Category Archives: Pregnancy

Pregnancy

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.

TMI? A home hCG test that detects pregnancy and estimates weeks since conception


CalendarI'm beginning to wonder what could possibly come next. Last month, Swiss Precision Diagnostics (via its Procter & Gamble partner) unveiled its newest product in consumer diagnostics: the Clearblue Advanced Pregnancy Test with Weeks Estimator. This urine hCG test determines pregnancy status but also provides an estimate of the number of weeks since ovulation. The device has been cleared by the FDA and will be availabe in the US in September, 2013.

It works like other qualitative urine hCG tests but the body of the device contains two test strips that capture and detect the hormone in the urine sample. Detection of hCG is accomplished by the appearance of a colored band at a specific location on the test strip.

One test strip is designated "high sensitivity" and can detect a low concentration of hCG (detection limit of 10 IU/L). This test strip determines pregnancy status (pregnant or not pregnant). The other test strip has "low sensitivity" and detects higher concentrations of hCG and is used to estimate the number of weeks since conception. An optical reader housed within the device determines the color intensity of the test strips and a digital display reports the results as 1) Not pregnant; 2) Pregnant 1-2 weeks; 3) Pregnant 2-3 weeks; or 4) Pregnant 3+ weeks. The device reports an invalid result if there is a malfunction due to the device itself or operator error.

Clearblue with Estimator

Note that the weeks estimate is based on hCG concentration which is not how a pregnancy is usually dated. Physicians calculate gestational age by the day of the last menstrual period (LMP) so the device's estimate will be about 2 weeks less than one based on the LMP.

In its press release, Procter & Gamble states that the test is "more than 99 percent accurate in detecting pregnancy from the day of the expected period, and it is approximately 93 percent accurate in estimating how many weeks based on time since ovulation." I wanted to know what studies were done to support those claims but I received no response when I reached out to the individual at Procter & Gamble identified in the press release. However, there are some data included in the FDA's decision summary:

  • An early pregnancy study was conducted using 100 urine samples collected from non-pregnant women expecting to become pregnant. These samples were collected on days -6 to 1+ relative to the day of expected period. 99.0% of the devices gave a "pregnant" result by day zero (the day of the expected period).
  • A clinical study was conducted using samples from 153 volunteers with singleton pregnancies to evaluate performance of the “Weeks Estimator” feature compared to actual gestational age (method not identified). Agreement of “Weeks Estimator” with actual gestational age ranged from 45-99% (bold-faced emphasis is mine).

The bolded statement above is ambiguous but it likely is supposed to mean that the device is accurately able to estimate the true week of gestational age 45-99% of the time. If so then there are several questions that need to be answered. For example, what was the source of the range? Was it derived from different studies or from different gestational ages? What was the median gestational age? Is the device more accurate at certain gestational agess? Also, the range doesn't indicate how inaccurate the device can actually be. That is, when it is wrong how wrong is it? 1 week, 2 weeks, 3 weeks, or even more?

Given that the "Weeks Estimator" is highly variable, it is likely that Procter & Gamble will include language (similar to what the FDA stated) to caution the consumer that "the 'Weeks Estimator' is meant solely as an estimate for the consumer and is not intended as a substitute for a doctor’s clinical diagnosis. The ‘Weeks Estimator’ is not intended for multiple pregnancies. The estimate provided by the device may be inaccurate in these cases."

From a practical perspective, while women may want/need to know when they conceived, this is not the device to accomplish the job. It seems to provide highly variable information that, at best, is just a curiosity. Procter & Gamble says “confirming pregnancy is a life-changing moment in any woman’s life, and it sparks so many immediate questions like 'when did I get pregnant?'" True, but necessary? I'm not convinced.

Trimester-Specific Reference Intervals for TSH


Thyroid tests

In September 2011, The American Thyroid Association (ATA) published new guidelines on the diagnosis and management of thyroid disease during pregnancy and postpartum.  There are many recommendations in the guidelines, but I wanted to highlight one in particular.

