Application of bead-based assays for flow cytometry analysis

To date microsphere-based assays in flow cytometry have focused on the detection of antibody or antigen. Most studies have been research based to evaluate the performance of the technique relative to conventional techniques. However, there have not been any carefully controlled studies of the sensitivity and specificity, as well as analytic sensitivity of the FMIA technique. As such, it is difficult to document advantages of this tecnique clearly. The data suggest that FMIA is considerably more sensitive than conventional techniques, and the ability to analyze for multiple analytes in one sample dilution is attractive. This ability to simultaneously analyze for multiple samples is primarily dependent upon the size difference as sensed by FALS of the microspheres. However, it is also possible to use microspheres of the same size but that differ in either fluorescence or RALS signal. If microspheres of the same size are used but one fluoresces red and the signal in the assay uses a green fluorochrome, then the two microspheres can be separated by their red fluorescence. Using this technique, one can increase the number of microspheres that can be used in an assay. It is also possible to use microspheres of the same size but with different abilities to scatter the incident light at right angles. The use of these microspheres is then similar to the nonfluorescent versus red microspheres. By the judicious combination of microsphere size, it is possible to easily differentiate eight different microspheres. With the addition of a fluorescebt dye and/or differences in right-angle light-scatter capabilities, the number of different microspheres that can be used simultaneously becomes quite large. In practice, the number of microspheres that can be differentiated is no doubt greater than the number of analytes that need to be assayed in one assay.Although the apparent increase in sensitivity and the ability to simultaneously detect and quantitate numerous analytes are important attributes of FMIA, there are drawbacks to this method. Although the FMIA lends itself well to one-step no-wash procedures, when wash steps are necessary they are time-consuming and ineffecient. Most wash steps in FMIA use centrifugation of the microspheres to remove them from the reagent. There is a significant loss of microspheres in these wash steps, which are time-consuming. There are studies ussing vacuum filtration of the suspension to separate the microspheres from the reagents. A number of different groups are pursuing an automated or semiautomated method for the efficient washing and reagent delivery system for FMIA. Commercial systems are being developed that may allow for the easier handling of these reagents.Numerous groups are investigating the use of microspheres and flow cytometry primarily in immunoassay development. The procedure has the advantages of the simultaneous yet discrete analysis of multiple analytes and the inherent increase in sensitivity using fluorescence over other signals. There will no doubt be wider applications     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

Nonparametric flow cytometry analysis.

A nonparametric statistical test for the analysis of flow cytometry derived histograms is presented. The method involves smoothing and translocation of data, area normalization, channel by channel determination of the mean and S.D., and use of Bayes' theorem for unknown histogram classification. With this statistical method, different sets of histograms from numerous biological systems can be compared.     

Multi-user system for analysis of data from flow cytometry.

A new program is described for the analysis of DNA histograms from flow cytometry. The fundamental model representing the cell population is similar to one described previously. It assumes the population is grouped into compartments, each consisting of cells having approximately the same DNA content. After staining the cells with an appropriate fluorochrome, the fluorescence distribution of cells within each compartment is assumed to be Gaussian. In the present algorithm, the parameters of the model can either be computed directly by the program from the data, or can be specified as input by the user. When synchronous cell populations lacking distinct G1 and G2/M phases are analyzed, the parameter values must first be obtained using an appropriate control. Percentages of cells in the various compartments are computed using a gradient search method described by Bevington.     

Modified histogram subtraction technique for analysis of flow cytometry data

Analysis of flow cytometry histogram data by the subjective selection of an integration window can be a tedious and time-consuming task and is often inaccurate. A new method for automated calculation of the percent positive from immunofluorescence histograms is presented. This new method is a modification of the currently used method of channel-by-channel histogram subtraction. Its accuracy is compared to that of the channel-by-channel histogram subtraction method and to another currently used automated method, which selects an integration window by finding the channels that contain the most fluorescent 2% of a control histogram. The new histogram subtraction method is objective, easy to use, and is more accurate than other currently used automated analysis methods. PASCAL source code is given for each method of analysis.     

Instrument for real-time pulse-shape analysis of slit-scan flow cytometry signals

An instrument is described which analyses shapes of fluorescence profiles generated by particles passing through the focussed laser beam of a flow cytometer. The output signal of this pulse-shape analyzer is used as input for the signal processing electronics of a commercial flow cytometer system. The instrument detects dips in pulse-profiles; a shape parameter named Pulse Dip Index (PDI) is defined as the ratio of the integrated signal from the beginning of the pulse until the first dip, relative to the integrated signal of the complete profile. This PDI is similar to the Centromeric Index of chromosomes. The composition of aggregates in mixtures of fluorescent particles of different sizes was evaluated by PDI analysis. In our experiments the PDI was determined within 30 microseconds from the onset of the pulse-profile and particles with a specified morphology of interest were selected for on-line registration of their profiles as digitized pulse-shapes. In a cell sorter system, the PDI can be used as a parameter for sorting.     

Chromosome analysis by high illumination flow cytometry

Fluorescence measurements from metaphase chromosomes of the Chinese hamster, stained with propidium iodide excited at high illumination irradiance, completely resolve each chromosome type. The measurements are performed in a specially designed flow cytometer that achieves high irradiance (4 MW/cm2) by using high power laser output (2 W at 488 nm) focused to small spot size (1% irradiance variation over 2 microns). The coefficient of variation of each chromosome peak is near 1.5%. Saturation of the fluorescence transition and photobleaching, two consequences of high irradiance, are shown to occur. Even with a nonlinear dependence of fluorescence upon illumination irradiance, fluorescence retains a proportional response to chromosome type; each chromosome peak maintains a consistent ratio to the others at every irradiance. No perturbation of fluorescence by the optical or geometrical properties of the chromosomes is evident. The advantages of high irradiance illumination are an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories on the precision. These benefits improve the resolution of chromosome analysis by flow cytometry, particularly the resolution of smaller chromosomes.     

