Selecting an appropriate hCG test for managing gestational trophoblastic disease and cancer.

Human chorionic gonadotropin (hCG) is a glycoprotein composed of 2 dissimilar subunits, alpha and beta, joined non-covalently. This hormone is not only heterogeneous in peptide structure but also in combination of subunits and in the structure of carbohydrate side chains. Common hCG-related molecules in serum samples include regular hCG, hyperglycosylated hCG (ITA), nicked hCG, nicked ITA, hCG missing the beta-subunit C-terminal extension, free alpha-subunit, free beta-subunit, free beta-subunit missing the C-terminal extension, hyperglycosylatedfree beta-subunit and nickedfree beta-subunit. The same molecules plus beta-core fragment are present in urine samples. While ITA and regular hCG predominate in pregnancy samples, any one of these multiple hCG-related molecules may be the principal source of immunoreactivity in gestational trophoblastic disease, gestational trophoblastic neoplasm, choriocarcinoma and placental site tumor cases as well as in testicular cancer and germ cell tumor. As such it is critical to appropriately detect all these isoforms in the management of these diseases. Only 2 tests, the DPC Immulite (DPC, Inc., Los Angeles, California) and U.K. RIA (radioimmunoassay) (used at Charing Cross Hospital, London) appropriately detect all these hCG-related molecules. False positive hCG results are a major problem in the management of gestational trophoblastic disease and cancer. A particular problem is observed with the Abbott AxSym test. This test is flawed in design. It should be avoided in the management of gestational trophoblastic disease and cancer. As shown in a blind study, a proportion of false positive samples in the Abbott AxSym test (Abbott Laboratories, Inc., Chicago, Illinois) can also be false positive with the U.K. RIA; none are false positive with the DPC Immulite test. Results clearly show that the DPC Immulite/Immulite 2000 is the only appropriate assay for monitoring patients with gestational trophoblastic disease or cancer.

Sialic acid-deficient invasive trophoblast antigen (sd-ITA): a new urinary variant for gestational Down syndrome screening.

BACKGROUND: Invasive trophoblast antigen (ITA), also known as hyperglycosylated hCG, is a glycosylation variant of hCG produced by invasive cytotrophoblast cells. Publications show that ITA is potentially a sensitive test for Down syndrome screening, reportedly detecting 38 and 80% of cases at a 5% false-positive rate in the first and second trimesters of pregnancy respectively. ITA has six oligosaccharides varying greatly in terminal sialic acid content (0-19 residues), leading to wide-charge heterogeneity. With the objective of developing an improved Down syndrome screening test, we examined the charge, or sialic acid variants, of ITA. METHODS: Twenty urine samples were collected from the 16th to 18th week of gestation from 10 patients with trisomy 21 fetuses, demonstrated by karyotype analysis, and from 10 patients with normal karyotype pregnancies. Proteins in each sample were separated by preparative isoelectric focusing. Fractions were collected, pooled into the pl ranges of 3.0 to 4.0, 4.1 to 5.0, 5.1 to 6.0 and 6.1 to 7.0 and assayed for ITA. RESULTS: A difference in the distribution of ITA charge variants was demonstrated between cases and controls, most apparent in the less acidic (pl 5.1-7.0) range. The controls and Down syndrome samples contained 26.7 +/- 14% versus 45.6 +/- 20%, respectively, of the less acidic form of ITA (P = 0.02). CONCLUSIONS: The ITA in Down syndrome cases is composed of a much higher proportion of less acidic ITA. This could be the major source of elevated levels of ITA in Down syndrome pregnancies. It is postulated that the development of a new assay specifically measuring ITA that is deficient in sialic acid might enhance the utility of the already highly sensitive screening tests for Down syndrome.

Between-method variation in human chorionic gonadotropin test results.

BACKGROUND: Results on sera and calibrators vary 1.4- to 2.3-fold among commercial human chorionic gonadotropin (hCG) assays. The relative contributions of calibrators, standards, hCG charge isoforms, and major structural variants to this variation have not been quantified. METHODS: Purified hCG was separated by isoelectric focusing into four fractions with pI ranges of 3-4, 4-5, 5-6, and 6-7. These four fractions together with pure hCG, hyperglycosylated hCG, hCG beta-subunit (hCGb), nicked hCG, and hCGb core fragment (hCGbcf) were tested in nine commonly used commercial serum assays for hCG. The compositions of pure hCG preparations, standards, and commercial hCG preparations were determined by immunoassay. RESULTS: The three pure hCG preparations and the four hCG charge isoforms each showed parallel responses in the nine commercial hCG assays. Although wide variations were found in the detection of hCG structural variants by the nine assays (range for hyperglycosylated hCG, 468-1544 IU/L; for hCGb, 3187-5535 IU/L; for nicked hCG, 2736-4240 IU/L; and for hCGbcf, <2-130 IU/L), this did not correlate with the between-method variation observed in results for the three pure hCG preparations. Commercial preparations of hCG and calibrators showed great variation in their content of hCG structural variants (from 34% to 100% intact hCG). CONCLUSIONS: Intermethod differences in hCG results were not explained by changes in responses attributable to hCG charge isoforms or to hCG structural variants, but wide variation was observed in concentrations of hCG structural variants in calibrators and in detection of these structural variants. Differences in assay specificity and in composition of the calibrators are the most likely sources of between-method variation.

