Micronuclei assay by laser scanning cytometry

BACKGROUND: The micronuclei (MN) assay is used to assess the chromosomal/mitotic spindle damage induced by ionizing radiation or mutagenic agents in vivo or in vitro. Because visual scoring of MN is cumbersome semi-automatic procedures that relay either on flow cytometry or image analysis were developed: both offer some advantages but also have shortcomings. METHODS: In the present study laser scanning cytometer (LSC), the instrument that combines analytical capabilities of flow and image cytometry, has been adapted for quantitative analysis of MN. The micronucleation of human breast carcinoma MCF-7 and leukemic HL-60 and U-937 cells was induced by in vitro treatment with mitomycin C. Cellular DNA was stained with propidium iodide (PI), protein was counterstained with fluorescein isothiocyanate (FITC). Two approaches were used to detect MN: (a) the threshold contour was set based on the data from the photosensor measuring red fluorescence of PI and MN were identified on the bivariate PI versus PI/FITC fluorescence distributions by their characteristic position; (b) the threshold contour was set on the data from the sensor measuring FITC fluorescence which made it possible, using the LSC software dedicated for FISH analysis, to assay both the frequency and DNA content of individual MN within each measured cell. RESULTS: The capability of LSC to relocate MN for visual examination was useful to confirm their identification. Visual identification of MN combined with their multiparameter characterization that took into an account their DNA content and protein/DNA ratio made it possible establish the gating parameters that excluded objects that were not MN; 93.3+/-3.3 events within the selected gate were MN. It was also possible to successfully apply FISH software to characterize individual cells with respect to quantity of MN residing in them. The percentage of MN assayed by LSC correlated well with that estimated visually by microscopy, both for MCF-7 (r = 0.93) and HL-60 cells (r = 0.87). CONCLUSIONS: LSC can be used to obtain unbiased estimate of MN frequencies. Unlike flow cytometry, it also allows one to characterize individual cells with respect to frequency and DNA content of MN residing in these cells. These analytical capabilities of LSC may be helpful not only to score MN but also to study mechanisms by which clastogenic agents induce MN.     

Clinical applications of laser scanning cytometry

This study reviews existing and potential clinical applications of laser scanning cytometry (LSC) and outlines possible future developments. LSC provides a technology for solid phase cytometry. Fluorochrome-labeled specimens are immobilized on microscopic slides that are placed on a conventional epifluorescence microscope and analyzed by one or two lasers. Data comparable to flow cytometry are generated. In addition, the position of each event is recorded, a feature that allows relocalization and visualization of each measured event. The major advantage of LSC compared with other cytometric methods is the combination of two features: (a) the minimal clinical sample volume needed and (b) the connection of fluorescence data and morphological information for the measured event. Since the introduction of LSC, numerous methods have been established for the analysis of cells, cellular compartments, and tissues. Although most cytometric methods use only two or three colors, the characterization of specimens with up to five fluorochromes is possible. Most clinical applications have been designed to determine ploidy and immunophenotype; other applications include analyses of tissue biopsies and sections, fluorescence in situ hybridization, and the combination of vital and nonvital information on a single-cell basis. With the currently available assays, LSC has proven its wide spectrum of clinical applicability in slide-based cytometry and can be introduced as a standard technology in multiple clinical settings.     

