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.

Reibergrama para evaluar la síntesis intratecal de Lectina de unión a Manosa / Reibergram to evaluate the Mannan binding Lectin intrathecal synthesis

INTRODUCCIÓN: la dinámica particular de las proteínas derivadas del cerebro en el líquido cefalorraquídeo es diferente a la dinámica de las proteínas derivadas de la sangre. OBJETIVO: describir los datos empíricos de la lectina de unión a manosa y brindar una interpretación teórica de la dinámica de esta proteína a través de la confección un nuevo reibergrama. MÉTODOS: la lectina de unión a manosa en suero y líquido cfalorraquídeo, fue medida en 40 adultos normales a través de un ensayo inmunofluorométrico. El criterio diagnóstico estuvo basado en; muestras controles (pacientes normales) y muestras de pacientes con enfermedades que cursaron con disfunción de barrera sangre-líquido cefalorraquídeo. RESULTADOS: el coeficiente de correlación entre la lectina de unión a manosa en el líquido cefalorraquídeo y en el suero, fue muy bajo. El reibergrama de la lectina de unión a manosa se diseñó de acuerdo con procedimientos previos. CONCLUSIONES: bajo cualquier condición de barrera sangre-líquido cefalorraquídeo, el reibergrama puede identificar la ocurrencia de síntesis intratecal de lectina de unión a manosa.

BACKGROUND: The dynamics of brain derived proteins in cerebrospinal fluid is different from the dynamics of blood-derived proteins. Aim: To describe the empirical data for mannan binding lectin and gives a theoretical interpretation of the dynamics of this protein in cerebrospinal fluid through a new reibergram. METHODS: Serum and cerebrospinal fluid mannan binding lectin were measured in 40 normal adults by immunofluorometric assays. The diagnostic criteria were based in; normal control samples defined clinically and diseases with blood-cerebrospinal fluid barrier dysfunction. RESULTS: Correlation coefficient between cerebrospinal fluid MBL and serum MBL was very low. Mannan binding lectin reibergram was designed according with previous procedures. CONCLUSION: Under all conditions of the blood-cerebrospinal fluid barrier, the reibergram can identify the occurrence of intrathecal mannan binding lectin synthesis.

Development, characterization and clinical validation of new sensitive immunofluorometric assay for the measurement of serum thyroglobulin / Desenvolvimento, caracterização e validação clínica de um novo ensaio sensível para a dosagem da tiroglobulina sérica

OBJECTIVE: In the last decade, data published stressed the role of highly-sensitive thyroglobulin (Tg) assays in the follow-up of differentiated thyroid carcinoma (DTC) patients. The present study describes a new, highly-sensitive Tg assay, compares it with an available commercial assay, and validates it in the follow-up of DTC patients. SUBJECTS AND METHODS: The immunofluorometric high-sensitivity Tg assay is based on monoclonal and polyclonal antibodies produced at our laboratories. It was validated in 100 samples of 87 patients with DTC submitted to total thyroidectomy, 87% of whom also received radioiodine. For correlation, all samples were also tested using a commercial Tg assay (Beckman Access) with functional sensitivity (FS) of 0.1 ng/mL. RESULTS: The new method showed FS of 0.3 ng/mL. The correlation between the two methods was good (r = 0.74; p < 0.0001). The diagnostic sensitivity was 88.9%, and it was increased to 100% when combined with neck US. CONCLUSION: This new, high-sensitivity Tg assay presented a good correlation with Beckman Access assay and with the clinical outcome of the patients. The continuous availability of a validated assay is an additional advantage for long term follow-up of DTC patients. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

