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PubMed1998

[Flow cytometric DNA analysis and MR imaging of salivary gland tumors].

We correlated the histopathology of 32 cases of salivary gland tumors with MR imaging and flow cytometric DNA analysis. All malignant tumors were invasive and/or had an ill-defined margin. Fifty-seven percent of the pleomorphic adenomas were shown as a high signal intensity area on T2 weighted images. Fifty-six percent of Warthin tumors were shown as a low or iso signal intensity area on T2 weighted images associated with the cystic portion. Sixty-seven percent of malignant tumors were characterized by DNA aneuploidy and/or a total percentage of the S-phase fraction plus the G2 + M fraction higher than 10%. It was less than 10% and the Ki-67 positive fraction was higher than 20% in all pleomorphic adenomas. The Ki-67 positive fraction was less than 20% in 78% of Warthin tumors. The histopathology of all malignant tumors, 86% of the pleomorphic adenomas, and 89% of Warthin tumors was correctly predicted by the combination of MR images and flow cytometry. We suggest that the MR images combined with flow cytometric analysis of fine needle aspiration-derived materials is useful in the pre-operative histopathologic diagnosis of salivary gland tumors.

Adenolymphoma↗
PubMed2006

Extracting quantitative information from tissue--an industrial perspective.

The focus of this article is to provide an overview of the current technologies for the pharmaceutical and biotech industry. Disease processes express themselves in the functional and structural disturbance of cellular systems. Cells and their metabolites constitute the building blocks of tissues and entire organisms. Studying the spatial and temporal phenotype of disease processes in tissues at the cellular level reveals a multitude of information about the progress and status of a disease. Detailed exploration of tissues by slide-based cytometry is an important source of information about disease processes. Technological and analytical advances allow us to shed a new light on tissues and to come to a better understanding of the complexity of disease processes. Dealing with complex multidimensional datasets from tissue samples requires an advanced approach to image processing and data management. The increase in computing power and the continuing research into imaging algorithms allow us to improve the exploration of the data content of tissues.

Animals↗
PubMed1995

Flow and image cytometric DNA measurements in fine needle aspiration samples of prostatic neoplasms.

OBJECTIVE: To compare the DNA content measured by flow cytometry (FCM) and image analysis (IA) from prostatic fine needle aspiration (FNA) samples. STUDY DESIGN: A total of 48 samples were studied. FCM was performed on propidium iodide-stained nuclei according to the Vindelov method, and image analysis was performed on Feulgen-stained slides. Positive FNA results were grade (1-3) and compared with Gleason grades. RESULTS: Aneuploidy was closely related to positive FNA results (P < .01). DNA histograms were found to be aneuploid in all grade 3 carcinomas (n = 13) by IA and in 11 cases (84.6%) by FCM. Grade 2 carcinomas (n = 9) were found to be aneuploid with both methods. In grade 1 carcinomas (n = 10), 2 cases exhibited IA aneuploid profiles, whereas all FCM cases were diploid. Aneuploid profiles were more often associated with high Gleason scores than were diploid ones (P < .01). Among the 16 patients with negative FNA results, two cases had tetraploid DNA profiles related to contaminating seminal vesicle cells. The difference in DNA measurements reached 10.4% but was not statistically significant. CONCLUSION: These findings show that the two methods, as applied to prostatic FNA samples, give comparable results and that seminal vesicle cells may be responsible for false tetraploid profiles.

Biopsy, Needle↗
PubMed1996

Cytometry with DAPI-stained tumor imprints. A reliable tool for improved intraoperative analysis of lung neoplasms.

OBJECTIVE: To determine the feasibility and potential use of application of 4'-6-diamidino-2-phenylindole dihydrochloride-stained imprints taken from primary lung carcinomas for intraoperative frozen section service. STUDY DESIGN: Tumor imprints taken from intraoperative sections were stained with the fluorochrome DAPI, and the integrated nuclear fluorescence (INF) was measured using an automated image analyzing system. The 40 cases studied comprised all major lung carcinoma types as well as intrapulmonary metastases. The measured features included percentage of tumor cells > 3C and > 5C, S-phase-related tumor cell fraction, 2 CV standard deviation and INF entropy. RESULTS: Test series with imprints of tumor-free lymph nodes revealed a minimum staining time of five minutes at 40 degrees C that led to reproducible staining intensities and size of the nuclear area. No significant differences were apparent in comparison to concomitantly performed measurements of the integrated optical density of "classical" Feulgen-stained specimens for each case. The fluorometric results could be submitted to the surgeon with 15-18 minutes (20 maximum). CONCLUSION: The data indicate that cytometric DNA measurements can be performed intraoperatively with reliable quality within time schedules acceptable for intraoperative surgical strategies.

