"Hook-Like Effect" Causes False-negative Point-of-Care Urine Pregnancy Testing in Emergency Patients

Richard T. Griffey, MD, MPH; Caleb J. Trent, MD; Rebecca A. Bavolek, MD; Jacob B. Keeperman, MD; Christopher Sampson, MD; Robert F. Poirier, MD

Disclosures

J Emerg Med. 2013;44(1):155-160. 

In This Article

Discussion

POC tests usually employ monoclonal antibodies or a combination of mono- and polyclonal antibodies to detect the intact hCG hormone in urine. These so-called "sandwich assays" consist of a fixed solid-phase antibody attached to some matrix and a separate free or soluble antibody to which a "reporter" label is attached. These antibodies bind different epitopes of an antigen (here, hCG), forming a "sandwich," which then results in a visible signal that the user can read to indicate the test is positive (Figure 2).

Figure 2.

Sandwich assay schematic demonstrating a normal positive test. Molecules of intact human chorionic gonadotropin (hCG) are recognized by both solid-state antibodies fixed to a matrix and by soluble free-floating antibodies with an affixed reporter label to form a "sandwich," resulting in a positive test.

Human chorionic gonadotropin is a glycoprotein comprised of α and β subunits that is produced in the placenta after implantation. The β-subunit of hCG is what is measured in pregnancy tests, as the α subunit can also be found in other hormones such as luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone. The β subunit exists in several forms, including hyperglycosylated hCG (H-hCG), nicked hCG, free β subunit, the core fragment of β-hCG (hCG-βcf), and others (Figure 3). The relative fractional concentrations of these forms change throughout pregnancy.[6] For example, H-hCG is the primary, if not sole form of hCG produced after implantation, and accounts for up to as much as 60% of hCG found at 4 weeks, dropping to < 5% in the second and third trimesters.[7] By contrast, hCG-βcf is high in mid-pregnancy urine.[8]

Figure 3.

Common human chorionic gonadotropin (hCG) variants. Adapted from Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997;43(12):2233–43 (15).

Urine and serum hCG concentrations vary widely during pregnancy, with urine concentrations measuring approximately half that of the corresponding serum fractions, depending on the hydration status of the patient. Reported thresholds for detection in POC tests are between 15 and 100 mIU/mL.[7] In the week after a missed menstrual period, serum hCG concentrations can range broadly from 6 to 19,950 mIU/mL.[9] One study reports that although POC tests routinely claim a "99% detection rate," their sensitivity in detecting pregnancy at 1 week, in fact, ranges only up to 97%.[10] In addition, various POC and OTC test kits may not detect certain hCG variants as well as others, and sometimes not at all. One study reported that a majority of urine pregnancy tests had poor sensitivity for H-hCG, which typically is the highest concentrated variant of hCG present in early pregnancy.[6,7,11] This has led to recognition of the potential for false-negative urine qualitative testing in very early pregnancy.

A known failure mode for pregnancy detection by urine hCG POC tests occurs when hCG is in large excess and no dilution step is performed to wash the excess hCG variant away. The excess hCG form can simultaneously saturate the fixed, solid-phase antibody and the labeled, soluble antibody, preventing sandwich formation (Figure 4). This "hook effect phenomenon" results in absence of a signal, and thus a false-negative result. The paradoxical decrease in the curve plotting an increased signal with increased concentration of antigen gives the appearance of a hook, hence the name (Figure 5).[6,9] In POC urine hCG tests, the hook effect is generally not observed until hCG concentrations reach 1,000,000 mIU/mL, as is seen in gestational trophoblastic disease. However, the "hook effect" can also cause false-negative results in normal pregnancies when an excess of hCG is present.

Figure 4.

The hook-effect phenomenon, where a human chorionic gonadotropin (hCG) variant (here, hCG-βCF) is in excess, saturating both the fixed and the free-floating, labeled antibodies to the exclusion of other variants, such as intact hCG. This prevents sandwich formation and results in a false-negative test.

Figure 5.

Hypothetical curve for an assay where (A) increasing concentration of an antigen results in a stronger signal, and (B) where increasing concentration of antigen results in a "hook effect," where the signal paradoxically decreases.

A similar but distinct "hook-like" effect was hypothesized to occur when one of the antibodies in the sandwich configuration is unable to bind a particular hCG variant such as hCG-βcf, whereas the other antibody avidly binds this fragment. Under normal conditions, a sandwich can still form when the two antibodies both bind to other hCG variants present, resulting in a positive test. However, when the variant (here, hCG-βcf) is in high concentration, it will saturate the antibody that recognizes it to the exclusion of other variants present. Because the other antibody in the sandwich assay does not bind hCG-βcf, sandwich formation (both fixed and soluble antibodies binding the hCG variant) cannot occur, resulting in a false-negative test (Figure 6).[8] This hypothesized phenomenon was recently confirmed when previously unavailable purified hCG-βcf was added to a pregnant patient's urine and resulted in a negative test.[6,12]

Figure 6.

This schematic demonstrates the "hook-like" effect where a human chorionic gonadotropin (hCG) variant such as hCG-βCF is present in excess in an assay where this variant is recognized by one but not both of the antibodies. Here, hCG-βCF is not recognized by the fixed antibodies, but saturates the free-floating, labeled antibodies, preventing them from binding with intact hCG and forming a sandwich with the fixed antibodies. This results in a false-negative test.

Although uhCG POC tests perform well under conditions where hCG variants are present in typical concentrations, such as in early pregnancy, false negatives can result when hCG-βcf is in excess, as can happen later in the first trimester. This example is particularly important because hCG-βcf is the major hCG beta subunit molecule in the urine from about 5–8 weeks of gestation until term, and accounts for up to 90% of the urine test activity in mid-pregnancy.[13] This same phenomenon presumably can occur with different hCG variants given similar conditions. Although hCG-βcf is found almost exclusively in the urine, theoretically this "hook-like" effect can occur in serum as well as urine tests. When a false-negative test is suspected and thought to be a result of the "hook-like" effect, repeating the test after diluting the urine can often resolve the issue. In our laboratory, dilutions for qualitative results are performed with normal saline at both 1:2 and 1:5 concentrations.

Concern over the discovery of these false-negative uhCG results in our institution resulted in switching from one POC device (OSOM hCG Combo Test; Genzyme Diagnostics, Cambridge, MA) to another (hCG Combo SP Brand Rapid Test device; SP hCG Rapid Test, Cardinal Health, Dublin, OH). Interestingly, this same "hook-like" phenomenon eventually was demonstrated to occur with certain lots of the new test kits.[12] Our institution postulates that the "hook-like" effect phenomenon resulting in false-negative uhCG POC results could be an issue for a wide variety of test kits currently on the market.[12] In the case of the latter occurrence, changes in the specificity of the polyclonal antibody used in new lot numbers likely resulted in false-negative test results.

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