Unforeseen effect of thyroxine binding globulin when using the microencapsulated antibody method to determine free thyroxine (FT4): misleading results due to circulating unsaturated thyroxine binding globulin

The effect of varying concentrations (0-52 mg/l) of purified thyroxine binding globulin (TBG) on the microencapsulated antibody method for free thyroxine was investigated. The results demonstrated that the free thyroxine values were strongly influenced by the concentration of thyroxine binding globulin in the samples. The standard curve could no longer be distinguished at a concentration of purified thyroxine binding globulin of 52 mg/l. In the clinical application, we observed that the values obtained using the microencapsulated antibody method were significantly higher than the expected values in patients receiving triiodothyronine treatment after total thyroidectomy (theoretically nil) and in patients with untreated primary hypothyroidism with negligible thyroxine (less than 12.9 nmol/l). These false positive values are considered to be due to the methodological problem mentioned above, i.e. the microcapsule membrane is not efficient and therefore must be improved. Consequently, any data based on this method should be interpreted with caution.     

Serum free thyroxine concentrations in normal euthyroid subjects and ones with high serum thyroxine binding globulin concentration

Serum free thyroxine (fT4) was assayed by a commercial fT4 method in 30 normal euthyroid subjects, 19 pregnant females, 13 euthyroid subjects with high thyroxine binding globulin (TBG) and three with low or undetectable serum TBG concentration. In a number of these fT4 was also calculated on the basis of the application of the law of mass action to the binding situation. In states in which TBG was altered for congenital reasons both the experimentally determined and calculated fT4 were not significantly different from their respective means in the normal euthyroid population. Pregnant females had both lower experimental and theoretical free T4 concentrations. It is inferred from these data that TBG concentration per se is without effect on serum fT4 concentration.     

A comparison of commercially available luminescence enhanced enzyme immunoassays with in-house non-radioisotopic assays for thyroxine binding globulin and total thyroxine

Commercial luminescence enhanced enzyme immunoassays (Amersham-Amerlite) for thyroxine binding globulin (TBG) and total thyroxine (TT4) were compared with the in-house methods (TT4--Abbott TDx, TBG--Immunoluminometric Assay (ILMA)). The experimental groups consisted of 108 healthy euthyroid blood donors, 165 non-selected thyroid outpatients, 44 tumour bearers and 84 haemodialysis patients. Total thyroxine/thyroxine binding globulin quotients were constructed as an index of thyroid function. The luminescence enhanced enzyme immunoassays were precise (interassay coefficients of variation less than 10% in the range 5-45 mg/l thyroxine binding globulin and 20-100 micrograms/l for total thyroxine) performed similarly to the in-house methods in the differentiation of eu-, hypo- and hyperthyroidism on the basis of total thyroxine/thyroxine binding globulin quotients. Although statistically significant differences often occurred in comparisons of the in-house method with the luminescence enhanced enzyme immunoassays, these only gave rise to thyroid status differences in two cases out of 273, where the in-house method gave a hyperthyroid, the luminescence enhanced enzyme immunoassay a euthyroid answer, when taken from the total thyroxine/thyroxine binding globulin quotients. The luminescence enhanced enzyme immunoassays performed as well as the in-house methods, and quality assessment data were comparable with their radioimmunological counterparts.     

An evaluation of serum thyroxine binding globulin as a routine test of thyroid function.

Serum thyroxine binding globulin (TBG) values were correlated with other thyroid function test results and with the clinical condition in 680 patients. The estimation of serum TBG was helpful in the evaluation of thyroid status only in those patients who were either acutely ill, were taking the contraceptive pill, or were pregnant. Further, the derived index, thyroxine: TBG ratio, proved to be a better diagnostic index than the thyroxine:tri-iodothyronine uptake ratio and in many cases would have avoided the use of more expensive and time-consuming tests.     

Preparation of a monospecific antiserum to thyroxine binding globulin for its quantitation by rocket immunoelectrophoresis

A simple technique is described for the preparation of a high titre monospecific antiserum to human serum thyroxine-binding globulin (TBG). This allows quantitation of the protein by rocket immunoelectrophoresis in 6 h with a day-to-day coefficient of variation of 2.9%. Using this technique serum concentrations of TBG have been determined in groups of patients in good health, in pregnancy and in women on oral contraceptive therapy     

The measurement of thyroxine in urine using a competitive protein binding technique

The competitive protein vinding assay for the measurement of thyroxine in blood serum was modified for the measurement of thyroxine in urine. Samples of urine of 1 to 5 ml volume containing 0 to 5 ng/ml could be assayed with 100% recovery, and above this range, up to 10 ng, recoveries were higher due to non parallelism with the standard curves. Tests carried out using porcine serum albumin indicate that results obtained by the method are not likely to be affected by proteinuria. The cross reaction with triiodothyronine was 25%. Analysis of urine samples stored at 25 degrees C gave higher values than those stored at +4 degrees C or --20 degrees C over similar periods. These increases at 25 degrees C were of the same magnitude as those obtained by acid hydrolysis of urinary thyroxine conjugates.     

