Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations

Bocsi, József; Varga, Viktor Sebestyén; Molnár, Béla; Sípos, Ferenc; Tulassay, Zsolt; Tárnok, Attila

doi:10.1002/cyto.a.20061

Cited by 27 publications

(25 citation statements)

References 32 publications

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“…Although FCM is a proven and reliable highthroughput technology, its major disadvantages for rare event analysis are that high numbers of cells are needed for accurate quantitation, and there is no capacity for morphological analysis (65,66). It has also been suggested that FCM may have limited sensitivity for CTC analysis in human blood samples (67). However, we have found that it has sufficient sensitivity (10 25 ) for use in our animal models when combined with immunomagnetic enrichment, perhaps because of the much smaller overall blood volume of a mouse relative to a human (39).…”

Section: Discussionmentioning

confidence: 99%

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

et al. 2008

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The inability to sensitively detect metastatic cells in preclinical models of cancer has created challenges for studying metastasis in experimental systems. We previously developed a flow cytometry (FCM) method for quantifying circulating tumor cells (CTCs) in mouse models of breast cancer. We have adapted this methodology for analysis of tumor dissemination to bone marrow (BM) and lymph node (LN), and for analysis of these samples by laser scanning cytometry (LSC). Our objective was to implement these methodologies for characterization of tumor cell dissemination in preclinical models of cancer metastasis. Human cancer cells were injected into mice via mammary fat pad (MFP; spontaneous metastasis), tail vein (TV; targets lung), or intracardiac (IC; targets bone) routes. At several time points postinjection (4 h to 8 weeks), mice were sacrificed and blood, LNs, and BM were collected. Samples were immunomagnetically enriched and labeled with human leukocytic antigen-fluorescein isothiocyanate and CD45-PE antibodies (FCM/LSC), and propidium iodide (FCM) prior to quantitative analysis. Following MFP injection, CTCs increased over time, as did disseminated cells to the LN. Interestingly, tumor cells also spontaneously disseminated to BM, peaking at 2 weeks postinjection. Following TV injection, CTCs were initially high but decreased rapidly by 1 week before increasing to peak at endpoint. Combined with an observed concurrent increase in disseminated cells to LN and BM, this suggests that tumor cells may shed into the circulation from lung metastases that establish following initial cell delivery. Following IC injection, CTCs increased over time, peaking at 4 weeks. Tumor cells in the BM (most prevalent site of metastasis after IC injection) remained at moderate levels until peaking at endpoint. Combined use of FCM and LSC allows sensitive quantification of disseminated tumor cells in preclinical models of metastasis. These methods will be valuable for future studies aimed at testing new therapeutics in the metastatic setting. ' International Society for Advancement of CytometryKey terms breast cancer; metastasis; preclinical animal models; tumor cell dissemination; flow cytometry; laser scanning cytometry BREAST cancer is a leading cause of morbidity and mortality in women (1,2), primarily because of the failure of effective clinical detection and management of metastatic disease in distant sites such as lymph node (LN), bone, lung, liver, and brain (3,4). The metastatic process is comprised of a series of sequential steps, and cancer cells must successfully complete each step in order to give rise to a metastatic tumor. These steps include dissemination of cancer cells from the primary tumor into the bloodstream (intravasation), survival in the circulation, arrest and extravasation into the secondary site, and initiation and maintenance of growth to form clinically detectable metastases (3-7). Breast cancer cells may also disseminate from the primary tumor through the lymphatic system, although the lac...

