SEARCH · PubMed Health

Results for “Flow Cytometry”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources

PubMed1988

Detection of an early cytomegalovirus antigen with two-color quantitative flow cytometry.

An early cytomegalovirus (CMV) antigen was detected with a monoclonal antibody by two-color fluorescent flow cytometry. With the aid of a human diploid fibroblast cell strain, FLOW 2000, infected with the AD169 strain of CMV, the viral antigen and the DNA content of infected or uninfected cells were measured. There was no evidence of change in the cell-cycle distribution of the infected cells. The viral antigen was detected within 30 minutes following virus adsorption at 0.1 and 1.0 plaque-forming units/cells; and the percentage of positive cells increased with time and viral dosage. All stages of the cell cycle were susceptible to viral infection and the average fluorescence was greater than the background fluorescence. Flow cytometry detected the viral antigen earlier than conventional immunofluorescent microscopy and cell culture for CMV cytopathological effect (CPE). Ten bronchoalveolar lavages assayed by flow cytometry and conventional diagnostic procedures demonstrated that flow cytometry might be useful in early diagnosis for CMV infection.

Antibodies, Monoclonal↗

PubMed2005

Intraoperative detection of lymph node micrometastasis with flow cytometry in non-small cell lung cancer.

OBJECTIVE: We sought to determine whether cytokeratin-positive cells can be detected as markers of lymph node metastasis by using flow cytometry within a time frame suitable for intraoperative decision making in non-small cell lung cancer. METHODS: Five lymph nodes from each of 20 patients with non-small cell lung cancer were randomly selected for study. Each node was divided longitudinally into 3 pieces: one piece for flow cytometry, one for immunohistochemical staining, and the last for conventional hematoxylin and eosin staining. In both flow cytometry and immunohistochemistry, cytokeratin-positive cells were detected with the fluorescein isothiocyanate-conjugated anti-cytokeratin antibody AE1/AE3. RESULTS: Cytokeratin-positive nodes were detected by means of flow cytometry within 40 minutes. Eight (8%) of the 100 lymph nodes from 4 (20%) of the 20 patients were deemed positive for metastasis on the basis of conventional histologic examination. By contrast, 33 (33%) lymph nodes from 13 (65%) patients were deemed positive on the basis of immunohistochemical cytokeratin staining, and 38 (38%) lymph nodes from 14 (70%) patients were deemed positive on the basis of flow cytometric cytokeratin-positive cell detection. All nodes deemed positive for metastasis on the basis of conventional and immunohistochemical methods were also positive on flow cytometry. CONCLUSIONS: Flow cytometry enables rapid intraoperative diagnosis of nodal metastasis in patients with non-small cell lung cancer. Flow cytometric detection of cytokeratin-positive cells within lymph nodes correlates with their immunohistochemical detection, and its level of sensitivity is greater than that of conventional histologic staining and about equal to that of immunohistochemical staining.

Biomarkers, Tumor↗

PubMed1995

Comparison of ploidy analysis by flow cytometry and image analysis in hydatidiform mole and non-molar abortion.

Determination of DNA ploidy is useful in the diagnosis and classification of hydatidiform mole. Most reports of ploidy analysis in molar tissue have used DNA flow cytometry. Although image analysis cytometry offers theoretical advantages over flow cytometry, there have been few reports of ploidy analysis by image analysis in hydatidiform mole. We selected 47 cases and measured DNA ploidy by flow cytometry and image analysis cytometry in complete hydatidiform mole, partial hydatidiform mole and non-molar abortion. The two cytometry modalities were compared using kappa statistics. There was reasonable overall agreement between the two modalities (kappa = 0.69) and when ploidy was stratified into diploid/polyploid and triploid categories there was near perfect agreement (kappa = 0.93). Aneuploid cell populations, which were not evident on flow cytometry, were identified by image analysis in a significant proportion of complete and partial hydatidiform moles and in a small number of non-molar abortions. Flow cytometry and image analysis cytometry yield comparable ploidy information, useful in the diagnosis and classification of hydatidiform mole. Image analysis cytometry offers greater sensitivity in the detection of small non-diploid cell populations but the significance of this latter finding is uncertain.

