HCG REFERENCE SERVICE
FALSE POSITIVE OF PHANTOM HCG TESTS

  
   Look at the latest finding with hCG false positive results hCG Reference Service Report 2002

   To understand the concept of false positive or phantom hCG results, we look carefully at the principal and mechanism of the hCG assay (click here to go to principal of hCG test & discordant results). Most hCG assays or pregnancy tests used today are "sandwich assays". Sandwich assays use at least two animal antibodies raised against hCG (SEE FIGURE 1, BELOW). Usually assays use a mouse monoclonal antibody against one site on the hCG molecule, and a mouse monoclonal, or a sheep, rabbit or a goat polyclonal antibody against a second distant site on the hCG molecule. One of the antibodies, the capture antibody, is in a solid phase permanently attached to a tube, plate, membrane or bead. The second antibody, the tracer antibody, is labeled in with radioactivity, with an enzyme, with a dye, a lanthanide or other identifier. This antibody is in the liquid phase (SEE FIGURE 1, BELOW). Blood (serum) is added to the assay system. After a short incubation period the hCG binds both the solid phase and liquid phase antibodies linking them. In this way it forms a sandwich or bridge between the solid support or capture antibody and the tracer antibody with the attached label. In this way the label becomes immobilized. After washing away the excess tracer antibody the amount of label attached to the solid support is measured. This is directly proportional to the amount of hCG.

FIGURE 1. Device with solid phase capture antibody to one site on hCG, and liquid phase tracer antibody (label shown by red star) to second or distant site on hCG.

    Unfortunately, hCG is not the only molecule that can form a bridge or sandwich between the solid phase antibody, capture antibody (either a IgG, IgA or IgM), and liquid phase antibody, tracer antibody (either a IgG, IgA or IgM). Humans can form antibodies against their own human antibodies (IgG against IgG, IgA against IgG, IgM against IgG etc.). Human antibodies are very similar to animal antibodies, so that these human antibodies against antibodies can bind animal as well as human antibodies (heterophilic antibodies). Small amounts of heterophilic antibodies in the blood can form sandwiches or links between the tracer and capture antibodies (they bind different non-specific sites on the capture and tracer antibodies) (SEE FIGURE 2, BELOW). After washing away the excess tracer, the amount of label or tracer attached to the solid support is measured (SEE FIGURE 3, BELOW). 
    In most instances hCG assays really measure hCG (>99% of cases). In which case the number measured is representative of the amount of hCG in the serum sample. When  heterophilic antibodies are present they can bind the animal antibodies used in the assay, also forming the sandwiches or links. This causes false positive of phantom hCG results.

FIGURE 2. Serum is added to the device containing capture (solid phase) and tracer (liquid phase) antibodies. The serum sample, instead of containing hCG contains small amounts of HAMA or heterophilic antibodies. After a short incubation, the HAMA or heterophilic antibodies form sandwiches or bridges between the capture and tracer antibodies.

FIGURE 3. The device is washed and excess tracer antibody is removed. The amount of tracer (label, shown by red stars) is measured. In true positive cases the amount of tracer is representative of the amount of hCG. In false positive or phantom cases it is representative of something less relevant, HAMA or heterophilic antibodies.

   Phantom or false positive hCG results can persist for many years, giving false hCG result in the range of 10 to 630 mIU/ml). Data accrued by the hCG Reference Service suggest that the false positivity (or heterophilic antibodies) may slowly dissipate. 
    The hCG Reference Service has identified 42 cases of phantom or false-positive hCG. For more information see the hCG Reference Service Report 2002. These 42 women erroneously diagnosed with  or gestational trophoblastic diseases or choriocarcinoma because of false positive hCG levels. Physicians had trouble interpreting the persistent low hCG levels. The initial thought was always pregnancy. After ruling out normal pregnancy (ultrasound), ectopic or tubal pregnancy was considered as the source of the positivity in the hCG test. After ruling out ectopic pregnancy (laparoscopy), and after ruling out hydatidiform mole (D&C), less benign sources of hCG were considered such as gestational trophoblastic disease or choriocarcinoma. Remember, the only symptom was positivity in an hCG test (10 to 630 mIU/ml). With this in mind, severe measures taken to tackle this potential cancer. Most of the 42 cases underwent chemotherapy (methotrexate and or EMACO), major surgery (hysterectomy, oophorectomy, and other procedures). This is all because of phantom or false positive hCG tests. Phantom or false positive hCG results need to be considered as a possible explanation for continuous elevated hCG levels, in individuals with no pregnancy and no other symptoms. 
      An unknown number of women are treated each year for ectopic pregnancy (methotrexate chemotherapy or surgery), based solely on a persistent phantom hCG level in the absence on intrauterine pregnancy (show by ultrasound). Phantom or false positive hCG should be ruled out in any individual with elevated hCG levels in the absence of intrauterine pregnancy.
       Special assays are required to detecting and proving the presence of phantom or false positive hCG. This can be done at the hCG Reference Service. Help may also be available from other sources, such as the manufacturer of the hCG test used or possibly by the clinical laboratory (click here for other important contacts). Phantom or false positive hCG may be demonstrated by the presence of hCG positivity in a serum sample (10 - 630 mIU/ml), with the complete absence of positivity in a parallel urine sample (while hCG is present in serum and urine, antibodies are only present in serum samples). Phantom hCG can be confirmed by the demonstration of loss of the hCG activity after treatment of a serum sample with a heterophilic antibody blocking agent (Scantibodies Inc. HBT). Phantom hCG can also be confirmed by the finding of widely (more than 5-fold) variable results in different commercial hCG tests, and by the finding of false positive results with other similar principal serum tests.
    It is noteworthy, that 36 of the 42 cases  were being managed by their physician using one brand of serum hCG test (over 40 brands are sold in the USA). This was the Abbott AxSym hCGß test. This test appears to be much more apt to giving false positive hCG results than other brands.  

REFERENCES

1.    Cole LA. Phantom hCG and phantom choriocarcinoma. Gynecol Oncol, 71:325-329, 1998.

2.    Cole, L.A., Rinne, K.M., Shahabi, S., and Omrani, A. False positive hCG levels leading to unnecessary surgery and chemotherapy, and needless occurrences of diabetes and coma. Clin Chem, 45:313-314, 1999.

3.    Rotmensch, S., and Cole, L.A. False diagnosis and needless therapy of presumed malignant disease in women with false-positive human chorionic gonadotropin concentrations. The Lancet 355:712-715, 2000.