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PubMed1998

Pleural carcinosis confirmed by adjuvant cytological methods: a case report.

We describe a case of a 43-yr-old woman presenting a progressive pleural effusion. The patient was known to have an acute myeloid leukemia in complete remission after allogenic bone marrow transplantation. Suspicion of pleural carcinosis was raised cytologically and confirmed by immunocytochemistry, DNA-cytometry, and atomic force microscopy. We emphasize the use of these additional methods for the distinction of adenocarcinoma cells in effusions from reactive mesothelial cells.

Acute Disease↗
PubMed2003

Immune consequences of pediatric and adult cardiovascular surgery: report of the 7th Leipzig workshop.

Cardiovascular surgery in children and adults is among the most common types of interventions in the western hemisphere for innate and acquired defects. In the recent decades, the risk of cardiovascular surgery has been reduced substantially. Nevertheless, open heart surgery is risky for the patient and can lead to postoperative complications such as postpericardiotomy syndrome, capillary leak syndrome, or multiple organ failure. To gain further understanding into the response to cardiovascular surgery, it is necessary to join forces from several disciplines of medicine and natural sciences. Interdisciplinarity is the basic concept of the Leipzig Workshop. The consensus of the workshop was that cardiovascular surgery with cardiopulmonary bypass induces a systemic antiinflammatory response due to (a) elimination of activated cells, (b) compensatory reaction to a local proinflammatory responses, (c) interleukin-10 release, (d) anesthetics and medication, and (e) leukocyte extravasation. The subsequent proinflammatory reaction is the response to surgical trauma modulating the antiinflammatory reaction. Novel therapeutic approaches include the introduction of autologous endothelial progenitor cells from the peripheral blood into the sites of injury. The analysis of immune response and outcome prediction require novel analytical tools that allow fast, accurate, and quantitative determination of the desired parameters in a multiplexed manner (i.e., cytomics), such as flow cytometric microbead array assays and slide-based cytometry. The major goal is predictive medicine by cytomics, i.e., the individualized risk assessment by analyzing the cytome in combination with sophisticated data pattern recognition. These developments may lead to individualized therapy for the benefit of the patient and cost reduction.

Adult↗
PubMed2004

A murine model of ex vivo angiogenesis using aortic disks grown in fibrin clot.

The rat aortic ring model is well utilized for evaluation of angiogenesis. We report here an alternative assay employing an ex vivo mouse aorta angiogenesis model that can be extensively manipulated and serially evaluated using digital-assisted image analysis. Mouse aortas were harvested, cut into 2-mm disks, and cultured in fibrin matrix with growth media. Radial vascular outgrowths arose from the cut edge of the aortic disk and were digitally photographed and serially quantified. A variety of culture conditions were evaluated to determine their ability to alter angiogenesis in this model. Vessel outgrowth became apparent on day 3 and continued through day 10 with linear growth occurring between days 3 and 6. Increasing concentrations of serum from 0% to 40% resulted in stimulation of angiogenesis after day 3. Suramin and endostatin dramatically inhibited angiogenesis, which was more profound when applied at day 0 than when linear growth could be identified (day 3). Cells isolated from vessel outgrowths were predominantly endothelial in origin by immunocytochemistry and FACS analysis. We demonstrate that angiogenesis in an ex vivo murine model can be easily quantified using digital image analysis, responds appropriately to stimulation and inhibition, and exhibits differential results based on time of inhibitor administration. Antiangiogenic agents may be most effective if administered before development of accelerated vessel growth.

Animals↗
PubMed1998

Blastic variant of mantle-cell lymphoma: cytomorphologic, immunocytochemical, and molecular genetic features of tissue obtained by fine-needle aspiration biopsy.

