A statistical analysis of flow cytometric determinations of phagocytosis rates.

Uptake of particles by phagocytosing cells is a process that exhibits variability of its rate. This variability is inherent in the mechanism of particle uptake and in the mechanisms that determine the distribution of physiological states within a population of phagocytosing cells. When numbers of particles ingested by cells are determined flow cytometrically an additional measurement variability is superimposed on and interacts with the aforementioned biological variability. In one method of determining population phagocytosis parameters, which involves fitting theoretical equations to experimental time course data on the fractions of cells which have ingested 0, 1, 2, ... particles, the effects of measurement variability are circumvented, although this usually has the cost of not using all the sample data obtained. However, in a second, simpler, method which is based on determining the time course of the number of particles ingested by an average cell, measurement variability is not circumvented and its effects must be considered. An analysis of the combined effects of biological and measurement variability on the results obtained with the simpler method is presented in this paper. Experimental results for phagocytosis of latex microspheres of uniform size and fluorochrome content by populations of the ciliate Tetrahymena pyriformis show that, for this system, measurement variability is entirely negligible in comparison with biological variability. This conclusion might not apply to other systems, however, and situations which might make measurement variability of some significance are mentioned in the paper. The equations given can be used for the analysis of such situations.     

A numerical analysis model for interpretation of flow cytometric studies of ex vivo phagocytosis

The study of ex vivo phagocytosis via flow cytometry requires that one distinguish experimentally between uptake and adsorption of fluorescently labeled targets by phagocytes. Removal of the latter quantity from the analysis is the most common means of analyzing such data. Because the probability of phagocytosis is a function of the probability of adsorption, and because partially quenched fluorescence after uptake often overlaps with that of negative controls, this approach is suboptimal at best. Here, we describe a numerical analysis model which overcomes these limitations. We posit that the random adsorption of targets to macrophages, and subsequent phagocytosis, is a function of three parameters: the ratio of targets to macrophages (m), the mean fluorescence intensity imparted to the phagocyte by the internalized target (alpha), and the probability of phagocytosis per adsorbed target (p). The potential values of these parameters define a parameter space and their values at any point in parameter space can be used to predict the fraction of adsorption(+) and [adsorption(-), phagocytosis(+)] cells that might be observed experimentally. By systematically evaluating the points in parameter space for the latter two values and comparing them to experimental data, the model arrives at sets of parameter values that optimally predict such data. Using activated THP-1 cells as macrophages and platelets as targets, we validate the model by demonstrating that it can distinguish between the effects of experimental changes in m, alpha, and p. Finally, we use the model to demonstrate that platelets from a congenitally thrombocytopenic WAS patient show an increased probability of ex vivo phagocytosis. This finding correlates with other evidence that rapid in vivo platelet consumption contributes significantly to the thrombocytopenia of WAS. Our numerical analysis method represents a useful and innovative approach to multivariate analysis.     

Use of flow cytometric methods for single-cell analysis in environmental microbiology

Flow cytometry (FCM) is emerging as an important tool in environmental microbiology. Although flow cytometry applications have to date largely been restricted to certain specialized fields of microbiology, such as the bacterial cell cycle and marine phytoplankton communities, technical advances in instrumentation and methodology are leading to its increased popularity and extending its range of applications. Here we will focus on a number of recent flow cytometry developments important for addressing questions in environmental microbiology. These include (i) the study of microbial physiology under environmentally relevant conditions, (ii) new methods to identify active microbial populations and to isolate previously uncultured microorganisms, and (iii) the development of high-throughput autofluorescence bioreporter assays.     

Statistical analysis and biological interpretation of the flow cytometric heterogeneity observed in bacterial axenic cultures

Histogram comparison and meaningful statistics in flow cytometry is probably the most widely encountered mathematical problem in flow cytometry. Ideally, a test for determining the statistical equality or difference of flow cytometric distributions will identify the significant differences or similarities of the obtained histograms. This situation is of particular interest when flow cytometry is used to study the heterogeneity of axenic bacterial populations. We have statistically measured the heterogeneity of successive cytometric measures, the modifications produced after 20 transfers from the same culture, and the differences between 20 subcultures of identical origin. The heterogeneity of the bacterial populations and the similarity of the obtained 360 histograms were analysed by standard statistical methods. We have studied bacterial axenic cultures in order to detect, quantify and interpret their cytometric heterogeneity, and to assess intrinsic differences and differences produced by laboratory manipulations. We concluded that the standard axenic cultures have a considerable intrinsic cellular and molecular heterogeneity. We suggest that the heterogeneity we have detected basically has two origins: cell size diversity and cell cycle variations.     

