Modifications of interphasic NORs as a diagnostic parameter of atypical lesions of the female breast.

Seventy-five breast samples including normal tissue, hyperplastic, metaplastic, atypical and neoplastic lesions were employed for the determination of interphasic Nucleolar Organizer Regions (NORs) modifications and Proliferating Cell Nuclear Antigen (PCNA) immunoreactivity. Interphase NORs were quantitatively and qualitatively modified in atypical lesions and breast carcinomas, whereas only modifications in the Ag-NORs count were found in benign samples. Our results investigated the nature of interphase NORs in the hope of finding a use for their evaluation in the diagnosis and biological clarification of breast epithelial atypia.

Morphological changes in a hyperfunctioning thyroid adenoma after percutaneous ethanol injection: histological, enzymatic and sub-microscopical alterations.

Ultrasound-guided ethanol injection (PEI) is an effective treatment of autonomously functioning thyroid nodules (AFTN) but little is known about the tissue lesions induced by alcohol. We report morphological changes of an AFTN which was removed after successful PEI treatment. At histological evaluation, the adenoma showed multiple areas of coagulative necrosis with eosinophilic ghost follicles, hemorrhages, small vessel thrombosis and a wedge-shaped hemorrhagic infarction. The peripheral adenomatous tissue and the surrounding thyroid parenchyma were free of regressive changes and/or lympho-monocytic inflammatory reactions. Enzyme histochemistry confirmed the hyperfunctioning character of the lesion and showed reduction of succinic dehydrogenase and cytochrome-c-oxydase activity in the viable tissue surrounding the central necrotic areas. By electron microscopy, the hyperfunctioning follicular epithelium showed submicroscopical alterations of the adenomatous cells surrounding the areas of coagulative necrosis. These results suggest that control of hyperthyroidism, after PEI, is due to: a) directly induced, irreversible damage (coagulative necrosis and vascular thrombosis with hemorrhagic infarction) in the central areas of the lesion; b) potentially reversible damage (reduction of intracellular enzyme activity and ultrastructural changes) in the peripheral areas.

[A case of nodular pseudosarcomatous fasciitis treated with "presuture" technique]. / Su di un caso di fascite nodulare pseudosarcomatosa trattato con tecnica di "presutura".

This paper reviews a case of nodular (pseudosarcomatous) fasciitis. It consists of a rare benign lesion of the soft tissues. The rapidity of its growth and the cellular features represent a diagnostic challenge when compared with that of a sarcoma. The patient, a 36 year old female, 20 days before surgical treatment, noticed the growth of a nodular lesion on the left forearm skin. After echotomography and ultrasound guided needle biopsy diagnosis of nodular pseudosarcomatous fasciitis was established and the patient underwent surgical treatment. The nodular lesion, noncapsulated, (diameter 2.5 x 1.2 cm) greyish-white colored, with slimy consistency, and myxoid character, was microscopically composed by atypical fibroblasts, with myxoid ground substance. The nodular lesion was removed using the "pressure" technique by Serge Baux of Rothschild Hospital in Paris using two Kirschner threads pulling the skin surrounding the biopsy wound. By this technique was possible to achieve a rapid skin expansion. The nodular pseudosarcomatous fasciitis remains a very difficult lesion to recognise unless the aid of a biopsy.

Interphasic nucleolar organizer regions expression and cell kinetics evaluation during gastric carcinogenesis induced by nitrosoguanidine in the rat.

