An improved assay of thyrotropin in dried blood samples on filter paper as a screening test for neonatal hypothyroidism.

A method for measuring thyrotropin (TSH) in eluates of dried blood samples on filter paper was evaluated and improved as a screening test for neonatal hypothyroidism. A linear relationship between the volume of eluate and the TSH value and good recoveries of endogenous TSH (104%) and added TSH (89%) were obtained, indicating that TSH in dried blood was extracted well by overnight elution and determined accurately by radioimmunoassay. The TSH content in the central portion of a spot was less (71%) than that in the peripheral portion. The TSH in dried blood samples on filter paper was stable at 4 degrees C, room temperature (about 25 degrees C) or 37 degrees C for 1 month. The TSH values of eluates were correlated with those of whole blood (r = 0.90) and serum (r = 0.81). Cases of primary hypothyroidism cound be readily differentiated from normal subjects by this method, even when eluates of their blood were combined with those of normal blood for assay of TSH.     

A sensitive and practical immunoradiometric assay of thyrotropin

We describe a two-site immunoradiometric assay for thyrotropin (TSH) in serum, based on use of two monoclonal antibodies directed against two separate antigenic determinants on the TSH molecule. One antibody is immobilized on polystyrene beads; the other is radioiodinated by a modified Chloramine T method. The detection limit of the assay is 0.02 milli-int. unit/L. The working range (CV less than 10%) is from 0.1 to greater than 50 milli-int. units/L. The log mean concentration of TSH in sera collected from 100 euthyroid subjects between 08:00 and 11:00 hours was 1.9 milli-int. units/L, the range 0.4-5.4 milli-int. units/L. Values for hyperthyroid patients and thyroid-cancer patients being treated with thyroxin were much lower than those for euthyroid persons. Results by this new assay correlated excellently with those by our conventional radioimmunoassay (r = 0.99) and also with a sensitive immunofluorometric TSH method (Delfia TSH) (r = 0.99).     

Highly sensitive immunoenzymometric assay for human thyrotropin

A sensitive assay procedure for immunoenzymometric assay of serum thyrotropin (TSH) was developed by making several modifications of the Enzymun-Test TSH kit (Boehringer, Mannheim GmbH). Serum samples were first incubated in plastic tubes precoated with monoclonal antibodies specific to the beta subunit of human TSH. After the tubes were washed, the TSH bound to the tubes was detected with peroxidase-conjugated polyclonal antibodies to TSH. The sensitivity of the assay was 0.2 milli-int. unit/L, and the intra-and interassay CVs were less than 10%. Analytical recovery was 96 to 106%. The normal basal range of TSH was 0.5 to 4.8 milli-int. units/L. The basal levels of TSH in all but one of 48 thyrotoxic patients with Graves' disease were less than 0.2 milli-int. unit/L, clearly different from those of normal subjects. Thyrotoxic patients in early normal pregnancy showed TSH concentrations of 1.7 to 2.9 milli-int. units/L by conventional double-antibody radioimmunoassay, possibly from cross reactivity with human choriogonadotropin, but undetectable TSH by this method. Measurement of basal TSH by this sensitive assay can be used as an initial screening test for thyroid dysfunction.     

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.     

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.     

Serum thyroglobulin concentrations in the first weeks of life as measured with an immunoluminometric assay

In addition to the determination of thyrotropin (TSH) on the 5th day of life as a screening parameter for congenital hypothroidism, serum thyroglobulin concentrations were first measured with a commercially available immunoradiometric assay in those cases presenting with elevated thyrotropin levels. As this thyroglobulin assay required at least 400 microliter serum for a duplicate determination, it was decided to employ a sensitive immunoluminometric assay (detection limit 50 amol/tube) instead, the amount of serum needed being reduced to 100 microliter, the sensitivity to under 3 micrograms/l. We describe the serum thyroglobulin concentrations determined by the immunoluminometric assay in both cord and venous blood and in both full-term and pre-term babies divided into 4 main groups with respect to thyroid function. The criteria for the groups with thyroid dysfunction were determined as a result of a) isolated thyrotropin elevation measured in the dry blood spot test (thyrotropin above 4 mU/l), b) lowered thyroxine:thyroxine binding globulin ratio together with elevated thyrotropin both prior to and under substitution with L-thyroxine. In full-term babies thyroglobulin levels in serum fell steadily over the first months of life. The same effect was seen in pre-term babies. The effect of L-thyroxine on the suppression of serum thyroglobulin levels appeared to be dose-dependent, as newborns from experimental criterium group a) (defined above) showed no suppression of serum thyroglobulin levels when under partial substitution with L-thyroxine (median 84 micrograms/l), whereas those in group b), i.e. with full L-thyroxine substitution showed a noticeable suppression of serum thyroglobulin (median 27 micrograms/l).     

