Evaluation of a sensitive immunoenzymometric assay for thyrotropin suitable for emergency and paediatric use.

This short communication compares a luminescent-labelled immunometric assay (BeriLux) with an enzyme-labelled immunometric assay (NovaPath) for thyrotropin determinations. The NovaPath assay lends itself to paediatric use, because it only uses 25 microliters sample, and to emergency use, because the main incubation can be reduced to 30 minutes. The correlation between both kits was acceptable (r = 0.825, n = 167 data pairs) although the NovaPath kit gave lower values than the BeriLux it (Median-BeriLux 1.00 mU/l, NovaPath 0.70 mU/l, p less than 0.001 = Wilcoxon Test). From 167 sera (range less than 0.01-44 mU/l) 3 gave discrepant values, being euthyroid in the BeriLux kit and hyperthyroid in the NovaPath kit. All 3 patients were undergoing thiamazole (Favistan) therapy at the time of sampling. The coefficients of variation were lower in the BeriLux kit (intraassay less than 4.5%, interassay less than 5.5% for the range 0.1-50 mU/l) than in the NovaPath kit (intraassay less than 7.5%, interassay less than 12.5%). This reflects the larger dynamic range (signal/noise ratio) of the BeriLux kit.     

Evaluation of a new chemiluminescence technique for human thyrotropin (BeriLux hTSH): diagnostic value of five immunometric assay methods.

A new commercially available human thyrotropin immunochemiluminometric assay (ICMA) kit was evaluated. The BeriLux assay (Hoechst Co., Germany) was compared with two other non-radioisotopic methods (AIA-1200 and IMx) and two other immunoradiometric assays (RIA-gnost TSH IRMA and EIKEN IRMA kits) in 32 normal subjects and 104 patients with Graves' disease, divided into seven groups: 1) untreated hyperthyroidism; 2) hyperthyroidism during treatment; 3) euthyroid with negative thyroliberin test (subclinical hyperthyroidism); 4) euthyroid with low thyroliberin test; 5) euthyroid with normal thyroliberin test; 6) euthyroid with high thyrotropin level (subclinical hypothyroidism); and 7) primary hypothyroidism. Patients in groups 2-6 were undergoing treatment with mercazole and propylthiouracil. The new immunoluminometric assay (ILMA) BeriLux kit was shown to have a remarkably improved analytical and clinical sensitivity. The minimal detectable level of thyrotropin in the assay was 0.006 mU/l. The precision was 2.8% and 6.1% at 0.093 +/- 0.003 mU/l and 0.028 +/- 0.002 mU/l, respectively, whereas the precision of the other methods was above 17.2% and 59.4% respectively. Seven patients from the untreated hyperthyroid group were given 500 micrograms thyroliberin i.v. (the thyroliberin test). The thyrotropin pattern before and after thyroliberin administration was always less than 0.006 mU/l with the BeriLux kit, whereas the other methods showed random fluctuations indicating their low accuracy at this concentration. Using the BeriLux kit, 7 of the 16 overt hyperthyroid patients undergoing treatment showed a measurable thyrotropin level below 0.01 mU/l but a negative thyroliberin test.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of common matrices for assay standards on performance of 'ultra sensitive' immunometric assays for TSH. Report of a joint WHO/IFCC collaborative study.

This report describes the results of a collaborative study organized by a joint working group of IFCC and WHO and involving nine manufacturers of TSH immunometric assay kits. The study was designed to determine whether a calibrator with a common matrix gives better between-laboratory agreement for calibration of serum samples than the various kit calibrators, and to assess various materials for their suitability for use as common matrices. Kit calibrators or calibrators consisting of the IRP for TSH made up in two common matrices, (1) serum from patients with untreated thyrotoxicosis or (2) serum taken from subjects treated with suppressive doses of triiodothyronine, gave similar results for the between-laboratory variation of estimates of TSH concentration for a range of serum samples. Dose-response curves for the two calibrators in 'common' matrices were similar to one another and to those for the kit calibrator. However, the occurrence of non-specific serum effects is shown by the comparison of results for these calibrators with results for calibrators made up in a third common matrix, serum treated with wheat germ lectin. Dose-response curves for this calibrator were dissimilar to those for the other calibrators and between-laboratory variation for estimates in terms of this latter calibrator showed a substantial increase. Moreover, although the between-laboratory variances for estimates of the TSH concentration in terms of each of these calibrators (except those made up in serum treated with in the wheat germ lectin) were similar for any one sample from five hyperthyroid patients, the variances were not consistent between samples, even for samples with similar mean TSH concentrations. These results suggest that a major factor in the between-laboratory variation, especially in the region near 'zero dose', is sample-related, and is caused by particular samples interacting differently with different assay systems. In general, it would appear that for the well controlled 'ultra-sensitive' TSH immunometric assay kits, included in this study, between-laboratory agreement of estimates of the TSH concentration in serum samples is not likely to be substantially improved by use of a common matrix for the standards.     

