Comparison of serum thyrotrophin concentrations determined by a third generation assay in patients with various types of overt and subclinical thyrotoxicosis.

OBJECTIVE: Recent increases in the sensitivity of methods used to measure TSH, especially third generation assays, have enabled separation of partial from complete pituitary suppression in patients with thyrotoxicosis. We have investigated the use of a sensitive chemiluminescent enzymeimmunoassay in the differential diagnosis of thyrotoxicosis. DESIGN AND PATIENTS: Serum TSH concentrations were determined by chemiluminescent enzymeimmunoassay in patients with various types of overt and subclinical thyrotoxicosis. RESULTS: The assay was highly sensitive with an analytical sensitivity of 0.0016 mU/l. Among 45 hyperthyroid patients with untreated Graves' disease, 37 (82.2%) showed undetectably low levels (< 0.002 mU/l). Serum TSH in the remaining 8 patients (17.8%) was 0.003-0.005 mU/l. In contrast, TSH was undetectably low in only 5 (20.0%) of 20 patients with painless thyroiditis and in 2 (12.5%) of 16 patients with subacute thyroiditis. Eleven (55.0%) with painless thyroiditis and 12 (75.0%) with subacute thyroiditis had TSH values greater than 0.005 mU/l (0.006-0.032 and 0.006-0.228 mU/l, respectively; normal range 0.5-5.0 mU/l). Serum TSH levels were subnormal in 10 of 12 patients with euthyroid ophthalmic Graves' disease, including 4 with undetectably low levels. Among 11 patients with an autonomously functioning thyroid nodule 6, including 1 with normal free T4 and free T3 and 2 with normal free T3, showed TSH values less than 0.002 mU/l. No significant correlation was observed between serum free T4 or free T3 and TSH concentrations in thyrotoxic patients. Together with the incomplete suppression of TSH observed in those with destructive thyroiditis, this suggests that the grade of TSH suppression was influenced by the duration of illness at the time of blood sampling. CONCLUSION: The third-generation TSH assay is useful for the differential diagnosis of various types of thyrotoxicosis, especially between Graves' disease and destructive thyroiditis.

Subclinical Graves' disease as a cause of subnormal TSH levels in euthyroid subjects.

In order to elucidate causes of subclinical thyrotoxicosis, we reviewed records of thyroid function tests obtained in our hospital between 1990 and 1992 showing normal thyroid hormones and subnormal TSH levels, and analyzed underlying clinical conditions of the patients. Of 186 patients with normal T4 and/or free T4 and normal T3 and/or free T3 but subnormal TSH (< 0.1 mU/l) levels in serum, 150 were under treatment with antithyroid drugs for hyperthyroid Graves' disease or with thyroid hormones for hypothyroidism. Twelve were in remission after treatment for Graves' disease, and 4 had destructive thyroiditis. Of the remaining 20 patients, 4 had autonomously functioning thyroid nodule (AFTN), 9 had euthyroid ophthalmic Graves' disease (EOG), and 7 had diffuse goiter without apparent ophthalmopathy (DG). When thyroid stimulating antibodies (TSAb) were measured in the last 3 groups of the patients, they were detected in none with AFTN but in all patients with EOG and DG. These 7 DG patients without ophthalmopathy had a clinical feature showing unstable thyroid functions, changeable to euthyroidism, overt hyperthyroidism and even hypothyroidism during follow-up. In conclusion, TSAb measurement is useful for detection of subclinical Graves' disease in euthyroid subjects with subnormal TSH levels in serum.

Clinical significance of measurements of antithyroid antibodies in the diagnosis of Hashimoto's thyroiditis: comparison with histological findings.

Measurements of antithyroglobulin and antimicrosomal (antiperoxidase) antibodies have been performed widely for the clinical diagnosis of autoimmune thyroid diseases. The present study was designed to compare these antibody titers with histological findings of the thyroid in patients with diffuse goiter who were suspected of having Hashimoto's thyroiditis. One hundred and ten euthyroid or hypothyroid patients (10 males and 100 females; age 48 +/- 15 (SD) years old) with diffuse goiter were studied for the measurement of antithyroglobulin and antimicrosomal or antiperoxidase antibodies by a hemagglutination technique (TGHA and MCHA, respectively) and by a newly developed radioassay (TgAb and TPOAb, respectively). The antibody titers were compared with the histological findings obtained by needle biopsy. TgAb, TPOAb, TGHA, and MCHA were detected in 80 (96.4%), 61 (73.5%), 37 (44.6%), and 54 (65.1%) of 83 patients with histologically proven Hashimoto's thyroiditis, respectively, but in only one (3.7%) of 27 patients without any inflammatory changes in the biopsy specimen. In 55 patients with negative TGHA and MCHA, the TgAb positivity was more closely associated with the histological diagnosis of Hashimoto's thyroiditis than the TPOAb positivity was, the incidence of each antibody in Hashimoto's thyroiditis being 89.7% (26/29) and 27.6% (8/29), respectively. In conclusion, the histological diagnosis of Hashimoto's thyroiditis can most precisely be predicted by the newly developed radioassay for TgAb.

