Caveats in the use of archival cell material for DNA ploidy analysis by image cytometry.

Monolayer preparation from paraffin-embedded tissue from archival biopsy specimens older than 10 years was shown in this study to be more difficult to deal with than recent biopsies. We have studied the disseminative effect of several enzymes on archival biopsies from laryngeal epithelium, as well as mechanical dissemination. Thirty minutes incubation in protease 0.5% in 37 degrees C without syringing provided the best cell yield. Hydrolysis and Schiff staining conditions were also studied. Cell loss was calculated by stereological methods and found to be high in most cases. Cell loss during various steps in monolayer production was examined and we found no proof of selective cell loss. Large variations in Feulgen-Schiff staining intensities were observed, especially in biopsy specimens older than 10 years. The use of reliable internal (intrinsic) control cells is advocated since interspecimen variations in the diploid integrated optical density (IOD) can be expected. Although DNA measurements on archival biopsy material should be treated with caution, we demonstrate that meticulous studies of the preparation procedures will make it possible to obtain reliable histograms from old, archival biopsies.

Chemical composition and morphology of welding fume particles and grinding dusts.

Elemental composition and morphology of pure manual metal arc (MMA) welding fumes, pure grinding dust, and combined fume/dust air samples were collected and determined separately under semilaboratory conditions. The base material was stainless steel. The purpose of the present study was to create a "synthetic" work situation under semilaboratory conditions by combining one grinding period and two MMA welding periods and comparing these results with results during welding in a workshop. The duty cycles of pure welding and of pure grinding were also observed. A comparison was also made between metal inert gas (MIG) and MMA welding on stainless steel as well as a nickel-rich alloy under regular conditions. The amount of collected material was determined by weighing the membrane filters before and after exposure, and the element contents were determined by atomic spectroscopy. Other transmission electron microscopy (TEM) filters were used for TEM and computer-image analysis, in which the amount of collected material and its morphological characteristics were observed. The arcing time and the consumption of filler material were estimated for different kinds of electrodes. Chemical analysis showed that the contents of manganese and total chromium were lower in grinding dust than in welding fumes. The contents of hexavalent chromium, Cr(VI), in grinding dust were undetectable. Samples collected in welding shops where concomitant grinding was performed contained about 30% less Cr(VI) than those collected under laboratory conditions during welding only. The sizes and shapes of the particles depend on the welding process and distance of collection from the plume of the fume. To compare laboratory experiments with regular welding situations, the experiment must resemble industrial welding.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic alterations of the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q in human breast carcinomas.

Fifty-nine primary breast carcinomas and 11 metastases were examined to identify genetic alterations in the tumour suppressor gene regions 3p, 11p, 13q, 17p, and 17q. Loss of heterozygosity (LOH) was frequently observed on chromosome arms 17p (p144D6 lost in 75%, pYNZ22.1 in 55%, and TP53 in 48% of the primary tumours), 13q (RBI lost in 40% of the primary tumours), and 17q (pRMU3 lost in 35%, pTHH59 in 29%, and NM23HI in 26% of the primary tumours). Loss of all the markers except p144D6 was observed even more frequently in the metastases. Pairwise comparisons for concordance of allele losses on 17p indicated that there might be two genes on 17p implicated in breast cancer development; the TP53 gene and a gene located close to the p144D6 and pYNZ22.1 markers. LOH of the RBI gene was associated with LOH of pYNZ22.1 and p144D6, but not with LOH of TP53. LOH of RBI and TP53 was associated with occurrence of ductal carcinomas, RBI and p144D6 losses with tumour size, and p144D6 losses with positive node status as well. LOH of TP53 and the three 17q markers NM23HI, pTHH59, and pRMU3 was most frequently observed in tumours from postmenopausal women. p144D6 losses occurred most frequently in progesterone receptor-negative tumours, whereas pTHH59 losses occurred most frequently in oestrogen receptor-negative tumours. LOH of the investigated loci was not associated with ERBB2 protooncogene amplification, with positive family history of breast cancer, or with survival.

Subunit assembly and active site location in the structure of glutamate dehydrogenase.