Thyrotropin enzyme-immunoassay in dried blood spots: a spectrophotometric method for neonatal thyroid screening

We describe an enzyme-immunoassay with photometric endpoint determination, suitable for the measurement of thyrotropin (TSH) in dried blood spotted on filter paper. Using reagents of a commercially available test kit provided for the measurement of thyrotropin in 200 microliter serum, we have adapted the method to the determination of thyrotropin in blood spots containing ca. 10 microliter blood. This was achieved by prolongation of the assay time from 3 to 20 hours, and by increasing the amount of enzyme-antibody complex. Precision and sensitivity of the blood spot assay are comparable to those of our in-house thyrotropin RIA, and the RIA/EIA correlation coefficient is 0.987 (n = 150). The advantages of EIA are the simplicity of the photometric end point determination (although strict time control has to be observed in order to avoid drifts in results), the long stability of reagents, and the non-isotopic label. The method therefore appears to be a suitable alternative to thyrotropin RIA for the determination of thyrotropin in neonatal thyroid screening.     

Immunoenzymatic quantification of low concentrations of thyrotropin

We evaluated an immunoenzymatic assay (Abbott HTSH EIA) for thyrotropin (TSH) as a tool for detecting hyperthyroidism and for monitoring thyroid hormone suppressive therapy in patients with nodular goiter, thyroid carcinoma, and hypopituitarism. We also tested with thyroliberin (TRH), to determine the correlation between peak and basal TSH in suppressed patients. For comparison, we used a nonequilibrium radioimmunoassay optimized for maximum sensitivity (J Clin Endocrinol Metab 1975;41:676). Hyperthyroid patients with values for either or both triiodothyronine and thyroxin above the normal reference interval had Abbott assay values less than or equal to 0.2 milli-int. unit/L, clearly below the Abbott assay normal range, as determined in 116 euthyroid subjects. We detected one-third of the suppressed patients (greater than or equal to 0.3 milli-int. unit/L) with RIA, 69% with the Abbott assay (TSH greater than or equal to 0.04 milli-int. unit/L). Only 20% of patients with undetectable basal TSH values in the Abbott assay responded to TRH with a detectable peak TSH value; the peak TSH value after TRH was proportional to the basal TSH value. A single basal TSH measurement by the Abbott HTSH EIA should be adequate for monitoring the degree of thyroidal suppression in thyroid-hormone-treated patients.     

Enzyme immunoassay of thyroxin-binding globulin

We describe a new double-antibody enzyme radioimmunoassay for thyroxin-binding globulin (TBG) in serum. A TBG-beta-D-galactosidase complex was prepared with m-maleimidobenzoyl N-hydroxysuccinimide ester. The mean analytical recovery of TBG added to serum was 99%, sample volume and TBG value were linearly related, and values for TBG determined by this method and those determined by radioimmunoassay correlated well (r = 0.98). Coefficients of variation averaged 7.6% within assay, 6.6% between assay. The measurable range of TBG in the serum was 3.3 to 52 mg/L, but higher TBG concentrations could be measured by diluting the test serum with TBG-free serum. Mean (and SD) serum TBG concentrations as determined by this method were as follows: for 20 normal subjects, 21.2 (3.7) mg/L; for 10 pregnant women, 50.1 (9.5 mg/L; and for 10 patients with TBG deficiency, not detectable.     

