Estudio comparativo de la determinación de vitamina D por dos inmunoensayos / Comparative study of the determination of vitamin D by two immunoassays / Estudo comparativo da determinação de vitamina D por dois imunoensaios

La vitamina D es un metabolito que tiene gran importancia en la actualidad, debido al descubrimiento de nuevas funciones. La mejor manera de medir los niveles de vitamina D en el organismo es medir los niveles de 25(OH)vitamina D, pero hoy en día no existe estandarización entre métodos y, por lo tanto, existe gran variabilidad entre ellos. En los laboratorios clínicos los métodos más utilizados son los inmunoensayos y sólo ADVIA Centaur Vitamina D Total assay e IDS-iSYS 25-Hydroxy Vitamin D pasaron la primera fase del Vitamin D Standardization Certification Program (VDSCP) diseñado por el Centre for Diseases Control (CDC). En este estudio se compararon estos 2 inmunoensayos, pero a pesar de haber pasado la primera fase del VDSCP los resultados reflejaron que sigue existiendo variabilidad entre métodos y, por lo tanto, no son intercambiables, siendo necesario definir valores de referencia para cada método a la espera que el proceso de estandarización termine y exista buena correlación entre los diferentes métodos.

Currently, Vitamin D is a highly important metabolite as a result of the discovery of its new functions. The best way to measure the levels of vitamin D in the body is by measuring the levels of 25(OH)vitamin D, but currently there is no standardization between methods and therefore there is a great variability between them. In clinical laboratories, immunoassays are the most used methods, and only ADVIA Centaur Vitamin D Total assay© and IDS-iSYS 25-Hydroxy Vitamin D©passed the first phase of the VDSCP designed by the CDC. In this research study, these 2 immunoassays were compared, and although both passed the first phase of the VDSCP, the results showed that there still exists variability between methods. Therefore, they are not interchangeable, being it necessary to define some reference values for each method until the standardization method is finishe, and there is a good correlation between methods.

A vitamina D é um metabólito que tem muita importância na atualidade, devido ao achado de novas funções. A melhor maneira de medir os níveis de vitamina D no organismo é medir os níveis de 25(OH)vitamina D, mas hoje em dia não existe padronização entre métodos e, por isso, existe uma grande variabilidade entre eles. Nos laboratórios clínicos, os métodos mais utilizados são os imunoensaios e só a ADVIA Centaur Vitamina D Total assay e IDS-iSYS 25-Hydroxy Vitamin D, passaram a primeira fase do Vitamin D Standardization Certification Program (VDSCP) desenhado pelo Centre for diseases control (CDC). No estudo foram comparados esses dois imunoensaios, porém, apesar de ter passado a primeira fase do VDSCP os resultados mostraram que continua existindo variabilidade entre métodos e, por isso, não são intercambiáveis, sendo necessário definir valores de referência para cada método à espera de que o processo de padronização finalize, e exista boa correlação entre os diferentes métodos.

Normalización de un método inmunoquímico para detectar Papilomavirus humano tipo 16 en lesiones cérvico-uterinas / Standardization of immunochemical method for detection of type 16 human papilomavirus in cervix uterine lesions

Introducción: la infección por Papilomavirus Humano (PVH) es la condición necesaria para la aparición y desarrollo del cáncer cérvico-uterino. Los genotipos de alto riesgo oncogénico son los causantes de este tipo de neoplasia y dentro de ellos el más frecuente es el PVH 16, que se encuentra aproximadamente en el 60 % de los casos. Los métodos de diagnóstico comerciales resultan costosos para países con escasos recursos económicos, lo que sugiere la búsqueda de alternativas empleando protocolos sencillos y baratos. Objetivos: normalizar un método inmunoquímico para la detección del antígeno L1 de PVH tipo 16 en muestras cérvico-uterinas de pacientes con lesiones intraepiteliales escamosas y determinar la coincidencia entre el método normalizado y la Reacción en Cadena de la Polimerasa en Tiempo Real (RCP-TR), como técnica de referencia, para estimar la utilidad de dicho método en el diagnóstico de la infección por este genotipo viral. Métodos: se compararon tres procedimientos de inmunotinción (Indirecto de inmunoperoxidasa en dos pasos, Estreptavidina-Biotina y Amplificación por polímero) respecto a sensibilidad analítica, tinción inespecífica de fondo y tiempo de terminación, para la detección de la proteína L1 de PVH 16 en líneas celulares derivadas de carcinomas cervicales humanos y en muestras cérvico-uterinas utilizadas como controles. El protocolo normalizado se aplicó a muestras cérvico-uterinas de mujeres entre 30 y 59 años, 82 con lesiones intraepiteliales cervicales y 10 sin antecedentes de alteraciones citológicas, a las que además se les determinó PVH 16 mediante RCP-TR. Resultados: el procedimiento de Estreptavidina-Biotina resultó el más sensible y específico. La coincidencia entre el método inmunoquímico y la RCP-TR fue de un 98,6 por ciento, la sensibilidad fue de un 98,57 por ciento y la especificidad de un 91,67 por ciento, con valores predictivos negativo y positivo por encima del 90 por ciento. Conclusiones: se demostró la validez del método inmunoquímico como prueba confirmatoria de la infección por PVH 16. Dicho método probó ser sensible, sencillo y no requiere de una compleja infraestructura para detectar PVH 16 en muestras cervicales. Además, esta técnica permite obtener información rápidamente y evita el uso de métodos invasivos(AU)

