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Results for “Laser Scanning Cytometry”

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At least 19 recordsLinked to original sources

PubMed1998

Five-color immunophenotyping plus DNA content analysis by laser scanning cytometry.

Laser scanning cytometry is a new laboratory technology similar to flow cytometry but with advantages for certain clinical and research applications. To date, laser scanning cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens. Several other interesting applications are also in development. In general, advantages of laser scanning cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells-allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the laser scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most laser scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology.

DNA↗

PubMed1993

[Analytic and preparative laser scanning cytometry].

Laser scanning cytometry for analysis and preparation is viewed by some as a blend between flow cytometry and image analysis, since it allows to measure and localize fluorescence and to obtain morphologic or morphometric information on adherent or suspended cells or tissues. However, laser scanning cytometry has additional capabilities such as kinetic studies (slow or rapid) of live cells or measurement of fluorescence recovery after photoblinding. Most of these studies can be performed with great accuracy on localized zones by adding a confocal microscope system and performing three-dimensional image reconstruction. Studies on some of the novel possibilities of laser scanning cytometry are very scant.

Calcium Channels↗

PubMed2000

Multiparameter analysis of human epithelial tumor cell lines by laser scanning cytometry.

Laser scanning cytometry (LSC) is a relatively new slide-based technology developed for commercial use by CompuCyte (Cambridge, MA) for performing multiple fluorescence measurements on individual cells. Because techniques developed for performing four or more measurements on individual lymphoid cells based on light scatter as a triggering parameter for cell identification are not suitable for the identification of fixed epithelial tumor cells, an alternative approach is required for the analysis of such cells by LSC. Methods for sample preparation, event triggering, and the performance of multiple LSC measurements on disaggregated fixed human cells were developed using normal lymphocytes and two human breast cancer cell lines, JC-1939 and MCF-7, as test populations. Optimal conditions for individual cell identification by LSC were found to depend on several factors, including deposited cell density (cells per unit area), the dynamic range of probe fluorescence intensities, and intracellular distribution of the fluorescent probe. Sparsely deposited cells exhibited the least cell overlap and the brightest immunofluorescent staining. Major advantages of using DNA probes over a cytoplasmic immunofluorescent protein marker such as tubulin for event triggering are that the former exhibit greater fluorescence intensity within a relatively sharply demarcated nuclear region. The DNA-binding dye LDS-751 was found to be suboptimal for quantitative DNA measurements but useful as a triggering measurement that permits the performance of simultaneous fluorescein isothiocyanate-, phycoerythrin-, and indodicarbocyanine-based measurements on each cell. A major potential advantage of LSC over flow cytometry is the high yields of analyzable cells by LSC, permitting the performance of multiple panels of multicolor measurements on each tumor. In conclusion, we have developed and optimized a technique for performing multiple fluorescence measurements on fixed epithelial cells by LSC based on event triggering using the DNA-binding dye LDS 751. Although not ideal for quantitative measurements of cell DNA content, the large Stokes shift of this dye permits the performance of three or more additional fluorescence measurements on each cell.

Breast Neoplasms↗

PubMed2004

Comparison of flow cytometry and laser scanning cytometry for the analysis of CD34+ hematopoietic stem cells.

BACKGROUND: Characterization of hematopoietic stem cells (HSCs) by laser scanning cytometry (LSC) was compared with conventional flow cytometry (FCM). The method was evaluated for application in the development of advanced cell culture substrates that were supposed to support the ex vivo expansion of HSC. For this purpose, adherent HSCs were grown in culture on thin polymer films coated with reconstituted collagen I fibrils and subsequently analyzed by LSC. METHODS: CD34+ HSCs were isolated from cord blood by immunomagnetic separation and cultivated on polymer films coated with reconstituted collagen I fibrils. Cell surface antigens (CD34, CD29) were stained with antibodies, and nuclei were labeled with a DNA stain (TO-PRO-3 iodide) that does not interfere with the fluorochromes of the antibodies. Fluorescence intensity of the adherent cells was measured by means of LSC. Before and after in vitro expansion for time periods of up to 7 days, suspension cells were analyzed with LSC and FCM. RESULTS: LSC-based analysis enabled reliable quantification of CD34+ cells with bright antigen expression before cell culture. At this stage, LSC and FCM data for CD34 expression at given HSC samples largely coincided. After in vitro expansion, LSC data deviated from FCM data for cells with dim CD34 antigen expression, whereas the fluorescence intensity of the CD29 antigen remained comparable. The deviation between LSC and FCM data for CD34dim was attributed to the better resolution of weak fluorescence by FCM. Based on the preceding evaluation of the method, LSC analysis could be applied to characterize HSCs cultivated on collagen I-coated polymer films without detachment of the cells from the substrate. CONCLUSIONS: LSC-based analysis allows for the automated evaluation of adherent HSCs. Although resolution of weakly expressed antigens can be achieved more precisely with FCM, the method provides a valuable tool to study interactions of HSCs with bioartificial substrates.

