AgNOR Staining Method Research Articles

Diagnostic Utility of Modified Method of AgNOR Staining in the Evaluation of Benign and Malignant Effusions

An exact identification of malignant cells in fluid by cytological examination is a well-known diagnostic challenge. One of the common problems is to distinguish reactive mesothelial cells from malignant cells. Conventional smears reported as ‘suspicious for malignancy’ indicate that the suspicious cases could not be classified with certainty as to whether they were reactive mesothelial cells or malignant cells. It poses problem in clinical staging of tumor, treatment and prognosis of malignancy. The purpose of the study was to determine the role of modified method of AgNOR staining in the evaluation of benign and malignant effusions. This cross-sectional study was conducted in the Department of Pathology, BIRDEM General Hospital, Dhaka, from July 2019 - June 2021. A total of 115 cases of effusion were included. All the samples were centrifuged and then smears were prepared from the deposit followed by staining with Hematoxylin & Eosin stain, Papanicolaou stain and AgNOR stain. At first the diagnosis was made on conventional smear method. Then the findings were compared and analyzed by modified AgNOR staining method. In malignant cells, the mean AgNOR count was 5.59±1.05 (±SD) and the AgNORs were multiple and irregular in shape. On the other hand, in benign cells the AgNORs were comparatively larger, single dots with a mean count of 1.31±0.48.The AgNOR count method has definite role in differentiating benign from malignant effusion. This method has supportive value which can be utilized in differentiating malignant effusions from the benign ones, especially in suspicious cases.

Prospects for the use of modern methods of staining nucleolar organizers regions of cells for the diagnosis of diseases in animals

Relevance.In recent years, issues of human and animal oncology have been considered in a broad aspect as a separate interdisciplinary science. Some diseases are more common in animals than in humans. That is why the selection of the necessary methods of purposeful value is required. In connection with the above data, the relevance of the work lies in the consideration of various methods of using NOR cells for the diagnosis of diseases of various etiologies in animals.Methods and results.In order to determine a particular disease, it is necessary not only to carefully study the available histopathological methods, but also to develop new ones. The comparability of human, bovine, sheep, and other mammalian gene maps opens up the possibility of applying methods used in medicine for animals (for example, FISH analysis with human DNA probes). Argyrophilic proteins associated with NOR ofcells are widely used in diagnostic pathology of a different nature. AgNOR staining method is considered as a proliferation marker. The use of the NOR method is promising in the differentiation of malignant and benign diseases. This method consists of counting the number of nuclei, determining the area and size of the nucleoli. Thus, methods based on the calculation of NOR are the most promising for diagnosing various pathologies in animals, including taking into account the prognosis of diseases.

How Useful is the AgNOR Staining Method for the Diagnosis of Salivary Gland Tumors?

Objectives: The purpose of this study was to determine the numbers and mean areas of the AgNOR dots in the 3 most common salivary gland tumors. Materials and Methods: One hundred and seventy paraffin blocks histologically diagnosed as pleomorphic adenoma, adenoid cystic carcinoma, and mucoepidermoid carcinoma were selected from the archives of the pathology department of Loghman hospital, Tehran, Iran and then cut and stained using the standard AgNOR method. The number and area of AgNOR dots were evaluated. Chi-square and Pearson correlation (2-tailed) tests were used for statistical analysis. The significance level was P < 0.05. Results: The chi-square test showed significant differences between tumor types and the numbers of AgNORs (P = 0.000) and between tumor types and areas of AgNORs (P = 0.000). Conclusions: The AgNOR technique can be used to diagnose salivary gland tumors.

