Proteinase K treatment improves RNA recovery from thyroid cells fixed with liquid-based cytology solution.

OBJECTIVE: Fine-needle aspiration biopsy (FNAB), an important diagnostic tool given its simplicity, safety, and cost-effectiveness, is fast becoming a popular procedure in the diagnosis of thyroid diseases. Generally, cells isolated from biopsies are transferred directly to microscope slides to prepare smears for cytopathological examination; however, the technical difficulties of this procedure often cause poor reproducibility, which limits the accuracy of diagnostic results. Liquid-based cytology (LBC), in which isolated cells are collected in a fixative solution, is advantageous in that it facilitates the preparation of homogenous cytological specimens. However, LBC has not been applied to molecular diagnoses, such as RNA expression-based diagnosis, mainly because of difficulties in cell recovery and RNA isolation. This study was aimed to improve RNA extraction from papillary cancer-derived K1 cells and thyroid FNAB specimens suspended in LBC solutions. RESULTS: K1 cells suspended in CytoRich-Red and CytoRich-Blue, fixatives for LBC, were efficiently recovered by trapping to glass-fiber filters. Importantly, subsequent Proteinase K treatment was essential for efficient RNA extraction from the fixed cells. This finding was also applicable to RNA extraction from CytoRich-Red-fixed thyroid FNAB specimens processed in the same way. Consistently, U6 small nuclear RNA was detected in these RNA samples by reverse transcription-polymerase chain reaction.

Probabilistic cumulative dietary risk assessment of pesticide residues in foods for the German population based on food monitoring data from 2009 to 2014.

Cumulative dietary risks for the German population owing to pesticide residues in foods were assessed using food monitoring and consumption data. Based on grouping principles for cumulative assessment groups (CAG) as defined by the European Food Safety Authority, probabilistic modelling gave cumulative long- and short-term dietary exposures relevant to the nervous and thyroid system. Compound specific toxicological reference values were considered to assess the total margins of exposure (MoEs) for each CAG, allowing an assessment of the cumulative dietary consumer risk. For the German population, no public health concerns were identified for 6 of 11 CAGs. For three CAGs high uncertainties remained, since MoEs were less than the usually required threshold of 100 for the upper confidence interval of the modelling uncertainty. For two CAGs relevant to the nervous and thyroid system, possible health risks cannot be excluded with the selected approach. Most potent risk drivers were chlorpyrifos and the group of dithiocarbamates (expressed as propineb). For regulatory decisions on possible cumulative dietary health risks the limitations of the published approaches and the absence of harmonized data sources for robust refinements have to be considered. Future research to reduce this high uncertainty is considered necessary in this area.

Reduction in clonogenic survival of sodium-iodide symporter (NIS)-positive cells following intracellular uptake of (99m)Tc versus (188)Re.

PURPOSE: Cellular radionuclide uptake increases the heterogeneity of absorbed dose to biological structures. Dose increase depends on uptake yield and emission characteristics of radioisotopes. We used an in vitro model to compare the impact of cellular uptake of (188)Re-perrhenate and (99m)Tc-pertechnetate on cellular survival. MATERIALS AND METHODS: Rat thyroid PC Cl3 cells in culture were incubated with (188)Re or (99m)Tc in the presence or absence of perchlorate for 1 hour. Clonogenic cell survival was measured by colony formation. In addition, intracellular radionuclide uptake was quantified. RESULTS: Dose effect curves were established for (188)Re and (99m)Tc for various extra- and intracellular distributions of the radioactivity. In the presence of perchlorate, no uptake of radionuclides was detected and (188)Re reduced cell survival more efficiently than (99m)Tc. A(37), the activity that is necessary to yield 37% cell survival was 14 MBq/ml for (188)Re and 480 MBq/ml for (99m)Tc. In the absence of perchlorate, both radionuclides showed similar uptakes; however, A(37) was reduced by 30% for the beta-emitter and by 95% for (99m)Tc. The dose D(37) that yields 37% cell survival was between 2.3 and 2.8 Gy for both radionuclides. CONCLUSIONS: Uptake of (188)Re and (99m)Tc decreased cell survival. Intracellular (99m)Tc yielded a dose increase that was higher compared to (188)Re due to emitted Auger and internal conversion-electrons. Up to 5 Gy there was no difference in radiotoxicity of (188)Re and (99m)Tc. At doses higher than 5 Gy intracellular (99m)Tc became less radiotoxic than (188)Re, probably due to a non-uniform lognormal radionuclide uptake.

