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PubMed2002

Velocity-independent microfluidic flow cytometry.

Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.

Bacteriophage lambda↗

PubMed2000

The application of flow cytometry to histocompatibility testing.

Flow cytometry is a powerful technique that enables the sensitive and quantitative detection of both cellular antigens and bound biological moieties. This article reviews how flow cytometry is increasingly being used as histocompatibility laboratories for the analysis of antibody specificity and HLA antigen expression. A basic description of flow cytometry principles and standardisation is given, together with an outline of clinical application in the areas of pre-transplant cross-matching, antibody screening, post-transplant antibody monitoring and HLA-B27 detection. It is concluded that flow cytometry is a useful multi-parametric analytical tool, yielding clinical benefit especially in the identification of patients at risk of early transplant rejection.

Animals↗

PubMed1999

Uses and future applications of flow cytometry in immunotoxicity testing.

Flow cytometry is an emerging technology that has numerous applications to immunotoxicity testing. The use and development of high-speed single-cell laser-based assays capable of quantitation of fluorescence, light scatter, and electrical impedance measurements can provide important information on xenobiotic-induced toxicity in defined target cell populations. The purpose of this article is to briefly review established and emerging immunotoxicology assays that use flow cytometry. In the coming years it is likely that many new flow cytometry assays will be developed and validated that will improve the sensitivity and perhaps specificity of immunotoxicity testing. Since flow cytometry is readily adaptable to high-throughput screening, it is also likely that this technology will increasingly find its place in the preclinical testing of drugs and chemicals in the pharmaceutical and chemical industries.

Animals↗

PubMed2000

Current and future applications of flow cytometry in aquatic microbiology.

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Animals↗

PubMedDate not supplied

Basics of flow cytometry.

The basics of flow cytometry are described. Cytometry is the measurement of cells; the flow cytometer performs this measurement quickly and reproducibly. Single cells in suspension are presented to a light source through hydrodynamic focusing. The resultant scattered light and fluorescence are collected and converted to electrical pulses, which are digitized for computer analysis. Frequency distributions can be viewed as single- or multiple-variable histograms for evaluating cell measurements. The versatility of flow cytometry is demonstrated by the types of samples it can analyze, characteristics it can measure, and applications in multiple laboratory sciences. Flow cytometry has been adapted easily and rapidly from a research tool to a clinically important procedure.

Clinical Laboratory Techniques↗

PubMed1997

Diagnosis of intraocular lymphoma by flow cytometry.

PURPOSE: To evaluate flow cytometry of vitreous cellular specimens as a means of diagnosing intraocular lymphoma and ocular inflammatory disease. METHODS: We undertook a retrospective, observational study of hematopoietic cell-surface markers in 20 patients with vitreous cellular infiltration in whom lymphoma was considered in the differential diagnosis. Immunophenotyping of vitreous cells obtained by vitrectomy was performed by flow cytometry using antibodies directed against specific cell-surface antigens, including ones associated with B-lymphocyte and T-lymphocyte lymphomas and activated inflammatory cells. Smears were examined cytologically. Cytofluorography was compared with the cytopathologic diagnosis and with final diagnosis. RESULTS: With flow cytometry, a diagnosis of intraocular lymphoma was confirmed in two of four patients with known lymphoma, one of whom had recurrent disease after radiation, and not confirmed in two patients who had had prior treatment with radiation or corticosteroids. In six patients with no prior diagnosis of lymphoma, five were diagnosed with lymphoma on the basis of cytofluorography. Thus, seven (70%) of 10 patients with intraocular lymphoma were diagnosed by cytofluorography compared with three (30%) of 10 with lymphoma diagnosed by cytology. With flow cytometry, 10 patients with uveitis or intraocular infections were distinguishable from patients with lymphoma by lack of a monotypic population and, in some cases, by elevated CD4:CD8 ratios and a high percentage of activated cells. CONCLUSIONS: Cytofluorography of vitreous cells is an effective alternative or adjunct to cytology. Information can be gained from specimens that are uninterpretable by routine cytology. The optimal technique for diagnosis may vary among institutions.

Adult↗

PubMed1996

Improved kinetic analysis of cytosolic free calcium in pressure-sensitive neuronal cells by fixed-time flow cytometry.

