The role of a new polyclonal competitive thyroglobulin assay in the follow-up of patients with differentiated thyroid cancer with structural disease but low levels of serum thyroglobulin by immunometric and LC-MS/MS methods.

PURPOSE: The aims of this study were to assess the role of an in-house competitive thyroglobulin assay (Tg-c) in the follow-up of metastatic differentiated thyroid carcinoma (DTC) patients who presented underestimated Tg measurements by immunometric assays (Tg-IMA) and to compare the results with IMA and LC-MS/MS Tg methods. METHODS: This prospective study included 40 patients. Twenty-one with metastatic disease: 14 had Tg-IMA levels inappropriately low or undetectable (eight patients with positive and six with borderline TgAb) and seven had high Tg-IMA levels. Nineteen had an excellent response to therapy. The competitive assay employs a polyclonal antibody produced in rabbits immunized with human Tg, Tg labeled with biotin, and for the solid phase separation, a monoclonal anti-rabbit IgG antibody adsorbed to microtiter plates. RESULTS: All 14 patients with structural disease and underestimated levels of Tg-IMA presented detectable Tg-c levels. The median Tg-c level in the group with positive TgAb was 183 µg/L (range: 22-710 µg/L), and 58 µg/L (range 23-148 µg/L) in the borderline TgAb group. The levels of Tg-LC-MS/MS were detectable in some patients (range < 0.5-18 µg/L). All seven patients with high Tg-IMA presented also high levels of Tg-c. Only 2/19 patients with excellent response had Tg-c levels above the functional sensitivity. CONCLUSIONS: The competitive assay was able to detect Tg in all patients, even in the presence of serum TgAb, and may be an option in patients with underestimated Tg-IMA and relevant structural disease.

Two Thyroid Stimulating Hormone assays correlated in clinical practice show disagreement in subclinical hypothyroidism patients.

Thyroid Stimulating Hormone (TSH) levels are related to the pituitary gland's ability to detect thyroid hormone concentration. Many studies have analyzed the correlation between TSH and T4, demonstrating a complex system correlation. This complex system may vary among different TSH levels and patients. OBJECTIVES: The main purpose of this study is to assess the correlation and agreement of serum TSH measured with two assays in different settings. DESIGN & METHODS: We evaluated healthy individuals as well as subclinical or overt hypothyroid patients. Eighty participants had TSH levels measured by Cobas Roche Elecsys 600 (Roche Diagnostics) and Abbott Architect I 2000 (Abbott Diagnostics). The TSH methods correlations were established with Pearson's correlation, and the strength of the agreement was determined by the McBride scale. The paired Student's t-test was applied to evaluate TSH values from both methods. The one-sample t-test was used to evaluate the difference between TSH values. The agreement was also assessed by a Bland-Altman plot. A regression analysis was applied to the correlation between TSH and T4. RESULTS: There was a significant difference in TSH values measured by the two methods (p<0.01). Our results demonstrated a poor correlation for TSH in the euthyroid (r: 0.888, p<0.01) and the subclinical hypothyroid (r:0.886, p<0.01) range. The Bland-Altman plot demonstrates that the majority of the TSH values fell between the lines of equality. There were few differences in the values in the normal upper range and slightly above that range (from a TSH: 3.25 to 6.36mUI/L). The level of correlation between TSH assays remains high in all scenarios for age (r≥0.951), BMI (r≥0.962), anti-TPO antibodies (r: 0.977) or levothyroxine use (r: 0.970). CONCLUSIONS: TSH measurement is essential to access thyroid function. Although the overall agreement between the methods is substantial, there was a poor agreement in the normal upper range and close above. The disagreement observed reinforces the difficulty in using different assays in clinical practice. The better correlation with fT4 and the reference range used by Cobas assay allowed the best clinical performance.

Novel immunoassay for TSH measurement in rats.

Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats.