Mantel-cell lymphoma (MCL) is a rare type of non-Hodgkin's lymphoma that has a moderately aggressive clinical course, generally between that a low-grade and intermediate-grade lymphomas. However, a small subset of MCLs, the so-called "blastic" variant, exhibits a poor prognosis and an aggressive clinical course. We describe a case of blastic MCL that occurred in a 64-yr-old man and that was diagnosed and accurately subclassified as blastic MCL on the basis of an fine-needle aspiration (FNA) biopsy. The aspirate smears showed a monotonous population of intermediate-sized lymphocytes with irregular nuclear contours, finely dispersed nuclear chromatin, and inconspicuous nucleoli. Material was obtained by FNA for ancillary studies (immunocytochemical stains, flow cytometry, cytogenetics, image analysis, and molecular studies) that supported the diagnosis of blastic MCL. Surgical biopsy confirmed the diagnosis. These findings underscore the utility of FNA in diagnosing lymphomas, particularly when the cytomorphologic examination is combined with appropriate ancillary studies.

Biopsy, Needle↗
PubMed1997

Automatic volumetric capillary cytometry for counting white cells in white cell-reduced plateletpheresis components.

BACKGROUND: As the benefits of white cell (WBC)-reduced blood components become increasingly apparent, the need has arisen for a simple, automated WBC-counting technique that is sensitive to low WBC concentrations. Automated volumetric capillary cytometry was evaluated for its ability to quantify residual WBCs in WBC-reduced plateletpheresis components. STUDY DESIGN AND METHODS: The volumetric capillary cytometry system evaluated uses a laser to excite fluorescent dye-labeled nucleated cells. The number of nucleated cells per microliter is reported. Four studies were performed: linearity, precision of results near the value of 5 x 10(6) WBCs per unit, the limit of detection, and correlation to the Nageotte manual counting method. RESULTS: Assay values correlated to expected values (range, 0-125 WBC/microliter) with an r2 > 0.99. In the range of 5 x 10(5) WBCs per unit the CV was 8.5 percent, and concentration differences of 0.15 log10 were detectable. The limit of detection was 1.0 WBCs per microliter (95% upper confidence limit). The assay correlated to the Nageotte method with an r2 of 0.98, slope of 1.0, and y-intercept of 2.0 WBCs per microliter. Assay results were 10 to 15 percent higher than Nageotte results, in samples with values near 5 x 10(6) WBCs per unit. Technician time per sample was 2 to 3 minutes. CONCLUSION: Volumetric capillary cytometry is precise and sensitive to small differences in WBC concentration in the range of clinical interest. The device provides an efficient new method for quality assurance and control of WBC-reduced plateletpheresis products.

Blood Component Removal↗
PubMed2006

Slide-based cytometry for cytomics--a minireview.

In the postgenomic era, to gain the most detailed quantitative data from biological specimens has become increasingly important in the emerging new fields of high-content and high-throughput single-cell analysis for systems biology and cytomics. Areas of research and diagnosis with the demand to virtually measure "anything" in the cell include immunophenotyping, rare cell detection and characterization in the case of stem cells and residual tumor cells, tissue analysis, and drug discovery. Systemic analysis is also a prerequisite for predictive medicine by genomics, proteomics, and cytomics. This issue of Cytometry Part A is dedicated to innovative concepts of system wide single cells analysis and manipulation, new technologies, data analysis and display, and, finally, quality assessment. The manuscripts to these chapters are provided by cutting edge experts in the fields. This overview will briefly highlight the most important aspects of this continuously developing field.

Animals↗
PubMedDate not supplied

Quantitative description of morphologic changes effected by antileukemic agents in L1210 leukemia cells.

The effect of antileukemic agents is explained mainly by the inhibition of DNA synthesis, which may reflect the structural alteration of the target cells. To identify such cellular alterations, in particular morphologic changes associated with the exposure to antileukemic agents were investigated in the murine leukemic L1210 cell line using flow cytometry and image analysis. Cells were cultured with eight kinds of antileukemic agents in cytostatic concentrations which are clinically achievable. Living cells were observed in culture without fixation or staining. Two parameters related to cell size and shape were measured simultaneously by the image analysing computer. These parameters provided us with different criteria for characterizing the antimetabolites, DNA-strand-damage inducers and other agents tested. Such morphologic studies may make it possible to estimate the characteristics of various antileukemic agents.

Animals↗
PubMed2004

Implementation of DNA cytometric measurements in fine-needle aspiration biopsy diagnostics of breast disease.

