Heterogeneity of the MtTW15 mammosomatotropic tumor. I. Light microscopic evaluation of cell types by means of immunocytochemistry,morphometric quantitation,fluorescence cytophotometry and radioimmunoassay

Large MtTW₁₅ pituitary tumors produced 200- to 800-fold elevations in serum growth hormone (GH) and prolactin (PRL) levels. Female tumor hosts showed doubling in body weight, milk secretion, and a 2-fold hepatosplenomegaly. Pituitaries of host animals were reduced by about 50% in both weight and concentrations of GH and PRL. Large tumors were well-encapsulated, multinodular and showed variable amounts of necrosis and hemorrhage. Cytofluorometric analysis revealed a range of 100-fold in nuclear DNA content of tumor parenchymal cells which were chromophobic, pleomorphic and frequently mitotic. Concentrations of hormones in tumors were less than in normal pituitaries and highly variable with the ratio of GH/PRL ranging up to 30-fold within the same tumor. Immunostaining and linear scanning quantitation showed that about 50% of the tumor cells contained immunodetectable hormones. Comparison of immunostained adjacent sections showed that hormone-containing tumor cells were pleomorphic, unequally distributed within nodules, lacking in distinctive identifying morphological characteristics and that they contained GH or PRL but not both hormones simultaneously. Collectively our results show that large MtTW₁₅ tumors are comprised of a markedly heterogeneous population of tumor cells and they suggest that the hormone-containing cells are monohormonal secreting tumor cells which can produce GH or PRL but not both hormones.     

Heterogeneity of the MtTW15 mammosomatotropic tumor. I. Light microscopic evaluation of cell types by means of immunocytochemistry, morphometric quantitation, fluorescence cytophotometry and radioimmunoassay.

Large MtTW15 pituitary tumors produced 200- to 800-fold elevations in serum growth hormone (GH) and prolactin (PRL) levels. Female tumor hosts showed doubling in body weight, milk secretion, and a 2-fold hepatosplenomegaly. Pituitaries of host animals were reduced by about 50% in both weight and concentrations of GH and PRL. Large tumors were well-encapsulated, multinodular and showed variable amounts of necrosis and hemorrhage. Cytofluorometric analysis revealed a range of 100-fold in nuclear DNA content of tumor parenchymal cells which were chromophobic, pleomorphic and frequently mitotic. Concentrations of hormones in tumors were less than in normal pituitaries and highly variable with the ratio of GH/PRL ranging up to 30-fold within the same tumor. Immunostaining and linear scanning quantitation showed that about 50% of the tumor cells contained immunodetectable hormones. Comparison of immunostained adjacent sections showed that hormone-containing tumor cells were pleomorphic, unequally distributed within nodules, lacking in distinctive identifying morphological characteristics and that they contained GH or PRL but not both hormones simultaneously. Collectively our results show that large MtTW15 tumors are comprised of a markedly heterogeneous population of tumor cells and they suggest that the hormone-containing cells are monohormonal secreting tumor cells which can produce GH or PRL but not both hormones.     

Functional classification of cell types in the growth hormone- and prolactin-secreting rat MtTW15 mammosomatotropic tumor with ultrastructural immunocytochemistry

The aim of this study was to identify the neoplastic endocrine cells which contain growth hormone (GH) and prolactin (PRL), in the MtTW₁₅ mammosomatotropic tumor, with ultrastructural immunocytochemistry. We used tumors recovered after 5 to 11 weeks of tumor development, from normal (untreated) rats and from rats treated with the progestin medroxyprogesterone acetate (MPA)—a stimulator of GH secretion in these tumors. Immunocytochemical staining was done with the peroxidase-antiperoxidase technique on ultrathin sections of tumor that had been fixed in glutaraldehyde and postfixed in osmium tetroxide. Immunospecific staining for PRL was found over small (150 nm) secretion granules, whereas staining for GH was deposited on the larger secretion granules (250 nm). Tumors from MPA-treated rats contained profuse numbers of neoplastic cells with large, GH-positive granules. Immunocytochemical staining for GH and PRL was also found in crinophagic, lysosome-like inclusions, particularly in cells that contained many secretion granules. The results support the hypothesis that GH and PRL are produced by separate neoplastic endocrine cell types in the MtTW₁₅ mammosomatotropic tumor, and demonstrate the value of ultrastructural immunocytochemical analysis for functional classification of cell types in chromophobic pituitary adenomas.     

