Influence of chronic lymphocytic thyroiditis on the risk of persistent and recurrent disease in patients with papillary thyroid carcinoma and elevated antithyroglobulin antibodies after initial therapy / Influência da tireoidite linfocítica crônica sobre o risco de doença persistente e recorrente em pacientes com carcinoma papilífero de tireoide e anticorpos antitireoglobulina elevados após a terapia inicial

Abstract Introduction In patients with papillary thyroid carcinoma who have negative serum thyroglobulin after initial therapy, the risk of structural disease is higher among those with elevated antithyroglobulin antibodies compared to patients without antithyroglobulin antibodies. Other studies suggest that the presence of chronic lymphocytic thyroiditis is associated with a lower risk of persistence/recurrence of papillary thyroid carcinoma. Objective This prospective study evaluated the influence of chronic lymphocytic thyroiditis on the risk of persistence and recurrence of papillary thyroid carcinoma in patients with negative thyroglobulin but elevated antithyroglobulin antibodies after initial therapy. Methods This was a prospective study. Patients with clinical examination showing no anomalies, basal Tg < 1 ng/mL, and elevated antithyroglobulin antibodies 8-12 months after ablation were selected. The patients were divided into two groups: Group A, with chronic lymphocytic thyroiditis on histology; Group B, without histological chronic lymphocytic thyroiditis. Results The time of follow-up ranged from 60 to 140 months. Persistent disease was detected in 3 patients of Group A (6.6%) and in 6 of Group B (8.8%) (p = 1.0). During follow-up, recurrences were diagnosed in 2 patients of Group A (4.7%) and in 5 of Group B (8%) (p = 0.7). Considering both persistent and recurrent disease, structural disease was detected in 5 patients of Group A (11.1%) and in 11 of Group B (16.1%) (p = 0.58). There was no case of death related to the disease. Conclusion Our results do not support the hypothesis that chronic lymphocytic thyroiditis is associated with a lower risk of persistent or recurrent disease, at least in patients with persistently elevated antithyroglobulin antibodies after initial therapy for papillary thyroid carcinoma.

Resumo Introdução Em pacientes com carcinoma papilífero de tireoide e com tireoglobulina sérica negativa após a terapia inicial, o risco de doença estrutural é maior entre aqueles com anticorpos antitireoglobulina elevados em comparação com pacientes sem anticorpos antitireoglobulina. Outros estudos sugerem que a presença de tireoidite linfocítica crônica está associada a um menor risco de persistência/recorrência do carcinoma papilífero de teireoide. Objetivo Este estudo prospectivo avaliou a influência da tireoidite linfocítica crônica sobre o risco de persistência e recorrência do carcinoma papilífero de tireoide em pacientes com tireoglobulina negativa, mas com anticorpos antitireoglobulinas elevados após a terapia inicial. Método Esse foi um estudo prospectivo, no qual foram selecionados pacientes com exame clínico sem anomalias; tireoglobulina basal < 1 ng/mL e anticorpos antitireoglobulina elevados 8-12 meses após ablação. Os pacientes foram divididos em dois grupos: Grupo A, com tireoidite linfocítica crônica no exame histológico; Grupo B, histologicamente sem tireoidite linfocítica crônica. Resultados O tempo de seguimento variou de 60 a 140 meses. Doença persistente foi detectada em 3 pacientes do Grupo A (6,6%) e em 6 do Grupo B (8,8%) (p = 1,0). Durante o seguimento, as recidivas foram diagnosticadas em 2 pacientes do Grupo A (4,7%) e em 5 do Grupo B (8%) (p = 0,7). Considerando tanto a doença persistente quanto a recorrente, doença estrutural foi detectada em 5 pacientes do Grupo A (11,1%) e em 11 do Grupo B (16,1%) (p = 0,58). Não houve nenhum caso de óbito relacionado à doença. Conclusão Nossos resultados não apoiam a hipótese de que a tireoidite linfocítica crônica esteja associada a um menor risco de doença persistente ou recorrente, pelo menos em pacientes com anticorpos antitireoglobulina persistentemente elevados após a terapia inicial do carcinoma papilífero de tireoide.

Arterial stiffness and 25-hydroxyvitamin D levels in chronic kidney disease patients

Summary Objective: Arterial stiffness refers to arterial wall rigidity, particularly developing in central vessels. Arterial stiffness increases in early stage chronic kidney disease (CKD), and it is a strong predictor of cardiovascular and all cause mortality. Vitamin D has beneficial effects on blood pressure, vascular endothelial function and arterial stiffness. 25-hydroxyvitamin D (25(OH)D) deficiency is quite common worldwide and in the CKD population. We aimed to evaluate the prevalence of 25(OH)D deficiency and its relation with arterial stiffness in CKD. Method: Our study included 101 patients (51 male, 50 female), with stages 3B-5 CKD not on dialysis. A single-cuff arteriograph device (Mobil-O-Graph) was used to evaluate arterial stiffness parameters of pulse wave velocity (PWV) and augmentation index (Alx@75). The patients were divided into two groups: group I vitamin D non-deficient [25(OH)D > 15 ng/mL] and group II vitamin D deficient [25(OH)D ≤ 15 ng/mL]. Results: Overall, the mean 25(OH)D level was 14.1±7.9 ng/mL and 70 patients (69.4%) were vitamin D deficient. The mean Alx@75 value was significantly higher in group II (28.6±10.8% vs. 23.3±13.5%, p=0.038). PWV was higher in group II, but the difference was not significant. Group II exhibited significantly lower serum albumin (p<0.001), hemoglobin (p=0.005), calcium (p=0.041) and estimated glomerular filtration rate (eGFR) (p=0.041), but significantly higher 24-hour proteinuria (p=0.011) and more females (p=0.006). Vitamin D was negatively correlated with Alx@75 augmentation pressure, parathyroid hormone, proteinuria and body mass index, and positively correlated with albumin, hemoglobin, eGFR, calcium and transferrin. 25(OH)D was independently associated with Alx@75 (beta=-0.469, p=0.001) and albumin (beta=0.447, p=0.002). Conclusion: In CKD patients 25(OH)D deficiency was common, particularly in females. Level of 25(OH)D was independently associated with Alx@75.