An increased number of interphasic nucleolar organizer regions containing ribosomal cistrons associated with argyrophilic proteins (AgNORs) has been described in human malignant tumor cells. In this study variations in AgNOR numbers have been compared with changes of cell kinetics, evaluated by the mitotic count (MC) and bromodeoxyuridine labeling index (BrdU LI), during gastric carcinogenesis induced with N-methyl-N'-nitro-N-nitrosoguanidine (NG) in rats. Significant differences (2 P < 0.005) in AgNOR mean numbers, evaluated in the antral isthmic cells, in MC mean values and BrdU LI, evaluated in the whole antral cellular population, were found when comparing areas of acute gastritis, atrophy and hyperplasia in NG-treated rats with the normal mucosa in controls. No differences were observed in MC and BrdU LI between normal antrum and carcinoma cells which showed an AgNORs mean number lower than in the isthmic cells of controls (2 P < 0.005). Moreover, significant correlations were found comparing changes in AgNOR numbers with MC (r = 0.89, P < 0.001) and BrdU LI (r = 0.66, P < 0.001) in different lesions. These data show that evaluation of AgNOR numbers does not allow the identification of malignant cells in NG-induced gastric carcinoma. However AgNOR quantification seems to be a reliable index of cell kinetics and related well with the cellular dividing fraction.

Demonstration of oestrogen and progesterone receptors in freeze-dried, paraffin-embedded sections of breast cancer.

Immunohistochemical evaluation of oestrogen and progesterone receptors is of importance in evaluating human breast tumours. Staining techniques can be performed on snap-frozen, cryostat-cut tissues or, as recently reported, on formalin-fixed, paraffin-embedded tissues. These methods are, however, limited by several drawbacks, including difficulties in retrospective studies and in storage of the material, and the relatively high frequency of false negative results for chemically fixed specimens. We therefore investigated the application of freeze-drying technology to assess the feasibility and reliability of this technique as an alternative method for diagnostic breast pathology. Morphological and immunohistochemical studies were performed on snap-frozen, freeze-dried and paraffin-embedded tissue obtained from 16 cases of benign and malignant breast neoplasms. Our results showed good preservation of tissue morphology, similar to standard formalin fixation, and excellent preservation of antigenic reactivity of nuclear receptors, comparable to that obtained with cryostat sections. We therefore suggest that freeze drying and paraffin embedding of frozen tissue blocks is equivalent or even preferable to formalin fixation for the demonstration of oestrogen and progesterone receptors, at least in the case of small tumours.

A comparative study of histopathology, hormone receptors, peanut lectin binding, Ki-67 immunostaining, and nucleolar organizer region-associated proteins in human breast cancer.

The current study was performed on 71 cases of human female breast cancer and compares the results of five morphologic methods developed for the detection of estrogen receptors (ER), progesterone receptors (PgR), lectin Peanut agglutinin (PNA) binding sites, monoclonal antibody Ki-67 immunoreactivity, and the mean number of argyrophilic nucleolar organizer regions (Ag-NOR). All the parameters were evaluated on serial cryostat sections representative of a closely related, if not identical, neoplastic population. A significant positive correlation was found between the occurrence of estrogen, progesterone, and peanut receptors and between Ki-67 immunoreactivity, mean number of NOR, and mitotic index. Furthermore, ER, PgR, and PNA receptors showed a significant, inverse correlation with Ki-67 immunoreactivity, mitotic index, and mean number of Ag-NOR. These results provide further data that support the hypothesis that (1) progesterone and PNA receptors are estrogen-induced and indicate a metabolic response of the target cells to functioning estrogen receptors; (2) the mean number of NOR reflects the cell kinetics of the tumor; and (3) metabolic differentiation of neoplastic cells is inversely correlated to the proliferation index.

Expression of interphasic nucleolar organizer regions in normal, dysplastic and neoplastic colorectal mucosa.

