Purification of anti-thyroglobulin IgG from human serum.

The high prevalence of subjects in the general population and among patients with thyroid diseases who are positive for serum anti-thyroglobulin antibodies, together with the inconsistency of hybridoma techniques to obtain large amounts of anti-thyroglobulin antibodies, prompted us to prepare anti-thyroglobulin antibodies from human serum. An anti-thyroglobulin antibody positive serum was absorbed with CNBr-activated sepharose 4B conjugated to thyroglobulin and anti-thyroglobulin antibodies eluted by acid pH (3.0), basic pH (10.7) and detergent (SDS 3 g/l or 7 g/l). Preliminary experiments carried out with rabbit anti-human thyroglobulin allowed us to purify an Ig-fraction that retained its anti-thyroglobulin activity by immunoprecipitation and tanned red cell haemagglutination. When subjected to agarose gel electrophoresis and Silver stain, this fraction has the mobility of rabbit lg. Further experiments were carried out using Hashimoto's serum (tanned red cell haemagglutination 1:40620). pH 3.0 was found to give the best yield of stable antibodies. An IgG fraction was eluted at a concentration of 10+/-1.2 mg/l of serum. This fraction has the same electrophoretic mobility as human IgG and is close to pure human anti-thyroglobulin antibody. A dose-response curve was built up at a concentration range between 0.016 mg-2 mg of IgG anti-thyroglobulin antibody per litre. The slope of the curve parallels that of a dilution curve of the whole serum, suggesting that the purified antibodies are representative of the whole antibody population. In conclusion, we provide a method for preparing considerable amounts of highly purified anti-thyroglobulin antibodies from human serum for application in clinical medicine and basic research, and in particular which provides a standard for measuring anti-human thyroglobulin serum antibodies by weight rather than the presently used standard expressed in U/ml. Furthermore, this reagent could act as an ideal immuno-vector for the diagnosis and therapy of differentiated thyroid carcinoma.

Lipoprotein(a) increase associated with thyroid autoimmunity.

Conflicting results have been reported regarding serum lipoprotein(a) (Lp(a)) concentrations in patients with hypothyroidism. We addressed the question whether thyroid autoimmunity could be associated with elevated Lp(a) values independent of the thyroid status. Lp(a) was measured by ELISA in 30 males, 29 premenopausal and 30 postmenopausal females positive for thyroid peroxidase- and/ or thyroglobulin-antibody (T-Abs) and normolipidemic, screened out respectively from 428 male donors, 162 premenopausal donors and 108 postmenopausal females; they were compared with 65 males, 72 premenopausal and 48 postmenopausal females, negative for thyroid antibodies, normolipidemic and matched for age. T-Abs-positive male donors showed serum Lp(a) concentrations significantly increased compared with males without T-Abs (mean 19.7 +/- 15.9 vs 12.7 +/- 17.5 mg/dl; median 17.0 vs 4.0 mg/dl; Mann Whitney U test: P = 0.0000). In premenopausal females no difference could be found between T-Abs-positive and T-Abs-negative subjects (mean 13.2 +/- 16.1 vs 12.3 +/- 13.9 mg/dl; median 5.2 vs 8.7 mg/dl), suggesting an Lp(a) lowering effect of estrogens. The study was, therefore, extended to postmenopausal females. Significantly elevated Lp(a) levels were found in 30 postmenopausal females with T-Abs when compared with 48 postmenopausal females without T-Abs (40.0 +/- 34.2 mg/dl vs 20.7 +/- 19.3 mg/dl; median 32.0 vs 18.0 mg/dl; Mann Whitney U test: P = 0.0002). Finally, 21 postmenopausal, normolipidemic, autoimmune hypothyroid patients on L-thyroxine and euthyroid compared with 48 postmenopausal females without T-Abs also showed increased serum levels of Lp(a) (mean 27.0 +/- 16.8 mg/dl vs 20.7 +/- 19.3 mg/dl, median 25.0 vs 18 mg/dl; Mann Whitney U test: P = 0.0024). Thyrotropin levels in all subjects and patients were within the normal range. In conclusion, our results in males and postmenopausal females with T-Abs and euthyroid show an association between thyroid autoimmunity and increased levels of Lp(a), while the results obtained in premenopausal females suggest that estrogens might interfere with the Lp(a) increase related to thyroid autoimmunity.

Thyroglobulin determined in thyroid fine needle aspiration biopsies by radial immunodiffusion and electroimmunodiffusion.