BACKGROUND: For this study, the diagnostic protocol included fine-needle aspiration biopsy (FNAB) and DNA cytometric measurements. The objective of the study was to improve the diagnostic sensitivity of FNAB. METHODS: Sixty-eight FNAB samples were stained using the Feulgen stain, and DNA histograms were produced by selecting the nuclei of cell groups and free cells separately. RESULTS: Among 28 samples that were classified as definitely benign (n = 17 samples) or atypical but benign (n = 11 samples), DNA cytometry generally showed diploid/peridiploid peaks. Three of those samples were associated later with carcinoma. In those samples DNA cytometry did not improve sensitivity. Among moderately atypical samples (n = 17 samples), 8 samples were diagnosed later as carcinoma. DNA cytometry helped the diagnostic procedure in 62.5% of those samples. Among 21 samples that were classified as highly suspicious (n = 10 samples) or definitely malignant (n = 11 samples), DNA cytometry generally showed atypical DNA histograms (20 of 21 histograms), and DNA cytometry supported the diagnosis of carcinoma in 95.2%. Histograms that were based on free cells frequently were more abnormal compared with histograms that were based on cell groups. Histologically verified benign lesions also could show abnormalities in DNA histograms. Accepting wider gates than are used normally for primarily Feulgen stained samples on these restained samples resulted in improved specificity, efficiency, and predictive values. CONCLUSIONS: DNA cytometry has a potential to support the differential diagnosis of breast lesions, and sampling of free cells increases sensitivity. Benign breast lesions (fibrocystic disease, fibroadenoma) included DNA-cytometrically abnormal cell clones and showed tendencies toward polyploidy, which should be included in the diagnostic criteria.

Biopsy, Needle↗
PubMed2001

Laser scanning cytometry: a novel method for the detection of platelet--endothelial cell adhesion.

BACKGROUND: Adherence of platelets to endothelial cells may be a significant event in the development of vascular thrombosis. Existing models, which examine platelet-endothelial cell interactions, compromise endothelial cell integrity or use radioactivity to identify platelets that adhere to endothelial cells. We report a novel method for in vitro detection of platelet-endothelial cell adhesion that allows endothelial cells to remain as an intact monolayer and for visualization of individual platelets. METHODS: Fluorescently labeled platelets were incubated with a confluent monolayer of endothelial cells. Laser scanning cytometry (LSC) identified platelets bound to endothelial cells based on their fluorescent signals. RESULTS: LSC detection of platelets reliably reproduced well-described findings of thrombin-induced platelet-endothelial cell adhesion. Results demonstrating reduced adhesion with a glycoprotein IIb-IIIa-specific blocking monoclonal antibody confirmed the specificity of the LSC detection of platelet-endothelial cell adhesion. CONCLUSIONS: LSC is a novel method for detecting platelet--endothelial cell adhesion. Its advantages over other methods are: (a) endothelial cells remain undisturbed and adherent throughout; (b) the ability to detect individual bound platelets and subpopulations; (c) the ability to store images and slides and then relocate, revisualize, and reanalyze individual cells or cell populations of interest; and (d) no radioactivity.

Adenosine Diphosphate↗
PubMed2002

[Comparative DNA cytometric investigations on aneurysmal bone cysts and giant cell tumors].

Giant cell-rich bone lesions consist of tumor-like lesions and true neoplastic giant cell tumors. In this study it was investigated whether DNA cytometry may contribute to the differential diagnosis between aneurysmal bone cysts and giant cell tumors. Statistically significant differences in the frequency of tetraploid stemlines were detected. Nevertheless, the knowledge of age, localization and radiological signs in addition to morphological findings are essential to distinguish between these lesions.

Adolescent↗
PubMed2002

Characteristic pattern of genetic aberrations in ovarian granulosa cell tumors.