Determination of cortisol and cortisone in human saliva by a liquid chromatography-tandem mass spectrometry method / Determinação de cortisol e cortisona na saliva por método baseado em cromatografia líquida e espectrometria de massas em tandem

Objective Salivary cortisol measurement plays an important role in the evaluation of adrenal function. Its high correlation with free serum cortisol, the easy of sampling and the limited presence of interfering steroids, generated multiple recent studies of its application, in special in the screening of adrenal hyperfunction. In this paper we present our experience in the development of a high pressure liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for salivary cortisol and cortisone measurement. Materials and methods For this study we used 181 saliva samples from our routine diagnostic laboratory. The HPLC-MS/MS method was based on a Waters Quattro Premier tandem mass spectrometer with an electrospray probe. After derivatization with hydroxylamine transitions monitored included cortisol and cortisone. An in-house radioimmunoassay (RIA) was used for salivary cortisol results comparison. Results Functional sensitivity was 24 ng/dL for cortisol and linearity from 24 to 1929 ng/dL. Saliva cortisol values obtained in the 181 samples presented a median of 52 ng/dL with 5‐95% percentile of 24 and 374 ng/dL. With the RIA the results were 86, 25 and 436 ng/dL, respectively, with values for RIA being significantly higher (P<0.0001) and high correlation (r=0.8312, P<0.0001). Cortisone measured in 159 samples showed a median of 278 ng/dL, with 5‐95% percentile of 100 and 1,133 ng/dL. Correlation with cortisol values was significant (r=0.820, P<0.0001). Conclusion We conclude that the HPLC-MS/MS method compares favorably with the RIA for salivary cortisol measurement, with the additional possibility of concomitant cortisone measurement and the evaluation of 11βHSD2 activity. .

Objetivo A dosagem de cortisol salivar é uma metodologia que vem tendo crescente aceitação no estudo da função adrenocortical. Sua alta correlação com a fração livre sérica, facilidade de coleta e presença limitada de interferentes têm originado múltiplas publicações, em especial no screening de pacientes suspeitos de hiperfunção. Neste trabalho apresentamos nossa experiência no desenvolvimento de metodologia baseada em cromatografia líquida e espectrometria de massas (HPLC-MS/MS) para a medida de cortisol e cortisona salivares. Materiais e métodos Para este estudo utilizamos 181 amostras de saliva de nossa rotina diagnóstica. A metodologia de HPLC-MS/MS baseou-se num espectrômetro de massas Waters Quattro Premier. Após derivatização com hidroxilamina, as transições monitoradas incluíram cortisol e cortisona. Um radioimunoensaio (RIE) in house foi empregado para comparação. Resultados A sensibilidade funcional para cortisol foi de 24 ng/dL, com linearidade entre 24 e 1,929 ng/dL. Os valores de cortisol obtidos nas 181 amostras apresentaram mediana de 52 ng/dL, com percentis 5‐95% de 24 e 374 ng/dL. Com o RIE, os resultados foram 86, 25 e 436 ng/L, respectivamente, com os valores obtidos no RIE significativamente mais elevados (P<0,0001), e alta correlação (r=0,8312, P<0,0001). Cortisona, medida em 159 amostras, mostrou mediana de 278 ng/dL, com percentis 5‐95% entre 100 e 1.133 ng/dL. A correlação com os valores de cortisol foi significativa (r=0,820, P<0,0001). Conclusão Concluímos que o método baseado em HPLC-MS/MS compara-se favoravelmente com o RIE para a medida de cortisol salivar, com a possibilidade adicional da medida concomitante de cortisona e avaliação da atividade da enzima 11βHSD2. .

Análise de um radioimunoensaio iodado para determinação de 11-deoxicortisol / Analysis of an iodide radioimmunoassay for 11-deoxicortisol measurement

Objetivo : Nosso objetivo foi comparar duas técnicas de dosagem do 11-desoxicortisol: a técnica de radioimunoensaio iodado, a qual foi validada neste trabalho, e a cromatografia líquida de alta performance seguida por espectrometria de massa em tandem (LC-MS/MS), sendo a última considerada o padrão-ouro para dosagem dos hormônios esteroides. Materiais e métodos : Para a comparação entre os resultados de 11-desoxicortisol, foram selecionadas 88 amostras. Resultados : A sensibilidade analítica do radioimunoensaio foi de 0,30 ng/mL, com linearidade e perfil de precisão inadequado (34% das amostras com CV ≥ 20%). Das 88 amostras selecionadas, apenas 54 apresentaram resultados mensuráveis em ambos os métodos. A comparação desses resultados, por meio da regressão de Deming, resultou em um coeficiente de correlação de 0,610, inclinação de 3,751, intercepção de 0,145, evidenciando a pobre correlação entre os resultados e a superestimação dos resultados pelo RIA. Conclusão : Concluímos que o método de dosagem de 11-desoxicortisol por radioimunoensaio iodado apresentou resultados inadequados nos diversos parâmetros avaliados, inviabilizando sua utilização como método de dosagem do 11-desoxicortisol. Arq Bras Endocrinol Metab. 2014;58(3):232-6 .