DNA, Neoplasm↗
PubMed2005

Nephrogenic adenoma of the urinary bladder.

INTRODUCTION: The nephrogenic adenoma (NA) is a benign metaplastic lesion of the urothelium and is attributed to chronic irritation of the mucosa, by injury, infection, stone disease or intravesical instrumentations. We present our experience on this morbid entity, its clinical appearance in the urinary bladder, its frequency and relapses. Furthermore we reviewed the related recent literature and focused on its potential to neoplastic degeneration and the value of the new diagnostic modalities. PATIENTS AND METHODS: Four patients with NA of the urinary bladder are presented. The papilloid or polypoid formations observed by the cystoscopy were identified after the TUR, as NA of the urinary bladder. Their mean follow-up was 3.5 years. RESULTS: Remission of the symptoms was observed after TUR in all patients. Three out of four patients presented 1-7 relapses, while in one case, after seven NA relapses, a urothelial carcinoma of the bladder was diagnosed. CONCLUSIONS: Unlike histological features, the clinical - endoscopic characteristics of NA are non-specific. Even if it is not definitely considered like a premalignant condition, NA has to be followed up frequently and long lasting, because of its high recurrence rate. The combination of Cytology, Flow cytometry, DNA image analysis and Fluorescence in situ hybridization of bladder washings or voided urine, are of high value in monitoring NA of the urothelium.

Adenoma↗
PubMed1998

Static cytometry and neural networks in the discrimination of lower urinary system lesions.

OBJECTIVES: To investigate the potential of morphometry and artificial intelligence tools for the discrimination of benign and malignant lower urinary system lesions. METHODS: The study group included 50 cases of lithiasis, 61 cases of inflammation, 99 cases of benign prostatic hyperplasia, 5 cases of in situ carcinoma, 71 cases of grade I transitional cell carcinoma of the bladder (TCCB), and 184 cases of grade II and grade III TCCB. Images of voided urine smears stained by the Giemsa technique were analyzed by a custom image analysis system. The analysis gave a data set of features from 45,452 cells. A learning vector quantizer (LVQ)-type neural network (NN) was used to discriminate benign from malignant cells on the basis of the extracted morphometric and textural features. The data from 13,636 randomly selected cells were used as a training set and the data from the remaining 31,816 cells made up the test set. Similarly, in an attempt to discriminate at the patient level, 30% of the cases randomly selected were used to train an LVQ NN and the remaining 329 cases were used for the test. RESULTS: The application of the LVQ NN enabled the correct classification of 95.42% of the benign cells and 86.75% of the malignant cells, giving an overall accuracy rate of 90.63%. At the patient level, the LVQ NN enabled the correct classification of 100% of benign cases and 95.6% of malignant cases, giving an overall accuracy rate of 97.57%. CONCLUSIONS: NNs combined with image analysis offer useful information in the discrimination of benign and malignant cells and lesions of the lower urinary system.

Diagnosis, Differential↗
PubMed1989

Discrepancies in ploidy determination due to specimen sampling errors.

Two techniques are described to enhance the detection of low frequency aneuploid cells in automated cell analysis. One method concerns a cell preparation technique; the other is focused on specific cell selection at the measurement level. The cell preparation method has been designed to select and process the tumour areas in paraffin blocks and can be used for image as well as for flow cytometry. The technique uses incident fluorescence microscopy for visual inspection of the surface of the fluorescently stained tissue block to select the specific tumour parts. Using image cytometry, it is shown that in tissue sections with very small tumour foci and many normal cells, aneuploidy could only be detected after enrichment of the cell sample with the specifically selected areas. The cell selection at the measurement level is directed towards detection of low frequency aneuploid cells on microscope slides using the specific capacities of LEYTAS (Leyden Television Analysis System). With this system, cells of interest can be selected by means of minimum size and intensity thresholds. In addition to measurement of the total cell population, all cells above a minimum DNA value can thus be specifically selected and measured. The advantage of both enrichment techniques is the possibility to detect and measure aneuploid cell lines in cases where normal, diploid cells dominate the paraffin tissue.