Telecytology: a tool for quality assessment and improvement in the evaluation of thyroid fine-needle aspiration specimens.

The objective of this study was to investigate the role of telecytology as a tool with increased quality standards in the optimal evaluation of thyroid fine-needle aspiration specimens prepared by the ThinPrep(R) technique (Cytyc Co., Boxborough, MA). The study was performed on 252 adequate specimens of 157 patients referred to the Cytopathology Department of University Hospital "Attikon" for preoperative evaluation of thyroid nodules. In all cases, surgical excision followed the initial cytological diagnosis. Three diagnostic categories of cytological reports were used. All cases were confirmed by histological diagnosis of surgical specimens. Ten characteristic images from each case were transferred via file transfer protocol to password-protected accounts for remote review by four independent cytopathologists. In addition to diagnosis, reviewers also commented on overall digital image quality. Contributor's and reviewer's diagnoses were collected, recorded and statistically evaluated. No significant difference in diagnostic accuracy could be detected between the diagnoses proffered on the basis of digitized images and conventional slides. Telecytology is a prompt and valid method for quality assessment and proficiency testing and can be integrated into daily workflow. The use of liquid-based cytology ensures that additional material is preserved for ancillary studies (if necessary) and that a sufficient number of replicate microscope slides can be produced. The use of telecytology in the daily workflow will ensure the reproducibility of cytological diagnoses and make feasible the production of digital educational material. Besides diagnostic accuracy, the implementation of a diagnostic telecytology system requires consideration of numerous financial, legal, professional, and ethical issues.

Calculation of electron dose to target cells in a complex environment by Monte Carlo code "CELLDOSE".

BACKGROUND: We used the Monte Carlo code "CELLDOSE" to assess the dose received by specific target cells from electron emissions in a complex environment. (131)I in a simulated thyroid was used as a model. METHODS: Thyroid follicles were represented by 170 microm diameter spherical units made of a lumen of 150 microm diameter containing colloidal matter and a peripheral layer of 10 microm thick thyroid cells. Neighbouring follicles are 4 microm apart. (131)I was assumed to be homogeneously distributed in the lumen and absent in cells. We firstly assessed electron dose distribution in a single follicle. Then, we expanded the simulation by progressively adding neighbouring layers of follicles, so to reassess the electron dose to this single follicle implemented with the contribution of the added layers. RESULTS: Electron dose gradient around a point source showed that the (131)I electron dose is close to zero after 2,100 microm. Therefore, we studied all contributions to the central follicle deriving from follicles within 12 orders of neighbourhood (15,624 follicles surrounding the central follicle). The dose to colloid of the single follicle was twice as high as the dose to thyroid cells. Even when all neighbours were taken into account, the dose in the central follicle remained heterogeneous. For a 1-Gy average dose to tissue, the dose to colloidal matter was 1.168 Gy, the dose to thyroid cells was 0.982 Gy, and the dose to the inter-follicular tissue was 0.895 Gy. Analysis of the different contributions to thyroid cell dose showed that 17.3% of the dose derived from the colloidal matter of their own follicle, while the remaining 82.7% was delivered by the surrounding follicles. On the basis of these data, it is shown that when different follicles contain different concentrations of (131)I, the impact in terms of cell dose heterogeneity can be important. CONCLUSION: By means of (131)I in the thyroid as a theoretical model, we showed how a Monte Carlo code can be used to map electron dose deposit and build up the dose to target cells in a complex multi-source environment. This approach can be of considerable interest for comparing different radiopharmaceuticals as therapy agents in oncology.

Citología tiroidea: métodos complementarios / Thyroid cytology: complementary methods