Antigens, CD34↗

PubMed1998

Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.

The determination of recombinant adenoviral (rAd) infectivity and p53 protein expression is important for the evaluation of rAd vectors containing the p53 gene (rAd-CMV-p53) for gene therapy. We have previously reported that rAd5-CMV-p53 vectors can be assessed for infectivity and concomitant p53 protein expression in single- and two-color assays using intracellular staining methodology and flow cytometric analysis. We have compared the flow cytometry-based assays for rAd infectivity (hexon protein) and p53 protein expression with the new slide-based laser scanning cytometry (LSC). We report that LSC analysis of rAd-CMV-p53-infected human 293 cells correlated very well with flow cytometric analysis across a wide range of viral infectivity for both infectivity assessment (r2 = 0.97) and p53 protein expression (r2 = 0.96). Absolute values for infective titer and p53 protein expression titer from an rAd5-CMV-p53 production batch were similar and within experimental error with the two different analytical methods. Finally, bivariate format analysis of rAd-CMV-p53-infected cells revealed comparable results between LSC and flow cytometric analysis. LSC is a reliable and useful tool for the intracellular staining for adenoviral hexon protein expression for determining infectivity and for p53 protein expression from an expressed p53 transgene.

Adenoviruses, Human↗

PubMed2001

Automated evaluation of frequencies of aneuploid sperm by laser-scanning cytometry (LSC).

BACKGROUND: Laser-scanning cytometry (LSC) allows fast automated scoring of fluorescence signals directly on microscopic slides. Frequencies of spontaneous aneuploidies in murine and human sperm were evaluated by using this new LSC technique. Rapid detection may be of great interest in reproductive toxicology, as certain chemicals act as aneugens during meiosis, increasing the production of aneuploid germ cells. Materials and Methods Selected chromosomes were detected by using fluorescence in situ hybridization (FISH) and fluorochrome-labeled DNA-probes. Sperm chromatin was counterstained with propidium iodide. By scanning across the slide, fluorescence signals within sperm nuclei were detected and counted. RESULTS: In murine sperm, the frequencies of disomies for chromosomes 8 and X were 0.019% and 0.021%, respectively. The automated assessment in human sperm resulted in disomy frequencies of 0.061% and 0.090% for chromosomes 13 and X, respectively. These results were comparable to data obtained from the same samples by manual microscopic scoring and to literature data. CONCLUSIONS: Frequencies of genotypically abnormal sperm were not significantly different between automated and manual scoring. In conclusion, sperm aneuploidy was reliably determined and disomic sperm were successfully relocated by LSC. By virtue of rapid and reliable analyses, LSC has the powerful potential to replace manual microscopic FISH analysis in molecular cytogenetics.

Aneuploidy↗

PubMedDate not supplied

Slide-based laser scanning cytometry.

OBJECTIVE: To show that laser scanning cytometry (LSCM) can provide data equivalent to flow cytometry (FCM) data and furnish a number of benefits, including cell relocation for visualization and several additional measurement features that may make it more suitable than FCM for pathology laboratories. STUDY DESIGN: A laser scanning cytometer, the LSC, was developed. Several instruments, at sites in the United States and Japan during the last two years, provided data characterizing the instrument and its usefulness. RESULTS: Data describing the sensitivity, precision, accuracy, utility of added measurement features and cell relocation capabilities of the LSC are presented. The data illustrate the applicability of the LSC to multiparameter DNA ploidy studies, resolution of phases of the cell cycle and cytogenetics. CONCLUSION: Because it is microscope based and measures cells on a slide, not in a flow chamber; records the position of each cell on the slide; and has higher resolution, LSCM provides a number of benefits that may make it more suitable than FCM for pathology laboratories.

Animals↗

PubMed2002

Applicability of laser scanning cytometry to study paediatric alveolar macrophages.

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

Antigens, CD↗

PubMed1996

DNA ploidy analysis by laser scanning cytometry (LSC) in colorectal cancers and comparison with flow cytometry.