[Clinical evaluation of TPA-assay kit using monoclonal antibodies].

The clinical utility of a newly-developed radioimmunometric assay kit for tissue polypeptide antigen (TPA) was evaluated focusing on the serum diagnosis of gastrointestinal cancers. Serum TPA concentrations of normal subjects, patients with various benign and gastrointestinal malignant diseases were measured using this kit. From the TPA concentrations of normal subjects, the cut-off value was determined to be 70 U/l, which was 40 U lower than the previous kit. It showed a fairly high false positive rate in patients with liver dysfunction, so care should be taken in evaluating patients with liver disorders. It showed high positive rates in patients with wide range of gastrointestinal cancers, confirming the wide-range usefulness of this assay. Cancer patients with advanced stages and recurrences showed a higher positive rate and also higher serum TPA concentrations than in the early stages, indicating that this TPA assay is especially useful in the evaluation of therapeutic effects and in the detection of recurrent diseases.

[A new one-step, labeled-antibody assay for measuring free thyroid hormone concentrations].

We describe one-step labeled-antibody assays for measuring free T4 and free T3 concentrations in serum, based on a novel principle (Amerlex-MAB). Free T4 or free T3 in the sample competes with a molar excess of a cross-reactant (T3 or T2, respectively), chemically coupled to magnetizable polymer particles, for binding to 125I-labeled monoclonal anti-T4 or anti-T3 antibody, respectively. 125I radioactivity bound to the solid phase is inversely proportional to the serum free T4 or free T3 concentration. This one-step assay apparently proceeds to equilibrium after 30 min at 37 degrees C. Within- and between-assay precision (CV) was < 5.7% for free T4 or 6.2% for free T3. The reference range was between 0.98 and 1.77 ng/100 ml for free T4 and between 2.8 and 4.6 pg/ml for free T3. The measurement of free T4 and free T3 concentrations could clearly discriminate hyperthyroid and hypothyroid patients from euthyroid subjects. These values correlated closely to those obtained by an analog radioimmunoassay (Amerlex-M). This method is free from interference by major T4-binding proteins in serum, showing improved performance, compared to the analog radioimmunoassay, with sera from NTI patients with low serum albumin concentrations or anti-thyroid hormone antibodies. We expect these assays to be clinically useful for the evaluation of thyroid functions.

[Basic and clinical evaluation of a novel immunoradiometric assay kit for free thyroxine].

We have evaluated a new immunoradiometric assay for free thyroxine (T4) utilizing thyroxine-coated particles and radioiodinated monoclonal anti-T4 antibodies. The assay results were reproducible with coefficients of variation no more than 3.1% for intraassay and no more than 6.7% for interassay. Serum free T4 values for 48 healthy subjects distributed at 1.13 +/- 0.19 ng/dl (mean +/- SD). In various thyroid diseases, the results with the new kit compared well with two other measurements (T4-analog RIA and equilibrium dialysis RIA). Altered concentration of thyroxine binding globulin did not significantly affect this assay, while anti-thyroxine autoantibodies caused factitiously elevated free T4 values in some cases. In pregnancy, free T4 concentration tended to decline mildly in late stages. About 20% of hypoalbuminemic samples from patients with nonthyroidal illnesses had free T4 concentration more than 2.00 ng/dl, probably due to renal failure or to the use of diuretics, heparin, or other drugs which may increase serum free T4 fraction in vivo. In conclusion, this new free T4 IRMA kit is convenient and reproducible, and suitable for routine measurement of clinical material.

Dissociation in serum CA125 concentrations measured by different monoclonal antibodies.