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

BACKGROUND: Flow cytometry (FCM) and laser scanning cytometry (LSC) are the routine techniques for fluorescent cell analysis. Recently, we developed a scanning fluorescent microscopy (SFM) technique. This study compares SFM to LSC (two slide-based cytometry, SBC, techniques) and FCM, in experimental and clinical settings. METHODS: For the relative cell-frequency determinations, HT29 colorectal cancer cells and Ficoll separated blood mononuclear cells (FSBMCs) were serially diluted (from 1:1 to 1:1,000) and measured by each of the three techniques. For the absolute cell number determinations (only for SBC) FSBMCs were smeared on slides, then HT29 cells were placed on the slide with a micromanipulator (5-50 cells). Tumor cells circulating in the peripheral blood were isolated by magnetic separation from clinical blood samples of colorectal cancer patients. All samples were double-stained by CD45 ECD and CAM5.2 FITC antibodies. For slides, TOTO-3 and Hoechst 33258 DNA dyes were applied as nuclear counter staining. RESULTS: In the relative cell frequency determinations, the correlations between the calculated value and measured values by SFM, LSC, and FCM were r(2) = 0.79, 0.62, and 0.84, respectively (for all P < 0.01). In the absolute cell frequency determinations, SFM and LSC correlated to a high degree (r(2) = 0.97; P < 0.01). CONCLUSIONS: SFM proved to be a reliable alternative method, providing results comparable to LSC and FCM. SBC proved to be more suitable for rare-cell detection than FCM. SFM with digital slides may prove an acceptable adaptation of conventional fluorescent microscopes in order to perform rare-cell detection.     

Analysis of ploidy in hypopharyngeal cancer by laser scanning cytometry on fine needle aspirate biopsies.

AIM: To test laser scanning cytometry (LSC) for the analysis of ploidy in squamous cell carcinoma of the hypopharynx (SCCH) and to develop a routine application for minimal samples such as fine needle aspirate biopsies (FNABs). METHODS: From 11 individuals 30 FNABs of primary tumors (n=11) and lymphatic metastases of SCCH (n=11) and non-metastatic lymph nodes (n=8) are analyzed by LSC. This microscope based instrument scans the cells after immobilization on a glass slide and after double staining of cytokeratin and DNA. The location of each cell is stored with the fluorescence data. Therefore the morphology of every cell can be documented by re-staining with H & E; and re-localization on the slide. Additionally, aliquots are Feulgen-stained for image cytometry in 8 specimens. RESULTS: The diploid reference peak is identified taking leukocytes as internal standard. The DNA-index of the carcinoma cells ranges from 0.4 to 3.8. Comparison with image cytometry shows good correlation (r=0.89). CONCLUSION: LSC provides a reliable and objective way to determine the ploidy of SCCH pre-operatively. Colour figures can be viewed on http://www.esacp.org/acp/2003/25-2/gerstner.htm.     

Determination of ploidy and steroid receptor status in breast cancer by laser scanning cytometry

BACKGROUND: Measurements on DNA content and steroid receptor status in breast cancer are of great clinical interest. Objective determination of estrogen and progesterone receptor expression should help to define the lowest levels of positivity still responding to adjuvant antihormonal therapy. For this purpose, a simple protocol for laser scanning cytometry is presented. METHODS: Analysis of 54 routine breast cancer samples was performed by laser scanning cytometry (LSC). To obtain single cell preparations from fresh tumor tissue, slides were prepared using the Cervisoft cytological device. Exact determination of tumor cell DNA content was done by referring to the CD45-positive tissue leukocyte fraction as the internal diploid reference cell population. Steroid receptor-expressing cells were detected by indirect immunolabeling. RESULTS: Indirect immunofluorescence allowed the best quantification of both the estrogen and progesterone receptor-expressing cell fractions by LSC. The number of receptor-expressing cells could be given in percentage. For comparison, the 10% cutoff value was used to determine receptor positivity. CONCLUSION: LSC enabled a simple, reliable, and inexpensive determination of DNA index and steroid receptor expression in breast cancer specimens by objective criteria.     

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.     