Novel immunoassay for TSH measurement in rats

ABSTRACT Measuring thyroid hormones is an important aspect for the study of metabolism and for monitoring diseases in both human and animal models. The traditional method for hormone measurement in rats is the radioimmunoassay (RIA). However, the RIA is associated with some practical disadvantages, including the use of radioactive material, the need for specialized equipment and expert staff, the short shelf-life of kits according to the half-life of the radioisotope and high costs. The objective of this study was to develop a new cost-effective method for measuring TSH levels in rats that avoids the use of radioactive material. We developed an in-house competitive immunoassay using a reference standard, polyclonal antibody produced in rabbits and biotinylated antigen. This method was tested in 64 Wistar rats that were divided into a control group (n = 41) and a group with hypothyroidism (n = 23). Our assay demonstrated an analytical sensitivity of 0.24 ng/mL (n = 12) and an intra-assay coefficient of variation (CV) of 8.9% for sera with TSH levels of 1.5 ng/mL and 13.2% for sera with TSH levels of 17.5 ng/mL (n = 14). The inter-assay CV was 13.5% for sera with TSH levels of 1.4 ng/mL and 14.5% for TSH levels of 18.2 ng/mL (n = 5). The analysis of mean TSH levels in control rats (5.06 ± 0.5701) and hypothyroid rats (51.09 ± 5.136) revealed a statistically significant difference (p < 0.001) between the groups. This method showed good sensitivity, can be automated and is low-cost compared with RIA. Our method offers a viable alternative for TSH measurement in rats.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance

ABSTRACT Objective We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. Subjects and methods Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. Results There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. Conclusion A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Prevalence of pancreatic autoantibodies in non-diabetic patients with autoimmune thyroid disease and its relation to insulin secretion and glucose tolerance.

OBJECTIVE: We evaluated the prevalence of glutamic acid decarboxylase (GADA) and tyrosine phosphatase-protein antibodies (IA2A), their titers and their relation to first phase insulin response (FPIR) and glucose tolerance in autoimmune thyroid diseases (ATDs) patients. SUBJECTS AND METHODS: Graves' disease (GD; n = 181) and Hashimoto's thyroiditis (HT; n = 143) patients in addition to healthy controls (n = 93) were studied. Secondly, FPIR and oral glucose tolerance tests (OGTT) were performed in 11 anti-pancreatic islet-cell (+) and in 20 anti-pancreatic-cell (-) patients. RESULTS: There was a non significant trend for higher prevalence of GADA positivity in GD vs HT (7.2% vs 2% p = 0.06), but the GADA titers were higher in HT. We also did not find a significant difference in IA2 prevalence (0.7% vs 0.0%) between these two groups or compared to the control group. In the subsequent analysis, low FPIR was found in 10% of these patients but without statistical difference for OGTT between pancreatic antibody-positive and -negative patients. CONCLUSION: A trend for greater prevalence of GADA was observed for GD patients than for HT or control. However, the titers of these autoantibodies were higher in HT patients, but there was no significant relation to insulin secretion and glucose tolerance at that moment and stage of autoimmune diseases.

Clinical impact of thyroglobulin (Tg) and Tg autoantibody (TgAb) measurements in needle washouts of neck lymph node biopsies in the management of patients with papillary thyroid carcinoma

ABSTRACT Objectives The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. Materials and methods We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patient’s serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. Results The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. Conclusions Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant.

Clinical impact of thyroglobulin (Tg) and Tg autoantibody (TgAb) measurements in needle washouts of neck lymph node biopsies in the management of patients with papillary thyroid carcinoma.

OBJECTIVES: The presence of thyroglobulin (Tg) in needle washouts of fine needle aspiration biopsy (Tg-FNAB) in neck lymph nodes (LNs) suspected of metastasis has become a cornerstone in the follow-up of patients with papillary thyroid carcinoma (PTC). However, there are limited data regarding the measurement of anti-Tg antibodies in these washouts (TgAb-FNAB), and it is not clear whether these antibodies interfere with the assessment of Tg-FNAB or whether there are other factors that would more consistently justify the finding of low Tg-FNAB in metastatic LNs. MATERIALS AND METHODS: We investigated 232 FNAB samples obtained from suspicious neck LNs of 144 PTC patients. These samples were divided according to the patient's serum TgAb status: sTgAb- (n = 203 samples) and sTgAb+ (n = 29). The TgAb-FNAB levels were measured using two different assays. Tg-FNAB was also measured using two assays when low levels (< 10 ng/mL) were identified in the first assay of the metastatic LNs from the sTgAb+ samples. RESULTS: The TgAb-FNAB results were negative in both assays in all samples. Low levels of Tg-FNAB were identified in 11/16 of the metastatic LNs of the sTgAb+ patients and 16/63 of the sTgAb- patients (p < 0.05) using assay 1. The measurement of the Tg-FNAB levels using assay 2 indicated additional metastases in 5 LNs of the sTgAb+ patients. CONCLUSIONS: Factors other than the presence of TgAb-FNAB may contribute to the higher number of metastatic LNs with undetectable Tg-FNAB in the sTgAb+ group. In addition, the measurement of Tg-FNAB using different assays was useful to enhance the diagnosis of metastatic LNs, particularly when cytological and Tg-FNAB results are discordant.

