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PubMed2004

Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays.

BACKGROUND: Sperm DNA integrity (SDI) is an important factor in the prognosis of male fertility. Here we compare the toluidine blue (TB) image cytometry test, recently proposed by us for SDI assessment, with two other tests-the sperm chromatin structure assay (SCSA) and the terminal nick-end labelling (TUNEL) assay. METHODS: Sperm samples from 35 men were evaluated for standard sperm parameters and subjected to the TB test and SCSA. Eighteen of the 35 samples were also subjected to the TUNEL assay. RESULTS: The proportion of sperm cells with abnormal DNA integrity assayed by the TB test correlated strongly with the proportion of abnormal cells detected by the SCSA and TUNEL assay (rho=-0.84 and rho=0.80, P<0.001, respectively). Furthermore, the fractions of abnormal cells by the TB test corresponded closely to the sum of two SCSA parameters, the DNA fragmentation index (DFI) and the fraction of highly DNA-stainable cells (HDS) (medians 33.0 versus 32.0%, P=0.6). CONCLUSIONS: Abnormal cells in a TB test correspond to the sum of DFI and HDS fractions in the SCSA. TB-positive cells may represent sperm with fragmented DNA and/or abnormal chromatin structure. Because the TB test is an easy and inexpensive method, its potential use as a routine test for sperm DNA integrity, complementary to standard semen parameters, should be investigated further.

Chromatin↗
PubMed1996

Preoperative fine needle aspiration from human breast cancer is a valuable sampling material for progesterone receptor and cytometric DNA analysis.

In a breast cancer series (n = 54), preoperative fine needle aspiration (FNA) was compared with biopsy at primary surgery as a source of material for the determination of progesterone receptor (PgR) content by enzyme immuno assay. The respective results manifested a strong correlation (r(s) = 0.82). The fact that PgR content was usually higher in FNA samples than in the corresponding biopsy samples and the finding that 11% of the tumours were PgR positive in FNA but PgR negative in the corresponding biopsy samples suggest a greater proportion of malignant cells to be obtained with FNA than in surgical biopsy. In another breast cancer series (n = 50), corresponding comparisons for DNA flow cytometry showed concordance in ploidy status (diploid vs. non-diploid) in 84% of cases and a strong correlation in S-phase fraction values (r(s) = 0.70). At DNA image cytometry, concordant results (Auer I + II vs. Auer III + IV) were obtained in 87% of the cases. To sum up, FNA seems to be a useful sampling technique for PgR determination and DNA cytometry.

Aneuploidy↗
PubMed1989

Quantitative morphology of primary and metastatic tumors: comparative studies on tissue sections.

Feulgen-stained tissue sections of 84 invasive ductal carcinomas of the breast (IDC), 30 colorectal adenocarcinomas (ACC), 30 gastric adenocarcinomas (ACG), 10 lymph node metastases of squamous cell carcinomas (SCC) and 11 lymph node metastases of malignant melanomas (MM) were investigated by image cytometry at a spatial resolution of 0.25 micron. At least 150 tumor cell nuclei were measured in each case. By means of multivariate statistical analysis the specificity of the nuclear image for different histological tumor types could be demonstrated in lymph node metastases of SCC and MM. Nuclear image analysis therefore may be a useful tool for pathologists in making the differential diagnosis on metastases of unknown primary tumors. For IDC, ACG and ACC the metastatic phenotype could be identified by nuclear image analysis of the primary tumors. The prognostic significance of the nuclear image has been proved in adenocarcinomas of the stomach. Features of chromatin structure played an important role in both the diagnosis of doubtful histological tumor types and the prediction of lymph node involvement or prognostic outcome.

Cell Nucleus↗
PubMed1986

A simple method to select specific tumor areas in paraffin blocks for cytometry using incident fluorescence microscopy.