Abortion, Spontaneous↗

PubMed1992

Rapid assessment of bacterial viability by flow cytometry.

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDA than with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.

Bacterial Physiological Phenomena↗

PubMed2001

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry.

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Adult↗

PubMed2000

A practical approach to multicolor flow cytometry for immunophenotyping.

Through a series of novel developments in flow cytometry hardware, software, and dye-chemistry it is now possible to simultaneously measure up to 11 distinct fluorescences and two scattered light parameters on each cell. Such advanced multicolor systems have a number of advantages over current two- and three-color flow cytometric measurements. They provide a large amount of novel information for each sample studied, an exquisitely accurate quantitation of even rare cell populations, and allow identification and characterization of novel cell subsets. In particular, this technology is proving crucial to identifying functionally homogeneous subsets of cells within the enormously complex immune system; such identification and enumeration is important for understanding disease pathogenesis. However, multicolor flow cytometry comes with a new and sometimes difficult set of technical problems that must be overcome by users to derive meaningful results. In this manuscript, we describe the basic aspects of multicolor flow cytometry, including the technical hurdles and artefacts that may occur, and provide some suggestions for how to best overcome these hurdles. While inspired by the 11-color technology that we currently use, these principles apply to all flow cytometric experiments in which more than one fluorescent dye is used.

Antibodies, Monoclonal↗

PubMed1995

Use of a spherical multiparameter transducer for flow cytometry.

Fused silicon dioxide, multiparameter flow transducers with 50 microns internal square cross section and approximately 60 microns length can simultaneously measure DC and RF impedance as well as fluorescence and multiple-angle light scattering. A spherical version of such a transducer was mounted in an EPICS CVA flow-cell housing and was installed on a research prototype equipped with an argon-ion laser. The signal that was produced by the spherical transducer with EPICS DNA-Check beads was 1.73 times greater than that produced with the standard cylindrical flow cell. Similarly, with EPICS Immuno-Brite beads, the average ratio was 1.96. The Coulter impedance and light-scattering measurements were similar to those produced with the conventional cylindrical outside flow cell, although the internal cross section of the sphere was square and that of the cylinder was circular. The theoretical arguments of Leif and Wells have been demonstrated to be correct. At present, monolithic, spherical fused-silica transducers are the optimal design for combined electrooptical, multiparameter flow cytometry analyzers.

Blood Cells↗

PubMed1982

Flow cytometry and cell proliferation kinetics.

Flow cytometric techniques are presented which allow to determine parameters of cell proliferation kinetics by means of histogram sequences after special manipulations of the cell culture under investigation: (a) In the stathmokinetic method metaphase blocking agents are applied which allow the cells of the population to continue progression through interphase and accumulate at 4C DNA content. The development of DNA specific histograms during this process is analysed as to the G1 phase duration and the fraction of nonproliferating cells. (b) In the BUdR/Hoechst method the suppression of Hoechst fluorescence after BUdR incorporation during S phase is taken as a means for inducing a temporal change of histogram shapes without perturbing the cell cycle progression of the population. This temporal development of histogram shapes is analysed as to phase duration, whole cycle time and fraction of nonproliferating cells. (c) By combining the BUdR/Hoechst technique with a simultanous DNA specific stain and analysing with a two-parametrical flow cytometer, more information is obtained from each histogram after BUdR incorporation: The location of cells in the cycle at the beginning of the experiment, the cycle stage at cell harvest, and from this the distance and velocity of progression through the cycle during drug incubation. By introduction of these dynamic methods flow cytometry has become a powerful tool for the study of cell proliferation kinetics in culture.

Autoradiography↗

PubMed1993

Optimal detection of apoptosis by flow cytometry depends on cell morphology.