Evaluation of Proliferative Activity in Oral Squamous Cell Carcinoma by the AgNOR Staining Method

Objetivo: avaliar a atividade proliferativa celular das regioes organizadoras nucleolares (AgNORs) em uma serie de casos de carcinoma espinocelular da cavidade bucal (CECB) e correlacionar esta atividade com o grau histologico do tumor, definida pela Organizacao Mundial da Saude (OMS). Metodologia: vinte casos de CECB estudados foram obtidos a partir de diagnostico do Servico de Diagnostico Histopatologico da UPF. Dados relativos a idade, sexo e origem etnica dos pacientes, alem da localizacao da lesao na cavidade bucal, foram obtidos a partir dos relatorios histopatologicos. A tecnica de AgNOR foi utilizada para analise histoquimica dos tumores. Resultados: houve predominio de CECB em pacientes do sexo masculino na sexta decada de vida. O labio foi o local mais afetado e o grau histologico I foi o predominante. O numero medio de NORs por nucleo na amostra foi de 2,00 (desvio padrao: 0,58). O teste de correlacao de Pearson mostrou que o numero de NORs no CECB foi diretamente correlacionado com o grau histologico do tumor (p = 0,021). Conclusao: o metodo AgNOR foi util como uma ferramenta de diagnostico e indicador de prognostico do CECB, quando combinado com a analise histopatologica convencional. Palavras-chave: AgNOR; Proliferacao Celular; Diagnostico; Carcinoma Espinocelular; Tratamento.

Demonstration of decreased activity of dorsal raphe nucleus neurons in depressed suicidal patients by the AgNOR staining method

Background Suicide and depression are closely related yet distinct phenomena. In both these phenomena, research has focused on central serotonergic system disturbances. The dorsal raphe nucleus (DRN) is the main source of serotonergic innervation of limbic structures crucial for the regulation of emotionally influenced behaviour. Methods The study was carried out on paraffin-embedded brains from 23 depressed patients (12 suicides and 11 non-suicides) and 26 matched controls without mental disorders. The karyometric parameters of DRN neurons were evaluated by the AgNOR silver staining method. Results The significant effect of suicide on the nuclear area was found in the cumulative analysis of all DRN subnuclei (ANOVA, P = 0.032). A decreased mean value of this parameter was observed in the suicides group versus controls ( t-test, P = 0.032). This effect was especially pronounced in the violent suicide victims ( t-test, P = 0.001), who also demonstrated a decreased AgNOR area versus controls ( t-test, P = 0.007). No significant effect of depression or polarity on AgNOR parameters was found. Limitations A major limitation of this study is relatively small case number. A further limitation is given by the lack of data on drug exposure across the whole life span. Conclusion Our findings suggest that hypoactivity of DRN neurons is a distinct phenomenon in depression, specific only for suicidal subgroup of depressed patients.

Assessment of Nucleolar Organizer Regions (NORs) in Proliferative Conditions of the Liver

To overcome the diagnostic dilemma in proliferative conditions of the liver which sometimes pose a problem to the working pathologist especially when the material is inadequate, a special staining technique (AgNOR) has been applied. By using this technique, nucleolar organizer regions were counted which determine the proliferative status of the cells. This prospective study included 65 cases of randomly selected liver core and fine needle aspiration biopsies. AgNOR staining was performed on formalin-fixed, paraffin-embedded tissue sections NOR dots were counted in 100 randomly selected hepatocytes at x100 oil immersion objective, and the mean count per cell was calculated for each case. Statistical analysis was done by using the Mann Whitney U test. AgNOR count results were later compared with the histologic diagnosis. The study revealed a gradual increase in mean AgNOR counts from normal liver through cirrhosis to hepatocellular carcinoma. The difference in NOR counts was significant in these three groups. The hepatocellular carcinomas were graded according to the Edmondson-Steiner histological grading system. The Grade I hepatocellular carcinomas show AgNOR counts ranging between 5-6/cell, a score which is much higher than in the normal liver, where it ranges between 1.2-2.0/cell. This technique can be used to assess the lesions where the distinction between normal liver and Grade I hepatocellular carcinoma is difficult with the use of routine methods. AgNOR counts in normal liver and chronic hepatitis cases were insignificant, but there was an appreciable difference between cases of chronic hepatitis, cirrhosis and hepatocellular carcinoma. In view of the results of this study, the AgNOR staining method is found to be a useful diagnostic tool to differentiate between normal liver, cirrhosis and hepatocellular carcinoma and also to precisely discriminate between cases of normal liver and Grade I hepatocellular carcinoma.