A simple method is described for the selection of tumor areas in paraffin blocks for cytometry. The surface of a paraffin block is stained with the fluorescent dye DAPI. By means of incident fluorescence microscopy on the cut surface of a total block, the tissue can be visualized. Location of the tumor area with the aid of conventional histopathological criteria is feasible using the adjacent section after hematoxylin-eosin staining as a diagnostic guideline. Once the position of the tumor area is determined, a small hollow bore, which is screwed in the objective holder, is pressed in the tissue. The depth is controlled by the microscrew of the microscope. After retraction of the bore, the block is removed, and a thick section is cut. The selected area can be processed for cytometry separately from the remaining tissue. The technique can be used either to enrich the sample to be analyzed with tumor cells or to analyze histopathologically different tumor compartments. Both flow and image cytometry can make use of this selection technique.

Breast↗
PubMed2000

Analysis of apoptotic cells by flow and laser scanning cytometry.

A large number of flow cytometric methods to identify apoptotic cells and analyze morphological, biochemical, and molecular changes that occur during apoptosis have been developed. These methods are also applicable to the laser scanning cytometer (LSC), a microscope-based cytofluorometer that combines advantages of flow and image cytometry and that, by offering a possibility of assessment of cell morphology, is of particular utility in analysis of apoptosis. Apoptosis-related changes in cell morphology associated with cell shrinkage and condensation of cytoplasm and chromatin are detected by measurements of the intensity of light scatter of the laser beam in the forward and 90 degrees angle directions. Changes in plasma membrane composition and function are analyzed by its altered permeability to certain dyes and by the appearance of phosphatidylserine, which reacts with annexin V-fluorochrome conjugates on the external surface of the membrane. Decrease in mitochondrial transmembrane potential is measured with several fluorochromes of the rhodamine or carbocyanine family. DNA fragmentation is detected either by measurement of cellular DNA content after elution of the degraded DNA from the cell before or during the staining procedure or by in situ labeling DNA strand breaks. Apoptotic cells are then recognized either on the basis of their reduced DNA-associated fluorescence as the cells with fractional DNA content ("sub-G1 cells"), or as the cells with an extensive number of DNA breaks, respectively. Advantages and limitations of the preceding methods are discussed and their adaptation to LSC is presented.

Annexin A5↗
PubMedDate not supplied

Past and present concepts in flow cytometry: a European perspective.

The development of flow cytometric instrumentation, methods and research concepts in Europe has been a continuous driving force for the general scientific advancement in this area over the years. This review addresses early European concepts of continuing interest with regard to instrumentation, data analysis, clinical and eperimental DNA analysis, cell function and microbiology at their worldwide first appearence while flow cytometric immunology and immunophenotyping will be covered separately. Flow cytometry represents an efficient approach to the enormous complexity of molecular cell architecture and cell function by the analysis of apparent molecular cell phenotypes in heterogeneous cell samples. The present merger of flow and image cytometry into the method independent cytomics discipline increases the potential of cell analysis very significantly. It opens the way for predictive medicine as well as for predictive cytopathology and predictive cytology in everyday clinical and medical practice. Current progress is driven by joint advances in molecular fluorescence technologies and instrument development. This complements the analysis of genome sequence information in an efficient way.

Apoptosis↗
PubMed1988

Comparison of absorption measurements of DNA stain content by utilizing video and scanning image cytometers.

After staining with the Feulgen reaction, the DNA stain contents of 155 mouse bone marrow cells and 22 adjacent chicken erythrocytes were measured by absorption image cytometry by utilizing two different systems--a scanning cytometer and a video cytometer. In the scanning cytometer (M85 microdensitometer, Vickers Instruments, Malden, MA), a spot of light was scanned across the cell. In the video cytometer (TAS Plus, E. Leitz, Rockleigh, NJ), the microscope field, which may contain several nuclei, was imaged onto a Plumbicon video camera. With each system, cells were scanned, digitized into their elementary pixels, and analyzed to determine their integrated absorbance. Comparison of the DNA stain contents of the same G0/G1 bone marrow cells and chicken erythrocytes, as measured by video and scanning cytometry, showed that both techniques gave comparable results; scanning cytometry is more precise. The coefficients of variation of the measurements for the G0/G1 bone marrow cells and for the chicken erythrocytes were 5.9% and 7.0%, respectively, when measured by video cytometry at the absorption peak (584 nm), compared to 4.1% and 3.5%, respectively, for the same cells when measured by scanning cytometry off the absorption peak (615 nm). The video-based measurements were relatively lower than the scanning measurements for darkly stained cells; this suggests that glare and other optical errors which increase with stain darkness caused greater systematic errors in the video cytometer than they did in the scanning cytometer.