The three-dimensional crystal structure of the NAD(+)-linked glutamate dehydrogenase from Clostridium symbiosum has been solved to 1.96 A resolution by a combination of isomorphous replacement and molecular averaging and refined to a conventional crystallographic R factor of 0.227. Each subunit in this multimeric enzyme is organised into two domains separated by a deep cleft. One domain directs the self-assembly of the molecule into a hexameric oligomer with 32 symmetry. The other domain is structurally similar to the classical dinucleotide binding fold but with the direction of one of the strands reversed. Difference Fourier analysis on the binary complex of the enzyme with NAD+ shows that the dinucleotide is bound in an extended conformation with the nicotinamide moiety deep in the cleft between the two domains. Hydrogen bonds between the carboxyamide group of the nicotinamide ring and the side chains of T209 and N240, residues conserved in all hexameric GDH sequences, provide a positive selection for the syn conformer of this ring. This results in a molecular arrangement in which the A face of the nicotinamide ring is buried against the enzyme surface and the B face is exposed, adjacent to a striking cluster of conserved residues including K89, K113, and K125. Modeling studies, correlated with chemical modification data, have implicated this region as the glutamate/2-oxoglutarate binding site and provide an explanation at the molecular level for the B type stereospecificity of the hydride transfer of GDH during the catalytic cycle.

Low-grade cribriform ductal carcinoma in situ of the breast. Fine needle aspiration cytology in three cases.

This paper presents the cytologic features of fine needle aspiration biopsy specimens from three cases of ductal carcinoma in situ characterized by small and uniform tumor cells growing in a predominantly cribriform pattern without comedo necrosis (low-grade cribriform ductal carcinoma in situ). On cytology, most of the tumor cells were clustered in three-dimensional ductal structures. Occasionally in the clusters the tumor cells were seen bordering central lumina, quite similar to the architecture in histology. A few single tumor cells and no myoepithelium were seen. The background was clear or slightly hemorrhagic, without necrosis. The tumor cells were uniform and had a cylindroid shape, with round or oval nuclei. Morphometrically the mean largest nuclear diameter was 1.5-1.6 times that of a red blood cell. The chromatin was finely granular, with a minute nucleolus and slight condensation along the nuclear membrane. In cut sections all three tumors showed strong immunoreactivity for neuron-specific enolase. Unless the cribriform growth pattern is recognized in the smear, the cytologic diagnosis of this entity is difficult.

Comparison between flow cytometry and image cytometry in ploidy distribution assessments in gynecologic cancer.

The DNA content in 37 tumors from 34 women with gynecological cancer was measured by flow cytometry (FCM) and interactive image cytometry (ICM). Agreement was obtained in 81% of cases as regards ploidy levels, but seven tumors (19%) showed different ploidies. Of these, five were classified as diploid by FCM but either aneuploid (three cases) or polyploid (two cases) by ICM. Two other tumors were aneuploid by ICM but polyploid (one case) and unclassifiable (one case) by FCM. All tumors classified as aneuploid by FCM were also aneuploid by ICM, and all tumors classified diploid by ICM were also diploid by FCM. Of six patients whose tumors were classified as euploid (five diploid and one polyploid) by FCM but classified as aneuploid by ICM, five relapsed, and three of these have died of disease. On the basis of these findings, it is concluded that ICM must be performed in cases classified as diploid by FCM to ensure that small subpopulations of aneuploid tumor cells are not overlooked.

Chromosome 13 alterations in osteosarcoma cell lines derived from a patient with previous retinoblastoma.

Various sublines of cells established from an osteosarcoma that developed in a patient (O.H.) with previous bilateral retinoblastoma were examined for different restriction fragment-length polymorphisms of chromosome 13q, as well as for rearrangements of the retinoblastoma gene using a cDNA probe. The independently established sublines were used to help separate primary and secondary events taking place in tumorigenesis of the osteosarcoma of this patient. Information from the present DNA analysis, taken together with data from cytogenetic and enzymatic studies on chromosome 13 in the cell lines, revealed both common and distinct genetic changes on chromosome 13q. The common changes may indicate the nature of the first and second mutational events in the development of the osteosarcoma. The first, constitutional cancer predisposing mutation seemed to be a base mutation or a small deletion/insertion, and the second event involved a deletion of a larger part of the long arm of chromosome 13. The distinct genetic changes included other deletion and duplication events of chromosome 13q. The existence of multiple sublines with different genetic constitutions provides improved possibilities for gaining insight into the nature of the genetic lesions leading to tumor formation, as these may reflect the clonal variation present in the primary tumor. We also demonstrate the difficulty of inferring from single tumor cell isolates to properties of the primary tumor.