Introduction: Human Papillomavirus (HPV) infection is the necessary condition for the occurernce and development of cervical cancer. The high oncogenic risk genotypes are the responsible for this type of neoplasia and the most frequent is HPV 16 that affects roughly 60 percent of cases. Commercial kits for HPV detection are expensive for resource-poor countries, which suggests the search for alternative throguh non-expensive simple protocoles. Objectives: to standardize an immunochemical method for the detection of HPV 16 L1 antigen in cervical samples of patients with squamous intraepithelial lesions and to determine the diagnostic coincidence between the immunochemical method and the real-time polymerase chain reaction to estimate the usefulness of this method for the detection of cervical infection with this viral genotype. Methods: three immunostaining methods (Two-Step Indirect Immunoperoxidase, Labelled Streptavidin-Biotin and Enhanced Polymer) were compared in terms of analytical sensitivity, nonspecific background staining and time of completion, for the detection of protein L1 of HPV-16 in a cell line derived from human cervical carcinoma and clinical samples from uterine cervix. The optimized protocol was applied to 82 cervical samples from women aged 30-59 years with squamous intraepithelial lesions and to 10 samples of sexually active women without previous signals of positive cytology. The presence of type 16 HPV was also detected with the aid of RT-PCR. Results: the Streptavidin-Biotin system was the most sensitive and specific. The diagnostic agreement between the immunochemical method and the real-time polymerase chain reaction reached 98.6 percent, sensitivity was 98.57 percent and specificity was 91.67 %, with positive and negative predictive values above 90 percent. Conclusions: the validity of the immunochemical method as a confirmatory test for infection by HPV-16 has been demonstrated. The normalized immunochemical method proved to be a sensitive, simple, relatively fast method to detect HPV from clinical samples of cervical cells. Furthermore, this method provides information quickly, avoiding the use of invasive methods in patients(AU)

Immunohistochemical study of polycystin-1 in dentigerous cysts.

Background: The alterations involved in step-wise transformation of a dental follicle to dentigerous cyst (DC) is not clearly known. Primary cilium and its protein have been hypothesized to be associated with DC. Mutation of a ciliary protein, polycystin‑1 (PC1) is associated with autosomal dominant polycystic kidney disease. This study was performed to assess the immunohistochemical expression of PC1 between DC and postfunctional follicular tissue (PFFT). Materials and Methods: Thirty‑one consecutive PFFT and 15 DC formed the study group. The PFFT and DC tissues were stained with antibody against PC1. Statistical Package for Social Service was used to analyze data. Descriptive statistics and Student’s Chi‑square test were appropriately used. P ≤0.05 was taken as significant. Results: Fifteen DC (100%) and 7 (22.58%) PFFT were positive for PC1. The difference was statistically significant (P = 0.000). PC1 expression was observed in the cytoplasm with varying intensity. Discussion and Conclusion: All PC1 positive epithelial cells’ cytoplasm stained diffusely. Abnormal cytoplasmic expression of PC1 in all positive epithelial lining indicates that the PC1 probably is associated with cystic transformation.