M918V RET mutation causes familial medullary thyroid carcinoma: study of 8 affected kindreds.

Germline mutations in codon 918 of exon 16 of the RET gene (M918T) are classically associated with multiple endocrine neoplasia type 2B (MEN 2B) with highly aggressive medullary thyroid cancer (MTC), pheochromocytoma and a unique phenotype. The objectives of this study are to describe the rare M918V RET mutation discovered in 8 MTC kindreds from Brazil lacking the MEN 2B phenotype classically observed in M918T patients and to investigate the presence of a founder effect for this germline mutation. Eight apparently sporadic MTC cases were diagnosed with the germline M918V RET mutation. Subsequently, their relatives underwent clinical and genetic assessment (n = 113), and M918V was found in 42 of them. Until today, 20/50 M918V carriers underwent thyroidectomy and all presented MTC/C-cell hyperplasia; the remainder carriers are on clinical follow-up. None of the M918V carriers presented clinical features of MEN 2B. Their clinical presentation was heterogeneous, and the age at tumor diagnosis ranged from 24 to 59 years. Lymph node metastases were present in 12/20 patients, and presumable distant metastases in 2/20; in contrast, we observed a carrier of up to 87 years of age without evidence of MTC. Ethnographic fieldwork and haplotype analyses suggested that the founder mutation first settled in that area fifteen generations ago and originated from Portugal. Our study is the first to demonstrate the RET M918V mutation co-segregating in 8 familial MTC kindreds with validated evidence of a founder effect. We suggest that M918V MTC should be clinically considered an American Thyroid Association (ATA) moderate-risk category.

High frequency of vitamin D receptor gene polymorphism FokI in Brazilian Type 1 diabetes mellitus patients with clinical autoimmune thyroid disease.

BACKGROUND: Polymorphisms of vitamin D receptor (VDR) gene have been studied as genetic markers of type 1 diabetes mellitus (T1DM) and some studies have reported associations with autoimmune thyroid disease. The aim of this study was to evaluate the relationship between VDR FokI polymorphism (rs10735810), thyroid autoimmunity and thyroid dysfunction (TD) in Brazilian T1DM. METHODS: One-hundred-eighty T1DM patients were evaluated for age, duration of diabetes (DDM), positivity to TPO Antibody (TPOA), GAD Antibody (GADA), IA2 Antibody (IA2A) and fasting serum C-peptide (FCP) according to diagnosis of TD. PCR-RFLP analyses were carried out for VDR polymorphism FokI. RESULTS: TPOA positivity (80.0 vs. 25.0 %, p < 0.001) and GADA positivity (56.0 vs. 30.3 %, p = 0.01) were higher in T1DM patients with TD with the same age and DDM than the group without TD, with no difference of FCP and IA2A positivity. We observed higher prevalence of VDR FokI in T1DM with TD (ff and Ff 73.9 % with TD vs. 52.7 % without TD, p = 0.05). Positivity to TPOA and presence of FokI polymorphism were significantly associated with the concurrence of TD in T1DM patients (OR 18.1; CI 3.7-87.0; p < 0.001). CONCLUSIONS: The VDR FokI polymorphism (rs10735810) was associated to persistence of GADA, TPOA positivity and TD in Brazilian T1DM. Positivity to TPOA and VDR polymorphism FokI were strongly associated with concurrence of T1D and TD. These data collaborate to understanding the joint susceptibility genes for TD in T1DM.

Macrocalcitonin Is a Novel Pitfall in the Routine of Serum Calcitonin Immunoassay.

