Osteopontin is histochemically detected by the AgNOR acid-silver staining

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.     

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.     

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.     

AgNOR proteins from morphologically intact isolated nucleoli.

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by "standard" methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.     

Standardized AgNOR analysis as a prognostic parameter in endometrial carcinoma, endometrioid type

OBJECTIVE: To evaluate interobserver reproducibility of histologic grade in endometrial adenocarcinomas of endometrioid type (EC), to assess the relationships between nuclear grade and the amount of argyrophilic nucleolar organizer region (AgNOR) proteins and to determine the prognostic value of AgNOR proteins and the main clinicopathologic parameters. STUDY DESIGN: Architectural and nuclear grading were independently assessed by two pathologists in 64 formalin-fixed, paraffin-embedded surgical samples of EC obtained from an equal number of patients (age range, 38-84 years; mean, 63.5). Interobserver agreement was determined using the kappa statistic; discrepant cases were reviewed, and a consensus was reached. Standardized AgNOR analysis was performed according to the guidelines of the Committee on AgNOR Quantification, measuring the mean area of AgNORs per nucleus (NORA) by an image analysis system. RESULTS: The kappa values for interobserver agreement were substantial for architectural grading and moderate for nuclear grading. When NORA values were compared to the nuclear grade assessed by different observers, the most significant linear correlation (r = .713, P < .001) was found for the nuclear assessment obtained by consensus of the two pathologists. Moreover, statistical analysis allowed discrimination of architectural grade 1 from grade 2 and 3 EC. By the Kaplan-Meier method, the prognosis was worse for patients with higher NORA values (> 4.212 micron 2), while, by Cox multivariate analysis, AgNOR quantity emerged as an independent prognostic variable. CONCLUSION: Use of standardized AgNOR analysis may be an additional and objective tool in the assessment of histologic grade as well as a reliable method of determining prognosis in EC.     

Improvement in the Specificity of the Silver Staining Technique for Ag NOR-ASSOCIATED Acidic Proteins in Paraffin Sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiffs reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

Nucleolar organizer region-associated proteins in cancer cells. Quantitative investigations on gliomas, meningiomas, urinary bladder carcinomas and pleural lesions.

Using a modified silver staining technique, we investigated nucleolar organizer region-associated proteins (AgNORs) in paraffin sections of 156 neoplastic tissues and other lesions, including gliomas (n = 41), meningiomas (n = 20), urinary bladder carcinomas (n = 58), and neoplastic and reactive lesions of the mesothelium of the pleural cavity (n = 37). We found significant differences in the mean number and area of AgNORs per nucleus between nonanaplastic and anaplastic astrocytomas. In meningiomas AgNOR analysis may be useful to distinguish between mostly benign tumors (grade 1 tumors) and atypical ones. Urinary bladder carcinomas exhibited a statistically significant increase in both AgNOR number and area as the grade of malignancy increased. Diagnostically useful differences in the AgNOR configuration between inflammatory and neoplastic processes were found for mesothelial lesions. In general, a higher grade of malignancy correlated with an increase in the AgNOR number. This was accompanied by an increase in the total AgNOR area per nucleus, irrespective of whether the size of the individual AgNORs had changed.     

Morphometric analysis of AgNORs in nonkeratinizing carcinoma and adjacent normal epithelia in the nasopharynx

OBJECTIVE: To evaluate the proliferative activity of different types of nonkeratinizing carcinoma and adjacent normal epithelia in the nasopharynx by the quantitative assessment of argyrophilic nucleolar organizer region (AgNOR) proteins. STUDY DESIGN: Silver staining of nucleolar organizer regions (NORs) was applied to 70 paraffin sections of nonkeratinizing carcinoma in nasopharyngeal biopsies. Fifty-four of the 70 cases had differentiated nonkeratinizing carcinoma (DNC), and the remaining 16 had undifferentiated carcinoma (UC). Nineteen of these 70 samples proved to contain, besides carcinoma, normal epithelia (NE), which was used as a control. The epithelial cells and cancer cells were analyzed for their AgNOR features by image cytometric analysis. RESULTS: As compared with normal epithelia, significant differences were found in mean nuclear area, AgNOR count, mean AgNOR area, AgNOR area ratio and AgNOR area/count ratio between NE and DNC (P < .05) and in mean nuclear area, mean AgNOR area and AgNOR area/count ratio between NE and UC (P < .001). Further, the differences in mean nuclear area, mean AgNOR area and AgNOR area/count ratio were statistically significant between DNC and UC. CONCLUSION: The evaluation of AgNORs is a useful histologic assessment of rapidity of cell proliferation in malignant and benign lesions and demonstrated that UC had more rapidly proliferative activity than DNC in this study.     

