Elecsys Testosterone Assay Evaluated

Serum testosterone (T) is one of the most commonly requested human steroids. In women, measurement of serum T concentration is useful in evaluating hirsutism, alopecia, and menstrual disorders. In boys and men, its utility is related to the investigation of testicular dysfunction. It has also been used to monitor the treatment of patients with congenital adrenal hyperplasia, as well as the antiandrogen therapy of patients with prostate cancer (1)(2)(3). In recent years, some laboratories measured T using RIAs with or without in-house extractions (4). Anomalous high results were reported with some commercial direct assays for T, which were related to interference of binding globulins (5)(6). Later, partially and fully automated assays were developed. These automated assays are convenient because they do not require any solvent extraction before immunoassay (7)(8). In 1997, Boehringer Mannheim (BM) launched a fully automated method for their Elecsys® 2010 system, which develops electrochemiluminescence immunoassays (ECLIAs) and provides short incubation times. We report our analytical and clinical evaluation of this assay performed over an 8-week period, following the guidelines of the ECCLS document (9). Samples were assayed in singletons, according to the manufacturer’s instructions. Routine patient sera were used for linearity, recovery, method comparison studies, and clinical validation (following the procedures approved by the ethical committee of our hospital). The system can develop both competitive and sandwich-format electrochemiluminescent assays, and in particular, the Elecsys T assay is based on the competition principle. A calibration curve is instrument-generated specifically for each analyte by a 2-point calibration, and a master curve is provided via the reagent bar code. The minimum detectable concentration of the Elecsys 2010 T assay is 0.069 nmol/L, as reported by the manufacturer. We find it more useful to evaluate the precision profile of the T assay. Sera from young children (all of them with low T) were used to prepare 10 serum-pool samples with T concentrations of 0.069–1.041 nmol/L. These samples were tested on 10 different nonconsecutive days in 1 month. Two different reagent lots and two software versions of the Elecsys 2010 system were used during this study. The functional sensitivity, defined as the lowest concentration of analyte measured reproducibly with an interassay variation of 20% or less (10), was 0.236 nmol/L (Fig. 1 A). Functional sensitivity of the ECLIA T assay and comparison with RIA. (A) Functional sensitivity (CV, 20%) for the ECLIA T assay (0.236 nmol/L), including the prediction lines of the mean (IC 95%). (B) Linear regression analysis of the comparison between the ECLIA and RIA T assays, including a plot (inset) of individual values <10 nmol/L: T-ECLIA (nmol/L) = −0.117 + 0.977 T-RIA (nmol/L); r2 = 0.986. Two BM human serum-based controls [Precicontrol Universal (PU; cat. no. 1731416) at two different concentrations (PU1 and PU2)] and different human serum pools at clinically relevant analyte concentrations were analyzed 20 times a day for the intraassay imprecision study and daily over 20 different nonconsecutive days for 1 month for the interassay imprecision study. Intraassay CVs were 1.2%, 2.0%, and 0.24% at mean T concentrations of 0.12, 0.70 (PU1), and 45 (PU2) nmol/L, respectively. Interassay CVs were 5.7%, 2.6%, 2.9%, and 1.1% at mean T concentrations of 0.86 (PU1), 9.0, 25, and 47 (PU2) nmol/L, respectively. The sera from three men with high concentrations of T were diluted 1:2, 1:5, and 1:10 with the Universal BM diluent (cat. no. 1732277). The evaluations were made by the percentage of difference between the expected and observed values of each sample. Observed values were 102–113% of those expected. Different amounts of a serum containing a known high concentration of T were added to three serum samples with different concentrations of T. The observed values were 98–114% of those expected. Two samples with low T concentrations were assayed immediately after a sample of high T concentration; no carryover problem was found in this case or when we measured the 131 samples for the method comparison study that ranged from 0.069 to 50 nmol/L, as was expected in an automated system that changes its tips and cuvettes with every sample. Generally, hemolysis, lipemia, and icterus are less significant for immunoassays or competitive binding assays than for the classic colorimetric analytical methods. We have not evaluated these specimen-based interferences, which usually have a method-specific nature and are not usual in a method like the ECLIA T assay. Nevertheless, many different categories of serum samples were tested in the following correlation studies. For this analyte, no difference between the Elecsys 2010 and the comparative assay was observed in patient serum samples with increased bilirubin, rheumatoid factor, immunoglobulins, triglycerides, or hemolyzed sera. Serum samples from 131 subjects were chosen by their T concentration to cover the Elecsys T assay analytical range (0.069–52 nmol/L) and also to include T at clinically relevant concentrations. Among these subjects we included 10 hypogonadal children, 32 patients with prostate cancer after antiandrogen therapy, 28 hirsute women, and 14 females with polycystic ovarian syndrome. These samples were tested by the Elecsys, and the results were compared with those obtained with the previous routine RIA assay of our laboratory (Spectria-Orion Diagnostica). The relation between the ECLIA and the RIA T is shown in Fig. 1B (11). The regression analysis showed a slope of 0.996, an intercept of −0.324, and a correlation coefficient of r = 0.977 (r=0.986; Sy‖x = 1.385). The 95% confidence interval [IC 95% (b), 0.957–0.998] showed that the slope was slightly different from 1.0, but the 95% confidence interval of the intercept [IC 95% (a), −0.450−0.216] was not statistically different from zero. We also evaluated whether the results obtained by the two methods could be interchangeable in two selected groups of patients. The first group (G1) consisted of 32 males with prostate cancer under treatment with antiandrogen therapy. The second (G2) included 28 hirsute women and 14 females with polycystic ovarian syndrome. We calculated the linear regression for both groups, finding a better correlation for the males (r = 0.798) than for the females (r = 0.696). These differences cannot be attributed to differences in the ranges of values for men and women, and may reflect interference from binding globulins and also cross-reactivity with structurally related compounds in the T immunoassays. We did not have the opportunity to validate this T assay with a reference method such as gas chromatography-mass spectrometry (7). We evaluated the agreement between the ECLIA and the RIA for classification, using a reference interval of 0.694–2.776 nmol/L in women. All the men in G1 were expected to have T <0.9 nmol/L (3). The concordances of the two methods were 96% for G1 and 80% for G2. Taking both groups together (74 subjects), the two methods provided the same classification in 69 (89%) of the cases. The Elecsys 2010 assay for T in serum is appealing for use in the routine laboratory. The assay is automated and rapid, giving the first result in 18 min, with 58 additional results in the following hour. The calibration curve was stable for at least 2 months, in contrast to the RIA for T, which requires a new calibration curve in each protocol. In addition, the T assay allows the use of nonradioactive reagents and offers an extended range, allowing a minimal number of sample dilutions. The Elecsys T assay showed a high degree of reproducibility; linearity and comparison studies were satisfactory and we found no carryover problem. It clearly separated hyperandrogenic women from healthy ones, and hypogonadal or androgen-blocked patients from healthy men. These characteristics tend to give quite a good diagnostic potential and reinforce the T assay as a cost-effective front-line androgen function test. Laboratorio de Hormonas, Servicio de Análisis Clínicos, Hospital General Universitario de Alicante, C/Maestro Alonso 109, 03010 Alicante, Spain We thank Boehringer Mannheim, Barcelona, Spain, for supplying the T assay reagents free of charge.