CONTEXT: Calcitonin (CT) is a sensitive marker of medullary thyroid carcinoma (MTC) and is used for primary diagnosis and follow-up after thyroidectomy. However, persistently elevated CT is observed even after complete surgical removal without evidence of a recurrent or persistent tumor. OBJECTIVE: To investigate the presence of assay interference in the serum CT of MTC patients who are apparently without a structural disease. PATIENTS AND METHODS: We studied three index MTC cases for CT assay interference and 14 patients with metastatic MTC. The CT level was measured using an immunofluorometric assay. Screening for assay interference was performed by determination of CT levels before and after serum treatment with polyethylene glycol. Additionally, samples were analyzed by chromatography on ultra-performance liquid chromatography and protein A-Sepharose. RESULTS: Patients with biochemical and structural disease showed CT mean recovery of 84.1% after polyethylene glycol treatment, whereas patients suspected of interference showed recovery from 2-7%. The elution profile on UPLC showed that the immunometric CT from these three patients behaved like a high molecular mass aggregate (>300 kDa). Additionally, when these samples were applied to the protein A-Sepharose, CT immunoreactivity was retained on the column and was only released after lowering the pH. CONCLUSIONS: For the first time, our results show the presence of a novel pitfall in the CT immunoassay: "macrocalcitonin." Its etiology, frequency, and meaning remain to be defined, but its recognition is of interest and can help clinicians avoid unnecessary diagnostic investigations and treatment during the follow-up of MTC.

Low iodine diet does not improve the efficacy of radioiodine for the treatment of Graves’ disease

Objective Consuming a low-iodine diet (LID) is a widely accepted practice before administering radioiodine (131I) to evaluate and to treat thyroid disease. Although this procedure is well established for the management of patients with differentiated thyroid cancer, its use in patients with benign disease is unclear. So, we aimed to evaluate the influence of a LID on the outcome in patients with Graves’ disease (GD) treated with131I. Subjects and methods We evaluated 67 patients with GD who were divided into 2 groups: one group (n = 31) consumed a LID for 1-2 weeks, and the second group (n = 36) was instructed to maintain a regular diet (RD). Results The LID group experienced a 23% decrease in urinary iodine after 1 week on the diet and a significant 42% decrease after 2 weeks on the diet. The majority (53%) of the patients in the LID group had urinary iodine levels that were consistent with deficient iodine intake. However, there was no difference in the rate of hyperthyroidism’s cure between the LID and the RD groups 6 months after 131I therapy. Furthermore, the therapeutic efficacy did not differ in patients with varying degrees of sufficient iodine intake (corresponding urinary iodine levels: < 10 μg/dL is deficient; 10-29.9 μg/dL is sufficient; and > 30 μg/dL is excessive). Conclusion In the present study, we demonstrated that although a LID decreased urinary iodine levels, those levels corresponding with sufficient or a mild excess in iodine intake did not compromise the therapeutic efficacy of131I for the treatment of GD.

Low iodine diet does not improve the efficacy of radioiodine for the treatment of Graves' disease.

OBJECTIVE: Consuming a low-iodine diet (LID) is a widely accepted practice before administering radioiodine (131I) to evaluate and to treat thyroid disease. Although this procedure is well established for the management of patients with differentiated thyroid cancer, its use in patients with benign disease is unclear. So, we aimed to evaluate the influence of a LID on the outcome in patients with Graves' disease (GD) treated with 131I. SUBJECTS AND METHODS: We evaluated 67 patients with GD who were divided into 2 groups: one group (n = 31) consumed a LID for 1-2 weeks, and the second group (n = 36) was instructed to maintain a regular diet (RD). RESULTS: The LID group experienced a 23% decrease in urinary iodine after 1 week on the diet and a significant 42% decrease after 2 weeks on the diet. The majority (53%) of the patients in the LID group had urinary iodine levels that were consistent with deficient iodine intake. However, there was no difference in the rate of hyperthyroidism's cure between the LID and the RD groups 6 months after 131I therapy. Furthermore, the therapeutic efficacy did not differ in patients with varying degrees of sufficient iodine intake (corresponding urinary iodine levels: < 10 µg/dL is deficient; 10-29.9 µg/dL is sufficient; and > 30 µg/dL is excessive). CONCLUSION: In the present study, we demonstrated that although a LID decreased urinary iodine levels, those levels corresponding with sufficient or a mild excess in iodine intake did not compromise the therapeutic efficacy of 131I for the treatment of GD.

