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PubMed1994

Identification of two human sperm populations using flow and image cytometry.

Flow cytometric studies of human sperm from fertile men display a constant and characteristic bimodal nonartifactual DNA pattern confirming the existence of two distinct populations. The main population is represented by a peak followed by a shoulder ("marginal population"). The appearance of this marginal population fluctuates with either freezing and thawing or with Percoll gradient centrifugation. We have analyzed both the main and marginal sperm populations by flow cytometry after cell sorting, laser scanning cytometry, light microscopic evaluation, and their sensitivity to DNase digestion. We have observed that the marginal population detected in fertile men represents a sperm group altered in the nuclear condensation, yielding unstable chromatin which appears more stainable with propidium iodide.

Cell Separation↗
PubMed2006

Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry.

BACKGROUND: Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities. METHODS: Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation. RESULTS: Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible. CONCLUSION: The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.

Antigens, CD19↗
PubMed2005

Preoperative detection of laryngeal cancer in mucosal swabs by slide-based cytometry.

The aim of our study was to evaluate slide-based cytometry in screening for laryngeal cancer using swabs a minimally invasive approach. Laser scanning cytometry (LSC) was used for the multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumour cells. Histograms with DI < 0.95, 1.05 < DI < 1.9, and 2.1 < DI were defined as DNA aneuploid. After subsequent haemotoxylin-eosin (HE)-staining, single cells were re-localised and an analysis by conventional cytology was performed. Additionally, routine histopathology of parallel biopsies was obtained in all cases. Fifty one swabs from 49 lesions were analyzed. Seven and 17 swabs, were classified as insufficient for LSC and cytology, respectively. One and two benign lesions, were misclassified as malignant, respectively. Out of 34 malignant lesions, LSC detected 25 and cytology 14. LSC was superior to cytology in all of the statistical parameters tested. This pilot study demonstrates the validity of LSC for the preoperative detection of malignancy in laryngeal tumours using swabs.

Algorithms↗
PubMed2006

Role of ancillary techniques in diagnosing and subclassifying non-Hodgkin's lymphomas on fine needle aspiration cytology.

Non-Hodgkin's lymphomas (NHL) are tumours of the lymphoid cells. During the process of development of lymphoid cells, neoplasia may evolve at any point. Neoplastic cells usually carry the imprint of cell of origin at the stage of origin. Various types of NHL may have similar morphology with wide variation in origin, immunophenotype and other biological features. Different ancillary laboratory techniques may help to overcome the limitations of morphology in this aspect. The commonly used ancillary techniques in lymphomas are immunocytochemistry (IC), flow cytometry, Southern blot (SB) technique, polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH). In addition, laser scanning cytometry (LSC) and DNA microarray technologies are in the research phase. Various laboratory techniques are used for immunophenotyping, demonstration of monoclonality, identification of chromosomal translocation, assessment of cell kinetics and expression of mRNA in the tumour cells. Flow cytometry helps in rapid immunophenotying of NHL and it has an added advantage over IC in recognizing the co-expression of CD markers. Fine needle aspiration cytology (FNAC) combined with flow immunophenotyping may help us to diagnose and subclassify certain NHLs, such as follicular lymphoma and mantle cell lymphoma, which were previously recognized as pure morphological entities. Loss of morphology is one of the important limitations of flow cytometry. LSC can overcome this limitation by studying morphology along with the immunophenotyping pattern of individual cells. Chromosomal changes in NHL can be identified by SB, PCR and FISH. Molecular diagnosis of NHL helps in diagnosis, subclassification, prognostic assessment and even in planning of therapy. DNA microarray is a relatively newer and promising technology. It gives information about the expression of several thousands of genes in a tumour in a single experiment. In the near future, FNAC combined with ancillary techniques may play a major role in diagnosis, subclassification and management of lymphomas.

Biopsy, Fine-Needle↗
PubMed2004

Quantitative histology by multicolor slide-based cytometry.