Two flow cytometric techniques were used to measure rapid transient changes in [Ca2+] in the neuronal cell line NH15-CA2. Using on-line injection, the cell suspension and stimulating solution are mixed and delivered to the detection point by a rapid increase in sample pressure. In NH15-CA2, injection of medium alone resulted in [Ca2+]i increase. Using the fixed-time method, where cells are maintained at constant pressure, no [Ca2+ ]i, increase was observed with medium alone. These results show that a rapid pressure increase alone alters the [Ca2+]i in NH15-CA2 cells. Both methods showed similar kinetics of [Ca2+], in response to bradykinin but the fixed-time method was found to be better for determination of the percentage of responsive cells.

3T3 Cells↗

PubMed1987

Viable sorting of intact multicellular spheroids by flow cytometry.

A flow cytometric method has been developed for sorting viable, intact multicellular spheroids in order to obtain uniformly-sized populations with diameters in the range of 50-100 microns. A FACS II instrument was modified for this purpose by installing a 200-microns-diameter exit orifice and by making adjustments in the sheath flow, oscillator frequency, and number of droplets sorted. Polystyrene microspheres (44 and 88 microns diameter) and 41-96-microns-diameter spheroids could be sorted and recovered with 70-100% efficiency, an improvement over previous reports. Unstained, viable spheroids were simultaneously analyzed for small-angle forward light scatter, 90 degree light scatter, and autofluorescence using a 488-nm laser operating at 100 mW. Analysis of the data demonstrated a considerable variation in both the 90 degrees light scatter and the autofluorescence signals for a given forward angle light scattering signal. By setting narrow sort windows on the forward angle light scattering signal and either the 90 degree light scatter or autofluorescence signals, uniformly spherical spheroid populations could be recovered. These sorted populations had coefficients of variation of the mean diameter in the range of 5-9%. This represents a variation of less than one cell diameter, and is a major improvement over any other technique. There was no significant difference in the subsequent growth rates of sorted spheroids compared to the unsorted spheroids. This technique will apply when uniform populations of small spheroids are required, such as investigations of the contact effect or in the initiation of growth curve studies.

Animals↗

PubMed1993

Phase-resolved fluorescence lifetime measurements for flow cytometry.

A flow cytometer capable of measuring fluorescence lifetimes by the phase shift method has been built and evaluated. Under optimal conditions, the resolution of the fluorescence lifetime measurement is shown to be under 200 picoseconds. Pulse intensity variations are normalized using limiting amplifiers and electronic filtering. Normalization of signal intensities provides a lifetime measurement that is independent of fluorescence intensity over at least a 50-fold (17 dB) range in fluorescence intensity. The fluorescence lifetimes of unbound dye, fluorescent beads, cells stained with ethidium bromide, propidium iodide, and phycoerythrin-conjugated monoclonal antibodies have been measured. The fluorescence lifetimes measured for these particles are well correlated with lifetime measurements made using a standard fluorimeter. Cells stained with ethidium bromide and propidium iodide at various nucleotide-to-dye ratios are shown to exhibit similar behavior to static cuvette measurements. The fluorescence lifetime parameter is also shown to resolve phycoerthyrin fluorescence from propidium iodide fluorescence.

Cells, Cultured↗

PubMed1993

Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry.

A flow cytometric method has been developed that uses phase-sensitive detection to separate signals from simultaneous fluorescence emissions in cells labeled with fluorochromes having different fluorescence decay lifetimes. By CHO cells were stained with propidium iodide (PI) and fluorescein isothiocyanate (FITC). These dyes bind to DNA and protein and the fluorescence lifetimes of the bound dyes are 15.0 and 3.6 ns, respectively. Cells were analyzed as they passed through a modulated (sinusoidal) laser excitation beam. Fluorescence was measured using only a long-pass filter to block scattered laser excitation light and a single photomultiplier tube detector. The fluorescence detector output signals were processed by dual-channel phase-sensitive detection electronics and the phase-resolved PI and FITC signals were displayed as frequency distribution histograms and bivariate plots. By shifting the phase of one detector channel reference signal by pi/2 + phi 1 degrees and the phase of the other detector channel reference signal by - pi/2 + phi 2 degrees, where phi 1 and phi 2 are the phase shifts associated with the PI and FITC lifetimes, the PI and FITC signals were separately resolved at their respective phase-sensitive detector outputs. This technology is also applicable to suppressing background interferences caused by cellular autofluorescence, unbound/free dye, nonspecific dye binding, and Raman and Rayleigh scattering.

Animals↗

PubMed1995

Urinary sediment analyzed by flow cytometry.