Comparative evaluation of serum thyroxine, free thyroxine and thyrotropin determinations in screening of thyroid function

We assessed a highly sensitive immunoradiometric thyrotropin (TSH) assay in screening thyroid dysfunction in 130 consecutive outpatients from a department of medicine and 224 patients from a municipal health centre. In addition to clinical examination, three routine tests were done: a thyroxine radioimmunoassay, an analogue-based free thyroxine assay and an immunoradiometric TSH assay. Triiodothyronine and the TRH test were done, if the findings were discrepant. Discrepancy existed in 24% of cases. The TSH assay had no false negative results (sensitivity 100%). Therefore TSH could screen all patients with thyroid dysfunction. Free thyroxine was the most specific assay (specificity 96%), but many subclinically or overtly hypothyroid patients would have been missed, if that assay had been used alone. We conclude that TSH(IRMA) is the best first-line measurement for thyroid dysfunction testing among outpatients. An abnormal TSH result alone is not diagnostic, but should be followed by the measurement of thyroxine or free thyroxine.     

Evaluation of simultaneous measurement of lutropin and follitropin with the SimulTROPIN radioimmunoassay kit

We evaluated the analytical performance of the "SimulTROPIN" (Becton-Dickinson Inc.) radioimmunoassay for the simultaneous measurement of lutropin (LH) and follitropin (FSH) in human serum. Dose response, linearity, analytical recovery, sensitivity, and reagent stability were all acceptable. Cross reactivity with other structurally related hormones [choriogonadotropin (CG) and thyrotropin (TSH)] was minimal. A normal reference interval was established for nonmidcycle, ovulatory women. We confirm the low degree of CG and TSH cross reactivity with LH and FSH reported by Becton Dickinson and conclude that this assay is an acceptable method for laboratory use in the simultaneous quantification of these analytes.     

Value of free thyroxine (FT4), free triiodothyronine (FT3), and sensitive thyrotropin (TSH) assay in the assessment of optimal thyroxine therapy

We describe a sensitive thyrotropin (TSH) assay in the evaluation of thyroxine replacement therapy. Patients taking varying amounts of L-thyroxine replacement doses were studied using a thyrotropin-releasing hormone (TRH) test and a sensitive TSH assay as the indices of optimal replacement therapy. There were no differences in the mean thyroxine doses of those patients who had a normal TRH response and those who had a flat response. Similarly there were no significant differences in the serum free thyroxine (FT4), free triiodothyronine (FT3), or total triiodothyronine (TT3) levels between these two groups. The patients in both groups had normal basal serum TSH values as measured by a standard, HTSH RIABEAD (Abbott) method. However, serum TSH values, as measured by a sensitive TSH3 MAIACLONE (Serono) immunoradiometric method, were subnormal in all patients with flat TRH responses. The TSH (Serono) assay provided the best single blood test of optimal thyroxine replacement.     

Development and utility of a monoclonal-antibody-based, highly sensitive immunoradiometric assay of thyrotropin

Using improved selection techniques, we isolated four monoclonal antibodies with high affinity for human thyrotropin (Ka = 1.6 X 10(8) to 2.6 X 10(10) L/mol). We used two of these in an immunoradiometric assay (IRMA) that also incorporates a novel phase-separation technology (Sucrosep TSH IRMA, Boots-Celltech). This assay's very low detection limit for TSH (0.03-0.08 milli-int. unit/L) and wide working range (0-250 milli-int. unit/L) allow the differential diagnosis of hypothyroid, euthyroid, and hyperthyroid patients. We compare the utility of this IRMA with that of a RIA for patients with various thyroid disorders. As determined by IRMA, a normal concentration of TSH in serum excludes hyperthyroidism or hypothyroidism, whereas an undetectable serum TSH concentration (less than 0.08 milli-int. unit/L) accurately predicts an abnormality in thyroid gland function.     

Circulating human antibodies as a cause of falsely depressed ferritin values

We have previously reported spuriously elevated values for serum thyrotropin (TSH) measured by immunoradiometric assay (IRMA). We tested those sera showing interference with the TSH assay in a ferritin IRMA which used 125I-rabbit antiferritin antibody and solid phase goat antiferritin antibody as reagents and spleen ferritin as standard. In this assay, we obtained falsely low ferritin values that were corrected by addition of 0.2% non-immune rabbit serum to the labelled antibody. Two other radioimmunoassays gave results with these sera that were not falsely lowered. The interference was shown to be due to human IgG reactive with rabbit serum, the specificity of the interfering antibody being similar in all affected sera. Human antibodies directed against immunoassay reagents may lead to spuriously increased or decreased immunoassay results depending on the specific reagents involved.     

