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.
In February, I wrote about interfering antibodies being the cause of false-positive hCG results from blood samples. As a follow-up, I thought it would be a good idea to talk about what the lab can do to investigate the potential problem.
As a reminder: false-positive hCG results are often attributed to the presence of an interfering antibody in the blood sample. Such was the case with Jennifer Rufer who was misdiagnosed with cancer due to a false-positive hCG blood test. It's important to know that laboratories are likely not able to independently identify when an interfering antibody is present. An interfering antibody should be suspected whenever the clinical picture of the patient fails to match the laboratory (in this case, hCG) results. When laboratorians are asked to investigate the possibility of a false-positive hCG result there are several ways to do so but the key piece of information to note is that the lab has to be notified that there is the suspicion of an erroneous hCG result. This is because the laboratory is nearly always unaware of the clinical picture of the patient. We need to rely on our clinical colleagues to alert us to the possibility that an hCG test result is potentially incorrect.
So, what can the lab do when asked if an hCG blood test result is falsely increased? There are several approaches the lab can take to investigate:
- Perform an hCG test on a urine sample obtained from the patient. Because hCG is excreted in the urine, the detection of hCG in a urine sample indicates that the result from the blood sample is likely accurate. Interfering antibodies aren't excreted into the urine and so if hCG is detected in the urine then the blood test result is probably really due to the presence of hCG.
- Perform a dilution of the blood sample. Because interfering antibodies are reactive against the hCG assay reagents, the expected response to sample dilution is typically not observed. That is, if the sample is diluted by a factor of 1:2, the hCG concentration should decrease by a factor of 2 if the hCG molecule is truly present. Failure to observe the expected decrease in concentration supports the presence of an interfering antibody.
- Repeat the hCG test using a different method. Interfering antibodies may be reactive against antibodies derived from a specific animal species (e.g. against mouse antibodies). If the hCG test is repeated using a different method and the results are considerably different then that can suggest the presence of an interfering antibody. Importantly, the alternative method selected should be one that uses antibodies derived from a different species of animal than the test in question.
- Treat the sample with blocking agents. Blocking agents are commercially available that can be used to remove (by adsorption) potentially interfering antibodies from the sample. If the hCG results after treatment is considerably different from the result of the untreated sample, then interfering antibodies may be present. The definition of considerably different is not well defined although many labs use a difference of 50% to alert them to the possibility that interfering antibodies are present.
I recommend that laboratories use more than one of these investigations rather than rely on a single one. As the Rufer case so clearly demonstrates, erroneous laboratory results can result in serious harm to patients. Together with our clinical colleagues, it our responsibility to do all that we can to assure that laboratory test results can be correctly interpreted.
Today I gave a lecture on hCG testing to 4th year medical students at the University of Utah. Part of that lecture included the tragic ordeal of Jennifer Rufer. In 2001, she was awarded nearly 16 million dollars because of a misdiagnosis of cancer from a false-positive pregnancy test. I previously wrote about false-positive pregnancy tests from urine specimens. This post deals with false-positive results from blood tests.
The same things that can cause false-positive results from urine specimens can also cause false-positive results from blood samples. It might help to review those first. There is another cause of false-positive hCG test results that is unique to blood samples: interfering antibodies.
First, a quick summary:
- Human blood normally contains many different types of antibodies: molecules made by our immune system that identify and neutralize invaders such as bacteria and viruses.
- Animals make antibodies, too, and their antibodies are used to make the components of hCG tests. For example, mouse antibodies that recognize human hCG molecules are used in tests that measure the concentration of hCG. One mouse antibody binds to one part of the hCG molecule (capture antibody) and a second mouse antibody binds to a different part of the hCG molecule. That second mouse antibody has a signaling molecule attached to it. We can measure the strength of that signal and that signal strength will be equal to the amount of hCG that it is bound to:
- Some people's immune systems make antibodies that react against animal antibodies. These "anti-animal" antibodies can cause false-positive hCG test results because they connect the capture antibody and the signaling antibody together even when hCG is not present, like this:
This was the cause of Jennifer Rufer's false-positive pregnancy test. Unfortunately for her, the false-positive result was recognized for what it was only after she had been treated for what her doctors thought was an aggressive type of cancer called choriocarcinoma.
It's uncertain how many people have interfering antibodies in their blood. Some estimate the frequency as high as 10-20%, other studies put that number much lower at <1%. It doesn't really matter how many people have them but what does matter is how doctors and laboratorians work together to identify this problem when it occurs. That's easier said than done.
Why? Because the laboratory that does the testing is nearly always unaware of the medical history of the patient so it's practically impossible for the lab to know if an hCG result is inconsistent with the patient's condition. The lab has to rely on the doctor to question an hCG test result when it don't fit what they are seeing in their patient. If the hCG result doesn't fit the clinical picture and the doctor calls the lab for help, there are investigations the lab can do to help identify if an interfering antibody is present or not. But without that call questioning the result, the lab doesn't have reliable mechanisms for identifying all cases of interfering antibodies.
In an earlier post, I wrote about the different types of tests used to detect hCG. While the results of These test are usually very accurate, false positive results can and do occur. Here I discuss some reasons why a urine pregnancy test might give a false positive result.
First, though, a few comments about what a false positive result actually is. Pregnancy tests are designed to detect hCG;the hormone produced by the placenta during pregnancy. If the hormone is truly present in the urine and the test detects it, then that result is a true positive result. In other words: hCG present = true positive test result. However, because hCG is usually only produced during pregnancy,many people consider a positive result to mean that a woman is pregnant. There's a big difference between those two meanings! To summarize:
- Pregnancy tests detect hCG
- hCG is usually (but not always) produced during pregnancy
- A positive pregnancy test indicates that hCG is present (even if the hCG is being made by something other than the placenta)
With that in mind, let's explore the causes of false positive urine pregnancy test results.
- Missed reaction time: The result of a pregnancy tests should be read when the manufacturer instructions say to read it. If the result is read after that time, a faint line is sometimes visible (possibly due to evaporation of the urine) that might be interpreted as a positive result. This would truly be a false positive result because hCG is not present.
- Biochemical pregnancy: A biochemical pregnancy is a real pregnancy but is one that ends very early after conception. If a pregnancy test is performed near the time of the expected period (or even several days later), a positive result can be obtained. This is actually not a false positive result because hCG is present and it was detected by a test designed to detect it! Even though the pregnancy has ended, hCG can still be detected in the urine for a few, or even several, more days. Biochemical pregnancies are quite common although many aren't even detected because the miscarriage occurs before the woman even knew she was pregnant and so had no reason to perform a pregnancy test. Indeed, the term "biochemical pregnancy" came into use only after urine pregnancy tests became sensitive enough to detect hCG close to the day of the expected period. Prior to that, the tests were not able to detect that rather low amounts of hCG that are associated with a biochemical pregnancy.
- hCG from sources other than the placenta: Although hCG is most commonly made only during pregnancy, there are other times that it is produced. Certain cancers sometimes make hCG; most notably a family of tumors collectively called trophoblastic tumors. The pituitary gland in the brain can also produce hCG although this is more common in women who have gone through menopause. Finally, people taking hCG as a weight-loss aid (a waste of money, by the way) or as part of fertility treatments can also have detectable hCG in their urine.