Interlaboratory comparison of assessments of Alzheimer disease-related lesions: a study of the BrainNet Europe Consortium.

This interlaboratory study evaluated the reproducibility of the assessments of neuritic plaques and neurofibrillary tangles (NFTs)--the hallmark lesions of Alzheimer disease--and compared the staining between the BrainNet Europe centers. To reduce the topography-related inconsistencies in assessments, we used a 2-mm tissue microarray (TMA) technique. The TMA block included 42 core samples taken from 21 paraffin blocks. The assessments were done on Bielschowsky and Gallyas silver stains using an immunohistochemical (IHC) method with antibodies directed to beta-amyloid (IHC/Abeta) and hyperphosphorylated tau (IHC/HPtau). The staining quality and the assessments differed between the participants, being most diverse with Bielschowsky (good/acceptable stain in 53% of centers) followed by Gallyas (good/acceptable stain in 57%) and IHC/Abeta (good/acceptable stain in 71%). The most uniform staining quality and assessment was obtained with the IHC/HPtau method (good/acceptable stain in 94% of centers). The neuropathologic diagnostic protocol (Consortium to Establish a Registry for Alzheimer Disease, Braak and Braak, and the National Institute of Aging and Reagan [NIA-Reagan] Institute) that was used significantly influenced the agreement, being highest with NIA-Reagan (54%) recommendations. This agreement was improved by visualization of NFTs using the IHC/HPtau method. Therefore, the IHC/HPtau methodology to visualize NFTs and neuropil threads should be considered as a method of choice in a future diagnostic protocol for Alzheimer disease.

Procedures for specific detection of silver-stained nucleolar proteins on western blots.

Nucleolar organizer region (NOR)-silver staining of the chromosomes and nucleoli is a method that enables the detection of proteins associated with the ribosomal genes. We adapted the most commonly used cytochemical NOR-silver staining techniques to Western-blotted proteins of HeLa cells, mimicking the silver staining of cells in situ, and testing several parameters that may influence the in situ reaction. Two of these techniques, both one-step methods with colloidal developers, were standardized to obtain reproducible results. The specificity of NOR staining is documented by: (a) only a few bands are revealed among the many proteins detected by total proteins staining on gels or blots; two major groups of bands are found around 100 KD and 40 KD that could correspond at least in part to nucleolin and B23 nucleolar proteins; (b) the silver staining of bands was not the result of the high relative protein concentrations; and (c) the same number of NOR-silver-stained bands was observed across a large range of protein concentrations. The reaction appeared to be specific for a subset of nucleolar proteins, because the same bands were observed with the use of nucleolar, nuclear, or total cell protein extracts, and the silver grains observed in electron microscopy were clearly confined to the nucleolar fibrillar centers and dense fibrillar component. The efficiency of the reaction was not modified by any of the tested fixative pre-treatments except that involving methanol. The presented standardization of NOR-silver staining on Western blots allows the characterization of the Ag-NOR proteins and their specific regions responsible for silver staining of the nucleolus.

Quality assurance and reproducibility of high-resolution two-dimensional electrophoresis and silver staining in polyacrylamide gels.

Qualitative data from high-resolution two-dimensional electrophoresis (2DE) have become clinically useful in immunoglobulin clonality analysis, in resolution of ambiguities in immunofixation typing of paraproteins, and in genetic typing of serum proteins. Since 1986, the authors have been evaluating the College of American Pathologists Reference Preparation for Serum Proteins (RPSP) as a quality control material for 2DE because (1) it is prepared exclusively from pooled human sera, (2) the pool yields a reference pattern of mixed heterozygosity for genetic markers, and (3) RPSP is widely available as a lyophilized preparation that currently serves in the authors' laboratory as a qualitative quality control preparation and that may become a quantitative quality control material or external quality assessment material for 2DE. Using the ISO-DALT 2DE system and silver-staining, the peptide patterns were examined in 11 lots of RPSP and compared with fresh serum and with each other. Consistent differences in the 2DE pattern between RPSP and fresh serum included the presence of freeze-thaw peptides, the presence of degradation spots of apolipoprotein A-I, and the diminution of apolipoprotein spot intensities in RPSP. All lots of RPSP yielded clear identification of the eight serum proteins used for quality control calculations. Run-to-run coefficients of variation for a single lot of RPSP for four parameters of 2DE spot location and gradient reproducibility were comparable with band location reproducibility for the one-dimensional procedures of serum protein electrophoresis and lactate dehydrogenase isoenzyme electrophoresis. It is concluded that the reproducibility, that is, imprecision, of 2DE is the same as one-dimensional clinical electrophoresis techniques and that either RPSP or pooled fresh serum can serve as a satisfactory internal quality control material.

