Administrative Office
St. Joseph's Hospital Site, L301-10
50 Charlton Avenue East
HAMILTON, Ontario, CANADA L8N 4A6
PHONE: (905) 521-6141
FAX: (905) 521-6142
http://www.fhs.mcmaster.ca/hrlmp/
Issue No. 60
QUARTERLY NEWSLETTER
December, 2001
PREGNANCY TESTS REVISITED 2002
FALSE POSITIVE AND FALSE NEGATIVE PREGNANCY TESTS: RECURRENT PROBLEMS
Chorionic gonadotrophin (CG) previously termed both human chorionic gonadotropin (hCG) and beta hCG (b -hCG) is the
glycoprotein hormone synthesized in the syncytiotrophoblastic cells of the placenta. Two subunits (alpha and beta) are
noncovalently linked to form the functional intact molecule and is structurally similar to the pituitary glycoproteins; FSH, LH,
and TSH. Intact chorionic gonadotropin (CG) exists in serum and urine as three major species: regular/normal CG, a
glycosylated molecule, so called nicked CG in which peptide bonds in the beta chain are broken, and recently identified
hyperglycosylated CG (H-CG).
MEASUREMENT OF CHORIONIC GONADOTROPHIN i.e. PREGNANCY TESTS
Measurement of CG (serum or urine) is confounded by three factors: 1. existence of different molecular species/fragments in
patient samples, 2. different antibody specificities, and 3. composition differences between standard materials. Today most
methods are standardized against the third International Standard (3rd IS).
Normal Pregnancy CG Levels: Detectable levels of CG (approx. 5 IU/L) are present at 8-11 days after conception (the third
week of pregnancy as measured from the LMP). Levels in approximately half of pregnant women reach 25 IU/L on the first
day of their missed period. Peak levels average approx. 100,000 IU/L at 8-10 weeks. By the end of the second trimester a
90% reduction has occurred. Levels are roughly constant during the third trimester.
With current sandwich (double antibody) immunoassays using monoclonal/polyclonal antibodies, the technology now exceeds
the clinical need. Sandwich technology uses one antibody as the capture molecule with the second antibody providing the
signal molecule, resulting in excellent specificity and sensitivity, giving assays capable of measuring <1 IU/L of CG.
Qualitative CG assays have sensitivities between 10 and 100 IU/L while quantitative assays have sensitivities between 1 and
10 IU/L (or better).
Clients want an assay indicating pregnancy at the first missed menses (four weeks gestation) or earlier (within days of
implantation). Increased sensitivity increases false positive and false negative results. Early termination (first trimester) due to
non viable fetus or faulty implantation is common (10-20% of pregnancies) and is the major cause of "false" positives.
FALSE NEGATIVE PREGNANCY TESTS
Home pregnancy testing kits claim 99% diagnostic accuracy for pregnancy, usually positive on the first day of the first missed
menstrual period. Such is not the case. The claim is based upon in vitro studies using regular CG but does not allow for
hyperglycosylated CG which may reduce diagnostic accuracy to 50%. Due to the rapid rise in CG levels in early pregnancy,
repeat testing a week later will almost invariably give the correct answer.
Hyperglycosylated CG (H-CG): a large variant of the CG molecule (relative molecular mass 41 - 42,000 vs. 36-37,000 for
regular CG) is the principal form of CG in serum and urine at the first missed menstrual period. Home pregnancy test kits use
antibodies raised and calibrated against CG (not H-CG) and are much less sensitive to H-CG than to CG. Butler et al (1)
tested 15 of 22 across the counter CG kits and found 9 of the 15 were much less sensitive to the hyperglycoslated form of CG
(H-CG).They conclude that over the counter pregnancy tests are not 99% accurate and there is need for improvement.
