Analyzer-interpreted pregnancy tests: the significance of borderline hCG levels in urine specimens

Aug. 18, 2014

Qualitative urine visual pregnancy tests are increasingly being replaced by analyzer-read chromatographic immunoassay (CIA) tests that determine human chorionic gonadotropin (hCG) in specimens. Accurate and consistent analyzer-read pregnancy test results offer significant advantages over traditional methodology and reporting. Since no visual interpretation or manually transcribed documentation is required, the chance for error is greatly diminished. hCG test instrumentation connected via laboratory or hospital information systems (LIS/HIS) automatically report hCG results that can be reviewed immediately and acted upon, thus facilitating the current trend towards testing and immediate healthcare provision in decentralized, ambulatory settings. 

Human chorionic gonadotropin

Human chorionic gonadotropin (hCG) is a 40kDa glycoprotein hormone secreted by the developing placenta shortly after fertilization. It is composed of two subunits α and β. The α subunit is common for hCG, follicle stimulating hormone (FSH), luteinizing (LH), and thyroid stimulating hormone (TSH). The β subunit is distinguishing for hCG, hence the nomenclature “βhCG” test, which implies reduced cross-reactivity with the other hormones mentioned above.

In normal pregnancy, hCG can be detected in serum as early as six to seven days following conception. Recent studies suggest that urine hCG concentrations are approximately one-half of, or less than one-half of, corresponding serum hCG concentrations. Thus, hCG can likely be reliably detected in urine as early as 14 days after conception (approximately 28 days since the last menstrual cycle). hCG approximately doubles in concentration about every two days until it peaks at approximately 8 to 10 weeks after the last menstrual period.1 The appearance of hCG soon after conception and its subsequent rise in concentration during early gestational growth make it an excellent marker for the early detection of pregnancy.

Hormone levels 25 mIU/mL are usually reported as a positive qualitative indication of pregnancy. Urine samples containing 5 mIU/mL usually indicate a negative result for pregnancy. A borderline result is generated by some assays when the hCG level is between 5 and 25 mIU/mL. Samples reported as borderline are considered indeterminate, and clinicians should request a repeat test within 48 to 72 hours or obtain a quantitative serum hCG. However, some assays report values in the 5 to 25 mIU/mL range as positive for pregnancy. This sometimes can cause confusion for the patient when she is tested later on and the result is negative.  

False-positive results can lead to inappropriate treatment. It has been established that serum hCG increases with age in non-pregnant women.2 In fact, Snyder et al. suggest that a cut-off of 14.0 mIU/mL should be used when interpreting hCG results in women >55 years of age. So it is apparent how some assays that give a positive result at low values can lead to misinterpretation. It is better to test two days later and see if the hCG level is rising as expected for a normal pregnancy. Using an indeterminate interpretation allows for better analysis of the situation that led to low hCG levels. In fact Cole, in a letter to the editor of Clinical Chemistry,3 states:

“Realistically, for pregnancy to be considered likely, hCG results need to exceed concentrations of at least 20 IU/L. For a successful term pregnancy, hCG should then at least double every two days until eight weeks of gestation. In cases in which persistently low hCG results are detected, malignancies may be considered. Trophoblastic malignancies make intact hCG dimer, whereas other malignancies primarily produce the free subunit of hCG (beta subunit). Both are detected by modern intact hCG assays. Pituitary production of hCG, or extreme cases of background hCG, always must be considered, particularly in women who are peri- or postmenopausal.”

Although analyzer-read hCG tests are not intended to detect conditions other than pregnancy, a number of conditions other than pregnancy, including trophoblastic disease and certain non-trophoblastic neoplasms, can cause borderline levels of hCG. Borderline hCG results are in no sense diagnostic, but may, especially when results do not fit a clinical picture, indicate to clinicians that further investigation is needed. 

Borderline hCG

Borderline hCG levels can occur in the following conditions or situations:

  • Early normal pregnancy
  • Ectopic pregnancies, when hCG levels rise at a significantly slower rate than in normal pregnancy
  • Trophoblastic diseases, including an ovarian tumor or hydatidiform mole (A hydatidiform mole is a mass that can form inside the uterus at the start of a pregnancy.)
  • Increased hCG levels in healthy, non-pregnant post-menopausal women
  • Passively acquired hCG from a blood transfusion
  • Undetected early spontaneous abortion (A miscarriage may also be called a “spontaneous abortion.” Around half of all fertilized eggs die and are lost, that is, aborted spontaneously, usually before the woman knows she is pregnant. Among women who know they are pregnant, the miscarriage rate is about 15% to 20%.4)
  • hCG levels that may remain detectable for up to several weeks following delivery, miscarriage, or hCG injections (from IVF treatment)
  • Interfering antibodies in the medications of patients on antibody therapies
  • Heterophile antibodies. (The presence of a heterophile antibody can interfere with immunoassay tests such as hCG. Heterophile antibodies are human antibodies with the ability to bind to animal immunoglobulins, thereby possibly interfering with the interaction between animal-derived antibodies and the antigen [hCG in our discussion], which are components of all immunoassays. They are commonly referred to as human anti-animal antibodies [HAAA]. Human anti-mouse antibodies [HAMA] belong to this category. They can create both false-positive and false-negative results.)5

Verifying hCG levels

The discussion above leads one to conclude that hCG testing for pregnancy is not as simple as it appears on the surface. hCG itself is not a simple molecule, nor does it exist in one form. Furthermore, a single test may lead one to a wrong conclusion diagnostically. That is why when a test presents with low levels of hCG it is better to verify the diagnostic conclusion than to just call the result positive, as some assays would have you do. In fact, Snyder states:2

“Because hCG measurements have two clinical applications, false-positive hCG results not only cause clinical confusion, but in several instances have led to harmful therapeutic interventions.”

Lawrence J. Crolla, PhD, is Adjunct Assistant Professor in the Department of Pathology, Loyola University, Stritch School of Medicine. This article was prepared using a grant from Siemens Diagnostics.


  1. Karine Chung, MDS. Defining the rise of serum HCG in viable pregnancies achieved through use of IVF. Human Reproduction. 2006;21(3): 823-828.
  2. Snyder JA, Haymond S, Parvin CA, Gronowski AM, Grenache DG. Diagnostic considerations in the measurement of human chorionic gonadotropin in aging women. Clin Chem. 2005; 51(10):1830-1835.
  3. Cole LA. “Background” human chorionic gonadotropin in healthy, nonpregnant women. Clin Chem. 2005;51(10):1765-1766.
  4. Storck S. Miscarriage. 2012; Accessed July 8,2014.
  5. Publication, M.R.S. The ghost in the assay tube: heterophile antibody interferences in immunoassays – an ever-recurring but often forgotten problem. In Mayo Reference Services Publication. 2003, Mayo Reference Services Publication.1-4 Accessed July 8, 2014.