Interpretations of five monoclonal immunoassays of lutropin and follitropin: effects of normalization with WHO standard

Five mono(oligo)clonal immunometric assays for lutropin (LH) and follitropin (FSH)--bioMérieux, IRE-Medgenix, Serono Diagnostics, Diagnostics Products Corp. (DPC), and LKB--were evaluated in comparison with two polyclonal RIAs (DPC and Amersham). Detection limits varied from 0.04 to 0.32 int. unit/L and 0.06 to 0.86 int. unit/L for LH and FSH, respectively. Intra- and interassay precision (CV) at three concentrations varied from 2.0% to 29.8%, showing that not all kits tested gave acceptable results, especially for LH. Linearity and parallelism were acceptable, except for the DPC FSH kit and the bioMérieux LH kit. High-dose "hook" effects were seen in some kits at LH concentrations of 250 int. units/L, but not in the FSH kits up to concentrations of 350 int. units/L. Reagents in some kits cross-reacted with choriogonadotropin. The clinical validity of the assays was tested in 25 pre- and 25 postmenopausal healthy women and in 66 patients with polycystic ovary disease. In contrast to FSH, LH values varied significantly not only between polyclonal and monoclonal assays but also between the various monoclonal assays, despite the fact that all manufacturers state that their kits are calibrated on the same standards: WHO International Reference Preparation (IRP) 68/40 for LH and 78/549 for FSH. We normalized the results by using new WHO standards: IRP 80/552 for LH and IRP 83/575 for FSH. This decreased significantly the between-kit differences in LH results for individuals. The much-used LH/FSH ratio greater than 3 for diagnosing patients with polycystic ovary disease is not valid when monoclonal assays are used, and is kit-dependent. However, using the normalized results yields a "new" LH/FSH ratio, which is kit-independent and differs significantly between patients and healthy subjects.     

Clinical application of a sensitive non-isotopic immunometric assay of thyrotropin

We have evaluated an immunometric assay of thyrotropin (TSH) based on enhanced chemiluminescence signal; its detection limit is 0.06 milli-int. unit/L. Values in 101 clinically euthyroid subjects with normal thyroid hormone concentrations ranged from 0.39 to 6.83 milli-int. units/L. TSH in 15 hypothyroid patients ranged from 10.3 to greater than 200 milli-int. units/L, whereas in 31 hyperthyroid subjects with increased concentrations of free thyroxin and free triiodothyronine, TSH was undetectable serum of all but one subject. Of 32 clinically and biochemically euthyroid patients with goiter, two had undetectable serum TSH and six had values below the normal range. In 19 clinically euthyroid patients from an intensive-care unit, TSH was undetectable in two and below the normal range in another two. This immunometric chemiluminescence assay distinguishes thyrotoxic from euthyroid subjects, but caution is required in interpreting TSH values alone in subjects with goiter or nonthyroidal illness.     

Evaluation of five high-sensitivity American thyrotropin assays

We compared five commercial immunometric kits for thyrotropin (TSH) manufactured in the United States--Abbott, Diagnostic Products, Hybritech, Nichols, and Becton Dickinson--and one sensitive "in-house" radioimmunoassay for their ability to monitor TSH levels in patients with hyperthyroidism and during thyroid hormone suppression therapy. With these five methods, we measured TSH concentrations is serum specimens from the following categories of patients: euthyroid (N = 99), hyperthyroid (N = 51), hypothyroid (N = 50), and thyroid cancer and nodular goiter on thyroid hormone suppression therapy (N = 33). The immunometric methods were similar in assay sensitivity and precision; the in-house radioimmunoassay was less sensitive than all other assays studied. The simultaneous correlation of TSH values obtained from all diagnostic categories was more than 90% among the assays studied. The immunometric assays clearly distinguished hyperthyroid from euthyroid patients and quantitated TSH levels in the subnormal range.     