The SimulTRAC FT4/TSH assay evaluated as a first-line thyroid-function test

We evaluated the SimulTRAC FT4 57Co/TSH 125I dual-isotope assay for the simultaneous measurement of free thyroxin (FT4) by radioimmunoassay analog techniques and of thyrotropin (TSH) by immunoradiometry. Inter- and intra-assay CVs were less than 10% over the entire range tested except for 15.9% at the lowest FT4 concentration. Results obtained by the SimulTRAC assay allowed complete differentiation of 85 hyperthyroid patients and 35 hypothyroid patients from normal subjects. However, such estimations of FT4 or TSH concentrations occasionally were misleading for assessing thyroid status in various clinical conditions. We conclude that the SimulTRAC assay has the same inherent disadvantages possessed by FT4 analog and TSH immunoradiometric assays; however, where results of one of the simultaneous assays may be misleading, the results provided by the other may indicate the underlying pathology without requiring an additional assay.     

Determination of thyrotropin in serum by time-resolved fluoroimmunoassay evaluated

We evaluated a new, highly sensitive time-resolved fluoroimmunoassay for thyrotropin (TSH) in serum. This direct immunometric "sandwich"-type assay involves two monoclonal antibodies against TSH, one immobilized, the other labeled with europium. Extremely high specific activity of the label and the use of labeled antibody in large excess make the method sensitive enough to measure TSH values falling below the normal reference interval. The standard curve is nearly linear over a wide range of TSH concentrations (standard concentrations range from 0.25 to 324 milli-int. units/L). The lowest concentration detectable was 25 micro-int. units/L. The CV for the assay was less than 6% at 0.5 milli-int. unit/L or higher, 11.3% at 0.1 milli-int. unit/L. For a CV of 10% the lower limit of the working range would be around 0.1 milli-int. unit/L. The interassay CV was 6.7 to 11.8% for TSH concentrations of 0.31 to 19.6 milli-int. units/L. The 95% confidence interval for sera from 111 healthy persons was 0.6-3.8 (range 0.3-3.8) milli-int. units/L. For hyperthyroid patients and thyroid cancer patients treated with thyroxin after thyroidectomy, serum TSH values were all below the reference interval (most were less than 25 micro-int. units/L).     

Antineutrophil autoantibodies in Graves'' disease. Implications of thyrotropin binding to neutrophils.

The hyperthyroidism of Graves' disease may be caused by autoantibodies to thyrotropin (TSH) receptors. We have found that patients with this disease have autoantibodies to neutrophils as well, which can be displaced by TSH. Using a radiochemical opsonic assay, we found serum antibodies against homologous neutrophils in 6 of 11 Graves' patients. With a staphylococcal protein A-binding assay, we detected circulating antibodies to homologous neutrophils in 10 of 20 patients, while finding cell-bound antibody on autologous neutrophils in 7 of 8 (including 2 with negative serum tests). Use of human 125I-TSH in a radioligand binding assay revealed that TSH bound to neutrophils rapidly (maximum binding within 10 min at 22 degrees C, pH 7.4), specifically (less than 20% nonspecific binding), and reversibly. Adding TSH to the radiochemical assay resulted in a dose-dependent inhibition of opsonic antibody activity in serum from patients with Graves' disease. In contrast, TSH did not inhibit antibody activity of serum from patients with immune neutropenia not associated with thyroid disease. Our findings suggest a basis for the association of Graves' disease with neutropenia. Furthermore, the discovery of such antineutrophil antibodies in Graves' disease permits detection of cell-bound antibody when free antibody is not present.     

