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PubMed1993

DNA ploidy of ameloblastoma and ameloblastic carcinoma of the jaws. Analysis by image and flow cytometry.

Ameloblastomas, the second most common odontogenic tumor, behave as benign locally aggressive tumors. Ameloblastic carcinomas, on the other hand, show histologic features of malignancy and may metastasize. By image cytometric analysis of Feulgen-stained sections from decalcified, formalin-fixed, paraffin-embedded tumors, we compared nuclear DNA content of 17 primary and five recurrent ameloblastomas and five ameloblastic carcinomas. Of the primary ameloblastomas, 14 (82%) were diploid; three (60%) of the five recurrent ameloblastomas were diploid. No significant difference in ploidy between primary and recurrent ameloblastomas or among plexiform, follicular, and acanthomatous types of ameloblastoma was demonstrated. Of the five ameloblastic carcinomas, four (80%) were aneuploid; ploidy was not significantly correlated with the incidence of metastasis. Flow cytometry was performed on three carcinomas; 100% concordance between image and flow cytometric data was seen. Aneuploidy is significantly more common in ameloblastic carcinomas than in ameloblastoma and is a strong predictor for malignant potential.

Adult↗
PubMed1990

Comparative assessment of proliferation and DNA content in breast carcinoma by image analysis and flow cytometry.

Although tumor DNA content and proliferation are usually determined by flow cytometry (FCM), quantitative microscopic image analysis is a viable alternative technique that also provides important histologic correlations. To compare these methods, we measured DNA content and proliferation in 54 consecutive breast cancers and 15 benign breast lesions by FCM and IA. DNA content determination was concordant in 49 of 54 cancers measured by FCM and IA. Four of the discordant cases were aneuploid by IA and diploid by FCM. There was good correlation between the DNA index (DI) measured by FCM and IA (r = 0.89, P less than 0.0001). Proliferation was assessed by IA quantitation of Ki-67 and PCNA/Cyclin antibody staining, as well as by flow cytometric S-phase fraction (SPF). Ki-67 positivity was greater in breast cancer than in benign controls (21.6% +/- 13.1% vs. 7.9% +/- 5.6% [P less than 0.0001]), as was PCNA/Cyclin positivity (10.2 +/- 6.7% vs. 2.7 +/- 2.5% [P less than 0.0001]). S-phase fraction measured by FCM was 7.9% +/- 5.7% for carcinomas and 3.17% +/- 2.1% for benign controls (P less than 0.003). Ki-67 and Cyclin staining, as well as SPF, were significantly increased in aneuploid compared to diploid tumors, and increased staining was associated with worsening nuclear grade. There were significant correlations between SPF and Ki-67 staining (r = 0.48, P less than 0.0001) and SPF and Cyclin staining (r = 0.48, P less than 0.0001). We conclude that FCM and IA provide comparable measurements of DNA content, although occasional discrepancies occur. Image analysis provides a valuable alternative method for assessing tumor cell proliferation and may offer certain advantages over FCM.

Antibodies↗
PubMed1993

Image analysis DNA cytometry of bladder cancer.

In a retrospective analysis, the DNA histograms of 65 paraffin-embedded bladder carcinomas from radical cystectomy specimens (stage pT1-pT4a, pN0, pN1, pN2) were analyzed using an automated image analysis system (Leytas 2). Automated image analysis was able to characterize invasive bladder carcinoma as being either diploid, polyploid, or aneuploid. Within the group of aneuploid tumors, the DNA content of the stem-cell line allowed further subtyping of the tumors; hypotriploid, hypertriploid, hypertetraploid, and even hyperpentaploid tumors could be distinguished. Comparing different sites of identical tumors, the DNA histogram was found to be a stable and reproducible tumor characteristic. The various tumor types differed significantly in prognosis. This technique can also be applied to smears of urine sediment or transurethrally resected tumor chips. In the case of superficial tumors, DNA cytometry defines those tumors which are potentially invasive, requiring careful follow-up and/or early aggressive treatment.

DNA, Neoplasm↗
PubMed1992

Comparison of image analysis with flow cytometry for DNA content analysis in pigmented lesions of the skin.