Objective : Our aim was to correlate 11-deoxycortisol levels obtained by two currently available techniques for 11-deoxycortisol measurement: radioimmunoassay, and high performance liquid chromatography followed by tandem mass spectrometry (MS/MS). The latter is the gold standard method for steroid hormone measurement. Materials and methods : We selected 88 samples and the results of these two methods were compared by Deming regression. Results : The analytical sensitivity of the RIA was 0.30 ng/mL, with inadequate linearity and inadequate precision profile (34% of the samples had a CV ≥ 20%). From the selected samples, 54 had measurable levels of 11-deoxycortisol in both methods and were used in the comparison. The comparison of RIA with LC-MS/MS showed an overestimation of the results by RIA. The correlation coefficient was 0.610; linear regression slope was 3.751; and the intercept was 0.145, indicating a poor correlation between the two methods. Conclusion : We concluded that 11-deoxycortisol measured by radioimmunoassay, despite a good analytical sensitivity, showed very low specificity, precluding its use as a reliable method for 11-deoxycortisol measurement. Arq Bras Endocrinol Metab. 2014;58(3):232-6 .

In utero and lactational exposure to dioxin altered the reproductive function in female rat offspring before and after puberty

To evaluate effects of in utero and lactational 2,3,7,8-tetrachlorodibenzo-rho-dioxin [TCDD] exposure on the reproductive function in female rat offspring, before and after puberty. The pregnant Sprague Dawely rat administered 0, or 1.0 rag TCDD/kg on Gestation Day [GD] 8 and 15. Female offspring were examined at the post-weanling before puberty on posnatal day [PND] 21 and in young adult stage of development on PND42. Ovulation assessment, radioimmunoassay for serum gonadotropins, steroids and histo-morphmetric analysis to the ovaries were evaluated. The analysis included a count, measurement and classification of preantral and antral follicles throughout the entire ovary on PND 21. The results indicate that TCDD treatment significantly reduced the ovulation rate, serum gonadotropins, steroids levels and the number of antral and preantral follicles of certain size classes. The histopathological examination revealed small preovulatory follicles displaying an atretic morphologic difference among the ovaries of rats exposed to TCDD treatments. These data support the hypothesis that TCDD results in adverse effects on female reproductive function. However, the age of animals before or after puberty play an important role in the difference between results. Moreover, TCDD exposure on the GD 8 or 15 has a great concern in the results observed

Desenvolvimento de um radioimunoensaio de dexametasona no soro como subsídio para o diagnóstico da síndrome de Cushing / Development of serum dexamethasone radioimmunoassay to corroborate Cushing's syndrome diagnosis

INTRODUÇÃO: O teste de supressão com 1 mg de dexametasona (TSDx) é amplamente empregado no rastreamento da síndrome de Cushing (SC) dada sua elevada acurácia diagnóstica. A SC é um distúrbio endocrinometabólico resultante do hipercortisolismo crônico com característica de ausência de supressão do cortisol no TSDx. OBJETIVO: Desenvolver um radioimunoensaio (RIE) para a dosagem de dexametasona (Dx) no soro para complementar o TSDx. MÉTODOS: Foram imunizados três coelhos com o conjugado dexametasona-21-hemissuccinato-BSA para escolha do melhor anticorpo e o RIE foi desenvolvido de acordo com as recomendações da RE 899/2003 da Agência Nacional de Vigilância Sanitária (ANVISA). Analisamos 96 voluntários, sendo 67 submetidos ao TSDx e 29 não, e 12 pacientes com SC, estudados na Universidade Federal de São Paulo (UNIFESP) e na Santa Casa de Misericórdia de São Paulo. RESULTADOS: O anticorpo contra Dx selecionado mostrou boa especificidade, coeficiente de variação (CV por cento) intra e interensaio < 20 por cento, exatidão de 93,8 por cento e dose mínima detectada de 19,5 ng/dl. A concentração de Dx no soro foi semelhante nos voluntários e pacientes com SC (ausência/supressão do cortisol): 205 a 703 ng/dl e 174 a 661 ng/dl (intervalo de confiança [IC] 95 por cento), respectivamente; os valores foram indetectáveis naqueles que não se submeteram ao teste. DISCUSSÃO: O anticorpo empregado apresenta boa afinidade e especificidade para quantificar a Dx no soro. O RIE mostrou reprodutibilidade e eficiência na determinação dos níveis séricos de Dx durante o TSDx. CONCLUSÃO: O presente RIE para a dosagem de Dx no soro é acurado e confiável, permitindo estabelecer uma faixa de referência de valores para subsidiar a interpretação do TSDx.

INTRODUCTION: The 1 mg dexamethasone suppression test (DxST) is widely used to screen Cushing's syndrome (CS) due to its high diagnostic accuracy. CS is an endocrine-metabolic disorder caused by hypercorticism, which is characterized by the absence of cortisol suppression in DxST. OBJECTIVE: To develop a radioimmunoassay (RIA) for the measurement of serum dexamethasone (Dx) to complement DxST. METHODS: Three rabbits were inoculated with dexamethasone-21-hemisuccinate-BSA in order to choose the best antibody. Serum Dx RIA was performed according to RE 899/2003 (Agência Nacional de Vigilância Sanitária [ANVISA]) regulations. Serum samples from 96 volunteers from Universidade Federal de São Paulo (UNIFESP) and Santa Casa de Misericórdia de São Paulo were analyzed, 67 of which were submitted to DxST and 29 were not. There were 12 patients with CS. RESULTS: The Dx antibody chosen showed good specificity. Intra- and interassay CV were < 20 percent with 93.8 percent accuracy and the lowest detection limit was 19.5 ng/dl. Serum Dx concentration was similar among both volunteers and CS patients (absence of cortisol suppression): 205 to 703 ng/dl and 174 to 661 ng/dl (95 percent CI), respectively. Values were undetectable among those that were not submitted to the test. Discussion: The anti-Dx antibody shows high specificity and reliability to quantify serum Dx in DxST. The Dx RIA presented reproducibility and reliability in the determination of serum Dx levels during DxST. CONCLUSION: The current RIA for serum Dx is accurate and reliable, which permits to establish a reference value range to substantiate DxST interpretation.