Translocation of Bax to mitochondria during apoptosis measured by laser scanning cytometry

BACKGROUND: During induction of apoptosis, the pro-apoptotic member of the Bcl-2 protein family (Bax) undergoes translocation to the mitochondria. The translocation, which leads to accumulation of Bax in the mitochondrial intermembrane space, appears to be the critical event determining release of cytochrome c to cytosol: the latter event triggers the irreversible steps of apoptosis, namely, the activation of caspases and the initiation of the degradation of many proteins. The aim of this study was to utilize the morphometric capabilities of the laser scanning cytometer (LSC) and adapt this instrument to detect and measure in situ the process of translocation of Bax to mitochondria. METHODS: Human breast carcinoma MCF-7 cells growing on microscope slides were treated with the DNA topoisomerase I inhibitor, camptothecin (CPT). CPT is known to induce apoptosis preferentially of S-phase cells. The cells were fixed and permeabilized on the slides, their DNA was stained with propidium iodide (PI), Bax was detected immunocytochemically with the fluoresceinated antibody, and red and green fluorescence emission was measured by the LSC. RESULTS: Prior to induction of apoptosis, Bax was uniformly and diffusely dispersed in the cell nucleus and cytoplasm. Its translocation and accumulation in mitochondria in cells undergoing apoptosis were detected and measured by the LSC as the increase in intensity of maximal pixel of Bax immunofluorescence. Bivariate analysis of DNA content versus maximal pixel of Bax fluorescence revealed that the CPT-induced Bax translocation into mitochondria was preferential to S-phase cells. Total cellular Bax immunofluorescence measured by flow cytometry, however, was increased in all phases of the cycle without a preference to S-phase cells. CONCLUSION: Changes in abundance and localization of particular proteins that undergo translocation within the cell, leading to their altered local density, may be conveniently detected by the LSC by taking advantage of its morphometric capabilities. Measurement of total cellular Bax immunofluorescence by flow cytometry combined with analysis of its translocation by LSC revealed that apoptosis of S-phase cells induced by CPT was unrelated to overall Bax abundance per cell but correlated with its accumulation in mitochondria.     

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.     

Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry

BACKGROUND: Caspase activation is a critical early step in the onset of apoptosis. Cell-permeable fluorogenic caspase substrates have proven valuable in detecting caspase activation by flow cytometry. Nevertheless, detection of early low-level caspase activation has been difficult using conventional area or peak fluorescence analysis by flow cytometry, despite the apparent presence of these cells as observed by microscopy. We describe a method utilizing maximum fluorescence pixel analysis by laser scanning cytometry (LSC) to detect early apoptotic cells. METHODS: The PhiPhiLux-G(1)D(2) caspase 3/7 substrate was used in combination with DNA dye exclusion and annexin V binding to identify several stages of apoptosis in EL4 murine thymoma cells by both traditional flow and LSC. LSC analysis of maximum pixel brightness in individual cells demonstrated an intermediate caspase-low subpopulation not detectable by flow or LSC integral analysis. LSC analysis of caspase activity was then carried out using the larger UMR-106 rat osteosarcoma cell line to determine if this apparent early caspase activity could be correlated with localized, punctate caspase activity observed by microscopy. RESULTS: The caspase-low subpopulation found in apoptotic EL4 cells was also observable in UMR-106 cells. Relocation to cells with low fluorescence due to caspase activity and subsequent examination by microscopy demonstrated that these latter cells indeed show punctate, highly localized caspase activation foci that might represent an early stage in caspase activation. CONCLUSIONS: Cells with low-level, localized caspase expression can be detected using maximum pixel analysis by LSC. This methodology allows an early step of apoptotic activation to be resolved for further analysis.     

Human lymphocyte subpopulations identified by using three-color immunofluorescence and flow cytometry analysis: correlation of Leu-2, Leu-3, Leu-7, Leu-8, and Leu-11 cell surface antigen expression