OBJETIVO: Na última década, estudos mostraram a importância dos ensaios de tiroglobulina (Tg) com melhor sensibilidade funcional no seguimento dos pacientes com carcinoma diferenciado de tiroide (CDT). Neste estudo, descrevemos o desenvolvimento de um novo ensaio de Tg de alta sensibilidade, que foi validado no seguimento de pacientes com CDT e correlacionado com um ensaio comercialmente disponível. SUJEITOS E MÉTODOS: O ensaio imunofluorométrico de Tg baseia-se em anticorpos, um monoclonal e um policlonal desenvolvidos em nosso laboratório. Avaliamos 100 amostras de soro de 87 pacientes com CDT submetidos à tiroidectomia total, sendo que 87% deles também receberam 131I. A Tg foi dosada também em ensaio comercial (Beckman Access). RESULTADOS: A correlação entre os dois métodos foi de 0,74 (p < 0,0001). O novo ensaio mostrou uma sensibilidade funcional de 0,3 ng/mL. A sensibilidade diagnóstica foi de 88,9%, que aumentou para 100% quando associada ao ultrassom cervical (US). CONCLUSÃO: O novo método de dosagem de Tg mostra boa correlação com o ensaio comercial Beckman Access e com a evolução clínica dos pacientes. O novo ensaio será fundamental no seguimento dos nossos pacientes com CDT. Arq Bras Endocrinol Metab. 2012;56(9):658-65.

Weight-adjusted neonatal 17OH-progesterone cutoff levels improve the efficiency of newborn screening for congenital adrenal hyperplasia / Determinação de pontos de corte para 17OH-progesterona de acordo com o peso ao nascimento melhora a eficiência da triagem neonatal da hiperplasia adrenal congênita

OBJECTIVE: To evaluate weight-adjusted strategy for levels of neonatal-17OHP in order to improve newborn screening (NBS) efficiency. SUBJECTS AND METHODS: Blood samples collected between 2-7 days of age from 67,640 newborns were evaluated. When N17OHP levels were > 20 ng/mL, and a second sample was requested. We retrospectively analyzed neonatal-17OHP levels measured by Auto DELFIA- B024-112 assay, grouped according to birth-weight: G1: < 1,500 g, G2: 1,501-2,000 g, G3: 2,000-2,500 g and G4: > 2,500 g. 17OHP cutoff values were determined for each group using the 97.5th, 99th, 99.5th and 99.8th percentiles. RESULTS: 0.5 percent of newborns presented false-positive results using the cutoff level > 20 ng/mL for all groups. Neonates of low birthweight made up 69 percent of this group. Seven full-term newborns presented congenital adrenal hyperplasia (CAH) and, except for one of them, 17OHP levels were > 120 ng/mL. Only the 99.8th percentile presented higher predictive positive value (2 percent), and lower rate of false-positives in all groups. CONCLUSIONS: We suggest the use of 99.8th percentile obtained by weight-adjusted N17OHP values of healthy newborns to reduce the rate of false-positive results in NBS.

OBJETIVO: Avaliamos retrospectivamente os valores da 17OHP ajustados para o peso ao nascimento para melhorar a eficiência da triagem neonatal. SUJEITOS E MÉTODOS: 67.640 recém-nascidos com amostras coletadas entre 2-7 dias de vida. Uma segunda amostra foi solicitada na presença de testes com valores da 17OHP > 20 ng/mL. 17OHP dosada pelo método DELFIA- B024-112 e correlacionada com o peso ao nascimento: G1 < 1.500 g, G2 1.501-2.000 g, G3 2.000-2.500 g e G4 > 2.500 g. Pontos de corte da 17OHP foram determinados para cada grupo usando os percentis 97,5th, 99th, 99,5th e 99,8th. RESULTADOS: Falso-positivos ocorreram em 5 por cento dos resultados com o ponto de corte > 20 ng/mL, dos quais 69 por cento eram prematuros. Sete recém-nascidos apresentaram deficiência da 21-hidroxilase e, exceto em um, os valores da 17OHP foram > 120 ng/mL. Somente o valor da 17OHP do 99,8th apresentou maior valor preditivo positivo (2 por cento) e menor índice de falso-positivos. CONCLUSÕES: Sugerimos o valor da 17OHP do 99,8th ajustado para o peso ao nascimento para se reduzir a taxa de resultados falso-positivos da triagem neonatal.