Ten highly sensitive thyrotropin assays compared by receiver-operating characteristic curves analysis: results of a prospective multicenter study

We report a prospective multicenter study, undertaken to compare the efficacy of 10 highly sensitive thyrotropin assay kits for the diagnosis of hyperthyroidism. Performances of the kits were compared with a reference diagnosis based on clinical examination, pertinent biological tests, and determination by an independent laboratory of the concentrations in serum of free triiodothyronine and free thyroxin. No thyrotropin determination was used in establishing this reference diagnosis. Receiver-operating characteristic curves were obtained for results from 600 patients (217 hyperthyroid and 383 euthyroid) by each kit. Even though analyses were performed out of the working range of most kits, the clinical correlation was nevertheless excellent. The best results corresponded to a sensitivity of 97.5% associated with a specificity of 96.1% and were significantly better than those obtained with all other kits. Results of this comparison depended greatly on the heterogeneity of the "normal"/"abnormal" categories. When only diffuse hyperthyroidism was considered, sensitivity and specificity were improved for all kits, and there was no significant difference among the five best kits.     

The role of precision in determining the performance of a thyrotropin assay in diagnosing hyperthyroidism

We examined the relationship between analytical sensitivity, precision at the lower limit of the reference interval, and diagnostic performance in hyperthyroidism for one radioimmunoassay and five immunometric assay kits for thyrotropin. The analytical sensitivity of these kits extended from 0.05 to 1.56 milli-int. units/L. Diagnostic efficiencies of the immunometric assays, in discriminating between euthyroidism and hyperthyroidism, ranged between 93% and 98%. There was a highly significant correlation (r = 0.99, P less than 0.001) between analytical sensitivity and diagnostic efficiency. The between-assay coefficients of variations, at the lower limit of the reference interval, ranged from 26% to 87%. There was no correlation (r = 0.36) between precision, at this concentration, and diagnostic efficiency. We conclude that analytical sensitivity and not precision is the major determinant in controlling the diagnostic performance of a thyrotropin assay in hyperthyroidism.     

凝血活酶参考品国际敏感度指数的标定

目的标定出凝血活酶参考品（ＷＲＰ），为国内凝血活酶产品国际敏感度指数（ＩＳＩ）的标定提供依据。方法以凝血活酶国际参考品为标准（ＩＲＰ），按照世界卫生组织（ＷＨＯ）推荐的凝血活酶国际参考品的使用方法进行测定，计算出被标定凝血活酶参考品的ＩＳＩ值和标定ＩＳＩ值的变异系数（ＣＶ）。结果标出的凝血活酶参考品的ＩＳＩ值为１．３５，标定ＩＳＩ值的ＣＶ为４．３％。符合ＷＨＯ的要求（ＣＶ≤５％）。结论用ＩＲＰ标定的凝血活酶参考品可作为国内凝血活酶试剂ＩＳＩ值标定的标准。     

Ten commercial kits compared for assay of thyrotropin in the normal and thyrotoxic range

I developed a standard procedure for assessing sensitivity, intra-assay precision, parallelism of sample dilutions, and assay drift, using a single trial assay kit. I used this to compare the performance of 10 commercial kits with an in-house radioimmunoassay for human thyrotropin. No one kit stood out as clearly superior overall. Differences in calibration between kits were evident in a comparison of thyrotropin concentrations measured in clinical samples, and by direct comparison of standards from different kits in a single assay. However, all kits gave clinically consistent results with respect to their published normal reference interval. Moreover, the performance of human thyrotropin assay kits has improved during the 14 months of this study.     

Serum thyrotropin as measured with a one-step monoclonal-antibody radioimmunometric assay compared with two commercial radioimmunoassay kits

We compared results obtained with a commercial immunoradiometric assay kit for human thyrotropin in which monoclonal antibodies are used (Tandem -R TSH (ONE STEP) ImmunoRadioMetricAssay; Hybritech Inc.) with those obtained with two commercial radioimmunoassay methods: GAMMA-DAB [125I]HS-hTSH RIA (Clinical Assays) and Thyro-SHure TSH Diagnostic Kit (Nuclear Medical Laboratories). The correlation of all results obtained in the Hybritech assay with those of the two commercial RIAs exceeded 95%. Mean values for 100 euthyroid samples measured in the Hybritech assay were lower than for the other two methods. The separation between hyperthyroid and euthyroid patients was much clearer with the Hybritech assay than with the RIA methods. The Hybritech assay was far more sensitive than the Clinical Assays or the Nuclear Medical Laboratories assay.     