Isoelectric focussing of human thyroxine binding globulin (thyropexin) and human prealbumin (transthyretin).

Two batches of the highly purified thyroid hormone-binding plasma proteins, human thyropexin and transthyretin, which were prepared in gram quantities for use in animal experiments, were subjected to analysis by isoelectric focussing. Under these conditions, it was observed that human transthyretin was composed of two components. This was presumably due to the use of 8 mol/l urea. The preparations of both human transthyretin and human thyropexin contained some products of decomposition which probably arose in the course of the purification processes and, in addition, possibly also contained some normal genetic variants of human thyropexin. In spite of the alterations, both protein preparations largely retained their thyroid hormone-binding capacity, which is essential for in vivo studies on the re-entry of thyroid hormones from the extravascular space into the circulation. For therapeutic use in thyrotoxicosis, human transthyretin seems to be preferable to human thyropexin.     

Automated measurement of lipoprotein(a) by immunoturbidimetric analysis

Summary Immunoturbidimetric analysis of lipoprotein(a) in plasma or serum was developed for use on the Roche COBAS FARA II and COBAS MIRA clinical chemistry analyzers. The components of the assay are: (1) buffer consisting of 2.25% polyethylene glycol in phosphate-buffered saline, 0.2% gelatin, and a surfactant; (2) fractionated goat anti-human lipoprotein(a) igG: (3) five standards with lipoprotein(a) concentrations ranging from 0.05 to 1.0 g/l; (4) two controls with concentrations of approximately 0.2 and 0.5 g/l. The analyzer delivers sample and buffer, incubates the reaction mixture at 37°C for 5 min, delivers neat lipoprotein(a) antibody, and incubates for an additional 10 min. The lipoprotein(a) concentration of samples is calculated by the COBAS DENS (Data Evaluation for Non-linear Standard Curves) option by fitting the standard curve values to a four-parameter logit-log curve model. Total imprecision results (CV%) for the FARA II and MIRA were under 11% (NCCLS protocol EP5-T). The assay is linear beyond the highest calibrator to 2.6 g/l. No interference was observed for plasminogen up to 2.3 g/l, apolipoprotein B up to 4.36 g/l, hemoglobin up to 10 g/l, bilirubin up to 4.0 g/l, and triglycerides up to 4.36 g/l. Comparison with a double monoclonal ELISA used at the North-west Lipid Research Laboratories yielded:R=0.970, slope=1.013, andy-intercept=0.00009 (n=37). comparison with a commercially available ELISA kit for lipoprotein(a) yielded:r=0.987, slope-1.243, andy-in-tercept=0.024 (n=40). This assay provides rapid, accurate, and precise screening of lipoprotein(a) in serum or plasma.     

A rapid method for the measurement of sex hormone binding globulin capacity of sera

A simple rapid method for the measurement of sex hormone binding globulin (SHBG) capacity, using 0.2ml of serum, is described. [4-¹⁴C] Testosterone is used as the saturating ligand at one dose level for all samples except pregnancy sera. Albumin bound [4-¹⁴C] testosterone is separated from the globulins with ammonium sulphate and the labelled globulin fraction counted. Pre-treatment of sera with a charcoal suspension under defined optimum conditions removes endogenous steroids associated with the binding proteins without affecting the binding [4-¹⁴C] testosterone to SHBG. The method shows good reproducibility with a coefficient of variation of 4% for male and 4.7% for female serum. Association constants measured for normal male, female and pregnancy sera were 3.6 × 10⁸ M^?1 , 3.2 × 10⁸ M^?1, and 1.7 × 10⁸ M^?1 at 20°, respectively.Normal males gave SHBG capacities of 5.0 ± 0.36 × 10^?8 M/1; females, 7.80 ± 1.24; prepubertal children, 7.01 ± 1.8. Women with amenorrhoea and hirsute women with polycystic ovaries had the lowest SHBG capacities of all, at 4.19 ± 0.75 and 4.06 ± 0.97 respectively. Post-menopausal women also had low levels, 5.25 ± 1.25. Five males with selective hypogonadotrophic hypogonadism had a mean value of 5.69 ± 0.84 which was significantly higher than the normal males. Two patients with Kallman's syndrome had elevated SHBG capacities. Two patients with panhypopituitarism and one patient with Klinefelter's syndrome had low SHBG capacities when compared with normal males. One patient with growth hormone deficiency had a normal SHBG capacity. Pregnancy sera had the highest capacities at 29.03 ± 3.76. No day to day variation in SHBG capacity could be demonstrated over a three day period in the sera from ten normal males.     