Silver-binding nucleolar organizer region (Ag-NOR) expression in interphasic nuclei was studied in normal, dysplastic and neoplastic colorectal mucosa at the light microscope level and by means of an image analyser (IBAS II). Both methods showed a progressive increase in the mean number of Ag-NOR sites per nucleus from mild dysplasia to invasive carcinoma. Ag-NOR counts differed significantly in the various classes of lesions (P less than 0.001), except between moderate and severe dysplasia (P greater than 0.05). Severe dysplasia showed a mean number of NORs lower than that for invasive carcinoma though an overlap in the respective frequency distributions was observed. The mean of the variances of the mean dot areas per cell nucleus (pooled variance) also showed a step-wise increase from normal to neoplastic lesions, indicating a greater variability in NOR size as a characteristic of malignant cells. A similar increase was observed in the percentage of nuclear area occupied by Ag-NORs. The mean area per silver-stained dot was also measured in the different classes of lesions by IBAS II. Data obtained showed no significant differences among the values. In conclusion, the wide overlap between the frequency distributions does not allow consideration of the Ag-NOR count alone to be a reliable marker of malignant transformation in a single cell. It appears that the study of Ag-NOR number needs to be evaluated together with dot anisometry in order to be a useful criterion in distinguishing the biological behaviour of neoplastic lesions in colorectal mucosa.

Telephonic transmission of cardiotocographic recordings from a remote facility to an obstetric unit.

The Authors describe their experience in telephonic transmission of cardiotocographic recordings. With a Corometrics 116 and 410 unit installed in a remote facility, 61 CTG recordings were tele-transmitted and received by a Toshiba personal computer. The concordance between original and transmitted CTG record was excellent and a very low percentage of signal loss during transmission (3.2%) was found. The advantages of telephone transmission of CTG in peripheral areas are stressed.

Use of freeze-drying technology for the study of renal biopsies.

Adequate handling of renal biopsies requires processing for light and electron microscopy, as well as for immunohistochemical study. Problems of adequate sampling are frequently encountered, and tissue size can be insufficient, since morphologic examination requires chemical fixation, while immunofluorescence study is currently performed on snap-frozen, cryostat-cut tissue. The application of freeze-drying technology on 20 renal needle biopsies has been investigated to assess the feasibility and reliability of the technique as a routine diagnostic tool in renal pathology. Morphologic as well as immunohistochemical studies were performed on freeze-dried, paraffin-embedded specimens, including immunofluorescence and PAP techniques to detect immunoglobulins, complement fractions, fibrinogen, collagen types, and laminin. Our results showed good preservation of tissue morphology, similar to standard formalin fixation. Moreover, absence of diffusion artifacts and intensity of immunohistochemical reactions were comparable to what is obtained with cryostat sections. We therefore suggest that freeze-drying before paraffin embedding is, at least for diagnostic purposes, preferable to formalin fixation; unfixed, cryostat-cut sections can still be informative and should be used whenever tissue is available in selected cases, and/or in experimental work.

Non-specific binding of immunoglobulins and protein A-Gold complex with cytoplasmic granules of human mast cells.

A non-specific staining of mast cells by a number of proteins and immunoglobulins has been observed using different immunocytochemical techniques and different antisera at light and electron microscopy level. This finding has been related to an ionic binding of cationic charges, such as aminic groups with an high pI, to the strongly anionic residues of mast cell granules. This hypothesis is supported by the lack of non-specific binding achieved using negatively charged chromogens (i.e. FITC--or Rhodamine-conjugated antibodies) or using cationic dyes before the immunohistochemical reaction. It is therefore stressed that adequate and accurate negative controls must always be carried out in order to get correct interpretations of the staining results.

Silver staining of interphase nucleolar organizer regions in cytologic smears previously stained by the Papanicolaou and May-Grünwald-Giemsa techniques.