We evaluated laser scanning cytometry (LSC) by comparing nuclear DNA ploidy determined by LSC and by flow cytometry (FCM) in 77 samples of human colorectal cancer from 48 patients. Both methods revealed an aneuploid peak in 30 (62.5%) of the cases, although two samples that were aneuploid by LSC were diploid by FCM and two others were diploid by LSC and aneuploid by FCM. The concordance rate for nuclear DNA ploidy was 91.7% in the 48 patients and 87.0% for the 77 samples. The DNA index was also highly correlated between two methods (r2 = 0.97, P < 0.001). We concluded that LSC provides DNA histograms equivalent to FCM for surgical specimens and has potential clinical application in pathology.

Aneuploidy↗

PubMedDate not supplied

Multiparameter immunophenotypic analysis of fine needle aspiration biopsies and other hematologic specimens by laser scanning cytometry.

OBJECTIVE: To test the new laboratory technology of laser scanning cytometry with respect to immunophenotyping of all types of hematologic and lymphoreticular specimens and particularly those of limited size, such as fine needle aspiration biopsies and hypocellular body fluids. STUDY DESIGN: Over the course of two years, 343 hematologic and lymphoreticular specimens of all types were immunophenotyped by laser scanning cytometry using methodologies modified from those of conventional flow cytometric immunophenotyping. Results for all cases were corroborated with histology and/or cytology and, for some cases, immunohistochemistry and/or flow cytometric immunophenotyping. RESULTS: Over 98% of the 343 cases were successfully immunophenotyped by laser scanning cytometry. These included many hypocellular specimens, such as 38 fine needle aspiration biopsies and 33 body fluid specimens. CONCLUSION: Laser scanning cytometry is a new laboratory technology with several significant advantages relative to flow cytometry for immunophenotypic analysis of hematologic malignancy. The laboratory techniques are simplified, and antibody usage is reduced by 80%. Even more important, full-panel immunophenotyping with multiple antibodies can be performed on specimens as small as 50,000 cells total, making the technology particularly relevant to cytopathology. After immunophenotypic analysis, specimens can be stained for light microscopic examination, and individual cells meeting user-defined antigenic or physical characteristics can be automatically relocalized.

Adult↗

PubMed1997

Multiparameter analysis of DNA content and cytokeratin expression in breast carcinoma by laser scanning cytometry.

OBJECTIVE: The objective of this study was to test a new laboratory technology, laser scanning cytometry, for the purpose of performing multiparameter DNA content analysis of breast carcinomas. DESIGN: We developed a simplified method of multiparameter DNA content analysis using cytokeratin expression to positively gate epithelial cells. Over 300 consecutive cases of breast carcinoma were analyzed by multiparameter laser scanning cytometry. The first 73 cases were analyzed in parallel by single parameter flow cytometry. SETTING: The Department of Pathology, Christ Hospital and Medical Center, Oak Lawn, Ill. SPECIMENS: Three hundred eighteen consecutive cases of breast carcinoma presenting between March 1994 and December 1995. MAIN OUTCOME MEASURES: Outcome measures included the percentage of cases for which DNA content analysis could be successfully performed given the limitations of specimen size. Additionally, for the first 73 cases, laser scanning cytometry results were compared with flow cytometry results. RESULTS: All of the first 73 cases were successfully analyzed by laser scanning cytometry, but for 8 cases (11%) there was insufficient material for flow cytometry. Correlation of DNA content for the remaining 65 cases analyzed in parallel by the two methods was nearly perfect (p = .994). Five seemingly discrepant cases highlighted the importance of cytokeratin gating of epithelial cells by any technique, as well as other advantages specific to laser scanning cytometry, such as the ability to examine individual cells microscopically and correlate cytologic morphology with DNA content results. CONCLUSIONS: Laser scanning cytometry is a promising new technology for DNA content analysis of solid tissue tumors. Further work needs to be performed to validate the prognostic potential of the laser scanning cytometric assay results and to generate methodologies aimed at providing highly objective determinations of tumor cell S-phase fraction.

Biomarkers, Tumor↗

PubMed1998

Cell cycle effects and induction of apoptosis caused by infection of HL-60 cells with human granulocytic ehrlichiosis pathogen measured by flow and laser scanning cytometry.