Proliferating cell nuclear antigen (PCNA), p53 and MDM2 expression in Hodgkins disease

CONTEXT AND OBJECTIVE: Tumor cells in Hodgkins disease (HD) express cell proliferation markers that are evaluated according to the oncogenes involved or the expression of their proteins. Correlations between the protein expression grade and clinical data are now important for disease prognosis. DESIGN AND SETTING: This was a retrospective analysis on proliferating cell nuclear antigen (PCNA), p53 and MDM2 (murine double minute-2) expression using immunohistochemistry, on formalin-fixed, paraffin-embedded tissues from diagnostic biopsies on 51 patients with HD. The study was conducted at the Division of Hematology and Transfusion Medicine, Hospital São Paulo, Universidade Federal de São Paulo. METHODS: Antigen expression was evaluated as the proportions of positive Hodgkin and Reed-Sternberg (HRS) cells and reactive lymphocytes (L), which were compared using Spearman correlation coefficients. The Friedman test was used for comparisons between the markers. The Pearson test was used to investigate associations between marker expression and clinical and laboratory parameters, marrow involvement, complete remission (CR) and overall survival (OS) rates. RESULTS: There was overexpression of antigen proteins in HRS, in relation to L (p < 0.001). In HRS, MDM2 was higher than p53 and PCNA (p < 0.003), while the latter two were equivalent. In L, p53 was lower than MDM2 and PCNA (p < 0.001), while the latter two were equivalent. There was no relationship between protein expression and clinical and laboratory variables or outcome. CONCLUSIONS: PCNA, p53 and MDM2 are tumor markers for HD, but showed no clinical or prognostic significance in our analysis.

CONTEXTO E OBJETIVO: As células tumorais da doença de Hodgkin (HD) são positivas para marcadores de proliferação celular que são analisados por seus genes e respectivas proteínas. A correlação entre a expressão destas proteínas e os parâmetros clínico-laboratoriais são, no momento, de importância para o prognóstico da doença. TIPO DE ESTUDO E LOCAL: Estudo retrospectivo da expressão do antígeno de proliferação celular (PCNA) e da p53 e MDM2 em tecidos obtidos ao diagnóstico, fixados por formol, embebidos em parafina de 51 pacientes com HD. O trabalho foi realizado na Divisão de Hematologia e Transfusão, Hospital São Paulo, Universidade Federal de São Paulo. MÉTODOS: As expressões antigênicas foram analisadas através da proporção de células de Hodgkin e células de Reed Sternberg (HRS) e linfócitos reacionais (L) positivos. A intensidade de expressão de cada proteína foi comparada entre L e HRS através do coeficiente de Spearman. A comparação da PCNA, p53 e MDM2 em L e HRS se fez pelo teste de Fiedman. As correlações entre variáveis clínico-laboratoriais, comprometimento da medula óssea, taxas de sobrevida geral e remissão clínica com as proteínas em HRS se fizeram pelo coeficiente de Pearson. RESULTADOS: Houve superexpressão das três proteínas em células HRS comparadas aos L (p < 0,001). Nas células HRS, a MDM2 foi maior que a p53 e a PCNA (p < 0,003), que foram equivalentes. Nos L, a p53 foi menor que a MDM2 e a PCNA (p < 0,001), que foram equivalentes Não houve relação entre as expressões das proteínas com as variáveis clínico-laboratoriais e sobrevida. CONCLUSÕES: PCNA, p53 e MDM2 são marcadores tumorais na HD, porém não mostraram significado clínico-prognóstico em nossa análise.

Solid tumors of childhood.

OBJECTIVE: Cancer is one of the leading causes of death in children. There is the need to have the histologic review of malignancies in children from the Indian sub-continent. METHODS: In the present study, malignant tumors received over 12 years were reviewed and re-classified according to classifications based on prognosis. RESULTS: A total of 472 tumors were received over 12 years. Of these 318 were benign and 154 malignant. The commonest malignant solid tumor was lymphoma followed by pediatric renal tumors. The sarcomas included bone tumors, Rhabdomyosarcoma and synovial sarcoma. There were 13 germ cell tumors, 10 retinoblastomas and six neuroblastomas. CONCLUSION: The review revealed that a definite diagnosis or classification was not assigned in 21 cases in the original reporting. Of these 14 could be assigned a definite category on review and immuno-staining. These included five non-Wilms sarcomas, four Rhabdomyosarcomas, three Ewing's sarcoma/PNETs and two Synovial sarcomas. The study also revealed an unexpected high percentage (11%) of epithelial malignancies in children.

