Differences in Recognition of the 1st WHO International Reference Reagents for hCG-Related Isoforms by Diagnostic Immunoassays for Human Chorionic Gonadotropin Catharine M. Sturgeon, 1* Peter Berger, 2 Jean-Michel Bidart, 3 Steven Birken, 4 Chris Burns, 5 Robert J. Norman, 6 and Ulf-Håkan Stenman, 7 on behalf of the IFCC Working Group on hCG BACKGROUND: The 1st WHO International Reference Reagents (IRRs) for 6 human chorionic gonadotropin (hCG)-related molecular variants, highly purified and calibrated in substance concentrations by the IFCC Working Group for hCG, permit experimental eluci- dation of what commercially available hCG methods measure in molar terms and enable assessment of their fitness for clinical purposes. METHODS: Pools containing known amounts of the IRRs spiked into normal human serum were issued to participants through the UK National External Quality Assessment Service for hCG for a period of 7 years. Among 16 assays used, 4 recognized only hCG, whereas 6 recognized hCG and its free -subunit (hCG), and 6 recognized hCG, hCG, and the beta core fragment. RESULTS: Differences in calibration of current hCG as- says are moderate. Mean recovery of the current Inter- national Standard (IS), hCG IS 75/589, was 107% (range 93% to 126%), whereas that of the IRR 99/688 for hCG was 139% (range 109%–164%). Between- method variation for the latter (CV 12.3%) was also greater than for IS 75/589 (CV 8.8%). Recognition of hCG varied markedly (CV 37%). Most assays overes- timated it, but 2 RIAs produced results that were slight underestimations. Recognition of the beta core frag- ment was even more variable (CV 57%) and was closest to equimolarity for the RIAs. CONCLUSIONS: Assays for hCG show considerable vari- ation in their recognition of various forms of hCG, and this variablility is the most important cause of method- related differences in hCG results in serum and an even more important cause of method-related differences in urine measurements. Equimolar recognition of the major hCG isoforms is essential if between-method comparability for hCG is to be improved. © 2009 American Association for Clinical Chemistry A prerequisite for improved standardization of immu- noassay methods is precise knowledge of what is being measured in clinical samples, which frequently contain heterogeneous mixtures of related molecules (1, 2 ). To this end the IFCC Working Group for human chori- onic gonadotropin (hCG) 8 established an unambigu- ous and user-friendly nomenclature that describes hCG and its 6 most important isoforms, and subse- quently prepared the 1st WHO International Reference Reagents (IRRs) for these 6 isoforms (1, 3 ). The 6 iso- forms were prepared by use of previously developed purification methods, which included hydrophobic interaction chromatography and reversed-phase HPLC (3). The high purity and homogeneity of the preparations—as confirmed by results of amino acid and sequence analyses, carbohydrate composition, electrophoretic patterns, and immunoassay studies— subsequently enabled their calibration in substance concentrations (i.e., molar units) (Table 1) (4, 5 ). The use of substance concentrations addresses a major limitation of earlier International Standards (IS) and International Reference Preparations (IRP) for hCG-related molecules, namely the 3rd and 4th hCG WHO IS (75/537 and 75/589) (which are essentially identical), hCG 1st IRP (75/551), and hCG 1st IRP 1 Department of Clinical Biochemistry, Royal Infirmary, Edinburgh, UK; 2 Institute for Biomedical Aging Research, Austrian Academy of Sciences, Innsbruck, Austria; 3 Department of Clinical Biology, Institut Gustave-Roussy, Villejuif, France; 4 Department of Obstetrics and Gynecology, College of Physicians and Surgeons of Columbia University, NY, NY; 5 National Institute of Biological Standards and Control, Potters Bar, UK; 6 Research Centre for Reproductive Health, Department of Obstetrics and Gynaecology, University of Adelaide, The Queen Elizabeth Hospital, Woodville, Australia; 7 Department of Clinical Chem- istry, Helsinki University Central Hospital, Finland. * Address correspondence to this author at: Department of Clinical Biochemistry, Royal Infirmary of Edinburgh, Edinburgh EH16 4SA, United Kingdom. Fax 44-131-242-6882; e-mail C.Sturgeon@ed.ac.uk. Received January 29, 2009; accepted April 24, 2009. Previously published online at DOI: 10.1373/clinchem.2009.124578 8 Nonstandard abbreviations: hCG, human chorionic gonadotropin; IRR, Interna- tional Reference Reagent; IS, International Standard; IRP, International Refer- ence Preparation; hCGn, nicked hCG; cf, core fragment. Clinical Chemistry 55:8 1484–1491 (2009) Endocrinology and Metabolism 1484 Downloaded from https://academic.oup.com/clinchem/article/55/8/1484/5629203 by guest on 13 June 2022