Arnaud Agin

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.

Efficacy of a new blocker against anti-ruthenium antibody interference in the Elecsys free triiodothyronine assay.

Following the introduction of a new formulation of the Elecsys free triiodothyronine (fT3) assay from Roche Diagnostics (Meylan, France) in our laboratory in November 2003, we experienced an increased frequency of high fT3 results not associated with low thyrotropin (TSH) values (1). The Elecsys fT3 assay is a competitive immunoassay involving a specific anti-T3 sheep monoclonal antibody labeled with a ruthenium complex, T3-biotin, and streptavidin-coated microparticles. The occurrence of a high fT3 result without a low TSH level is rare. Apart from the statistical 2.5% of euthyroid patient results found slightly above the upper fT3 limit, this association can be found in sera from patients treated with T3 or triiodothyroacetic acid (2, 3) (these treatments are not frequently used in our patients) or in sera containing an interfering substance: heterophilic antibodies, anti-T3 antibodies (4), anti-ruthenium antibodies (involved in the Elecsys assay) or interfering drugs such as non-steroidal antiinflammatory drugs (5). We report here the results obtained for 15 sera collected between May 2004 and March 2006. In these samples, high Elecsys fT3 results could not be confirmed with the Ria-gnost fT3 radioimmunoassay from CIS biointernational (Gyf-surYvette, France). Run in a two-step format, this assay is free from anti-T3 antibody interference (4). Riagnost fT3 results were within the reference interval (Table 1). The procedures were in accordance with the Helsinki Declaration of 1975 and the subsequent 1996 amendments. No drug known to possibly interfere in fT3 assays identified from clinical notes, interference from hete-

Plasma copeptin, a possible prognostic marker in cirrhosis

Copeptin, secreted stoichiometrically with vasopressin, demonstrated its prognostic role in various diseases other than cirrhosis.

Extensive study of human insulin immunoassays: promises and pitfalls for insulin analogue detection and quantification.

Abstract Background: Over the last few decades, new synthetic insulin analogues have been developed. Their measurement is of prime importance in the investigation of hypoglycaemia, but their quantification is hampered by variable cross-reactivity with many insulin assays. For clinical analysis, it has now become essential to know the potential cross-reactivity of analogues of interest. Methods: In this work, we performed an extensive study of insulin analogue cross-reactivity using numerous human insulin immunoassays. We investigated the cross-reactivity of five analogues (lispro, aspart, glulisine, glargine, detemir) and two glargine metabolites (M1 and M2) with 16 commercial human insulin immunoassays as a function of concentration. Results: The cross-reactivity values for insulin analogues or glargine metabolites ranged from 0% to 264%. Four assays were more specific to human insulin, resulting in negligible cross-reactivity with the analogues. However, none of the 16 assays was completely free of cross-reactivity with analogues or metabolites. The results show that analogue cross-reactivity, which varies to a large degree, is far from negligible, and should not be overlooked in clinical investigations. Conclusions: This study has established the cross-reactivity of five insulin analogues and two glargine metabolites using 16 immunoassays to facilitate the choice of the immunoassay(s) and to provide sensitive and specific analyses in clinical routine or investigation.

Use of insulin immunoassays in clinical studies involving rapid-acting insulin analogues : Bi-insulin IRMA preliminary assessment