Improved immunodetection of human papillomavirus E7

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell line which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.

Cuantificación de tirotropina. Ensayo inmunorradiométrico versus inmunoquimioluminimétrico / Quantificación de tyrotropin. Immunoradiometric versus inmmunochemoheminometric assays

Con el propósito de evaluar métodos opcionales al ensayo inmunorradiométrico -establecido como método de referencia en medicina nuclear- en la cuantificación de analitos cuya concentración en sangre es en nano o picogramos/mL, se comparó la determinación de tirotropina por ensayo inmunoquimioluminimétrico. Por los métodos mencionados se analizaron simultáneamente 36 muestras séricas de pacientes atendidos en consulta externa del Hospital de Especialidades, Centro Médico Nacional Siglo XXI, seleccionados en forma aleatoria. Los resultados obtenidos mediante la aplicación de parámetros estadísticos demostraron excelente correlación entre ambos métodos. A través del análisis de la concentración sérica de tirotropina por los dos procedimientos, se obtuvo el mismo número de pacientes eutiroideos y de pacientes con valores anormales. De acuerdo con los resultados se puede concluir que ambos métodos poseen sensibilidad y especificidad similares y determinan la misma concentración sérica de tirotropina.

Paracoccidioides brasiliensis: a mycologic and immunochemical study of two strains

Os autores estudaram do ponto de vista micologico, imunoquimico e de sua biologia molecular, duas amostras de Paracoccidioides brasiliensis, uma isolada do solo, no municipio de IBIA (MG) por Silva-Vergara et al (1996,1998) denominada IBIA e outra, BAT, cultivada de um caso humano de paracoccidioidomicose em Ribeirao Preto (SP) por Freitas da Silva (1996). Tais amostras apresentam colonias cotonosa (M) e leveduriforme (L ou Y), sendo patogenicas para cobaios inoculados por via testicular, produzindo orquite granulomatosa e/ou supurativa. Do ponto de vista imunoquimico, atraves de provas de Imunodifusao dupla, Imunoeletroforese e Western Blotting, foi demostrada a presenca da gp43

Comparative evaluation of immunochemical methods for estimation of albumin in microalbuminuria.

Three immuno assays namely radioimmunoassay (RIA), radial immunodiffusion (RID) and rocket immunoelectrophoresis (RIE) were compared for their performance and utility. The accuracy limits of the methods were compared and also between methods using RIA as the reference. Urine samples, from known diabetic patients with albumin concentration ranging from 2.5 mg/l to 120 mg/l were analysed by the three methods. The mean differences were only 0.91 mg/dl and 0.5 mg/dl respectively for RID vs RIA and rocket vs RIA which is not statistically significant. Excellent correlation was seen between RIA and RIE (r = 0.98) and also between RIA and RID (r = 0.97). Compared to RID, RIE required less time and was more precise. RIA is suited for assaying large sample loads yet not suited for laboratories receiving samples occasionally. For a small pathological laboratory with limited facility rocket electrophoresis may be the most suitable method taking into consideration accuracy, time and cost.

Desarrollo de un ELISA de alta detectabilidad y especificidad para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos / Development of a quantitative ELISA to determination of gladins in food aimed to coeliac patients

La enfermedad celíaca (EC) es una enfermedad gastrointestinal crónica de muy alta incidencia en nuestro país. Se estima que puede afectar 1 de cada 300 habitantes. En individuos susceptibles, la patología es provocada por la ingestión de mínimas cantidades de prolaminas de los cereales: trigo, triticale, cebada y centeno. Su único tratamiento es una estricta dieta libre de dichas proteínas. La Organización Mundial de la Salud (OMS) establece que un alimento puede ser considerado como apto para consumo por enfermos celíacos sólo si su contenido de gluten es inferior a 1 mg/100 g de producto seco. La detección precisa de estas proteínas requiere entonces de métodos de alta detectabilidad y especificidad para discriminar entre las proteínas nocivas y las de otros vegetales frecuentemente usados como reemplazo en la formulación de los alimentos para estos pacientes. En este trabajo, se muestra el desarrollo de un ELISA con anticuerpos policlonales, para la cuantificación de gliadinas en alimentos destinados a enfermos celíacos. Se empleó un diseño de ELISA competitivo secuencial con un antisuero obtenido en conejos que detecta selectivamente las prolaminas tóxicas. Este inmunoensayo presenta un nivel de detección de 0,1 mg de gluten/100 g de producto y cumple con los niveles de detectabilidad aconsejados por la OMS. Mediante el ELISA descripto se han analizado una gran variedad de muestras comerciales, pudiendo cuantificar las prolaminas incluso en alimentos que sufrieron tratamientos térmicos durante su fabricación

