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Technical Briefs |
1 Department of Biochemistry and Hormonology, Hôpital Antoine Béclère, 157 rue de la Porte de Trivaux, 92141 Clamart cedex, France
aauthor for correspondence: fax 33-1-45374745, e-mail joelle.taieb{at}abc.ap-hop-paris.fr
Rapid steroid hormone immunoassays often agree poorly, especially at normal and low concentrations (1)(2)(3)(4). These problems result from low assay specificity, inadequate standardization, and poor optimization of the methods over the large range of concentrations seen clinically (5)(6)(7). These systems are often unsuitable for clinical applications that require a low detection limit, such as the following: (a) estradiol measurements in men [<110 pmol/L; (<30 pg/mL)] or children [from <18 pmol/L to 165 pmol/L (<5 pg/mL to 45 pg/mL)] (8) and evaluation of down-regulation by gonadoliberin analogs before in vitro fertilization and embryo transfer (IVF-ET) programs; (b) progesterone determinations during ovarian stimulation, with values <3.2 nmol/L (<1 ng/mL) on the day of human chorionic gonadotropin administration predictive for pregnancy in IVF-ET (9)(10); (c) testosterone assays for children [from <0.35 nmol/L to 5 nmol/L (<0.1 ng/mL to 1.5 ng/mL)] and women [<2.4 nmol/L (<0.7 ng/mL)] (11)(12). Furthermore, limits of detection determined with the zero calibrator are generally far below the lowest concentration that can be reliably quantified in human serum [functional sensitivity (13)(14) or limit of quantitation (LOQ) (15)].
In this study, we analyzed and compared detection limits and functional sensitivities for nine estradiol (E2) and eight progesterone (P) immunoassays.
Between 1997 and 2001, we tested nine automated multianalyte systems for E2 and/or P measurements: ACS-180 (Bayer Diagnostics), Advia-Centaur (Bayer Diagnostics), Vitros ECi (Ortho-Clinical Diagnostics), Architect i2000 (Abbott Laboratories), Kryptor (Brahms), Immuno-1 (Bayer Diagnostics) for E2 and P; IMx (Abbott Laboratories), Elecsys 2010 (Roche Diagnostics) for E2; and AxSYM (Abbott Laboratories) for P. All of these nonisotopic immunoassays are based on competitive methods and involve detection by direct (Architect i2000, Advia-Centaur, ACS-180) or indirect (Vitros ECi) chemiluminescence, electrochemiluminescence (Elecsys 2010), fluorescence (IMx, AxSYM), spectrophotometry (Immuno-1), or Trace® technology (Kryptor). We have also studied one direct RIA for E2 and P (Coatria 125I; Bio-Mérieux).
We determined the detection limit, defined as the concentration at 2 SD above the mean signal value of the zero calibrator (free of analyte) from each assay (measured 10 times within a single analytical run), with respect to the concentration for another calibrator concentration. If no zero calibrator was included in the calibration set (most of the systems studied required master curve calibration carried out by the manufacturer and required only two calibrators to adjust the master curve), we asked the manufacturer to supply it. We determined the functional sensitivity (not usually determined by the manufacturer), defined as the lowest concentration of analyte that can be measured with a run-to-run imprecision (CV) of 20% (13). The interassay precision profile was used to determine, for each analyte and each assay, the concentration corresponding to CV of 20%. This profile was determined with pools of sera covering the calibration curves, analyzed (once for each analytical run) over 30 days with two different lots of reagent, according to the protocol of Spencer et al. (14).
Detection limits were 11 pmol/L (3 pg/mL; Kryptor) to 77 pmol/L (25 pg/mL; Architect i2000) for E2 and 0.19 nmol/L (0.06 ng/mL; Kryptor) to 0.17 ng/mL (0.54 nmol/L; AxSYM) for P (Table 1
). All immunoassays except E2 Architect and E2 IMx had detection limits close to 37 pmol/L (10 pg/mL) for E2 and close to 0.32 nmol/L (0.1 ng/mL) for P. The values obtained in our study were close to those given by the manufacturers (cited in package inserts). Only E2 Architect i2000 had a detection limit (77 pmol/L; 25 pg/mL) different from that given by the manufacturer (<66 pmol/L; <18 pg/mL). However, Architect i2000 did not give results under the detection limits programmed into the analyzer: E2, <66 pmol/L (<18 pg/mL); P, <0.32 nmol/L (<0.1 ng/mL).
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Functional sensitivities were 20 pmol/L (5.5 pg/mL; Kryptor) to 169 pmol/L (46 pg/mL; Architect i2000) for E2 and 0.32 nmol/L (0.1 ng/mL; Kryptor) to 1.43 nmol/L (0.45 ng/mL; ACS-180) for P.