Animals↗
PubMed2002

Detection of in situ activation of transglutaminase during apoptosis: correlation with the cell cycle phase by multiparameter flow and laser scanning cytometry.

BACKGROUND: One of the hallmarks of apoptosis is activation of tissue transglutaminase (Tgase; also called transglutaminase type 2 [TGase 2]). Its activation causes cross-linking of cytoplasmic proteins, making them insoluble and presumably less immunogenic. Several biochemical and cytochemical methods to detect activity of TGase 2 exist, but none has been adapted for multiparameter flow or image cytometry. METHODS: Apoptosis of HL-60 or U-937 leukemic cells was induced by camptothecin, tumor necrosis factor alpha, hyperthermia, or the cytotoxic RNase onconase. Two different approaches to detect TGase 2 activation were developed: (a) the unfixed cells were treated with 4',6'-diamidino-2-phenylindole, and sulforhodamine 101 in solutions of nonionic detergents; (b) the TGase 2 substrate fluoresceinated polyamine cadaverine (F-CDV) was administered into the cultures for several hours before cell harvesting. The cells were then fixed and their DNA counterstained with propidium. Cellular fluorescence was measured by flow or laser scanning cytometry. RESULTS: (a) Exposure of nonapoptotic cells to detergents caused their full lysis, resulting in preparation of isolated nuclei devoid of cytoplasm. Conversely, the cross-linking of cytoplasmic protein by activated TGase 2 in apoptotic cells provided resistance to detergents: the nuclei or nuclear (chromatin) fragments of apoptotic cells remained attached to the cytoplasmic protein, embedded within the proteinaceous "shell." Such cells were identified by their high protein content: intensity of fluorescence after staining with the protein fluorochrome sulforhodamine 101 was markedly higher than that of isolated nuclei. (b) Activation of TGase 2 was also detected by virtue of intense cell labeling with fluoresceinated polyamine cadaverine. Interestingly, in many cells apoptosis progressed without evidence of activation of TGase 2, suggesting that this event may not be a prerequisite for completion of apoptosis. CONCLUSIONS: Activation of TGase 2 can be detected simply by cell resistance to detergents or in situ reactivity with F-CDV. Both methods allow one to correlate activation of TGase 2 with the cell cycle position. However, because activation of TGase 2 is not always detected during apoptosis, the lack of the activation cannot be considered a marker of nonapoptotic cells. Hence, an apoptotic index based solely on TGase 2 activation may underestimate incidence of apoptosis.

Apoptosis↗
PubMed1991

Clinical applications of cytometry: 5th annual meeting.

The 5th annual Clinical Applications of Cytometry meeting was held September 12-15, 1990 in charleston, SC. The theme which emerged repeatedly throughout the meeting was the need to take full advantage of the quantitative power of cytometry to provide the most useful clinically relevant diagnostic and prognostic information. Greater quantitative power is based on careful and reproducible standards and quality control. The same principles, albeit with somewhat different approaches, apply to cell surface immunofluorescence analysis, DNA measurements, and image cytometry assessments. Monoclonal antibody probes against oncogenes, others against lymphokines within the Golgi, and a novel fluorogenic substrate designed to quantitate the activity of a mitochondrial enzyme were exciting developments described at the meeting.

Autoimmune Diseases↗
PubMed1990

Which oral white lesions will become malignant? An image cytometric study.

