An innovation in flow cytometry data collection and analysis producing a correlated multiple sample analysis in a single file

The problems associated with rapid analysis and interpretation of data from multicolor immunofluorescence panels have been a formidable barrier to their routine use. Using present flow cytometry concepts, a panel of 11 tubes each containing multiple phenotypic markers or controls requires postdata acquisition manipulation of many multiparameter histogram and listmode files. We have developed a method that compresses all of the information from such a panel into a single listmode data file during run time. A single data file is used to record the entire phenotypic analysis for a particular patient or series within an experiment. This is accomplished by the incorporation of a tube identifier parameter (TIP) as well as the fluorescence and light scatter parameters normally collected. The TIP can then be used for gating discrimination of any tube or set of tubes within a panel. When the TIP is correlated with the PRISM parameter the entire patient phenotypic image can be represented within a single two-parameter histogram we have called a phenogram. This phenogram can be generated in real time, providing on-line preprocessing of a complex multicolor experiment. By examining the image created by the phenogram it is possible to rapidly flag abnormalities such as incorrect gating. This procedure was carried out on an EPICS Elite flow cytometer in its standard configuration with the addition of hardware to provide an input for the TIP.     

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body’s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).     

Localization of monoclonal B-cell populations through the use of Komogorov-Smirnov D-value and reduced chi-square contours

In this study three different flow cytometric analysis techniques are woven together into a single system that permits improved detection of small percentages of monoclonal B cells in a milieu of either normal blood leukocytes or bone marrow cells. This analysis is an extension of the concept of clonal excess, which is used to detect the presence of a tumor that is a clonal expansion of B cells expressing either kappa or lambda light chains. The technique also utilizes "multiple listmode processing," which is defined in this context as the simultaneous analysis of two or more listmode files that share one or more common parameters. This type of data structure enables the segmentation of two parameter light scatter displays into regions from which numerous kappa and lambda histograms subsequently can be analyzed for their respective Komogorov-Smirnov D-values or R-values (reduced chi-square value). The final technique makes use of a calculated parameter display system. Superimposed on the light scatter dot density plot are D-value or R-value contours. The contours target the location of the population that is abnormal, thus providing information for setting optimal bitmap gates for clonal excess studies, other phenotypic analyses, or cell sorting. In experiments using model systems, the sensitivity of this assay is estimated to be between 0.25% and 2.5%. The technique's distribution information and sensitivity may prove useful for staging, treatment monitoring, and relapse detection of B-cell leukemia and lymphoma. This application illustrates the potential of combining multiple listmode processing and calculated parameter display to expand the effective dimensionality of listmode data.     

Design and characterization of a compact dual channel virus counter.

BACKGROUND: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the "gold standard." Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. METHODS: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels. RESULTS: The instrument was fully characterized, which included analysis of the data acquisition rate, sampling time, flow rate, detection efficiency, linear dynamic range, channel cross-talk, and the limit of detection. Baculovirus samples were analyzed and the results were compared with concentrations obtained by a one-channel flow cytometer and plaque assay. CONCLUSIONS: The dual channel virus counter yields a representative value for the concentration of active viruses in an unpurified sample when compared with plaque assay and a one-channel flow cytometer. The technique is rapid (within minutes), requires only minimal sample preparation and minimum sample size (approximately 100 microl).     

Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

Neutrophil function screening in patients with chronic granulomatous disease by a flow cytometric method.

Neutrophils from patients with chronic granulomatous disease (CGD) fail to produce a significant oxidative burst following stimulation. We have evaluated the use of flow cytometry and the dye 2',7'-dichlorofluorescein diacetate (DCF) for routine screening for deficiencies of neutrophil oxidative burst. A range for DCF fluorescence for phorbol myristate acetate stimulated and non-stimulated neutrophils was established based on data from 52 healthy adults. Samples from three patients with suspected neutrophil dysfunction, three patients with X-linked CGD, and one patient with autosomal recessive (AR) CGD were evaluated with both the DCF assay and the quantitative nitroblue tetrazolium dye reduction (NBT) test. For the DCF test, the ratio of mean fluorescence intensity of stimulated to non-stimulated neutrophils was less than 5 for CGD patients and from 16 to greater than 50 for healthy individuals. With the DCF test, two populations of neutrophils could be identified in samples from four carriers of X-linked CGD, although two carriers of AR CGD had NBT and DCF results in the normal range. Our data suggest the DCF test is a sensitive and convenient method for detecting CGD.     

