an affected pregnancy) may choose cfDNA testing but they caution that conventional screening tests are more appropriate. This document replaces an earlier opinion, published in 2012, which clearly stated that cfDNA screening tests should not be offered to the general obstetrical population because they are considered to be at low-risk.

So ACOG went from recommending that cfDNA testing not be performed on low-risk women to say that they may choose cfDNA testing. Why the subtle change? Well, as ACOG correctly notes, the landscape of cfDNA is changing rapidly. New studies are published frequently and those that have examined the performance of cfDNA tests in  low-risk women have reported that the test performs just as well in them as it does in high-risk women.

However, they make an important point about a metric that doesn't get the attention it deserves. The positive predictive value (PPV). See here for background. Because the prevalence of fetal aneuploidy in low-risk women is lower than it is in high-risk women, a "positive" or "abnormal" test result in low-risk women is more likely to be a false-positive result. For example, a positive result in a 25-year-old woman gives a 33% chance that the fetus is affected but that chance increases to 87% in a high-risk woman.

The report also calls out the "no result" problem. cfDNA tests fail to produce a result in 1-8% of samples tested, usually due to a low amount of fetal DNA in the blood sample. It's becoming clear that women with samples that fail to produce a result are at increased risk of having an affected fetus. According to ACOG, these women she be offered diagnostic testing such as fetal karyotyping using amniotic fluid obtained by amniocentesis.

Other notable points contained within the updated guidance include:

  • Caution about not routinely performing microdeletion screening (offered by some labs) because it has not been fully validated in clinical studies.
  • Clearly indicating that a negative or normal result does not rule out the possibility of an affected fetus.
  • Providing genetic counseling to patients about test limitations and that decisions such as pregnancy termination should not be based on these screening tests.
  • A reminder that cfDNA tests do not screen for neural tube or ventral wall defects

This certainly won't be the final say that ACOG has on cfDNA aneuploidy screening tests. Indeed, they state that "It will be critical to remain abreast of this rapidly changing technology to provide patients with the most effective, accurate, and cost-conscious methods for aneuploidy screening."

False negative pregnancy tests: the story continues


This post was written by Robert D. Nerenz, PhD, an assistant professor at the University of Kentucky, in Lexington.

Neg pregnancy testIn previous posts, we have discussed false negative pregnancy test results caused by hCG beta core fragment (hCGβcf), the predominant form of hCG found in urine after six weeks of pregnancy. False negative pregnancy tests can result in undesirable outcomes (including loss of pregnancy) if inappropriate treatment is given. In a recent study evaluating the performance of devices used in a hospital setting, 9 of 11 devices were found to be susceptible to false negative results when used to test urine solutions containing hCGβcf concentrations observed in normal pregnancy.

After our study was published, we were frequently asked whether these devices actually performed poorly in clinical practice or if the results we observed only occurred in a controlled laboratory environment. To address this question, we searched the database of medical device malfunctions reported to the FDA (Manufacturer and User Facility Device Experience, or MAUDE) to find reports of false negative pregnancy test results in a clinical setting. Using the search term “MAUDE pregnancy false negative” we found 707 reports between 2000 and 2014 that described false negative urine hCG results in women shown to be pregnant at the time of testing. 91 different POC hCG devices were described from 14 manufacturers, including 10 of the 11 devices evaluated in our initial screening study.

Based on the description in the MAUDE report, we subdivided the false negatives by potential cause. In 433 reports the cause of the false negative result was unknown.  132 were likely because the hCG concentration was so low that it was below the limit of detection for the test device. Of these 132, nine were associated with adverse events. We also found 142 reports that were likely due to hCGbcf hook effect and ten (or 7%) of those were associated with adverse events, including delayed prenatal care, delayed treatment of ectopic pregnancy, performance of inappropriate imaging studies and even surgery leading to loss of pregnancy. In addition to the reports documented on the FDA website, it is virtually certain that many more false negative results have occurred and have gone unreported to the FDA.

The fact that false negative pregnancy test results occur relatively frequently in clinical practice was an important finding because it highlighted the limitations of currently available devices and emphasized that this problem is not limited to one or two devices. Rather, the fact that the vast majority of pregnancy test devices performed poorly in our study and were reported to generate false negative results in clinical practice indicates that this is a much larger problem.