A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells

Flow cytometry has provided a powerful tool for analyzing bacteria-host cell associations. Established approaches have used bacteria, labeled either directly with fluorochromes or indirectly with fluorescently conjugated antibodies, to detect these associations. Although useful, these techniques are consistently unable to include all host cells in the analysis while excluding free, aggregated bacteria. This study describes a new flow cytometry method of assessing bacterial adherence to host cells based on direct fluorescent labeling of both bacteria and host cells. Eukaryotic host cells were labeled with PKH-26, a red fluorescent dye, and bacteria were labeled with fluorescein isothiocyanate, a green fluorescent dye. The red host cells were gated and the mean green fluorescence intensity (MFI) of these red cells was determined. We used MFI values obtained from control samples (unlabeled and labeled host cells with unlabeled bacteria) to eliminate contributions due to autofluorescence. The final MFI values represent fluorescence of host cells resulting from the adherent bacteria. Because all red fluorescent cells are analyzed, this method includes all the eukaryotic cells for analysis but excludes all free or aggregated bacteria that are not bound to target cells.     

Rapid titer assay of adenovirus containing green fluorescent protein gene using flow cytometric analysis

The plaque assay has been widely used for titration of adenovirus (AdV). However, it takes usually 2-3 weeks, so this slow assay often impedes bioprocess development of large-scale AdV production. In this study, we developed a rapid AdV titration assay that can be done within a day. Further, unlike the plaque assay, this assay does not require a laborious serial dilution of samples. This rapid assay can be achieved by using green fluorescent protein (GFP) as a marker gene and flow cytometric analysis. It yields a good correlation between infectious titer of AdV harboring GFP and flow cytometric parameters such as average green fluorescence intensity or % of infected cells. Taken together, this rapid assay will facilitate bioprocess development for efficient large-scale AdV production.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Staining procedures for flow cytometric monitoring of bacterial populations

Dual or multiple parameter flow cytometric analysis is developing into a powerful method for characterizing microbial populations. The distinguishing of the populations only by assignment of size/shape measurements by scattered ligth renders as not satisfactory. To differentiate between the cells, the employment of a specific fluorescence marker is absolutely necessary. Methods are presented for the flow cytometric determination of DNA and the polymer poly-β-hydroxybutyrate (PHB) content in three different bacterial strains. The measurement of the 3β-hydroxysterol content enables the differentation between yeast and bacterial organisms in mixed microbial populations. Monitoring the ratio of live to dead bacterial cells in soil or water samples, e.g. in pure culture systems, is shown.     

Rapid flow cytometric studies of Borrelia burgdorferi phagocytosis by human polymorphonuclear leukocytes

The interactions between a strain of Borrelia burgdorferi and human polymorphonuclear leukocytes were studied by flow cytometry in the presence of specific or non-specific opsonizing factors. The capacity of the borrelias to stimulate leukocyte metabolism was also investigated. The results indicated that a low phagocytosis by isolated purified polymorphonuclear leukocytes did occur in the presence or absence of specific antibodies. Within whole blood the percentages of phagocytosting leukocytes increased in the presence of non-specific opsonizing factors. No stimulation of the oxidative metabolism stimulated by Borrelia was observed and PMA or zymosan stimulation of leukocytes was inhibited by the spirochaetes.     

Cell-by-cell flow cytometric analysis of chromosomes

The authors discuss various aspects of a recently developed method permitting a detailed flow cytometric analysis of the individual cell karyotypes such as instrumentation, histochemistry, data proceeding algorithms. Possible drawbacks of the method and the ways of their overcoming are considered. Results of analysis of the Chinese hamster cells are presented that illustrate the possibilities of the method, including the metaphase chromosome distribution according to their fluorescence intensity, the analysed cell distribution according to their chromosomes number, the table in which the individual cell karyotypes are distributed according to their fluorescence. The results obtained show that the developed method may be successfully used for investigating chromosomal iNstability and heterogeneity of the mammalian cells.     