A flow cytometer for the automated analysis of urinary sediment was designed, and its performance was examined by the evaluation of 821 specimens. Auramine O, a dye for DNA and RNA, was used for the staining of the sediment. Urine (5 ml or more) was processed by the instrument for sediment analysis. Conventional microscopic analysis was done for comparison. The RBC count, the WBC count, and the number of bacterial cells, epithelial cells, and casts found by the flow cytometer and by microscopy were compared. Correlation was high for all these results. The overall sensitivity, specificity, and efficiency (accuracy) in the items analyzed were 84.7%, 57.8%, and 67.2%, respectively. One hundred specimens could be analyzed by the instrument per hour. The instrument seemed useful for screening for urinary tract disorders to identify specimens that should be analyzed microscopically in routine laboratories.

Adolescent↗

PubMed1990

Simultaneous measurement of neutrophil, lymphocyte, and monocyte glutathione by flow cytometry.

A flow cytometric method for quantitation of glutathione (GSH) was applied to simultaneous analysis of the major leukocyte types in peripheral blood. Cellular thiols (predominantly GSH) were stained with monochlorobimane (MCIB), and thiol fluorescence was measured with a flow cytometer. The fluorescence of the thiols closely reflected the GSH content, as measured by a specific glutathione reductase assay. Fluorescence of individual cell types could be measured after delineating those cells by their light-scatter characteristics, utilizing dual-angle light scatter for discrimination. By this means, GSH contents of 12.5 +/- 2.0 nmol/10(7) neutrophils, 14.5 +/- 2.7 nmol/10(7) monocytes, and 5.0 +/- 1.0 nmol/10(7) lymphocytes were found. The results obtained for neutrophils with the flow cytometer were virtually identical with those obtained with chemical assay in purified samples of neutrophils, indicating the validity of the flow cytometric method.

Cell Separation↗

PubMed1998

Some characteristics of a cellular receptor for virulent infectious bursal disease virus by using flow cytometry.

A flow cytometric virus binding assay that directly visualizes the binding of infectious bursal disease virus (IBDV) to its target cells was established. The chicken B lymphoblastoid cell line, LSCC-BK3, which is permissive for IBDV infection, bound high levels of the virus. Another B lymphoblastoid cell line, LSCC-1104-B1, bound low levels of the virus, although it was nonpermissive. No virus binding was detected in nonpermissive T lymphoblastoid cell lines. In the binding assay to heterogeneous cell populations of chicken lymphocytes, IBDV (a highly virulent OKYM strain) bound to 94% cells in the lymphocytes prepared from the bursa of Fabricius, 37% cells in those prepared from the spleen, 3% cells in those prepared from the thymus, and 21% cells in those prepared from the blood. Most of the cells, which bound the virus, were surface immunoglobulin M (SIgM)-positive, but a small number of them were SIgM-negative. Additionally, the binding of IBDV to the LSCC-BK3 cells was affected by treatment of the cells with proteases and N-glycosylation inhibitors. These findings may indicate that the IBDV host range is mainly controlled by the presence of a virus receptor composed of N-glycosylated protein associated with the subtle differentiation stage of B-lymphocytes represented mostly by SIgM-bearing cells.

Animals↗

PubMed1986

A fluorescence NK assay using flow cytometry.

A flow cytometric NK assay was developed in which the K562 targets were labelled with the fluorogenic substrate, carboxyfluorescein diacetate (c'FDA). This new assay compared favourably with results obtained using the conventional 51Cr-release assay. c'FDA was not toxic to target cells and did not inhibit lysis. The assay permits the evaluation of various aspects of NK activity such as the activity of NK-enriched, IFN-alpha-activated, and ALG-inhibited populations. The assay can be used in place of 51Cr-release, and has the advantages of being able to directly monitor target cell lysis of reducing overall assay time, and the avoidance of radioisotope usage.

Adult↗

PubMed1995

Dual analyte assay based on particle types of different size measured by flow cytometry.

Simultaneous flow cytometric assays have been developed for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), with internal determination of sample related non-specific binding (NSB). The assays use particles of 7.5, 6.5 and 5.5 microns diameter coated with, respectively, monoclonal antibodies specific for AFP, hCG or an epitope normally not present in serum. The different particle types were identified simultaneously by light-scatter measurements as their specific immunofluorometric responses were determined. The NSB in the simultaneous assay of AFP and hCG was increased by approximately 30% compared to corresponding single analyte assays. The working range of the dual analyte assays was 0.6-2000 kIU/l for AFP and 6-10,000 IU/l for hCG. No significant interference from the presence of the other analyte was observed in the measurement of either AFP or hCG. The 95% confidence interval for the ratio of dual over single analyte assay results was [0.81, 1.11] for AFP and [0.88, 1.16] for hCG.

