Flaw in many home pregnancy tests can return false negative results

But a degraded form of the hormone also can be found in the urine, and in some devices the first antibody will bind to the degraded form. The amount of the degraded form, called hCG core fragment, goes up as pregnancy progresses. The more of the fragmented hormone that is around, the more likely the first antibody will accidentally capture the fragment instead of the intact hormone. However, the signal antibody does not respond to the fragment so it does not change color when that happens, and therefore you get a negative result even though the hormone might be present.

Why Did a Second Line Appear Later on My Pregnancy Test?

A delayed second line on a pregnancy test might mean a false positive. Here’s what you need to know.

Updated on April 5, 2024
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Understanding how pregnancy tests work is key to interpreting the results. When you take a home pregnancy test, you look at the results window to see whether there are two lines or just one. The first line is the control line. If that’s all you see, your result is negative. But two lines in the testing window mean that you’re pregnant.

Pregnancy test results are only valid for 10 minutes. Sometimes, however, you may get a negative result, then look at the pregnancy test later in the day to see that a positive line has magically appeared.

There are a few reasons why this can happen. However, in most cases, the original reading of the test (“not pregnant”) is typically the correct one. Here’s what you need to know about a delayed second line on a pregnancy test.

How Do Pregnancy Tests Work?

Home pregnancy tests measure a hormone called human chorionic gonadotropin (hCG) in your urine. Your body starts producing hCG after a fertilized egg implants itself in your uterus, about six to ten days after ovulation. Your body only produces hCG during pregnancy, so even the faintest suggestion of a second line indicates that you have likely conceived.

To take a pregnancy test, direct your urine stream over the stick or collect your urine in a sterile container and dip the stick into it for about 20 seconds. Check the test again in about three minutes to see whether one or two lines appear. Note that different tests have different instructions, so you should always follow the manufacturer’s instructions for your specific test.

Michela Buttignol | Getty Images

Interpreting Pregnancy Test Results

You can say for sure that you’re expecting if you see a positive result on your pregnancy test within 3 to 10 minutes. Remember that it doesn’t matter how faint that line is, because your body only produces the pregnancy hormone hCG while you’re pregnant.

Sometimes you’ll see a faint line if you test on the earlier side and the hCG is still building up in your system. A line that gets progressively fainter over days of testing may indicate that you’re miscarrying, so reach out to your provider if this happens.

A positive pregnancy test can almost always be considered reliable. However, this may not be true if you’re taking certain fertility drugs. Medications used to trigger ovulation may contain hCG. Until they clear from your system, you could get a positive result when you’re not pregnant.

What Happens If a Positive Line Appears Later?

The instructions on most pregnancy tests will advise you to read the results within a certain window of time. This usually spans up to 10 minutes. If you see a positive result beyond this time frame, you may be left second-guessing the results.

However, always remember that a pregnancy test result is only accurate within 10 minutes. After that, it’s best to throw the test away to avoid confusion.

Am I Seeing Evaporation Lines?

If urine splashes onto the pregnancy test window, it can dry and leave streaks. If one of these streaks appears around the spot where a positive result would appear, it can look like a second line.

The easiest way to differentiate between a positive test result and evaporation line is to check your test within the allotted time. A true positive will appear within two to 10 minutes, while urine won’t dry and form streaks until some time later.

What If My Positive Line Is Faint?

If you follow all of the instructions correctly, you may notice a thin, faint pregnancy test line that appears within a few minutes. There are several possible explanations for this line’s faintness.

• You may have taken the test too early in your cycle. If you test earlier than 10 to 12 days past ovulation, the hCG hormone may not be high enough to produce more than a very faint line. The best time to test is after your period is late, or 2 weeks after you ovulated.
• Your urine might be diluted, skewing the test’s results. You can dilute your urine sample if you’re drinking too many liquids; this is why experts recommend testing first thing in the morning.
• Some pregnancy tests have lower sensitivity levels, so they’ll show fainter lines earlier on. Although it’s not necessary, you can try retesting with a different type of test if you want to see a darker line. Look for “early results” indicated on the test label. Be sure to also check the pregnancy test expiration date.
• If your positive result is appearing lighter each day, this could mean that you are having an early miscarriage.

Remember that many factors can impact how light or dark your positive line is, such as using different test brands, drinking different amounts of fluids, or testing at different times of the day. Reach out to your health care provider if you have any concerns about your pregnancy.

False Negative Pregnancy Test Results

While it’s nearly impossible to get a false positive result, false negatives can certainly happen. Even if you’re pregnant, your hCG levels may sometimes be too low for detection in your urine. This is more common if you test before your period is due, but it can also happen right around the time of your missed period.

Many of the same factors that cause a faint line can also cause a false negative, such as diluted urine or lower sensitivity tests. If you still haven’t gotten your period, wait a few days and test again. HCG builds rapidly in your system, so you may see a positive soon after seeing a false negative.

Always use a sample from your first-morning urine (which is more likely to have higher concentrations of hCG.

How to Handle a Positive Pregnancy Test

If you get a positive pregnancy test, contact your health care provider or search for an OB-GYN in your area. They’ll likely ask you to come in sometime around 7 weeks past the first day of your last menstrual period. At this visit, your provider will conduct an ultrasound to confirm the pregnancy.

If your test is negative, wait for your period to come. If you want to get pregnant, you can start trying again shortly thereafter.

If you take a pregnancy test and you’re not sure what it’s telling you, you can always test again the next day, or see a doctor for a pregnancy blood test. Sometimes this is the best way to get rid of any stress surrounding a confusing test result.