BACKGROUND: In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about altercations during diseases (leukemia, HIV, AIDS) and their therapeutic regime, as well as in experimental settings. METHODS: We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct and indirect immunofluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single-(Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single-(FITC) up to fourcolor (FITC/PE/PECy5/APC) stained specimen. RESULTS: Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring a identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. CONCLUSIONS: We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Automation↗
PubMed2004

Quantitative histology by multicolor slide-based cytometry.

BACKGROUND: In lymphatic organs, the quantitative analysis of the spatial distribution of leukocytes by tissue cytometry would give relevant information about alterations during diseases (leukemia, HIV, AIDS) and their therapeutic regimen, as well as in experimental settings. METHODS: We have developed a semiautomated analysis method for laser scanning cytometry (LSC) termed "multiple thresholding," which is suitable for archived or fresh biopsy material of human lymph nodes and tonsils. Sections are stained with PI for nuclear DNA and up to four antigens using direct or indirect immunofluorescence staining. Measurement is triggered on DNA-fluorescence (argon laser, Ar) or on specific cell labeling. Due to the heterogeneity of cell density, measurements are performed repeatedly at different threshold levels (low threshold: regions of low cellular density, germinal center; high threshold: dense regions, mantle zone). Data are acquired by single- (Ar) or dual-laser excitation (Ar-HeNe) in order to analyze single- (FITC) up to four-color (FITC/PE/PECy5/APC) stained specimen. RESULTS: Percentage and cellular density of cell-subsets is quantified in different microanatomical regions of the specimen. These data were highly correlated with manual scoring of identical specimens (r(2) = 0.96, P < 0.0001). With LSC, semiautomated operator-independent immunophenotyping in tissue sections of lymphatic organs with up to three antibodies simultaneously is possible. CONCLUSIONS: We expect this tissue cytometric approach to yield new insight into processes during diseases and help to quantify the success of therapeutic interventions.

Automation↗
PubMed2006

A simple correction for cell autofluorescence for multiparameter cell-based analysis of human solid tumors.

BACKGROUND: Corrections that have been proposed to minimize the unwanted contribution of cell autofluorescence to the total fluorescence signal often require either specialized instrumentation or the sacrifice of a data channel so as to perform a measurement that can be used to correct for autofluorescence in individual cells. Here we propose a simple cell by cell correction for autofluorescence that is suitable for multiparameter laser scanning cytometry (LSC) studies in human solid tumors that relies on the ratio of mean autofluorescence to mean total cell fluorescence (mean Flauto/mean Fltotal). This approach assumes a correlation between the autofluorescence component and the total signal in individual cells. This correction does not require specialized instrumentation, and does not sacrifice a data channel in multiparameter studies. A potential disadvantage is that errors may be introduced by the assumption of a correlation between the two components of the total fluorescence signal in individual cells in samples in which no such correlation exists. METHODS: Distributions of cell autofluorescence and total Her-2/neu cell fluorescence were obtained separately by LSC in three human breast cancer cell lines and in three samples of primary human lung cancer. In the breast cancer cell lines, autofluorescence measurements and Her-2/neu measurements were also obtained on the same cells. RESULTS: We show that there is a partial correlation between autofluorescence and total Her-2/neu/FITC fluorescence in individual cells in the three breast cancer cell lines. We also show that the results of a ratio-based autofluorescence correction agree with those based on a true cell by cell correction. Computer simulation studies suggest that in samples with no correlation between the autofluorescence component and the true probe/dye fluorescence component, the ratio correction produces robust estimates of the mean true fluorescence signal, with relatively small but systematic underestimates of the coefficient of variation of such measurements under conditions commonly encountered in the measurement of human solid tumors. CONCLUSIONS: A simple cell by cell correction for autofluorescence based on the ratio of mean Flauto to mean Fltotal can be applied in cell samples in which there is a correlation between cell autofluorescence and true probe/dye fluorescence in individual cells. In cell samples that lack this correlation, or in which it is not known whether such a correlation exists, this correction can be used with the reservation that there is a systematic but relatively small underestimation of the degree of variability of the measurements.