Proliferation in non-Hodgkin's lymphomas and its prognostic value related to staging parameters.

In malignant lymphomas, cell kinetics has shown to be related with histologic type as well as with the clinical behaviour. The aim of our study was to investigate the relevance of cell proliferation parameters on overall survival in non-Hodgkin's lymphomas as well as their relationship with prognostic factors such as International Prognostic Index (IPI). We performed DNA-flow-cytometry (S-phase fraction and detection of DNA-aneuploidy) as well as cytologic examination and the AgNOR technique in material obtained by fine needle aspiration of lymph nodes at diagnosis. The majority of the patients were stage IV by Ann Arbor and intermediate risk by IPI (42/55). When analyzing all patients together, histologic type by the WHO classification, IPI and the presence of a DNA-aneuploid clone could not separate well patients with a different survival. For all patients, univariate Cox analysis revealed S-phase (SPF) and AgNOR parameters to be of prognostic value. In the multivariate analysis, however, only SPF remained in the final model. Yet, when stratifying for DNA-ploidy, only the total number of AgNORs/nucleus was an independent parameter. Looking only at the DNA-diploid cases, the AgNOR pattern remained the most important parameter, whereas for the DNA-aneuploid cases this was true for SPF. When studying patients with B large cell lymphoma separately, only DNA-ploidy was a prognostic factor. In summary, cell kinetic parameters reveal important prognostic information in NHL patients. Furthermore, DNA-aneuploidy seems to interfere with the analysis of the AgNOR pattern.

A reliable method for Golgi staining of retina and brain slices.

Although the classical Golgi method is a powerful means for structural analysis of the brain, it is generally considered to be an unpredictable technique making anatomists wary of using it. Often, even when successful staining has occurred, deposits of silver chromate crystals on the surface of the tissue obscure examination. This paper describes a simple procedure for Golgi impregnation of retina and brain slices so that good, even staining is obtained and crystal formation is avoided. The most outstanding feature of the method is the consistency of results. This consistency is due to two factors: (1) the accurate determination of the optimal chromation by measuring the rise of pH in the solutions and (2) the uniform penetration of dichromate and silver nitrate to the specimen by using a freely hanging, sandwiching technique. We suggest that the method described here can be applied to other parts of the nervous system and will be a reliable way to identify and better classify new cell types.

The use of a sodium tungstate developer markedly improves the electron microscopic localization of zinc by the Timm method.

The Timm's sulfide-silver method is frequently used for the demonstration of the mossy fiber bundle or sprouted mossy fibers in the normal or epileptic hippocampal dentate gyrus. Under the light microscope the results are excellent, but the ultrastructure is considerably impaired and the silver grains produced are too large as compared to the sizes of intra-synaptic structures. The present study was meant to test a series of physical developers containing, instead of gum arabic, sodium tungstate as protective colloid. One of them left the ultrastructure fairly intact and produced small, round silver grains, making it possible to precisely locate zinc in mossy terminals. With this method, it could be demonstrated that zinc is contained inside synaptic vesicles in the resting axon terminals of granule cells. As a consequence of prolonged sodium sulfide perfusion, zinc is released from synaptic vesicles and enters the synaptic cleft.

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 staining and quantification.

Nucleolar organiser regions (NORs) are defined as nucleolar components containing a set of argyrophilic proteins, which are selectively stained by silver methods. After silver-staining, the NORs can be easily identified as black dots exclusively localised throughout the nucleolar area, and are called "AgNORs". The NORs' argyrophilia is due to a group of nucleolar proteins, which have a high affinity for silver (AgNOR proteins). A number of studies carried out in different tumour types demonstrated that malignant cells frequently present a greater AgNOR protein amount than corresponding non-malignant cells. Moreover, in cancer tissues AgNOR protein expression was found to be strictly related to the cell duplication rate. Over the past 12 years, the "AgNOR method" has been applied in tumour pathology for both diagnostic and prognostic purposes. However, the lack of a standardised silver-staining protocol has led to much misinterpretation of actual structures evaluated in individual studies. Indeed, the absolute AgNOR scores reported by different authors for the same types of tumour are scarcely comparable and the results produced by these investigations sometimes seem to be conflicting. In order to achieve definitive standardisation of the AgNOR method and produce comparable data in all laboratories, the "International Committee on AgNOR Quantitation" was founded, and during the first Workshop "AgNORs in Oncology" held in Berlin in 1993 guidelines for AgNOR protein evaluation were first defined. The present paper discusses the main technical aspects of NOR silver-staining, and critically evaluates the methods commonly employed for AgNOR protein quantification in routine cyto-histopathology.