Flow cytometry has recently become a choice technique for the quantitative analysis of apoptosis. Monoparametric DNA analysis usually allows identification of apoptotic cells as a "subdiploid" peak. Progression through apoptosis leads to chromatin condensation, nuclear fragmentation and eventually to cell disruption. Thus, a major problem for the flow cytometric analysis of apoptotic populations is discrimination between debris and apoptotic cells. Here we demonstrate that the best parameter on which to make such a distinction is the DNA content, no matter what type of cell is studied. In contrast, discrimination between apoptotic, non-apoptotic cells, and debris is possible on the basis of scattering signals only in few selected cases, depending on the morphology of the intact cells.

Animals↗

PubMed1995

Detection of platelet-associated antibodies by flow cytometry in hematological autoimmune disorders.

We describe our experience in the evaluation of platelet-associated immunoglobulins (PAIg) by flow cytometry in comparison to solid-phase assay in patients affected by idiopathic thrombocytopenic purpura and by Evans syndrome. Results show that the analysis of PAIg by flow cytometry is easy and reliable and correlates well with data obtained by the solid-phase technique. In addition, flow cytometry allows the evaluation of samples containing small numbers of platelets (< 20,000/mm3); the analysis is objective, not influenced by personal experience. Moreover, flow cytometry appears simple enough to be performed in a routine laboratory, and data might be retrieved to perform batch analysis. Our results appear to indicate that PAIg flow cytometry might be a sensitive tool for the evaluation of patients with autoimmune thrombocytopenia.

Autoantibodies↗

PubMed2003

[Changes of peripheral blood CD45RA+ cells after hematopoietic stem cell transplantation: evaluation with four-color flow cytometry].

OBJECTIVE: To investigate the alteration of peripheral blood naive T cells in adult patients after hematopoietic stem cell transplantation (HSCT). METHODS: Peripheral blood samples were collected from 49 patients requiring stem cell transplantation before and 15, 30, 90, and 180 d after transplantation, respectively. Dynamic changes of CD4(+)CD45RA(+)/CD8(+)CD45RA(+) cell counting were measured using flow cytometry. RESULTS: Flow cytometry results of 90 samples showed that CD8(+)CD45RA(+) cells recovered to normal level 30 d after transplantation, whereas CD4(+)CD45RA(+) cells remained low and kept a slow pace of gradual increase over the 6 months following the transplantation. More rapid recovery of the transport and regeneration function of the thymus cells was achieved in patients receiving hematopoietic stem cell autotransplant than in those with allotransplant. CONCLUSION: Measurement of the changes of CD4(+)CD45RA(+)/CD8(+)CD45RA(+) cell ratio using flow cytometry may provide insights into the molecular picture of the thymus regeneration function in association with immune reconstruction process in patients after stem cell transplantation. The number of newly generated T cells transported from the thymus can be a putative marker of immune reconstruction after transplantation.

Adolescent↗

PubMed2006

Identification of organ-specific T cell populations by analysis of multiparameter flow cytometry data using DNA-chip analysis software.

BACKGROUND: The analysis of cells from multiple experimental groups by multiparameter flow cytometry leads to the generation of complex data sets, for which adequate analysis tools are not commonly available. We report here that software designed for transcriptomics applications can be used in multiparameter flow cytometry. METHODS: Lymphocytes isolated from nine different mouse organs were stained and subjected to 10-parameter flow cytometry. The resulting data set contained 594 different T cell subsets per organ per mouse and was organized into a so-called flow cytometry array (FCA). RESULTS: Computation of a hierarchical tree revealed that lymph nodes and spleen were populated by similar T cell subsets, while T cells from peripheral organs displayed a diverse subset composition. Furthermore, organ-specific T cell subsets were identified. CONCLUSIONS: This new FCA concept in flow cytomics proved to be a valuable tool for the fast and unbiased analysis of complex multiparameter flow cytometry data sets. It can be used for assessing disease progression and therapeutic intervention, and for the association of disease-related biomarkers on the protein level.

Animals↗

PubMed2000

[Flow cytometry for modern pediatric diagnosis].

