Between-Method Variation in Human Chorionic Gonadotropin Test Results

Abstract

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 ␤-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 resultswere 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

Standardization of Assays for Human Chorionic Gonadotropin

“…The concentration measured, 130 IU/L, corresponds to 0.374 nmol/L hCG, and this corresponds to 3.74 g/L hCG␤cf, i.e., similar to the nominal concentration, 4 g/L. Thus, with the exception of hCG␤cf, no variant is grossly over-or underestimated by any of the assays studied (8 ). This comparison also demonstrates the advantage of using molar concentrations to express values for various forms of hCG (5 ).…”

“…The authors draw the conclusion that use of impure assay calibrators in combination with variable recognition of hCG subunits and other variants is an important cause of the variation. Analysis of several commercial hCG preparations revealed that most of them contain degraded forms, subunits, and fragments (8 ), and earlier studies have shown that rather crude hCG preparations are used as calibrators (4 ).…”

hCG, Hyperglycosylated hCG, Pituitary hCG, Cancer hCG and Fetal hCG

“…specific antibody which is O-linked oligosaccharide sensitive, leading to poor detection of hCG-2 [40,41].…”

“…The existence of the six semi-independent molecules was demonstrated by structure studies [9,10,46]. Bold underline poor detection 6 of 9 5 of 9 6 of 9 7 of 9 5 of 9 5 of 9 7 of 9 7 of 9 1 of 9 7 of 9 6 of 7 LH crossreactivity 0.01% 0.01% 0.01% 0.01% 0.32% 0.01% 0.01% 0.01% 0.04% 0.53% 0.01% Table 9: Detection of serum hCG-related molecules by different automated total hCG tests (40,41). ßCTP is ß-subunit C-terminal peptide, RIA is radioimmunoassay.…”

Forensic pregnancy testing: a special case in molecular diagnostics

“…The underlying principle of all such tests lies in the immunodetection of proteins that are specifically produced as result of pregnancy. Most often, the human chorionic gonadotropin (hCG) hormone, which is released from the placenta a few days after conception, is employed and detected via immunoassays [2,3]. However, there is at least one field of potential application of molecular pregnancy diagnostics where the detection principle of the currently available tests appears problematic owing to the amount and kind of material available: the forensic field.…”