To decrease the occurrence of false negative pregnancy test results, contributions from multiple different groups will be required. First, the FDA should insist that device manufacturers market devices that generate positive results in all pregnant women, including those with high urine concentrations of hCGβcf. Second, clinicians at large hospitals should request that pregnancy testing be performed on serum using a quantitative assay, especially in patients with abdominal pain, vaginal bleeding or other symptoms that strongly suggest the patient might be pregnant. Quantitative serum assays can generate results in less than an hour, can detect lower concentrations of hCG than point-of-care test cartridges and are not affected by hCGβcf because hCGβcf is not present in serum. Lastly, laboratorians should work to decrease the time required to generate test results in order to make quantitative testing more appealing to clinicians. At institutions where urine point-of-care testing must be performed, laboratorians should evaluate all available options and select the device that provides an optimal combination of sensitivity and lack of susceptibility to interference caused by elevated concentrations of hCGβcf.

Unfortunately, currently available pregnancy test devices present a risk to patients. It is our hope that a coordinated effort from the FDA, manufacturers, clinicians and laboratorians will eliminate that risk.

Confusion over NIPT invites catastrophe


Timing is everything. A week after I wrote about false-positive NIPT results, the Boston Globe published an article titled "Oversold prenatal tests spur some to choose abortions" written by Beth Daley of the New England Center for Investigative Reporting. The article describes non-invasive prenatal testing (NIPT) using relatively new cell free DNA tests with a focus on women who have experienced receiving incorrect results.

The article focuses on one woman who had Sequenom's MaterniT21 PLUS test that indicated her fetus had trisomy 18 or Edwards syndrome. She initially considered immediately terminating the pregnancy and her doctor helped her locate a physician who could perform the procedure the next day. Only hours later did her doctor caution her to consider diagnostic testing which confirmed the fetus did not have trisomy 18. Additional cases in the article tell of one woman who experienced a false-positive result (confirmed by diagnostic testing) but so trusted the results of the DNA test that she aborted her pregnancy anyway and a woman who experience the trauma of a false-negative result.

Stories like this indicate a clear lack of understanding regarding the limitations of NIPT and demonstrate that physicians and consumers don't always appreciate the fact that these are screening tests. In a post on its blog about the Globe article, the Society for Maternal Fetal Medicine emphasizes just that by stating "It is important for providers to remember that cell free DNA is a screening test, and does not have the diagnostic accuracy of amniocentesis." They also point out that doctors who order DNA-based screening tests need to understand the test characteristics and they emphasize the role of genetic counseling for women who undergo screening for aneuploidy. The Society's statement was the focus of a follow-up piece by the New England Center for Investigative Reporting.

Whether aneuploidy screening is performed using DNA-based tests or by traditional biochemical screening, it is a screening test. Neither are diagnostic tests. Abnormal results from any screening test must be followed up by diagnostic testing to confirm (or not) the results of the screening test. To be misinformed on this basic fact of laboratory medicine is to flirt with disaster.

Improved Qualitative Pregnancy Devices


In the past, we have blogged about false negative urine qualitative hCG tests in both point-of-care (POC) hospital devices and over-the-counter (OTC) devices due to the presence of high concentrations of hCGbcf. We feel this represents a real problem for patients and clinicians trying to diagnose pregnancy and could results in harm to mother and/or fetus.

It is our understanding that the FDA is requiring device manufacturers to address this problem in any new devices going through the FDA approval process. However, it is up to manufacturers if they want to voluntarily change their existing devices. Hence we have urged manufacturers to modify their devices to eliminate false negatives due to hCGbcf.

Recently, we were made aware of two manufacturers that had apparently modified their qualitative pregnancy devices: The Cen-Med Elite Plus One-Step Pregnancy Test (a hospital POC device) and the First Response Early Result OTC device (an over-the-counter device). In order to evaluate these modifications we compared the old and new devices using the screening test we have developed previously.   Our results demonstrated that indeed, the new version of each device perform better than the previous version.  Both original devices demonstrated significantly diminished signal when 500 pmol/L hCG was tested in the presence of 500,000 pmol/L hCGbcf. However the modified devices gave faint or clear positive signals in the presence of the same hCG concentrations (see figure). Figure for Blog
It is clear that improvement of qualitative urine hCG devices is possible and we encourage all manufacturers to design devices that are not inhibited by hCGbcf. 