Assessment of the Effect of Two Distinct Restricted Iodine Diet Durations on Urinary Iodine Levels (Collected over 24 h or as a Single-Spot Urinary Sample) and Na(+)/I(-) Symporter Expression.

INTRODUCTION: A restricted iodine diet (RID) may be recommended for depletion of the whole-body iodine pool in patients with differentiated thyroid cancer referred for radioiodine treatment or a whole-body scan. Evaluation of the iodine pool is possible through urinary iodide (UI) measurements, which can be collected in 24-hour (24U) or spot urinary (sU) samples. However, the minimum period required for an RID to lower the iodine pool, the measurement of iodine in sU samples as a iodine pool marker, and the influence of the iodine pool on Na(+)/I(-) symporter (NIS) expression are debatable in the literature. OBJECTIVES: To compare the effects of 15- and 30-day RID on UI measurements in 24U and sU samples and the impact of RID on NIS expression. METHODS: Thyroidectomized patients went on a 15- or 30-day RID and collected 24U and sU samples before and after the RID. Twenty healthy individuals were evaluated for mRNA NIS expression before and after the RID. RESULTS: Of 306 patients, only 125 properly complied with both the RID and 24U collection. We observed a correlation between sU and 24U UI before the RID (n = 306, ρ = 0.47, p < 0.001), after a 15-day RID (n = 79, ρ = 0.49, p < 0.001), and after a 30-day RID (n = 46, ρ = 0.73, p < 0.001). There was a significant decrease in UI after the RID. The median UI measurement was 275 µg/l at baseline and 99 and 80 µg/l after a 15- and 30-day RID, respectively. There was a significant increase in NIS expression after a 15-day RID. CONCLUSIONS: A 15-day RID is sufficient to deplete the iodine pool. sU can replace 24U UI as a marker for assessing the iodine pool. NIS expression was increased after a 15-day RID.

Adult exposure to bisphenol A (BPA) in Wistar rats reduces sperm quality with disruption of the hypothalamic-pituitary-testicular axis.

Reproductive physiology involves complex biological processes that can be disrupted by exposure to environmental contaminants. The effects of bisphenol A (BPA) on spermatogenesis and sperm quality is still unclear. The objective of this study was to investigate the reproductive toxicity of BPA at dosages considered to be safe (5 or 25mg BPA/kg/day). We assessed multiple sperm parameters, the relative expression of genes involved in the central regulation of the hypothalamic-pituitary-testicular axis, and the serum concentrations of testosterone, estradiol, LH and FSH. BPA exposure reduced sperm production, reserves and transit time. Significant damage to the acrosomes and the plasma membrane with reduced mitochondrial activity and increased levels of defective spermatozoa may have compromised sperm function and caused faster movement through the epididymis. BPA exposure reduced the serum concentrations of testosterone, LH and FSH and increased the concentration of estradiol. The relative gene expression revealed an increase in gonadotropin releasing hormone receptor (Gnrhr), luteinizing hormone beta (Lhb), follicle stimulating hormone beta (Fshb), estrogen receptor beta (Esr2) and androgen receptor (Ar) transcripts in the pituitary and a reduction in estrogen receptor alpha (Esr1) transcripts in the hypothalamus. In this study, we demonstrated for the first time that adult male exposure to BPA caused a reduction in sperm production and specific functional parameters. The corresponding pattern of gene expression is indicative of an attempt by the pituitary to reestablish normal levels of LH, FSH and testosterone serum concentrations. In conclusion, these data suggest that at dosages previously considered nontoxic to reproductive function, BPA compromises the spermatozoa and disrupts the hypothalamic-pituitary-gonadal axis, causing a state of hypogonadotropic hypogonadism.

Daily exposure to silver nanoparticles during prepubertal development decreases adult sperm and reproductive parameters.