An improved flow cytometric assay for somatic mutations at the glycophorin A locus in humans

An improved method has been developed for the glycophorin A assay for somatic cell mutations in humans. The new assay, named the "BR6" assay, can be performed on a commercially available, single-beam flow cytometer, in contrast to the previously described 1W1 assay that required a dual-beam flow sorter. A modified cell labeling method developed for the BR6 assay results in improved separation of normal and mutant phenotype cells, as well as eliminating some cellular artifacts that affected the 1W1 assay. Parallel measurements on samples from 17 normal donors showed that the BR6 assay yields comparable variant cell frequencies and improved measurement precision compared with the 1W1 assay. A detailed analysis of three individuals who showed large differences in background variant frequency with the 1W1 assay confirmed that these differences could also be detected with the BR6 assay. A dramatically elevated variant cell frequency was seen with the BR6 assay of an individual exposed to a high level of ionizing radiation in an accident at Goiania, Brazil.     

Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is employed in biological dosimetry to determine the dose of radiation to an exposed individual from the frequency of micronuclei (MN) in binucleated lymphocyte cells. The method has been partially automated for the use in mass casualty events, but it would be advantageous to further automate the method for increased throughput. Recently, automated image analysis has been successfully applied to the traditional, slide-scoring-based method of the CBMN assay. However, with the development of new technologies such as the imaging flow cytometer, it is now possible to adapt this microscope-based assay to an automated imaging flow cytometry method. The ImageStream^X is an imaging flow cytometer that has adequate sensitivity to quantify radiation doses larger than 1 Gy while adding the increased throughput of traditional flow cytometry. The protocol and analysis presented in this work adapts the CBMN assay for the use on the ImageStream^X. Ex vivo-irradiated whole blood samples cultured for CBMN were analyzed on the ImageStream^X, and preliminary results indicate that binucleated cells and MN can be identified, imaged and enumerated automatically by imaging flow cytometry. Details of the method development, gating strategy and the dose response curve generated are presented and indicate that adaptation of the CBMN assay for the use with imaging flow cytometry has potential for high-throughput analysis following a mass casualty radiological event.     

Photon-burst analysis in two-photon fluorescence excitation flow cytometry.

We studied the use of a dramatically reduced testing zone in combination with two-photon excitation and photon-burst analysis in high-throughput rare-event detection simulation using a modified flow cytometer. Two-photon excitation measurements were performed with a mode-locked titanium:sapphire laser. Fluorescence emission was measured with a photon-counting avalanche photodiode. Measured signal was analysed offline by autocorrelation and burst detection methods. Test samples were composed of full blood and orange fluorescent polystyrene nanospheres mixed in full blood. Results show that two-photon fluorescence excitation and time-correlation analysis provide a good signal-to-noise ratio for rare-event particle detection in a turbid sample environment.     

Eight-parameter PC-AT based flow cytometric data system

An 8-parameter flow cytometric data system is described using an IBM-AT compatible personal computer (PC) and a commercial analog to digital conversion (ADC) board. A dedicated pulse processing interface adapts the flow cytometric pulses to the ADC board and controls the number of parameters to be taken up and the trigger conditions. The trigger thresholds are automatically held at a level immediately above the noise level. For the timing of kinetic measurements a linear voltage ramp of adjustable rise time is available. A built-in precision voltage source can be used for an overall calibration. The data system is operated by software written in assembly language. Data may be collected and processed in 1-8-parameter listmode or 1-3-parameter histogram mode. Functions are available for graphical color displays, numerical integration, multiparameter gating, and printing.     