Three-color immunofluorescence has been used to determine the co-expression of cell surface antigens on human peripheral blood lymphocytes. Monoclonal antibodies or avidin were coupled to either FITC (green), phycoerythrin (orange), or Texas Red (red) fluorochromes. These three fluorochromes could be independently measured by using a dual laser FACS IV system equipped with an argon ion laser (488 nm) and a dye laser (600 nm). Human peripheral blood lymphocytes were stained with the following combinations of reagents: (1) FITC anti-Leu-11a + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; (2) FITC anti-Leu-11a + PE anti-Leu-3a + TR avidin/biotin anti-Leu-7; (3) FITC anti-Leu-8 + PE anti-Leu-2a + TR avidin/biotin anti-Leu-7; and (4) FITC anti-Leu-11a + PE anti-Leu-2 + TR avidin/biotin anti-Leu-8. The light scatter, green fluorescence, orange fluorescence, and red fluorescence signals for each sample were stored by a Consort 40 PDP/11 computer in list mode files. Sequential reanalysis of the data directly demonstrated the existence of several unrecognized subpopulations of lymphocytes. Previously, we reported that the anti-Leu-7 and anti-Leu-11 antibodies can be used to identify discrete subsets of human NK cells with distinct functional capacities. In this report, we show that these subsets can be further subdivided on the basis of Leu-8 and Leu-2 expression. Thus, these studies illustrate how multicolor and multiparameter flow cytometry can further our understanding of cellular heterogeneity within this group of lymphocytes.     

DNA ploidy analysis of urinary tract epithelial tumors by laser scanning cytometry

OBJECTIVE: To evaluate a rapid and simple method for DNA content analysis of urinary tract epithelial tumors with laser scanning cytometry (LSC). STUDY DESIGN: The subjects were 25 patients (37 specimens) who underwent surgery for urinary tract epithelial tumors. Tissue specimens of such tumors were frozen immediately after tumor resection and stored at -80 degrees C until used. Touch preparations were made and fixed in ethanol at room temperature. The cell nucleus was stained with propidium iodide solution containing RNase, and DNA ploidy was analyzed by LSC. Nuclear debris and overlapping nuclei were gated out by special statistical filters. In LSC, a normal diploid reference peak was determined by observing lymphocytes morphologically on the computer display of the instrument and/or under the microscope. RESULTS: DNA ploidy could be evaluated in all tumor tissues. The time it took from preparing the tumor specimen to the last measurement was about 40 minutes at the shortest, and measurement of all the specimens was completed within one hour. The coefficient of variation was 2.8-7.8% (mean, 4.4%). All eight specimens (100%) at grade 1 (G1) were DNA diploid, but 20% and 85.7% of the G2 and G3 cells, respectively, were DNA aneuploid. In total, 15 of the 37 specimens were DNA aneuploid. All 17 pTa-pT1 specimens (100%) were DNA diploid, but 100% and 50% of the T2 and T3 tumors, respectively, were DNA aneuploid. CONCLUSION: One can now supplement a morphologic diagnosis with useful information using LSC of touch preparations of tumors obtained at surgery or of imprints of archived, frozen specimens. LSC provides excellent DNA histograms for surgical specimens and has great potential for clinical application in pathology.     

"Liquidless" cell staining by dye diffusion from gels and analysis by laser scanning cytometry: potential application at microgravity conditions in space

BACKGROUND: Conventional staining of cells or tissue sections on microscope slides involves immersing the slides into solutions of dyes then rinsing to remove the unbound dye. There are instances, however, when use of stain solutions is undesirable-e.g., at microgravity conditions in space, where the possibility of accidental spill (many dyes are known carcinogens) introduces health hazard. Likewise, transporting bulk of liquid stains and rinses may be burdensome in certain situations such as field expeditions or combat. METHODS: The "liquidless" staining procedure is proposed in which the dyes are contained in thin strips of hydrated polyacrylamide or gelatin gels that have been presoaked in the stain solutions. Fluorochromes that have affinity to DNA (propidium iodide, PI; 4,6-diamidino-2-phenylindole, DAPI, Hoechst 33342) or to protein (sulforhodamine 101) were used to saturate the gels. The gel strips were placed over the prefixed cells or tissue sections deposited on microscope slides and relatively low (20 g/cm2) pressure was applied to ensure the contact. The cells were also stained by using commercially available mounting media into which DAPI or PI were admixed. Intensity of fluorescence of the PI stained cells was measured by laser scanning cytometry (LSC). RESULTS: Satisfactory cell and tissue staining, with minimal background, was achieved after 10-20 min contact between the cells and gels. Optimal concentrations of the dyes in the solutions used to presoak the gels was found to be 2-4-fold higher than the concentrations used routinely in cytometry. The measurements of intensity of cellular fluorescence by LSC revealed that the staining of DNA was stoichiometric as reflected by the characteristic cellular DNA content frequency histograms with distinct G1, S, and G2/M cell populations and 2:1 ratio of G2/M to G1 peak fluorescence. Individual gels can be saturated with more than a single dye-e.g., to obtain differential DNA and protein staining. Cell staining with DAPI or PI in the gelatin-based mounting media led to high fluorescence background while staining with DAPI in "aqueous" medium was satisfactory. CONCLUSIONS: Relatively fast staining of cells or tissue sections on microscope slides can be achieved by nonconvective dye diffusion using hydrated gels permeated with the dyes, applied to cells at low pressure. The quality of the staining provided by this methodology is comparable to conventional cell staining in dye solutions.     

Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence) as a separate parameter

BACKGROUND: The cytometric methods of bivariate analysis of cellular RNA versus DNA content have limitations. The method based on the use of metachromatic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the use of an instrument that provides two-laser excitation, including the ultraviolet (UV) light wavelength. METHODS: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposited on microscope slides and fixed. DNA and double-stranded (ds) RNA were stained with propidium iodide (PI) and protein was stained with BODIPY 630/650-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measured with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and after the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted from the fluorescence intensity of the same cell measured prior to RNase treatment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the cell fluorescence measured after RNase treatment or against the protein-associated BODIPY 630/650-X or FITC fluorescence. RESULTS: The scattergrams were characteristic of the RNA versus DNA bivariate distributions where DF represented cellular ds RNA content and fluorescence intensity of the RNase-treated cells, their DNA content. The distributions were used to correlate cellular ds RNA content with the cell cycle position or with protein content. CONCLUSIONS: One advantage of this novel approach based on the recording and plotting of DF is that only the RNase -specific fraction of cell fluorescence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell constituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wavelength detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained with other fluorochromes of the same-wavelength emission but targeted (e.g., immunocytochemically) for different cell constituents.     

Unique analytical capabilities of laser scanning cytometry (LSC) that complement flow cytometry

Flow cytometry has become an indispensable instrumentation in many disciplines of biology and medicine. There are some limitations of flow cytometry, inherent to the fact that the cells are measured in flow, which limit its usefulness in some applications. The microscope-based laser scanning cytometer (LSC) has many features similar to flow cytometry but few restrictions of the latter and therefore it is useful in many new applications. This review briefly outlines the applications that are unique to LSC, particularly related to its morphometric capabilities and the possibility of cell relocation. Potential future applications of LSC are also discussed.     

Simultaneous analysis of steady-state intracellular pH and cell morphology by automated laser scanning cytometry.

BACKGROUND: Cytosolic pH (pHi) changes are critical in cellular response to diverse stimuli, including cell survival and death signaling. The potential drawback in flow-based analysis is the inability to simultaneously visualize the cells during pHi measurements. Here, the suitability of laser scanning cytometer (LSC) in pHi measurement was investigated. AIM: Using the two extensively reported pH-sensitive fluorescent probes, 2,7-bis(2-Carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) and 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester, we evaluated the potential of automated LSC as a platform for simultaneous determination of pHi and cell morphology. The effect of a variety of buffer systems-commonly employed for pHi measurements-on cell morphology before pH clamping with the ionophore, nigericin, was also assessed. METHODS: Measurement of cytosolic pH was performed using pH-sensitive fluorescent probes BCECF-AM and SNARF-1. pH clamping was carried out using nigericin and samples were analyzed on the LSC or CyAn ADP Flow Cytometer. RESULTS: The pHi clamping conditions were optimized as 140 mM potassium and 10 microM nigericin. The suitable buffers used for pH clamping: 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, 20 mM MES and 140 mM KCl, 1 mM MgCl2, 2 mM CaCl(2).2H2O, 5 mM glucose, and 20 mM Tris. Results obtained with the LSC strongly correlated with those obtained by flow cytometry. CONCLUSION: We report here that LSC is an excellent and highly reproducible platform for pHi determination, and provides the added advantage of simultaneous imaging of cells before, during, and after pH measurements.     