Human granulocytic ehrlichiosis (HGE) is an occasionally severe and even fatal disease caused by an agent closely related to Ehrlichia equi and Ehrlichia phagocytophila, which is transmitted by ticks. Little is known about the pathogen itself, which only very recently has been isolated. The agent can be cultivated in vitro because it replicates in human promyelocytic leukemic HL-60 cells. Using multiparameter flow cytometry and laser scanning cytometry (LSC) we have investigated changes in HL-60 cells following their infection with the pathogen. Its presence within the infected HL-60 cells was detected and its intracellular level measured inmmunocytochemically using antibodies obtained from HGE-infected patients. The percentage of the infected cells measured by flow cytometry or LSC correlated well with the estimates by microscopy on the Giemsa-stained specimens. In the infected cultures, the cells had diminished levels of cyclins D3 and E as well as the cyclin dependent kinase inhibitor p21WAF1/CIP1 and were arrested predominantly in G0/1. The apoptosis-associated regulatory proteins were also affected by cell infection: expression of Bcl-2 was decreased in the infected cells whereas expression of Bax become more variable, with some cells showing higher levels of this protein. The infected cells developed numerous DNA strand breaks characteristic of apoptosis. The presence of the pathogen was also detected by LSC in cells from peripheral blood of the infected patients; after relocation and visual inspection ("CompuSort") the pathogen-positive cells were identified as leukocytes. This unique ability of LSC to detect, quantify, and visualize HGE in infected cells made this instrument particularly useful to measure the degree of infection in peripheral blood of the patients and study effects of the infectious agent on the cell cycle and apoptosis of the host cells.

Apoptosis↗

PubMed2006

Laser scanning cytometry in human brain slices.

BACKGROUND: The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. METHODS: We adapted this technique to immunohistochemical labelled human brain slices. RESULTS: We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. CONCLUSIONS: In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.

Alzheimer Disease↗

PubMedDate not supplied

Laser scanning cytometry evaluation of MART-1, gp100, and HLA-A2 expression in melanoma metastases.

Assessment of antigen expression by solid tumors has relied predominantly on immunohistochemistry, flow cytometry, and more recently quantitative real-time polymerase chain reaction. However, all these techniques present intrinsic limits. The laser scanning cytometer, by combining the properties of light and fluorescence microscopy with those of laser cytometry, can quantitatively and objectively analyze hypocellular samples such as fine-needle aspirates on an individual cell basis. To validate the fidelity of laser scanning cytometry for quantitative immunophenotyping of fine-needle aspirates, the authors measured the expression of the melanoma-associated antigens MART-1 and gp100 as well as HLA-A2, a HLA class 1 restriction element associated with their recognition by melanoma-specific T cells. Expression of melanoma antigens and HLA was measured by laser scanning cytometry and immunohistochemistry in fine-needle aspirates from melanoma metastases. In addition, transcription levels of both melanoma antigens were recorded by quantitative real-time polymerase chain reaction. A quantity of less than 1,000 cells per sample (average 682 cells) was sufficient for the analysis. Laser scanning cytometry estimates correlated with those of immunohistochemistry and quantitative real-time polymerase chain reaction for MART-1 and gp100. A good correlation in HLA-A2 detection by laser scanning cytometry and immunohistochemistry was also observed. Moreover, the laser scanning cytometer could discriminate subsets of cells from the same lesion with heterogeneous melanoma antigen expression, leading to the observation that cells with a DNA index greater than 2.5 expressed significantly less gp100. Thus, laser scanning cytometry yields detailed information on protein expression in individual cells and represents a new tool for dissecting the immune response in the tumor microenvironment.

Antigens, Neoplasm↗

PubMed2002

Evaluation of neuronal cell death by laser scanning cytometry.

We developed a method in which laser scanning cytometry (LSC) is applied to evaluate cell viability. Neuronal cell death induced by glutamic acid, serum potassium deprivation and 3-nitropropionic acid was studied in cerebellar granule cells by neutral red assay (NR) and LSC, using propidium iodide (PI) as fluorescent dye. PI labeled the nuclei of dead neurons and increased fluorescence was measured using a laser scanning cytometer. Similar levels of damage for each injury were detected by NR or LSC. The protocol presented here, provides a fast and sensitive assay for the analysis of neuronal viability using LSC, and can be used to study new neuroprotective drugs in neuronal cell cultures.

Animals↗

PubMed2006

Laser scanning cytometry: principles and applications.