Aneuploidy↗
PubMed2002

Application of laser scanning cytometry followed by epifluorescent and differential interference contrast microscopy for the detection and enumeration of Cryptosporidium and Giardia in raw and potable waters.

AIMS: The main goal of this study was to validate a new laser scanning cytometry method (ChemScanRDI) that couples immunofluorescence detection with differential interference contrast (DIC) confirmation, against manual microscopic enumeration of Giardia and Cryptosporidium (oo)cysts. This study also assessed the basic performance of the new Association Française de Normalisation (AFNOR) NF T 90-455 method for Giardia and Cryptosporidium (oo)cyst enumeration with respect to (oo)cyst yield, linearity, repeatability, influence of turbidity and detection limit in raw and potable waters. METHODS AND RESULTS: The new standard method relies on cartridge (Envirocheck) filtration, immunomagnetic separation purification, immunofluorescence staining and detection followed by DIC confirmation. The recovery was 30-50% for both parasites at seeding levels from 30 to 230 (oo)cysts. The method is linear from 0 to around 400 seeded (oo)cysts and the yield does not significantly vary for turbidity levels from 10 to 40 Formazin Nephelometric Units (FNU). The results were obtained using manual microscopic enumeration of the (oo)cysts. The ChemScanRDI yielded counts that were at least equivalent to those obtained using manual microscopy for both parasites in raw and potable water concentrates, for seeding levels of 10-300 or 10-100, respectively. The purification and labelling method proposed by the supplier of theChemScanRDI (Chemunex) reached very similar recoveries to the AFNOR protocol (70-86% in both cases). CONCLUSIONS: Laser scanning cytometry can be used as a more standardized alternative to manual enumeration as part of the new AFNOR standard method. SIGNIFICANCE AND IMPACT OF THE STUDY: By using laser scanning cytometry instead of manual microscopy, laboratories could circumvent the limitations of manual microscopy, namely: low sample throughput, operator subjectivity and operator fatigue. The study further supports the drive to incorporate laser scanning cytometry in the standard methods for Giardia and Cryptosporidium enumeration.

Animals↗
PubMed2003

Cytometry of cell cycle regulatory proteins.

Immunocytochemical detection of cyclins, inhibitors of cyclin-dependent kinases and other cell cycle regulatory proteins, combined with analysis of cell cycle position (DNA content) by multiparameter cytometry offers unique analytical possibilities. This review focuses on applications of the methodology that yield information on the abundance, modification or interactions of these proteins in relation to cell cycle progression that cannot be obtained by other methods. Particular attention is given to the use of multiparameter cytometry in analysis of the mechanism by which antitumor drugs affect cell cycle progression and induce cell cycle phase-specific apoptosis.

Animals↗
PubMed2003

Fetal nucleated red blood cells in peripheral blood of pregnant women: detection and determination of location on a slide using laser-scanning cytometry.

OBJECTIVE: The purpose of the study was to assess the feasibility of analysis of fetal nucleated red blood cells (NRBC) present in the maternal circulation by laser-scanning cytometry. METHODS: CD71-positive cells were obtained by magnetic cell sorting of peripheral blood of pregnant women after density centrifugation. Immunofluorescence for the Hbgamma-chain was combined with fluorescent staining of DNA (TO-PRO-3) and fluorescence in situ hybridization (FISH) with a Y-chromosome specific probe. The cells were scanned on a slide using a laser-scanning cytometer (LSC). Events double positive for Hbgamma and TO-PRO-3 were relocated and their morphology and FISH reactivity were visually assessed. Determination of male fetal sex with LSC was compared with findings from amniocentesis. RESULTS: In 8/15 pregnancies with male fetuses and in 0/9 with females (apart from one case with a male/female twin pregnancy), we detected Y-chromosome-positive NRBC. In pregnancies with female fetuses, Y-chromosome-positive cells other than NRBC were found in all women who had previously given birth to male babies, whereas women with no abortion and no male babies in their history did not present with Y-chromosome-positive non-NRBC. CONCLUSION: On the basis of automatic relocation of once-defined cells of fetal origin from the current pregnancy, laser-scanning cytometry is likely to facilitate repeated (poly-)FISH analysis and single-cell PCR for noninvasive prenatal diagnosis.