Flow cytometry, a modern diagnostic method that enables to evaluate the morphology and some function of cells, has an increasing value in clinical medicine. Apart from its establish place in diagnostic of hematopoetic diseases, there is a growing evidence for its usefulness to childhood diseases especially in establishing diagnosis of inherited and acquired immunodeficiencies. Physics rules, functioning and design of flow cytometer were shortly described in this paper.

Child↗

PubMed1985

Heterogeneous distribution of antigens on human platelets demonstrated by fluorescence flow cytometry.

We have used fluorescence flow cytometry to analyse cell-to-cell variability in the density of platelet ABH, Ii, Lewis, P, P1A1, Bak,a and HLA class I antigens. Human IgG and IgM antibodies were used in a two-stage assay with goat FITC-conjugated antihuman IgG (H&L) antibody as the label, followed by single cell analysis of 10 000 platelets per sample using a 256-channel fluorescence flow cytometer (Becton-Dickinson FACS Analyser). Computer analysis of fluorescence intensity histograms for mean and peak channel and coefficient of variation shows that the degree of heterogeneity in platelet antigen density varies with each particular blood group. The broad fluorescence distribution curves with oligosaccharide antigens (CVs: A = 53, B = 40, I = 44, Lea = 40, P = 40) indicate that these antigens possess a greater variability in the number of sites per cell compared to the more homogeneous distribution of P1,A1 BaK,a and HLA (CVs: P1A1 = 24, HLA = 30). These findings may partly account for the mechanism by which transfusion of ABO-incompatible platelets results in a biphasic survival curve, with a period of early rapid removal of those platelets with a high density of antigen sites, followed by a relatively normal survival curve for those platelets that possess only a few or no antigen sites. In contrast, P1A1 and HLA sites are less variable in number from one platelet to another in a given donor, and immune-mediated removal would be more likely to approximate a single exponential curve.

Blood Group Antigens↗

PubMed1996

Quantitation of hemopoietic cell antigens in flow cytometry.

Using appropriate standards immune flow cytometry permits quantitative analysis of the expression levels of cellular antigens. This information can be gained on an absolute basis and reported in terms of numbers of molecules per cell. This offers clues for reaching time-to-time and lab-to-lab comparability of immuno-fluorescence data. After briefly reminding the characteristics of available standards this paper provides an overview of the main relevant candidate molecules for flow cytometric quantitation on hemopoietic cells and reviews the major fields of application in immuno-hematology.

Animals↗

PubMed1993

Assessment of human sperm acrosome reaction by flow cytometry: validation and evaluation of the method by fluorescence-activated cell sorting.

OBJECTIVE: To determine the applicability of flow cytometry to assess human sperm acrosome reaction. DESIGN: Prospective evaluation of semen samples incubated overnight for the development of spontaneous acrosome reaction or exposed to the calcium ionophore A23187 (5 microM) for 3 hours for induction of the acrosome reaction. SETTING: University-affiliated tertiary care center. PATIENTS: Normal healthy volunteers. INTERVENTIONS: The spermatozoa were stained with fluorescein isothiocyanate (FITC)-labeled pea agglutinin. The labeled samples were assessed visually and also subjected to analytic flow cytometry and fluorescence-activated cell sorting. MAIN OUTCOME MEASURES: Acrosome reaction assessed visually and by flow cytometry. RESULTS: Flow cytometric analysis showed a single peak of FITC fluorescence in the washed semen samples. A second peak of lower FITC fluorescence intensity was noted after overnight incubation or exposure to A23187, suggesting loss of fluorescence, which indicated the occurrence of the acrosome reaction. There was a statistically significant correlation between the assessment by the two methods (n = 41). However, although the mean difference between the methods was small (2.49%), the difference between the two methods for each individual sample can vary between -24% to +29%. When the sperm cells were subjected to cell sorting based on green fluorescence intensity, reanalysis and visual scoring verified that the low intensity peak contained a majority of acrosome-reacted spermatozoa (77.52% +/- 2.39%). CONCLUSION: These results validated the flow cytometric method for assessment of acrosome-reacted spermatozoa. Although flow cytometry is more objective and less time consuming when many samples are assessed at the same time, the visual method remains a useful and practical procedure in the clinical andrology laboratory.