Breast Neoplasms↗
PubMed2006

Prediction of upper aerodigestive tract cancer by slide-based cytometry.

AIM: To evaluate slide-based cytometry in screening for and following up of carcinoma of the upper aerodigestive tract using swabs for a minimal-invasive approach. METHODS: Laser scanning cytometry (LSC) was used for multiparametric analysis of cells stained for cytokeratin and DNA to determine the DNA-index (DI) of the tumor cells. Histograms with 0.95 < DI < 1.05 and 1.9 < DI < 2.1 were defined as DNA euploid and any other DI as DNA aneuploid. After subsequent HE-staining, single cells were relocalized in order to document morphology. Conventional cytology was also performed on a subset of the slides. Routine histopathology of parallel biopsies served as gold standard in all cases. RESULTS: 115 swabs from 109 patients were obtained from the entire upper aerodigestive tract. 16 swabs were classified as insufficient for LSC. In the remaining 99 specimens, 1 benign lesion was misclassified as malignant, while 61 of the 75 malignant lesions were correctly identified. This corresponds to predictive values of 98.4% and 62.2% for the detection of malignant and benign samples by LSC. CONCLUSION: This pilot study demonstrates the validity of LSC screening for the identification of tumor malignancy in the upper aerodigestive tract from swab collected cytological material.

Humans↗
PubMed2006

Iterative restaining as a pivotal tool for n-color immunophenotyping by slide-based cytometry.

AIM: Slide-based cytometry (SBC) allows to "ask a cell a second time." We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). METHODS: PBLs primarily stained for CD-markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single-cell level. RESULTS: We analyzed six virtual immunostainings by "recycling" the PE-channel for four CD-markers. CONCLUSION: Iterative restaining might prove to be a pivotal tool for n-color immunophenotyping exclusive to SBC concepts.

Antibodies↗
PubMed2004

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells.

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.

Apoptosis↗
PubMed2005

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations.

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining beta-cell viability. For these reasons we have developed novel methods for the specific assessment of beta-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of beta-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.

Apoptosis↗
PubMed2005

Global genomic changes induced by two-stage carcinogen exposure are precancerous alterations in non-transformed human liver epithelial THLE-3 cells.

Global genomic changes, including DNA aneuploidy, may be necessary for carcinogenesis; however, such genomic changes in precancerous cells have not been studied extensively. To identify early global genotypic changes associated with precancerous lesions, a non-transformed human liver epithelial cell line, THLE-3, was treated with benzo[a]pyrene or N-methyl-N-nitro-N-nitrosoguanidine, then by 12-O-tetradecanoyl-phorbol-13-acetate, resulting in morphological transformation of cells. We examined genotypic changes of the transformed cells by laser scanning cytometry, fluorescence in situ hybridization, and comparative genomic hybridization. Transformed fusiform cells displayed tetraploidy, chromosomal instability, DNA copy number aberrations. Cells with these changes were still in the precancerous stage. However, it is suggested that these global genomic changes including tetraploidization provide cells with genetic alterations leading to cancer.

Benzo(a)pyrene↗
PubMedDate not supplied

Tracking lymphocytes in vivo.

The ability to track antigen (Ag)-specific lymphocyte populations in vivo has greatly increased our understanding of the location and functional status of these cells throughout the course of an immune response. Recent technical advances have enhanced researchers' capability to follow migration, activation and cellular interactions of Ag-specific lymphocytes in situ. It is now possible to monitor changes in T cell subsets, co-stimulatory molecules, and chemokine expression within the physiological context of secondary lymphoid organs. Furthermore, the Ag-presenting cell-T cell interaction can be studied,thus dissecting the role and timing of Ag presentation of particular dendritic cell subsets in the initiation of the immune response. The capacity to adoptively transfer small populations of Ag-specific T lymphocytes has also increased our knowledge of the physiologically important role of regulatory T cells in autoimmunity and immunosuppression. New fluorescence imaging techniques such as multicolor video microscopy, laser scanning cytometry, and multiphoton tissue imaging have provided new ways in which researchers can track cellular changes within Ag-specific lymphocytes in vivo. This review summarizes some of the ways in which these techniques have led to discoveries in the role of signaling cascades, cell cycle progression, and apoptosis in maintaining an Ag-specific immune response.