overview of various labeled assays used in medical laboratory diagnosis immune and non-immune assays

In this review, some light is thrown on various labeled immunoassays that depend on antigen-antibody [Ag-Ab] reactions, including immunofluorescence, radioimmunoassay, and enzyme immunoassay [EIA or ELISA]. Their definitions, principles, and applications are described, then they are discussed chronologically to show their stepwise development that led finally to full automation. Enzyme labeled immunoblot assays [Western blot, blot spot, and recombinant immunoblot assay], and luminescence [bioluminescence and chemiluminescence] are also discussed chronologically. Labeled assays, that do not involve Ag-Ab reaction but rather, utilizing biotin-steptavidin [BS] interaction and probe-target DNA interaction, and described, together with their applications for DNA/RNA detection and genotyping. Finally, included in the discussion were some luminescent labeled techniques that utilitze the immune Ag-Ab reaction together with non-immune BS reaction, such as the luminescent oxygen channeling immunoassay, and its commercialized AlphaLISA, both eliminate the washing steps without sacrificing high sensitivity, or wide dynamic range

relation between maternal vitamin D levels and their neonates

In recent years, despite of the numerous preventive and therapeutic strategies now available, vitamin D deficiency has resurfaced as a global health problem among infants and children. There has been a resurgence of nutritional rickets in children in many developing countries and some developed countries. We evaluate the magnitude of vitamin D deficiency among Kuwaiti mothers and their neonates to provide adequate basis for recommendation to prevent or at least minimize vitamin D deficiency effects. We determined vitamin D levels among mothers and their neonates to detect if their relationship, if any to infantile rickets. One hundred twenty eight full term pregnant mothers and their neonates were selected from medical consultation center in Kuwait. All mothers had normal vaginal delivery. On the day of delivery 2.5ml of maternal blood and 2.5 of cord blood samples were withdrawn .Serum 25-hydroxyvitamin D[25 OHD] was determined in duplicate by radioimmunoassay using lncstar kit. A total 128 mother-neonates pairs were selected from medical consultation center The mean [ +/- SD] age and parity of the mother were 24.7[4,6], 2.48 [1.51] respectively. The mean [ +/- SD] 25OHD levels of mothers their neonates were 17.5 [6.8] ng/ml, 8.1[6,1]ng/ml respectively. Our results demonstrated that vitamin D of the mothers and neonates are highly correlated About 60%of neonates and 45%of their mothers are vitamin D deficient on the day of delivery

Validação de conjuntos diagnósticos comerciais para dosagem sérica de progesterona e estradiol em jaguatirica (Leopardus pardalis) e gato-do-matopequeno (Leopardus tigrinus / Progesterone and estradiol commercial kits validation in serum of ocelots (Leopardus pardalis) and tigrina (Leopardus tigrinus)

Este estudo teve como objetivo validar os conjuntos diagnósticos m comerciais DPC (Coat-A-Count - Diagnostic Products Corporation/USA), em fase sólida, para dosagem de progesterona e DSL (Diagnostic System Laboratories INC/USA), em fase líquida, para dosagem de estradiol, por radioimunoensaio, em soro de Leopardus pardalis (n=5)e Leopardus tigrinus (n=4), antes (15-30 dias) e após (24-28 horas) tratamento com gonadotropinas exógenas (hCG/ Novormon® eeCG/ Vetecor®; pFSH/Folltropin-V® e pLH/Lutropin-V®). A concentração sérica para Leopardus pardalis variou 0,005 a 0,151ng/ml para estradiol e 0,15-37,22ng/ml para progesterona. A concentração sérica para Leopardus tigrinus variou 0,012-0,147ng/ml para estradiol e 0,06-34,09ng/ml para progesterona. A sensibilidade mínima detectada foi 0,004 ng/ml para progesterona e 0,00014ng/ml para estradiol. Para progesterona o coeficiente intra-ensaio baixo foi 2,58% e alto6,36%, já o coeficiente inter-ensaio baixo foi 0,67% e alto 5,55%. Para o estradiol o coeficiente intra-ensaio baixo foi 0,69% e alto 4,15%,sendo o coeficiente inter-ensaio baixo 1,40% e alto 3,00%. Paralelismo foi encontrado para progesterona e conjunto comercial DPC com r = 0,96 para Leopardus pardalis e r = 0,99 para Leopardus tigrinus. Para estradiol em conjunto comercial DSL paralelismo foi encontrado com r = 0,98 para Leopardus pardalis e com r = 0,99 para Leopardus tigrinus. Com estes resultados podemos concluir que o conjunto diagnóstico comercial DPC para dosagem de progesterona e DSL para dosagem de estradiol foram validados para utilização em soro de Leopardus pardalis e Leopardus tigrinus, podendo ser utilizado como ferramenta no manejo reprodutivo destas espécies visando à conservação.