Subcellular localization, redistribution and photobleaching of sulfonated aluminum phthalocyanines in a human melanoma cell line.

Intracellular localization, intracellular translocation and photobleaching following non-lethal laser microirradiation of the fluorescing derivatives of sulfonated aluminum phthalocyanines (Al-PcSn, n = 1-4) in a human melanoma cell line (LOX) were studied by means of confocal laser scanning microscopy (LSM) and image processing. Use of confocal microscopy allowed 3-dimensional information to be obtained. Both Al-PcS1 and Al-PcS2 localized diffusely in the cytoplasm of the cells, while Al-PcS3 and Al-PcS4 exhibited a granular pattern in the extranuclear fraction of the cells. None of the Al-PcSn family was observed in the nuclei of the cells except that a small fraction of fluorescence was occasionally detected in nuclei of some cells treated with Al-PcS1 and Al-PcS2. Furthermore, exactly the same granular localization patterns and positions in the same cells were found after incubation initially with Al-PcS3 (or Al-PcS4) followed by acridine orange (AO) which emits red fluorescence from lysosomes of cells. Thus, the granular fluorescence of Al-PcS3 and Al-PcS4 is confined to the lysosomes of the LOX cells. Non-lethal laser exposure of cells incubated with high concentrations of the 2 dyes resulted in a translocation of the dyes from the lysosomes to the whole cytoplasm and an increase in total intracellular fluorescence intensity. Finally, a small fraction of the dyes localized into the nuclei of the cells. The laser exposure of cells incubated with low concentrations of the lysosomally localized dyes resulted in an increase in the intracellular fluorescence intensity with no translocation of the dyes. Under all conditions, high laser exposure resulted in a decrease in the total intracellular fluorescence intensity.

Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy.

By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as TPPS1 (tetraphenylporphine monosulfonate), TPPS2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), AlPCS1 (aluminium phthalocyanine monosulfonate) and AlPCS2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h postinjection and the fluorescence was still observable 120 h postinjection. The more hydrophilic dyes such as TPPS3 (tetraphenylporphine trisulfonates), TPPS4 (tetraphenylporphine tetrasulfonates), and AlPCS4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h postinjection. 120 h postinjection no significant fluorescence of these dyes could be seen in the tumors. P-II (Photofrin II), 3-THPP [tetra(3-hydroxyphenyl)porphine], TPPS2o (tetraphenylporphine disulfonates with the sulfonate groups on opposite rings) and AlPCS3 (aluminum phthalocyanine trisulfonates) had a combined localization pattern, i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern, although some fluorescence could be seen in the tumorous stroma. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.

Location of P-II and AlPCS4 in human tumor LOX in vitro and in vivo by means of computer-enhanced video fluorescence microscopy.

The patterns of in vitro intracellular and in vivo intratumoral localization of Photofrin II (P-II) and aluminum phthalocyanine tetrasulfonate (AlPCS4) in human melanoma LOX were studied by means of computer-enhanced video fluorescence microscopy (CEVFM). The hydrophobic drug P-II localized diffusely in the perinuclear fraction of the cytoplasm of the LOX cells cultivated in vitro. Light exposure did not result in any observable change in the localization pattern. The hydrophilic dye AlPCS4 was distributed as granular and grain patterns in the cytoplasm before light exposure, in exactly the same pattern as that of acridine orange incubated in the same cells, which is known to emit red fluorescence from lysosomes, thus indicating that AlPCS4 was also primarily localized in the lysosomes of the LOX cells. After light exposure the distribution of the intracellular AlPCS4 fluorescence was altered and the intensity increased. In vivo, P-II had a combined cellular localization pattern (i.e. a strongly cytoplasmic membrane-localizing pattern and a weakly intracellular distribution pattern) and an extracellular distribution pattern in the tumor tissue, while the AlPCS4 fluorescence was seen mainly in the stroma of the tumor. The total fluorescence intensity of P-II and AlPCS4 in the LOX tumor tissue at different times after injection was quantitatively determined by means of CEVFM.