Animals↗
PubMed2004

Micropatterning of endothelial cells by guided stimulation with angiogenic factors.

Micropatterning technology holds significant promise in the development of micro/nanomedical devices. The precise control of cell position and migration is important in several applications. For example, the optimal design of implantable devices depends on the implant material's micro-and nano-texture, which influences the response of nearby tissue, including the microvessels. Therefore, we were interested in endothelial cell positioning and colonization on specific surface domains in the size range of microvasculature. To this end, endothelial cells were seeded in microfabricated grooves and exposed to vascular endothelial growth factor (VEGF), which plays a key role in the angiogenic response. Patterned silicon wafers with grooves of 50 microm width and depth and 150 microm groove spacing were used. Each patterned region had two semicircular ports at either end, one of which was used to seed human retinal endothelial cells (HREC) and the other to house VEGF embedded in Matrigel. After 1 week, cells were fixed and analyzed by laser scanning cytometry (LSC). Our results shows that we can control HREC seeding and positioning in surface grooves and that the speed of colonization of the grooves can be manipulated by local VEGF application. We were able to quantify this effect, showing that HREC relocate inside the grooves twice as fast in response to VEGF stimulation, compared to control conditions, at a speed of 3.14 +/- 0.01 and 1.55 +/- 0.01 microm/min, respectively. Our approach could be used towards the fabrication of "designer" substrates or devices that not only allow patterned cell growth, but also permit dynamic cell repositioning.

Angiogenesis Inducing Agents↗
PubMed2005

Apoptosis and autophagy induced by TGF-B1 in bovine mammary epithelial BME-UV1 cells.

Mammary gland growth and involution is based on a dynamic equilibrium between proliferation and apoptosis of mammary gland epithelial cells (MEC). TGF-beta1 is an important antiproliferative and apoptogenic factor for mammary gland epithelial cells, acting in auto/paracrine matter and thus considered an important local regulator of mammary tissue involution. So far the studies on mammary gland involution concerned only apoptosis as a type I of MEC programmed cell death (PCD). Autophagy is known to be type II of PCD and this paper is the first, supporting evidence for the TGF-beta1-induced autophagy in bovine mammary epithelial cell line BME-UV1, as a distinct to apoptosis type of PCD. Laser scanning cytometry and confocal microscopy were used for analysis of MAP1 LC3 and Beclin 1 expression - two proteins considered being the most reliable biochemical markers of autophagy. The significant increase of MAP1 LC3 and Beclin 1 expression in cells treated with TGF-beta1 (2 ng/ml) was observed. Ultrastructural observation in electron microscopy revealed that autophagy is not only alternative, but also complementary to apoptosis type of cell death in TGF-beta1-treated bovine MEC. It was manifested by typical morphological features of apoptosis (cell shrinkage, margination and condensation of chromatin) and autophagy (autophagosomes, autophagic vacuoles) in the same cell.

Animals↗
PubMed2006

HER2 expression as a potential marker for response to therapy targeted to the EGFR.

Since human epidermal growth factor receptor 2 (HER2) is known to participate with the epidermal growth factor receptor (EGFR) in mitogenic signalling, we hypothesised that HER2 overexpression might indicate responsiveness to EGFR targeted therapies. MCF7 breast cancer cells transfected with the HER2 gene were subcloned to establish a set of genetically related cell lines expressing graded levels of HER2 by immunoblot analysis. The subcloned cell lines and parental MCF7 cells were characterised by their growth characteristics, and cell by cell patterns of EGFR, HER2 and HER3 expression as well as levels of phosphorylated mitogen-activated protein kinase (MAPK) and AKT by laser scanning cytometry (LSC). Growth inhibition assays were used to characterise response to EGFR targeted therapy, and to determine the relationship between therapeutic response and levels of tyrosine kinase expression. The levels of growth inhibition of AG1478 and of the AG1478-trastuzumab combinations were correlated with levels of HER2 expression among the different cell lines. Among EGFR, HER2 and HER3, HER2 overexpression was the best single predictive marker, but combinations of two markers provided additional predictive information.