Intracellular location of mycoplasmas in cultured cells demonstrated by immunocytochemistry and electron microscopy.

Mycoplasma fermentans (strain 'incognitus') was incubated with HeLa cells for up to 96 h. After 24 h, mycoplasma organisms were demonstrated intracellularly by immunocytochemistry using mule anti-M. fermentans antiserum and gold labelling on ultrathin sections of both Lowicryl K4M and Araldite-embedded HeLa cells, the latter being treated with hydrogen peroxide. The Araldite-embedded cells were fixed with glutaraldehyde and osmium tetroxide in the presence of ruthenium red to stain the mucopolysaccharide surface components of both the procaryotic and eucaryotic cells. Intracellular localization of some M. fermentans organisms was confirmed by exclusion of ruthenium red from their membranes. Various numbers of mycoplasma organisms were seen per cell and occasionally some were within vacuoles, the membranes of which were also unstained by ruthenium red. The PG18 strain of M. fermentans and a strain of M. hominis were also detected intracellularly using similar methodology and homologous mule or rabbit antisera. The occasional presence of both apparently normal and some denser degenerate mycoplasmas in the same cell may indicate gradual degradation by phagolysosomal digestion.     

Heterogeneity of bronchial lumen mast cells which are homogeneous by electron microscopy

Bronchial lumen mast cells were obtained by bronchial lavage from rhesus monkeys and fractionated on a discontinuous metrizamide gradient. Those cells identified as pleomorphic by standard microscopy sedimented in the higher concentrations of metrizamide, while the round cells were dispersed through all gradient fractions. Electron microscopic studies of the pleomorphic and round cells demonstrated that these were morphologically identifiable as mast cells. Although all the cells appear to be mast cells, they are heterogenous in that mast cells in the higher concentrations of metrizamide had higher concentrations of histamine. By contrast, the mast cells in the lower concentrations of metrizamide had greater biologic activity as indicated by greater release of histamine following an immunologic stimulus. The heterogeneity of bronchial lumen mast cells shown by light microscopy, histamine content, and histamine releasibility may be due to the presence of subpopulations of mast cells or changes in a single cell line reflecting differential maturation.     

Neuroendocrine carcinomas: Role of immunocytochemistry and electron microscopy

Neuroendocrine tumors occur in many sites of the body and can present significant diagnostic problems when poorly differentiated. To identify a tumor as neuroendocrine, pathologists commonly use either immunocytochemistry or electron microscopy. In this report, the various immunocytochemical reagents are reviewed along with the ultrastructural features of neuroendocrine tumors. Site-specific variations in neuroendocrine tumors are discussed. A cost-effectiveness evaluation was performed on tumors from one laboratory which showed that electron microscopy was a less expensive diagnostic modality if more than three antibodies were necessary to arrive at the correct pathological diagnosis.     

Characterization of cholinergic neurons in the rat neostriatum. A combination of choline acetyltransferase immunocytochemistry, Golgi-impregnation and electron microscopy

Immunocytochemistry with a monoclonal antibody against choline acetyltransferase has been used to characterise cholinergic neurons in the rat neostriatum. The light microscopic morphology, ultrastructure and synaptic input of these neurons was compared to that of the three types of large neuron found in Golgi preparations of the striatum. The cholinergic neurons are large and have long infrequently branching dendrites. Two of the immunoreactive neurons were also Golgi-impregnated and showed characteristics of the "classical" large neurons of the striatum. Examination in the electron microscope revealed that the synaptic input to perikarya and proximal dendrites is sparse, thus distinguishing them from another large type of neuron, found in the ventral regions of the striatum, whose dendrites and perikarya are ensheathed in synaptic boutons. It is concluded that one of the three morphologically distinguishable classes of large neuron in the striatum is a cholinergic neuron.     