Localization of potent photosensitizers in human tumor LOX by means of laser scanning microscopy.

By means of laser scanning fluorescence microscopy the intratumoral localization patterns of several photosensitizers in LOX tumors in nude mice were studied. Lipophilic dyes such as P-II (Photofrin II), 3-THPP tetra(3-hydroxyphenyl)porphin, TPPS1 (tetraphenylporphine monosulfonate), TPPS2a (tetraphenylporphine disulfonates with the sulfonate groups on adjacent rings), A1PCS1 (aluminium phthalocyanine monosulfonate) and A1PCS2 (aluminium phthalocyanine disulfonates) localized mainly in tumor cells. The fluorescence intensity of these dyes increased from 4 h to 48 h post-injection and the fluorescence was still observable 120 h post-injection. The more hydrophilic dyes such as TPPS2o (tetraphenylporphine disulfonates with the sulfonates groups on opposite rings), TPPS3 (tetraphenylporphine trisulfoantes), TPPS4 (tetraphenylporphine tetrasulfonates), A1PCS3 (aluminium phthalocyanine trisulfonates) and A1PCS4 (aluminium phthalocyanine tetrasulfonates) localized mainly extracellularly in the tumorous stroma. The fluorescence intensity of these dyes decreased from 4 h to 48 h post-injection. 120 h post-injection no significant fluorescence of these dyes could be seen in the tumors. The data are discussed in relation to what is known about the in vivo photosensitizing efficiency of some of the dyes.

The selection of thin epithelial layers: a method for single cell preparations of very small biopsies from the vocal cords.

Precancerous lesions in biopsy material very rarely contain more than a thin epithelial layer of atypical cells overlaying stromal parts. This makes it difficult to produce cell monolayers for cytometry from archival material and necessitates a technique which excludes the stromal areas. We have developed a method by which it is possible to separate epithelial layers from the stroma with a thickness of 100-200 microns, using fluorescence microscopy and dissection under a stereo microscope. The stroma can be used as an internal reference in IOD measurements. Our method insures maximum control when dealing with very small lesions of interest (down to 0.02 mm2) that have to be separated from the rest of the biopsy. The method has been applied to normal, dysplastic and cancerous epithelial parts of biopsies of the vocal chords.

Three-dimensional structure of the surface layer of Wolinella recta.

The three-dimensional structure of the crystalline surface layer (S-layer) of Wolinella recta ATCC 33238T, a gram-negative, anaerobic periodontopathogen, was determined to 3.8 nm resolution by electron microscopy and digital image processing. The S-layer protein is closely associated with the outer bacterial membrane, and shows p6 symmetry with lattice spacing and thickness of 21 nm and 15 nm, respectively. The funnel-shaped subunits consist of 6 heavy domains located round a common base at the sixfold axis, and communicate with the adjacent subunits through a lighter domain at the threefold axis (M6C3 arrangement).

Image analysis of endodontic radiographs: digital subtraction and quantitative densitometry.

In this study computerized image analysis procedures were applied to endodontic radiographs. Kontron IBAS 2000 is a commercially available image analysis system with processing routines applicable to radiograph digitizing and transformations. The system was evaluated for: its ability to harmonize blackening and contrast in endodontic radiographs; its ability to compensate for angulation distortion of sequential exposures of individual teeth; its potential for application of digital subtraction methods; and its use in automated gray-level analyses of diseased and healthy bone areas in endodontic radiographs. The Kontron IBAS 2000 system proved suitable for all applications. However, the specificity of the subtraction procedure was limited by some inherent problems in the harmonization of blackening and in the subtraction process itself. On the other hand, automated gray level measurements proved to be a robust method for unbiased and quantitative assessment of healing of apical periodontitis.

Characterization of the morphological properties of welding fume particles by transmission electron microscopy and digital image analysis.