Radial immunodiffusion and electroimmunodiffusion were used to measure thyroglobulin, the main component of thyroid colloid, in thyroid fine needle aspiration biopsies. A linear relationship was established between precipitation ring diameter and thyroglobulin concentration by radial immunodiffusion (0.5-3.0 g/l), and between "rocket" height and thyroglobulin concentration by electroimmunodiffusion (0.1-2.0 g/l). A nearly complete correlation was observed between the two methods (r = 0.97). In radial immunodiffusion the ring diameter is dependent on time of diffusion and on the antiserum concentration in the agar gel. In this study, the observation time was standardised at 48 h, and the rabbit anti-thyroglobulin serum concentration at 26 ml/l. The intrathyroidal concentration of thyroglobulin was determined by radial immunodiffusion and the thyroid find needle aspiration biopsy of 45 thyroid tumours with different cytological-laboratory- and clinical diagnoses. It was found that in colloid nodules or cysts thyroglobulin is markedly higher than in euthyroid nodular goitre (13.7 +/- 11.9 g/l vs. 1.35 +/- 0.8 g/l, p = 0.005). In conclusion radial immunodiffusion and electroimmunodiffusion are precise, easy to perform, low cost, non polluting methods, which do not require high sample dilution (in contrast, high sample dilution is necessary for measurement of thyroglobulin in thyroid fine needle aspiration biopsy by radial immunodiffusion). Measurement of thyroglobulin in thyroid fine needle aspiration biopsy provides a quantitative estimate of colloid, an important marker in the differential diagnosis of thyroid nodules.

Population survey of thyroid autoimmunity in Italy. Three year follow up.

Thyroid autoimmunity and primary hypothyroidism have been evaluated in a 3 year interval in 4 samples of general population from Bologna, Napoli, Palermo, Rome and Cagliari. The average prevalence of Mic-Abs was in males 4.1% and in females 10%. A significantly higher prevalence of Mic-Abs was found in the Cagliari sample where it reaches in males 6.1% and in females 20.4% (p less than 0.0005). Likewise Tg-Abs have been found increased in Cagliari (males: 4.6% vs 10.7%; females: 10.0% vs 16.6%; p less than 0.01). In the 3 year follow up Mic-Abs titre increases significantly whereas the Tg-Abs titre remains substantially unchanged. In the initial screening 1.65% of subjects were found to be latent (1.2%) or overt (0.45%) hypothyroids. Hypothyroidism was particularly frequent in the Cagliari sample and was constantly associated with Mic-Abs. In the 3 year follow up only 1 of 8 latent hypothyroids became overt.

Monoclonal autoantibody to thyroglobulin as a possible vector in immunodiagnosis and immunotherapy of differentiated thyroid cancer.

Differentiated thyroid carcinoma synthetize and secrete thyroglobulin. During its biosynthesis this antigen becomes expressed in the microvilli-bearing surface of carcinoma cells. Attempts have been carried out to target, with specific antithyroglobulin antibodies, the membrane bound absorption thyroglobulin in cancer cells for in vivo diagnosis and therapy. In the serum of patients with autoimmune thyroid diseases a high concentration of antithyroglobulin antibodies is frequently found (1-3 mg/ml). Their purification by immunoabsorption and dissociation is hampered by a low recovery and partial denaturation. It has been recently reported that about 1% of sera from Hashimoto's thyroiditis bear in their electrophoretogram a "myeloma-like protein". In the present report we describe in the serum of a Hashimoto patient a myeloma-like IgG which is an antithyroglobulin autoantibody with restricted functional and structural properties. The serum concentration of this myeloma-like IgG was found to be 40 mg/ml with a capacity of 6.5 nM of human thyroglobulin/mg IgG. The light chain composition was determined to be mostly of the lambda type. The clonal analysis of this myeloma IgG carried out by isoelectrofocusing, immunoblotting and autoradiography resulted in the recognition of several distinct clones, two of which were prominent at pH 8.7 and 7.8. By this technique and in view of the high serum concentration of this myeloma-like IgG, single clones of antithyroglobulin autoantibody can be easily obtained in high yields and without denaturation from human serum. This reagent could offer an ideal immunovector to target membrane-bound thyroglobulin of cancer cells.

Restricted clonality of antithyroglobulin antibodies in an M-component of Hashimoto's serum.

In serum of a 70 y. female with Hashimoto's thyroiditis we have identified an M-component IgG which is predominantly composed by a single class of antihuman thyroglobulin antibody. Our assumption is supported by Scatchard analysis and by the high concentration of lambda chains in serum and IgG fraction which are absorbed by h-thyroglobulin. Attempts to dissociate the monoclonal antibody treating the immunoprecipitate at acidic pH, were unsuccessful; only a few percent (3.7) of the antibodies could be released from immunoprecipitate and those were polyclonal. In order to explain the immunoprecipitating reaction of the monoclonal antibody, it is suggested that the antibody is reacting with a repetitive structure of the h-thyroglobulin molecule.

Hypothyroidism and thyroid autoimmunity in acute myocardial infarction (AMI).