Acrosome↗

PubMed2005

Analysis of baculovirus aggregates using flow cytometry.

Aggregation of viral particles represents a significant problem for baculoviral stock processing and storage. Aggregation may also affect the results of viral particle counting. A method using flow cytometry was previously developed in our lab to measure the concentration of baculovirus particles produced in insect cell cultures. In the present study, the use of the flow cytometry method was extended to the detection of baculovirus aggregates. Flow cytometry analysis of freshly prepared baculovirus stocks, stained with SYBR Green, generally exhibited a single unimodal distribution; while, baculovirus stocks stored at 4 degrees C for a few months exhibited a bimodal distribution of the fluorescent intensity signal. The bimodal distribution was associated with a decrease in the size of the original viral population and an emergence of a new viral population with a high fluorescence intensity. Treatment of these samples with an endonuclease (Benzonase) confirmed that the new population observed in the flow cytometry analysis is not free cellular DNA. Filtration through 0.22 and 0.45 microm membranes of the stored samples prior to flow cytometry analysis confirmed that the high fluorescence intensity population involved particles larger than a single baculovirus. Exposing freshly amplified baculovirus stocks with a unimodal distribution to a pH of 5.3, a condition known to induce aggregation, showed the emergence of a second population with a bimodal distribution. These results suggest that flow cytometry analysis could be used to detect baculovirus aggregates. The aggregates were associated with high fluorescence intensity populations and the mean green fluorescence intensity of these populations could be used as an indicator of the mean aggregate size.

Animals↗

PubMed1998

DNA ploidy, proliferative activities, and immunophenotype of malignant lymphoma: application of flow cytometry.

BACKGROUND: To explore the flow cytometric diagnosis of malignant lymphoma, we examined the deoxyribonucleic acid (DNA) ploidy, proliferative activities, and immunophenotype of surgical biopsy- and fine-needle aspiration (FNA)-derived materials. Our goal was to determine the possibility of making a diagnosis of malignant lymphoma by flow cytometric analysis of FNA-derived materials. METHODS: The DNA ploidy and proliferative indices including the percentage of S-phase fraction (SPF), G2 + M fraction (G2M), and Ki-67-positive fraction (Ki-67) were analyzed on the fresh materials from 84 consecutive patients with suspected malignant lymphoma. Flow cytometric analysis of surface antigens was simultaneously performed. Fourteen of the patients underwent FNA and subsequent surgical biopsy of the same lymph nodes for flow cytometric analysis. RESULTS: The proliferative indices of intermediate-grade non-Hodgkin's lymphomas (NHL) (n = 28) and high-grade NHL (n = 23) were significantly higher than those of the reactive hyperplasia (n = 25). The total for SPF + G2M of 6% was a satisfactory threshold for differentiating these NHL from reactive hyperplasia (sensitivity of 84%, specificity of 88%, and accuracy of 86%). However, low-grade NHL (n = 3) and Hodgkin's lymphoma (HL, n = 5) could not be discriminated by employing this parameter. DNA aneuploidy was seen in 13 of the 28 intermediate-grade NHL and 8 of the 23 high-grade NHL, whereas it was not seen in 25 reactive hyperplasia, 3 low-grade NHL, and 5 HL. The percentage of CD19-positive cells in B-cell NHL or CD3-positive cells in T-cell NHL was significantly higher compared with those for reactive hyperplasia. The percentage of CD16 + CD56-positive cells in natural killer (NK) cell NHL was extremely high, with a mean of 91.8%. Flow cytometric results for FNA-derived materials showed excellent correlation with those for surgical biopsy-derived specimens. CONCLUSIONS: Analyses of DNA ploidy, proliferative activities, and immunophenotype by flow cytometry (FCM) are useful for diagnosing intermediate- and high-grade NHL. Fine-needle aspiration is a less invasive approach than surgical biopsy, and, when combined with FCM, it may have a place in the diagnosis of NHL.

Biopsy, Needle↗