TSH RIA Research Articles

12 HYPOTHYROIDISM NEONATAL SCREENING IN CORD BLOOD, EVALUATION OF 2 TSH ASSAYS

Cord blood neonatal screening for congenital hypothyroidism was carried out from October 1991 to December 1992 in Maternidad Ramón Sardá. It has been determined by TSH RIA. A total of 7365 dried blood samples from newborns were tested. Two hypothyroids (incidence 1:3682)and 2 transient hypothyroids(incidence 1: 3682) and 6 false positives (with TSH between 30 and 50 uU/ml) were detected. The recall rate was 0.14%versus 0.04%that is the rate we have in our screening after 48 hs of life. In order to decrease the recall rate in cord blood 76 samples from newborns were tested in parallel with RIA (DPC) and IRMA (ICN). The interassay coefficients of variation of RIA and IRMA were 20% and 5%, with a detection limit of 25 uU/ml and 2.5 uU/ml respectively. Distribution of TSH concentration (uU/ml) determined by RIA and IRMA was a follows:RIA results were lower than IRMA(p< 0.001)(Wilcoxon's test). No correlation was found between the 2 mothods (r:0.13 Spearman rank). We suggest that IRMA neonatal assay represents a true alternative to RIA and has better sensitivity and specificity for cord blood samples allowing to decrease false positive results.

Analysis of pulsatile secretion of thyrotropin and growth hormone in the hypothyroid rat.

To characterize the role of TRH in the generation of TSH pulsatility as well as the effect of hypothyroidism on episodic GH secretion, blood was constantly withdrawn (30-60 microliters/min) from rats treated with 0.02% methimazole in the drinking water for 8-10 days. This treatment significantly reduced circulating levels of both T3 and T4 and elevated plasma TSH; however, since thyroid hormone titers were still detectable (T3, 39.6 +/- 5.3 vs. 89.8 +/- 5.3 ng/dl in euthyroid animals), methimazole-treated rats were referred to as being mildly hypothyroid. TSH was found to be secreted in secretory bursts, consisting of one to several peaks in these rats. Pulsar analysis of TSH secretory profiles revealed a mean pulse frequency of 2.8 pulses/h, a mean pulse amplitude of 10 ng/pulse, and a mean pulse duration of 0.2 h. Euthyroid rats exhibited similar fluctuations of circulating TSH levels; however, due to the variability of the TSH RIA in the range of euthyroid TSH titers, no significant pulsatility was detected by Pulsar. Mean plasma TSH levels in eu- and hypothyroid rats were 2.3 +/- 0.3 and 14.6 +/- 1.8 ng/ml, respectively. To confirm that the TRH antiserum (TRH-AS) used in the present study for passive immunization had sufficient binding capacity to absorb endogenous TRH release, euthyroid rats were pretreated with either normal rabbit serum or TRH-AS, followed by the injection of clonidine (100 micrograms/kg BW, iv). This alpha 2-adrenergic agonist caused a significant (P < 0.01) 12.7-fold rise in plasma TSH levels in normal rabbit serum-treated animals, which was completely abolished by TRH-AS pretreatment, indicating that clonidine stimulates TSH secretion via activation of hypothalamic TRH release. When TRH-AS was slowly infused into hypothyroid rats that were sampled frequently for the detection of TSH pulsatility, it caused a significant (60.3%; P < 0.01) decrease in mean TSH levels, with TSH titers approaching euthyroid concentrations 1 h after the infusion of TRH-AS. The antiserum treatment also caused the disappearance of statistically significant (Pulsar) TSH secretory pulses. Mild hypothyroidism shifted the GH secretory profiles from a low frequency, high amplitude in euthyroid animals to a high frequency, low amplitude pattern in hypothyroid rats. Mean GH levels in hypothyroid rats were 76% lower than those in euthyroid controls. These findings show that TSH is secreted in a pulsatile fashion in the hypothyroid rat and that TRH is predominantly responsible for the generation of TSH pulsatility.(ABSTRACT TRUNCATED AT 400 WORDS)