On multiparameter data analysis in flow cytometry

Increasing numbers of parameters that are accessible to simultaneous measurement in flow cytometric instruments, combined with the extremely large sample sizes common in flow cytometry, make it necessary to examine methods of multivariate statistics for their applicability to problems of visualization and quantitative analysis of flow cytometric data. This article describes some approaches to dimensionality reduction that appear well suited for data sets obtained by flow cytometry.     

The analysis and interpretation of DNA distributions measured by flow cytometry

A principal use of flow cytometers is for the measurement of fluorescence distributions of cells stained with DNA specific dyes. A large amount of effort has been and is being expended currently in the analysis of these distributions for the fractions of cells in the G1, S, and G2 + M phases. Several methods of analysis have been proposed and are being used; new methods continue to be introduced. Many, if not most, of these methods differ only in the mathematical function used to represent the phases of the cell cycle and represent attempts to fit exactly distributions with known phase fractions or unusual shapes. In this paper we show that these refinements probably are not necessary because of cell staining and sampling variability. This hypothesis was tested by measuring fluorescence distributions for Chinese hamster ovary and KHT mouse sarcoma cells stained with Hoechst-33258, chromomycin A3, propidium iodide, and acriflavine. Our results show that: a) single measurements can result in phase fraction estimates that are in error by as much as 40% for G2 + M phase and 15-20% for G1 and S phases; b) different dyes can yield phase fraction estimates that differ by as much as 40% due to differences in DNA specificity; c) the shapes of fluorescence distributions and their interpretation are very dependent on the dye being used and on its binding mechanism.     

Chromosome banding analysis by slit-scan flow cytometry

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers.     

Expression of glycosphingolipids in lymph nodes of mice lacking TNF receptor 1: biochemical and flow cytometry analysis

The expression of gangliosides and neutral glycosphingolipids (GSLs) in the lymph nodes of mice lacking the gene for the tumour necrosis factor-alpha receptor p55 (TNFR1) has been investigated. GSL expression in the tissues of mice homozygous (TNFR1-/-) or heterozygous (TNFR1+/-) for the gene deletion was analysed by flow cytometry and high-performance thin-layer chromatography (HPTLC) followed by immunostaining with specific antibodies. HPTLC immunostaining revealed that lymph nodes from TNFR1-/- mice had reduced expression of ganglioside GM1b and GalNAc-GM1b, neolacto-series gangliosides, as well as the globo- (Gb3, Gb4 and Gb5) and ganglio-series (Gg3 and Gg4) neutral GSLs. Flow cytometry of freshly isolated lymph node cells showed no significant differences in GSL expression, except for the GalNAc-GM1b ganglioside, which was less abundant on T lymphocytes from TNFR1-/- lymph nodes. In TNFR1-/- mice, GalNAc-GM1b+/CD4+ T cells were twofold less abundant (3.8% vs 7.6% in the control mice), whereas GalNAc-GM1b+/CD8+ T cells were fourfold less abundant (5.0% vs 20.2% in the control mice). This study provides in vivo evidence that TNF signalling via the TNFR1 is important for the activation of GM1b-type ganglioside biosynthetic pathway in CD8 T lymphocytes, suggesting its possible role in the effector T lymphocyte function.     

Optimizing optical flow cytometry for cell volume-based sorting and analysis

Cell size is a defining characteristic central to cell function and ultimately to tissue architecture. The ability to sort cell subpopulations of different sizes would facilitate investigation at genomic and proteomic levels of mechanisms by which cells attain and maintain their size. Currently available cell sorters, however, cannot directly measure cell volume electronically, and it would therefore be desirable to know which of the optical measurements that can be made in such instruments provide the best estimate of volume. We investigated several different light scattering and fluorescence measurements in several different cell lines, sorting cell fractions from the high and low end of distributions, and measuring volume electronically to determine which sorting strategy yielded the best separated volume distributions. Since we found that different optical measurements were optimal for different cell lines, we suggest that following this procedure will enable other investigators to optimize their own cell sorters for volume-based separation of the cell types with which they work.     

The curvHDR method for gating flow cytometry samples

Background  

High-throughput flow cytometry experiments produce hundreds of large multivariate samples of cellular characteristics. These samples require specialized processing to obtain clinically meaningful measurements. A major component of this processing is a form of cell subsetting known as gating. Manual gating is time-consuming and subjective. Good automatic and semi-automatic gating algorithms are very beneficial to high-throughput flow cytometry.     

Parametric analysis of histograms measured in flow cytometry

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.     

DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.     

Isolation of murine microglial cells for RNA analysis or flow cytometry

There is increasing interest in the isolation of adult microglia to study their functions at a morphological and molecular level during normal and neuroinflammatory conditions. Microglia have important roles in brain homeostasis, and in disease states they exert neuroprotective or neurodegenerative functions. To assay expression profiles or functions of microglia, we have developed a method to isolate microglial cells and infiltrating leukocytes from adult mouse brain. This protocol uses a digestion cocktail containing collagenase and dispase, and it involves separation over discontinuous percoll gradients. Isolated cells can be used for RNA analysis, including RNase protection analysis (RPA), quantitative RT-PCR, high-density microarray, proteomic or flow cytometric characterization of cell surface markers or adoptive transfer. Cell isolation can be completed in less than 4 h.