The new immunoradiometric assay for CA125 (CA125II assay) uses the monoclonal antibody M11 as an immunoadsorbent. The epitope recognized by M11 is different from the OC125 epitope. Monoclonal antibodies 130-22 and 145-9 recognize an epitope designated as CA130 on the molecule expressing the OC125 epitope. Similarity of M11 epitope to the epitope of anti-CA130 antibodies and dissociation of antigen levels measured by the original CA125 assay and new CA125II assay were examined. Anti-CA130 antibodies partially competed with M11 for the M11 epitope. Among more than 20,000 serum samples we found 12 patients in whom the serum CA125 concentration measured by the CA125II assay was different from that measured by the original assay. In 11 out of 12 patients the CA125 concentration was moderately or extremely high by the original assay but very low by the CA125II assay. Eight of the 11 patients had benign disease, one had no apparent disease and two had cancer. The antigen level determined by CA130 assay was very low in all the 11 patients. In one patient the CA125II assay showed a higher antigen level than the original assay or CA130 assay. The heterogeneity of the epitope expression could cause the dissociation of CA125 levels measured by the different monoclonal antibodies.

Serum cytokeratin-19 measured by cyfra21-1 assay in patients with gastrointestinal and gynecologic malignancy.

The serum fragment of cytokeratin 19 was measured in 130 patients with gastrointestinal, breast and gynecologic cancer and 62 patients with benign disease using CYFRA21-1 immunoradiometric assay. Patients with advanced cancer showed the highest level of serum CYFRA21-1. When the cut-off level was set at 2 ng/ml, 7 of 61 patients with earlier stage cancer were positive whereas 45 of 69 patients with advanced cancer had serum CYFRA21-1 levels over 2 ng/ml. In benign gastrointestinal and gynecologic diseases, 4 of 66 patients were positive. Retrospective evaluation revealed a good correlation between the serum CYFRA21-1 concentration and clinical course. CYFRA21-1 may be a useful tumor marker for monitoring of gastrointestinal and gynecologic malignancies.

[Evaluation of a cytokeratin 19 assay kit "BALL ELSA CYFRA21-1"].

We evaluated "BALL ELSA CYFRA21-1" kit, an immunoradiometric assay kit for cytokeratin 19. Monoclonal antibodies KS19-1 and BM19-21 are used for immunoadsorbent and indicator, respectively. There was no problems in reproducibility, dilution test and recovery test. Minimum detectable concentration was 0.42 ng/ml. The antigen measured by this kit was immunologically cross-reactive with tissue polypeptide antigen (TPA) and CYFRA21-1 concentration was closely correlated with TPA concentration in patient's serum. One of twenty-six healthy subjects showed serum concentration over a cut-off value of 2.0 ng/ml. Serum CYFRA21-1 concentration elevated in 5 of 10 esophageal cancer patients, 5 of 10 gastric cancer patients, 7 of 10 colorectal cancer patients and 6 of 10 pancreatic cancer patients. Positive rate in patients with benign disease including hepatopathy was low. BALL ELSA CYFRA21-1 kit is reliable and CYFRA21-1 could be a useful tumor marker in gastrointestinal cancer.

Similarity and co-expression of tumour-associated antigens recognised by different monoclonal antibodies.

The concentration of carcinoembryonic antigen (CEA), CA130, CA125, SLX, CA19-9, SPan1, and tumour-associated glycoprotein 72 (TAG-72) in the culture supernatant of 15 cancer cell lines and in the sera of 58 cancer patients was measured, and the co-expression of these antigens was examined by double determinant immunoradiometric assays. The high correlation coefficient of the concentrations and significant binding in the double determinant assays indicated a close relationship between CA125 and CA130 and between CA19-9 and SPan1. There was variable binding of the 125I-labelled anti-SLX, anti-CA19-9, and anti-SPan1 antibodies to anti-CA130 beads that had been pre-incubated with the culture supernatants, suggesting the presence of the epitopes of SLX, CA19-9, and SPan1 on the molecule expressing CA130. Similarly, the epitopes of SLX, CA19-9, and SPan1 could be present on the molecule expressing CEA. 125I-labelled anti-CA19-9, anti-SLX, and anti-TAG-72 antibodies were bound in variable proportions to anti-CA130 beads or to anti-CEA beads that had been pre-incubated with patients' sera. However, CEA and CA130 were not expressed on the same molecule, either in the culture supernatant, or in patients' sera. In conclusion, the carbohydrate epitopes of CA19-9, SPan1, SLX, and TAG-72 could be present on the molecule recognised by the anti-CA130 or anti-CEA antibody; however, the epitopes of CA130 and CEA did not co-exist on the same molecule.

[Rapid measurement of human parathyroid hormone-(1-84) by immunoradiometric assay for use in intraoperative determination of hyperfunctioning parathyroid glands].