The morphological characteristics of welding fume particles have been determined using transmission electron microscopy (TEM) and automatic image analysis (AIA). Two personal samples and one background sample were collected using a new, easy to handle sampling method, during tungsten inert gas (TIG) and manual metal arc (MMA) welding on Inconel in the same shop. The collection method gave samples which were suitable for TEM and AIA. Electron micrographs were taken in a transmission electron microscope and further analyzed using an image analysis unit. Aggregates composed of many individual particles were analyzed both for the parameters of the aggregate and for the parameters of the individual particles by using an algorithm based on a grain boundary reconstruction technique. The morphological parameters allowed the welding fume's particulate matter to be divided into three types - here called small, medium, and large - with a somewhat unclear distinction between medium and large. Medium and large particles occur either as individual particles or as clusters of approximately spherical particles with average diameters of 0.07 and 0.15 microm, respectively. Small particles occur almost exclusively as long chains or lace-like structures of aggregates of particles, often in the range of 5-10 microm. The aggregates have an average projected area of 2.6 x 10-3 microm2 and are composed of several hundred individual particles.

Ultrastructural and electron immunohistochemical features of medullary thyroid carcinoma.

An ultrastructural study, both morphological and immunohistochemical, has been carried out on eight thyroglobulin-positive and nine thyroglobulin-negative medullary carcinomas of the thyroid. The morphometric analysis of granule size showed that all tumours contained cells with small granules and cells with medium size granules, whereas eight tumours had additional cells with large granules. The small granules had an electron dense core, while the medium and large sized granules were both pale-cored and dense-cored. The cells with small, medium or large secretory granules were all immunoreactive for calcitonin and CGRP. No ultrastructural differences were observed between thyroglobulin-positive and thyroglobulin-negative cases of medullary carcinoma of the thyroid.

Characterization of chromatin structure by image analysis--a method for the assessment of changes in chromatin organization.

This paper describes an image analysis technique for the assessment of changes in chromatin organization in ultrathin tissue sections. Transmission electron micrographs of tissue sections were analyzed by a SEM-IPS image processing unit (Kontron) at a total magnification of 7,500. The boundaries of each nucleus and each nucleolus were defined interactively, and the grey level threshold between heterochromatin and euchromatin was determined. The grey level distribution and the total area of each nucleus was measured. In addition, the total area, number, and individual areas of heterochromatin particles and nuclei were measured. Based on these measurements, a number of different variables have been defined, and several of these have proved to discriminate between normal and malignant cells.

Two types of mitochondrial crystals in diseased human skeletal muscle fibers.

Mitochondrial crystalline inclusions, frequently found in mitochondrial myopathies, were analyzed by crystallographic techniques and computer-aided image processing. It could be shown that these structures were real crystals. There are two distinct types of crystal, which can be distinguished by shape, size, and pattern. So-called type I crystals are usually present in the intracristal space, whereas the type II crystals are preferentially located in the intermembrane space between outer and inner mitochondrial membranes. The unit cell dimensions were found to be 38 x 34 x 8 nm for the type I crystals and 20 x 17 x 8 nm for the type II crystals. These results strongly suggest that the crystals are composed of macromolecules, presumably proteins. Arguments are presented that indicate that type I crystals occur only in type 1 muscle fibers and type II crystals in type 2 muscle fibers.

The crystal structure of glutamate dehydrogenase from Clostridium symbiosum at 0.6 nm resolution.

The structure of glutamate dehydrogenase from Clostridium symbiosum has been solved by single-crystal X-ray-diffraction studies at 0.6 nm resolution by using a combination of isomorphous replacement and molecular averaging. The electron-density map reveals that this glutamate dehydrogenase is a hexameric oligomer, arranged in 32 symmetry, of cylindrical appearance and dimensions, of length 10.8 nm and radius 4.4 nm. From an analysis of this map each subunit appears to contain some 55% alpha-helix and is organized into two distinct globular domains separated by a deep cleft. The subunits associate using the domain closest to the 32-symmetry point, making intimate contacts around the threefold and twofold interfaces. The second domain shows structural homology to the NAD-binding domain of other dehydrogenases, and difference Fourier analysis has shown that the NAD is bound in both a structurally equivalent position and a similar conformation to that observed for those related enzymes.