HCG REFERENCE SERVICE
SPECIALIZED TESTS AT HCG REFERENCE SERVICE

 

   A parallel serum (2 x 10 ml red-top) and urine sample (spot urine, >10ml) are requested. While the hCG Reference Service is strictly a consulting program, 30 to 50 specialized immunoassays are run on the serum and urine sample to help interpret the the nature and source of hCG and related molecules. The number of assays and types of test are tailored to fit the patients circumstances. While a fee is required for the consultation, no specific charge is made for these tests.

   Tests include commercial hCG immunoassays (FDA approved). The hCG Reference Service currently uses the DPC Immulite hCG test as its first information point to evaluate serum and urine samples. This test was chosen because it detects similarly all known forms of hCG and its break down products present in serum and urine samples in pregnancy, cancer and trophoblastic disease (regular hCG, nicked hCG, hyperglycosylated hCG, hCG missing the ß-subunit C-terminal peptide, free ß-subunit, nicked free ß-subunit, free ß-subunit missing the CTP, and urine ß-core fragment). This test also has given no false positive results in clinical practice. This test is described and compared with other in the hCG Reference Service Report 2002. In addition to running the DPC Immulite hCG test on untreated serum samples, we run the DPC Immulite on serum samples treated with a heterophilic antibody blocking agent (Scantibodies Inc. HBT). This blocks heterophilic antibody interferences with tests. Heterophilic antibodies are seemingly the most common cause of false positive hCG results.

   The hCG Reference service runs 5 specialized tests, detecting specific forms of hCG. In most cases, serum and urine samples are tested undiluted, and at 1/2 and 1/5 dilutions in the DPC Immulite hCG and in each of the 5 specialized immunoassays, developed to aid in the consultation. The 5 specialized immunoassays are all in-house (non FDA approved) 96 well plate sandwich-type assays. The properties and specificities of these 5 assays have been described in publications (references 1-6, below). The assay all use pure hCG standards calibrated by amino acid analysis. In addition, a first International Reference Preparation is tested. Results for intact hCG assays (assays a. and b.) are presented in ng/ml and mIU/ml. Results for other assays are given as ng/ml and as molar equivalents of hCG in mIU/ml. When needed, we run special high sensitivity tests, detecting <0.5 mIU/ml of hCG-related molecule immunoreactivity. The high sensitivity assays use coated star tubes, using 0.5 ml serum or urine in each tube (in duplicate), and an extra-long 18 hour incubations.

assay a. Intact hCG-1. This assay detects hCG only (regular hCG, nicked hCG, hyperglycosylated hCG and hCG missing the ß CTP. The sensitivity ~2 mIU/ml (high sensitivity test ~0.5 mIU/ml). In addition, assay a. is performed with serum treated with a heterophilic antibody blocking agent (Scantibodies Inc. HBT). This blocks heterophilic antibody interferences with tests. Heterophilic antibodies are seemingly the most common cause of false positive hCG results.

assay b. Nicked hCG only. This is a new test specific for the nicked or inactive form of hCG only (hCG cleaved at a single point in the ß-subunit, either between ß47-48, 43-44 or 44-45). Sensitivity ~3 mIU/ml.

assay c. Hyperglycosylated hCG  (Invasive Trophoblast Antigen or ITA) only. This is a new test specific for a carbohydrate variant of hCG with larger sugar side chains, produced by invasive trophoblast cells in early pregnancy, persistent trophoblast disease, choriocarcinoma and germ cell cancers). Sensitivity ~4 mIU/ml (high sensitivity test ~1 mIU/ml).

assay d. hCG free ß-subunit only. The free ß-subunit is secreted directly by trophoblast cells and by cancer cells, it is also produced by the slow dissociation of regular hCG, or more rapid split of nicked or hyperglycosylated hCG. Sensitivity ~2 mIU/ml (molar equivalents of hCG). In addition, assay d. is performed with serum treated with a heterophilic antibody blocking agent (Scantibodies Inc. HBT). This blocks heterophilic antibody interferences with tests.

assay e.   hCG ß-core fragment only. ß-core fragment is the terminal degradation product of hCG. It is only normally detected in urine samples. The finding of ß-core fragment in serum, but not urine, is consistent with the presence of HAMA (human anti-mouse antibodies) or heterophilic antibodies which cause phantom or false-positive hCG results. Sensitivity ~1 mIU/ml, high sensitivity test ~0.3 mIU/ml (molar equivalents of hCG). In addition, assay e. is performed with serum treated with a heterophilic antibody blocking agent (Scantibodies Inc. HBT). This blocks the heterophilic antibody interference.