DIAGNOSIS OF ECTOPIC PREGNANCY
CG levels in normal pregnancies increase >66% every two days (doubling time <2 days); while in ectopic pregnancy CG
levels increase <66% every two days (doubling time >2 days). Pelvic ultrasound at 5-6 weeks post LMP shows a gestational
sac and at 7-8 weeks a fetal heartbeat. Kadar combined ultrasound and CG levels using a Discriminatory CG Zone (6500
IU/L). Above 6500 IU/L pelvic ultrasound is diagnostic for intrauterine pregnancy; if no shadow is found a diagnostic
laparoscopy is recommended. However, transvaginal ultrasound is now the standard diagnostic test for ectopic pregnancy
(2). The superior resolution allows earlier detection of ectopic and intrauterine pregnancy using a CG cutoff between 1000
and 2000 IU/L.
OTHER SOURCES OF CHORIONIC GONADOTROPIN
Serum CG levels are required for diagnosis and monitoring of trophoblastic disorders (hydatiform mole, choriocarcinoma,
etc.), and should be continued until levels are undetectable for a year. Approximately half of patients with testicular tumors
have detectable CG levels. These malignancies may produce intact CG, free alpha subunit, free beta subunit (or a
combination) which serve as markers for treatment.
Non Trophoblastic Malignancies: Approximately 20% of patients with non trophoblastic/non testicular tumors have
detectable serum CG (usually <20 IU/L). The highest incidence occurs in gynecological (ovarian, breast, and cervical
carcinoma) head and neck, breast, lung, gastrointestinal tract tumors and malignant melanoma. Detection of even low CG
levels raises the concern of an undiagnosed non trophoblastic malignancy. However, in most malignancies CG testing is not
helpful; one exception being the alpha subunit as a marker of pancreatic endocrine tumors.
Low CG levels in Normal Individuals: Highly sensitive IRMA (sandwich) assays demonstrate low levels of CG in some
normal males. Some menopausal females demonstrate even higher levels, usually from 5 to 25 IU/L.
FALSE POSITIVE RESULTS - "DISCORDANT" OR "PHANTOM" CG
Increasing use of high sensitivity assays for CG has resulted in increased occurrence of test results that are difficult to
interpret. "Discordant" CG results are relatively low (usually <30 IU/L, but occasionally up to 200 IU/L) and should be
suspected in the following situations. The positive result is inconsistent with clinical information (i.e. no evidence of
pregnancy, trophoblastic disease or malignancy). Analysis in competitive binding assays at increasing dilutions yield
increasingly higher CG values when corrected for dilution. The result is negative in at least one alternate quantitative assay
for CG (i.e. utilizing different technology) with comparable sensitivity. Finally, the sequential CG level does not change
significantly with time for a given procedure for a given patient.
The circulating substance measured by the assays may be intact molecule, free alpha subunit, free beta subunit, fragment
molecules or the hyperglycosylated molecule. Additionally, circulating compounds may interfere variably (positive or negative)
in the different assays; these include rheumatoid factor, heterophile antibodies, Human Antimouse Antibodies (HAMA) and
CG bound to immunoglobulin.
The issue that remains is whether or not there is an early malignancy which is not clinically apparent. This consideration has
resulted in some individuals having a laparotomy or a laparascopy for diagnosis of potential malignancy consequent to a false
positive (i.e. discordant) but reproducible serum CG result.
The Hamilton Regional Laboratory Medicine Program currently uses the Abbott AXSYM total b -hCG assay for serum CG
assays. It is standardized against the Third International Standard 75-537 and detects both the intact whole molecule and the
free Beta subunit down to a level of 2 IU/L. The urine pregnancy test is the Abbott Test Pack Plus which detects only the
intact whole molecule at a level of 25 IU/L or greater. The medical staff are asked to keep these limitations in mind and
contact the laboratory if there are any diagnostic difficulties.
REFERENCES
1. Butler SA et al. Clin Chem 2001; 47:2131-2136 2. Ankum WM. BMJ 2000; 321:1235-1236.
A.W. (George) Luxton, B.Sc., MD, FRCPC, FCACB
Henderson Hospital, 905-527-4322, Ext. 42066
Clinical Chemistry
Hamilton Regional Laboratory Medicine Program
M. Donahoe, B.Sc., ART
Hamilton General Hospital, 905-527-4322, Ext. 46130
Clinical Chemistry
Hamilton Regional Laboratory Medicine Program