Anomalous thyrotrophin in a newborn infant due to anti-thyrotrophin antibody

A baby was found to have an elevated thyrotrophin (TSH) value on neonatal screening but was clinically euthyroid. Repeat TSH measurements confirmed the elevated levels. Investigation of the mother revealed a similar finding with elevated TSH, normal free thyroxin value and euthyroid clinical state. TSH values using the Farmos TSH RIA kit were 3.5 MU/L for the mother and 7.8 MU/L for her son. This suggested an interference in the previous TSH assays. Measurement of anti-thyrotrophin receptor antibody also produced anomalous results of -57 U/L in the mother and -15 U/L for the son (reference range -13 to 13 U/L). The presence of an anti-TSH antibody was determined by incubating serum with I-TSH followed by immunoglobulin separation with protein-A sepharose. After centrifugation and washing the immunoglobulin bound I-TSH was counted in a gamma counter. This showed binding of 39.5% of tracer in the mother compared with less than 2% In controls. The interfering antibody was acquired by the son transplacentally. On follow up the interference has remained in the mother’s serum but has decreased in her son’s. TSH antibodies causing anomalous TSH values are rare and the reason for their development in the mother unknown. A baby was found to have an elevated thyrotrophin (TSH) value on neonatal screening but was clinically euthyroid. Repeat TSH measurements confirmed the elevated levels. Investigation of the mother revealed a similar finding with elevated TSH, normal free thyroxin value and euthyroid clinical state. TSH values using the Farmos TSH RIA kit were 3.5 MU/L for the mother and 7.8 MU/L for her son. This suggested an interference in the previous TSH assays. Measurement of anti-thyrotrophin receptor antibody also produced anomalous results of -57 U/L in the mother and -15 U/L for the son (reference range -13 to 13 U/L). The presence of an anti-TSH antibody was determined by incubating serum with I-TSH followed by immunoglobulin separation with protein-A sepharose. After centrifugation and washing the immunoglobulin bound I-TSH was counted in a gamma counter. This showed binding of 39.5% of tracer in the mother compared with less than 2% In controls. The interfering antibody was acquired by the son transplacentally. On follow up the interference has remained in the mother’s serum but has decreased in her son’s. TSH antibodies causing anomalous TSH values are rare and the reason for their development in the mother unknown.

Thyrotropin (TSH)-Releasing Hormone-Stimulated TSH Release and TSH Concentration in the Guinea Pig Pituitary, as Determined by a Heterologous Radioimmunoassay*

Little is known regarding how the guinea pig (GP) compares with the rat in terms of TSH economy. To develop a heterologous RIA for GP TSH, rabbits were injected with GP TSH. In one rabbit (anti-gpTSH-8), antibodies that bound 125I-labeled bovine (b) TSH and rat (r) TSH but not 125I-labeled bLH or rPRL were generated. The binding of anti-gpTSH-8 to [125I]bTSH was inhibited in a parallel manner by bTSH over a range of 0.047-5.42 ng, rTSH over a range of 0.24-25 ng, and dilutions of GP pituitary extracts. This system, with bTSH as the standard, was employed as the basis for a heterologous TSH RIA (GP TSH RIA). The cross-reactions of rTSH and bLH in the GP TSH RIA were 45% and 7%, respectively. Rat and bovine FSH, GH, and PRL had little or no cross-reaction. GP pituitaries were incubated in vitro and dosed with LHRH and TRH. The GP TSH RIA detected an 11-fold increase in TSH in the medium in response to TRH and no change in immunoreactivity in response to LHRH. In contrast, a RIA for bLH detected a 25-fold increase in LH in the medium in response to LHRH and no increase in response to TRH. The TSH content in GP pituitaries was significantly lower than that in the rat (GP, 16.8 +/- 1.6 ng/mg; rat, 80.3 +/- 6.2 ng/mg; P less than 0.05) as were serum TSH concentrations (GP, 0.8 +/- 0.4 ng/ml; rat, 4.5 +/- 1.1 ng/ml; P less than 0.05). Thyroid hormone administration (T4 Rx) in both GP and rat produced a significant reduction in pituitary TSH content (GP control, 4.8 +/- 0.4 ng/mg; T4 Rx, 2.1 ng/mg; P less than 0.05; rat control, 52.4 +/- 4.0 ng/mg; T4 Rx, 20.5 +/- 1.6 ng/mg; P less than 0.05) and TSH release (GP control, 9.4 +/- 2.3 ng/ml; T4 Rx, 4.3 +/- 1.5 ng/ml; P less than 0.05; rat control, 357 +/- 81 ng/ml; T4 Rx, 133 +/- 27 ng/ml; P less than 0.05) from incubated hemipituitaries. Thyroidectomy in the rat was associated with a decrease in pituitary TSH content, but no change in pituitary content was found in thyroidectomized GPs. These studies demonstrate the feasibility of estimating GP TSH with a heterologous RIA that employs polyvalent antiserum against GP TSH as the first antibody and bTSH as the tracer and standard.(ABSTRACT TRUNCATED AT 400 WORDS)

