We compared the analytical and clinical performance of two free-thyroxine (FT4) assays--a solid-phase radioimmunoassay, Spectria, and a time-resolved fluoroimmunoassay, Delfia, both of them two-step methods--with the performance of a direct radioimmunoassay, Nichols, to measure FT4 concentration in equilibrium dialysate of undiluted serum. The three assays showed comparable analytical performance. We tested clinical utility in sera from 135 healthy subjects with and without thyroxine-binding abnormalities and in 61 patients with and without thyroidal illnesses. We found significant differences for FT4 measured by different assays in sera from the same euthyroid patients. To explain the differences, we studied the influence of temperature on performance and calibration. Most important was the neglected fact that the association constant for the binding of thyroxine to thyroxine-binding globulin decreases when the temperature rises from 20 to 37 degrees C, causing a doubling of FT4. The two-step assays, if performed at room temperature without a well-defined calibration, can give misleading FT4 concentrations. This is the case when sera from patients with thyroxine-binding abnormalities are measured against kit standards, made up in normal human sera. If an assay is to reflect the in vivo FT4 concentration at body temperature in all types of samples, it should be performed at body temperature. For practical reasons 37 degrees C is recommended, and reference values should be defined at 37 degrees C. The same might be valid for other free-hormone assays.

SUMMARY. Three sensitive assays (Behring, Organon and LKB-Pharmacia) for measurement of serum thyrotropin (TSH) were evaluated. All three assays showed good precision, sensitivity, linearity and for practical purposes negligible high dose hook effects. The correlations between the assays were excellent, but due to standardisation and/or matrix effects there were incomplete recoveries in two kits and the kits showed systematic differences of up to 20% in the TSH values obtained.The clinical utility of the three assays was investigated in patients with thyroidal and non-thyroidal illnesses and in healthy subjects with thyroxine binding abnormalities.According to the results of the analytical and clinical evaluation, all three kits are acceptable and reliable as first-line thyroid function tests. SAMPLES AND CONTROL SERASerum samples from all subjects and serum pools containing high (range 5-27 ml.I/L) and undetectably low concentrations of TSH were, after collection, stored in aliquots at -18°C.The human TSH reference preparations (WHO 1 st IRP (MRC: 68/38) and WHO 2 nd IRP (MRC: 80/558» were obtained from the

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We describe a modification of the second-derivative spectrophotometric assay for carboxyhaemoglobin in blood. Using the original procedure we have often observed a time-dependent change in the signal leading to unreliable results. By using phosphate-buffered-saline (pH 7.4) instead of ammonia to dissolve the deoxygenating agent (sodium dithionite), we obtained stable and reproducible second-derivative signals from which the percentage carboxyhaemoglobin in the patients' samples can be calculated.

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