    The hCG Reference Service investigates the nature of the hCG present in parallel serum and urine samples (regular hCG, nicked hCG, hyperglycosylated hCG, hCG missing the ß-subunit C-terminal peptide, free ß-subunit, nicked free ß-subunit, free ß-subunit missing the CTP, and urine ß-core fragment). From the nature of the hCG in serum and urine, published information and our data bank, inferences are made about the origin of the hCG-related molecules (pregnancy, benign trophoblast disease, persistent trophoblast disease or choriocarcinoma, germ cell or other cancer, phantom or false-positive hCG).

    Elevated proportions of hyperglycosylated hCG (>10% of total) are associated with persistent trophoblastic disease or choriocarcinoma (reference 7, below). Primarily low concentrations of regular hCG (and usually no significant hyperglycosylated hCG) are present in individuals with quiescent gestational trophoblastic disease or with unexplained elevated hCG (see hCG Reference Service Report 2002). The finding of free ß-subunit in blood (no hCG) and ß-core fragment in urine may be associated with germ cell, bladder or other non-trophoblastic malignancy (references 3 and 8, below). The finding of predominantly nicked hCG is linked to clearance following evacuation of a mole or treatment of choriocarcinoma (reference 9, below). The finding of hCG-related molecules in serum but not urine, more than 5-fold varying hCG concentration in different immunoassays, of serum hCG results blocked by heterophilic antibody blocking agent HBT, and of ß-core fragment immunoreactivity in serum, are indicators of false-positive or phantom hCG (reference 5 and 10, below, see hCG Reference Service Report 2002).

REFERENCES

1.    Cole, L.A., Kardana, A., Park S-Y., Braunstein, G. The deactivation of hCG by nicking and dissociation. J Clin Endocrinol Metab 76:704-10, 1993.

2.    Kardana, A., Cole, L.A. Human chorionic gonadotropin b -subunit nicking enzymes in pregnancy and cancer patient serum. J. Clin. Endocrinol. Metab.,79:761-767, 1994

3.    Cole, L.A., Tanaka, A., Kim, G.S., Park, S-Y., Koh, M.W., Schwartz, P.E., Chambers, J.T., and Nam, J-H. Beta core fragment (b -core / UGF / UGP), a tumor marker: Seven year report. Gynecol. Oncol., 60:264-270, 1996.

4,    Cole L. Immunoassay of hCG, its Free Subunits and Metabolites. Clin Chem 43:2233-2243, 1997.

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

6.    Cole, L.A. Shahabi, S., Rinne, K.M., Oz, U.A., Bahado-Singh, R.O., Mahoney, M.J. Urinary Screening Tests for Fetal Down Syndrome: II. Hyperglycosylated hCG. Prenat Diagn, in press, 1999.

7.     Elliott M, Kardana A, Lustbader J, Cole L. Carbohydrate and peptide structure of the alpha- and beta-subunits of hCG from normal and aberrant pregnancy and choriocarcinoma. Endocrine 7:15-32, 1997.

8.    Alfhan, H., Haglund, C., Roberts, P., and Stenman, U.H. Elevation of free ß-subunit of human choriogonadotropin and core b fragment of choriogonadotropin in serum and urine of patients with malignant pancreatic and biliary disease Cancer Res. 52, 4628-4633 (1992).

9.    Cole L, Kohorn E, Kim G. Detecting and monitoring trophoblast disease: New perspectives in measuring hCG levels. J Reprod. Med. 39:193-200, 1994.

10.    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.

11.    Birken, S., Krichevsky, A., O’Connor, J., Schlatterer, J., Cole, L.A., Kardana, A., and Canfield, R. Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient. Endocrine J., in press, 1999.