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Section: Discussionmentioning

confidence: 99%

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

et al. 2008

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“…Thus, it is possible to be cytometric (acquiring high number of data on a high number of individual cells) even when images are collected with fluorescent microscope. User friendly software, relocation possibility seems to be an attractive alternative for laser scanning cytometry (LSC) (53)(54)(55).…”

Section: Technology Developments Concerning Slidementioning

confidence: 99%

Cytomics and nanobioengineering

Pierzchalski¹,

Robitzki²,

Mittag³

et al. 2008

Cytometry Part B Clinical

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The finding that an individual's genome differs as much as by many million variants from that of the human reference assembly diminished the great enthusiasm that every disease could be predicted based on nucleotide polymorphisms. Even individual cells of an organ may be specifically equipped to perform specific tasks and that the information of individual cells in a cell system is key information to understand function or dysfunction. Therefore, cytomics received great attention during the last years as it allows to quantitatively and qualitatively analyzing great number of individual cells, cell constituents, and of their intracellular and functional interactions in a cellular system and also giving the concept of analysis of these data.Exhaustive data extraction from multiparametric assays and multiple tests are the prerequisite for prediction of drug toxicity. Cytomics, as novel approach for unsupervised data analysis give a chance to find the most predictive parameters, which describe best the toxicity of a chemical. Cytomics is intrinsically connected to drug development and drug discovery.Focused on small structures, nanobioengineering is the ideal partner of cytomics, the systems biological discipline for cell population analysis. Realizing the idea ''from the molecule to the patient'' develops and offers chemical compounds, proteins, and other biomolecules, cells as well as tissues as instruments and products for a wide variety of biotechnological and biomedical applications.The integrative nanobioengineering combining different disciplines of nanotechnology will promote the development of innovative therapies and diagnostic methods. It can improve the precision of the measurements with focus on single cell analysis. By nanobioengineering and whole body imaging techniques, cytomics covers the field from molecules through bacterial cells, eukaryotic tissues, and organs to small animal live analysis. Toxicological testing and medical drug development are currently strongly broadening. It harbors the promise to substantially impact on various fields of biomedicine, drug discovery, and predictive medicine.As the number of scientific data is rising exponentially, new data analysis tools and strategies like cytomics and nanobioengineering take a lead and get closer to application. Bionanoengineering may strongly support the quantitative data supply, thus strengthening the rationale for cytomics approach. q

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“…The LSC setup is built around a conventional epifluorescence microscope (11)(12)(13)(14). The scanning fluorescence microscope is a computer-controlled technique with an automated object scanner and data storage (15,16).…”

Section: Slide-based Cytometrymentioning

confidence: 99%

Regenerative and predictive medicine of cardiovascular disease: The 9th Leipziger workshop and the 2nd international workshop on slide based cytometry

et al. 2005

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Slide‐based cytometry (SBC) and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform but also for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows restaining and subsequent reanalysis of the specimen. These separate measurements of the same specimen can be fused to one data file (merging), thus increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a feature typical of SBC, can be of integral importance for cytometric analysis. Due to this feature, artifacts can be excluded and morphology of measured cells can be documented. Predictive medicine aims at the detection of changes in patient's state before the manifestation of the disease or its complications. Such instances concern multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high‐risk patients undergoing cancer cytostatic therapy. Early anti‐infectious or antishock therapy and curative chemotherapy in combination with stem cell transplantation may provide better chances of patients' survival at concomitant cost containment. Predictive medicine that guides early individualized decrease or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). Regenerative medicine concerns patients who have diseased and injured organs and may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Neovascularization is promoted by bone marrow–derived endothelial progenitor cells that lead to the formation of entirely new vessels into ischemic tissue. With this knowledge, many therapeutical borders can be skipped. Diseases formerly uncontrolled can be corrected with stem cells to provide causal healing with regeneration processes. The 9th Leipziger Workshop combined with the 2nd International Workshop on SBC aimed to offer new methods in image cytometry and SBC for solutions in clinical research. It moved toward practical applications in clinics and the clinical laboratory. This development will be continued in 2005 at the upcoming Leipziger Workshop and the 3rd International Workshop on SBC. © 2005 Wiley‐Liss, Inc.

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Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations

Cited by 27 publications

References 32 publications

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry

Cytomics and nanobioengineering

Regenerative and predictive medicine of cardiovascular disease: The 9th Leipziger workshop and the 2nd international workshop on slide based cytometry

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