Laser-based microscopic approaches: application to cell signaling in environmental lung disease.

Cell-imaging approaches using new laser-based technologies have a wide applicability to thefields of pathology and cell biology. Here, we present the application of several of these techniques, including confocal scanning laser microscopy (CSLM), laser scanning cytometry (LSC), and laser capture microdissection (LCM), to studies of cell signaling by environmental agents in lung disease. Using both cells in culture and lung tissue, we show that these technologies are powerful tools for understanding signal transduction cascades elicited by toxic agents, such as oxidants and asbestosfibers, and their relationship to the development of cell injury and proliferation, responses leading to lung disease and/or repair.     

A new approach for the analysis of testicular cells using a laser scanning cytometer.

The laser scanning cytometer (LSC) is a new laboratory tool that offers increased sensitivity and specificity compared to traditional technology. By combining the properties of the advantages of flow cytometry and immunohistochemistry, LSC-based analysis allows the automated evaluation of testicular cells in general and meiosis in particular. Testicular cell smears with previous staining by propidium iodide were analyzed by LSC. The results were compared with those for flow cytometry. LSC is a new, applicable methodology for analyzing spermatogenesis schedule.     

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.     

A practical method to determine the amount of tissue to analyze using laser scanning cytometry.

BACKGROUND: Laser scanning cytometry (LSC) is a new technology similar to flow cytometry but generates data from analysis of successive microscopic fields. Unlike its use in other applications, LSC-generated data are not random when used for tissue sections, but are dependent on the microanatomy of the tissue and the distribution and expression of the protein under investigation. For valid LSC analysis, the data generated requires the evaluation of a sufficient tissue area to ensure an accurate representation of expression within the tissue of interest. METHODS: In this report, we describe a simple and common sense method for determining the area of tissue required for sound LSC analysis by tracking the variation in the measure of target expression with increasing number of fields until it approaches zero. RESULTS: This approach was used to evaluate the expression of immunohistochemical markers with differing tissue distributions in liver (PMP70, CYP1A2, and Ki67 positive macrophages) and a colorectal adenocarcinoma (activated caspase-3 positive cells), which exhibited diffuse, regional (centrilobular), random, and irregular distribution patterns respectively. CONCLUSIONS: Analyses of these markers demonstrated that the amount of tissue area required to reach a steady measure of a parameter increased with increasing variability of the tissue distribution.     

Automated laser scanning cytometry: a powerful tool for multi-parameter analysis of drug-induced apoptosis.

BACKGROUND: Simultaneous analysis of multiple intracellular events is critical for assessing the effect of biological response modifiers, including the efficacy of chemotherapy. Here we used the automated laser scanning cytometry (LSC) for multi-parameter analysis of drug-induced tumor cell apoptosis. MATERIALS: Using 2-mercaptopyridine-N-oxide-hydrate sodium salt, or the commonly used chemotherapeutic agents etoposide and camptothecin, we performed simultaneous analyses of apoptosis-related morphological features as well as fluorescence-based biochemical changes in a 96-well format. RESULTS: We demonstrate the scope of LSC as a platform for comparing multiple variables between different cell populations, distinguishing unique events at a single cell level within a sample population, and enabling simultaneous screenings in a single assay at multiple dosages and time-points. CONCLUSION: These data underscore the power of LSC for simultaneous multi-parameter analysis, which could have implications for screening or assessing the efficacy of drug responses in heterogeneous cell populations and at the single cell level.