Françoise Gasser

Clinical Evaluation of Nine Free Thyroxine Assays: Persistent Problems in Particular Populations

Abstract Over the past decade, numerous papers have addressed the various methodological problems encountered with free thyroxine (FT4) assays. We evaluated the clinical performance of nine FT4 assays in five centres, using a panel of 310 sera: 156 from euthyroid controls; 27 from hyperthyroid patients; 34 from untreated hypothyroidism; 22 from patients with renal failure; 30 from women in the last trimester of pregnancy; 23 from patients on thyroid substitutive therapy; and 18 from patients treated with amiodarone. Only three methods showed a Gaussian distribution of FT4 concentrations. Reference ranges were calculated using the 2.5th and 97.5th percentiles. A significant difference was observed between FT4 values in men and women. The areas under the receiver operating characteristic (ROC) curves ranged from 0.996 to 1 for hyperthyroidism and from 0.973 to 1 for hypothyroidism. In sera from patients with renal failure and from pregnant women, method-dependent biases were observed and confirmed with dilution experiments. In conclusion, current FT4 assays show good performance regarding the diagnosis of overt dysthyroidism. Nevertheless, FT4 measurements are still vulnerable to method-dependent artefacts in particular populations such as patients with renal failure and pregnant women.

Determination of Free Triiodothyronine by Six Different Methods in Patients with Non-Thyroidal Illness and in Patients Treated with Amiodarone:

We performed a methodological comparison of free triiodothyronine (FT3) estimates in patients with liver cirrhosis and renal failure. Patients were classified in terms of severity of illness on the basis of their total triiodothyronine, total thyroxine and reverse triiodothyronine profiles. FT3 levels, measured in direct dialysis, microchromatography, labelled analogue and two-step immunoextraction assays were significantly (P < 0·01) lower than the control group in all patient categories. However, FT3 measured by a labelled antibody radioimmunoassay was significantly reduced only in the most severely ill sub-group of patients. In a further group of patients on long-term amiodarone therapy for cardiac disease all FT3 methods, with the exception of the labelled antibody radioimmunoassay and an analogue method, yielded significantly (P < 0·01) reduced levels. A significant negative association between FT3 and subject age was demonstrated for all methods except the labelled antibody radioimmunoassay, and a weak but significant negative correlation between log thyrotropin and FT3 was only seen with this assay. Three methods demonstrated a correlation (P < 0·02) with albumin levels in patients with the ‘low T3 syndrome’. In this group, albumin had a predictive value (P ≤ 0·02) for four out of six assays as determined by stepwise variable selection. Our findings suggest that users of FT3 assays should exercise caution in interpreting results in non-thyroidal illness and amiodarone treated patients, as there are method-related differences in the profiles obtained.

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-

Improved Specificity of a New Direct Assay for Urinary Cortisol: Application in Corticoid Treated Patients

Abstract Twenty four hours urinary free cortisol (UFC) excretion has been determined in 35 eucortisolic control patients, in seven of them before and after tetracosactide (Synacthen®) stimulation and in 18 patients treated by anti-inflammatory steroids. Results of the new direct Immunotech RIA (DIm) were compared to those of the INCSTAR RIA kit with (ECA) or without methylene chloride extraction (DCA). In controls DIm UFC (106.2±45.8 nmol/24h) was significantly lower than DCA UFC (397±119 nmol/24h) and than ECA UFC (127±49 nmol/24h). After tetracosactide stimulation, median of DCA/DIm ratio decreased from 3.61 to 1.88 whereas ECA/DIm ratio did not change significantly (1.31 to 1.06). In treated patients most DCA and ECA results were over the upper limit of controls but only 5 DIm results were increased. DIm assay showed good specificity and practicability and may be used with benefit in the evaluation of the adrenal gland function.

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.