Recommendation 2

"If trimester-specific reference ranges for TSH are not available in the laboratory, the following reference ranges are recommended: First trimester, 0.1-2.5 mIU/L; second trimester, 0.2-3.0 mIU/L; third trimester, 0.3-3.0 mIU/L."

 These reference intervals are lower than the non-pregnant reference intervals. This is due to the fact that hCG has mild thyroid-stimulating ability.  Therefore, hCG stimulates the thyroid and suppresses TSH. This is most apparent during the first trimester (7-11 weeks) when hCG is at its highest concentration. TSH concentrations actually decrease, although usually not below the normal, non-pregnant, reference interval.

This means that hypothyroidism during pregnancy needs to be defined using these pregnancy-specific reference intervals. Overt hypothyroidism is defined as decreased fT4 with TSH > 2.5 mIU/L. Subclinical hypothyroidism is defined as serum TSH 2.5-10 mIU/L with normal fT4. The ATA recommends treating overt hypothyroidism, but not subclinical hypothyroidism, unless women are also positive for anti-TPO antibodies. When patients are treated, the goal is to achieve the trimester-specific reference intervals listed above.

Interestingly, the ATA recommends that women who are taking T4 therapy have their dose adjusted to achieve a TSH concentration of 2.5 mIU/L before pregnancy. This reduces the risk of hypothyroidism during the first trimester. Likewise once women who are on T4 therapy get pregnant, their T4 therapy should be adjusted to keep them within the pregnancy-specific TSH reference intervals and serum TSH should be monitored approximately every 4 weeks during the first half of pregnancy. Serum TSH should also be checked again between weeks 26 and 32.

Similarly, according to the ATA, euthyroid (normal functioning thyroid gland) women who are not on T4 replacement therapy but are TPO antibody positive, should also have serum TSH monitored every 4 weeks during the first half of pregnancy and again between weeks 26 and 32.

Should I get my iodine measured during pregnancy?


Salt2

The short answer is no, but let me explain why.

Iodine is necessary for the production of the thyroid hormones T3 and T4. A deficiency of iodine leads to decreased production of these hormones and can cause goiter (enlargement of the thyroid) and hypothyroidism.

During pregnancy, a number of normal changes occur that involve the thyroid gland and the need for iodine including:

  1. hCG is similar in structure to TSH, the hormone that stimulate the thyroid gland, and so hCG can also stimulate the thyroid gland;
  2. There is an increased demand for T3 and T4; and,
  3. Clearance of iodine through the kidneys is increased.

In areas where there is iodine deficiency, pregnancy is associated with a 20-40% increase in the size of the thyroid gland. In areas where iodine is replete, like the United States, the thyroid increases in size by only around 10% during pregnancy.

Because of these changes, dietary iodine requirements for pregnant women are higher than they are for non-pregnant women. If iodine intake was adequate before pregnancy, women should have sufficient iodine stores and therefore have no difficulty meeting the needs for iodine during pregnancy and lactation. If their iodine intake was not sufficient, it can result in overt hypothyroidism which is associated with miscarriage, stillbirth, and, in very severe cases, cretinism (characterized by severe mental retardation and deafness). Iodine deficiency is the leading cause of preventable mental retardation worldwide. According to public health experts, iodization of salt may be the world's simplest and most cost-effective measure available to improve health

While the U.S. is an iodine replete country, some studies have suggested that women of reproductive age may be at risk of iodine deficiency.  This might make one think that iodine status should be determined in these women. Iodine status is usually assessed by measuring urine iodine concentrations. However, there is significant day-to-day variation in urine iodine excretion, such that measurement in a single individual is not useful. Urine concentrations are most useful to assess the iodine status of a whole population.

In 2011, the American Thyroid Association (ATA) published guidelines for the diagnosis and management of thyroid disease during pregnancy and postpartum. In these guidelines, the ATA recommends that all pregnant and lactating women ingest a minimum of 250 ug of iodine daily. For U.S. women that means supplementing their diet with a daily oral supplement that contains 150 ug of iodine (optimally potassium iodide).