Automated measurement of lipoprotein(a) by immunoturbidimetric analysis.

Immunoturbidimetric analysis of lipoprotein(a) in plasma or serum was developed for use on the Roche COBAS FARA II and COBAS MIRA clinical chemistry analyzers. The components of the assay are: (1) buffer consisting of 2.25% polyethylene glycol in phosphate-buffered saline, 0.2% gelatin, and a surfactant; (2) fractionated goat anti-human lipoprotein(a) IgG; (3) five standards with lipoprotein(a) concentrations ranging from 0.05 to 1.0 g/l; (4) two controls with concentrations of approximately 0.2 and 0.5 g/l. The analyzer delivers sample and buffer, incubates the reaction mixture at 37 degrees C for 5 min, delivers neat lipoprotein(a) antibody, and incubates for an additional 10 min. The lipoprotein(a) concentration of samples is calculated by the COBAS DENS (Data Evaluation for Non-linear Standard Curves) option by fitting the standard curve values to a four-parameter logit-log curve model. Total imprecision results (CV%) for the FARA II and MIRA were under 11% (NCCLS protocol EP5-T). The assay is linear beyond the highest calibrator to 2.6 g/l. No interference was observed for plasminogen up to 2.3 g/l, apolipoprotein B up to 4.36 g/l, hemoglobin up to 10 g/l, bilirubin up to 4.0 g/l, and triglycerides up to 4.36 g/l. Comparison with a double monoclonal ELISA used at the Northwest Lipid Research Laboratories yielded: R = 0.970, slope = 1.013, and y-intercept = 0.00009 (n = 37). Comparison with a commercially available ELISA kit for lipoprotein(a) yielded: r = 0.987, slope = 1.243, and y-intercept = 0.024 (n = 40). This assay provides rapid, accurate, and precise screening of lipoprotein(a) in serum or plasma.     

Validation of an automated immunoturbidimetric assay for measurement of plasma D-dimer.

The performance characteristics and diagnostic accuracy of a new rapid automated quantitative immunoturbidimetric D-dimer assay, Auto-Dimer (Biopool, Ume?, Sweden) were evaluated in a population of 135 outpatients with suspected deep-vein thrombosis of a lower limb. Enzyme-linked immunosorbent assay was used as the reference method. The Auto-Dimer assay showed good reproducibility. The correlation between Auto-Dimer and enzyme-linked immunosorbent assay was high (r = 0.91, p < 0.05) and agreement in classifying patients above or below the cut-off was good (kappa coefficient 0.74, 95% CI 0.62-0.86). At a cut-off of 340 microg/l the sensitivity and specificity of Auto-Dimer were high (100% and 61%, respectively). These data show that Auto-Dimer is a reliable screening test for exclusion of deep-vein thrombosis. The assay could be included in prospective patient management studies in order to obtain further information on its clinical usefulness.     

Assessment of serum thyroxine binding capacity-dependent biases in free thyroxine assays

BACKGROUND: Free thyroxine (FT4) assays may exhibit biases that are related to serum T4 binding capacity (sBC). We describe two tests that can be used to assess the presence and magnitude of sBC-dependent biases in FT4 assays. METHODS: We used a direct equilibrium dialysis FT4 assay as the reference method and compared the results obtained with those of the FT4 assays under investigation, in patient sera having a wide range of sBC. We then compared the expected and observed FT4 results for sera diluted with an inert buffer. Because serum dilution causes a predictable decrease in sBC, an increasingly negative bias on progressive dilution is indicative of a sBC-dependent bias. RESULTS: The automated FT4 assay investigated (Vitros FT4) showed no demonstrable sBC-dependent bias by either test. CONCLUSION: These two tests can be used to screen for sBC-dependent biases in FT4 assays.Copyright 1999 American Association for Clinical Chemistry