Malignant cells are known to display a distinctive argyrophilic nucleolar organizer region (Ag-NOR) protein distribution compared with distribution in their benign counterparts. In this study, Ag-NOR staining was applied to smears from reactive and malignant serous effusions. The smears were either unstained or had been previously stained with the Papanicolaou or May-Grünwald-Giemsa (MGG) technique. The purpose of this study was to assess the feasibility and usefulness of Ag-NOR staining in diagnostic cytopathology and to set up a procedure that could be used on prestained smears in retrospective studies. The one-step silver staining method was applied to prestained smears after a weak destaining step in trichloracetic acid and to unstained smears after a postfixation step in Carnoy's fluid. Positive results were observed on both destained and unstained preparations, MGG-stained smears showing a better visualization of the silver deposits than Papanicolaou-stained smears. Major differences in size, shape, and distribution patterns of the Ag-NOR-positive granules were observed between neoplastic and reactive cells. Furthermore, the mean number of silver-stained dots per cell was significantly higher in malignant cells (33.32 +/- 2.23) than in reactive mesothelial cells (9.71 +/- 0.66). These data indicate that this Ag-NOR technique can be profitably applied to prestained cytologic smears to assist in the diagnosis of malignancy and that the technique has the advantage that cellular morphology and silver staining can be evaluated on the same slide.

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.

Membranous nephropathy associated with giant lymph node hyperplasia. A case report with histological and ultrastructural studies.

A young woman presented with nephrotic syndrome due to membranous nephropathy and a localized form of giant lymph node hyperplasia (Castleman's disease) occurring as a large intra-abdominal mass. Five months after surgical removal of the mass, only mild proteinuria persisted. Twenty months later a second kidney biopsy showed a near-normal morphology. Six years later the patient was free of symptoms and had normal urinalysis. Reviewing the pertinent literature, it seems to be the first case of a biopsy-proven cure of membranous nephropathy in a patient with membranous nephropathy and Castleman's disease.

Presence of an alpha-galactolipid on the cell surfaces of endothelial cells of human kidney.

The presence of an alpha-galactolipid was investigated with a peroxidase-labelled lectin from Griffonia simplicifolia (GSA-I) with specific binding for terminal alpha-D-galactose residues. Normal kidney tissue was obtained from patients undergoing nephrectomy for renal neoplasms. For light microscopy, tissue was snap-frozen; 4 microns-thick sections were briefly fixed in paraformaldehyde and incubated with GSA (0.025 mg ml-1). The peroxidase activity was developed with 3-amino-9-ethylcarbazole. Adjacent sections were stained at the same time after lipid extraction with 3:1 (v/v) chloroform/methanol. For electron microscopy, 0.2-0.5 mm-thick paraformaldehyde-fixed blocks, with or without lipid extraction, were stained with peroxidase-labelled GSA. The label was developed with diaminobenzidine and osmium tetroxide. Some structures, such as tubular epithelia, stained both in lipid-extracted and non-extracted tissues, suggesting that glycoproteins were most likely involved. In addition, tissue stained immediately after fixation showed GSA reactivity on endothelial cell surfaces of intertubular capillaries and larger vessels. In lipid-extracted tissues, however, tubular epithelium was still positive for GSA but endothelial cells failed to stain. These findings suggest that a glycolipid, bearing a terminal alpha-galactose residue, is present on the endothelial cells in human kidney and possibly on tubular epithelia. Our data may explain the preferential storage of alpha-galactolipid in endothelial cells of patients with Fabry's disease and other biological phenomena such as Escherichia coli adhesion.

Ultrastructural localization of sulphate glycoconjugates in the human glomerular capillary wall using the high iron diamine method.

The distribution of fixed anionic sites within glomerular capillary walls has been studied in man by applying two ultrastructural histochemical methods--the high iron diamine and dialysed colloidal iron methods--to tissue chopper sections and to isolated glomeruli obtained from surgical fragments of renal tissue. By using the high iron diamine method we have been able to demonstrate that in man, too, there are sulphate (possibly heparan sulphate proteoglycan) sites preferentially located in the lamina rara esterna of the basement membrane and in the cell coat of the urinary surface of podocytes. Non-sulphate (high iron diamine-negative, dialysed colloidal iron-positive) anionic sites have been identified not only in the glycocalyx of the epithelial and endothelial cells but also in the laminae rarae of the basement membranes, where they show a more extensive distribution pattern than sulphate sites. The proposed methods seem particularly suitable for the study of human renal tissue; they could, in fact, provide useful information about the behaviour of the various anionic components of the glomerular capillary wall in pathological conditions.