The DNA ploidy of 85 melanocytic skin lesions was determined by flow cytometry (FCM) and interactive image analysis (IA) using nuclear extracts of paraffin-embedded tissue. Of the 85 lesions analyzed, 43 were malignant melanomas in different stages of evolution, 15 were dysplastic nevi, 11 were Spitz nevi, and 16 were other types of nevi. Some of the last had features of congenital nevi. Within the melanoma category, there was 42% aneuploidy by FCM versus 56% by IA. Of those melanomas aneuploid by FCM, all but one were aneuploid by IA. All dysplastic nevi, 10/11 Spitz nevi and 15/16 other nevi were diploid by both methods. One of the 16 nevi from the "other types" category was tetraploid by IA but diploid by FCM. A single Spitz nevus was tetraploid by FCM but diploid by image analysis. While our results suggest that interactive IA is potentially a more sensitive method than FCM for detecting aneuploidy in cutaneous pigmented lesions, it remains to be shown whether this will translate into better prognostic assessment of the biologic behavior of melanocytic neoplasms than provided by flow cytometric ploidy analysis.

Aneuploidy↗
PubMed1993

Comparative analysis of DNA ploidy and proliferative index in fine needle aspirates of non-Hodgkin's lymphomas by image analysis and flow cytometry.

To determine the application of image analysis (IA) to fine needle aspiration (FNA) of non-Hodgkin's lymphomas, 78 cases, categorized according to the Working Formulation, were analyzed and compared with those analyzed by flow cytometry (FCM). Aspirated material was immediately fixed in either IA fixative (31 cases) or acetone, stored at -70 degrees C (47 cases) for periods up to 48 months and postfixed in IA fixative. IA assessed DNA index (DI), ploidy balance, degree of hyperdiploidy and proliferation index (PI). DNA index, % S + G2M and RNA index were obtained by acridine orange FCM. There were no significant differences in measured variables between fresh and stored cases. Statistically significant differences between low, intermediate and high grade lymphomas were present in all evaluated variables except DNA ploidy (DI) (P = .074) measured by IA. DI, however, showed a very high correlation with the DI obtained by FCM (r = .94). PIs, although generally lower, also had a high correlation with S + G2M (r = .85). We conclude that results obtained by IA were comparable to results by FCM and that frozen material can be used to retrospectively evaluate ploidy and proliferation. IA is especially suitable for the evaluation of low-volume specimens obtained by FNA.

Biopsy, Needle↗
PubMed1996

3D presentation of the nuclear cell features in quantitative cytometry.

The use of an image cytometer in analysis of smears and needle aspirates provides valuable information to a cytologist. It allows to combine the overall impression, formed by visually inspecting the cells, with measured and numerically expressed nuclear cell features. Both types of information can be used efficiently only if presented to the expert in an appropriate way. Cell images (as they are seen with a microscope) are easily analysed by the experts. However, measured nuclear features can not be presented as a list of numerical values. Instead, an user interface should be developed, providing graphical presentation of the nuclear features. It should show as much information as possible and provide a comprehensive link between nuclear features and cell images. The user interface described in this paper shows nuclear features in three dimensions. It is based on a perspective projection of the three dimensional feature space onto a two dimensional surface. It allows the user to dynamically change the perspective, i.e., to look at the virtual three dimensional structure from different viewpoints. Each nucleus is represented by a single object in the three dimensional space. When an object in the three dimensional feature space is selected, the image (or the visual appearance) of the corresponding cell is shown. When a nucleus image is selected, its position in the feature space is highlighted. This provides an interconnection between nuclear cell features and cell images allowing simultaneous analysis of both types of information.

Cell Nucleus↗
PubMed1990

DNA ploidy in breast lesions. A comparative study using two commercial image analysis systems and flow cytometry.

DNA ploidy determinations on a series of 24 breast specimens were performed independently utilizing flow cytometry (FCM) and two separate commercially available computerized image analysis systems for image cytometry (ICM). The tissues analyzed were obtained from 20 carcinomas, 2 benign neoplasms and 2 benign reductive procedures. The results showed a close correlation between the DNA indices (DIs) obtained by all methods in 14 of the 24 cases. In four cases, all methods showed aneuploid peaks, but with differing DIs. In six cases (two benign and four malignant) FCM showed diploidy while ICM showed peridiploid cell populations. The results obtained with the two image analysis systems were in agreement for 20 of the 24 cases. ICM is an acceptable alternative to FCM for reproducible ploidy analysis. ICM-based measurements have the advantage of the visual discrimination of abnormal cells and therefore may have a greater sensitivity in identifying small aneuploid populations. Populations with DIs in the range of 1.0 to 1.3 need to be assessed carefully in ICM-based determinations due to the potential that these "aneuploid" peaks may represent shifted diploid populations.

Breast Diseases↗
PubMed2006

Analysis of CD36 expression on human monocytic cells and atherosclerotic tissue sections with quantum dots: investigation by flow cytometry and spectral imaging microscopy.