Free prolactin determinations in hyperprolactinemic men with suspicion of macroprolactinemia

BACKGROUND The Elecsys prolactin (PRL) assay reacts more strongly with macroprolactin than the Centaur PRL assay. We evaluated Elecsys direct and free PRL measurements vs. Centaur direct PRL measurements, in sera with and without macroprolactin. METHODS PRL was measured using Elecsys and Centaur direct assays and the Elecsys assay in the supernatant obtained after PEG precipitation (free PRL) in 34 sera from 34 hyperprolactinemic male subjects (Elecsys direct PRL>434 mIU/l) classified, according to the PRL recovery after PEG precipitation, as: negative, i.e. without predominant macroprolactin (recovery %>50, n=12), positive (recovery %<40, n=18) or indeterminate (n=4). RESULTS The positive bias between Elecsys direct and Centaur PRL results was clearly influenced by the presence of macroprolactin and the mean bias between Elecsys free and Centaur prolactin values was near zero in the negative and positive groups. Among 14 patients from the positive group presenting clinical conditions possibly ascribable to hyperprolactinemia, Elecsys free and Centaur PRL levels were normal in seven and increased in five. In the negative and positive groups considered together, Elecsys free PRL agreed well with Centaur prolactin (28/30). The poor concordance observed for the indeterminate samples underlined the heterogeneity of macroprolactin. CONCLUSION Elecsys free PRL determination can be used to reduce the marked influence of macroprolactin in this assay.

Evaluation of Elecsys® free triiodothyronine assay: relevance of age-related reference ranges

OBJECTIVE This study aimed at evaluating Elecsys free triiodothyronine (FT3) assay performed on an Elecsys 2010 system, while paying special attention to age relationship in euthyroid subjects. DESIGN AND METHODS FT3 levels were measured in 149 euthyroid control subjects aged between 2 and 92 years old, 33 hyperthyroid and particular euthyroid patients: female in the last 3 months of pregnancy (n = 30), nonthyroidal ill hospitalized in medical (NTlm, n = 31), or intensive care units (NTlc, n = 31) and amiodarone-treated (n = 27). RESULTS FT3 was inversely related to age in controls (r = -0.67). Three reference ranges were used: below 20 years 4.5-9.0 pmol/L, between 20 and 60 years 3.9-7.2, and over 60 years 2.4-6.5. Compared to age-matched controls, FT3 decreased in pregnancy, NTlm, NTlc, and amiodarone groups. Use of age-related reference ranges improved the specificity markedly in amiodarone patients and to a lesser extent in NTlm and TClc patients. CONCLUSIONS The reliability of the Elecsys FT3 assay was found to be satisfactory for clinical use, when the age of patients was taken into account.

Analytical and clinical evaluation of a new one-step non-analogue radioimmunoassay for serum-free thyroxine

We evaluated analytically and clinically the new one-step non-analogue free thyroxine (FT4) assay (Amerlex-MAB from Amersham), using a labelled monoclonal thyroxine-specific antibody as tracer, in comparison with the Gammacoat two-step FT4 kit (Baxter). Analytical performances of the new kit were excellent: within and between run coefficients of variation were < 5% in the working range. Clinical sensitivities for hypo- and hyperthyroidism were comparable for both kits (FT4 Amerlex-MAB 95% confidence interval: 12–25 pM). When serum was supplemented with albumin we observed a slight decrease in FT4 values measured by both kits. When oleate was added to serum we noted a moderate increase with the Amerlex-MAB kit up to 10 mM oleate added and a much more marked increase with the two-step kit. Results obtained with patients from particular euthyroid populations, known to have low albumin or high free fatty acids concentrations or to have perturbed FT4 results when measured by an analogue-based method, agreed with those of the in vitro studies. With these patients the specificity of the Amerlex-MAB FT4 results was good but slightly decreased compared with the two-step FT4 method, except for heparin-treated patients who were all classified according to their euthyroidal status (17/17 instead of 13/17 with the two-step kit).

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.