Technical and clinical performance of six sensitive immunoradiometric assays of thyrotropin in serum.

We evaluated the analytical and clinical performance of six commercial immunoradiometric assay kits for thyrotropin (TSH) in serum in 218 subjects, with and without thyroid dysfunction. Detection limits of the six kits were lower (from 0.07 to 0.25 milli-int. unit/L) than that of conventional TSH RIA (0.7 milli-int. unit/L). Precision was adequate over a wide range of concentrations, although interassay CVs at very low concentrations were good for only two kits (7.3% and 13.1%). Results by all the kits correlated to about the same degree with the TSH RIA (r = 0.92 to 0.98). All showed a positive correlation between both the basal and post-thyroliberin (TRH)-stimulation values for TSH (r = 0.78 to 0.88), and all showed similar euthyroid reference ranges for basal concentrations of TSH. With four of the six kits we could clearly distinguish most of the hyperthyroid patients from healthy euthyroids; however, basal and post-TRH TSH values were not sufficient to discriminate among groups of patients with different grades of thyroid hyperfunction.

Current Management of the Patient with Autonomously Functioning Nodular Goiter

Autonomously functioning thyroid nodules (AFTNs) are presumably independent of TSH for growth and function and appear "hot" on scintiscan because they selectively concentrate radionuclide to a greater extent than the remaining thyroid gland, which is controlled by the normal TH-TSH feedback mechanism. Such autonomously functioning tissue may occur in "patchy" areas, as a solitary nodule, or as multiple nodules (classic Plummer's disease), with the mass of hyperfunctioning tissue and the related secretion of thyroid hormones determining whether the patient is euthyroid or hyperthyroid. Important diagnostic tests include a 99mTc thyroid scan, T4 RIA, T3 uptake, FTI, TSH RIA, and occasionally T3 RIA ("T3 thyrotoxicosis"). Solitary autonomous nodules in adult patients characteristically progress slowly over many years, with toxicity rarely developing in nodules less than 2.5 cm in diameter and occurring primarily in nodules 3 cm or larger and in older patients. The decision to treat a solitary nodule depends upon the size and degree of function of the nodule and the patient's age. Surgery and radioactive iodine are effective therapies. Hyperfunctioning thyroid nodules in children and adolescents (under age 18) have a more rapidly progressive course than those in adults and should be treated by thyroid lobectomy at the time of diagnosis. Subtotal thyroidectomy is the preferred treatment for most patients with toxic multinodular goiter, because it achieves prompt control of the hyperthyroidism and removes the goiter. Radioiodine therapy and long-term antithyroid drug therapy are alternative forms of treatment for patients who are poor surgical risks or who develop recurrent hyperthyroidism following thyroid surgery.