OBJECTIVE: To demonstrate CD36 expression with quantum dots (QDs) 525 and/or 605 on human monocytic U937 cells and atherosclerotic tissue sections by means of flow cytometry (FCM) and/or confocal laser scanning microscopy (CLSM). STUDY DESIGN: U937 cells and tissue sections were analyzed by means of FCM and/or CLSM. FCM was performed, using different ultraviolet (UV) and visible (488/532 nm) excitation modes. In the visible mode, fluorescence intensities of QDs, phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were compared. Three-dimensional (3-D) sequences of images were obtained by spectral analysis in a CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, providing factor curves and images. Factor images are the result of the FAMIS image processing method, which differentiates emission spectra from 3D sequences of images. In CLSM analysis, preparations are screened in a UV excitation mode to optimize the possibilities of QDs and have the benefit of 4',6-diamino-2-phenylindole or Hoechst 33342 counterstaining of nuclei. RESULTS: FCM and CLSM revealed CD36 expression by means of QDs 525 and/or 605. Fluorescence intensity of PE and of FITC was higher than that of QDs 525 and of 605. As factor curves and images show the red emission of QDs 605 only, subsequent reliable identification and localization of CD36 was obtained. CONCLUSION: QDs 525 and 605 are useful to analyze antigenic expression. Following FCM, which is well adapted to detect fluorescence emission of QDs in the UV or visible excitation mode, CLSM and subsequent spectral analysis assess more specific characterization of QD fluorescent emissions.

Arteries↗
PubMed2006

Flow cytometry and spectral imaging multiphoton microscopy analysis of CD36 expression with quantum dots 605 of untreated and 7-ketocholesterol-treated human monocytic cells.

OBJECTIVE: To evaluate CD36 expression with quantum dots 605 (QDs 605) on untreated and 7-ketocholesterol (7KC)-treated monocytic U937 cells by flow cytometry (FCM) and confocal and multiphoton laser scanning microscopy (CLSM). STUDY DESIGN: Cells were analyzed by CLSM, following flow cytometric quantification of CD36 expression and 7KC uptake. Image sequences were obtained by spectral analysis in monophoton and multiphoton CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm to differentiate emission spectra. In CLSM analysis, cell deposits were screened in ultraviolet excitation modes to optimize the possibilities of QDs 605 and have the benefit of nuclei counterstaining by DAPI. RESULTS: FCM and CLSM reveal the expression of CD36 by means of QDs 605. FCM provides information on 7KC uptake. CLSM provides the localization of 7KC vs. DAPI. As factor curves and images show the red, narrow emission of QDs 605 vs. violet and blue emissions of 7KC and DAPI, respectively, a reliable identification of CD36 is obtained. CONCLUSION: QDs 605 are useful tools to perform antigenic expression in FCM and CLSM. Moreover, CLSM and subsequent spectral analysis provide a more specific characterization of QDs 605 fluorescent emission in the UV excitation mode and a simultaneous identification of 7KC.

Algorithms↗
PubMed2000

Changes in the motility, morphology, and F-actin architecture of human dendritic cells in an in vitro model of dendritic cell development.

An in vitro model has been developed for analyzing the two developmental phases of human dendritic cell (DC) migration. Employing the age of the culture and the addition of GM-CSF, IL-4, and serum to regulate cellular phenotype, and glass coated with acid-precipitated human plasma proteins to facilitate persistent DC translocation, the model produces three sequential in vitro phenotypes with the following suggested in vivo counterparts: (1) DCs recently isolated from blood, which are highly polar and motile, and reflect the behavior of "undifferentiated" DCs that must extravasate from the blood stream and migrate into peripheral tissue; (2) large, nonmotile, stellate DCs, which reflect the highly "differentiated" signature phenotype of DCs in peripheral tissue, whose function is to capture foreign antigens; and (3) the large, motile "dedifferentiated" DCs, which reflect the behavior of "veiled cells" that have captured an antigen, retracted dendritic processes, migrated out of peripheral tissue, and are in the process of transporting a captured antigen to a proximal draining lymph node for presentation to T cells. Computer-assisted motion analysis of the three sequential phenotypes and fluorescent staining of F-actin reveal three unique behavioral states and unique cellular architecture consistent with inferred in vivo function. This in vitro model should serve as a starting point for elucidating the cues and molecular mechanisms involved in the regulation of DC differentiation and motility.

Actins↗
PubMed1998

Synovial sarcoma. Evaluation of prognosis with emphasis on the study of DNA ploidy and proliferation (PCNA and Ki-67) markers.

