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Results for “Flow Cytometry”

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PubMed1991

Identification and comparison of CD34-positive cells and their subpopulations from normal peripheral blood and bone marrow using multicolor flow cytometry.

Four-color flow cytometry was used with a cocktail of antibodies to identify and isolate CD34+ hematopoietic progenitors from normal human peripheral blood (PB) and bone marrow (BM). Mature cells that did not contain colony forming cells were resolved from immature cells using antibodies for T lymphocytes (CD3), B lymphocytes (CD20), monocytes (CD14), and granulocytes (CD11b). Immature cells were subdivided based on the expression of antigens found on hematopoietic progenitors (CD34, HLA-DR, CD33, CD19, CD45, CD71, CD10, and CD7). CD34+ cells were present in the circulation in about one-tenth the concentration of BM (0.2% v 1.8%) and had a different spectrum of antigen expression. A higher proportion of PB-CD34+ cells expressed the CD33 myeloid antigen (84% v 43%) and expressed higher levels of the pan leukocyte antigen CD45 than BM-CD34+ cells. Only a small fraction of PB-CD34+ cells expressed CD71 (transferrin receptors) (17%) while 94% of BM-CD34+ expressed CD71+. The proportion of PB-CD34+ cells expressing the B-cell antigens CD19 (10%) and CD10 (3%) was not significantly different from BM-CD34+ cells (14% and 17%, respectively). Few CD34+ cells in BM (2.7%) or PB (7%) expressed the T-cell antigen CD7. CD34+ cells were found to be predominantly HLA-DR+, with a wide range of intensity. These studies show that CD34+ cells and their subsets can be identified in normal PB and that the relative frequency of these cells and their subpopulations differs in PB versus BM.

Adult↗

PubMed2005

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Blood Preservation↗

PubMed2003

High throughput screening of G-protein coupled receptors via flow cytometry.

The molecular assemblies of signal transduction components, for example kinases and their target proteins or receptor-ligand complexes and intracellular signaling molecules, are critical for biological functions in cells. To better understand the interactions of these molecular assemblies and to screen for new pharmaceutics that could control and modulate these types of interactions, we have focused on developing high throughput approaches for the analysis of G-protein coupled receptors via flow cytometry. Flow cytometry offers a number of advantages including real-time collection of multicomponent data, and together with improvements in sample handling, the high throughput sampling rate is up to 100 samples per minute. For our targets, assemblies of solubilized GPCRs, a screening platform of a dextran bead has proven to be flexible, allowing different surface chemistries on the beads. The bead can be either ligand-labeled or have epitope-linked proteins attached to the bead surface, enabling several molecular assemblies to be constructed and analyzed. A major improvement with this system is that for screening ligands for GPCRs the underlying mechanism of action for these compounds can be investigated and incorporated into the definition of a 'hit'. Our current screening system is capable of simultaneously distinguishing GPCR agonists and antagonists.

Animals↗

PubMed1992

A statistical model and computer program to estimate association constants for the binding of fluorescent-labelled monoclonal antibodies to cell surface antigens and to interpret shifts in flow cytometry data resulting from alterations in gene expression.

Flow cytometry is used to obtain estimates for the distribution of fluorescent ligands bound to cell surface receptors throughout a cell sample. The equipment used provides light scattering parameters and also cell staining data in the form of dot plots and histograms of fluorescence intensities and the frequency of occurrence of particular fluorescence intensities. It is then assumed that fluorescence intensity is proportional to the number of labelled ligands bound to surface receptors. In this paper we present an outline of a statistical theory to account for the stretching and translation of such flow cytometry profiles which occur either as a result of alterations in gene expression, or from changing the sub-saturating concentration of fluorescent-labelled monoclonal antibodies or lectins used to stain the cells. We describe how the theory has been incorporated into two programs CSAFIT (cell surface antigen fit) and MAKCSA (make data to test CSAFIT). The program CSAFIT can be used to estimate two parameters, alpha and beta, by constrained non-linear regression analysis of the flow cytometry profiles. If the shift results from changes in the concentration of a staining agent then the estimates alpha and beta calculated by CSAFIT are functions of the ligand concentration, the ligand type and the cell line characteristics. They quantify the stretch and translation events that are encountered in flow cytometry. So when the parameter estimates alpha and beta are then further analysed as functions of ligand concentration, estimates for the average association constant K for the binding-site/ligand interaction can be obtained. This paper describes details of the development of programs CSAFIT and MAKCSA. We also discuss the distribution of parameter estimates calculated by CSAFIT and the overall performance of CSAFIT as assessed by simulation studies using data generated by MAKCSA.