Heterogeneity of carboxyl-terminal parathyroid hormone circulating forms in patients with hyperparathyroidism due to end stage renal disease / Heterogeneidade das formas carboxi-terminal circulantes de paratormônio em pacientes com hiperparatiroidismo devido à insuficiência renal crônica terminal

OBJECTIVE: To study carboxyl-terminal (COOH) parathyroid hormone (PTH) circulating forms in patients with hyperparathyroidism due to end stage renal disease (ESRD). METHODS: An immunometric assay that recognizes both intact and COOH PTH forms was developed. The assay, in conjunction with an intact assay, was used to measure PTH in serum samples obtained from 25 patients with hyperparathyroidism due to ESRD. Samples were also submitted to gel filtration chromatography in a Superdex® 30 1.6 x 60 cm column, and the PTH content in the elution tubes, measured using both assays. RESULTS: Values from 39.000 to 232.300 ng/mL (mean ± sd = 101.680 ± 45.330 ng/mL) were found using the COOH assay (PTH 39-84 was used as standard). Values obtained by the intact PTH assay ranged from 318 to 3.307 ng/mL (1.769 ± 693 ng/mL) with a correlation between assays of 0.462 (p = 0.02). The elution profile obtained using the COOH assay showed a preponderance of forms with MW ranging from 8.500 to 4.500 daltons. The profiles obtained from the 25 patients were very similar. CONCLUSIONS: In patients with hyperparathyroidism due to ESRD circulating PTH levels contain a broad range of molecular forms including COOH with MW ranging from 8.500 to 4.500 daltons. These forms are not recognized by the standard intact PTH assays. The correlation of these findings to the clinical aspects of bone disease in ESRD patients remains to be studied.

OBJETIVO: Estudar as formas carboxi-terminal (COOH) circulantes de paratormônio (PTH) em pacientes com hiperparatiroidismo devido à insuficiência renal crônica (IRC) terminal. MÉTODOS: Foi desenvolvido um ensaio imunométrico que reconhece formas intactas e COOH longas de PTH. Esse ensaio foi utilizado, em conjunto com um ensaio para molécula intacta de PTH, em amostras de 25 pacientes com hiperparatiroidismo devido à IRC. As amostras também foram submetidas à cromatografia de gel filtração em coluna de Superdex® 30 de 1,6 x 60 cm, e o conteúdo de PTH nos tubos de eluato foi medido, empregando-se os dois ensaios. RESULTADOS: Valores entre 39.000 e 232.300 ng/mL (média ± dp = 101,680 ± 45,330 ng/mL) foram obtidos usando-se o ensaio COOH (PTH 39-84 foi utilizado como padrão). Com o ensaio para PTH intacto, os valores distribuíram-se entre 318 e 3,307 ng/mL (1,769 ± 693 ng/mL) com correlação entre ambos de 0,462 (p = 0,02). O perfil cromatográfico obtido com o ensaio COOH mostrou predomínio de formas com PM entre 8.500 e 4.500 daltons. Os perfis cromatográficos dos 25 pacientes foram bastante semelhantes. CONCLUSÕES: Em pacientes com hiperparatiroidismo devido à IRC, os níveis circulantes de PTH contêm um espectro de formas moleculares que incluem formas carboxi-terminais, com PM entre 8.500 e 4.500 daltons. Essas formas não são reconhecidas pelos ensaios de rotina utilizados para a medida de PTH intacto. A correlação entre esses achados e os aspectos clínicos da doença óssea em pacientes com IRC necessita de maiores estudos.

Expression of tryptase and chymase in human lung tissue of anaphylactic shock / 法医学杂志

OBJECTIVE@#To explore the expression of tryptase and chymase in human lung tissue of anaphylactic shock and its value for forensic medicine.@*METHODS@#With ten carbon monoxide poisoning cases as control group, the levels of tryptase and chymase were observed by immunofluorescence and analyzed using the Image Analyze and the Image-pro plus 5.0.2. The positive mast cells were counted and the levels of the tryptase and chymase were calculated respectively.@*RESULTS@#There was a statistically significant difference (P < 0.05) for the tryptase and chymase concentrations in the lung tissue between the anaphylactic shock group and the control group.@*CONCLUSION@#The levels of the tryptase and the chymase expression are greatly increased in human lung tissue of anaphylactic shock, which may provide the morphological evidence and reference for the diagnosis of anaphylactic shock in forensic practice.