DNA-protein flow cytometric analysis in non-Hodgkin lymphoma

A flow cytometric study of DNA and protein contents was performed on cell suspensions obtained from 73 adult patients with non-Hodgkin lymphoma. Bivariate analysis identified a second subpopulation, not revealed by DNA determination, in 25% of the tumors. Protein heterogeneity was more frequently observed in diffuse than in nodular histology according the Rappaport classification and in high-grade than in low-grade malignancy tumors by the Kiel classification and the Working Formulation, but it was not related to ploidy or cell proliferative rate. The presence of an additional subpopulation, detected by protein analysis, defined as monoclonal by DNA analysis, could adversely affect clinical outcome in terms of response to treatment and overall survival.     

A novel flow cytometric assay for measurement of In Vivo pulmonary neutrophil phagocytosis

Background  

Phagocytosis assays are traditionally performed in vitro using polymorphonuclear leukocytes (PMNs) isolated from peripheral blood or the peritoneum and heat-killed, pre-opsonized organisms. These assays may not adequately mimic the environment within the infected lung. Our laboratory therefore has developed a flow cytometric in vivo phagocytosis assay that enables quantification of PMN phagocytosis of viable bacteria within the lungs of rats. In these studies, rats are injected transtracheally with lipopolysaccharide (LPS) to recruit PMNs to their lungs. They are then infected with live 5(-and 6) carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) labeled type 3 Streptococcus pneumoniae. Bronchoalveolar lavage is performed and resident alveolar macrophages and recruited PMNs are labeled with monoclonal antibodies specific for surface epitopes on each cell type. Three color flow cytometry is utilized to identify the cell types, quantify recruitment, and determine uptake of the labeled bacteria.     

Simultaneous flow cytometric method to measure phagocytosis and oxidative products by neutrophils.

We developed a rapid and sensitive two-color flow cytometric method which allows the simultaneous quantification of both the phagocytosis rate and the oxidative burst activation of polymorphonuclear leukocytes (PMNLs). The oxidation of hydroethidine (HE) to ethidium bromide (EB) was performed by the oxidative neutrophil products within the cells during the respiratory burst, which was stimulated by phagocytized fluorescein-labeled Staphylococcus aureus. By means of flow cytometry we measured red EB fluorescence emission together with green fluorescence, which was emitted by the ingested fluoresceinated bacteria. The fluorescence intensity was proportional to the number of bacteria ingested. Adherent bacteria were distinguished from the ingested ones. This two-color cellular staining permits measurement of two different functions of neutrophils in one step. This method could be of interest for the determination of the interactions between neutrophils and bacteria and for the investigations on infectious diseases in chronic granulomatous disease patients.     

Use of xantham gum to suspend large particles during flow cytometric analysis and sorting

In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity approximately 9-fold. For suspensions of multicellular spheroids 100-400 microns in diameter the measured sedimentation velocity was approximately 9 times slower than that in medium alone. Thus, spheroids of 100 microns diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.     

New method for the analysis of flow cytometric data

A numerical method for deriving the fractions of cells in different phases of the cell cycle from a single observed DNA histogram is presented. The observed histogram is regarded as a polluted version (containing allocation errors) of the true histogram. A mathematical model is used to describe the pollution process. A theoretical histogram, representing the true histogram, is constructed so that G1 cells are put into one channel and G2M cells into another; the distribution of S cells in between is approximated with a set of harmonic functions. This theoretical histogram is subsequently disturbed with Gaussian dispersion functions to stimulate the pollution, yielding a predicted histogram. Using a maximum likelihood estimation technique, the model parameters are adjusted iteratively, matching the predicted histogram to the actually observed one. With the final parameter values substituted, the corresponding final theoretical histogram is regarded as a reliable reconstruction of the true histogram. From the latter, the required percentages can be read directly. The advantage of this approach over other mathematical analysis methods is that it allows a wide range of different, continuous distributions for relatively few model parameters (thus featuring flexibility and realism and a diminished risk of encountering computational problems). In addition, estimation errors providing a measure of accuracy can be obtained. To test the method, it was used to analyze various observed histograms from the literature that have been obtained by either simulation or actual flow cytometric measurements. The method appeared to perform well, as compared to the reported results of several other methods of analysis applied to the same data.     