Controversy still exists regarding the validity of parameters commonly used in the evaluation of prognosis of patients with synovial sarcoma (SS). Forty-nine cases of previously untreated primary SS (23 females and 26 males, ranging in age from 7 to 81, with 31 tumors located in the lower extremity, 8 at the upper extremity and 10 at the trunchus), without regional lymph-node or distant metastases were studied. We investigated the relationship between (flow and image) DNA cytometry, proliferation activity, clinicopathologic parameters, and relapse-free and overall survival of the patients. The prognostic value of gender, age, duration of symptoms, location, compartmentalization, size, adequacy of surgical margins, residual tumor, adjuvant therapy, histologic subtype, extent of necrosis, glandular differentiation, calcification, and extent of hemangiopericytic areas, mitotic rate, amount of mast cells, blood vessel invasion, histologic (UICC and NCI) grades, DNA ploidy, percentage of cells in S and S+G2 phases, PCNA and Ki-67 labeling indices (LI), and TNM (UICC) stage of the tumors, were evaluated by univariate and multivariate (Cox hazard model) analyses. Short duration of symptoms (<12 months), biphasic SS, scarcity of mast cells (<10/10 HPF), high mitotic rate (> or =10/10 HPF), high histologic grade (grade 3), high PCNA-LI (> or =20%), high Ki-67-LI (> or =10%), DNA aneuploidy, and advanced TNM stage (stage III) were features associated with significantly shorter relapse-free and overall 5-year survival rates in the univariate analyses. Scarcity of mast cells, high mitotic rate, or high PCNA-LI were significant predictors of poor survival, in addition to TNM stage in the multivariate analyses. The amount of mast cells was inversely correlated with mitotic rate and PCNA-LI. Scarcity of mast cells, high mitotic rate, or high PCNA-LI are factors associated with poor prognosis, in addition to advanced TNM stage in patients with localized SS.

Adolescent↗
PubMed1992

[Image analysis of DNA cytometry in tumors of the upper gastrointestinal tract].

The prognostic influence on the DNA content was investigated in 189 patients (esophagus carcinoma n = 45, gastric cancer n = 103, pancreatic cancer n = 41) who underwent a curative resection. In a multivariate analysis the DNA content had a strong as well as an independent influence on the prognosis in esophagus cancer and in pancreatic carcinoma. In gastric cancer, the DNA content had no influence on the prognosis. These results show that the DNA content of the tumor cells, as a measurement of the numerous chromosomal aberrations, well reflects the aggressiveness of the tumor growth in esophagus- and pancreatic cancer. In these tumors it represents the decisive criteria for the prognostic judgement.

Esophageal Neoplasms↗
PubMed1998

Single-cell measurement of superoxide anion and hydrogen peroxide production by human neutrophils with digital imaging fluorescence microscopy.

Besides flow cytometry, fluorescence microscopy combined with computerized image analysis offers an alternative tool for assessing phagocyte oxidant generation at the single-cell level. This technique provides an opportunity for the direct visualization of cells and simultaneous measurement of cellular fluorescence intensity. Thus, we developed a simple method for the quantitative evaluation of intracellular superoxide anion and hydrogen peroxide production with image cytometry by using hydroethidine and dihydrorhodamine 123 dyes, respectively. Human neutrophils stimulated with phorbol dibutyrate and labeled by these fluorogenic substrates showed intense, well recognizable red or green fluorescence. The intensity of signals from individual granulocytes of cytospin preparations were quantitatively measured in digitized images. There was a great heterogeneity in response to the stimulus within the granulocyte population as shown by the integrated fluorescence intensity values. In agreement with the results of parallel flow cytometric experiments, this simple image analysis performed on cells of cytospin preparations was able to detect the defects in the oxidative metabolism of neutrophils from patients with cervix carcinoma. We demonstrated that even minor alterations in superoxide anion/hydrogen peroxide generation can be detected by image cytometry as efficiently as by flow cytometry. This result validates imaging microscopy as an alternative to flow cytometry in such experiments. In addition, the image cytometric technique allows the observation of the kinetics of free radical production in individual cell under adherent conditions. Therefore, we carried out image analysis of the oxidative burst of neutrophils adherent to uncoated glass and fibronectin- and type IV collagen-coated surfaces in response to stimulation with phorbol dibutyrate or N-formyl-methionyl-leucyl-phenylalanine. We elaborated a calibration technique for the quantitative measurement of the ethidium bromide generation mediated by superoxide anion within individual adherent granulocytes. The ethidium bromide production varied between 0.48 and 1.17 amol/cell/min.

Anions↗
PubMed2001

Detection of subgroups from flow cytometry measurements of heterotrophic bacterioplankton by image analysis.