Determinación de sensibilización alérgica a dermatofagoides en niños de 5 años y menores por fluoroinmuno ensayo-UniCAP / Dust mite allergic sensitization in children aged 1 month to 5 years-old using specific IgE determination by unicap fluoroimmunoassay

Dust mite sensitization is considered a major risk factor for pediatric asthma; however, there are few data about early sensitization in Chilean children available. This study aimed to investigate allergic sensitization to mites in infancy and early childhood. The patient population, 224 children aged 0 to 5 years with suspected respiratory allergy, was divided into 3 groups of age: 1 to 12 months-old, 13 to 35 months-old, and 3 to 5 years-old. Sensitization status was ascertained by assessing the specific IgE to mite by UniCAP fluoroimmunoassay (Pharmacia). Sixty eight (30.6 percent) patients were sensitized to mites. The frequency and level of sensitization increased with the age of the children. Our data support that avoidance measures in the domestic environment aimed at the primary prevention of mite-driven sensitization should be introduced at the earliest possible stage.

La sensibilización a dermatofagoides es un factor de riesgo para el desarrollo de asma. Hay escasa información sobre la sensibilización temprana en niños chilenos. Objetivo: Investigar la sensibilización a dermatofagoides en niños de 5 años y menores. Método: Revisión de resultados de IgE específica por ensayo UniCAP (Pharmacia) contra dermatofagoides de niños de hasta 5 años con sospecha de alergia respiratoria, divididos en 3 grupos de edad: 1-12 meses, 13-35 meses y 3-5 años. Resultados: Se estudió un total de 224 niños, entre 1 mes y 5 años de edad (x 2,65 años +/- 1,48 DS), 57,1 por ciento de sexo masculino. Su distribución según grupo etario fue: 66 en el grupo 1; 45 en el grupo 2 y 113 en el grupo 3. El 30,6 por ciento presentó sensibilización a dermatofagoides (18,2 por ciento grupo 1; 33,3 por ciento grupo 2 y 36,3 por ciento grupo 3). El grupo 3 presentó concentraciones de IgE específica significativamente más elevadas que los otros grupos. Conclusiones: La sensibilización a dermatofagoides es detectable en menores de un año, con frecuencia y nivel que aumentan en forma paralela a la edad.

Concentração sérica de paratormônio intacto em cães com insuficiência renal crônica / Serum concentration of intact parathormone in dogs with chronic renal failure

Estudou-se a ocorrência de hiperparatireoidismo secundário renal e determinaram-se as concentrações séricas de paratormônio intacto (PTHi-c), cálcio total e fósforo em 30 cães com insuficiência renal crônica (IRC) e em 40 cães sadios. Para a determinação do PTHi-c, foi utilizado o método imunofluorométrico, com o emprego de anticorpos anti-aminoterminal (extraídos de gema de ovo da galinha) e de anticorpos monoclonais anti-carboxiterminal (H5P10), marcados com Europium. As concentrações séricas de PTHi-c (717,23±469,13pg/ml no grupo IRC e 36,76±34,40pg/ml no grupo-controle; P=0,0001), cálcio total (11,46±2,03mg/dl no grupo IRC e 10,11±0,91mg/dl no grupo-controle; P=0,003) e fósforo (12,01±8,06mg/dl no grupo IRC e 4,33±0,74mg/dl no grupo-controle; P=0,0001) foram mais altas nos cães com IRC. Observou-se estreita correlação entre PTHi-c e fósforo (r=0,56; P=0,0006), o que não ocorreu entre PTHi-c e cálcio. Hipercalcemia em cães com alta concentração de PTHi-c demonstrou a possível ocorrência de hiperparatireoidismo terciário em 11 animais. A hiperfosfatemia pode indicar, de forma indireta, a ocorrência de hiperparatireoidismo secundário nos cães com IRC.

