Falsely Decreased Human Chorionic Gonadotropin (hCG) Results Due to Increased Concentrations of the Free β Subunit and the β Core Fragment in Quantitative hCG Assays

Grenache, David G.; Greene, Dina N.; Dighe, Anand S.; Fantz, Corinne R.; Hoefner, Daniel M.; McCudden, Christopher R.; Sokoll, Lori J.; Wiley, Carmen L.; Gronowski, Ann M.

doi:10.1373/clinchem.2010.143479

Cited by 29 publications

(14 citation statements)

References 23 publications

(36 reference statements)

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“…There are several different variants or isoforms of hCG, including intact hCG, nicked hCG, free β -subunit (hCG β ), nicked hCG β , and degradation products including β -subunit core fragment (hCG β cf) (9, 10). Almost all hCG β cf is produced during renal excretion and accounts for ≥50% of the hCG found in urine (4, 11, 12). …”

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confidence: 99%

Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Woldemariam

Butch

2014

Clinical Chemistry

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BACKGROUND Human chorionic gonadotropin (hCG) stimulates testosterone production by the testicles. Because of the potential for abuse, hCG is banned (males only) in most sports and has been placed on the World Anti-Doping Agency list of prohibited substances. Intact hCG, free β-subunit (hCGβ), and β-subunit core fragment (hCGβcf) are the major variants or isoforms in urine. Immunoassays are used by antidoping laboratories to measure urinary hCG. Cross-reactivity with isoforms differs among immunoassays, resulting in widely varying results. We developed a sequential im-munoextraction method with LC-MS/MS detection for quantification of intact hCG, hCGβ, and hCGβcf in urine. METHODS hCG isoforms were immunoextracted with antibody-conjugated magnetic beads and digested with trypsin, and hCGβ and hCGβcf unique peptides were quantified by LC-MS/MS with the corresponding heavy peptides as internal standard. hCG isoform concentrations were determined in urine after administration of hCG, and the intact hCG results were compared to immunoassay results. RESULTS The method was linear to 20 IU/L. Total imprecision was 6.6%-13.7% (CV), recovery ranged from 91% to 109%, and the limit of quantification was 0.2 IU/L. Intact hCG predominated in the urine after administration of 2 hCG formulations. The window of detection ranged from 6 to 9 days. Mean immunoassay results were 12.4-15.5 IU/L higher than LC-MS/MS results. CONCLUSIONS The performance characteristics of the method are acceptable for measuring hCG isoforms, and the method can quantify intact hCG and hCGβ separately. The limit of quantification will allow LC-MS/MS hCG reference intervals to be established in nondoping male athletes for improved doping control.

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Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Woldemariam

Butch

2014

Clinical Chemistry

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“…No high-dose hook effect is observed with hCG concentrations up to 2,000,000 IU/l in serum and the assay is not affected by the hCG variant effect. [11]…”

Section: 0 Materials and Methodsmentioning

confidence: 99%

“…hCG may not be detected if samples are collected in very early pregnancy and results may be falsely decreased due to the high-dose hook effect [11,12]. False positives results can occur when interfering antibodies react with the assay reagents.…”

Section: 0 Introductionmentioning

confidence: 99%

Analytical and clinical validation of the Immulite 1000 hCG assay for quantitative analysis in urine

Cate¹,

Moffett²,

Gronowski³

et al. 2013

Clinica Chimica Acta

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Background The Siemens Immulite hCG assay detects all major hCG variants in serum. Currently, this assay is only FDA approved for qualitative measurement of hCG in urine. Methods Complete validation of the hCG assay in urine was performed on the Siemens Immulite 1000 immunoassay platform. Reference intervals were established for females <55 y, females ≥55 y, and males 20–70 y. Results The limit of quantitation was 2.0 IU/l. The Immulite hCG assay was precise for measuring hCG in urine from pregnant patients with intra- and inter-assay imprecision of <11% CV. The assay was linear over a dynamic range of 2–2600 IU/l and 2–3500 IU/l for hCG and hCGβ respectively. The assay was non-linear for hCGβcf. No hook effect was observed at concentrations up to 1,200,000 pmol/l, for hCGβ or hCGβcf. The reference intervals were <2.0 IU/l for males, <2.2 IU/l for females <55 y, and <12.2 IU/l for females ≥55 y. Conclusion The Immulite 1000 hCG assay can accurately quantify hCG in urine.

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“…When hCG levels are high, both the capture and tracer antibodies saturate, and the signal response is decreased. The "hook effect" occurs when non-sandwiched tracer antibodies are washed away with the excess material resulting in a falsely low or negative test (Grenache et al, 2010). This may delay diagnosis and lead to mismanagement of patients.…”

Section: Contents Lists Available At Sciverse Sciencedirectmentioning

confidence: 99%

“…This hook effect was first described by Miles et al in 1976, and is more commonly seen in assays for prolactin and thyroid stimulating hormone. The Advia Centaur assay is used at our hospital, and has been shown in previous studies to have dose-dependent falsely decreased results at hCG concentrations greater than 940,000 (Grenache et al, 2010); this effect is usually not significant until concentrations are greater than those typically seen in malignancy. Current hCG assays measure non-nicked (biologically active) hCG and one of seven other combinations of hCG-related molecules, attributing to variation in different hCG assays (Cole, 1997).…”

Section: Contents Lists Available At Sciverse Sciencedirectmentioning

confidence: 99%

Abnormally low hCG in a complete hydatidiform molar pregnancy: The hook effect

Nodler

Kim

Alvarez

2011

Gynecologic Oncology Case Reports

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Falsely Decreased Human Chorionic Gonadotropin (hCG) Results Due to Increased Concentrations of the Free β Subunit and the β Core Fragment in Quantitative hCG Assays

Cited by 29 publications

References 23 publications

Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Immunoextraction–Tandem Mass Spectrometry Method for Measuring Intact Human Chorionic Gonadotropin, Free β-Subunit, and β-Subunit Core Fragment in Urine

Analytical and clinical validation of the Immulite 1000 hCG assay for quantitative analysis in urine

Abnormally low hCG in a complete hydatidiform molar pregnancy: The hook effect

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