DPC commercial kit (Coat-A-Count - Diagnostic Products Corporation/USA) and DSL commercial kit (Diagnostic System Laboratories INC/USA) were used for progesterone and estradiol radioimmunoassay validation in serum of two endangered Brazilian felids: Leopardus pardalis (n=5) and Leopardus tigrinus (n=4) before (15-30 days) and after (24-28 hours) exogenous gonadotrophins treatment(hCG/ Novormon® and eCG/ Vetecor®; pFSH/Folltropin-V® andpLH/Lutropin-V®). Variation of estradiol concentration was 0.005 -0.151 ng/ml and progesterone concentration was 0.15 - 37.22 ng/ml for Leopardus pardalis. Variation of estradiol concentration was 0.012 -0.147 ng/ml and progesterone concentration was 0.15 - 37.22 ng/ml for Leopardus tigrinus. Minimum sensibility detected for progesterone was 0.004 ng/ml and for estradiol was 0.00014 ng/ml. Progesterone intra-assay coefficient was 2.58% and 6.36% and inter-assay was 0.67% and 5.55%. Estradiol intra-assay coefficient was 0.69% and 4.15%,and inter-assay was 1.40% and 3.00%. Parallelism was used for kits validation. Progesterone and DPC commercial kit Parallelism wasr = 0.96 for Leopardus pardalis and r = 0.99 for Leopardus tigrinus. Estradiol and DSL commercial kit Parallelism was r = 0.98 for Leopardus pardalis and r = 0.99 for Leopardus tigrinus. In conclusion, these results showed that DPC commercial kit for serum progesterone dosages and DSL commercial kit for serum estradiol dosages can be used for Leopardus pardalis e Leopardus tigrinus. These findings are potentially valuable for the reproductive management and conservation of endangered felid populations.

Evaluation of preoperative predictive values of serum CA15-3 and CEA within Sudanese women with breast cancer

Objectives: Early detection of cancer comprises early diagnosis in symptomatic and screening of asymptomatic individuals.Our aim was to evaluate the significant values of carbohydrate antigen 15-3 (CA15-3) and/or carcinoembryonic antigen (CEA) in women with breast cancer.Design and setting: This case control study was conducted in Khartoum Teaching Hospital; Khartoum; Sudan. Application of such measurement may be helpful within screening and early detection efforts in such a country like Sudan with poor resources.Methods: We examined by serological radioimmuno-assay methods; significant elevation of CA15-3 and CEA serum samples obtained from 100 women of whom 40and 35were patients with histopathologically confirmed breast cancer and benign breast lumps respectively and the remaining 25were apparently healthy controls. Statistical analysis: Data were analyzed by using a computer SPSS program.Results: Among the 75 patients with breast lumps; 33 (44) and 31(37.3) showed high CA15-3 and CEA levels respectively. Of the 40 carcinomas; high expressions of CA15-3 and CEA were found among 28(70) and 24(60) respectively. Notably; only 2(8) of the controls showed lightly elevated CEA. Conclusions: The obtained Specificity of 85.7; 80and sensitivity of 70; 60for CA15-3 and CEA correspondingly; support the combined application of both markers in screening for breast cancer

Emprego da cromatografia líquida de alta eficiência na determinação de cortisol sérico em substituição à técnica de radioimunoensaio / High-performance liquid chromatography application for serum cortisol quantification as a substitute for radioimmunoassay

Introdução: A determinação de cortisol nos diferentes fluídos orgânicos tem sido aplicada como auxílio diagnóstico em distintas condições nosológicas em humanos, bem como empregada em estudos envolvendo pesquisa clínica. No intervalo de aplicação clínica, rotineiramente é determinado pela técnica de radioimunoensaio (RIE). Na determinação do cortisol urinário livre essa técnica vem sendo substituída peloemprego da cromatografia líquida de alta eficiência (HPLC), principalmente no diagnóstico da síndrome de Cushing. Já para a determinação do cortisol sérico não se têm evidências do emprego da cromatografia líquida em substituição a outras técnicas analíticas. Objetivos: O desenvolvimento de metodologia analítica empregando HPLC no modo fase reversa (RP-HPLC) para a determinação de cortisol sérico em substituição ao RIE visando à redução da geração de resíduos radioativos. Material e métodos: O cortisol foi quantificado diretamente empregando-se RP-HPLC em amostras de soro previamente extraídas com éter utilizando-se acetonido de triancinolona como padrão interno (PI). Utilizou-se coluna analítica BDS-Hypesil-C18® (125 x 4 mm, 5 μm), fase móvel composta de água e acetonitrila (72:28; v/v) a 1 ml/min e detecção a 243 nm. Resultados: O cortisol e o PI apresentaram tempo de retenção de 3,4 e 7,1 min, respectivamente. O coeficiente de variação (CV%) obtido no estudo da precisão foi menor que 10%, e a exatidão apresentou um desvio inferior a 4%. Discussão: O método mostrou-se eficaz e eficiente, com sensibilidade e linearidade na faixa estudada de 2,5 a 60 μg/dl. Conclusão: O método proposto substitui o RIE no intervalo de sua aplicação clínica.