Analytical evaluation of four sensitive assays of thyrotropin, including effects of variations in patient sampling

Four immunometric kits for thyrotropin with detection limits below 0.1 milli-int. unit/L were evaluated with respect to accuracy, precision, specificity, matrix effects, and high-dose "hook" effect. We also studied variations of values related to the patients' sex and age and time of the day and season when samples were collected. Correlation among the four methods was excellent, except for a few samples, and the interference in these samples could be abolished by adding mouse serum or "suppression medium." These phenomena can also be expected to occur in other immunometric assays involving monoclonal antibodies. With some modifications all tests are suitable for a clinical study of the usefulness of the thyrotropin assay as a primary test for thyroid function.     

A laboratory and clinical evaluation of an immunochemiluminometric assay of thyrotropin in serum

We evaluated an immunochemiluminometric assay for human thyrotropin. A chemiluminescent acridinium ester is used as a label, with magnetic-particle separation. The lower limit of detection of the assay (mean + 3 SD of the zero standard) was 0.07 milli-int. unit/L, with a working range of 0.5 to greater than 60.0 milli-int. units/L. Assay accuracy was good as judged from analytical recovery, analysis of external quality-assessment samples, and comparison with an enzyme-amplified immunoassay. There were no significant interferences or cross-reactivities. Twenty-four samples assayed showed aggregation of the magnetic particles. On re-assay, four of these samples showed a significant increase in the measured TSH by the luminescence assay. Assay time for 60 tubes was approximately 3.5 h with the use of a semi-automated luminometer. The reference interval, determined from data on 144 healthy euthyroid subjects, was 0.3-4.0 milli-int. units/L. Sixteen of 19 thyrotoxic patients showed clearly suppressed concentrations of thyrotropin in serum.     

An evaluation of kits for measuring thyrotropin in newborns

Several commercial radioimmunoassay kits are now marketed specifically for determination of thyrotropin (TSH) from whole-blood specimens collected on filter paper in neonatal screening programs. We have compared five kits in use in such screening programs in the United States. The reagent kits from Becton Dickinson, Neometrics, and Nuclear Medical Systems gave similar satisfactory results. That from Pharmacia was somewhat more difficult to use and gave greater coefficients of variation. Diagnostic Products' kit appeared to perform satisfactorily, but the analytical values obtained were significantly low, which may suggest erroneous calibrator values within the kit.     

Serum human chorionic gonadotrophin levels in early pregnancy

Serum hCG reference intervals for various gestational periods in normal pregnancies were determined using three commercial assays--two standardized against the WHO 2nd IS (Amersham Amerlex-M beta HCG RIA (AMX) and Abbott beta-HCG 15/15 (ABB] and one standardized against the WHO 1 IRP (Hybritech Tandem -E HCG (HYB]. Serial samples from patients with accurately determined gestational periods were analyzed. We correlated these assays to determine the validity of the common practice of interchanging values between assays using the same WHO standard and of converting 1st IRP values to 2nd IS values by a fixed factor. The slope of correlation between the two 2nd IS assays (AMX, ABB) was 1.43, r = 0.960; whereas between the 1 IRP assay (HYB) and the two 2nd IS assays the slopes were 1.67, r = 0.963 and 1.22, r = 0.971 for AMX and ABB, respectively. In a prospective study of 52 patients with normal pregnancies, serum beta-hCG values in 46% of samples taken at 28-35 days gestation fell below the lower limit of the reference curves supplied with the AMX kit. Ninety-two percent of samples were within the newly established intervals. These results indicate that supplier's reference limits may not be accurate; in addition, a common factor should not be used to convert values from one commercial kit to another.     