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.     

Improvement in the specificity of the silver staining technique for AgNOR-associated acidic proteins in paraffin sections

Some recently developed silver staining methods allow selective staining of acidic nucleolar proteins. Pretreating deparaffinized sections with Schiff's reagent improves the specificity of Goodpasture and Bloom's AgNOR staining (as modified by Kodama et al.) after aldehyde fixation.     

Argyrophilic nucleolar organizer regions

Silver staining techniques developed to demonstrate argyrophilic nucleolar organizer regions (Ag-NORs) have been widely applied in a variety of cell kinetic studies, using the mean number of AgNORs in tumour cells as a marker for malignancy of certain types of neoplasms. However, the AgNOR techniques currently available are not entirely satisfactory, as unspecific silver precipitates readily form in the sections. On the other hand, the contrast staining, may be so weak as to render identification of the AgNORs difficult. In the present study, some of the key factors influencing the outcome of AgNOR staining were evaluated in a more systematic way. A modified AgNOR staining procedure is now proposed, giving highly contrasting AgNORs with minimal unspecific silver precipitation, thus facilitating both manual and computerized counting. The new technique involves the use of microwave irradiation in order to shorten the processing time, the use of gelatin as a protective colloid, and a Farmer's solution to optimize the specificity of the technique.     

Quantitation of AgNORs by flow versus image cytometry.

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase-Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.     

Melanin bleaching in argyrophilic staining of AgNORs in pigmented lesions: a morphometric evaluation

OBJECTIVE: To establish a procedure that can effectively bleach melanin from pigmented lesions without affecting quantification of argyrophilic staining of nucleolar organizer regions (AgNORs). STUDY DESIGN: Twenty banal compound nevi, five from each of nonpigmented, slightly pigmented, moderately pigmented and heavily pigmented groups, were bleached by 10% H202 for periods of 0 (nonbleached controls) and 24 hours. AgNOR size and count parameters of nevomelanocytic nuclei were measured by video image analysis. Melanin bleaching using KMnO4 was also investigated. RESULTS: In all lesions treated with 10% H202 for 24 hours, the melanin was bleached effectively, with no qualitative change in AgNOR appearance. There were no significant differences in mean AgNOR number per nucleus (AgNOR number), mean individual AgNOR size (AgNOR size) or mean percentage of AgNOR area per nucleus (% nuclear area) between nonbleached and bleached sets in both the nonpigmented and slightly pigmented groups. However, disintegration of AgNOR dots was observed in those treated with 1% KMnO4 for 5, 10 and 15 minutes. There were significant decreases in AgNOR size (P = .002) and % nuclear area (P = .003) and increase in AgNOR number (P = .05) in the slightly pigmented group evaluated when treated with 1% KMnO4 for five minutes. CONCLUSION: Melanin in pigmented lesions can be bleached effectively with an H202 procedure without significantly affecting AgNOR staining properties in contrast to bleaching with KMnO4.     

Silver-stained nucleolar organizer proteins in chondrosarcoma.

Silver-stained nucleolar proteins (AgNORs) were counted in primary chondrosarcomas of three histologic grades and in metastatic chondrosarcomatous lesions in the lung. The AgNOR numbers of neoplastic cells in primary tumors increased stepwise from grade 1 (4.42 +/- 1.11) through grade 2 (4.94 +/- 1.31) to grade 3 (6.97 +/- 1.10). There was a significant difference in AgNOR numbers between grade 3 and both grades 1 and 2 (p less than 0.001). Furthermore, the mean number of AgNORs in metastatic lesions (9.75 +/- 0.83) was significantly higher than that in primary sites (p less than 0.001). The number of AgNORs therefore reflects the grade of the chondrosarcoma. The results in the present study indicate that silver colloid staining is a useful technique for determining the histologic grade and evaluating the proliferative activity of chondrosarcomas.     