False Negative Pregnancy Tests Still a Real Problem in Home and Hospital Devices


Neg pregnancy testWe have blogged in the past about false negative pregnancy tests due to hCG beta core fragment (hCGbcf).   After about 5 weeks of pregnancy (i.e. 3 weeks after the expected period) concentrations of hCGbcf, in urine, are higher than all other forms of hCG. Our group has shown previously that the concentration of hCGbcf can saturate one of the antibodies used in the point-of-care hospital pregnancy kits. As a result, test shows a negative result. The variant hook effect can be confirmed if testing shows a positive result after diluting the sample. This phenomenon is referred to as the "variant hook effect" and was reported to the FDA in 2009.

Recently, our group took this observation one step further and examined over-the-counter home pregnancy devices to see if they were subject to the same problem.  We examined six over the counter devices and selected two that seemed to be most affected by the variant hook effect. We then compared those two devices to the hospital device that we had made our original observations in four years ago, and to a hospital device that we thought performs best when compared to various other hospital pregnancy devices. Not surprisingly, we found that the over-the-counter home pregnancy devices are also subject to the variant hook effect. However, what was a surprise was that the hospital pregnancy devices were more affected by hCG beta core fragment than the home pregnancy devices!  Furthermore, despite the fact that the variant hook effect was reported to the FDA in 2009, manufacturers have not changed their devices to avoid this problem. To hear more about this paper you can listen to a podcast describing the findings.

Our laboratory is currently working to better define how much hCGbcf is required to cause the variant hook effect. We hope that this will help manufacturers to produce devices that avoid false negative results. In the meantime, several things need to be done:

  1. Physicians, nurses, and other health care professionals need to be educated about this problem-especially in the hospital setting.
  2. The variant hook effect should be made clearly visible in pregnancy test package inserts and they need to state that when a false negative is suspected, a simple dilution can yield a positive result if the patient is truly pregnant. This is very important for centers that have no alternative way of testing for pregnancy.
  3. Finally, in my opinion, quantitative serum hCG testing should be the preferred pregnancy test in centers where it is available. Serum testing is not subject to the variant hook effect because hCGbcf is not present in serum. Furthermore, quantitative serum assays are much more sensitive than the qualitative assays.

Wanted! A sensitive qualitative hCG test.


Today’s post is by a guest author, Dina N. Greene, Ph.D. Dr. Greene is a Scientific Director at Northern California Kaiser Permanente Regional Laboratories in Berkeley, CA. She discovered that qualitative hCG tests may not be as analytically sensitive as we all have come to believe and she shares her observations here. A report of her work has been published in Clinica Chimica Acta.
Neg pregnancy test
The assessment of very early pregnancy (from conception until about two weeks following the
expected menses) is dependent on the detection of hCG in serum or urine. In health care settings a urine sample is often the specimen of choice because it is convenient and usually easy to obtain.

When urine samples are tested for hCG they are most frequently tested using qualitative (yes/no) point-of-care (POC) devices. This type of testing is attractive because it is performed close to the patient and the test results can be obtained within minutes. In general, when challenged with urine or serum containing hCG these devices work well. However, what was not known was how sensitive these devices are for detecting very early pregnancy. That is, could pregnancy be ruled out if a qualitative POC test was negative?

To answer that question we completed a study that took a systematic approach to this question by testing urine and serum specimens collected from patients that spanned a wide range of hCG concentrations with two commonly used POC devices.

While many concentrations of hCG were represented in these samples, we purposefully skewed the specimens so that a large percentage (~30%) had concentrations of hCG expected to be seen only in very early pregnancy. The results were surprising.

We found was that the devices did not always detect hCG at the lowest detectable concentration claimed by the manufacturer (20 IU/L for urine and 10 IU/L for serum). In fact, we had many false-negative results when the urine concentration of hCG was as high as 200 IU/L or the serum hCG concentration was as high as 50 IU/L. We further showed that the urine specimens were collected from patients that were at approximately 4 weeks’ of gestation which, if calculated from the day of the last menstrual period, is close to the day of expected menses.

Anecdotally, medical providers at some institutions have recognized this phenomenon. If a sexually active woman is unsure of her pregnancy status, and the POC urine hCG test result is negative, the provider may encourage the patient to return for retesting in a few days. Alternatively, if the patient’s pregnancy status must be known urgently, the provider may collect a blood sample for quantitative serum hCG testing performed in the laboratory to confirm the negative POC test result.