Adult↗
PubMed2002

Applicability of laser scanning cytometry to study paediatric alveolar macrophages.

Laser scanning cytometry (LSC) generates quantitative information on immune receptor expression from cells cytocentrifuged onto a microscope slide. In children, the description of developmental changes in immune receptor expression on alveolar macrophages (AM) has been limited by the small number of cells recovered by bronchoalveolar lavage (BAL). The applicability of LSC to the study of AM from normal children was therefore assessed. AM were obtained by BAL of normal children following intubation prior to elective surgery. The ability of LSC to identify the cytoplasm of AM was assessed using either: 1) autofluorescence; 2) forward scatter; 3) nuclear staining with propidium iodide; or 4) a fluorescent-labelled monoclonal antibody to CD68, a pan-macrophage antigen. LSC could only reliably identify individual AM when stained with CD68. The sensitivity for detecting single whole AM using CD68 was 0.97 and the positive predictive value was 0.88, respectively, with excellent repeatability. In addition, a range of immunofluorescence parameters were generated for CD68. It is concluded that laser scanning cytometry is suited to the study of immune receptor expression from small numbers of paediatric alveolar macrophages, when CD68 is used for cell identification.

Antigens, CD↗
PubMed1998

Flow cytometric and quantitative image cell analysis of DNA ploidy in renal chromophobe cell carcinoma.

Flow cytometry and quantitative image cell analyses were performed on a series of 31 chromophobe cell carcinoma and the findings were compared with those from 14 clear cell carcinomas. Thirty of 31 chromophobe cell carcinomas had significant hypodiploid cell clones with both techniques. By contrast, none of the 14 clear cell carcinomas was hypodiploid. Using quantitative image cell analyses, four groups of nuclei with hypodiploid, diploid, hyperdiploid, and tetraploid/hypertetraploid DNA patterns were identified, and their relative proportions were compared. In most of the chromophobe cell carcinomas, the predominant nuclear pattern was hypodiploid, and in clear cell carcinoma, diploid nuclei were most frequent. The number of binucleated cells in chromophobe cell carcinomas varied from 1.40% to 23% (mean, 10.8%) whereas, in clear cell carcinoma, these varied from 0.4% to 9.2% (mean, 2.5%). Evaluation of DNA content of double hypodiploid nuclei in chromophobe cell carcinomas showed that their combined DNA content was essentially similar to that of single hyperdiploid nuclei, thus suggesting polyploidy resulting from the fusion of these nuclei. Polyploidy may indeed be the basis for nuclear heterogeneity in chromophobe cell carcinoma. Scatterplots generated by plotting nuclear DNA mass against nuclear area produced patterns that were distinctive for the two types of carcinoma. We believe that the comparative findings in this study provide a comprehensive understanding of the ploidy status of chromophobe carcinoma and that these findings may be used as supportive evidence for establishing the diagnosis of chromophobe cell carcinoma.

Adult↗
PubMed1987

Quantitative description of nuclear morphology in assessing resistance of sarcoma 180 to adriamycin.

Resistance to adriamycin generally is explained through changes of cell/drug interactions that possibly reflect structural alterations of intracellular targets. One of the main targets of adriamycin is believed to be nuclear chromatin. In order to recognize chromatin alterations, we studied cell nuclei morphology and chromatin structure by means of digital image analysis. The studies were performed in both adriamycin-sensitive and -resistant Sarcoma 180 cell lines which were cultured under growth-stimulated and nonstimulated conditions. Using specially developed methods, we extracted parameters characterizing geometrical, optical, and structural properties of the cell nuclei from light microscopical images. The latter parameters concerned microscopical appearances of condensed chromatin and were described by features of high-optical-density regions. The results demonstrated that the quantitative criteria applied enabled the discrimination of sensitive and resistant cells. The most important parameters are the nuclear size, number, distribution, and optical density of condensed chromatin regions. In addition, the criteria permit recognition of changes related to differences in the growth conditions of the cells. The data of the image analysis suggest that adriamycin resistance in Sarcoma 180 cells is associated with characteristic patterns of cell nuclear morphology which can be described with a sufficient number of appropriate parameters. The advantages of image analysis are evident when these results are compared with the flow cytometric findings. The conclusion is that structural features of nuclear chromatin provide information essential for the assessment of drug resistance.