We investigated the value of image cytometry in predicting the prognosis of oral epithelial lesions, whether or not they show dysplasia. Thirty-five oral epithelial lesions were studied retrospectively. Of these, 23 had later transformed to carcinoma and 12 had not. By means of the Leitz TAS image analyzer, 200 nuclei of epithelial cells and 20 nuclei of lymphocytes from each section were individually assessed for eight features related to shape and amount of stain and for six features related to chromatin pattern. The mean, standard deviation, and interquartile range of each feature were calculated, first for each section and then for each group. With the use of linear stepwise discriminant analysis we constructed a predictive model, which consisted of three variables related to chromatin pattern. The variables were mean margination, standard deviation of clumping, and standard deviation of condensation. In the jackknife classification, this model predicted the malignant potential of the lesions that later transformed to cancer with 86% predictive value and 83% sensitivity.

Adult↗
PubMed1995

Heterogeneity of the chromatin fine structure in DNA-diploid breast cancer cells.

The chromatin fine structure of Feulgen-stained DNA-diploid tumour cells was studied by means of image cytometry in fine-needle aspiration biopsies of 115 breast cancer patients. The investigation was focused on diploid, several subtypes of polyploid, and aneuploid tumours. Only the clearly separable diploid peaks of these tumours were measured cytometrically by an OPTIMAS-based image analysis system, which led to a set of 93 primary cytometric features in each diploid subpopulation. The results demonstrate that the diploid tumours are clearly different to the diploid compartments of various subtypes of polyploid and aneuploid tumours. Between those diploid subpopulations from polyploid tumours, chromatin structure differences also exist. A higher degree of similarity between polyploid and aneuploid tumours, compared to diploid, became evident. The actual structural differences between the diploid subpopulations indicate both increasing variances within the peak and increasingly coarse and contrasted, irregular chromatin structure with the occurrence of tetraploid, octoploid, and aneuploid DNA-histogram peaks. Therefore, the structural effects of an increasing genomic instability seems not to be restricted to the non-diploid cell populations.

Adenocarcinoma↗
PubMed1998

Prognostic significance of DNA cytometry of postirradiation cervicovaginal smears.

BACKGROUND: Cytologic sampling is performed routinely after radiotherapy for cervical carcinoma. The prognostic significance of postirradiation dysplastic and atypical cells is uncertain because of difficulties in distinguishing preneoplastic and cancerous changes from benign radiation changes. DNA cytometry studies may provide a more objective method of identifying significant lesions. METHODS: Postirradiation cervical carcinoma patients with cervical/vaginal smears containing atypical or dysplastic cells were identified prospectively. Papanicolaou smears were destained, restained with a Feulgen stain, and evaluated for DNA content using image cytometry. Pathologic and clinical records were monitored on each patient for evidence of recurrence or biopsy-proven dysplasia. RESULTS: Of 46 patients, 14 had been diagnosed on cytology as having atypical squamous cells, 4 as having atypical/suspicious cells, 12 with low grade squamous intraepithelial lesions (SIL), 3 with high grade SIL, and 13 with ungraded SIL. DNA histograms were classified as follows: 14 diploid, 19 polyploid, and 13 aneuploid. Cytologic diagnosis and histogram type were correlated significantly and both correlated with clinical outcome. The probability of either postirradiation dysplasia or recurrence was as follows: SIL, 82%; suspicious, 100%; polyploid, 79%; and aneuploid, 92%. Patients with atypical squamous cells of undetermined significance or diploidy most frequently had negative follow-up (57% each). All patients with both SIL and aneuploidy developed either dysplasia or recurrence. The stage of disease did not correlate with outcome or histogram pattern. CONCLUSIONS: DNA analysis of postirradiation cytologic smears demonstrating atypia or dysplasia may provide useful ancillary information. The presence of aneuploidy usually signifies either recurrence or dysplasia. Polyploidy most frequently occurs in dysplastic processes, whereas diploid histograms usually denote a benign disease course.

Adult↗
PubMed2000

Microbial analysis at the single-cell level: tasks and techniques.