Proliferative activity of the thyroid oxyphilic tumor cells estimated by means of quantitative analysis of silver stained nucleolar organizer regions (AgNORs).

UNLABELLED: Promising results of studies on different neoplasms, by means of morphometric analysis of argyrophilic nucleolar organizer regions (AgNORs), known as proliferative activity marker, made us undertake an attempt to evaluate of proliferative activity in Hürthle Cell Tumors using the same technique. 78 cases including 20 Hürthle Cell Carcinomas (HCC), 32 Hürthle Cell Adenomas (HCA) and 26 hyperplastic nodules with Hürthle Cell Metaplasia (HCM), diagnosed in the Department of Clinical Pathology, Medical Academy of Bialystok in the years 1990-2000, were subjected to analysis. For visualization of the NORs we used the D. Ploton et al. technique. Mean AgNOR count and NORDS (NOR distribution score--the percentage of nuclei with at least 5 argyrophilic granules) were estimated in each case in 100 randomly chosen nuclei. Non-parametric Mann-Whitney U-test was applied to determine statistically significant differences. RESULTS: Mean AgNoRs counts and NORDS values were 5.1 and 52%, 3.4 and 26%, 2.5 and 7% in HCC, HCA and HCM respectively. Statistically significant differences were found between AgNORs counts in carcinomas and adenomas (p<0.01), HCCs and HCMs (p<0.005) and betwveen NORDS in all groups (p<0.001).

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.

Nucleolar organizer regions in jaw tumours of cartilaginous origin.

Nucleolar organizer regions [NORs] are loops of DNA that transcribe to ribosomal RNA. They can be visualized as intranuclear black dots by histochemical staining with a colloid silver solution. Silver-stained nucleolar proteins [AgNORs] were counted in cases comprising of primary chondrosarcomas of three histologic grades, in chondromyxoid fibroma and in controls comprising of normal bone and cartilage tissues of the jaw bones. The AgNOR counts increased step-wisely from normal bone tissue [1.11 0.4], chondromyxoid fibroma [2.66 0.78], grade I chondrosarcoma [3.94 0.34], grade II chondrosarcoma [4.32 0.52], and grade III chondrosarcoma [5.54 0.44]. There was a statistically significant difference in the mean AgNOR counts between grade 1 and grade III chondrosarcoma [p < 0.05]. The mean AgNOR counts for benign cartilaginous [chondromyxoid fibroma] tumour was significantly lower than the mean, AgNOR count for malignant cartilaginous tumours [chondrosarcomas] [p < 0.05]. The results in the present study indicate that silver colloid staining is a useful technique for evaluating the proliferative activity of chondrosarcoma and benign cartilaginous tumour such as chondromyxoid fibroma.

Argyrophilic grains: a distinct disease or an additive pathology?

BACKGROUND: Argyrophilic grains (AG) are silver-positive spindle-shaped lesions found at postmortem. Their significance is controversial. OBJECTIVE: To determine clinical correlates of AG and MRI patterns of atrophy that could allow premortem recognition of this pathology. METHODS: Cases with AG were identified from a longitudinal study of aging and dementia. Clinical features were compared between subjects with and without dementia. Voxel-based morphometry (VBM) was used to assess patterns of grey matter atrophy in subjects compared to controls. Whole brain volumes (WBV) were compared across groups. RESULTS: Twenty-two cases (14 females; median age at death of 90 years; range: 70-101) with AG were identified. Eight of the 22 were demented. Those with dementia had higher Braak (p=0.02) and lower Mini-Mental State Examination (MMSE) (p=0.002). VBM demonstrated hippocampal atrophy in those with dementia (N=3) but no atrophy in those without (N=9). There was no difference in WBV between groups. CONCLUSION: AG is a feature of old age commonly occurring in non-demented subjects. In this age group, the presence of AG may reduce the threshold for dementia.

Silver stainings distinguish Lewy bodies and glial cytoplasmic inclusions: comparison between Gallyas-Braak and Campbell-Switzer methods.