Comparative study on diagnostic and prognostic value of FT4-level, TRH-test and TSH receptor binding assay in Graves disease (GD)

Twentyfour patients with GD, aged 4 to 18 years, were studied before, during and after antithyroid treatment. FT4-level was determined by Amersham RIA-kit (110 samples), TRH-test was carried out with RELEFACT (Hoechst) using Byk Mallinckrodt TSH RIA kit (56) and TSH receptor binding was detected by the assay of Shewring and Rees Smith (53). The diagnostic values of FT4-RIA and TRH-test were excellent in this material. The TSH receptor antibody results were positive (greater than 15%) in one or more samples of 15 patients out of 24, showing the nature of the disease. The prognostic value of the FT4-RIA is valuable only in short time prognosis: sometimes the relapse was indicated first by increase of the FT4-level. The bad prognosis of GD was predicted better by the TRH-test but good prognosis by the TSH receptor binding assay. The value of TRH-test and TSH receptor binding assay has been poor for choosing the adequate stopping time of antithyroid treatment in 50 % of cases.

Preparation of thyrotroph cells from adult male rat pituitary glands by centrifugal elutriation.

To study the metabolism of thyrotrophs and dynamics of TSH secretion in vitro, it is desirable to have a highly enriched population of thyrotrophs. For that purpose, centrifugal elutriation, a recently developed cell isolation method based on the size and density of cells, was used to prepare thyrotrophs from a cell suspension of adult male rat pituitary cells. Trypsin-dispersed cells (4-8 X 10(7] were loaded into the elutriation rotor (Beckman, JE-6) operating at 2800 rpm. Twelve cell fractions were collected at variable rotor speed (2000-2800 rpm) and increasing medium flow rate (10-103 ml/min). Cell recovery was 77-98%. The viability of the cells after elutriation was 90-95% based on trypan blue exclusion. Each fraction was analyzed for TSH, GH, and PRL content and for TRH-stimulated TSH release by RIA. Thyrotrophs were found predominantly in fractions 8-11 (flow rate 38-75 ml/min) based on TSH RIA. The mean TSH concentration in these fractions was 56 +/- 13.6 (+/- SD) microU/10(3) cells compared with 7.6 +/- 3.8 microU/10(3) cells in the initial cell suspension, representing a 7- to 8-fold enrichment of the thyrotrophs. Incubation with 20 nM TRH for 3 h increased the TSH release of cells eluted in fractions 8-11 by 3- to 5-fold; there was no significant increase in TSH release in fractions 3-6. Centrifugal elutriation may be used to prepare a uniform highly enriched thyrotroph fraction with excellent recovery from a suspension of rat pituitary cells. This technique should be valuable for study of the metabolism of thyrotrophs.

Clinical evaluation of routinely applied highly sensitive TSH-RIA

Forty-one routine TSH assays were carried out by applying newly developed, highly sensitive TSH RIA (T. Mori, et al., Folia Endocrinol. Jap., 56: 1231, 1980). B/B0 percent of the least standard point (0.156 microU/ml) was consistently lower than B0 and higher than that of 0.31 microU/ml. The distributions of assayed TSH concentrations in 1394 sera revealed that 34.8% of the total were in an undetectable range when measured by conventional method (less than 1.0 microU/ml), but this method picked up 16.4% (0.156-1.0 microU/ml), and only 5.4% exceeded the upper limit (20 microU/ml). TRH test results (500 micrograms i.v. bolus) in 45 cases of thyroid disorders with low or normal basal TSH revealed that the peak TSH of those with basal TSH of 0.156-1.0 microU/ml (8.98 +/- 4.15 microU/ml) was significantly different from those of less than 0.156 microU/ml or 1.0-3.2 microU/ml. Further, TSH concentrations in 19 patients after T3 administration (75 micrograms X 7 days) were 0.183 +/- 0.073 microU/ml, and all but one (0.43 microU/ml) showed values lower than the normal range (0.31-3.2 microU/ml). Thyroid hormone concentrations in cases with TSH of 0.156-1.0 microU/ml were limited in the ranges of less than 300 ng/dl T3 and/or less than 15 micrograms/dl T4, and these were considered to be the threshold of definite TSH inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Transient neonatal hyperthyrotropinemia: a factitious syndrome due to the presence of heterophilic antibodies in the plasma of infants and their mothers.