Flow cytometric analysis and optimisation for measuring phagocytosis in three Australian freshwater fish

The phagocytic activity of fish immunocytes has been measured by a wide range of methods, and has been used as a bio-indicator to assess the immunotoxicity of environmental pollutants and the efficiency of immunostimulants used in aquaculture. This study demonstrates the utilisation of a flow cytometric technique for measuring phagocytosis as an alternative to manual evaluations by light microscopy. Optimal conditions for the phagocytosis of latex beads were ascertained, including incubation period, cell:bead ratio and media components, for head kidney cells isolated from three native Australian fish that inhabit the Murray-Darling basin, i.e. silver perch (Bidyanus bidyanus), golden perch (Macquaria ambigua) and crimson-spotted rainbowfish (Melanotaenia fluviatilis). Thus, standardised protocols have now been established for future use in the immunotoxicity testing of xenobiotics in native Australian freshwater fish.     

Flow cytometric analysis of the uptake of Hoechst 33342 dye by human lymphocytes

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature-dependent with minimal uptake rate below 16 degrees C. The activation energy of dye transport was found to be 135 kJ/mol above 20 degrees C and about 20 kJ/mol below 16 degrees C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.     

Microcomputer experience in analysis of flow cytometric DNA distributions

The program described analyses DNA histograms obtained in flow cytometry using the Gaussians method. The program is written in BASIC to run on a low-cost microcomputer. It utilizes a simple strategy to obtain good estimates of the parameters required for reducing the problem to a task solvable with linear least-squares methods. Features of the program are flexibility, since it is possible to choose different options for parametrization and spacing of Gaussians, and the fact that the operator is not required to provide interactive inspection or inputting parameter values. The capability and velocity of the program, in all its options, are tested and compared on a series of different (not computer-simulated) histograms obtained in our flow cytometry laboratory. Our results suggest that a fresh approach to parametrization may be useful.     

Preparation of tissues for DNA flow cytometric analysis

A method for measuring DNA in tissue cells by flow cytometry utilizing a one step combination nuclear isolation-DNA fluorochrome staining procedure is described. A variety of cells and tissues, both in vivo and in vitro, was used to illustrate the universal nature of this technique. These included murine bone marrow, liver testicle, sarcoma brain tumor, rat pancreatic islets, human peripheral blood, colon mucosa, colon cancer, sarcoma and brain tumor tissues. A special nuclear isolation medium, which contained either of the DNA fluorochromes, 4',6-diamidino-2 phenylindole-2 HCl or propidium iodide, was utilized successfully to isolate single suspensions of DNA fluorochrome stained nuclei in a rapid (5-10 min), consistent manner from a variety of tissues and cells. Multiple sampling of the same tissue or comparison between whole tissues and their single cell isolates showed that a representative sample was being obtained.     

DNA flow cytometric analysis of mouse seminiferous epithelium

In order to provide a basis for quantitative studies of murine spermatogenesis, we performed a DNA flow cytometric analysis on the mouse seminiferous tubules isolated at defined stages of the epithelial cycle by transillumination-assisted microdissection. Accurate stage identification was performed by examining spermatids in the adjacent tubule segments by phase-contrast microscopy. For flow cytometry, suspension of nuclei of spermatogenic cells was obtained by detergent treatment of isolated seminiferous tubules, and fresh samples were stained with propidium iodide. DNA histograms of the 12 stages of the mouse seminiferous epithelial cycle varied in a stage-specific manner. DNA histograms of stages I-VIII of the cycle were characterized by a hypofluorescent haploid peak, the location of which changed with the decreasing DNA dye (propidium iodide)-binding capacity of elongated spermatids. The absence of the hypohaploid peak and the high ratio of the cells with 4C amount of DNA to the cells with 1C amount of DNA characterized stages IX-XI of the cycle. Stage XII showed a high 2C peak, owing to a large population of secondary spermatocytes arisen from the first meiotic division. By using fluorescent beads as an internal volume standard cell numbers in defined stages were determined. These data provide a basis for quantitative studies of mouse spermatogenesis.