Antibodies, Monoclonal↗

PubMed1990

Characterization of lymphocyte subpopulations in the blood of the cynomolgus monkey using flow cytometry.

This study was performed to assess the usefulness of flow cytometry in comparison with acid alpha-naphthyl acetate esterase (ANAE) staining for the enumeration of lymphocyte subpopulations in cynomolgus monkeys. For flow cytometry, use was made of mouse antihuman leukocyte monoclonal antibody (T11), recognized to undergo cross-reaction with monkey T lymphocytes or antimonkey IgG serum labelled with fluoroscein isothiocyanate. The percentages of lymphocyte subpopulations in mononuclear cells of thirty healthy female cynomolgus monkeys were 72.1 +/- 2.4% for T cells and 24.6 +/- 2.4% for B cells as assayed by flow cytometry evaluation, and 63.8 +/- 2.5% for T cells and 15.8 +/- 2.2% for B cells as determined by ANAE staining. Although the percentage of T cells shown by ANAE staining was significantly lower than that seen with flow cytometry, the coefficient of correlation indicated a close correlation in both T and B cell subsets between these two methods. In monkeys receiving 20 mg kg-1 cyclophosphamide daily for 14 days, counts of all leukocytes and T and B cells were decreased, whereas animals treated with 1.0 mg kg-1 muroctasin daily for 14 days showed higher monocyte and neutrophil counts without changes in T or B cells. These results suggest that flow cytometry evaluation has several advantages over ANAE staining with respect to rapidity and precision in toxicological studies using large numbers of monkeys.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1995

Anti-D quantification by flow cytometry: an alternative to the AutoAnalyser?

A flow cytometry method for quantifying levels of anti-D is described. The method is based on the indirect antiglobulin test and uses an FITC Fab anti-human IgG reagent for detection of bound anti-D. A standard curve is generated from the fluorescence measured by flow cytometry using the British Standard anti-D preparation 73/515. The fluorescence obtained from test samples by flow cytometry is converted into IU/mL anti-D, using the standard curve. In a limited study with 42 serum samples the assay showed good correlation with results obtained using the AutoAnalyser for sera with anti-D levels less than 100 IU/mL. The method is simple to perform and has potential as a replacement for the AutoAnalyser.

Autoanalysis↗

PubMed1997

Rapid assessment of antibiotic effects on Escherichia coli by bis-(1,3-dibutylbarbituric acid) trimethine oxonol and flow cytometry.

The effects of selected antibiotics on Escherichia coli were studied by flow cytometry with the fluorescent anionic membrane potential probe bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The actions of azithromycin, cefuroxime, and ciprofloxacin at five times the MIC on E. coli were compared by the traditional CFU assay and flow cytometry. Changes in viable counts of bacteria determined with DiBAC4(3) and by flow cytometry following treatment with the antibiotics showed trends similar to those found by the CFU assays. However, viable counts determined by flow cytometry following antibiotic treatment were 1 to 2 logs higher than those determined by the corresponding CFU assays. All the results obtained by flow cytometry were provided within 10 min after sampling, whereas the conventional CFU assay results took at least 18 h. The results indicated that flow cytometry is a sensitive analytical technique that can rapidly monitor the physiological changes of individual microorganisms following antibiotic action and can provide information on the mode of action of a drug. The membrane potential probe DiBAC4(3) provides a robust flow cytometric indicator for bacterial cell viability.