Effect of spatial arrangement of the basement membrane on cultured pleomorphic adenoma cells. Study by immunocytochemistry and electron and confocal microscopy

In a cell line from human pleomorphic adenoma (AP2 cells) we studied the response of these cells to basement membrane proteins. The culture was characterized as myoepithelial-like by transmission electron microscopy and immunocytochemistry. AP2 cells were grown in contact with a reconstituted basement membrane (Matrigel). Cells grown on Matrigel showed conspicuous phenotypic alterations, depending on how the substrate was applied. Cells grown on the top of Matrigel developed a dendritic phenotype, exhibiting thin, long and intercommunicating cytoplasmic extensions resembling normal myoepithelial cells. Cells grown inside Matrigel formed multi-layered clusters. Light, confocal and transmission electron microscopy showed that these clusters were formed by double-layered epithelioid cells delimiting luminal spaces. The cells facing the lumen were cuboidal, showing microvilli at the apical plasmalemmal and junctional complexes. The spatial arrangement of basement membrane is a key modulator of morphogenetic changes and cytodifferentiation of tumour myoepithelial cell lineage in culture.     

Characterization of glomeruli by immunohistochemistry and electron microscopy in a case of Wilms' tumor

A patient with an abdominal mass was found to have a Wilms' tumor. Light microscopic examination showed that, in addition to blastema, stroma, and tubules, the tumor contained an unusually large number of glomeruli. Glomerular basement membranes stained positively by immunohistochemistry for laminin, collagen type IV, fibronectin, and Goodpasture antigen. Staining for Goodpasture antigen was seen only after acid-urea treatment, which was similar to findings in fetal and infant glomeruli. Glomerular cells stained positively for actin, myosin, and desmin, but there was no staining for factor VIII or Ulex europaeus agglutinin I, indicating an absence of endothelial cells. These findings were supported by electron microscopy, which showed basement membrane material, visceral epithelial cells, and mesenchymal cells (presumably primitive mesangial cells) in glomeruli, but no patent capillaries or capillary endothelium. Hence, the glomeruli in this case of Wilms' tumor were immature and also showed aberrant glomerulogenesis.     

Structural studies of the RNA component of the poliovirus replication complex. II. Characterization by electron microscopy and autoradiography.

The endogenous RNA component of the purified poliovirus replication complex was characterized in the electron microscope after cytochrome spreading. This RNA species has a double-stranded RNA core equal in length to poliovirus replicative form RNA, with 0 to 6 single-stranded tails per molecule. DNase I, RNase, and base treatment confirmed the double-stranded RNA nature of these molecules, which are not observed in extracts from uninfected or infected, guanidine-inhibited cells. Electron microscope autoradiography verified that these double-stranded RNA structures are the site of in vitro and in vivo viral RNA synthesis. After RNA synthesis in vitro, the double-stranded core is unchanged, but the number of tails decreases and the branch points are localized towards the ends of molecules. These results demonstrate that the RNA component of the active purified replication complex is analogous to replicative intermediate RNA and favor the double-stranded model for the in vivo poliovirus replication complex.     

Characterization of four melanoma cell lines with electron microscopy, immunocytochemistry, cytogenetics, flow cytometry, and southern analysis.

Four cell lines established from human metastatic malignant melanoma, derived from four patients, were analyzed. Ultrastructurally and immunocytochemically, the cultured tumor cells had retained characteristic features of melanocytes and of the primary malignant melanomas. The genetic stability was investigated by repeated flow-cytometric and cytogenetic analyses over 24 months of continuous cultivation. The DNA indices ranged from 1.7 to 2.1 and were stable during the entire period. The same was true for the karyotypes, which had modal numbers ranging from 50 to 84. The most common types of abnormalities were: isochromosomes i(1q), i(9q), translocations (1;17) and (3;6), and other aberrations (1p+,4p+,5p+,11p+,11q-,11q+). Abnormalities involving chromosome 1 were present in all cell lines, but loss of genetic material from chromosome 1p was demonstrated in only one of four cell lines when tested by the Southern blotting technique using a lambda MS1 probe.     