Describir nuestra experiencia en la punción aspiración con aguja fina de tiroides utilizando ultrasonido tiroideo y/o control citológico inmediato para así evaluar la sensibilidad de los mismos en apoyo a este procedimiento. Se revisaron historias clínicas de 242 pacientes entre enero 2005 y abril 2007 en los cuales se realizaron 130 punciones guiadas por ultrasonido, 126 con control citológico inmediato y ambos en 26 pacientes; evaluando el número, tamaño y distribución de los nódulos, caraterísticas ecográficas, número de láminas, diagnóstico citológico y correlación con el diagnóstico definitivo. Se encontró mayor prevalencia de nódulos únicos entre 1 y 4 cm; ubicándose principalmente en el lóbulo derecho siendo más frecuentes las lesiones complejas. Para valorar los diagnósticos citológicos, fueron agrupados como maligno, hipercelular o hipocelular, consiguiendo porcentajes de 12 por ciento, 81 por ciento respectivamente. De las muestras hipocelulares 15 fueron guiadas por ultrasonido y 5 con control citológico inmediatos, en 3 casos se utilizaron ambos métodos. La sensibilidad de las punciones guiadas por ultrasonido fue de 88 por ciento y de los casos en los que se utilizó control citológico inmediato del 96 por ciento. El 20 por ciento de los pacientes fueron llevados a cirugía, siendo compatible el diagnóstico citológico con el definitivo en 88 por ciento de los mismos. La punción aspiración con aguja fina de tiroides es un método adecuado para suministrar información de los nódulos tiroideos, cuya sensibilidad aumenta al utilizar soporte citológico y ultrasonográfico. Estudios posteriores deben realizarse con el objetivo de valorar la relación costo beneficio en cada caso.

Standardized assessment of cell proliferation: the approach of the RITA-CEPA working group.

Since 1985, quantitative data on cell proliferation increasingly form an integral part in the risk assessment of many pharmaceutical, chemical, and agrochemical compounds. Technical guides for the conduct of cell proliferation studies are not readily available. The detection of cell proliferation relies almost exclusively on immunohistochemistry, which is rarely standardized between laboratories. Against this background pathologists formed the RITA-CEPA working group with the aim of elaborating standard guides for the conduct of cell proliferation studies. We present here, as examples, the general BrdU guide as well as the organ-specific guides for hepatocytes, thyroid gland follicular cells, and renal tubular epithelial cells in the rat. Today, RITA-CEPA has available guides for BrdU cell proliferation studies in 20 organs as well as for studies with PCNA as proliferation marker and for apoptosis studies with the TUNEL technique. The relevant information from more than 500 selected publications is organized in a database structure to make it easily traceable. New hardware and software used for image analysis are being assessed and are included in the information exchange practiced in the CEPA working group. Further fields of major relevance for cell proliferation studies, such as statistics and the validation of image analysis equipment in a GLP environment are investigated. A guide on the use of statistics in cell proliferation studies is in preparation. CEPA-group members meet regularly to exchange information. They use the electronic database for planning and interpretation of cell proliferation studies. CEPA will continue with the preparation of organ guides and expand the series for other proliferation markers, e.g. Ki-67. It promotes standardization of techniques applied in proliferation studies, especially immunohistochemistry and image analysis. Members of the CEPA-group have access to a comprehensive and steadily increasing knowledge base on all aspects of cell proliferation. Membership of the CEPA group is open for all industrial organizations who develop chemical, agrochemical, or pharmaceutical products.

Assessment of thyroidal "C" cell secretion in osteoporotic girls with Turner's syndrome. Basal and calcium-stimulated levels of total calcitonin, extractable calcitonin and katakalcin.

Osteoporosis is a common finding in Turner's syndrome. To test the hypothesis that calcitonin deficiency may contribute to bone mineral loss in Turner's syndrome, we studied basal and calcium-stimulated (2 mg/kg body weight in 5 min) levels of total calcitonin, extractable calcitonin and katacalcin in 15 girls with Turner's syndrome and osteoporosis. Fifteen age-matched healthy girls were studied as controls. Both basal calcitonin (total and extractable) and katacalcin values were not significantly different in patients with Turner's syndrome in comparison with those of the controls. The calcium stimulation test showed a similar "C" cell secretory reserve in both groups. The calculation of delta CT/delta iCa of total and extractable calcitonin and delta KC/delta iCa, which accounts for individual variations in serum ionized calcium increases, did not show any significant difference between girls with Turner's syndrome and controls. We conclude that calcitonin deficiency is not a causative factor of osteoporosis in girls with Turner's syndrome and that in this syndrome long-life estrogen deficiency does not impair "C" cell secretory activity.

Assessment of antibody dependent cell cytotoxicity in autoimmune thyroid disease using porcine thyroid cells.