Increased Nucleolar Organizer Regions in Osteoclast Nuclei of Paget’s Bone Disease

The etiology of Paget’s disease of bone is still unknown but several studies have reported a viral origin. At the electron microscopic level, characteristic nuclear and cytoplasmic inclusions have been found and mimic paramyxoviral nucleocapsids in osteoclasts (Oc). Sarcomatous degeneration is observed in 2% of pagetic patients. Nuclear organizer regions are parts of the nucleolus involved in the synthesis of ribosomes, and they contain nonhistone proteins that can be silver stained (AgNORs) on the interphasic nuclei. AgNOR number is known to correlate with the proliferative activity of the cell populations, whether normal or malignant. Cancer cells have an increased demand for (robosomal) rRNA and correlations have been found between AgNORs and proliferative antigens. We have adapted the AgNOR staining method to undecalcified bone biopsies at the light and TEM levels. Bone sections from 10 pagetic patients (without a previous bisphosphonate treatment) were stained for AgNORs. 10 patients having metabolic bone diseases associated with an increased Oc number (i.e., primary and secondary hyperparathyroidism) were used as controls. AgNORs appeared as black dots within the nucleoli of Oc nuclei and were easily numbered. A maximum of two or three dots could be seen in Oc nuclei from control subjects. In pagetic Oc, AgNOR number was greatly increased (6.80 ± 2.57 dots vs. 2.12 ± 1.07 in controls). TEM study also showed AgNOR in the nucleoli of pagetic patients’ Oc. The viral inclusions within the nuclei appeared faintly stained and could not be confused with AgNORs. The large number of AgNORs in the nuclei of pagetic Oc reflects the need for a greater abundance of ribosomes. With the pagetic Oc being highly active, the cytoplasmic synthesis of proteins is maximized (probably hydrolases involved in the matrix breakdown). An increase in AgNORs does not reflect the proliferative activity of the cell because Oc are made by the fusion of precursors. It is postulated that: (a) other mRNAs (of viral/oncogene origin) could be actively transcribed in pagetic patients and require more numerous ribosomes; or (b) a viral genome/oncogene promotes alteration of the nuclear/nucleolar mechanism.

Prognostic value of nucleolar organizer regions in supraglottic carcinoma

The relationship of nucleolar organizer regions (NORs) to proliferative activity and tumor progression was studied in 16 supraglottic carcinomas. The number of NORs stained by a silver colloid staining method (AgNOR staining method) was determined. The mean AgNOR number tended to be higher (but not significantly so) in poorly differentiated tumors. Moreover as T and N categories and stage of the tumor rose, the AgNOR number also rose, but not significantly. More interestingly, the mean AgNOR number was significantly higher in the presence than in the absence of lymph node metastasis. These studies indicate that the AgNOR number might be of clinical value as a predictor of lymph node metastasis of supraglottic carcinomas.

Diagnostic and prognostic value of nucleolar organizer regions in squamous-cell carcinomas of the maxillary sinus.

The clinicopathological implications of nucleolar organizer regions (NORs) were assessed in 25 squamous-cell carcinomas arising in the maxillary sinuses. NORs in the carcinoma tissue were stained with silver colloid and counted. As the histological differentiation of the tumors decreased, the mean AgNOR number rose significantly. The mean AgNOR number in T4 cancers was higher than that in T2 and T3 cancers, and the difference between T3 and T4 categories was significant. On the other hand, there were no significant differences in the AgNOR number between tumors of patients with and without recurrence after therapy, between those with different clinical stages or between those with good or poor response to treatment. Moreover, there was no significant difference in survival rates between patients with low and high AgNOR counts. These studies indicate that, in the collective examined, the AgNOR staining method was of no prognostic value for patients with squamous-cell carcinomas of the maxillary sinus.

Cytochemical and immunocytochemical study of coiled bodies in different cultured cell lines.

We analyzed by different cytochemical and immunocytochemical approaches the biochemical compositon of coiled bodies in three different cultured cell lines. Coiled bodies are stained by the AgNOR staining method and by the EDTA regressive staining method preferential for ribonucleoprotein (RNP). Using the in situ polyadenylate nucleotidyl transferase-immunogold technique or anti-RNA antibodies, we decisively demonstrated the presence of appreciable amounts of RNA in coiled bodies. Neither the in situ terminal deoxynucleotidyl transferase-immunogold technique nor anti-DNA antibodies revealed any DNA in coiled bodies. Coiled bodies thus appear as distinct regions of cell nuclei involved in some steps of RNA metabolism but not directly in RNA synthesis. Their relationships with the dense fibrillar component of the nucleolus and with interchromatin granule clusters are discussed.