The functional sensitivities of direct E2 and P immunoassays were two- to fourfold higher than the detection limits of these tests. A detection limit is generally defined by the manufacturer and cited alone in the package insert. This leads clinical laboratories to adopt an inappropriate detection limit on a patient’s report, and unresolved questions remain concerning the use of these methods for clinical situations requiring assays with high sensitivity (16).
Assuming that the same (or very similar) calibrators are used (gravimetric weighing of the pure analyte, dissolution in a suitable solvent before the preparation of serum calibrators, and testing against the isotope dilution–gas chromatography–mass spectrometry reference method), differences between assays lead to calibration having different matrix effects (ionic strength, pH, and protein concentration) (17).
Other possible reasons for the differences observed between assays are the characteristics of the different antibodies and their different affinities and titers, along with specificity (18)(19)(20). However, we observed no clear difference between assays using monoclonal antibodies (Architect i2000 for E2 and P; AxSYM, Immuno-1, ACS-180, and Advia-Centaur for P) and those using polyclonal antibodies (Coatria 125I, Vitros ECi, and Kryptor for E2 and P; Elecsys 2010, Immuno-1, IMx, ACS-180, and Advia-Centaur for E2). We found no difference among the results obtained by the isotope method (Coatria 125I) and chemiluminescence, electrochemiluminescence, fluorescence, or spectrophotometry methods, but we did find a difference between the results obtained with the Trace technology (Kryptor) and those obtained with all other methods. Trace seems to be the most sensitive detection system among the E2 and P immunoassays tested.
None of the E2 and P assays tested seemed to have the functional sensitivity required for the evaluation of E2 and P in sera from children or of E2 in sera from men. These assays have been optimized for clinical applications in which high concentrations are expected (e.g., E2 determination for the monitoring of ovarian stimulation), and it is important to consider functional sensitivities as the lowest measurable concentrations. However, these tests could be used for determinations in sera from women to evaluate down-regulation before (E2) and during (P) ovarian stimulation. In such cases, despite the low precision obtained in these concentration ranges (CV, 
10–15%), these rapid procedures are convenient for clinicians (results are available within 1 h). Although these automated systems are easy to use, with short cycle times and low costs, their use should be avoided for sera in which low concentrations are expected. We did not analyze testosterone in this study, but all the problems raised for E2 and P also apply to testosterone, especially in the concentration range found in women (12). We hope that manufacturers will soon agree to include the determination of functional sensitivity in package inserts to enlighten users concerning the limitations of these assays. Indeed, a collaborative study was recently established between an academic laboratory and industry for the standardization of steroid immunoassays (21)(22)(23).
Acknowledgments
We thank the following manufacturers for supplying the assay reagents and systems free of charge: Abbott Laboratories, Bayer Diagnostics, Bio-Merieux, Brahms, Ortho-Clinical Diagnostics, and Roche Diagnostics.
References
The following articles in journals at HighWire Press have cited this article:
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 M. M. Kushnir, A. L. Rockwood, W. L. Roberts, E. G. Pattison, W. E. Owen, A. M. Bunker, and A. W. Meikle Development and Performance Evaluation of a Tandem Mass Spectrometry Assay for 4 Adrenal Steroids Clin. Chem., August 1, 2006; 52(8): 1559 - 1567. [Abstract] [Full Text] [PDF]
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 J. P. Bilezikian What's Good for the Goose's Skeleton is Good for the Gander's Skeleton. J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1223 - 1225. [Full Text] [PDF]
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E. Orwoll, L. C. Lambert, L. M. Marshall, K. Phipps, J. Blank, E. Barrett-Connor, J. Cauley, K. Ensrud, S. Cummings, and for the Osteoporotic Fractures in Men Study Group Testosterone and Estradiol among Older Men J. Clin. Endocrinol. Metab., April 1, 2006; 91(4): 1336 - 1344. [Abstract] [Full Text] [PDF]
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M. M. Kushnir, A. L. Rockwood, W. L. Roberts, E. G. Pattison, A. M. Bunker, R. L. Fitzgerald, and A. W. Meikle Performance Characteristics of a Novel Tandem Mass Spectrometry Assay For Serum Testosterone Clin. Chem., January 1, 2006; 52(1): 120 - 128. [Abstract] [Full Text] [PDF]
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Z. Cao, T. A. Swift, C. A. West, T. G. Rosano, and R. Rej Immunoassay of Estradiol: Unanticipated Suppression by Unconjugated Estriol Clin. Chem., January 1, 2004; 50(1): 160 - 165. [Abstract] [Full Text] [PDF]
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J. Taieb, C. Benattar, R. Diop, A. S. Birr, and A. Lindenbaum Use of the Architect-i2000 Estradiol Immunoassay during in Vitro Fertilization Clin. Chem., January 1, 2003; 49(1): 183 - 186. [Full Text] [PDF]
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