Charge variants in serum and urine hCG.

BACKGROUND: All serum and urine pregnancy tests sold in the United States are calibrated against the WHO 3rd and 4th International Standards (3rd and 4th I.S.) of Human Chorionic Gonadotropin (hCG). These standards have been isolated from pregnancy urine; however, they are used to calibrate, and generate antibodies used in both urine and serum hCG tests. hCG molecules may vary in sialic acid content; this changes the acidity of the molecule. Published studies have shown that these carbohydrate elements may alter recognition of hCG in different serum and urine hCG tests. We investigated the charge variants of hCG in serum and urine samples, and in hCG standards. METHODS: Samples were analyzed by preparative isoelectric focusing. Charge variants of hCG were quantitated using the DPC Immulite hCG assay. RESULTS: A difference was observed in the proportion of charge variants in urine and serum samples. There was a significantly higher proportion of more-acidic variants in the urine samples. CONCLUSIONS: Urine-derived standards may not be representative of serum hCG and therefore may not be appropriate for calibrating serum assays. Variation among hCG test results when using different immunoassays has been a persistent problem for years. Additional studies are needed to focus on the molecular dissimilarity of urine and serum hCG, as well as other factors, to determine their significance and contribution to the problem of interassay variation when comparing hCG results.

Accuracy of home pregnancy tests at the time of missed menses.

OBJECTIVE: The purpose of this study was to investigate the validity of home pregnancy tests (HPTs) around the time of the missed menses. STUDY DESIGN: Levels of human chorionic gonadotropin (hCG) were determined in urine around the time of the missed menses, and 5th and 95th centiles were calculated. In a blind study evaluating 18 brands of HPTs, each was tested six times with five concentrations of hCG, 0 to 100 mIU/mL. Sensitivity was defined as the concentration at which six of six brands gave positive results, at the suggested reading time or at an extended time. RESULTS: It was estimated that a sensitivity of 12.5 mIU/mL was needed to detect 95% of pregnancies at the time of missing menses. Only 1 of 18 HPTs had this sensitivity. If faintly discernible results considered, 2 of 18 brands gave false-positive or invalid results. Clear positive results were given by only 44% of the brands at 100 mIU/mL hCG. If one accepted faintly discernible and extended reading times, all brands responded at 100 mIU/mL. A test with 100 mIU/mL sensitivity would detect approximately 16% of pregnancies at the time of missed menses. CONCLUSION: The utility of HPTs is questioned. Clinicians need to be aware of the limitations of current HPT brands.

Hyperglycosylated hCG (invasive trophoblast antigen, ITA) a key antigen for early pregnancy detection.

OBJECTIVES: Hyperglycosylated human chorionic gonadotrophin (hCG) is an hCG variant with extra-large O-linked oligosaccharides, produced by phenotypically invasive cytotrophoblast cells in choriocarcinoma and pregnancy. It is the principal form of hCG produced in the first weeks of gestation. We investigated the importance of hyperglycosylated hCG in pregnancy testing and its detection by current hCG tests. DESIGN AND METHODS: We measured the concentration of hyperglycosylated hCG and total hCG in 512 pregnancies throughout gestation. We assessed and compared the abilities of 14 commonly used commercial laboratory hCG tests and 18 home pregnancy tests to detect regular and hyperglycosylated hCG. RESULTS: Hyperglycosylated hCG is the principal source of hCG-related immunoreactivity in early pregnancy. In the week following missing menses, hyperglycosylated hCG measurements may be more sensitive than regular hCG measurements in detecting pregnancy. Of 14 commercial laboratory hCG tests, 3 appropriately detected hyperglycosylated hCG standard. Of 18 different home pregnancy products 11 poorly or very poorly detected this key antigen. CONCLUSIONS: Hyperglycosylated hCG may be the key molecule in the detection of early pregnancy. However, the majority of tests poorly detected or failed to detect this key antigen. New pregnancy tests are needed that either solely detect hyperglycosylated hCG or equally detect regular hCG and hyperglycosylated hCG.