Effects of long-term storage of filter paper blood samples on neonatal thyroid stimulating hormone, thyroxin and 17-alpha-hydroxyprogesterone measurements.

OBJECTIVES: Clinical laboratories store filter paper samples used in neonatal screening for various periods of time after performing hormonal measurements. However, due to lack of data concerning specimen stability, it is unclear for how long these samples should be stored. The objective of this study is to determine the stability and reproducibility of thyroid-stimulating hormone (TSH), thyroxine (T(4)) and 17-hydroxyprogesterone (17-OHP) measurements in filter paper blood samples stored for up to 60 months. METHODS: Two hundred and twenty-eight blood samples, drawn between the second and the fourth day of life, were divided into seven distinct groups and kept at 4-8 degrees C for one day or 2, 12, 24, 36, 48 or 60 months after basal hormonal measurements. In each group, TSH, T(4) and 17-OHP levels were initially assayed 24-48 hours after collection (basal) and repeated once at the end of storage timing. All the measurements were performed by time-resolved fluorometry (1235 AutoDELFIA, Wallac Oy, Turku, Finland). Repeated and basal levels of each hormone were compared within the same group by Student's paired t-test. Differences were considered significant at P < 0.05. RESULTS: Compared with basal measurements, TSH and T(4) levels declined significantly only when these hormones were re-assayed at 48 or 60 months of sample storage. In contrast, 17-OHP concentrations decreased earlier, starting at 24 months and continuing throughout the remaining period. CONCLUSION: Our data suggest that neonatal screening of filter paper samples kept at 4-8 degrees C are reliable for repeating the hormonal measurements when specimens are stored for up to one year, in the case of 17-OHP, or three years, in the case of T(4) and TSH.

The dynamics of glucose metabolism under calcineurin inhibitors in the first year after renal transplantation in nonobese patients.

BACKGROUND: The incidence of glucose metabolism disturbances after transplantation often is based on the use of hypoglycemic agents and not on the results of glucose tolerance tests (GTTs), which may camouflage the real incidence. A lack of information also exists regarding the profile of glucose metabolism during the first year after transplant. METHODS: Oral GTT along with insulin measurements and drugs pharmacokinetics were prospectively performed at days 30, 60, 180, and 360 after transplant to diagnose disturbances of glucose metabolism after renal transplantation, in nonobese patients receiving either tacrolimus (n=55) or cyclosporine (n=29), along with mycophenolate mofetil and steroids. RESULTS: The incidence of impaired glucose tolerance or diabetes mellitus reached a peak at 60 days and decreased at 1 year. It could not be adequately diagnosed using fasting plasma glucose in a decreased abnormal (>99 ng/mL) range. In both groups, insulin secretion, evaluated by the Homeostasis Model Assesment (HoMA-beta), decreased (P<0.005) from the condition of normal GTT (101+/-56%) to impaired glucose tolerance (72+/-35%) and diabetes mellitus (54+/-25%). In the cyclosporine group, insulin secretion was normal and stable throughout the study period, but in the tacrolimus group, insulin secretion recovered over time and was inversely correlated with tacrolimus exposure. Insulin resistance (HoMA-IR) did not change. CONCLUSIONS: This study shows the need to perform an oral GTT at 60 days and at the end of the first year of renal transplantation to adequately diagnose impaired glucose metabolism.

Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.

BACKGROUND: The identification of somatic mutations in tissues is often difficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We found that the identification of gsp mutation was not possible by direct sequencing and present a new approach that improves the identification of gsp somatic mutations. MATERIAL/METHODS: Genomic DNA was extracted from frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplified by PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR product did not reveal mutations, probably due to the small amount of mutant alleles. To improve this amount, the PCR products were re-amplified using as template the excised products of the mutant homoduplex and heteroduplex bands obtained at the first DGGE. RESULTS: This approach resulted in the enhancing of the mutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Direct sequencing of the second round PCR clearly identified the mutation R201C in the ovarian Leydig cell tumor. CONCLUSIONS: We have demonstrated a relatively rapid, convenient and reliable method to improve gsp somatic mutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutant homoduplex bands disclosed in a first DGGE followed by direct sequencing.

Estudo clínico, molecular e terapêutico de um caso de adenoma hipofisário produtor de TSH / Clinical, molecular and therapeuthic of a case of pituitary adenoma

O adenoma produtor de TSH é o mais rato dos adenomas hipofisários (<1 por cento) e deve ser suspeitado em todo paciente com quadro clínico de hipertireoidismo e nível sangüíneo de TSH detectável, dosado por ensaios ultrassensíveis. Descrevemos a história clínica de um homem de 38 anos, tratado por hipertireoidismo com drogas antitireoideanas durante três anos, antes de se estabelecer o diagnóstico de macroadenoma hipofisário secretor de tireotrofina. Apresentava níveis elevados de T3 (380mcg/dl). T4 (18,6mcg/dl) e TSH (19,8uU/ml), este último interpretado, inicialmente, como erro laboraotrial. A TC e RNM de hipófise evidenciaram um macroadenoma com expansäo para e supra-selar. Após compensaçäo do hipertireoidismo com antitiroideanos, o paciente foi submetido à adenomectomia transesfenoidal. A análise dos exons 8 e 9 pGS e dos exons 5 e 6 da pGi em DNA extraído do tecido hipofisário, mostrou migraçäo normal no gel em gradiente de denaturaçao, afastando presença de mutaçao ativadora no gene da proteína G na etiologia do tumor. A administraçäo aguda de octreotídeo promoveu queda significativa dos níveis de TSH. Seis dias após a cirurgia, os níveis e T3, T4 e TSH estavam normais. Após dois meses, entretanto, constatou-se recorrência clínica e bioquímica do hipertireoidismo, caracterizada por níveis elevados de T3 e T4 na presença de níveis sangüíneos inapropriadamente detectáveis de TSH. Segundo nosso conhecimento bibliográfico, este foi o primeiro caso relatado no Brasil.

Pseudo-hermafroditismo masculino por deficiência da 5-alfa-redutase: efeito da terapêutica com testosterona e diidrotestosterona

Os autores estudaram a resposta terapêutica com testosterona por via intramuscular e didrotestosterona por via intramuscular e diidrotesterona por via cutânea em altas doses em dois pacientes adultos portadores de pseudohermafroditismo masculino por deficiência de 5-alfa-redutase. Os pacientes apresentaram pequena resposta a terapêutica, sem normalizaçäo do tamanho do pênis tanto com o tratamento com testosterona como com a didrotesterona. Os autores concluem que a resposta terapêutica ao tratamento hormonal de adultos com deficiência da 5- alfa-redutase é discreta, embora näo possam afastar a possibilidade de haver resposta terapêutica satisfatória em idade pré-puberal