Background: The quantification of cortisol in different organic fluids has not only been applied to different humannosological conditions as a diagnostic aid but it has also been used in clinical research. In clinical application, cortisolis routinely measured by radioimmunoassay (RIA). In the determination of free urinary cortisol this technique has been replaced by the high-performance liquid chromatography mainly in the diagnosis of Cushing syndrome. As to serum cortisol determination, there is no evidence of the application of liquid chromatography as a substitute for other analytical techniques. Objective: The development of an analytical methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) to determine serum cortisol levels as a substitute for RIA in order to reduce radioactive waste. Material and methods: Cortisol was directly quantified by RP-HPLC in previouslyether-extracted serum samples. Triamcinolone acetonide was used as internal standard (IS). The chromatographic separation was developed in a BDS-Hypersil-C18® column (125 x 4 mm, 5μm) using water-acetonitrile (72:28; v/v) as mobile phase at 1 ml/min and steroid peaks were measured at 243 nm. Results: Cortisol and IS presented retention time of 3.4 and 7.1 min, respectively. The precision was less than 10% and accuracy was less than 4%. Discussion: The method was effective and efficient, with good sensitivity and linearity in the concentration range of 2.5 to 60.0μg/dl. Conclusion: The present methodology substitutes RIA at clinical application.

Screening for Y-chromosome microdeletions in infertile Indian males: utility of simplified multiplex PCR.

BACKGROUND & OBJECTIVE: Analysis of the microdeletions in the azoospermia factor (AZF) region of Y chromosome by PCR is an important screening tool in the work-up of infertile males opting for assisted reproductive techniques. In the present study, the Y chromosome microdeletions were analyzed by PCR using primers corresponding to 16 sequence tagged sites (STS) and three genes of the AZF region in infertile Indian men. Feasibility of developing a simplified multiplex PCR for screening of the Y chromosome microdeletions has been explored. METHODS: A total of 271 male subjects were analyzed, of which, 170 were infertile patients (51 oligospermic and 119 azoospermic) and 101 were fertile controls. Subjects showing normal karyotype only were included in the study. The semen analysis was done and plasma follicle stimulating hormone (FSH) concentrations were determined by radioimmunoassay. Testicular histopathology was analyzed by fine needle aspiration cytology (FNAC). RESULTS: Y chromosome microdeletions were observed in nine out of 170 (5.29%) infertile males all of whom were azoospermic. Of the nine subjects, two had deletions in AZFa, one in AZFb, three in AZFc and three in AZFb+c regions. No deletions were observed in the infertile severe oligospermic men (< 5 million sperm/ml semen) and fertile controls. No difference in the FSH concentrations of infertile patients with and without deletions (18.36 and 18.10 mIU/ml respectively) was observed. A clear relationship between Y chromosome microdeletions and testicular phenotypes could not be established. Two multiplex PCRs were designed using 7 STSs markers, which could detect Y chromosome microdeletions in infertile male subjects as efficiently as PCR based on larger number of PCR reactions. INTERPRETATION & CONCLUSION: The multiplex PCRs described in the present study may be a suitable, cost-effective and less time consuming method for screening the Y chromosome deletions in infertile males in routine clinical diagnosis and counselling prior to assisted reproduction.

Relation of serum estrogen level to endometrial abnormalities and uterine blood flow in cases of post menopausal bleeding

Post menopausal women are subject to various endometrial changes, this study was done to determine the relation between endometrial thickness, abnormalities and uterine blood flow, to the serum estrogen level in cases of postmenopausal bleeding. The study was carried out for Two years period May 2005-Apr 2007 including 50 patients with postmenopausal uterine bleeding, examined by transvaginal ultrasound to assess the endometrial thickness and echogenicity; Color Doppler imaging was used to evaluate blood flow velocity through both uterine arteries and Pulsatility Index [PI] to analyze the waveform; Serum estrogen level was measured to all cases using the Radio-immunoassay [RIA] technique, The normal value of serum estrogen in postmenopausal women is 5-25 pg/ml. Dilatation and curettage was done to study the endometrial and endocervical histopathological feature. TVS and measurement of serum E2 were used as a screening tests for the detection of postmenopausal endometrial hyperplasia and/or malignancy. Doppler flow mapping for the uterine arteries and histopathological evaluation further added as complementary parameters. Endometrial hyperplasia was reported in 22 cases [44%] and endometrial cancer in 12 cases [24%]. The hyperplasia group further classified into simple, complex and atypical. Simple hyperplasia recorded in 12 cases [24%], complex in 7 cases [14%], and atypical in 3 cases [6%]. Endometrial polyp was seen in 5 cases [10%], Endometrial atrophy was seen in 7 cases [14%], while fibroid was seen in 4 patients [8%]. Assessment of the endometrium by TVS and measurement of serum E2 should be used as a primary diagnostic simple screening tests for the detection of postmenopausal endometrial hyperplasia and/or malignancy; But they must be supplemented by Doppler sonography of the uterine arteries whenever the serum E2 level is above normal or endometrial thickness is more than 5 mm in order to detect cancer at an early stage

Produção de progesterona in vitro pelas células do corpo lúteo bovino ao longo da gestação / In vitro progesterone production from bovine corpus luteum throughout gestation