The occurrence of renal secondary hyperparathyroidism was studied and serum intact parathormone (PTHi-c), total calcium and phosphorus were measured in thirty dogs with chronic renal failure (CRF) and in forty healthy dogs. The imunnofluorometric method was used for the PTHi-c assay, using anti-aminoterminal antibodies (obtained from chicken yolk) and anti-carboxiterminal monoclonal antibodies (H5P10), marked with Europium. Mean value ± SD of serum concentration of PTHi-c was 717.23±469.13pg/ml in CRF group and 36.76±34.40pg/ml in control group (P=0.0001); for total calcium it was 11.46±2.03mg/dl in CRF group and 10.11±0.91mg/dl in control group (P=0.003); and for phosphorus it was 12.01±8.06mg/dl in CRF group and 4.33±0.74mg/dl in control group (P=0.0001). The highest values were observed in CRF dogs. A positive correlation between PHTi-c and phosphorus was observed (r=0.56; p=0.0006), and no correlation was detected between PTHi-c and total calcium. In dogs with CRF, hypercalcemia in presence of high level of serum PTHi-c showed a possible indication of tertiary hyperparathyroidism in 11 animals. In conclusion, hyperphosphatemia could indirectly demonstrate the occurrence of secondary hyperparathyroidism in CRF dogs.

Characterization of Orientia tsutsugamushi strains isolated in Shandong Province, China by immunofluorescence and restriction fragment length polymorphism (RFLP) analyses.

In order to identify the characteristics of the Sta56 gene of the 23 isolates of Orientia (O.) tsutsugamushi isolated in Shandong Province, indirect immunofluorescence assay (IFA) was used to identify the gene type of 23 strains O. tsutsugamushi isolated from scrub typhus patients, chigger mites, and rodents. Restriction fragment length polymorphism (RFLP) analysis was also used to analyze the restriction profiles of the Sta56 gene PCR amplification products of the 23 isolated strains of the O. tsutsugamushi; the results were compared with those acquired by nested PCR. By IFA, 21 of the 23 isolates belonged to the Gilliam type, and 2 to the Karp type. Using RFLP analysis, 21 strains had similar restriction profiles to the Japan Kawasaki strain, but they had no restriction site Hha I, and thus had some difference in gene sequence compared with the Japan Kawasaki strain. The other 2 strains had similar restriction profiles to Karp. These results were identical to that acquired by nested-PCR. In Shandong Province, the gene types of epidemic O. tsutsugamushi strains were similar to the Japan Kawasaki type, but had some differences in gene sequence. In addition, Karp also existed.

Comparison between the prospect cryptosporidium microtiter assay and the immunofluorescent test in the diagnosis of cryptosporidiosis

The immunofluorescent test utilizing rabbit antisera against cryptosporidium oocysts isolated from dairy calves was compared with the prospect cryptosporidium microtiter commercial assay, which is an enzyme immunoassay utilizing monospecific antibody to cryptosporidium specific antigen [CSA]. The former test proved to be more sensitive than the latter one in diagnosing human cases positive for cryptosporidiosis by the modified acid fast stain

Development of a magnetisable solid-phase fluoroimmunoassay for primaquine and carboxyprimaquine.

Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produce antiprimaquine antibodies in three sheep. The antisera obtained were characterised by the increase in fluorescence polarisation found upon binding to fluorescein-labelled primaquine prepared via same route. All sheep showed a good antibody response and one antiserum was coupled to magnetisable solid-phase particles to facilitate the separation of the antibody bound from free labelled antigen and the removal of interfering components which may be present in the sample. The fluoroimmunoassay requires addition of 100 microliters of standard or sample (urine or serum) to 100 microliters tracer (150 nmol/l) followed by 100 microliters of magnetisable solid-phase particles (12.5 g/l). After one hour incubation followed by the usual washing and eluting procedures, using a magnetic rack, the fluorescence of the supernatant was measured directly in a fluorimeter. Sodium salicylate was incorporated in the tracer solution to block the non-specific binding of tracer to the protein in serum samples. Cross-reactivity studies showed that the antibodies have high specificity for the 8-aminoquinoline nucleus but not to the 8-N-aminobutyl side chain. Thus carboxyprimaquine cross-reacted equally with primaquine and the assay can be used to measure their combined level. After extraction of primaquine from a basified sample with methylene chloride, the assay may be applied for the quantitation of either primaquine (in the organic phase) or its acidic metabolites including carboxyprimaquine (in the aqueous phase) separately. This approach was applied for the determination of total primaquine (primaquine and its metabolites) and extracted primaquine in urine samples following a single oral dose of 45 mg primaquine.(ABSTRACT TRUNCATED AT 250 WORDS)