Animals↗
PubMedDate not supplied

Multiple spectral parameter imaging in quantitative fluorescence microscopy. I: Quantitation of bead standards.

Digitized images from fluorescence microscopy may suffer from nonlinearity of camera response to illumination intensity, from uneven spatial response of the camera, and from uneven illumination of the microscopical field. Structureless fluorescence standard slides are used to characterize the camera response. This information permits the images to be corrected for all the above errors, in one operation. The computer algorithms are described, and also those for thresholding and finding objects. The method is illustrated by tests using fluorescent beads.

Algorithms↗
PubMed2001

Roles of DNA cytometry and detection of EBERs in predicting a diagnosis of nasopharyngeal carcinoma.

OBJECTIVE: To analyze the suitability of DNA cytometry and detection of Epstein-Barr virus (EBV)-encoded RNAs (EBERs) on nasopharyngeal brushings for predicting a diagnosis of nasopharyngeal carcinoma (NPC). STUDY DESIGN: Cytologic preparations in 66 cases suspicious for NPC were evaluated for NPC diagnosis in comparison with the histologic diagnosis. Based on cytologic examination, 38 cases containing cytologically proven cancer and 8 cases interpreted as cytologically negative for cancer with adequate cellularity in the smear specimens were analyzed for DNA ploidy with an image analyzer and for EBER expression by in situ hybridization (ISH). RESULTS: Based on the cytologic diagnosis, DNA aneuploidy analysis, DNA nondiploidy analysis and EBER detection demonstrated a sensitivity of 50%, 84% and 92%, respectively, with the same specificity, 100%, for predicting a diagnosis of cancer. Their negative predictive values were 30%, 57% and 73%, respectively. There was a significant difference between DNA aneuploidy analysis and EBER analysis in sensitivity (P < .001) and in negative predictive value (P < .05) but not between DNA nondiploidy analysis and EBER analysis even though EBER analysis showed a slightly higher value in both parameters (P > .1 and P > .5, respectively). CONCLUSION: ISH for EBERs in cytologic smears showed a role superior to that of DNA aneuploidy analysis in the diagnosis of NPC. Considering its advantages of simple experimental conditions and lower cost as compared with DNA measurement, EBER detection can play a practical and important diagnostic role in patients suspected of having primary NPC.

Adult↗
PubMed1996

Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur.

Analysis of Variance↗
PubMed2000

Mucoepidermoid penile carcinoma: clinical, histologic, and immunohistochemical characterization of an uncommon neoplasm.

We report a case of a mucoepidermoid penile carcinoma. The specimen was studied by immunohistochemistry and DNA cytometry. Mucoepidermoid and adenosquamous penile carcinoma are exceedingly rare variants of penile cancer, with very little follow-up data available. To evaluate the possible prognostic significance of these differentiation patterns, it is necessary to document more cases of this unusual tumor entity.

Aged↗
PubMed2005

Screening for apoptosis--classical and emerging techniques.

There has been a rapid development of cell-based assays and screening methods to identify promising apoptosis-inducing drug candidates for the treatment of cancer. Distinguishing between the complex processes involved in apoptosis and other forms of cell death requires information on both biochemical and morphological processes in the cell. Traditionally, many assays have been limited to measuring, for example, caspase activity using fluorogenic substrates. However, these screening assays provide only limited information on the complex processes involved in apoptosis. In this review we describe some of the available apoptosis assays amenable to high-throughput screening. In particular, image-based high-content screening assays to evaluate multiple biochemical and morphological parameters in apoptotic cells are described. Through combining the imaging of cells in microtiter plates with powerful image analysis algorithms, one can acquire deeper knowledge on multiple biochemical or morphological pathways at the single-cell level at an early stage in the development of novel anti-cancer drugs.

Animals↗