Category Archives: hCG Variants

hCG Variants

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.

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. 

Examination of eleven hospital hCG devices for false negative results


Beckman Icon 20In previous blog posts we have discussed false negative urine hCG tests due to high concentrations of hCG beta core fragment (hCGβcf), the predominant form of hCG found in urine after six weeks of pregnancy. High concentrations of hCGβcf saturate either one or both of the antibodies used in the test. This hCG variant effect prevents the development of a positive signal and generates a negative result despite the presence of intact hCG in the urine sample being tested.

The original studies were performed by adding increasing concentrations of purified hCGβcf to a urine sample obtained from a pregnant woman. The problem with this approach is that the relative abundance of hCG, hCGβcf and other hCG variants in the urine that may affect device performance vary between women. A standardized method, using defined concentrations of hCG, is needed to evaluate the performance of currently available devices.

In a recent publication, we describe a screening method that can be easily used to examine the effect of hCGβcf and have used this method to test eleven POC hospital urine hCG devices.

First, a wide range of purified hCG and hCGβcf concentrations were combined to prepare 2 solutions:

  • Solution A: 500 pmol/L (171 IU/L) intact hCG with 0 pmol/L hCGβcf and
  • Solution B: 500 pmol/L intact hCG with 500,000 pmol/L hCGβcf

These solutions were tested on two hCG devices, the results of which helped to define a screening method:  

Each device is tested with the two solutions and the intensity of the test bands are compared. If Solution B shows a lighter test band than solution A, then the device is susceptible to false negatives with hCGβcf.

Using these two solutions we compared the performance of 11 hospital urine hCG devices

  • SP hCG Combo Rapid Test, Cardinal Health
  • OSOM hCG Combo Test, Genzyme Diagnostics
  • hCG Combo, Alere
  • ICON 20 hCG, Beckman Coulter
  • ICON 25 hCG, Beckman Coulter
  • Elite Plus hCG, Cen-Med
  • Clinitest hCG Pregnancy Test, Siemens
  • hCG Urine Test, McKesson
  • QuickVue+ One-Step hCG Combo Test, Quidel Corporation
  • QuPID One-Step Pregnancy Test, Stanbio Laboratory
  • Sure-Vue Serum/Urine hCG-Stat, Fisher HealthCare

Interestingly, we found that only 2 devices were acceptable (i.e. not affected by the hCG variant effect). These were the Beckman Icon 20 (shown above) and the Alere hCG Combo devices. By contrast, the Genzyme OSOM and Cen-Med Elite Plus hCG devices were the most susceptible to false negative results due to hCGβcf (OSOM shown below). The remaining seven were moderately affected.

Osom

The paper also demonstrated that devices that gave the strongest signal with hCGβcf alone were those that were least likely to show a false negative effect.

The screening method can be used by device users and manufacturers to evaluate hCG devices for inhibition by hCGβcf. We hope that the results of this study will help healthcare providers make informed decisions about which hCG devices to select, especially in medical centers that are unable to perform rapid, quantitative measurements of hCG in serum.

However, while the availability of hCG test devices that are not affected by hCGβcf is certainly reassuring, quantitative serum measurement of hCG should still be the test of choice when available.

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.

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

The many faces of hCG


One of the things that I find interesting about hCG is that there is more to it than meets the eye.  We often talk about hCG as though it's a single molecule when, in fact, there are lots of different variants of hCG.  Here's a quick summary of what those different molecules are all about.

  1. Intact hCG: This is the variant that gets the job done.  That is, it's the biologically active form of the hormone that is made by the placenta.  It's job is to keep the blood progesterone concentrations high which is critical for maintaining pregnancy.  It's made up of two different protein subunits simply called alpha and beta.  The alpha subunit isn't unique to hCG because it's also part of three other completely different hormones: thyroid stimulating hormone, follicle stimulating hormone, and luteinizing hormone.  The beta subunit is unique to just hCG (the other three hormones also have their own unique beta subunit).  A complete, intact hCG molecule is made when the alpha and beta subunits are attached to each other.  During pregnancy, most of the hCG in the blood is intact hCG.
  2. Nicked hCG (hCGn): Nicked hCG is made when a chemical bond in part of the beta subunit is broken.  When that happens, the hormone loses is biological activity and the alpha and beta subunits will often come apart.  During pregnancy, nicked hCG makes up about 10% of the total hCG in the blood.
  3. Free Alpha Subunit of hCG (hCGα): It goes without saying that this is the alpha subunit all by itself.  It has no biological activity.  That is, it doesn't work and can't keep blood progesterone levels high.
  4. Free Beta Subunit of hCG (hCGβ): This variant of hCG is the free beta subunit without the alpha subunit and it also lacks biological activity.  During pregnancy, the free beta subunit makes up about 1% of all the hCG in the blood.  Also, this variant is sometimes made by certain types of cancers.
  5. Nicked Free Beta Subunit of hCG (hCGβn): This is similar to hCGn (#2 above) yet there is no alpha subunit attached to it and so it, too, has no activity.
  6. Beta Core Fragment of hCG (hCGβcf): This is the final breakdown product of hCG.  It is a small molecule that is made up of the center (core) part of the hCG beta subunit.  Like the other non-intact hCG variants, the beta core fragment is inactive.  However, unlike all of the other hCG forms that are present in both the blood and the urine of pregnant women, the beta core fragment is only present in the urine.  In fact, the urine of pregnant women contains more beta core fragment than any of the other hCG variants.
  7. Hyperglycosylated hCG (hCG-h): Intact hCG normally has carbohydrate (sugar) molecules attached to it.  Hyperglycosylated hCG is like intact hCG except it has even more carbohydrate molecules attached.  This hCG variant is the predominant form of hCG produced during the first few weeks of pregnancy but it is nearly undetectable by the end of the first trimester.

 Here is a figure of the forms of hCG that contain all or part of the beta subunit (adapted from Clin Chem 1997;43:2233-2243):

HCG_ASCP 2011
All of these forms of hCG (and probably others that we don't even know about) are produced during pregnancy and can be detected in the blood and urine of pregnant women (hCGβcf is only in the urine).  Future posts will discuss how these different hCG variants make detecting and measuring hCG a bit of a challenge.  Things are not always as straightforward as they seem!