Anti-Bacterial Agents↗

PubMed1989

Applications of flow cytometry in diagnostic pathology.

The growth of flow cytometry in diagnostic pathology has been aided by the development of instruments that can be incorporated into the pathology laboratory and new assays that have expanded the role of flow cytometry in clinical medicine. The evaluation of DNA content in solid tumors is rapidly becoming a routine procedure in surgical pathology; this topic is only briefly covered here and has been more completely reviewed elsewhere. Other assays are more completely discussed, including reticulocyte analysis, identification of antiplatelet and antineutrophil antibodies in immune cytopenias, and lymphocyte antibodies in organ transplant cross-matching. Mitogen cell cycle analysis, detection of nuclear antigens, and neutrophil function assays are also briefly covered. This review is intended to increase the awareness of how flow cytometry can be incorporated into the clinical pathology laboratory, beyond its traditional use in monoclonal antibody immunophenotyping.

Antibodies↗

PubMed1999

Bacteria genome fingerprinting by flow cytometry.

BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. METHODS: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary. RESULTS: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%. CONCLUSIONS: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.

Bacterial Typing Techniques↗

PubMed2002

Nozzle design parameters and their effects on rapid sample delivery in flow cytometry.

BACKGROUND: Rapid kinetic and high throughput flow cytometry are emerging as valuable tools in biotechnology research applications ranging from mechanistic analysis of molecular assemblies to high throughput screening. Many of these new applications have been made possible by improved sample delivery capabilities, focusing increased attention on fluidic issues associated with rapid sample delivery. METHODS: Using basic fluidic premises, we derived a model that predicted the effect of nozzle parameters during rapid sample delivery. We tested the model using the rapid mix flow cytometer and modifications were made to the equipment to optimize performance. RESULTS: The model predicted that shorter nozzles with wide exit orifices decrease the delay before initial particle analysis and the fluidic stabilization time. Experimental results confirmed this prediction and model-based modifications allowed analysis of particles within 55 ms or 600 ms after mixing, with or without electronic gating, respectively. CONCLUSIONS: The model along with modifications to commercial equipment will allow rapid mix flow cytometry to analyze reactions in time frames threefold shorter than previously possible. The model allows for nozzle design predictions that should allow for analysis in the millisecond time frame. Furthermore, these findings are general for all rapid delivery applications, including high throughput flow cytometry.

Automation↗

PubMed1995

Flow cytometry provides rapid and highly accurate detection of antisperm antibodies.

OBJECTIVE: Immunobead testing (IBT), the current standard for antisperm antibody detection, is time consuming and somewhat subjective. To overcome these limitations and maintain accuracy, we studied an immunofluorescent assay using flow cytometry. DESIGN: A validation study comparing flow cytometry to IBT in the detection of serum antisperm antibodies. SETTING: Flow cytometry laboratory. PATIENTS: Sera from 37 men after vasectomy (test) and sera from 35 fertile men (control). MAIN OUTCOME MEASURE: Test serum with and without immunoglobulin (Ig)G, IgA, and IgM antisperm antibodies as defined by IBT were analyzed by flow cytometry. Sensitivity and specificity of flow cytometry was calculated by defining the IBT as the true result. RESULTS: Flow cytometry identified 22 of 22 sera that were IgG positive (100% sensitivity), 12 of 14 sera that were IgA positive (86% sensitivity), and 4 of 4 sera that were IgM positive (100% sensitivity). Overall, 22 of 37 men were positive for antisperm antibodies. The flow cytometry correctly identified 71 of 71 negative sera (100% specificity). Fluorescence intensity values from the 37 study patients significantly correlated with immunobead binding to the head region and to the entire (more than one) region. CONCLUSIONS: Detection of IgG, IgA, and IgM antisperm antibodies by flow cytometry is highly sensitive and specific. In addition, flow cytometry is able to assess thousands of sperm rapidly and accurately, reducing sampling error and technical time.