Carbohydrate histochemistry of human Brunner's glands.

Brunner's glands are known to produce neutral mucins. In order to achieve a better knowledge of their carbohydrate profile, we used five peroxidase-labeled lectins on surgical specimens of human duodenum. This method allowed us to identify at least two different types of neutral mucins in Brunner's glands secretion, thus demonstrating a heterogeneous mucin production. The structure of terminal oligosaccharidic chains in these glycoproteins has also been hypothesized.

Ultrastructural histochemical investigations of "dense deposit disease". Pathogenetic approach to a special type of mesangiocapillary glomerulonephritis.

Dense deposit disease is characterized by the presence of intramembranous dense deposits; their constituents are unknown but immunological and biochemical studies have demonstrated that they contain no gamma-globulins or any other plasma protein. In order to clarify the nature of the dense deposits better, we investigated their most distinctive character, (marked electron-density) by means of ultrastructural histochemistry techniques using thin sections from Formaldehyde fixed, OsO4 postfixed and Epon embedded specimens collected for diagnostic electron microscopy. The dense deposits have a higher osmium affinity than the lamina densa of normal basement membranes, and the electron-density is strictly osmium-dependent suggesting the presence of a lipid component. Further data, obtained using an extraction method for lipids, seems to confirm our hypothesis.

Interphasic nucleolar organizer region distribution as a diagnostic parameter to differentiate benign from malignant epithelial tumors of human intestine.

The distribution of the interphasic nucleolar organizer regions (NORs) has been investigated in five hyperplastic polyps, five adenomatous polyps and fifteen colonic adenocarcinomas. The study was performed using electron microscopy and paraffin-embedded sections stained for Ag-NOR proteins. Malignant tumor cells were characterized by a large number of NORs which were small in size and showed a scattered distribution. Nuclei of both types of polyp had only a small number of large-sized NORs in a clustered distribution. In two adenomatous polyps, cells were also observed with an NOR distribution pattern intermediate between that of frankly benign and malignant lesions.

Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa.

The effect of adding cetylpyridinium chloride to the fixative on the preservation of sulphated glycosaminoglycans (SGs) was studied in human normal colonic mucosa. SGs were visualized at the ultrastructural level through the application of Spicer's High Iron Diamine (HID) technique followed by a post-fixation with potassium ferrocyanide-reduced osmium tetroxide. SGs were mainly localized in basement membranes of epithelium and capillary wall and along collagen fibers. The morphology of the reactive sites depended on the presence of cetylpyridinium chloride, SGs being granular in absence of the salt and more or less elongated when cetylpyridinium chloride was added to the fixative. We suggest that the use of cetylpyridinium chloride during fixation may help to preserve SG molecule at the ultrastructural level.

An improved periodic acid-thiosemicarbazide-osmium technique to reveal glycoconjugates at the molecular level in situ.

The periodic acid-thiocarbohydrazide or thiosemicarbazide-OsO4 method (Seligman AM, Hanker JS, Wasserkrug H, Katzoff L: J Histochem Cytochem 13:629, 1965) has been modified in order to obtain a periodic acid-Schiff (PAS)-like reaction for electron microscopy capable of visualizing structures at the molecular level in situ. Thiocarbohydrazide (TCH) and thiosemicarbazide (TSC) have been used dissolved in distilled water and bubbled with SO2. Treatment of previously oxidized thin sections with TCH (SO2) or TSC (SO2), followed by osmification, resulted in selective and very good staining of all the PAS-positive structures examined: glycogen, intestinal mucopolysaccharides, plasma membrane glycoproteins, basement membranes, Golgi apparatus, and collagen. The staining reaction was highly specific when TSC was used on thin sections from paraformaldehyde-fixed samples. The non-particulate end-reaction product made possible visualization of a periodic distribution of sugar residues in the 64-nm unit of collagen and the structural organization of the PAS-positive glycoconjugate components in the glomerular basement membrane.