In 1924, the Morton Salt Company began distributing iodized salt nationally, which is a good source of iodine. While iodized salt is the main source of iodine in the American diet, only ~20% of the salt Americans eat contains iodine!  Reasons for this include:

  1. Increase in popular designer salts like sea salt and Kosher salts (see photo below); Salt
  2. Iodized salt is not used in most fast and processed foods or in the production of commercial breads; and,
  3. Patient concerns about salt intake & hypertension. Good dietary sources of iodine include kelp seaweed, seafood (cod, sea bass, haddock, and perch are good sources) and dairy products.

In summary, if you are pregnant make sure you are taking a supplement that contains iodine, but do not worry about having your iodine concentration measured.

 

Assessing Ovarian Reserve


OvariesWomen in their mid to late 30s and early 40s with infertility constitute the largest portion of the total infertility population. These women are also at an increased risk for pregnancy loss. This reflects a decline in oocyte quality and a diminished ovarian reserve as a result of follicular depletion. Ovarian reserve is a term that is used to describe the capacity of the ovary to provide eggs that are capable of fertilization resulting in a healthy and successful pregnancy.

While there is no gold standard for assessing the ovarian reserve of individual women, its indirect determination has been used to help direct infertility treatment.

Serum concentrations of follicle-stimulating hormone (FSH) and estradiol on day 3 of the menstrual cycle have been the tests of choice for assessing ovarian reserve. Cycle day 3 is chosen because at this time the estrogen concentration is expected to be low, a critical feature, as FSH concentrations are subject to negative feedback from estradiol. In general, day 3 FSH concentrations >20 to 25 IU/L are considered to be elevated and associated with poor reproductive outcome.   FSH concentrations are expected to be below 10 IU/L in women with reproductive potential.  Concomitant measurement of serum estradiol adds to the predictive power of an isolated FSH determination. Basal estradiol concentrations >75-80 pg/mL are associated with poor outcome. 

Inhibin B is produced by the developing follicles and concentrations peak during the follicular phase. Concentrations of inhibin B can be used in conjunction with serum FSH and estradiol to assess ovarian function. As women age, serum FSH concentrations in the early follicular phase begin to increase. It has been suggested that this is due to a decline in the number of small follicles secreting inhibin B.  Because inhibin is produced by the ovaries, it is thought to be a more direct marker of ovarian activity and ovarian reserve than FSH. In addition, cycle day 3 inhibin B concentrations may demonstrate a decrease before day 3 FSH concentrations. 

Seifer et al reported that women undergoing in vitro fertilization (IVF) with day 3 inhibin B concentration <45 pg/mL had a pregnancy rate of 7% and a spontaneous abortion rate of 33% as compared to pregnancy rate of 26% and abortion rate of 3% in women with day 3 inhibin B concentrations of > 45 pg/mL. 

In recent years, anti-Mullerian Hormone (AMH) has been suggested to be a more useful predictor of ovarian reserve. AMH is expressed by the granulosa cells of the ovary during the reproductive years, and controls the formation of primary follicles by inhibiting excessive follicular recruitment by FSH. In 2005 Tremellen reported that plasma AMH concentrations start to drop rapidly by age 30, and are ~10 pmol/L by the age of 37. David has blogged previously about the use of AMH as a predictor of IVF outcome.   

Using a cut off value of 8.1 pmol/L, plasma AMH could predict poor ovarian reserve on a subsequent IVF cycle with a sensitivity of 80% and a specificity of 85%.     In 2008, Riggs and colleagues confirmed that AMH concentrations correlated the best with the number of retrieved oocytes relative to age, FSH, inhibin B, LH, and estradiol. 

High concentrations of AMH can also be present in women with polycystic ovarian syndrome (PCOS), a cause of female infertility.  Therefore, in PCOS patientsAMH should not be used alone, but should be combined with transvaginal ultrasound to count the number of follicles.