The heterogeneity of microorganisms themselves is orders of magnitude greater than the heterogeneity of perspectives from which they are contemplated by human observers. Even closely related species may exhibit marked differences in biochemistry and behavior, and, under many conditions, similar, striking heterogeneity may exist within a clonal population of organisms which, in the aggregate, occupy too small a region of space to be visible to the unaided human eye. Using methods of microscopy, microspectrophotometry, and cytometry developed and refined since the 1960s, it is now possible to characterize the physiology and pharmacology of individual microorganisms, and, in many cases, to isolate organisms with selected characteristics for culture and/or further analysis. These methods include fluorescent and confocal microscopy, scanning and image cytometry, and flow cytometry. Fluorescence measurements are particularly important in single-cell analysis; they allow demonstration and quantification of cells' nucleic acid content and sequence, of the presence of specific antigens, and of physiologic characteristics such as enzyme activity and membrane potential. Multiparameter cytometry, combined with cell sorting, provides insight into population heterogeneity and allows selected cells to be separated for further analysis and culture. The technology is applicable to a wide range of problems in contemporary microbiology, including strain selection and the development of antimicrobial agents.

Bacteria↗
PubMed1994

DNA ploidy in human colorectal adenomas.

The DNA content of 101 colorectal adenomas of various histologic types resected from 83 patients was determined by image cytometric measurements in order to investigate if a correlation between DNA ploidy and particular histomorphologic features exists and if DNA measurements can be of additional diagnostic value. Overall, 67 of 101 (66%) adenomas showed an aneuploid DNA distribution pattern, including 8 of 19 (42.1%) mildly atypical, 32 of 48 (66.7%) moderately atypical and 27 of 34 (79.4%) severely atypical adenomas. Correlating DNA content with the histologic type, 17 of 42 (40.5%) tubular, 28 of 37 (75.7%) tubulovillous and all 22 villous adenomas exhibited aneuploid DNA histograms. Aneuploidy was also observed more frequently in larger adenomas. The results show a good correlation between tumor size, histomorphologic features and DNA content. The most remarkable observation is that as many as 42% of the adenomas histomorphologically considered mildly atypical exhibited aneuploidy. Since aneuploidy has been demonstrated to indicate premalignant or malignant cellular alterations, DNA image cytometry is suggested for providing valuable additional information on the diagnosis of colorectal adenomas.

Adenoma↗
PubMed1994

Comparative assessment of DNA analysis in effusions by image analysis and flow cytometry.

Cytologic evaluation of body cavity fluids is useful to detect malignancy within the pleural and peritoneal spaces. A definitive diagnosis cannot always be made on cytologic evaluation alone. As malignant processes may show abnormal DNA content, DNA analysis of effusions may be useful. Therefore, we determined the DNA content of 37 effusions by flow cytometry (FC) and image analysis (IA) using the CAS 200. Of the 37 fluids evaluated, 18 were cytologically malignant, 15 benign, and four atypical. Overall, 22 fluids (60%) showed concordance between FC and IA. None of the benign fluids were aneuploid. All showed diploid histograms or diploidy with increased proliferating cells. Three of four atypical fluids had increased proliferating cells by either FC or IA, whereas one was diploid by both methods. Aneuploidy was detected in 13 malignant fluids: five were aneuploid by both methods and eight by only one method. IA identified aneuploidy in five of those eight cases, while three were identified by FC. Three of the cytologically malignant fluids were diploid by both methods, and two showed increased proliferating cells by IA and diploidy by FC. The specificity of both methods was 100%. However, the sensitivity of identifying a malignant fluid by aneuploidy is low, 44% for FC and 55% for IA. IA appears to identify small aneuploid populations more frequently than FC. The detection of aneuploidy in effusions is highly suggestive of malignancy, and the combination of both techniques gives the highest detection rate (72%). However, neither are as sensitive as traditional cytologic evaluation with the occasional use of additional histochemical stains.

DNA, Neoplasm↗
PubMed1999

[An information system for automated cytogenetic image processing: analysis of interphase chromatin of nerve cells of embryonic brain of rats with varying nervous system excitability].