As silver nanoparticles (AgNPs) have antimicrobial properties and potentiate the activity of some antibiotics, they are broadly used in both medical and nonmedical applications. In this study, prepubertal male Wistar rats were orally treated with 15 or 30 µg/kg/day AgNPs from postnatal day 23 (PND23) to PND58 and sacrificed at PND102. The acrosome integrity, plasma membrane integrity, mitochondrial activity and morphological alterations of the sperm were analyzed. Sexual partner preference, sexual behavior and the serum concentrations of FSH, LH, testosterone and estradiol were also recorded. The results were evaluated following the appropriate statistical analyses, and differences among the groups were considered significant when p < 0.05. AgNPs reduced the acrosome and plasma membrane integrities, reduced the mitochondrial activity and increased the abnormalities of the sperm in both treatment groups. AgNP exposure also delayed the onset of puberty, although no changes in body growth were observed in either treatment group. The animals did not show changes in sexual behavior or serum hormone concentrations. This study shows for the first time that prepubertal exposure to AgNPs causes alterations in adult sperm parameters. Importantly, the sperm appeared to be more sensitive to the toxic effects of AgNPs and demonstrated adverse effects following exposure to lower doses. Consequently, the effects of AgNPs on sperm should be considered in order to establish safety limits for the use of these particles.

Development and application of a novel sensitive immunometric assay for calcitonin in a large cohort of patients with medullary and differentiated thyroid cancer, thyroid nodules, and autoimmune thyroid diseases.

BACKGROUND: Serum calcitonin (sCT) is a useful biomarker for medullary thyroid cancer (MTC). Consensus has not been reached concerning sCT measurements in the evaluation of nodular thyroid disease (NTD). OBJECTIVE AND METHODS: We developed a new immunofluorometric assay for sCT and have validated it in samples from 794 patients [203 with MTC, 205 with autoimmune thyroid disease (ATD), 248 with NTD, 80 with differentiated thyroid cancer (DTC) 'free of disease', 58 with chronic renal failure (CRF)] and 178 normal individuals, including samples after pentagastrin tests and samples from the washout of 92 FNA procedures in patients with NTD or MTC. We also compared some samples from patients with low or high calcitonin levels using both this assay and the Nichols Institute Diagnostics (NID) assay. RESULTS: The assay's analytical sensitivity was 1.0 pg/ml. Considering MTC patients prior to surgery, the cut-off values for the 95% reference range were 11.1 pg/ml for males and 5.5 pg/ml for females and employing the ROC curve were 18.4 pg/ml for males and 7.8 pg/ml for females. sCT in patients with MTC was strongly correlated with disease status. Patients with NTD and ATD did not present false-positive results. sCT measurements were significantly correlated with age (excluding MTC and CRF). The NID test had a strong correlation with our assay. A hook effect was observed only with concentrations >200,000 pg/ml. CONCLUSIONS: We developed a novel sCT assay and validated it in healthy subjects, as well as in a large cohort of patients with MTC, NTD, ATD, DTC, and CRF.

Basal serum thyroglobulin measured by a second-generation assay is equivalent to stimulated thyroglobulin in identifying metastases in patients with differentiated thyroid cancer with low or intermediate risk of recurrence.

BACKGROUND: Guidelines for the follow-up of differentiated thyroid cancer (DTC) recommend the measurement of TSH-stimulated thyroglobulin (s-Tg) instead of basal Tg on T4 therapy (b-Tg). However, these guidelines were established using first-generation Tg assays with a functional sensitivity (FS) of 0.5-1.0 ng/ml. Current more sensitive second-generation Tg assays (Tg2G; FS 0.05-0.10 ng/ml) have shown that low-risk DTC patients with undetectable b-Tg rarely have recurrences. OBJECTIVES: This study was undertaken to compare b-Tg using a chemiluminescent Tg2G assay (Tg2GICMA; FS 0.1 ng/ml) with s-Tg in DTC patients with an intermediate risk of recurrence. METHODS: We evaluated 168 DTC patients with a low (n = 101) and intermediate (n = 67) risk of recurrence treated by total thyroidectomy (147 also treated with radioiodine), with a mean follow-up of 5 years. RESULTS: b-Tg was undetectable with the Tg2GICMA in 142 of 168 patients. s-Tg was <2 ng/ml in 138 of these 142 patients, and only 3 of these 138 (2%) presented metastases on cervical ultrasound (US). Of the 4 of 142 patients with s-Tg >2 ng/ml, 1 had cervical metastases seen after radioiodine. Furthermore, 26 of 168 patients presented detectable b-Tg with the Tg2GICMA; 17 of these 26 patients also presented s-Tg >2 ng/ml. In 10 of these 17 patients, metastases were detected. Cervical US or b-Tg were positive in 14 of 15 patients with recurrent disease. Globally, the sensitivity and negative predictive value of the Tg2GICMA plus US were 93 and 99%, respectively. CONCLUSION: b-Tg measured with a Tg2GICMA and cervical US, used together, are equivalent to s-Tg in identifying metastases in patients with DTC with a low or intermediate risk of recurrence.