The cytogenetic abnormalities of granulosa cell tumors (GCT) of the ovary are only partially known. Up to now, mainly numerical chromosomal aberrations have been described. Therefore we performed a comprehensive study on paraffin-embedded material of 20 GCT (17 adult, 3 juvenile; patient age between 16 and 78 y) combining comparative genomic hybridization (CGH); fluorescence in situ hybridization (FISH) using DNA-specific probes for chromosome 12, 17, 22, and X; DNA cytometry; and immunohistochemistry (inhibin, p53, Ki67). By DNA cytometry, 16 of 20 tumors (80%) were diploid. However, 6 of 16 diploid tumors (37%) showed aberrations by FISH. FISH revealed monosomy 22 in 8/18 cases (40%); trisomy 12 in 5/20 (25%); monosomy X in 2/20 (10%); and loss of chromosome 17 in one case (5%). The main findings by CGH were gains of chromosomes 12 (6 cases, 33%) and 14 (6 cases, 33%) and losses of chromosomes 22 (7 cases, 35%) and X (1 case, 5%), mostly comprising whole chromosomes or chromosome arms. Inhibin and p53 were expressed in 100% and 95% of the tumors, respectively. The Ki67 index ranged from 0% to 61%. Neither immunohistochemistry, nor DNA cytometry and molecular genetic analysis, provided statistically significant prognostic information. In summary, our study reveals a distinctive pattern of cytogenetic alterations in GCT. Our observations confirm earlier reports that trisomy 12 and 14 are frequent aberrations; however, monosomy 22 seemingly is even more prevalent.

Adolescent↗
PubMed1999

[Diagnosis and therapy of cervical squamous epithelial dysplasias].

Diagnosis of cervical dysplasia is based on colposcopy, cytology and histology. In case of suspect cytology, the management (controls only, extended diagnosis by histology or therapy without additional histological control) is dependent on colposcopic findings. Biopsy or endocervical curettings are necessary in cases of suspect ectocervical findings or endocervical lesions, respectively. As a rule, HE staining is sufficient for histological diagnosis. Yet, additional prognostic information is obtained by HPV-analysis and DNA-cytometry. Grading of dysplasia should be done according to the most recent WHO/ISGYP criteria. The exact diagnosis as to the grading and extension of dysplasia is the prerequisite of an effective individually adjusted therapy. For ablative therapy, gynecologists have to focus their attention on modern organ preserving surgery strategies (loop-excision).

Cervix Uteri↗
PubMed1999

Application of laser scanning cytometry to the analysis of chromosomal aberrations induced by benzo[a]pyrene in CHO-WBLT cells.

BACKGROUND: A recently developed laser scanning cytometry technique was applied to cytometric studies to detect rapidly stable chromosomal aberrations induced by a carcinogen in a Chinese hamster fibroblast cell line, CHO-WBLT. METHODS: Individual chromosomes were collected from metaphase cells by a syringe technique and spread on slides. The DNA content of each chromosome stained with propidium iodide was measured with a laser scanning cytometer (LSC). A characteristic DNA histogram, designated as the "laser scanning karyotype (LSK)," was obtained from about 20,000 chromosomes of CHO-WBLT cells. Each chromosome was confirmed morphologically under the microscope by using a "re-location" system built into the LSC. RESULTS: A total of 21 chromosomes, including marker chromosomes specific to the cell line, were assigned to 10 major peaks in the LSK, which was analogous to the karyotype demonstrated with the classical Q-banding technique. In contrast, clonal sublines isolated after exposure to the carcinogen benzo[a]pyrene showed LSKs different from those found in untreated control cells, and seven of 20 clones were found to be abnormal, with a small number of chromosomal translocations and/or deletions, which were confirmed by Q-banding. CONCLUSIONS: The laser scanning cytometry technique was employed to detect stable chromosomal aberrations in CHO-WBLT cells after treatment with benzo[a]pyrene. The results obtained with this technique were comparable to those obtained by Q-banding; therefore, this method may be useful for rapid primary screening to detect stable, abnormal karyotypes induced by environmental chemicals and/or radiation.

Animals↗
PubMed2000

The development of a cell array and its combination with laser-scanning cytometry allows a high-throughput analysis of nuclear DNA content.