It has been suggested that subjects with thyroid autoimmunity are more frequently affected by acute myocardial infarction (AMI), than the general population (Lancet ii, 155-158, 1977). Serum thyroid antibodies (microsomal and thyroglobulin) were measured in a cohort of 132 males admitted consecutively to the Coronary Unit of Clinica Medica II Univ. "La Sapienza" of Rome with AMI. In the AMI group the thyroid autoimmunity was twice as frequent as in an age matched random population (9.1 vs 17.4%), but the association was statistically weak (p less than 0.05). In the group over 60 y two cases of overt hypothyroidism were found and none in the control group. The prevalence of 2.5% is higher than that reported in previous surveys carried out in elderly populations. No differences have been demonstrated in concentration of serum cholesterol, triglycerides, apolipoprotein A and B between patients with and without thyroid autoimmunity, although the serum cholesterol of AMI patients and those with asymptomatic thyroiditis was significantly higher than that of the general population. It is concluded that the prevalence of thyroid autoimmunity and hypothyroidism are increased in AMI and migth thus contribute to development of hypercholesterolemia and/or immune mechanisms.

Low field (0.02 T) MR imaging of the whole body.

The diagnostic utility of extremely low field magnetic resonance (MR) (0.02 T) was evaluated in various parts of the body outside the head. The areas investigated included the spine (104 patients), kidneys (19 patients), female pelvis (21 patients), and hips (15 patients). The results were compared with those of other imaging modalities, e.g., ultrasound, CT, and scintigraphy. Available pulse sequences and typical examination times for different types of studies are reported. Poor spatial resolution and long imaging times limit the application of this technique to the whole body. Due to poor signal-to-noise ratio, image quality is not comparable with that obtained at higher field strengths. In spite of these restrictions it was possible to demonstrate lumbar disk herniations, to differentiate renal and ovarian cysts from tumors, and to detect early avascular necrosis of the femoral head. Extremely low field MR imaging may eventually be used in screening spinal pathology and in the diagnosis of early avascular necrosis of the femoral head. At present, this technique can not replace ultrasound or CT in abdominal examinations.

Magnetic resonance imaging of renal masses at 0.02 tesla.

To determine the usefulness of extremely low field magnetic resonance imaging (MRI) in the evaluation of renal masses 19 patients with 15 tumors (13 renal and 2 renal pelvic carcinomas) and 8 cysts were examined in a 0.02 tesla MRI unit. The findings were compared with results of computed tomography (CT) and ultrasound. Cavography was performed in 6 patients. MRI enabled differentiation between cysts and solid tumors. Tumor extension into the inferior vena cava could be demonstrated in one case and liver metastases in two patients. The image quality was inferior to that reported at higher field strengths and the tumors were more precisely staged by CT and ultrasound. At present, it is unlikely that low field MRI will play any substantial role in the evaluation of renal masses.

Low dose pelvimetry with biplane digital radiography.

The digital moving slit technique as used by most CT scanners for overview images, was used for antero-posterior and lateral views of the pelvis for pelvimetry. The method was evaluated in phantom experiments and clinical examinations were performed in 23 patients. The method was compared with conventional pelvimetry in 14 patients. Estimated ovarian dose was reduced by a factor of 14.2. The discrepancy in measurements of the pelvic diameters by computed tomography compared with conventional pelvimetry was considered to be without practical importance from an obstetric point of view. Digital pelvimetry is easier for the patient, faster and the need for repeated exposures is eliminated. Based on these facts it is suggested that digital pelvimetry should replace the conventional method whenever possible.

Assessment of residual urine volume during routine cystography and urography.

The residual urine volume after voiding was measured by a modified cardiac formula in patients who were examined by means of voiding cystography. The reliability of the formula was tested in an experimental situation. A high degree of accuracy in correlating computed volumes with instilled volumes was found.

Computer-aided multilayer design of optical filters with wide transmittance bands using SiO(2) and TiO(2).

Starting with the A B C B A multilayer design of Thelen, a computer-aided method is presented to replace the three-material by a two-material design. The method, which utilizes an optimized equivalent layer design, is illustrated by the two commonly used coating materials SiO(2) and TiO(2).

Low prevalence of thyroid antibodies in a Somalian population.

Prevalence rates of serum thyroid antibodies in a Somalian population have been compared with those of an Italian population. Thyroid antibodies have been evaluated by passive haemagglutination (microsomal antigen) and by 125I-thyroglobulin binding. In the Italian survey 13% of all subjects were positive for thyroid antibodies. A significantly lower prevalence of thyroid antibody positive subjects was found in the Somalian group (2,07%, P less than 0.001). It is suggested that the observed difference between Somalian and Italian populations could reflect nutritional or genetic causes which influence the antithyroid autoimmune response.