BACKGROUND: Flow cytometry is an invaluable tool for the analysis of large series of samples in aquatic microbial ecology. However, analysis of the resulting data is often inefficient or does not reflect the complexity of natural communities. Because bacterioplankton assemblages frequently fall into several clusters with respect to their cellular properties, these subgroups seem to be a promising level of abstraction. Image analysis was used to detect clusters from flow cytometry data. The method was tested on a bacterial community under heavy protozoan grazing pressure. METHODS: A bivariate histogram of flow cytometry data was transformed into a gray-scale image for image analysis. After low-pass filtration, regional maxima were delimited by a watershed algorithm. The resulting areas were then used as gates on the original measurements. RESULTS: Three clusters could be detected from the bacterial assemblage. Protozoan grazing had a strong impact on the bacterial community, which could be analyzed in detail at the level of individual subgroups. CONCLUSIONS: Investigation at the level of bacterial subgroups allowed a more detailed analysis than whole-community statistics and delivered essential and ecologically meaningful information. Image analysis proved to be an adequate tool to detect the subgroups without a priori knowledge.

Animals↗
PubMed1990

Methodological aspects on cytochemical DNA assessment of adenocarcinoma of the endometrium by means of image and flow cytometry using conventionally formalin-fixed and paraffin-embedded specimens.

Sometimes widely diverging results have been reported as regards the nuclear DNA ploidy pattern of adenocarcinomas of the endometrium. Since such discrepancies might be due to differences in the techniques applied, it seemed worthwhile to investigate this possibility in conventional uterine curetted specimens. In order to obtain a high incidence of tumours with cancer cell nuclei showing "aneuploid" DNA distribution pattern, a selection was made, so that only those adenocarcinomas that had led to a fetal outcome of the neoplastic disease were examined. The results of two image cytometric (ICM) techniques for cytochemical nuclear DNA assessments were compared. One was direct photographic cytometric measurements on Feulgen-stained sections; the other was densitometric assessments on isolated tumour cell nuclei of deparaffinised and disintegrated specimens. In 39 cases out of 43 the DNA ploidy pattern was the same by means of the two techniques. However, about half the numbers of the specimens (40 out of 83) were lost during the deparaffinisation and disintegration procedure. As far as could be found from a limited study on 20 (out of the 43) selected cases, these losses of specimens became even greater when the flow-cytometric (FCM) technique was applied on the deparaffinised specimens; about one third of these specimens were not possible to evaluate. In addition, in those where assessments by means of FCM could be made, the DNA ploidy pattern obtained differed from that of the two ICM techniques in not less than 80% of the cases. Broad peaks and high amounts of counts in the background in the DNA histograms indicated that most of the DNA assessments made by means of FCM on archival material of the present kind of curetted specimens of endometrial adenocarcinomas gave no reliable results. Consequently, differences in the techniques applied in cytochemical assessments of the nuclear DNA distribution pattern in endometrial carcinomas can explain the more or less controversial results reported from different laboratories.

Adenocarcinoma↗
PubMed1992

Quantitative DNA analysis and proliferation in breast carcinomas. A comparison between image analysis and flow cytometry.

The DNA content and proliferation in 100 invasive breast carcinomas were evaluated by computerized image analysis (IA) and flow cytometry (FCM). For DNA content, image analysis of Feulgen-stained slides of fresh tumor imprints were compared with flow cytometry of propidium iodide-stained disaggregated fresh tumor tissue. The DNA indices obtained by the two methods showed close correlation by linear regression analysis (r = 0.89, p less than .001). There were 44 (44%) diploid and 56 (56%) aneuploid tumors. There was agreement between the two methods in detection of aneuploidy in 81% of tumors. Image analysis required smaller tissue samples, permitted direct visualization and selection of tumor cells, and was more sensitive in detecting tetraploid and highly aneuploid cell populations. In contrast, flow cytometry histograms provided better resolution, and were more effective in detecting multiploid tumors and near-diploid aneuploid tumors. Aneuploidy was significantly related to various adverse prognostic parameters, namely, negative estrogen receptor, high mitotic rate, high histologic and nuclear grades. Proliferation was evaluated by measuring the FCM S phase fraction (SPF), and by image analysis quantitation of immunohistochemical staining using Ki-67 monoclonal antibody. SPF and Ki-67 count showed modest correlation (r = 0.42). Both SPF and Ki-67 count were significantly related to the mitotic rate, histologic and nuclear grades. Our results indicate that the two methods provide comparable results, but offer individual advantages and are complementary techniques in analyzing DNA ploidy and proliferation in breast carcinomas.

Adenocarcinoma, Mucinous↗