Women who are diagnosed with diminished ovarian reserve should be counseled regarding options such as oocyte donation or adoption.

Is the qualitative serum pregnancy test obsolete?


I’ve written several times about qualitative hCG tests in this blog.  As a reminder, qualitative tests can be performed using urine or serum samples.  Urine tests can be performed close to the patient or even at home because the urine sample requires no special processing.  However, when serum is the test really can’t be performed at home or at the point-of-care because the blood sample has to be centrifuged to first obtain the serum and centrifugation is usually only performed in the clinical laboratory.  Notably, clinical labs are often able to do quantitative hCG testing on serum, too.

So, if a lab can do qualitative and quantitative hCG testing on serum, why not just offer one test instead of two?  In other words, might the qualitative test be considered obsolete?  My lab recently published a study that addressed that question.

To answer that question we surveyed several hundred doctors and the survey results revealed the following:

  1. When requesting serum hCG tests, 49% of physicians preferred to order a qualitative rather than a quantitative test even though they believed quantitative tests were more accurate.
  2. Physicians preferred qualitative tests because they believed that they received the test results faster.

However, when we examined the turnaround time data, that last point was not supported.  There are a few definitions of turnaround time to consider.  Doctors consider it to be the time it takes to get a result after the sample is collected while laboratorians consider it to be the time it takes to produce the result after they receive the sample.

By the lab’s definition, qualitative tests were performed more rapidly than quantitative tests but there were no differences using the doctors’ definition of turnaround time.  That’s because the time it takes to transport the sample to the laboratory is known to contribute the most to delays in the total testing process.  So, although physicians believed they get results from qualitative tests more quickly, it doesn’t seem to be the case.

We also compared the analytical sensitivities of the two types of tests.  The qualitative test that we used had a claimed detection limit of 25 IU/L.  That is, a sample with an hCG concentration above 25 IU/L should produce a positive result.  Of the samples that gave a positive result, about 20% had an hCG concentration that was <25 IU/L which indicated that the qualitative test was more analytically sensitive than we expected it to be.  In my opinion, that’s a good thing.

Because we determined the actual pregnancy status of all the patients with a positive result, we were also able to determine how well the qualitative and quantitative tests performed at determining pregnancy status.  Both tests did quite well and showed high sensitivity and specificity.  That is, there were very few false-negative or false-positive results.  From a clinical perspective, a false-negative result is more concerning than a false-positive one because a pregnant patient who is incorrectly identified as not being pregnant risks being exposed to a medical intervention that could harm the fetus.  The false-negative rate was lowest, only 0.1%, when the qualitative test was evaluated against pregnancy status and the detection threshold of 25 IU/L.  The performance of the quantitative serum hCG test was identical.  So, both the qualitative and quantitative serum hCG tests do a very good job at ruling-out a possible pregnancy.

So, given this evidence, I would conclude that while qualitative hCG tests could be replaced by quantitative tests, there is really no compelling reason to do so.

Anti-müllerian hormone as a predictor of IVF outcome


Human egg Investigators at Brown University have reported that the serum concentration of a hormone made by the ovaries can predict the success of in vitro fertilization (IVF). Specifically, the higher amounts of the hormone were correlated to a greater number of eggs retrieved and the probability of achieving pregnancy.

The study measured blood concentrations of anti-müllerian hormone, or AMH. This is a rather interesting hormone. It plays an important role in both males and females but at different stages of human development.

  • In fetal development, AMH is synthesized by the testes of a male fetus and it functions to suppress the transformation of the müllerian ducts into the uterus and fallopian tubes. In the absence of AMH (i.e. a female fetus), the wolffian ducts fail to develop into ejaculatory ducts of the male reproductive system while the müllerian ducts complete their development.
  • AMH is synthesized by the female ovary but only after birth when it does not affect the development or maturation of the female reproductive tract. It increases slightly at puberty because of it functions to regulate the formation of ovarian follicles (eggs) by opposing the action of another hormone, follicle-stimulating hormone (FSH).