Chromatographic, immunochemical and electrophoretic studies of human uterotrophic placental factor (hUTPF)

The human uterotrophic placental factor (hUTPF) is a protein obtained from human term placentae and acts on uterine growth, mammary gland, and blastocyst development and implantation. In the present work, we further define some molecular characteristics of hUTPF using chromatographic, electrophoretic and immunochemical methods. It is concluded that in human term placenta a high molecular weight hUTPF is present, bound to albumin and immunoglobulins, which could represent a storage or transport form of this factor. hUTPF presents several molecular forms, one of them of 270 kDa and others of approximately 90 kDa and 27 kDa

Rhinosporidiosis: experience with sixteen cases

Rhinosporidiosis is a chronic granulomatous disease primarily of the anterior nares. It is histologically characterized by mucosal lymphoplasmacellular infiltrates, transepithelial elimination of nodular bodies [NB] and destruction of late stage NBs in histiocytic granulomata with central neutrophilic microabscesses. Early NBs are immunohistochemically positive for alpha 1-AT alpha 1-ACT, CEA, S100, fibronectin, amyloid-p-component, IgG, IgA, Clq and C3. Structures formerly regarded as "sporangia" [NB] and "spores" are believed to be lysosomal bodies loaded with indigestible residues to be cleared via transepithelial elimination. This is supported by immunohistochemical findings and ultrastructural demonstration of lysosomal bodies in early NBs but not in end-stage NBs which contain mostly amorphous electron dense materials. Immunopathology of the disease is discussed and in view of overwhelming evidence against a fungal etiology it is proposed to change the name to Seeber's disease

Immunohistochemical detection of keratin proteins in the surface epithelium adjacent to oral squamous cell carcinoma

The surface epithelium adjacent to 32 cases of oral squamous cell carcinoma were taken, fixed, processed and stained with routine H and E and antikeratin antibody [Dako Pat Kit K 522]. The surface epithelium adjacent to oral squamous cell carcinoma showed variable changes as normal hyperplastic, dysplastic epithelium including areas with leukoplakia and carcinoma in situ. The expression of keratin proteins in normal, hyperplastic and mild dysplastic surface epithelium showed a regular and regional distribution pattern. On the other hand, the whole thickness of the surface epithelium with moderate and severe dysplastic changes involving areas with leukoplakia and carcinoma in situ exhibited irregular immunostaining pattern where variable numbers of the basal and superbasal cells displayed positive reaction. The reaction lead to the suggestion that there was a correlation between the pattern of keratin distribution the degree of cellular differention as well as this marker can be considered as a helpful aid in the diagnosis of moderate and severe dysplasia including carcinoma in situ especially in small fragmented biopsies. Based on the staining intensity, the cell of leukoplakia can be also considered as highly differentiated cells whereas that of carcinoma in situ can be considered as less differentiated cells

Hidatidosis: un problema y desafío regional / Hydatidosis: a regional problem and challenger

La infección por el echinococcus granulosus es la principal zoonosis parasitaria que afecta a la IX Región de Chile, con tasas de incidencia en humanos entre 18,2 y 48 X 100.000, dependiendo de la zona estudiada y el método empleado para su cálculo. A nivel animal la incidencia es de 40 por ciento en bovinos, 39,5 en ovinos y 14,8 en porcinos sacrificados en la Plata Faenadora de Carnes de la ciudad de Temuco. Se estima e infiere que la zoonosis significa en la Región un gasto aproximado de U$A 300.000 anuales por recuperación en humanos y decomiso de vísceras, aceptando que existe subnotificación, que se desconoce la magnitud animal real en zonas rurales en que se sacrifican animales para el consumo doméstico sin revisión veterinaria y que el rendimiento en carne, lana, leche, etc. del animal parasitado es menor. A pesar de la magnitud del impacto no existe aún un Programa de Control Regional por lo que estamos organizando la II Jornada Nacional de Hidatidología con ese fín