Nucleolar organiser regions in colonic dysplasia. A preliminary study

As a preliminary investigation in the evaluation of the argyrophilic nucleolar organiser region (AgNOR) technique in colonic dysplasia, quantitation of AgNORs was carried out in biopsies of normal rectal mucosa and tubulovillous adenomas. The AgNOR counts in the lower third of the normal crypts were approximately twice those in the surface mucosa but there was no significant difference between counts in normal crypt bases and adenomas. It is concluded that the AgNOR technique is unlikely to be of value in the assessment of colonic dysplasia.     

Nucleolar organizer regions in the fine needle aspirates of lung tumours

Nucleolar organizer region associated proteins (AgNOR proteins) have been identified by means of an argyrophil technique in smears of 60 consecutive fine needle aspirates from lung tumours. AgNOR proteins were visualized as silver-positive granules distributed in loose, intranuclear aggregates. Variations in the number as well as differences in the distribution pattern of AgNOR granules were found among different types of tumours. Except for small cell lung carcinoma, the count of AgNOR granules increased when the differentiation of tumours decreased. In particular well differentiated tumours had relatively few AgNOR granules, distributed in cohesive aggregates. Poorly differentiated tumours had many AgNOR granules organized in loose clusters and small cell lung carcinoma had relatively few granules dispersed throughout the nucleoplasm, showing characteristics unique among all lung neoplasms. The application of the AgNOR technique in cyto-preparations is useful in discriminating between small cell and non-small cell lung carcinomas. Moreover, the pattern of distribution of AgNOR proteins may be of diagnostic value in the assessment of tumours displaying overlap in AgNOR counts.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.     

AgNOR count in resting cells (resting NOR) is a new prognostic marker in invasive bladder tumor.

PURPOSE: We have previously demonstrated that the AgNOR count in proliferating cells is a predictor of tumor recurrence in superficial bladder tumor (J. Urol. 162 (1999), 63-68). In the present study, we evaluate the type of AgNOR associated with cell cycles as a prognostic factor in invasive bladder tumor using a double staining technique employing both AgNOR and MIB-1 labelling. MATERIALS AND METHODS: Forty-four paraffin sections of invasive bladder tumors were stained simultaneously with AgNOR and MIB-1. The number of AgNORs in proliferating (MIB-1 positive) or resting (MIB-1 negative) cells were counted from a total of 100 nuclei. Correlations between MIB-1 associated AgNOR count and clinicopathological parameters were statistically analyzed. RESULTS: The AgNOR count in proliferating cells (proliferating NOR) was significantly higher than that in resting cells (resting NOR) (p<0.01). The resting NOR in tumors with distant metastases was significantly higher than that in tumors without metastases (p<0.05). Patients with a low resting NOR tumor had a better prognosis than those with a high resting NOR tumor, whereas the proliferating NOR was not associated with survival. Survival analysis revealed that the resting NOR was the most powerful prognostic marker in patients with invasive bladder tumor (p<0.05). CONCLUSIONS: Resting NOR had a predictive value in the prognosis of patients with invasive bladder tumor.     

Modified silver staining of nucleolar organizer regions to improve the accuracy of image analysis

Silver staining of nucleolar organizer regions (NORs) and their subsequent quantification by image analysis are used increasingly in human pathological specimens and experimental models. Because certain conditions determined by the type of tissue and/or its fixation render AgNOR segmentation for image analysis difficult due to insufficient contrast or nonspecific silver precipitation, we propose three improvements to the original technique to overcome these difficulties. Pretreatment with 7% nitric acid produced very distinct dark brown images of AgNORs on a yellow background. The gradient of background colors allowed easy discrimination of nucleolar, nuclear and cytoplasmic structures. Seven morphometric parameters related to number, size and shape of AgNORs were evaluated quantitatively by image analysis on sections pretreated with nitric acid and on adjacent sections treated with citrate buffer in a wet autoclave according to the most widely accepted method for image analysis of AgNOR. Both methods yielded similar results. A second improvement was achieved by coating the slides with 7% celloidin solution in ethyl alcohol-ether prior to AgNOR staining and acid pretreatment. This coating prevented nonspecific silver deposition on argyrophilic bacteria and other tissue debris in human vaginal smears that could make visualizing AgNOR sites difficult. Finally, placing sections face down on the staining solution prevents the formation of nonspecific silver precipitates. These procedures can be applied together or separately according to the requirements of the material to be evaluated.