Role of immunocytochemistry,electron microscopy,and DNA analysis in fine-needle aspiration biopsy diagnosis of Wilms' tumor

We reviewed the cytologic features and results of ancillary studies in eight fine-needle aspiration biopsies (FNAB) performed by posterior approach in 8 patients with unresectable Wilms' tumor (WT). Chemotherapy was given following the FNAB diagnosis of WT, which was confirmed subsequently by histologic examination of surgically resected specimens. Indications for FNAB included: unresectable tumor, bilateral disease, initial presentation with metastatic disease, uncertainty regarding tumor site, and documentation of recurrence. Cytologic examination revealed blastemal cells (8/8 aspirates), spindle cells (3/8 aspirates), and epithelial differentiation or tubules (3/8 aspirates). There was no cytologic evidence of anaplasia in any of the cases. Immunocytochemical studies on cell blocks and/or smears showed cytokeratin positivity in 5/8 and vimentin positivity in 5/5 of the aspirates in which these studies were performed. Focal positivity for neuron-specific enolase (NSE) was seen in 3/3 aspirates. Stains for actin and leukocyte-common antigen were negative (0/3 and 0/2 aspirates, respectively). DNA ploidy analysis of the aspiration material by flow cytometry revealed near-diploid populations in three aspirates. Electron microscopic findings helpful for diagnosis included: cell junctions, microvilli, flocculent basement membrane-like material, cilia, autophagolysosomes, and lack of neuroectodermal differentiation. Diagnostic morphologic pitfalls for an incorrect diagnosis of neuroblastoma included nuclear molding (all aspirates), pseudorosette formation (one aspirate), and focal NSE positivity (3/3 aspirates). None of the tumors showed anaplasia on histologic examination. Cytologic recognition of the triphasic cellular components of WT (blastemal cells, spindle cells, and epithelial cells) can be helpful for a correct diagnosis; however, in 5/8 aspirates in this study, only the blastemal component was present. In these cases, immunocytochemical stains and electron microscopy proved useful in arriving at a correct FNAB diagnosis of WT. However, NSE positivity can be a pitfall for a diagnosis of neuroblastoma if the radiologic, clinical, and other cytologic features are not clearly delineated. Presence of cytokeratin and vimentin positivity would be helpful in the diagnosis of WT in such instances. Diagn Cytopathol 1996; 14:101–107. © 1996 Wiley-Liss, Inc.     

Characterization of the functional stages of osteoclasts by enzyme histochemistry and electron microscopy.

To investigate the functional stages of osteoclasts, the ultrastructural histochemical distribution of the lysosomal enzymes [acid phosphatase (tartrate-sensitive) and neutral phosphatase], the plasma membrane enzymes [alkaline phosphatase, Ca(++)-ATPase, and alkaline ouabain-insensitive p-nitrophenylphosphatase (alkaline p-NPPase)], and the mitochondrial enzyme (cytochrome C oxidase) was evaluated in the chicken tibial metaphysis. Both active-appearing and detached (resting) osteoclasts were studied. Serial sectioning was used to identify detached osteoclasts which were present in the perivascular space. The ultrastructure of detached osteoclasts was similar to that of active osteoclasts, except for the lack of a ruffled border and clear zone, and an altered distribution pattern of small vesicles. Small vesicles were uniformly distributed in the cytoplasm of resting osteoclasts, whereas they were concentrated beneath the ruffled border of active osteoclasts. Alkaline p-NPPase, a marker enzyme for the basal ruffled border, was also apparent on the membrane of small vesicles. However, the vesicles did not possess Ca(++)-ATPase, a marker enzyme for the apical plasma membrane. These findings support the concept that small vesicles serve as a membrane reservoir for the ruffled border membrane. Pre-osteoclasts contained abundant mitochondria and lysosomes, prominent Golgi complexes, moderately developed endoplasmic reticulum, and lacked small vesicles. Pre-osteoclasts appear to fuse with osteoclasts which are attached to the bone surface, but not with detached osteoclasts. The small vesicles, from which the ruffled border arises, are absent from pre-osteoclasts, suggesting that they develop after fusion with pre-existing osteoclasts or after attachment to the bone surface. Alkaline p-NPPase appears to be a marker for differentiation of pre-osteoclasts to mature osteoclasts.     