葡萄糖氧化酶诱导肝细胞氧化应激模型的建立

目的:研究不同浓度葡萄糖氧化酶(GO)对人肝细胞L02氧化应激水平的影响,以确定建立肝细胞氧化应激模型的合适浓度。方法:用不同浓度GO干预L02肝细胞2h,MTT法检测细胞的存活率,流式细胞术检测细胞内活性氧簇(ROS),荧光强度(FI)来表示ROS水平。分光光度法检测检测细胞MDA、GSH,速率法检测细胞培养液LDH、AST和ALT的水平。结果:①随GO浓度增加,肝细胞的存活率逐渐降低,其中75U/L、100U/L和125U/L组存活率显著低于对照组(P<0.05)。②随GO浓度增加,MDA含量逐渐增高,其中50U/L、75U/L、100U/L、125U/L组MDA水平较对照组显著增高(P<0.05)。GSH水平随GO浓度增高而逐渐减低,各干预组较对照组均显著降低(P<0.05)。GO各干预组FI均较对照组显著降低(P<0.05)。③各干预组LDH活性均显著高于对照组(P<0.05),50U/L、75U/L、100U/L、125U/L干预组AST与ALT水平均较对照组显著增高(P<0.05)。结论:GO能引起的肝细胞氧化应激损伤有剂量依赖性,100U/L是建立肝细胞氧化应激的合适浓度。     

Kinetic diversity of Na+ channel bursts in frog skeletal muscle

Individual Na+ channels of dissociated frog skeletal muscle cells at 10 degrees C fail to inactivate in 0.02% of depolarizing pulses, thus producing bursts of openings lasting hundreds of milliseconds. We present here a kinetic analysis of 87 such bursts that were recorded in multi-channel patches at four pulse potentials. We used standard dwell-time histograms as well as fluctuation analysis to analyze the gating kinetics of the bursting channels. Since each burst contained only 75-150 openings, detailed characterization of the kinetics from single bursts was not possible. Nevertheless, at this low kinetic resolution, the open and closed times could be well fitted by single exponentials (or Lorentzians for the power spectra). The best estimates of both the open and closed time constants produced by either technique were much more broadly dispersed then expected from experimental or analytical variability, with values varying by as much as an order of magnitude. Furthermore, the values of the open and closed time constants were not significantly correlated with one another from burst to burst. The bursts thus expressed diverse kinetic behaviors, all of which appear to be manifestations of a single type of Na+ channel. Although the opening and closing rates were dispersed, their average values were close to those of alpha m and 2 beta m derived from fits to the early transient Na+ currents over the same voltage range. We propose a model in which the channel has both primary states (e.g., open, closed, and inactivated), as well as "modes" that are associated with independent alterations in the rate constants for transition between each of these primary states.     

Small-volume rapid-mix device for subsecond kinetic analysis in flow cytometry.

BACKGROUND: Rapid-mix flow cytometry has emerged as a powerful tool for mechanistic analysis of ligand binding, cell response, and molecular assembly. Although progress has come from improving sample delivery capabilities, little attention has been paid to the volumetric requirements associated with precious biological reagents. METHODS: By using programmable syringes, valves, and other fluidic components, we created a modular, precisely regulated rapid-mix device for the delivery of small-volume samples to the flow cytometer. The device was tested using a bead-based assay in which the binding kinetics between native biotin and fluorescein biotin-bearing beads were characterized. RESULTS: Bead suspensions and reagents paired in 35- to 45-microl aliquots were efficiently mixed by the device and delivered to the flow cytometer. Kinetic data associated with the fluorescein biotin beads were analyzed and used to calibrate the performance characteristics of the device in terms of sample delivery and mixing efficiency. CONCLUSION: The rapid-mix device is capable of detecting subsecond kinetics of biological reactions using microliter volume of samples. Dimensions of the device have been minimized, and the quantitative aspects of sample delivery and analysis have been optimized. Further, the modular design has been optimized for adaptation to a variety of experimental protocols.     