Interestingly, the package insert of one qualitative hCG POC device used in our study states “If a negative result is obtained, but pregnancy is suspected, another sample should be collected and tested 48-72 hours following.” Most other hCG POC devices provide a similar disclaimer. Although it is empirically recognized that false-negative results are possible in early pregnancy, most individuals (health care professionals and consumers alike) assume that this corresponds to the period of gestation that precedes hCG production. What our study showed is that hCG is present in the urine and serum of these women, but the concentration is too low for the POC devices to always detect reliably.

False-Positive Results in Point-of-Care Ovulation Prediction Devices Due to Very Low Concentrations of Human Chorionic Gonadotropin


Point of care devices which detect luteinizing hormone (LH) are used to predict ovulation and time intercourse in women who are trying to get pregnant. Women attending fertility clinics also commonly use these devices to time intrauterine insemination.  Although the hormones LH and hCG share 80% structural homology, cross reactivity in quantitative (laboratory) LH and hCG assays has not been a problem for many years due to the use of very specific antibody pairs. Many physicians and laboratorians assume that that specificity holds true for qualitative (home) devices as well.

Recently, a women undergoing fertility treatment at our institution detected a positive LH surge using an over the counter LH device despite the fact that she was later found to be pregnant. This made us ask the question "Could the over-the-counter ovulation kits cross react with hCG?"  Therefore, we undertook a study where we added purified hCG to saline and tested three home ovulation prediction devices [Clear Blue® (Swiss Precision Diagnostics, Geneva, Switzerland), First Response® (Church & Dwight, Princeton, NJ), and Walgreens® (Inverness Medical (now Alere), Waltham, MA)]. We found that all the devices we tested returned false positive results at hCG concentrations ranging from 10 to 10,000 mIU/mL.

The concentration of hCG at which devices were positive varied by brand. Both Clear Blue and Walgreens were clearly positive at hCG concentrations of 100 mIU/mL! Clear Blue was positive at an hCG concentration of 10 IU/L! Walgreens turned positive between 50 and 100 mIU/mL, and First Response turned positive between 5,000 and 10,000 mIU/mL. The LH concentration that caused positive results also varied by device brand (Table). Only Clear Blue was definitively positive at 50 mIU/mL of LH. First Response and Walgreens turned positive between 50 and 100 mIU/mL of LH.

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These devices may produce false positive results in women who are very early in pregnancy. At our institution, the reference interval for hCG in the first three to four weeks of pregnancy is 9 to 130 mIU/mL. Package inserts for some devices contain a cautionary statement that results obtained during pregnancy or administration of certain drugs including hCG may produce misleading results; however, it is clear that most physicians and laboratorians are unaware of the any potential cross-reactivity. This data is being presented at the American Association of Clinical Chemistry (AACC) meeting (Wednesday July 31, 2013, Poster B50) in Houston, and will be published in full later this year in Clinical Biochemistry doi.org/10.1016/j.clinbiochem.2013.07.017.

Fertility clinics and physicians that rely on home LH devices to detect an LH surge for the timing of intrauterine insemination should be aware that early pregnancy may cause false positive results on LH devices. Fertility clinics in particular should instruct their patients to use home LH devices with minimal hCG cross-reactivity.

DNA-based tests for Down syndrome screening show excellent clinical performance


The use of biochemical screening tests to identify pregnant women who are at high risk of having a fetus with Down syndrome is well established.  Biochemical screening began nearly 30 years ago and, over the years, the tests have evolved and improved.  Now there’s a new kid on the screening test block and it’s name is DNA.

The discovery of cell-free fetal DNA in maternal plasma in 1997 opened up new possibilities for Down syndrome and other aneuploidy screening protocols.  Rather than rely on biochemical testing to determine a biochemical phenotype, DNA-based tests have been developed that can detect the molecular pathology of aneuploidies (e.g. a fetus that has more than the expected 2 copies of chromosomes 21, 18, or 13; the cause of Down syndrome, Edwards syndrome, and Patau syndrome, respectively).

We’ve written about DNA-based screening tests before (here and here) and described the clinical performance of the Sequenom test.  Now, other clinical performance studies have been published for 3 of the 4 tests that are (or will be) commercially available.  As expected, all of them show excellent clinical performance.  As shown in the table below, the detection rates for trisomy 21 are greater than or equal to 99% with very low false-positive results.  Similar performance has been reported for trisomy 18 and 13.