Antibodies↗

PubMed1995

Determination of urinary calcium oxalate crystallization mechanisms and kinetics using flow cytometry.

This paper describes the application of flow cytometry to the determination of calcium oxalate crystallization kinetics and mechanisms in real urine. The technique has the unique advantage of simultaneously providing quantitative particle number-size distributions and qualitative data concerning particle structure and morphology. Twenty-four-hour urines from 10 healthy male subjects were treated with sodium oxalate and the ensuing crystallization of calcium oxalate was monitored by flow cytometry. In 7 specimens crystallization was accompanied by increasing size and by a fundamental change in particle morphology. This suggests that nucleation and aggregation occurred in these specimens. In the remaining 3 samples, increasing particle numbers occurred without any changes in size or morphology, indicating that nucleation was the sole mechanism in these specimens. These results demonstrate that flow cytometry is able to differentiate between nucleation, growth and aggregation mechanisms thereby making it an extremely useful analytical tool for stone researchers.

Adult↗

PubMed2002

Lamina propria plasma cells in inflammatory bowel disease: intracellular detection of immunoglobulins using flow cytometry.

This is the first application of flow cytometry for the detection of lamina propria plasma cells and their intracellular immunoglobulins in patients with inflammatory bowel disease compared to healthy controls. The study has been focused on the distribution of IgA, IgG, IgM and the four IgG subclasses. Plasma cells were detected as high CD38 positive cells. For fixation and permeabilisation a single step reagent, Ortho Permeafix, was used. By flow cytometry, in patients with inflammatory bowel disease compared to healthy controls, a higher percentage of IgG+ cells can be observed, in Crohn's disease also a higher percentage of IgM+ cells. Regarding the IgG subclass distribution, patients with Crohn's disease show an increase in IgG2+ cells, patients with ulcerative colitis an increase in IgG1+ and IgG3+ cells. These results do agree with and expand the results of earlier immunohistochemical and functional studies, which are favoured today. For the determination of lymphocyte subset proportions and the detection of intracellular antigens, flow cytometry provides a useful alternative to well-established immunohistochemical methods. By analysing a larger number of cells, this method is more reproducible and less prone to interobserver variations than immunohistochemistry, which needs the pre-selection of a mucosal area, the microscopic scoring of a limited number of cells and the circumvention of high background staining. The optimized flow cytometric protocol used in this study might be a promising tool for further investigations of various purposes.

ADP-ribosyl Cyclase↗

PubMedDate not supplied

Detection of P-glycoprotein in solid tumors by flow cytometry.

P-glycoprotein (P-gp) detection by FCM (flow cytometry) was correlated with P-gp detection by immunohistochemical staining or Western blotting in K562 cell lines variably resistant to doxorubicin (DOX). A strong correlation was noted between the flow cytometric data and the degree of resistance assessed by thymidine incorporation assay (TIA). Of the human carcinomas tested, 37.5% were P-gp positive by flow cytometry. The flow cytometry data correlated strongly with the immunohistochemical staining (p < 0.001). Thirteen of the 15 P-gp positive tumors were resistant to DOX and vincristine (VCR) on TIA. Thus, P-gp detection by FCM correlates strongly with tumor sensitivity to DOX and VCR (p < 0.001).

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2005

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.

Biotin↗

PubMed2004

Flow cytometry in the diagnosis of mediastinal tumors with emphasis on differentiating thymocytes from precursor T-lymphoblastic lymphoma/leukemia.

