Intermethod variability in TSH-receptor antibody measurement: implication for the diagnosis of Graves disease and for the follow-up of Graves ophthalmopathy.

BACKGROUND: We compared the analytical and clinical performance of 3 porcine thyroid receptor antibody (TRAb) methods (1 second- and 2 new third-generation systems) with the conventional TRAb assay based on the human recombinant TSH receptor (hTRAK). PATIENTS AND METHODS: We obtained sera from 86 patients with untreated Graves disease (GD) and 71 healthy controls. We measured TRAb concentrations by radioreceptor assay using the hTRAK (Brahms) or the porcine TSH receptor (pRRA) from Beckman-Coulter, by electrochemiluminescence immunoassay (ECLIA) with the Elecsys/Cobas (Roche), and by ELISA using the Medizym TRAb clone (Medipan). RESULTS: Between-run assay imprecision was < or =10% and < or =7.6% for hTRAK and ECLIA, but reached 14% and 14.9% for ELISA and pRRA, respectively. Maximal specificity and sensitivity close to 100% were obtained for hTRAK, ECLIA, and ELISA. pRRA failed to detect positive TRAbs in 5 GD patients. Although calibrated against the same reference standard 90/672, the assays displayed a high intermethod variability. The results were significantly higher by ECLIA and lower by ELISA and pRRA compared with hTRAK. Patients with ophthalmopathy had higher TRAb results by ELISA and pRRA than those without eye disease. CONCLUSIONS: Second- and third-generation TRAb assays had similar diagnostic sensitivities in the diagnostic evaluation of GD. Despite the use of the same reference standard for calibration, high intermethod variability in TRAb assay results was seen in untreated GD patients. Assay harmonization is necessary for correct interpretation in the follow-up of Graves ophthalmopathy.

Determination of interferon beta neutralizing antibodies in multiple sclerosis: improvement of clinical sensitivity of a cytopathic effect assay.

INTRODUCTION: Neutralizing antibodies (NAb) against interferon beta (IFNbeta) reduce treatment efficacy in patients with multiple sclerosis (MS). OBJECTIVE: We used the cross-reactivity of NAbs against IFNbeta-1a or IFNbeta-1b for improving the sensitivity of NAb measurement. PATIENTS/METHODS: The study included sera from 185 MS patients treated at least 12 months (T1) with IFNbeta-1a (Rebif; n=62 or Avonex; n=61) or IFNbeta-1b (Betaferon; n=62). NAbs were measured by CPE in all the sera using the WISH cell line infected by the bovine stomatitis vesicular virus. NAb titres were expressed in ten-fold reduction (TRU)/mL. NAb-positive titres at T1 were also measured 3 to 6 months later (T2). RESULTS: At T1, with the classical CPE assay using the IFNbeta type (1a or 1b) according to the molecule administered, 29/180 (15.7%) patients had positive NAb titres: 9/62 (14.5%), 14/62 (22.6%) and 6/61 (9.8%) subjects were treated with Betaferon, Rebif and Avonex, respectively. When IFNbeta-1a (Rebif or Avonex) was used, NAb-positive results were found in 33/185 (17.8%) patients. They included the 29 patients previously found positive with the classical CPE method and 7 other subjects treated with Betaferon and NAb-negative against IFNbeta-1b. All the 33 NAb-positive patients at T1 were positive 3 to 6 months later. CONCLUSION: IFNbeta-1a should be used for the NAb determination for an optimal evaluation of NAb-positive patients in MS patients treated with IFNbeta.

Neutralizing antibodies to interferon beta in multiple sclerosis: analytical evaluation for validation of a cytopathic effect assay.

BACKGROUND: Recent guidelines have recommended the use of validated assays for the measurement of neutralizing antibodies (NABs) to interferon beta (IFNbeta) in patients with multiple sclerosis (MS). In an attempt of validation, we studied the analytical performance of a bioassay based on antiviral cytopathic effect (CPE) using WISH cells and the vesicular stomatitis virus (WISH/VSV CPE). METHODS: NAB titres measured with the WISH/VSV CPE assay in 63 sera from IFNbeta-treated MS patients were compared to those obtained with the reference CPE method using A549 cells and the encephalomyocarditis virus. Binding antibodies (BABs) were measured using a capture ELISA as a screening test for NABs. RESULTS: No false-negative BAB was obtained in our patients. The between-run coefficients of variation (CVs) determined with log10 titres of the NIH anti-IFNbeta (G038-501-572) yielded good results (

Analytical performance of a new two-step ADVIA Centaur estradiol immunoassay during ovarian stimulation.

Measurement of estradiol is useful in women undergoing ovarian stimulation for in vitro fertilization and embryo transfer (IVF-ET). The analytical performance of a new two-step estradiol assay (ADVIA Centaur) estradiol-6 III from Bayer Diagnostics) was evaluated in 41 sera from 11 women undergoing ovarian stimulation. The results were compared to those obtained with two radioimmunoassays (RIAs; RIA Estradiol Immunotech IM 1663 from Beckmann Coulter and Coat-A-Count Estradiol from Diagnostic Products Corporation) and with one chemiluminescent immunoassay (CLIA; ADVIA Centaur estradiol-6). The ADVIA Centaur) estradiol-6 III assay was the most sensitive assay, with a functional sensitivity of 55 pmol/L. Within- and between-run coefficients of variation calculated for the new ADVIA Centaur assay ranged from 3.3% to 9%, which was better than the precision obtained for the other assays. A dilution test showed serum interferences when estradiol was measured in non-diluted samples. No statistical difference was observed between the estradiol results obtained in diluted sera with the new two-step ADVIA Centaur assay and those measured with the Immunotech RIA and the other CLIA. In conclusion, this new, two-step estradiol assay performed on the ADVIA Centaur system displays suitable sensitivity, precision and intermethod agreement with the Immunotech RIA for the measurement of serum estradiol concentrations in women undergoing ovarian stimulation and IVF-ET. For correct linearity, estradiol measurement should be performed on diluted samples.