DNA test performance

Table References: Genet Med 2011;13:913-920Genet Med 2012;14:296-305Obstet Gynecol 2012;119:890-901

By comparison, the detection rate of the best biochemical Down syndrome screening test (the Integrated test) is very good at 93%.  However, about 5% of all Integrated test results are false-positive.  A 5% false-positive rate may not seem very high but it is.  For example, consider a population of 100,000 pregnant women who choose Integrated testing in the second trimester.  The prevalence of Down syndrome in the second trimester is about 1 in 500 pregnancies so 200 of those 100,000 women will have a fetus with Down syndrome and 99,800 women (100,000 – 200) will have unaffected fetuses.  Of those 99,800 women with unaffected fetuses, 4,900 will have a false-positive Integrated test result.

Because the false-positive rate of the DNA-based tests is so low (about <0.2%), then if those same 100,000 women were screened there would be only 200 false-positive results, a 96% decrease!

Does this mean that DNA-based tests should replace biochemical screening tests?  Probably not but I’ll leave the explanation as to why for my next post.

Four reasons for a positive hCG test in the absence of pregnancy


“Why is the pregnancy test positive if she’s not pregnant?”

This is a question I’ve been asked several times and it’s a good one.  The query usually comes from a nurse, doctor, or other healthcare provider after performing a test for human chorionic gonadotropin (hCG) and getting a positive or elevated result that they did not anticipate.

Because hCG is a hormone normally produced during pregnancy, hCG tests are used to diagnose the pregnant patient.  That makes it easy to think of hCG tests as “pregnancy tests.”  While that’s not an inaccurate label for them, identifying hCG tests as pregnancy tests gives the impression that is all they are supposed to do.  Technically, hCG tests are designed to qualitatively detect and/or measure the hormone in urine or blood and there are other reasons besides being pregnant that can cause hCG to be present.

I can think of four different reasons why hCG could be present in a non-pregnant woman.

  1. Biochemical pregnancy.  A biochemical pregnancy occurs when a woman becomes pregnant yet has a spontaneous loss of the fetus before she even knew she was pregnant.  If hCG testing occurs before all of the hCG has been metabolized out of the body then hCG can be detected by a lab test.  This situation is not as uncommon as one might think for two reasons.  First, hCG tests are frequently performed in healthcare settings in order to identify the pregnant patient in order to avoid any medical interventions that are potentially harmful to a fetus.  Second, hCG tests are capable of detecting very low concentrations of the hormone.  The high frequency of testing combined with the analytical sensitivity of the tests means that biochemical pregnancies are easily detected.
  2. Pituitary hCG.  Although the placenta normally produces hCG during pregnancy, it can be made by the pituitary gland.  The pituitary gland is a small structure in the brain that secretes many different hormones that function to regulate many endocrine organ systems.  Interestingly, three hormones normally produced by the pituitary gland (thyroid stimulating hormone, follicle stimulating hormone, and luteinizing hormone) are structurally similar to hCG.  Pituitary hCG is more commonly detected in women greater than 55 years of age but can be detected in women as young as 41 years.  Non-pregnant women with pituitary hCG usually have low concentrations of hCG present in the blood and urine.  Importantly, concentrations of hCG produced by the pituitary gland don’t show the rapid increases that occur during pregnancy.
  3. Malignancy.  Cancer cells sometimes make hCG.  While many different types of cancer have been shown to make the hormone, it’s most commonly associated with the gestational trophoblastic diseases and certain types of germ cell tumors of the testes.  Because testicular tumors occur only in men, the question of detecting hCG in the absence of pregnancy is clearly not relevant.
  4. Interfering antibodies.  Some women have antibodies in their blood that can interfere with hCG tests and cause a positive or elevated result in the absence of hCG.  Only hCG tests performed on blood can be affected by this problem because the interfering antibody molecules aren’t normally present in the urine.  This can be a serious problem because some women have been mistakenly diagnosed with cancer due to the false-positive hCG test result and have undergone unnecessary treatments for it.  The frequency of this problem is difficult to know but it’s probably very low.  Over the last several years, the manufacturers of hCG tests have worked to minimize possible interference from these antibodies but nothing can be done to completely eliminate the problem.  When alerted, the laboratory can help to determine if an hCG test result is falsely positive due to this issue.

So, just because an hCG test result is interpreted as positive doesn’t automatically mean that a woman is pregnant.  There are very valid reasons for detecting hCG in the absence of pregnancy.  That said, when the hCG test result doesn’t match the clinical picture, the laboratory should still be asked that question!  When alerted to the discrepancy, the lab can help to investigate the problem and perhaps shed some light on the cause.

There is quite a bit more to say on each of those four causes but I’ll save those comments for future posts.

 

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