Rapid measurement of serum intact parathyroid hormone concentration was achieved by modification of an immunoradiometric assay for the hormone. Incubation of serum samples for 15 min at 37 degrees C under shaking gave optimal results in terms of assay variance and reproducibility: intra-assay CVs were less than 10% over the hormone concentrations of 11-1,600 pg/ml; intra- and inter-assay CVs for two control sera at different hormone levels were less than 12%. The minimal detectable hormone concentration was found at 27.8 pg/ml. The serum hormone levels of 43 subjects (31 health subjects, 9 patients with primary hyperparathyroidism, and 3 patients with secondary hyperparathyroidism) determined by either rapid or regular assay well correlated with each other (r2 = 0.979, p less than 0.001). In two patients with parathyroid adenoma serum intact PTH levels fell rapidly to 12.1% of the preoperative values 20 min after ligation of the vascular pedicle to the hyperfunctioning glands. We conclude that the modified assay protocol allows rapid, accurate, and simple estimation of intact PTH concentrations, and can be used as an intraoperative measure to aid both diagnosis and surgical cure of hyperparathyroidism.

Episodic fluctuation in serum intact parathyroid hormone concentration in men.

To evaluate the temporal features of physiological fluctuation in serum PTH concentration, we sampled peripheral blood at 4-min intervals for 24 h from five normal men (32.8 yr; range, 26-40 yr) and measured serum PTH levels using a two-site immunoradiometric assay with the exquisite sensitivity and specificity for human PTH-(1-84) (intact PTH). The resultant 24-h time series of serum intact PTH levels were assessed by contemporary techniques in chronophysiology for rhythmic and episodic peak detection. Cosinor analysis disclosed a significant circadian rhythm in serum intact PTH concentrations in all five men, with the mean circadian amplitude and acrophase of 7.2 +/- 4.4 ng/L and 2305 +/- 401 h, respectively (mean +/- SD; n = 5). No apparent fixed ultradian periodicity was found by autocorrelation and spectral analyses. Evaluation of episodic intact PTH pulsatility by Cluster analysis revealed 23.0 +/- 4.4 discrete PTH pulses/24 h (P less than 0.01 vs. signal-free noise), which occurred at an interpulse interval of 61.6 +/- 11.1 min. The average duration of a serum intact PTH peak was 42.8 +/- 7.3 min, and its mean incremental amplitude was 12.6 +/- 1.3 ng/L, which corresponded to a 31.8 +/- 5.2% increase above the preceding nadir. Discrete PTH peaks were separated by nonpulsatile valleys which lasted for 17.9 +/- 4.4 min. Cross-correlation between the time series of serum intact PTH and whole blood ionized calcium (Ca2+) was at its maximum (-0.5) at concurrent time points in three subjects, while significant positive correlation between serum intact PTH and simultaneous serum inorganic phosphorus concentrations was observed in four of five subjects. There was no apparent correlation between the levels of serum intact PTH and serum magnesium. Our data show that serum levels of intact PTH, the only biologically active form of PTH in the blood, is characterized by a significant circadian periodicity, spontaneous episodic pulsatility with distinct peak properties, and a significant temporal coupling with Ca2+ and inorganic phosphorus concentrations. We conclude that PTH secretion, as judged by the temporal pattern of serum intact PTH levels, is pulsatile in normal men.

Optimization and clinical assessment of a radioreceptor assay for thyrotropin-binding inhibitor immunoglobulins.

We tried to improve the sensitivity of a radioreceptor assay for thyrotropin-binding inhibitor immunoglobulins (TBII) by modifying assay conditions. About a twofold increase in sensitivity without a loss of reproducibility was obtained by prolonging the incubation of the receptors with test serum from 15 to 120 min before the addition of 125I-labeled thyrotropin. In 20 untreated, 49 treated patients with Graves' disease and 19 patients with euthyroid Graves' disease, TBII activities obtained using 120 min preincubation were significantly higher than those obtained using 15 min preincubation (p less than 0.005). No significant increase in TBII activities was observed in the presence of sera from patients with primary hypothyroidism (n = 17), simple goiter (n = 7), adenomatous goiter (n = 11), thyroid adenoma (n = 11) or cancer (n = 12). TBII were detectable in 15 (47%) of 32 triiodothyronine-nonsuppressible Graves' patients who were receiving maintenance antithyroid drug therapy using 120 min preincubation, while they were positive in only 6 patients (19%) using 15 min preincubation. The assay using a longer preincubation period was found to be sensitive, specific and useful for diagnosis and follow-up of Graves' disease.