To reveal a relationship between the genetically determined excitability of rats and the structural-functional chromosome organization in their neurone nuclei a computer information system has been developed to classify the neurone nuclei according to their specific DNA image cytometry features. The nuclear features, such as size, shape, and DNA content, are calculated, along with features describing characteristics of chromatin with the nucleus. The neural and glial cells are separated according to the rule based expert system approach with the use of a nuclear feature vector. The DNA image processing is performed and a feature vector consisting of normalized measures of the neurone nucleus chromatin region descriptors is extracted. The results indicate a relationship between the peculiarities of the nervous system and the structural-functional state of the chromosomal apparatus.

Animals↗
PubMed1991

Fluorescent tetradecanoylphorbol acetate: a novel probe of phorbol ester binding domains.

Protein kinase C (PKC) has a prominent role in signal transduction of many bioactive substances. We synthesized the fluorescent derivative, phorbol-13-acetate-12-N-methyl-N-4-(N,N'-di(2-hydroxyethyl)amino)-7-n itr obenz-2-oxa-1,3-diazole-aminododecanoate (N-C12-Ac(13)) of 12-O-tetradecanoylphorbol-13-acetate (TPA) to monitor the location of phorbol ester binding sites and evaluate its potential use as a probe of PKC in viable cells. The excitation maximum wavelength of N-C12-Ac(13) is close to 488 nm, facilitating its use in argon-ion laser flow and imaging cytometry. When incubated with 100 nM N-C12-Ac(13) at 25 degrees C, P3HR-1 Burkitt lymphoma cells accumulated the dye rapidly, reaching maximum fluorescence within 25 min, 20-fold above autofluorescence. Addition of unlabeled TPA significantly decreased the fluorescence of N-C12-Ac(13) stained cells in a dose-dependent manner indicating specific displacement of the bound fluoroprobe. Competitive displacement of [3H]-phorbol-12,13-dibutyrate ([3H]-PBu2) from rat brain cytosol with N-C12-Ac(13) gave an apparent dissociation constant (Kd) of 11 nM. N-C12-Ac(13) possessed biological activity similar to TPA. Like TPA (final concentration 65 nM) N-C12-Ac(13), at a lower concentration (51 nM), induced expression of Epstein-Barr viral glycoprotein in P3HR-1 cells, differentiation of promyelocytic HL60 cells, and caused predicted changes in the mitotic cycle of histiocytic DD cells. Microspectrofluorometric images of single cells labeled with N-C12-Ac(13) showed bright fluorescence localized intracellularly and dim fluorescence in the nuclear region, consistent with dye binding mainly to cytoplasmic structures and/or organelles and being mostly excluded from the nucleus. Because of the high level of non-specific binding of N-C12-Ac(13), this probe is not ideal for visualizing PKC in intact cells, but would be a valuable fluoroprobe to investigate the kinetic properties of purified PKC. Also, knowledge gained from these studies allows us to predict structures of fluorescent phorbols likely to have less non-specific binding and, consequently, be potentially useful for monitoring PKC in viable cells.

Animals↗
PubMed2000

Significance of DNA ploidy measurements in Spitz nevi.

In 28 Spitz nevi DNA content was determined by video-imaging cytometry. The nevi were selected for this study because of difficulties in differentiation from melanoma and heterogeneity of this type of nevus. Morphological features of Spitz nevi and differences helpful for differentiation between Spitz nevi and malignant melanoma were identified. DNA ploidy was measured in paraffin embedded and fresh tissue material from each patient and the results were comparable. The sample preparation process and video-imaging method are presented in this study. Twenty two (78.6%) lesions contained diploid cell populations, 5 (17.9%) aneuploid and 1 (3.6%) tetraploid cell population. A significant correlation was observed between DNA ploidy measured in fresh tissue and retrospective material. The results indicate the presence of abnormal DNA content in some of the lesions. This observation does not indicate that DNA ploidy cytometry is useful for the differentiation of Spitz nevi from malignant melanoma.

Adolescent↗