We developed a cell array device for analyzing cellular characteristics such as DNA ploidy, numerical chromosomal aberrations, and antigen expression in multiple specimens in a single experiment. Fifty (10 x 5) spots, 2 mm in diameter, were arrayed in an area of 30 x 16 mm on a glass slide, and approximately 1,000 cells were placed on each spot. To demonstrate the usefulness of the cell array, we measured nuclear DNA content using laser-scanning cytometry for DNA ploidy analysis in nine human tumor cell lines and normal lymphocytes. Combining the cell array with laser-scanning cytometry allows not only measurement of nuclear DNA content for 50 samples but also easy comparison of DNA ploidy among the samples in a single experiment. In addition, we used the cell array for fluorescence in situ hybridization using a DNA probe specific for the pericentromeric region of chromosome 11.

Cell Count↗
PubMed2006

Object-oriented image analysis for high content screening: detailed quantification of cells and sub cellular structures with the Cellenger software.

BACKGROUND: Detailed image analysis still is a considerable bottleneck for many cellular assays, and automated solutions to the problem are desirable. However, dealing with the complexity and variability of structures in cellular images makes detailed and reliable analysis a nontrivial task. METHODS: Therefore, based on the object-oriented image analysis approach, a novel image analysis technology, a flexible and reliable system for image analysis in cellular assays was developed. It contains a library of predefined, adaptable modules, each of them developed for a specific analysis task. The system can be configured easily by combining appropriate modules and adapting them interactively to the specific image data, if necessary. By representing cells and sub cellular structures within a network of interlinked image objects, a large number of parameters can be derived that describe shape, intensity, and relevant structural and relational aspects of any chosen class of structures. RESULTS: Thus, multi-parameter analysis and multiplexing are supported. A sample application based on this approach demonstrates that GFP signals can be distinguished based on their properties and the relative location within the cell.

Cell Membrane↗
PubMed1997

Ploidy profile of morphologically normal squamous epithelium adjacent to high grade cervical intraepithelial neoplasia.

We have investigated the ploidy profile of morphologically normal mucosa adjacent to high grade CIN (n = 16) and also from normal cervix (n = 18). DNA ploidy was assessed using flow cytometry and image analysis. All cases were diploid by both modalities. Our results show that morphologically normal squamous mucosa has a stable ploidy profile even when it lies adjacent to high grade CIN. This finding supports the view that high grade CIN is a neoplastic expansion of transformed cells rather than the result of a field change effect.

Epithelium↗
PubMedDate not supplied

[Study of the statistical characteristics of latex suspensions using the Magiscan-2 image analyser].

Characteristics of latex suspensions are studied in order to estimate their potential metrologic application. The study is carried out on the set "Magiscan 2" with the standard programs. Histograms for distribution of perimeters and particle diameters are determined, and the correlation between them is specified. Also presented are the histograms for distributing optical particle density. The correlation between optical parameters and the geometry of particles is found as well as the orientation and correlation between the optical density of particles and their location on the microscopic stage.

Flow Cytometry↗
PubMed1997

Two-photon fluorescence lifetime imaging microscopy of macrophage-mediated antigen processing.

Two-photon fluorescence lifetime imaging microscopy was used noninvasively to monitor a fluorescent antigen during macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and protease-dependent processing. Fluorescein-conjugated bovine serum albumin (FITC-BSA) served as the soluble exogenous antigen. As a relatively nonfluorescent probe in the native state, the antigen was designed to reflect sequential intracellular antigen processing events through time-dependent changes in fluorescence properties. Using two-photon lifetime imaging microscopy, antigen processing events were monitored continuously for several hours. During this time, the initial fluorescein fluorescence lifetime of 0.5 ns increased to approximately 3.0 ns. Control experiments using fluorescein conjugated poly-L-lysine and poly-D-lysine demonstrated that the increase in fluorescence parameters observed with FITC-BSA were due to intracellular proteolysis since addition of the inert D-isomer did not promote an increase in fluorescence lifetime or intensity. Comparisons of intravacuolar and extracellular FITC-dextran concentration suggested active localization of dextran in the vacuoles by the macrophage. In addition, the kinetics of degradation observed using two-photon microscopy were similar to results obtained on the flow cytometer, thus validating the use of flow cytometry for future studies.

Animals↗