The laser scanning cytometer (LSC) is the microscope-based cytofluorometer that offers a plethora of analytical capabilities. Multilaser-excited fluorescence emitted from individual cells is measured at several wavelength ranges, rapidly (up to 5000 cells/min), with high sensitivity and accuracy. The following applications of LSC are reviewed: (1) identification of cells that differ in degree of chromatin condensation (e.g., mitotic or apoptotic cells or lymphocytes vs granulocytes vs monocytes); (2) detection of translocation between cytoplasm vs nucleus or nucleoplasm vs nucleolus of regulatory molecules such as NF-kappaB, p53, or Bax; (3) semiautomatic scoring of micronuclei in mutagenicity assays; (4) analysis of fluorescence in situ hybridization; (5) enumeration and morphometry of nucleoli; (6) analysis of phenotype of progeny of individual cells in clonogenicity assay; (7) cell immunophenotyping; (8) visual examination, imaging, or sequential analysis of the cells measured earlier upon their relocation, using different probes; (9) in situ enzyme kinetics and other time-resolved processes; (10) analysis of tissue section architecture; (11) application for hypocellular samples (needle aspirate, spinal fluid, etc.); (12) other clinical applications. Advantages and limitations of LSC are discussed and compared with flow cytometry.

Cell Nucleus↗

PubMed1999

Analysis of apoptosis by laser scanning cytometry.

Flow cytometry techniques that are widely used in studies of cell death, and particularly in the identification of apoptotic cells, generally rely on the measurement of a single characteristic biochemical or molecular attribute. These methods fail to recognize cell death lacking that attribute, as in some examples of atypical apoptosis. Since apoptosis was originally defined by morphologic criteria, we suggest that for any new cell system the cytometry-defined apoptosis be confirmed by morphologic examination. This quality assurance measure is now provided by laser scanning cytometry (LSC). LSC measurements of cell fluorescence are precise and highly sensitive, comparable to flow cytometry (FCM), and can be carried out on cells on slides, permitting cell by cell correlation of fluorescence cytometry with visual microscopic morphology. In this report we describe adaptations of various flow cytometry techniques for detection of apoptosis by laser scanning cytometry. We also describe features unique to LSC that are useful in recognizing apoptosis. Hyperchromicity of DNA, reflecting chromatin condensation, is evidenced by high maximal pixel values for fluorescence of the DNA-bound fluorochrome. Mitochondrial probes that have been adapted to LSC to measure the drop in mitochondrial transmembrane potential that occurs early in apoptosis include rhodamine 123, 3,3'-dihexiloxadicarbocyanine [DiOC6(3)], and the aggregate dye 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). The changes in plasma membrane phospholipids and transport function, also early in apoptosis, are probed by a combination of the fluoresceinated annexin V and DNA fluorochromes such as propidium or 7-aminoactinomycin D. We also review methods of detection of apoptosis based on analysis of DNA fragmentation and their application to clinical oncology. Visual examination of the presumed apoptotic cells detected by cytometry makes it possible to discriminate those that are genuine from monocytes/macrophages that have ingested nuclear fragments via apoptotic bodies. Applications of flow cytometry and laser scanning cytometry in analysis of cell death are discussed and their respective advantages and disadvantages compared.

Annexin A5↗

PubMed2006

Guidelines for improving the reproducibility of quantitative multiparameter immunofluorescence measurements by laser scanning cytometry on fixed cell suspensions from human solid tumors.

BACKGROUND: Laser scanning Cytometry (LSC) is a versatile technology that makes it possible to perform multiple measurements on individual cells and correlate them cell by cell with other cellular features. It would be highly desirable to be able to perform reproducible, quantitative, correlated cell-based immunofluorescence studies on individual cells from human solid tumors. However, such studies can be challenging because of the presence of large numbers of cell aggregates and other confounding factors. Techniques have been developed to deal with cell aggregates in data sets collected by LSC. Experience has also been gained in addressing other key technical and methodological issues that can affect the reproducibility of such cell-based immunofluorescence measurements. METHODS AND RESULTS: We describe practical aspects of cell sample collection, cell fixation and staining, protocols for performing multiparameter immunofluorescence measurements by LSC, use of controls and reference samples, and approaches to data analysis that we have found useful in improving the accuracy and reproducibility of LSC data obtained in human tumor samples. We provide examples of the potential advantages of LSC in examining quantitative aspects of cell-based analysis. Improvements in the quality of cell-based multiparameter immunofluorescence measurements make it possible to extract useful information from relatively small numbers of cells. This, in turn, permits the performance of multiple multicolor panels on each tumor sample. With links among the different panels that are provided by overlapping measurements, it is possible to develop increasingly more extensive profiles of intracellular expression of multiple proteins in clinical samples of human solid tumors. Examples of such linked panels of measurements are provided. CONCLUSIONS: Advances in methodology can improve cell-based multiparameter immunofluorescence measurements on cell suspensions from human solid tumors by LSC for use in prognostic and predictive clinical applications.

Cell Aggregation↗