"Delfia" and "Amerlite": two sensitive nonisotopic immunoassay systems for assay of thyrotropin compared

We assessed the LKB "Delfia" (time-resolved dissociation-enhanced lanthanide fluoroimmunoassay) and the Amersham "Amerlite" (enhanced luminescent immunometry) assays of thyrotropin in serum. Both assays are sensitive (respective detection limits: 0.02 and 0.04 milli-int. unit/L) and have very good within- and between-batch precision over a wide range of thyrotropin concentrations. Results by the two methods correlate well (r = 0.992); the regression equation is: Amerlite = 0.915 Delfia - 0.33 milli-int. unit/L. The standard curve for the Delfia assay was linear, but that for the Amerlite assay showed some deviation from linearity below 0.5 milli-int. unit/L. Both assays have a negative bias in comparison with radiolabeled immunoradiometric assays, as judged by results for samples from the Quality Assurance Scheme. Both assays discriminate well between hyper-, hypo-, and euthyroid subjects, and results for thyrotropin for most patients with nonthyroidal illness were within the euthyroid reference interval. Both assays are convenient to perform and are based on systems that provide a viable alternative to radioimmunoassay.     

Heterophilic serum antibodies: a cause for falsely elevated serum thyrotropin levels

We report two cases of euthyroid patients with inappropriately elevated serum levels of thyrotropin (TSH) in whom investigation led to the detection of heterophilic antibodies. In addition, we compare the performance of two TSH immunometric assay systems in this setting. Even when monoclonal assay systems are used, TSH results must be critically interpreted when they are at variance with clinical and other biochemical findings.     

Advantages of sensitive assays for thyrotropin in the diagnosis of thyroid disorders

Using two new immunoradiometric assays for thyrotropin, we measured thyrotropin levels in serum of patients suffering from various clinically and biochemically diagnosed thyroid disorders, and in healthy controls. A thyroliberin stimulation test was performed in all patients and controls. Thyrotropin levels were measured using a solid phase IRMA in 339 patients and a coated tube sandwich assay in 152 patients. The lower limits of detection of 0.1 mU/1 (solid phase IRMA) and 0.02 mU/1 (coated tube sandwich assay) as well as the specificity of these two assays were superior to those of conventional thyrotropin assays. Basal thyrotropin values were clearly different between euthyroid controls and patients non responding to thyroliberin stimulation. However, the values for these two groups overlapped those for patients showing a subnormal increase upon stimulation with thyroliberin. The thyroliberin stimulation test in patients with autonomous adenomas, together with the measurement of thyrotropin using these sensitive assays, provides additional information in the low range below 1.0 mU/1,i.e. below the lower limit of detection of conventional double antibody radioimmunoassays.     

An automated immunoradiometric assay for human thyrotropin

In this two-site immunoradiometric assay for thyrotropin, developed for use in the "Kemtek 3000" automated radioimmunoassay system, commercially available monoclonal antibody to thyrotropin is labeled with 125I, and the solid-phase antibody is an IgG fraction of sheep antiserum to thyrotropin, covalently coupled to reprecipitated aminocellulose. There are two incubations, totalling 3 h, the sensitivity is 0.03 milli-int. unit/L. The mean thyrotropin value for 82 healthy euthyroid subjects was 1.7 milli-int. units/L (range 0.4-3.6). For 19 overtly clinically and biochemically hyperthyroid subjects the values ranged from undetectable to 0.2 milli-int. unit/L. In this assay, euthyroid and hyperthyroid subjects can be distinguished with assay of a single basal sample. The assay appears suitable for routine use as a first-line test of thyroid function.     

In vitro and in vivo effects of increased concentrations of free fatty acids on free thyroxin measurements as determined by five assays

To compare in vitro and in vivo effects of increased concentrations of free fatty acids (FFA) on free thyroxin (FT4) values, we measured FT4 in three pooled sera supplemented with oleate and in serum from 18 euthyroid patients before and after an infusion of fat emulsion (Intralipid). We used five FT4 RIA kits: two two-step methods [Gammacoat, Baxter (GC); Ria-gnost, Behring (RG)], two analog RIAs [Amerlex-M, Amersham (AM); Coat-Ria, BioMérieux (CR)], and one kit with labeled antibodies [Amerlex-MAB*, Amersham (AA)]. In vitro, at the maximum oleate addition of 5 mmol/L, FT4 increased when measured by the GC and RG kits, decreased by the AM kit, and showed no significant change by the CR and AA kits. In vivo, post-Intralipid, FFA concentrations rose significantly and the FT4 changes agreed with the results of the in vitro experiments, except for the RG kit, for which FT4 increased in only nine patients. We conclude that in vitro oleate addition is useful to predict the in vivo effect of increased FFA on FT4 values; moreover, in serum from euthyroid subjects with high concentrations of FFA, FT4 analyzed with the CR or AA kits should better agree with normal results for thyrotropin than FT4 values measured with the other kits.