Lewy bodies (LBs) of idiopathic Parkinson's disease and glial cytoplasmic inclusions (GCIs) of multiple system atrophy are pathological deposits both composed of phosphorylated alpha-synuclein woven into different filaments. Although both LBs and GCIs are considered to be hallmarks for each independent synucleinopathy, until now they could not be clearly distinguished on the basis of their biochemical or immunohistochemical features. We have examined possible differences in their argyrophilic features and their relation to synuclein-like or ubiquitin-like immunoreactivity (IR). Pairs of mirror sections from different brain areas were triple-fluorolabeled with an anti-alpha-synuclein antibody, an anti-ubiquitin antibody and thiazin red (TR), a fluorochrome that labels fibrillary structures such as Lewy bodies or neurofibrillary tangles. One of the paired sections was subsequently stained using the Campbell-Switzer method (CS), and the other by the Gallyas-Braak method (GB). By comparing of the same microscopic field on the paired fluorolabeled sections, subsequently silver-stained with either CS or GB, five different profiles of each structure could be determined: alpha-synuclein-like IR, ubiquitin-like IR, affinity to TR, argyrophilia with CS or GB. GCIs exhibited argyrophilia with both CS and GB but lacked affinity to TR. In contrast, LBs exhibited argyrophilia with CS but not with GB and some affinity to TR. These disease-specific profiles of argyrophilia were consistent, and were not influenced by areas or cases examined. Although immunohistochemical features of LBs and GCIs were similar in exhibiting IR for alpha-synuclein and ubiquitin, the contrast in their argyrophilic profiles may indicate possible differences in the molecular composition or conformation of alpha-synuclein. Even though these empirical differences still remain to be explained, awareness of this clear distinction is potentially of diagnostic and pathological relevance.

Standardized AgNOR analysis: its usefulness in surgical oncology.

Recent improvements both in the staining quality and computer-aided quantitation of silver-stained nucleolar organizer region (AgNOR)-associated proteins offer the possibility to reliably investigate these proteins on routinely processed archival material. This article deals with the historical background, the recent introduction of a standardized quantitation, the clinical relevance, and future perspective for AgNOR evaluation. It is specifically emphasized that AgNOR analysis after both standardized staining and computer-aided quantitation (as recommended by the Committee on AgNOR Quantitation of the European Society of Pathology) is now regarded as the gold standard whenever routinely formalin-fixed and paraffin-embedded material is investigated.

Development-dependent inheritance of 5-azacytidine-induced epimutations in triticale: analysis of rDNA expression patterns.

Genomic imprinting of rye origin rDNA sequences in triticale is modulated by DNA methylation responsible for ontogenic expression patterns of those sequences. Considering the dynamic nature of these phenomena, we evaluated the influence of plant development on the inheritance of modified rye rDNA expression patterns. DNA hypomethylation was induced in triticale by 5-azacytidine (5AC) treatments at distinct developmental stages of M1 plants, and expression patterns were analysed in M2. The activity of rye origin rRNA genes in progeny of untreated and 5AC-treated plants was evaluated by silver staining in meristematic root tip cells and in meiocytes at diplotene. In the progeny of 5AC-treated plants, a significant increase in rye rDNA expression was observed, contrasting with the residual activity in untreated plants. Significant differential effects of 5AC treatments were observed in M2 plants and correlated with the M1 plant developmental stage in which DNA hypomethylation was induced. Hypotheses to explain the origin of those differences are discussed here.

Standardized staining and analysis of argyrophilic nucleolar organizer region associated proteins (AgNORs) in radically resected colorectal adenocarcinoma--correlation with tumour stage and long-term survival.

Quantification of silver-stained nucleolar organizer region associated proteins (AgNORs) was introduced in histopathology as a marker of cellular and nucleolar activity. However, due to the poor staining quality obtained on routinely processed archival material, the method yielded controversial and sometimes non-reproducible results. The recent introduction of wet autoclave pretreatment has reliably improved AgNOR staining quality on routinely formalin-fixed and paraffin-embedded tissues. In the present study, 92 routinely processed colorectal carcinomas were investigated, applying this novel staining technique. Subsequent standardized morphometric analysis revealed, irrespective of common tumour staging or grading classifications, a statistically highly significant correlation between AgNOR parameters and clinical course. The usefulness of standardized AgNOR parameters for the independent prediction of patient survival was proven by uni- and multivariate analysis.

Nucleolar organizer regions argyrophilic associated proteins in cutaneous melanocytic lesions.

We applied a simple silver staining technique to visualize nucleolar organizer regions associated proteins (AgNORs) for the study of 47 melanocytic lesions (20 malignant melanomas, 5 dysplastic nevi, 4 Spitz nevi, 2 Reed and Gartman's fusiform nevi and 16 melanocytic nevi). A statistically significant difference existed between the numbers of AgNORs per cell in benign and malignant lesions as a group. However, some overlapping counts were found, limiting the usefulness of the technique in differentiating benign from malignant lesions in individual cases.