Acrylates↗

PubMed1983

Detecting radiation damage to human chromosomes by flow cytometry.

The flow karyotype profile of ethidium bromide-stained chromosomes from human peripheral blood lymphocytes has been analysed following exposure of lymphocytes to graded series of X-ray doses in vitro. Flow analysis offers the potential for rapid counting of chromosome abnormalities and it is shown that the level of background fluorescence, the distribution of fluorescence and the area of peaks associated with the larger chromosomes, are altered in a dose-related fashion following previous exposures of cultured lymphocytes to 50-400 rad. Moreover, parallel manual analysis of the incidence of chromosome aberrations in metaphase samples of the irradiated cells show a close correlation between flow karyotype profile distortion and aberration frequency. It is estimated that for any given irradiated blood sample doses above 100 rad could be detected with certainty.

Chromosomes↗

PubMed2000

A novel method for the simultaneous assessment of natural killer cell conjugate formation and cytotoxicity at the single-cell level by multi-parameter flow cytometry.

A flow cytometric assay for the combined measurement of cell-mediated cytotoxicity and conjugate formation has been developed. Cytolysis is detected by propidium iodide uptake. Target cells, effector cells and conjugates between targets and effectors are separated by post-culture immunophenotyping and their scatter profiles. Pre-assay staining of cells is thus not required. Each cluster of cells can be further examined at the single-cell level by simultaneously performed additional immunophenotyping. Two applications were established: the assessment of NK cell activity against K562 cells and the evaluation of LAK cell cytotoxicity against both K562 and Daudi cells. A comparison with the standard 51Cr release assay for the detection of NK cytotoxicity showed that the two assays were strongly correlated, but the sensitivity of the flow cytometric assay was significantly higher.

Chromium Radioisotopes↗

PubMed2003

In vitro detection of functional humoral immunocompetence in juvenile chinook salmon (Oncorhynchus tshawytscha) using flow cytometry.

A flow cytometric (FCM) assay for detection of immunomodulatory effects of environmental factors on the humoral response of chinook salmon (Oncorhynchus tshawytscha) is described and validated. This technique combines exposure of whole animals or leucocyte cultures to immunomodulatory agents/conditions with in vitro mitogenic activation of B-lymphocytes. The proportion of leucocytes undergoing blastogenesis following in vitro stimulation with lipopolysaccharide (LPS) is quantified by FCM analysis of forward and side scatter properties. In addition, binding of a fluorescein isothiocyanate labelled anti-rainbow trout immunoglobulin M monoclonal antibody (anti-RBT SIgM-FITC), quantified by FCM analysis, is used to determine the ability of the lymphoblasts to express surface immunoglobulin M (SIgM). Through a series of calibration steps, it was confirmed that anti-RBT IgM-FITC was specific for B-lymphocyte SIgM in chinook salmon. Binding of anti-RBT IgM-FITC to chinook salmon SIgM positive leucocytes was effectively blocked with salmon serum and an isotype control was established. B-lymphocytes were partially removed from a population of leucocytes through adherence to a nylon wool column, which then demonstrated a consequent reduction in anti-RBT IgM-FITC binding. Using anti-RBT IgM-FITC as a marker, the distribution of resting lymphocytes expressing SIgM in lymphoid tissues of juvenile chinook salmon was described. The mean percentage of SIgM positive cells in spleen, pronephros and blood were found to be 62.1 (+/-2.82), 34.8 (+/-1.86) and 56.7% (+/-4.7) of all viable leucocytes, respectively. In a time-course experiment for optimal in vitro activation of leucocytes for this assay, blastogenesis and up-regulation of SIgM expression of splenic leucocytes were observed through FCM by 4 days post in vitro stimulation with LPS, continued through 7 days, but was no longer visible by 10 days post stimulation. Using this assay, reduced expression of SIgM in splenic and pronephric B-lymphocytes was detected following in vitro exposure to physiologically relevant stress concentrations of cortisol in conjunction with mitogenic stimulation. This technique will be a useful addition to the assays already available in the rapidly growing field of fish immunology.

Animals↗