Antibodies, Monoclonal↗
PubMed2005

Development and validation of algorithms for measuring G-protein coupled receptor activation in cells using the LSC-based imaging cytometer platform.

BACKGROUND: A cell-based assay system (Transfluor) has been developed for measurement of G-protein coupled receptor (GPCR) activity by using cells transfected to express a fusion protein of arrestin plus green fluorescent protein (GFP) and the target GPCR. Upon agonist stimulation, the arrestin-GFP translocates to and binds the activated GPCR at the plasma membrane. The receptor/arrestin-GFP complexes then localize in clathrin-coated pits and/or intracellular vesicles. This redistribution of arrestin-GFP into condensed fluorescent spots is useful for visually monitoring the active status of GPCRs and its quantitation is possible with certain types of digital image analysis systems. METHODS: We designed two lines of image processing algorithms to carry out quantitative measurement of the arrestin-GFP movement on an inverted version of laser scanning cytometry (iCyte) as an imaging platform. We used a cell line expressing arrestin-GFP and the wild-type beta2-adrenergic receptor or a modified version of this receptor with enhanced affinity for arrestin. Each cell line was challenged with various concentrations of agonist. RESULTS: A dose-dependent signal was measured and half-maximal effective concentration values were obtained that agreed well with results determined by other methods previously reported. CONCLUSIONS: The results indicate that the combination of Transfluor, iCyte, and our algorithms is suitable for robust and pharmacologically relevant GPCR ligand exploration.

Algorithms↗
PubMed2006

Biological activities of a recombinant adenovirus p53 (SCH 58500) administered by hepatic arterial infusion in a Phase 1 colorectal cancer trial.

The major focus of intrahepatic arterial (IHA) administration of adenoviruses (Ad) has been on safety. Currently, there is little published data on the biological responses to Ad when administered via this route. As part of a Phase I study, we evaluated biological responses to a replication-defective adenovirus encoding the p53 transgene (SCH 58500) when administered by hepatic arterial infusion to patients with primarily colorectal cancer metastatic to the liver. In analyzing biological responses to the Ad vector, we found that both total and neutralizing Ad antibodies increased weeks after SCH 58500 infusion. The fold increase in antibody titers was not dependent on SCH 58500 dosage. The proinflammatory cytokine interleukin-6 (IL-6) transiently peaked within 6 h of dosing. The cytokine sTNF-R2 showed elevation by 24 h post-treatment, and fold increases were directly related to SCH 58500 doses. Cytokines TNF-alpha, IL-1beta, and sTNF-R1 showed no increased levels over 24 h. Predose antibody levels did not appear to predict transduction, nor did serum Ad neutralizing factor (SNF). Delivery of SCH 58500 to tumor tissue occurred, though we found distribution more predominantly in liver tissues, as opposed to tumors. RT-PCR showed significantly higher expression levels (P<0.0001, ANOVA) for adenovirus type 2 and 5 receptor (CAR) in liver tissues, suggesting a correlation with transduction. Evidence of tumor-specific apoptotic activity was provided by laser scanning cytometry, which determined a coincidence of elevated nuclear p53 protein expression with apoptosis in patient tissue. IHA administration of a replication defective adenovirus is a feasible mode of delivery, allowing for exogenous transfer of the p53 gene into target tissues, with evidence of functional p53. Limited and transient inflammatory responses to the drug occurred, but pre-existing immunity to Ad did not preclude SCH 58500 delivery.

Adenoviridae↗