O presente trabalho foi desenvolvido para testar a hipótese de que células luteínicas bovinas em cultivo, provenientes dos três terços de gestação, comportam-se da mesma maneira que células in vivo em relação à produção de P4. Foram coletadas amostras de corpos lúteos (CL) de 90 (n=3), 150 (n=3) e 210 (n=3) dias de gestação obtidos em abatedouro. Sob condições assépticas, as células foram mecanicamente dispersas e cultivadas em placas de 96 poços. Após 24 horas de cultivo foram feitas a lavagem dos poços e a adição do precursor pregnenolona. Os tratamentos foram realizados em octuplicata para cada tempo de tratamento (24, 48 e 96 horas) com três repetições de cada período gestacional. As amostras de meio de cultura e as células foram coletadas 24, 48 e 96 horas após adição do precursor e acondicionadas em freezer a -20ºC até o processamento. A progesterona foi dosada através de radioimunoensaio e o conteúdo protéico pelo método de Lowry. Os resultados foram analisados estatisticamente e considerados diferentes quando p<0.05. Foi observada maior produção de P4 aos 90 dias de gestação (35,277±0,075), posterior decréscimo aos 150 dias (28,820±0,231) e novo aumento aos 210 dias (32,777±0,099). A produção de P4 em células cultivadas por 24 horas foi maior (p<0,05) em células oriundas do grupo de 90 dias (2,912±0,047) quando comparado a 150 (2,669±0,137) e 210 dias (2,741±0,088). As 48 e 96 horas de cultivo, células luteínicas bovinas de 90 dias produziram mais P4 que células de 210 dias (2,934±0,029 e 2,976±0,121 respectivamente x 2,760±0,059 e 2,695±0,149, respectivamente; p<0,05), que por sua vez produziram mais do que células de 150 dias (2,334±0,084 para 48 horas e 2,205±0,136 para 96 horas). Aos 150 dias de gestação a produção de progesterona apresentou diminuição gradativa ao longo das 96 horas de cultivo. Essas diferenças podem ser explicadas pela expressão gênica diferencial de enzimas ou também de fatores presentes na cascata esteroidogênica...

The aim was to test the hypothesis that cultivated bovine luteal cells from three different thirds of pregnancy behave the same way as in vivo luteal cells relative to P4 production. Corpus luteum samples from days 90 (n=3), 150 (n=3) and 210 (n=3) of pregnancy were obtained at a local slaughterhouse. Under aseptic conditions cells were mechanically dispersed and cultivated in a 96 wells-plate. After 24 hours of culture, cells were washed and the precursor pregnenolone was added. Experiments were conducted eight times for each studied time period (24, 48 and 96 h) and three times for each gestational age. Culture medium and cells were collected after 24, 48 and 96 hours of precursor addition and kept frozen at -20ºC until processing. Progesterone was measured by RIA and protein content by Lowry's method. Results were statistically analyzed and considered different when p <0.05. A higher P4 production was observed on day 90 of gestation (35.277±0.075), then this production was decreased at day 150 (28.820±0.231) and increased again at day 210 (32.777±0.099). After 24 hours of culture, luteal cells P4 production reached maximum values in the group of 90 days (2.912±0.047) when compared to 150 (2.669±0.137) and 210 days (2.741±0.088). At 48 and 96 hours of culture, bovine luteal cells from day 90 of gestation produced more P4 than cells from day 210 (2.934±0.029 and 2.976±0.121 respectively x 2.760±0.059 and 2.695±0.149, respectively; p<0.05), which in turn, produced more P4 than cells from day 150 (2.334±0.084 for 48 h and 2.205±0.136 for 96 h). Luteal cells from day 150 of gestation presented a decreasing P4 production throughout the 96 hours of culture. These differences could be explained by differential gene expression of enzymes and/or factors belonging to the esteroidogenic cascade in accordance to the gestational period. The established luteal cell culture model could be used for further functional studies once P4 secretion pattern...

Efeito de diferentes protocolos de superovulação sobre a concentração plasmática de progesterona e de metabólitos lipídicos de vacas Nelore / Effect of different superovulation protocols on plasma progesterone concentration and lypidic metabolites in Nelore cows

Avaliaram-se as concentrações plasmáticas de triglicérides, colesterol, aspartato transaminase (AST) e progesterona (P4) em vacas Nelore não lactantes com elevado escore corporal, superovuladas com diferentes protocolos. Foram utilizados três grupos de animais, G1 (n=11), G2 (n=8) e G3 (n=5), superovulados com 500UI de FSH, 200mg e 180mg de FSH (hormônio folículo estimulante), respectivamente, em doses decrescentes, duas vezes ao dia, durante quatro dias. As amostras de sangue foram coletadas antes da superovulação (A), no terceiro dia da superovulação (B), no momento da inseminação artificial (C) e na coleta dos embriões (D). As concentrações de triglicérides, AST e colesterol foram verificados por espectrofotometria, e a de progesterona (P4) por radioimunoensaio. Não houve alteração (P>0,05) na concentração de triglicérides, AST e colesterol entre as amostras. Não houve efeito (P>0,05) do protocolo de superovulação sobre a concentração de triglicérides, AST e P4 nas diferentes amostras. O G2 apresentou menor concentração de colesterol (P<0,05) nas amostras A e B, possivelmente em razão da grande instabilidade dessa variável.

The purpose of this research was to evaluate the concentration of tryglicerides, cholesterol, aspartate transaminase (AST) and progesterone (P4) in embryo donor Nelore cows superovulated with different protocols. Twenty four donors were randomly distributed in three groups: group 1 (n=11), donors superovulated with 500UI of FSH and group 2 (n=8) and group 3 (n=5) respectively with 200mg and 180mg of FSH, in decreasing doses, twice a day, during four consecutive days. Blood samples were collected before superovulation (A), in the third day of superovulation (B), at the artificial insemination time (C) and at the embryo collection time (D). The concentrations of tryglicerides, aspartate transaminase (AST) and cholesterol were measured by spectrophotometry and progesterone (P4) by radioimmunoassay. There was no alteration (P<0.05) in the concentration of tryglicerides, AST and cholesterol among the samples. There was no effect (P>0.05) of the superovulation protocol on the concentration of tryglicerides, AST and P4 in the samples. In the samples A and B of group 2 the concentration of cholesterol was lower (P<0.05) than in groups 1 and 3, probably due to the instability of the parameter.