The Value of the AgNOR staining Method in Identifying Carcinoma in Situ Testis

An early and reliable diagnosis is necessary in order to have the chance of a curative therapy of Carcinoma in situ testis (Cis). Forty-six testicular biopsies were investigated to assess the value of the AgNOR staining method in comparison to placental alkaline phosphatase (PLAP) immunostaining. Both methods provided corresponding results and identical tumor cells were recognized in serial sections. The mean AgNOR counts per nucleus were 26.86 (19-52, SD: 2.68) for CIS cells, 8.18 (5-14, SD: 2.20) for spermatogonia and 12.96 (9-18, SD: 2.44) for Sertoli cells, without the counts overlapping within these three groups. Even single CIS cells are easily and reliably recognizable by their typical AgNOR pattern and their high AgNOR count per nucleus. The independent estimation of 9 testicular biopsies with the AgNOR staining method and the PLAP immunostaining correspondingly revealed 7 biopsies with CIS. Two biopsies lacked tumor cells. The AgNOR staining method can be recommended as an additional diagnostic tool in identifying CIS, because of the short and convenient staining procedure, low costs and the applicability on formalin-fixed and paraffin-embedded material.

AgNOR染色法と顕微蛍光核DNA定量を用いた喉頭癌の増殖能の検討-核DNA解析からみた情報

AgNOR染色法と顕微蛍光核DNA定量を用いた喉頭癌の増殖能の検討-核DNA解析からみた情報

A combined AgNOR-Feulgen staining technique.

We describe a new staining technique (H-Ag-S) which allows observation and counting of active nucleolus organizer regions (NORs) and evaluation of the amount of DNA in the same cell nucleus. The procedure consists of combining a modified AgNOR staining method with the Feulgen reaction. A sequential procedure is proposed, based on the determination of optimal staining conditions. The technique, which was designed to allow studies of correlations between the transcriptional activity of rDNA genes and the cell ploidy, was primarily developed for rat liver smears. It should be applicable to most biological preparations, but the optimal conditions might be variable.

Simultaneous high resolution localization of Ag-NOR proteins and nucleoproteins in interphasic and mitotic nuclei.

Silver stainable proteins of the Nucleolar Organizer Regions (Ag-NOR proteins) of human breast cancer tissues have been localized at the electron microscopical level with a new method which combines a simple and reproducible one step Ag-NOR staining method combined with an acetylation procedure. This new method allows the fine identification of nucleolar components, particularly those which are stained by silver. In order to determine the cytochemical nature of the components associated with Ag-NOR proteins, the EDTA regressive preferential staining procedure for ribonucleoproteins has been applied to sections. By this means the precise localization of the Ag-NOR proteins was studied simultaneously with that of ribonucleoprotein within interphasic nucleoli and mitotic chromosomes. In interphasic nucleoli, stainable Ag-NOR proteins were localized in fibrillar centres and part of the dense fibrillar component. No silver deposits were seen on perichromatin or interchromatin fibrils and granules. In metaphasic nuclei, Ag-NOR proteins were only found on roundish fibrillar ribonucleoprotein structures, which could correspond to secondary constrictions. No silver deposits were seen on the well defined ribonucleoprotein sheet surrounding the chromosomes. In telophasic nuclei, Ag-NOR proteins were seen on the central part of roundish ribonucleoprotein fibrillar structures integrated in decondensing chromosomes. These structures have been interpreted as the nucleolar organizer regions around which rRNA synthesis resumes. In interphasic and mitotic nuclei, Ag-NOR proteins were never found within condensed chromatin but always in association with ribonucleoprotein components. The new method proposed here appears to be a useful tool for the simultaneous study of the localization of ribonucleoprotein and Ag-NOR proteins during the cell cycle.