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Letters to the Editor |
University of North Carolina, School of Medicine, Department of Pathology and, Laboratory Medicine, Chapel Hill, NC
aAddress correspondence to this author at: CB#7525, University of North Carolina School of Medicine, Chapel Hill, NC 27599. Fax 919-966-9490; e-mail dgrenach{at}unch.unc.edu.
To the Editor:
Human chorionic gonadotropin (CG) is a heterodimeric glycoprotein hormone composed of noncovalently associated 
and ß 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 ß subunit is a component of nicked CG (CGn), CG ß-subunit (CGß), nicked CG ß-subunit (CGßn), and CG ß-core fragment (CGßcf).
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 ß 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 CGß (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), CGß (99/650), CGßn (99/692), CG (99/720), and CGßcf (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+ß assay on a Roche Elecsys® 2010 (Roche Diagnostics) immunoassay analyzer. This method uses specific monoclonal anti-ß capture and signal antibodies that recognize CG, CGn, CGß, CGßn, and CGßcf. 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®+ One-Step 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.
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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 CGß and CGßn. CGßcf was consistently detected by the Clinitest hCG and hCG Combo SP, whereas it was inconsistently detected by Quick-Vue. No device detected CG.
These data support the hypothesis that some qualitative CG devices detect nondimeric isoforms, despite being designed to detect only heterodimeric CG isoforms. Given the previously described purity of the WHO reference reagents (3), the most likely explanation for the detection of nondimeric isoforms is that the specificities of the antibodies used by device manufacturers are incompletely characterized and may react with numerous CG isoforms. Unfortunately, details of the selectivity of these antibodies are proprietary information.
Unless a clinical advantage is identified, there is limited motivation to demonstrate that pregnancy screening tests detect isoforms other than intact CG. Detection of other forms of CG may be important for early detection of pregnancy. McChesney et al. (4) reported that in urine samples collected during early pregnancy, intact CG is sometimes transiently undetectable and sometimes present in lower concentrations than nondimeric isoforms. Similarly, Butler et al. demonstrated that hyperglycosylated CG is the predominant isoform in early pregnancy urine and advocated that assays be optimized to detect it (5). Investigating whether the different selectivities (analytical specificities) of qualitative urine tests affect diagnostic sensitivity is part of an ongoing study that includes further characterization of the concentrations of CG isoforms in early pregnancy urine.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
References
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