Determinação do sexo de psitacídeos por radioimunoensaio (RIE) de esteróides sexuais a partir de excretas cloacais / Psittacine sex determination by radioimunoassay (RIA) of sex steroids using fecal samples

Para o presente estudo utilizaram-se amostras de excretas cloacais de 50 aves da família Psittacidae, previamente sexadas. Os andrógenos e estrógenos fecais foram extraídos com Tampão Fosfato Salino (PBS) e com uma solução PBS:Álcool Etilico (4:1) e a mensuração hormonal foi realizada em "kits" comerciais para radioimunoensaio no Laboratório de Dosagens Hormonais (LDH) do Departamento de Reprodução Animal (VRA) da Faculdade de Medicina Veterinária e Zootecnia (FMVZ) da Universidade de São Paulo (USP). O sexo de cada ave foi confirmado utilizando como parâmetro o intervalo de confiança (95%) da média dos valores transformados dos índices do fator testosterona e seus metabólitos. Setenta por cento das aves tiveram o sexo confirmado pela técnica do radioimunoensaio. Os resultados encontrados demonstram a necessidade da realização de mais estudos para a determinação do sexo de aves monomórficas por meio de técnicas não invasivas.

For the current study, it were used fecal samples from 50 psittacines, previously sexed by PCR from blood cells. The fecal androgens and estrogens metabolites were extracted with PBS (Phosfate Buffer Saline) or PBS:Ethil Alchool (4:1) and measured by comercial radioimunoassay kits at the "Laboratório de Dosagens Hormonais" of the "Departamento de Reprodução Animal" of the "Faculdade de Medicina Veterinária e Zootecnia" of the "Universidade de São Paulo". The sex determination of the birds were performed using the confidence interval (95%) for transformed androgens values. 70% of birds had the sex confirmed by radioimunoassay. The results showed that further studies for sex determination on monomorphic birds by non-invasive techniques are necessary.

The Clinical Significance of Serum and Urinary Neopterin Levels in Several Renal Diseases

Neopterin is a pyrazino-pyrimidine compound, and is known to be a marker associated with cell-mediated immunity in various diseases. We hypothesized that the levels of serum and urine neopterin would be elevated in renal disease, and would correlate with certain clinical parameters. We evaluated serum and urinary neopterin levels in patients with several renal diseases, including nephrotic syndrome (NS, n=19), chronic renal failure (CRF, n=8), end stage renal disease (ESRD, n=64) and controls (n=22). Serum neopterin was elevated in patients with CRF and ESRD, as compared to controls. Urinary neopterin levels were also found to be elevated in patients with CRF and ESRD, as compared to controls. Serum neopterin levels showed significant positive correlation with age, serum BUN and creatinine levels, and inverse correlation with WBC, hemoglobin, hematocrit, serum albumin and total iron binding capacity. Urine neopterin levels exhibited positive correlation with age and serum creatinine levels, and inverse correlation with WBC, hemoglobin, hematocrit, BUN and serum albumin. In conclusion, increased serum and urinary neopterin levels were found in some patients with renal disease and were correlated with certain clinical parameters.

Quantificação de esteróides fecais de fêmeas de onça-pintada (Panthera onca) mantidas em cativeiro: validação da técnica / Fecal steroid and quantification in captive jaguars (Panthera onca): validation of a method

Foi estudada a atividade ovariana de fêmeas de onça-pintada (Panthera onca; adultas n=2 e pré-púberes n=3) mantidas em cativeiro, pela extração e quantificação de estrógenos e progestinas fecais. Foram colhidas amostras fecais de 2-7 vezes por semana durante 16-18 meses. Foi realizada a validação dos radioimunoensaios em fase sólida, para progesterona e 17beta-estradiol, para uso em extratos fecais em onça-pintada. A concentração média de estrógenos fecais (ng/g de fezes secas) para o grupo dos animais pré-púberes foi de 10,97 (variando de 0,28 a 59,16) e para o grupo dos animais adultos foi de 68,99 (variando de 3,50 a 609,37). A concentração média de progestinas fecais (müg/ g de fezes secas) para o grupo dos animais pré-púberes foi de 0,26 (variando de 0,02 a 4,44) e para o grupo dos animais adultos foi de 0,85 (variando de 0,08 a 6,51).

Calcitonina pleural – Resultados preliminares e perspectivas / Pleural calcitonin - Preliminary results and future

Introdução: a calcitonina é um hormônio polipeptídico cuja secreção é originária, principalmente, das células C ou parafoliculares tiroideanas. Níveis elevados ocorrem em pacientes com doença não malígna do pulmão e nas seguintes doenças malignas: câncer de mama, carcinóide, hepatoma, hipernefroma, câncer de pulmão, gastrinoma, tumores gastrointestinais e o carcinoma medular da tireóide. O objetivo do presente estudo foi avaliar a utilidade diagnóstica da calcitonina, dosada no líquido pleural, como marcador tumoral para o diagnóstico diferencial na síndrome do derrame pleural. Pacientes e Método: três líquidos pleurais, provenientes de três pacientes, com causas de síndrome do derrame pleural confirmada. Radioimunoensaio, tendo iodo como marcador, foi utilizado para dosagem de calcitonina pleural (CALC-L). Resultados: os níveis encontrados para CALC-L em pacientes com derrame pleural parapneumônico complicado, carcinoma brônquico de células não pequenas e adenocarcinoma pleural foram 68pg/mL, 89pg/mL e 46pg/mL respectivamente. O valor normal no soro varia de 23 e 71pg/mL. Conclusão: ainda não foi possível calcular o rendimento da CALC-L para diagnóstico diferencial na síndrome do derrame pleural. O valor de referência no líquido pleural ainda não pode ser estabelecido