Detection of unexpected isoforms of human chorionic gonadotropin by qualitative tests.

To the Editor: Human chorionic gonadotropin (CG) is a heterodimeric glycoprotein hormone composed of noncovalently associated and subunits that are synthesized by trophoblastic tissue in pregnancy. Systemic modification and degradation of the intact CG molecule and subunits leads to molecular heterogeneity in the serum and urine (1 ). The subunit is a component of nicked CG (CGn), CG -subunit (CG ), nicked CG -subunit (CG n), and CG -core fragment (CG cf). Qualitative urine testing for CG is employed in point-of-care and laboratory settings because it is a rapid and effective pregnancy screen. These tests typically detect 20 to 25 IU CG/L and reportedly detect pregnancies at 8 to 12 days after conception (1 ). Most qualitative test devices are chromatographic immunometric assays that use antibodies that recognize distinct epitopes on the and subunits, enabling the detection of heterodimeric CG isoforms (CG and CGn). Recently, we reported unexpectedly positive qualitative urine pregnancy tests in a patient with an endometrial adenocarcinoma that synthesized only free CG (2 ). Consequently, we questioned the analytical specificity (selectivity) of qualitative urine pregnancy tests and investigated the detection of specific CG isoforms by 6 commonly used qualitative CG tests. The 4th International Standard of Chorionic Gonadotropin, Human (4th IS-CG) and purified 1st WHO reference reagent preparations of 5 CG isoforms were obtained from the National Institute for Biological Standards and Controls (Hertfordshire, UK). Ampoules containing lyophilized residues of 4th IS-CG (75/589), CGn (99/642), CG (99/650), CG n (99/692), CG (99/720), and CG cf (99/708) were reconstituted with 1.0 mL CG-free male serum or PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4) and then diluted 1:100 into urine from premenopausal, nonpregnant women. The 4th IS-CG served as a positive control for all test devices, and the 1st WHO reference reagent preparations were free from contamination (3 ). Total CG concentrations were determined in duplicate for the prepared urine samples using the hCG assay on a Roche Elecsys 2010 (Roche Diagnostics) immunoassay analyzer. This method uses specific monoclonal anticapture and signal antibodies that recognize CG, CGn, CG , CG n, and CG cf. We have validated this method for quantifying CG in a urine matrix (data not shown). Six commonly used qualitative CG devices were selected: SureVue Serum/Urine hCG-STAT (Fisher Scientific Co.), Clinitest hCG (Bayer HealthCare), Quick-Vue OneStep hCG Combo (Quidel Corp.), Osom Card Pregnancy Test (Genzyme Diagnostics), hCG Combo SP (Cardinal Health), and the ICON II HCG (Beckman Coulter). The manufacturers report that the methods detect 20 to 25 IU CG/L; other properties of the devices are shown in Table 1. Each device was tested and interpreted 5–10 times with the prepared urines, according to the manufacturer’s instructions. As shown in Table 1, all qualitative CG devices detected dimeric CG isoforms (CG and CGn) but only the Osom Card Pregnancy test did so exclusively. The remaining devices detected CG and CG n. CG cf was consistently detected by the Clinitest