Flow cytometry (FC) has become the routine technique in the evaluation of hematopoietic neoplasms. Since the anterior mediastinum is a frequent site of involvement by both primary and secondary lymphoma/leukemia, flow cytometry plays an important role in the evaluation of mediastinal masses. The present study reviews 100 flow cytometry cases from patients presenting with mediastinal lesions. In 5 cases (5%) flow cytometry was not diagnostic due to either insufficient cell yield or low viability. In the remaining cases (95/100) cell suspensions were adequate for flow cytometry evaluation. Results showed that in 31/32 (96.8%), 2/3 (66.7%), 7/9 (77.8%), 7/8 (87.5%) and 11/11 (100%) cases of B-cell lymphoma, T-cell lymphoma, carcinoma, T-ALL/LBL and thymoma/thymic hyperplasia, respectively, the diagnosis could be reached by flow cytometry alone. Excluding HL, the general sensitivity of FC in diagnosing mediastinal tumors was approximately 92%. Among the 100 cases, flow cytometry gave non-informative results in 3 cases of diffuse large B-cell lymphoma, 1 case of peripheral T-cell lymphoma, and 3 cases of carcinoma. No false positive results were encountered. The phenotypic pattern, especially surface CD3 expression versus forward scatter, reliably discriminated between immature thymocytes from thymoma/thymic hyperplasia from T-ALL/LBL. Flow methodology has the advantage of rapid turn-around time as well as high sensitivity, enabling patients with large anterior mediastinal masses and/or superior vena cava syndrome to begin treatment as promptly as possible. In experienced hands, flow cytometry plays a valuable and complementary role to histology and immunohistochemistry in diagnosing mediastinal tumors.

Adolescent↗

PubMed2005

Quantification of cell-cycle distribution and mitotic index in Hydra by flow cytometry.

The applicability of flow cytometry (FCM) to analyse cell-cycle distribution and mitotic cells in Hydra oligactis and Hydra vulgaris is demonstrated. The freshwater polyps H. vulgaris and H. oligactis are well-accepted animal models for studying cell proliferation, regeneration and differentiation. Disintegrated animals were labelled for FCM analysis according to the method of Nuesse et al. [(1990) Flow cytometric analysis of G(1) and G(2)/M-phase subpopulations in mammalian cell nuclei using side scatter and DNA content measurements. Cytometry 11, 813]. Proliferation and regeneration experiments, in the absence or presence of the oligopeptide head activator, were quantified. Cell-cycle analysis of different parts of the animals shows low proliferation in the head region and high proliferation in the gastric and foot regions. Cell-cycle analysis of different parts of Hydra, comparison of H. oligactis and H. vulgaris, as well as pharmacological treatment, yielded results that are in agreement with prior microscopic analysis. Our results demonstrate that FCM is an appropriate technique for quantifying proliferation in this animal model. It can be used for basic research on development, regeneration and differentiation as well as for innovative drug investigation and toxicology studies.

Animals↗

PubMed1992

The predictive value of flow cytometry and urinary cytology in the followup of patients with transitional cell carcinoma of the bladder.

To determine the predictive value of flow cytometric deoxyribonucleic acid (DNA) ploidy and urine cytology in patients with superficial transitional cell carcinoma of the bladder, a retrospective analysis was performed on 181 patients who presented for evaluation of presumed superficial transitional cell carcinoma of the bladder. Of the patients 91 were confirmed to have superficial transitional cell carcinoma and were systematically followed with cystoscopy, flow cytometry and urine cytology from 1984 until 1989. They underwent 637 evaluations (mean 7 evaluations per patient). At initial evaluation, flow cytometry had 81% sensitivity and 57% specificity, while urine cytology was 75% sensitive and 94% specific. During the followup flow cytometry was 76% sensitive and 36% specific. Urine cytology was less sensitive (40%) but more specific (81%) than flow cytometry in followup evaluation. These results were similar whether intravesical chemotherapy or bacillus Calmette-Guerin was administered. To ascertain whether false positive flow cytometry represented early detection of recurrent transitional cell carcinoma not apparent at cystoscopy, patients with positive flow cytometry and urine cytology were followed longitudinally. False positive flow cytometry and urine cytology were equally predictive of recurrent transitional cell carcinoma progressively with time. However, for any given examination flow cytometry was more likely to detect and predict recurrent transitional cell carcinoma. At 4 years the bladder transitional cell carcinoma incidence for false positive flow cytometry and urine cytology was 87% and 84%, respectively.

Adult↗