Antibody Dependent Cell Cytotoxicity (ADCC) appears to be involved in Autoimmune Thyroid Disease (AITD). Homologous system may trigger non-specific reactions which might obscure specific ADCC. Heterologous target cells may be useful for studying ADCC, provided relevant antigen(s) are expressed. We therefore tested the capacity of porcine thyroid cells to elicit ADCC reaction in the presence of sera from various patients with AITD. Porcine thyroid cells were used in a 4-hr chromium release assay in the presence of 1/10 heat inactivated human sera and human peripheral blood lymphocytes at a 30:1 effector-target ratio. There was a significant correlation (r = 0.64; P < 0.01) between ADCC activities tested on human or porcine thyroid cells. Serum or IgG effects on porcine thyroid ADCC were dose-dependent between 1/10 to 1/10,000 dilutions. Non-thyroid cell systems were unaffected by thyroid cytotoxic sera. Porcine thyrocyte susceptibility to ADCC peaked at the fourth day of culture and was enhanced by addition of TSH or TSH and methimazole in the culture medium. Using this heterologous system, we demonstrated ADCC activity in a significant proportion of patients with thyroiditis (14/19), Graves' opthalmopathy (19/44) or of mothers of children with congenital hypothyroidism (14/39) and in the children themselves (15/39). Discrepancies observed in some sera between ADCC activity and antithyroperoxidase antibody suggest that thyroperoxidase is not the only antigen involved in ADCC. These results indicate that porcine thyroid cells appear suitable for ADCC assay in patients with AITD. Also this system should be helpful to characterize the antigen-antibody involved.

Recombinant human thyroid-stimulating hormone: initial bioactivity assessment using human fetal thyroid cells.

We have assessed the bioactivity of newly available recombinant human TSH (rec-hTSH) using human fetal thyroid cells, with the longer term aim of assessing its use for clinical applications. Rec-hTSH caused a consistent and dose-related increase in thyroid monolayer cell cAMP release and human thyroglobulin (hTg) secretion, confirming its bioactivity. Repetitive studies (n = 5) allowed us to derive an estimated biopotency for the rec-hTSH preparation examined of 5.6 IU/mg compared to 10 IU/mg for commercially available bovine TSH for human use. The rec-hTSH had a bioimmune ratio of 0.55, similar to that of purified pituitary hTSH standards, Furthermore, rec-hTSH induced thyroid epithelial cell growth, as evidenced by a decrease in thyroid cell doubling time from 54 +/- 2.1 to 31 +/- 1.7 h (P less than 0.005). Hence, rec-hTSH is a potent glycoprotein hormone preparation when measured in a homologous human thyroid cell culture system. Rec-hTSH could serve as a future definitive International Standard and has the potential for a useful diagnostic and therapeutic reagent.

Assessment of the bioactivity of human and bovine thyrotrophin preparations using a porcine thyroid cell bioassay.

The biopotency of six preparations of thyrotrophin (TSH) has been compared in a highly sensitive in vitro porcine thyroid cell bioassay using iodide uptake as an endpoint. Three of these preparations were of human origin and three derived from bovine pituitary tissue. One human TSH preparation, the 2nd International Reference Preparation, 80/558, was used to calibrate the other five. The log dose-log response curves for all preparations were sigmoidal in shape. For the purpose of evaluation the central linear portions of the curves were compared. With all preparations the slopes in this region were very similar. The relative biopotencies of the bovine preparations (unit:unit) were at least five times those of the human standards when measured using the porcine thyroid cell bioassay. These findings emphasise the need to control the TSH standards employed in a variety of bioassays, particularly those used for between-laboratory comparison.

Bioassay of thyroid-stimulating immunoglobulins using human thyroid cell cultures: optimization and clinical assessment.

Primary monolayer cultures of human thyroid cells have been used to investigate the intracellular cyclic AMP response to thyroid-stimulating immunoglobulins (TSIg) prepared from the sera of patients with Graves' disease. In particular, attention was directed towards dose-response characteristics obtained under differing incubation conditions, and an optimized incubation procedure based upon conditions consistent with the maximal precision of TSIg measurement was developed. The magnitude of the cyclic AMP response to a tested TSIg preparation was shown to be greatest after the longest incubation period investigated, 16 h, for all doses of TSIg investigated. The greatest precision of TSIg measurement, as determined by the lowest relative error, was obtained at an immunoglobulin (Ig) dose of 1 mg/ml, and when incubated at this dose level for 16 h, all Ig-enriched fractions prepared from the sera of 28 newly-diagnosed patients with Graves' disease significantly (P less than 0.05) raised the intracellular cyclic AMP level of thyroid cell monolayers. After thionamide drug therapy, the incidence of TSIg detection declined to 42% (5 of 12 patients). TSIg bioactivity was also found in 9 of 14 (64%) patients with euthyroid exophthalmos and in 4 of 11 (36%) cases of non-toxic goitre. In contrast, Ig's prepared from the sera of normal euthyroid volunteers were devoid of TSIg bioactivity.