Electron microscopy of endothelial cells in culture: II. Scanning electron microscopy and OTOTO impregnation method

Summary A method is described whereby endothelial cells are treated with several cycles of osmium and thiocarbohydrazide then critical point dried. Cells grown on Formvar-coated gold grids can be examined directly by transmission electron microscopy, cells grown on glass strips are mounted on stubs for scanning electron microscopy and are scanned without further coating with gold or palladium.     

Distribution of dengue-2 antigens by electron immunocytochemistry.

The distribution of dengue-2 antigens was studied in infected monkey kidney cells (LLC MK2) using an indirect, horseradish peroxidase-conjugated immunoglobulin technique. This procedure allowed both light and electron microscopic examination of serial-step sections of individual cells cut in a plane perpendicular to the monolayer. Both virion and nonvirion antigens were identified on the plasma membrane with this technique. A diffuse cytoplasmic reaction product was also present. The intensity and distribution of the cytoplasmic reaction product was related to disruption of the plasma membrane.     

Early senile plaques in Alzheimer's disease demonstrated by histochemistry, immunocytochemistry, and electron microscopy

To clarify early pathologic changes in Alzheimer's disease, the brains from two cases from a single family with this disease were examined. A mother who died at age 75 with severe dementia showed an abundance of typical senile plaques, neurofibrillary tangles, and cerebrovascular amyloidosis. The senile plaque and cerebrovascular amyloid were strongly immunoreactive to anti-beta protein antibody. Her son manifested erratic and bizarre behavior, and was suspected of having committed suicide at age 52. His brain weight and macroscopic observations were normal; however, Bielschowsky's silver impregnation and methenamine silver stains showed numerous argyrophilic plaque-like lesions in the neocortex. They were weakly immunolabeled by anti-beta protein antibody, but lacked any abnormal neurites of Congophilic amyloid deposits. These lesions resembled the "type 3" immunoreactive lesions (previously reported by us in Alzheimer's disease and Down's syndrome) which seem to be an early stage of senile plaque formation. These putative early plaque lesions were also examined by methenamine silver electron microscopy, and were seen to consist of loose aggregations of irregular spindle-shaped structures with a heavy deposition of silver grains, with genuine amyloid fibrils not being apparent. It is believed that the accumulation of beta-protein immunoreactive material without amyloid fibril formation might be an initial step in the development of the senile plaque, and that the son, having extensive cortical involvement with type 3 plaque lesions, demonstrated clinical manifestations of less completely developed Alzheimer's disease.     

Hürthle-cell lesions of the thyroid: a combined study using transmission electron microscopy, scanning electron microscopy, and immunocytochemistry

Hürthle cell transformation found in 2 nodular goiters, 2 cases of Hashimoto's thyroiditis, 4 follicular adenomas, 3 follicular carcinomas, 2 papillary carcinomas and 1 anaplastic carcinoma were studied by transmission electron microscopy, scanning electron microscopy and immunocytochemistry. Ultrastructural features of Hürthle cells were identical in non-neoplastic and neoplastic lesions. Cells crammed with mitochondria, showing abnormalities in size, shape and content were prominent in most cases. The presence of distinct smooth-surfaced cells interspersed with cells with many microvilli is almost a pathognomonic scanning electron microscopic feature of benign and malignant Hürthle cell lesions. Most Hürthle cells stained positively for thyroglobulin in all cases, but no immunoreactivity for CEA and calcitonin was found.