Exfoliative cytology as a tool for monitoring pre-malignant and malignant lesions based on combined stains and morphometry techniques.

BACKGROUND: Prevention and early diagnosis have the greatest potential for public health and are the most effective method in the long-term to control oral cancer. The aim was to apply PAP staining together with AgNOR staining and morphometric analysis in oral exfoliative cytology, to determine the sensitivity and specificity of these methods in the detection of malignant changes for the purposes of both initial population monitoring and follow-up. METHODS: AgNOR, Papanicolau, and morphometric tests were conducted in samples of patients with oral cancer, oral potentially malignant disorders and controls (opposite side of lesions). Specificity and sensitivity values for each stain method and the curve under ROC area were estimated. RESULTS: The diagnostic variables which allowed greatest accuracy in identifying malignancy relative to the healthy control were cluster (76.92%), satellite (75.64%), and total (90%). The diagnosis was seen to be associated with PAP and total AgNOR, total AgNOR and PAP, total AgNOR and satellites and clusters, and total AgNOR nuclear area/cytoplasmic area ratio. CONCLUSIONS: The total number of AgNOR is a reliable marker for detecting neoplastic cells; this method increases sensitivity and specificity by decreasing the likelihood of false negatives or positives, as the accuracy obtained was 90%. It is also a low-cost, non-invasive, simple methodology that can be recommended to help the early detection of oral cancer and monitoring of patients with a first diagnosis of cancer.

Does microwaving enhance the Golgi methods? A quantitative analysis of disparate staining patterns in the cerebral cortex.

As a family of techniques, the Golgi methods have long been used for studying the morphology and structure of the central nervous system. Due to their capricious nature, many modifications have been employed to improve the reliability and quality of the technique, including the recent addition of microwave energy. In the present study, we evaluated the effectiveness of adding microwave energy to two Golgi methods: the Golgi-Cox method and the rapid Golgi method. These methods were selected for their widespread use in animal research and human postmortem studies. Control tissue was compared to tissue exposed to microwave energy for varying lengths of time during the chromating step of both methods. As assessed by stereological cell counts and qualitative observation, the addition of microwave energy improved the quality of the impregnations and the number of labeled profiles in both methods up to a specific limit of exposure. Surprisingly, increases in the number of profiles were often the result of increased non-neuronal staining at the expense of neuronal staining. This result appears to be due to the fact that different classes of labeled profiles displayed distinct staining time courses.

Correlation between silver-stained nucleolar organizer region area and cell cycle time.

BACKGROUND: The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. METHODS: After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. RESULTS: Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. CONCLUSIONS: The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units.

PCNA/AgNOR and Ki-67/AgNOR double staining in oral squamous cell carcinoma.

This study was performed on oral squamous cell carcinomas (OSCC) in order to investigate the relation between the number of interphase silver-stained nucleolar organizer regions (AgNORs) and the immunolabeling of proliferation-associated markers, using antibodies to Ki-67 and proliferating cell nuclear antigen (PCNA). Fifteen consecutive cases of oral squamous cell carcinoma were used and a double staining technique was performed in order to quantify the number of NORs in PCNA-positive and -negative cells as well as in Ki-67-positive and -negative cells. Our results showed a higher mean number of AgNORs in PCNA- and Ki-67-positive cells than in PCNA- and Ki-67-negative cells. We concluded that there is an association between cell proliferation and AgNOR score in OSCC.

Usefulness of AgNOR score in differentiating benign from malignant pulmonary aspiration cytology.

OBJECTIVE: Malignant cells are known to display greater argyrophil staining for nucleolar organizer regions (AgNORs) than for benign cells due to their active proliferation. In this study we assessed the diagnostic value of AgNOR staining on 47 fine needle aspiration cytologic specimens of lung previously stained with the May-Grünwald-Giemsa (MGG) method. METHODS: Cytologic specimens obtained from fine needle aspiration of the lung in 47 proven cases prestained with the MGG technique were destained and restained with the AgNOR method. Seventeen of them were benign and 30 malignant. To differentiate malignant from benign entities, the highest median value for AgNOR number (AgNOR score) obtained from the benign cases was chosen as a cutoff point (test specificity, 100%). RESULTS: AgNOR scores of malignant cases were significantly higher than those of benign cases (P < .001). There was no significant difference between two subgroups of benign diseases or among four subgroups of malignant diseases. The sensitivity of the AgNOR score was 93% (28/30) in providing a diagnosis of malignancy when the cutoff value was set at 6. CONCLUSION: The AgNOR technique may be of considerable value in aiding a diagnosis of malignancy, especially when the score is > 6.

Electroinmunofijación de orinas sin concentrar por coloración con metales pesados / Immunofixation electrophoresis of unconcentrated urines with heavy metal staining

Se describe una técnica sencilla y altamente sensible para la identificación inmunológica de proteínas en orina sin concentración previa, basada en la alta sensibilidad de las tinciones con metales pesados, como plata y oro, al estado coloidal (100 veces más sensible que el azul brillante de Coomassie (CBB) R y G 250). Posteriormente a la corrida electroforética convencional se efectuó una inmunofijación utilizando una cantidad de antisuero monoespecífico en el orden de 3,5 Al/cm² de gel. El contacto antígeno-antisuero se mantuvo durante 30 minutos y posteriormente se efectuaron lavados con sucesivos recambios de solución fisiológica y se aplicaron las coloraciones argénticas, áurica y con CBB R 250. Bajo las condiciones de trabajo utilizadas se lograron identificar satisfactoriamente las cadenas livianas k y ? monoclonales en proteinurias de Bence Jones (BJ) positivo, y la ß2 microglobulina en proteinurias de tipo tubular, con un límite de sensibilidad del orden de los 5 x 10-4ug de proteína/mm2 de gel. Esta técnica resultó de gran utilidad en el estudio del cuadro uroproteico y se presenta como un método simple, rápido, de alta sensibilidad y baja relación costo/beneficio, para el estudio de los distintos cuadros de proteinuria en muestras sin concentrar (AU)

Observer variation in AgNOR counts in neoplastic breast lesions.

OBJECTIVE: To determine interobserver and intraobserver variability of AgNOR quantitation in neoplastic lesions of the breast. STUDY DESIGN: Forty-five cases, 20 benign and 25 malignant lesions, were included in the study. Counts were performed on one slide from each case within a pre-marked area of about 1 cm2 in a standardized manner by two observers blind to the histopathologic diagnosis and independent of each other and repeated after two weeks. Interobserver and intraobserver agreement was assessed using the Bland-Altman method. RESULTS: Our results showed small mean interobserver and intraobserver differences but wide limits of agreement. CONCLUSION: Observer variation in AgNOR counts is too high for the method to be of any diagnostic or prognostic relevance.

Diagnostic value of AgNOR method in thyroid cytopathology: correlation with morphometric measurements.

A silver staining technique was applied to 51 thyroid smears. The numbers of silver-stained nucleolar organizer regions (AgNORs) were counted, and the mean AgNOR and nuclear area per cell were determined with an image analyzer. The mean AgNOR count per cell was significantly higher in malignant than in benign lesions, but there was a considerable overlap. The mean AgNOR area and the mean SD of the AgNOR area per cell were significantly higher in carcinomas than in benign lesions (P = 10-9 and P = 5 x 10-10, respectively) and there were only two and one benign cases, respectively, of overlap. A strong correlation was observed between the mean AgNOR area and the mean nuclear area (r = 0.88), the former being a better discriminator between benign and malignant lesions. The AgNOR technique may contribute to routine thyroid cytopathology.