An evaluation of six solid-phase thyrotropin (TSH) kits

This article describes an objective evaluation of six thyrotropin (TSH) kits. One was a radioimmunoassay kit taken for comparison, three were immunoradiometric assays and one was an immunoenzymometric assay. The laboratory internal immunoluminometric assay for thyrotropin was used to measure the concentrations of thyrotropin in the kit standards using a standard curve of WHO 68/38 international reference preparation in serum from a thyrotoxic patient as matrix. The in-house assay was used to demonstrate the "sensitivity" to citrated plasma and the fact that kit standards could only measure "correctly" when used in its own kit. The study was carried out in a "blind" way, the assayist and organiser not knowing from which of the four groups under test (blood donors - serum and citrated plasma, thyroliberin-test and thyroid outpatient clinic patients) the samples came until the study had been completed. The immunometric assay kits were able to differentiate statistically between euthyroid and untreated hyperthyroid patients, although one IRMA kit (Kit F) had a large "grey zone" where both euthyroid and hyperthyroid patients overlapped. Compound precision profiles covering the range 0-5 mU/l thyrotropin were good, a mean coefficient of variation under 5% within the range 0.5-5 mU/l being demonstrated by 3 immunometric assays. The immunometric assay kit with the most cumbersome methodology showed, as was to be expected, the worst precision. The euthyroid ranges for thyrotropin were similar in 3 immunometric assay kits using the WHO 68/38 reference material as calibrator (Kits C and E, 0.25-3 mU/l) and correlated well with one kit using the 2nd IRP (NIBSC 80/558) as calibration material (Kit D 0.33-4 mU/l), although the results were around 30% higher in Kit D. The second kit (Kit A) using the 2nd IRP material as calibrator gave identical values with the kits using the WHO 68/38 reference thyrotropin-preparation for calibration purposes. In a further kit (Kit F) it was stated that both thyrotropin international reference preparations gave rise to identical serum values when used as calibrators. The thyroliberin-test may have an additional role to play in monitoring returning pituitary function in thyrotoxic patients under treatment as the immunometric assay kits were easily able to measure a thyrotropin difference of 0.5 mU/l in the range 0-1 mU/l. The conventional radioimmunoassay (Kit B) was unable to match the precision and sensitivity of the better immunometric assay kits in the range under 1 mU/1.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of a sensitive enzyme immunoassay for human thyrotropin in the diagnosis of hyperthyroidism

A sensitive, simultaneous sandwich enzyme immunoassay for TSH was evaluated especially for its ability to distinguish hyperthyroid patients from the euthyroid population. A total of 140 patient samples was analzyed by this assay as well as with a two-step sandwich radioimmunoassay. The diagnostic sensitivity of the thyrotropin assay was 92.5% and the specificity was 88%. False negatives by thyrotropin assay included two patients with Graves' disease who were being treated with propranolol at the time of testing and one patient who was considered hyperthyroid while receiving synthroid. Twelve patients with elevated free thyroxine index levels were considered euthyroid and 50% of these had thyrotropin values that were undetectable; most were elderly patients with nonthyroidal illnesses. Although the thyrotropin enzyme immunoassay had good sensitivity and precision for the detection of hyperthyroidism, our data suggest the limitation of a single thyrotropin determination in establishing the euthyroid state, especially in elderly patients with associated nonthyroidal illnesses and hyperthyroxinemia.     

Use of sensitive immunoradiometric assay for thyrotropin in clinical practice

Although measurement of thyrotropin (thyroid-stimulating hormone; TSH) by radioimmunoassay was a major advance in the laboratory diagnosis of thyroid failure--replacing the time-consuming TSH stimulation test--it was not sufficiently sensitive to discriminate reliably between euthyroid and hyperthyroid patients. Measurement of the TSH response to thyrotropin releasing hormone (TRH) served this purpose, however. The recent development of TSH assays that are severalfold more sensitive and more specific than conventional radioimmunoassays has allowed distinction of euthyroid from hyperthyroid patients and eliminated the need for the TRH test. Although undetectable levels of TSH, compatible with hyperthyroidism, are occasionally noted in euthyroid patients with severe nonthyroidal illness and during the first trimester of pregnancy, false-positive results are less often recorded for TSH than for free or total thyroid hormone measurements. Measurement of TSH by sensitive immunoradiometric assay is currently the most useful first-line test of thyroid function in patients with suspected thyroid disease and, in addition, has a valuable role in monitoring the dose of thyroxine replacement therapy.