In a national screening program for neonatal hypothyroidism, our criterion for diagnosis is a plasma TSH value exceeding 40 microunits/ml on the fifth day of life. Excluding cases with obvious thyroid abnormalities, we identified a group of 14 neonates with unexplained plasma TSH elevation out of 10,261 screened. The mothers of these infants also exhibited high plasma TSH immunoreactivity; both mother and child in these instances were clinically euthyroid. The material responsible for this TSH immunoreactivity had the following characteristics. After parturition, it disappears from circulation within 2 months in the infants and within 4-6 months in the mother, its apparent molecular weight approximated 150,000 daltons, and its affinity for protein A-Sepharose and its binding properties suggested that the material was an immunoglobulin. Binding is observed typically for heterologous immunoglobulin and human TSH. The heterophilic character of these immunoglobulins explains their interference in many RIA systems, including the TSH RIA commonly used in neonatal screening programs for hypothyroidism.

Falsely elevated serum thyrotropin (TSH) in newborn infants: transfer from mothers to infants of a factor interfering in the TSH radioimmunoassay.

In a TSH screening program for congenital hypothyroidism we detected seven newborn infants with normal plasma T4 and T3 levels but high immunoassayable TSH. Similar findings were obtained in their mothers. Serial plasma dilution curves, with and without the addition of normal rabbit serum to the samples, showed that the result of TSH assay performed with antihuman TSH rabbit antiserum was falsely elevated in mothers and infants by an interfering factor. Follow-up of the infants demonstrated that the falsely elevated plasma TSH levels returned to normal within the first 6 months of life. On the contrary, plasma TSH levels remained high in the mothers. These results suggested a placental transfer of maternal antibodies. Indeed, the analysis of the mothers anamnesis revealed that all had previously received injections of a microbial vaccine cultured on a rabbit lung-containing medium. We conclude that placental transfer of a maternal antirabbit factor may cause an artefactual hyperthyrotropinemia in the newborn and the incorrect diagnosis of neonatal hypothyroidism. This can be avoided by the addition of normal rabbit serum or immunoglobulin to the TSH RIA tubes.

The influence of long-term low dose thyrotropin-releasing hormone infusions on serum thyrotropin and prolactin concentrations in man.

The purpose of the present study was to evaluate in man the relative thyrotroph and lactotroph response to a 48-h low dose constant TRH infusion. Before, during, and after the 75 ng/min TRH constant infusion, serum samples were obtained every 4 h in six euthyroid ambulating male subjects for measurements of TSH, PRL, T4, and T3. The TSH response, employing a specific and sensitive human TSH RIA, demonstrated a significant rise from the mean basal pre-TRH value of 2.35 +/- 0.64 microU/ml (+/- SEM) to 3.68 +/- 0.80 (P < 0.005) during the TRH infusion; this value fell below the basal level to 1.79 +/- 0.47 (P < 0.05) post infusion. Serum T4 values were increased above basal both during (P < 0.025) and after (P < 0.025) TRH infusion, whereas serum T3 values were not significantly changed throughout the entire study period. The daily TSH nocturnal surge was augmented in both absolute and relative terms during the first 24 h or the TRH infusion, unchanged during the second 24 h of infusion, and inhibited during the first postinfusion day. Other than a minimal increase in serum PRL during the first few hours of the infusion, no significant alteration in the mean basal concentration or circadian pattern of PRL secretion was evident during or after the low dose TRH infusion. These findings would indicate that 1) near-physiological stimulation of the pituitary with TRH produces a greater stimulation of TSH release than of PRL release and 2) the factor or factors producing the circadian TSH surge may not be mediated through fluctuations in endogenous TRH.

