The Pregnancy Lab

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):

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!