A ten-year clinical update of a large RET p.Gly533Cys kindred with medullary thyroid carcinoma emphasizes the need for an individualized assessment of affected relatives.

OBJECTIVE: Reviewing the clinical outcomes of a large kindred with a RET p.Gly533Cys mutation, 10 years after the first description of this kindred, has provided an important set of clinical data for healthcare decision-making. DESIGN AND PATIENTS: We identified 728 RET533 Brazilian relatives, spread out over 7 generations. We performed clinical examination, biochemical and imaging analyses in the proband and in 103 carriers. MEASUREMENT AND RESULTS: The proband has been followed without evidence of structural disease in the last 10 years but with elevated calcitonin. The clinical and surgical features of 60 thyroidectomized RET533 relatives were also described. Forty-six patients had MTC (21-72 years), and 11 patients had C-cell hyperplasia (CCH) (5-42 years). Twelve MTC patients with lymph node metastases had a tumour size of 0·7-2·8 cm. Calcitonin level and CEA were correlated with disease stage, and none of the patients presented with an altered PTH or metanephrine. A 63-year-old woman developed pheochromocytoma and breast cancer. Two other RET533 relatives developed lung squamous cell carcinoma and melanoma. CONCLUSIONS: A vast clinical variability in RET533 presentation was observed, ranging from only an elevated calcitonin level (3%) to local metastatic disease (25%). Many individuals were cured (42%) and the majority had controlled chronic disease (56%), reinforcing the need for individualized ongoing risk stratification assessment. The importance of this update relies on the fact that it allows us to delineate the natural history of RET 533 MEN2A 10 years after its first description.

Optimizing nucleic acid extraction from thyroid fine-needle aspiration cells in stained slides, formalin-fixed/paraffin-embedded tissues, and long-term stored blood samples / Otimização da extração de ácidos nucleicos de material de punção aspirativa por agulha fina de tiroide obtido de lâminas coradas, tecidos fixados em formalina e emblocados em parafina e amostras de sangue estocadas por longo período

OBJECTIVE: Adequate isolation of nucleic acids from peripheral blood, fine-needle aspiration cells in stained slides, and fresh and formalin-fixed/paraffin-embedded tissues is crucial to ensure the success of molecular endocrinology techniques, especially when samples are stored for long periods, or when no other samples can be collected from patients who are lost to follow-up. Here, we evaluate several procedures to improve current methodologies for DNA (salting-out) and RNA isolation. MATERIALS AND METHODS: We used proteinase K treatment, heat shock, and other adaptations to increase the amount and quality of the material retrieved from the samples. RESULTS: We successfully isolated DNA and RNA from the samples described above, and this material was suitable for PCR, methylation profiling, real-time PCR and DNA sequencing. CONCLUSION: The techniques herein applied to isolate nucleic acids allowed further reliable molecular analyses. Arq Bras Endocrinol Metab. 2012;56(9):618-26.

OBJETIVO: O isolamento adequado de ácidos nucleicos a partir de sangue periférico, lâmina corada de punção aspirativa por agulha fina, tecido fixado em formalina e emblocado em parafina e tecido fresco é fundamental para assegurar o sucesso de técnicas aplicadas em endocrinologia molecular, principalmente quando lidamos com amostras estocadas por longos períodos ou quando há impossibilidade de nova coleta de amostra de pacientes que perderam o seguimento. Neste trabalho, objetivamos otimizar as metodologias clássicas para a extração de DNA (salting-out) e RNA. MATERIAIS E MÉTODOS: Utilizamos proteinase K, choque térmico, dentre outras modificações, com o objetivo de aumentar a quantidade e a qualidade do material recuperado a partir das amostras descritas acima. RESULTADOS: Isolamos com sucesso DNA e RNA de tais amostras e o material obtido foi adequado para a realização de PCR, perfil de metilação, PCR em tempo real e sequenciamento de DNA. CONCLUSÃO: As técnicas aplicadas neste estudo para isolar ácidos nucleicos permitiram a realização posterior de análises moleculares consistentes e confiáveis. Arq Bras Endocrinol Metab. 2012;56(9):618-26.