The development of a sensitive radioimmunoassay for H-TSH and its clinical application (author's transl)

Using a commercially available h-TSH RIA kit (Daiichi), a sensitive RIA method was developed. When pooled serum from patients with active Graves' disease was used as a carrier, combined applications of a delayed addition of 125-I TSH and utilization of X2 diluted anti-TSH antibody resulted in the consistent detection of 0.156 microunits/ml level of serum TSH. Further, 0.078 microunits/ml level could also be detected when 200 microliter of serum samples were applied. However, usage of 200 microliter sample volume should be determined after further studies concerning possible non specific effects of human serum. Assay results of 29 patient sera by the sensitive assay correlated quite well (r = 0.953, Y = 0.97X-0.06) with those by the regular assay. Serum TSH in all of the 20 normal subjects were detectable and ranged from 0.31 to 3.2 microunits/ml giving mean values of 1.48 +/- 0.65 (s.d.) microunits/ml. Values less than 0.156 microunits/ml were observed in patients with untreated Graves' disease, acute stage of subacute thyroiditis, and panhypopituitarism, and in postoperative cancer patients under suppressive doses of T-4. In conclusion, this sensitive TSH RIA made clear discrimination of low and normal serum TSH possible, and the clinical application of this method was considered quite useful.

Evidence for a thyrotropin inhibitory effect of histamine in man.

Because of certain side effects of cimetidine therapy which may be hormonally mediated (e.g. gynecomastia), there has been recent interest in the possible endocrine effects of this H2 histamine receptor-blocking agent used in the treatment of peptic ulcer disease. Accordingly, the effect of chronic cimetidine therapy on anterior pituitary function was examined in 12 adult men with mild peptic ulcer disease. TRH and insulin-hypolycemic stimulation tests were performed by standard methods. Serum for TSH and PRL RIA was obtained after TRH; serum for GH, cortisol, and PRL RIA was obtained after insulin-induced hypoglycemia. In addition, serum for LH, FSH, testosterone, and PRL was obtained every 4 h for 24 h. After these baseline studies, 300 mg cimetidine were administered orally 4 times a day for 4--8 weeks and the studies were repeated as before. Chronic treatment with cimetidine caused a significant increase in the peak TSH response to TRH at 30 min (mean peak TSH value before cimetidine, 7.0 microU/ml; after cimetidine, 10.2 microU/ml; P less than 0.05) as well as a significant increase in the TSH area under the curve. There was no statistically significant effect of cimetidine on basal TSH or basal or stimulated PRL secretion. Cimetidine had no effect on the GH, PRL, or cortisol response to insulin-induced hypolycemia or the 24-h secretion of LH, FSH, testosterone, or PRL.

Radioimmunoassay of TSH by TSH RIA PAC (author's transl)

Radioimmunoassay of TSH by TSH RIA PAC (author's transl)

Systematic screening for congenital hypothyroidism using thyrotrophin (TSH)determination in dried blood samples on filter paper:report of an experience of nine months

Systematic screening for congenital hypothyroidism was started in Lyon in september 1976 (21073 births in 1975). This screening was coupled with the one for PKU, using the same dried blood samples on filter paper obtained on the 5 th day of life. TSH levels were determined by radioimmunoassay adapted for dried blood samples using Abott H TSH RIA Kit. 10 963 samples were analyzed. High TSH levels (20 to 175 μUI/ml) were recorded in 50 newborns (0, 4%) Pathological deliverie occured in most of these infants. A second TSH measurement was made in 47 of them and was found either normal (< 10 μUI/ml) in 43 cases or elevated in 4 cases during the first month of life (11, 12, 13, 110 μUI/ml). In 2 of these, TSH returned to normal within the 2 nd month of life. The third case is still under investigation. In the last case, the high control levels of TSH (53, 5 then 110 μUI/ml) contrasted with normal levels of T3 and T4 : a thyroid scintigram using 99 m Tc pertechnetate showed an ectopic thyroid at the base of the tongue. The infant could be treated from the 38 th postnatal day on.