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Parents uses only high-quality sources, including peer-reviewed studies, to support the facts within our articles. Read our editorial process to learn more about how we fact-check and keep our content accurate, reliable, and trustworthy.

1. Doing a Pregnancy Test. National Health Service, 2024
2. Strips of Hope: Accuracy of Home Pregnancy Tests and New Developments. Geburtshilfe Frauenheilkd, 2014
3. Improved Pregnancy Rate with Administration of HCG after Intrauterine Insemination: A Pilot Study.” Reproductive Biology and Endocrinology, 2010
4. What to Do About an Evaporation Line on a Pregnancy Test. Medical News Today, 2022
5. Guidance for over-the-counter (OTC) human chorionic gonadotropin (hCG) 510(k)s. U.S. Food and Drug Administration, 2024

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Flaw in many home pregnancy tests can return false negative results

Pregnancy tests can sometimes give a false negative result to women several weeks into their pregnancies, according to research by Ann Gronowski, PhD, a professor of pathology and immunology at Washington University School of Medicine in St. Louis. Her findings led the FDA to change its standards for evaluating new pregnancy tests, but old tests with the false-negative problem are still on the market.

Each year, women in the U.S. rely on some 20 million home pregnancy tests to learn potentially life-altering news. Despite marketing claims that such tests are 99 percent accurate, research at Washington University School of Medicine in St. Louis over the past decade has shown that up to 5 percent of pregnancy tests return results indicating a woman is not pregnant when, in reality, she is.

Makers of pregnancy tests advise that tests taken in the first week or two after conception could be inaccurate because pregnancy hormones may not have risen high enough to be detected. But Ann Gronowski, PhD, a professor of pathology and immunology, and of obstetrics and gynecology, and medical director of core laboratory services at Barnes-Jewish Hospital, discovered that pregnancy tests can also give incorrect results to women five weeks or more into their pregnancies, when hormone levels tend to be very high. She published the first paper describing this problem in 2009, and since then has continued studying and raising the alarm on this serious but under-recognized issue. Recently, she and colleagues published a paper in the journal Clinical Chemistry, in which they evaluated how likely several pregnancy devices were to give false negative results.

Q: How did you discover that some pregnancy tests produce false negative results?

About a decade ago, a woman came to the emergency department saying she was pregnant and was experiencing cramping and spotting – which can be signs of miscarriage – but the pregnancy test we gave her at the hospital came back negative. She was certain she was pregnant, so we performed a blood test and an ultrasound, both of which confirmed she was pregnant. Shortly afterward, we had a similar experience with a second patient, and at about the same time a colleague at Vanderbilt University called me with a similar observation. So we started investigating, and we published a paper describing the problem and its cause.

Q: Why do pregnancy tests sometimes return false negative results?

We use a kind of pregnancy test in the emergency department that is similar to what you can buy at a drugstore. These tests detect a pregnancy hormone called human chorionic gonadotropin (hCG) in a urine sample. One antibody captures the hormone in the urine, and the other serves as a signal. It has a color that becomes visible when it detects the correct hormone. That’s the second blue or pink line you see on a test, and it indicates a positive result (the first line is just there to show the device is working properly).

But a degraded form of the hormone also can be found in the urine, and in some devices the first antibody will bind to the degraded form. The amount of the degraded form, called hCG core fragment, goes up as pregnancy progresses. The more of the fragmented hormone that is around, the more likely the first antibody will accidentally capture the fragment instead of the intact hormone. However, the signal antibody does not respond to the fragment so it does not change color when that happens, and therefore you get a negative result even though the hormone might be present.

Q: How common is this problem?

It depends on the device. They all use different antibodies, and some are better at discriminating the full hormone from the fragment than others. We looked at 11 of the most commonly used hospital pregnancy tests to see if they were susceptible to false negatives when levels of the hormone fragment were high. Seven were somewhat susceptible, two were highly susceptible, and only two tests were not susceptible. The worst one gave false negatives in 5 percent of the urine samples of pregnant women tested. That was, unfortunately, the test we were using when that initial patient came in. Based on our research, we have switched to a test that does not have this false-negative problem.

Q: Are the makers of pregnancy tests doing anything to solve this problem?

Based on our work, the Food and Drug Administration has acknowledged the need for pregnancy tests that are not susceptible to false negatives. The FDA requires that all new tests generate a positive signal even when concentrations of the fragmented hormone are high. Unfortunately, this requirement does not apply to tests that were already FDA-approved.

Q: What can women do if they think they received a false negative on a pregnancy test?

What’s interesting is that some women discovered our research and have been posting in online pregnancy forums, saying that if you think you’re pregnant but the test comes back negative, dilute the urine with water and try again because sometimes the tests are wrong. And that can actually work. Dilution reduces levels of the hormone fragment enough that the first antibody is more likely to detect the intact hormone again. But I do not recommend this. The best test to detect pregnancy is a blood test. If there’s any doubt, I would recommend women talk to their physicians and request blood tests.

Related: Blood test is highly accurate at identifying Alzheimer’s before symptoms arise

When combined with age and genetic risk factor, test is 94% accurate

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Washington University School of Medicine’s 1,500 faculty physicians also are the medical staff of Barnes-Jewish and St. Louis Children’s hospitals. The School of Medicine is a leader in medical research, teaching and patient care, ranking among the top 10 medical schools in the nation by U.S. News and World Report. Through its affiliations with Barnes-Jewish and St. Louis Children’s hospitals, the School of Medicine is linked to BJC HealthCare.

Judy Martin Finch

Director of Media Relations