Abstract Background: In clinical studies involving rapid-acting analogues (RAAs), insulin immunoreactivity is frequently measured, including endogenous, regular insulin (RI) and RAA immunoreactivities. Such a procedure implies equivalent cross-reactivities of all insulins present in serum. Commercially available human insulin immunoassays have been widely used, but their limitations (including hemolysis and anti-insulin antibodies) were not fully investigated. The aims of our study were to compare cross-reactivities of RI and RAAs in buffer and in serum and to investigate insulin immunoassay pitfalls. Methods: Cross-reactivities were assessed using Bi-insulin IRMA (Schering Cis-Bio International) in phosphate-buffered saline (PBS)-1% bovine serum albumin (BSA) and in pools of sera spiked with RI and RAAs (lispro and aspart). To investigate the influence of hemolysis, a pool of sera spiked with RAA was mixed with a concentrated hemolysate (final hemoglobin concentration 10g/L) and incubated for 3h at room temperature. To determine interference by anti-insulin antibodies, insulin was removed using charcoal from 18 sera with anti-insulin antibodies and from 17 sera without detectable anti-insulin antibodies. These insulin-free samples were then spiked with RI and RAAs and the immunoreactivity was determined. Results: Compared with buffer, cross-reactivity in serum for RI, lispro and aspart was lower (35%, 29% and 26% lower, respectively). Hemolysis degraded almost all RI and RAAs contained in the serum (≥95%). Anti-insulin antibody interference was significant for RI and RAAs (p≤0.004) and correlated with anti-insulin antibody level in the serum (p≤0.001). Conclusions: In serum, RI and RAA cross-reactivities are slightly lower than in buffer. For RAA assessment, hemolysed samples should be discarded and anti-insulin antibodies should be removed from samples before immunoreactivity measurements. Clin Chem Lab Med 2006;44:1379–82.

Thyroid-stimulating hormone and free thyroxine on the ADVIA Centaur immunoassay system: A multicenter assessment of analytical performance.

OBJECTIVES We assessed the analytical performance of the TSH and FT4 assays on ADVIA Centaur in a multicenter national evaluation. DESIGN AND METHODS A precision study and a method comparison were performed. Reference values stated by the manufacturer were checked from 379 normal subjects. RESULTS For TSH and FT4, the intra-assay CVs were below 2.3 and 5.2%, respectively, and the inter-assay CVs below 4.4% and 7.2%, respectively. Therefore, the precision and reproducibility were acceptable. Bland-Altman bias plots revealed good correlation and agreement with Cobas assays. TSH and FT4 data yielded reference ranges of 0.64-3.24 mIU/L and 10.5-18.9 pmol/L, respectively. CONCLUSION These assays demonstrate reliable characteristics. The reference ranges obtained can be used for interpretation of thyroid function.

Should functional sensitivity of a new thyroid stimulating hormone immunoassay be monitored routinely? The ADVIA Centaur® TSH3-UL assay experience

OBJECTIVES We emphasize the importance of routine follow-up of subnormal TSH concentrations with QC materials. DESIGN AND METHODS The functional sensitivity (FS) of the ADVIA Centaur system TSH assay was assessed. We report the values yielded for QC materials in two clinical laboratories. RESULTS The FS was <0.02 mIU/L. The low-TSH QC (a serum pool) showed unacceptable between-lot imprecision (mean 0.0252 mIU/L, CV 22%). CONCLUSION We do encourage healthcare laboratories to constitute low-TSH serum pools to ensure that the results they report meet 3rd-generation criteria.

Effect of the oestrogen receptor antagonist fulvestrant on the cirrhotic rat lung

It has been postulated that cirrhosis‐related lung vasodilatation and the subsequent hepatopulmonary syndrome are partly explained by an increased estradiol level through an enhanced endothelial formation of nitric oxide (NO). In this study, we assessed whether the oestrogen receptor antagonist fulvestrant (F) improves cirrhosis‐related lung abnormalities. Cirrhosis was induced in rats by chronic bile duct ligation (CBDL). Four groups were studied: CBDL, CBDL+F, sham, and sham+F. Histological, immunohistochemical, and Western blot analyses were performed on lung samples. In the lung, the endothelial NO synthase and the nitrotyrosine protein expressions were increased in CBDL as compared to sham rats. Both parameters were significantly reduced by fulvestrant in the CBDL rats. Surprisingly, the level of pVASP (an indirect marker of NO formation and action) was decreased in CBDL rats, and fulvestrant had no effect on this parameter. The level of the vascular endothelial growth factor, the diameter of small lung vessels, and the number of macrophages were increased in CBDL lungs in comparison with sham lungs, and these parameters were unaffected by fulvestrant treatment. In conclusion, fulvestrant may not be relevant to improve lung abnormalities in cirrhosis because NO may not be biologically active and because key events contributing to the lung abnormalities are not affected by fulvestrant.