Because of its role in follicle development, AMH can be useful as a marker of ovarian function. In particular it seems to be a good indicator of the growing pool of follicles in the ovary and so reflects the number of eggs in the ovary. That’s exactly what the scientists at Brown University reported.

In 190 women undergoing IVF to achieve pregnancy, the concentration of AMH in a blood sample collected on the first day of ovarian stimulation with FSH was a strong predictor of favorable outcome. Women with the lowest concentrations of AMH had fewer eggs retrieved than those with higher concentrations. Also, the probability of achieving pregnancy was 2.5 times greater in the women with the higher AMH concentrations.

What might these results mean from a practical perspective? The authors of the study comment that IVF patients could be better counseled and IVF management strategies modified by the AMH results. IVF is expensive. Women who seek to achieve a pregnancy by this route are highly motivated to obtain a positive outcome and so they may undergo several IVF cycles in an effort to become pregnant. Those with a low AMH concentration may opt for only a single IVF cycle or seek adoption or other routes of parenthood rather than invest thousands of dollars into what may be an unachievable biological goal.

Laboratory testing in ectopic pregnancy investigation


What’s the leading cause of maternal death in the first trimester? That would be an ectopic pregnancy, which occurs when the fertilized egg implants someplace other than the inside of the uterus. In the vast majority of ectopic pregnancies the zygote implants in the fallopian tube, an organ that, unlike the uterus, can’t expand to accommodate the growing embryo. If not diagnosed early, an ectopic pregnancy can cause the fallopian tube to rupture that can result in massive internal bleeding and death.

 Diagnosing an ectopic pregnancy is challenging. The typical symptoms of abdominal pain and vaginal bleeding are not present in all patients and can be due to reasons other than an ectopic pregnancy. Also, the risk factors of ectopic pregnancy are absent in about half of all women who have an ectopic pregnancy. Fortunately there are laboratory tests that assist in identifying an ectopic pregnancy.

 hCG testing is first used to confirm that the patient is, indeed, pregnant. Although a rapid, point-of-care urine test may be used to detect hCG, I think it is much safer to perform a serum hCG test because it can detect lower concentrations of hCG than a urine test can. If the test is negative, that is hCG is not present, then the patient isn’t pregnant at all. If hCG is detected then pregnancy is confirmed but, all by itself, a single hCG test cannot identify a normal from an ectopic pregnancy.

 To do that, other investigations are commonly put to use:

  1. Transvaginal ultrasound. Ultrasound imaging is used to visually observe if an embryo is in the uterus or the fallopian tube but it can also be inconclusive if no embryo is observed anywhere. If the ultrasound is inconclusive then the concentration of serum hCG can be useful. That’s because an intrauterine pregnancy should always be able to be seen by ultrasound when the serum hCG concentration is greater than 1500 to 2000 IU/L. If that threshold is reached and no embryo is seen in the uterus then the patient is likely to have an ectopic pregnancy. If that threshold is not present at the initial evaluation, then serial hCG testing is considered.
  2. Serial hCG testing. Collecting more than 1 blood sample over time for hCG testing is helpful because in a normal pregnancy serum concentrations of hCG increase by 53% or more every 2 days. An increase that is less than 53% is cause for concern. That’s because an abnormal increase in indicates an abnormal pregnancy. In an ectopic pregnancy, hCG can show a normal or abnormal increase so an abnormal increase by itself it isn’t diagnostic of an ectopic pregnancy.

 These two tests are usually used together in an algorithm that looks something like this:

 

 

Ectopic algorithm

 

The take home message here is that, as helpful as lab tests are, the diagnosis of ectopic pregnancy cannot be accomplished by lab tests alone. All clinical data must be evaluated in order to arrive at the final diagnosis.

 

Investigating unexpectedly positive hCG test results


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:

  1. 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.
  2. 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.
  3. 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.
  4. 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.