Flow microsphere immunoassay-based method of virus quantitation.

A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach. Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples. After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer. The adsorption of virus to microspheres was examined under different experimental conditions. The flow cytometric assay was determined to be as accurate in detecting adenovirus as titering on 293 cells. The proposed method can be used to quantify virus in viral stocks and in biological samples.     

Cellometer image cytometry as a complementary tool to flow cytometry for verifying gated cell populations

Traditionally, many cell-based assays that analyze cell populations and functionalities have been performed using flow cytometry. However, flow cytometers remain relatively expensive and require highly trained operators for routine maintenance and data analysis. Recently, an image cytometry system has been developed by Nexcelom Bioscience (Lawrence, MA, USA) for automated cell concentration and viability measurement using bright-field and fluorescent imaging methods. Image cytometry is analogous to flow cytometry in that gating operations can be performed on the cell population based on size and fluorescent intensity. In addition, the image cytometer is capable of capturing bright-field and fluorescent images, allowing for the measurement of cellular size and fluorescence intensity data. In this study, we labeled a population of cells with an enzymatic vitality stain (calcein-AM) and a cell viability dye (propidium iodide) and compared the data generated by flow and image cytometry. We report that measuring vitality and viability using the image cytometer is as effective as flow cytometric assays and allows for visual confirmation of the sample to exclude cellular debris. Image cytometry offers a direct method for performing fluorescent cell-based assays but also may be used as a complementary tool to flow cytometers for aiding the analysis of more complex samples.     

Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.     

Flow cytometric platform for high-throughput single nucleotide polymorphism analysis

We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.     

Discrimination of viable and non-viable cells using propidium iodide in two color immunofluorescence

The relative ease with which a flow cytometer can perform simultaneous two color immunofluorescence to examine subpopulations of lymphoid cells has been well documented. Thus, flow cytometers equipped with only a single argon laser can be used to delineate various cell types by exciting both fluorescein- and phycoerythrin-conjugated antibodies to cell surface antigens. One problem that remains, however, is the artifactual staining of dead cells and clumps, which cannot be distinguished from viable cells on the basis of cell surface staining characteristics. We describe a method for simultaneous two color analysis or sorting of viable leukocytes which requires only a single laser. The method utilizes propidium iodide, which stains dead cells and thereby excludes such cells from the analysis. Using this method, as many as four viable cell types have been simultaneously analyzed in a single sample.     

Assessment of bovine sperm viability by MTT reduction assay

The MTT reduction assay depends on the ability of metabolically active cells to reduce the tetrazolium salt (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) to formazan. This study was conducted to examine and validate a simple and less costly MTT test to determine bovine sperm viability and compare the efficiency of this test with a flow cytometer. Fresh ejaculates from eight bulls were included in this study. Semen sample was diluted to 30x10(6) sperms/ml in a Hepes 0.1% BSA. The rates of MTT reduction were measured in microtiter plates after incubation for 1h at 37 degrees C using spectrophotometer (MS2 Reader) at wave length 550nm. Simultaneously split samples of the same semen were tested, using a flow cytometer for sperm viability, mitochondrial activity, and acrosomal integrity using SYBR-14, Rhodamine 123 and LysoTracker Green DNA-26, respectively. The correlation between the results of these tests was calculated using the Pearson correlation coefficients. The results revealed a strong correlation (P<0.001) between the results of MTT reduction rate and the results that simultaneously determined by flow cytometer, yielding correlation coefficients of r=0.950 for sperm viability, of r=0.926 for mitochondrial activity and of r=0.959 for acrosomal integrity. The same correlation coefficient was observed between the values of sperm viability calculated on the basis of MTT reduction rates and the results of flow cytometer. In conclusion, the MTT reduction test was found to be a reliable method in evaluating bovine semen viability and can be used successfully, especially in routine analysis, where practical aspects such as time, costs and practicability are important.