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Results for “Flow Cytometry”

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PubMed1987

The application of flow cytometry in the study of natural killer cell cytotoxicity.

A flow cytometric assay was developed to detect the cytotoxic function of natural killer (NK) cells. This procedure employed a single fluorochrome and forward angle light scatter to differentiate dead cells from live cells. When results obtained by flow cytometry were compared with 51Cr release assay, this assay showed high specificity and sensitivity. When propidium iodide was used to stain target cells killed by NK cells, we found that NK cell cytotoxicity was not cell cycle specific.

Cell Survival↗

PubMedDate not supplied

Flow cytometry evaluation of urokinase-type plasminogen activator receptor (UPA-R) in acute myeloid leukemia cells.

The aim of this study was to investigate by flow cytometry the expression of the UPA-R (Urokinase type plasminogen activator receptor-CD87) on the blastic population of AML and ALL patients in order to evaluate whether the presence of this molecule could be associated with peculiar clinical and biologic features of leukemic cells. Five different monoclonal antibodies (MoAbs) (clones: 3B10#; VIM5*; 109#; 68#; 100#) were used in order to detect the distinct forms of this cellular receptor. Cell reactivity varied significantly from case to case, also depending on the MoAb used for the flow cytometry analysis. In brief, 3B10# and VIM5* MoAbs were found to be positive in more than 90% of monocytes and neutrophils from healthy subjects, while the number of positive cells was decreased (60%) using the 109# MoAb. However, either 68# and 100# MoAbs recognised only a low number of blood monocytes and neutrophils (8-20%), while lymphocytes were unreactive with all the five UPA-R MoAbs. ALL cells were found to be CD87 negative in all cases. Blasts from AML showed a heterogeneous pattern of expression for the UPA-R MoAbs, being the reactivity strictly dependent on the MoAb used, and, to a higher extent, on the degree and type of maturation of the blastic cells. The number of blasts recognising 3B10# and VIM5* MoAbs was significantly higher than that reacting with the remaining MoAbs irrespective of the FAB subtype. Since proteolytic enzymes, like UPA, play a key role in the dissolution of the extracellular matrix, and in facilitating the cell egress from the bone marrow, it is conceivable that the expression of the UPA-R could contribute to the invasive properties and, possibly, metastatic potential of leukemic cells.

Acute Disease↗

PubMed2001

The use of flow cytometry as a tool for monitoring filament formation of fungi.

Flow cytometry (FC) has the ability to discriminate a variety of cell parameters including cell size and complexity, and fluorescence intensity. As yeast cells or fungal spores germinate they undergo a morphological transformation from round oval shaped cells to elongate filamentous forms. To date, monitoring these events has been performed using microscopic examination. Microscopic examination is a labor intensive process that examines a very small percentage of the total cell population. We have developed a method using FC that is rapid, simple to perform, and reproducible. The major advantages of FC include analysis of a larger number of cells, increased objectivity due to nonselective measurements of all cells in the population studied, and the computer related data analysis capability of the flow cytometer.

Candida↗

PubMed1995

Limitations of flow cytometry in the analysis of CD1a/HLADR+ human oral mucosal Langerhans cells.

Suspensions of human oral epithelial cells were stained with antibodies to CD1a and HLADR conjugated with fluorochromes and analysed by flow cytometry with the aim of purifying double-labelled Langerhans cells, a population comprising approximately 2% of the cell total. Whole suspensions had high levels of autofluorescence and a wide range of forward and right angle scatter properties. The mean percentage of CD1a/HLADR+ cells was 2.1%, though the double-labelled cells did not form a discrete group and the percentages of positive cells using control antibodies were similar. Density gradient centrifugation prior to flow cytometry did not facilitate Langerhans cell identification within the suspension. The results indicate flow cytometric analysis of minority cell populations (such as Langerhans cells) within oral epithelium is limited by the autofluorescence of physically heterogeneous keratinocytes, and emphasize the importance of controls in studies of oral epithelium which use this method.

Adult↗

PubMed1992

[Proliferative activity in gastric cancer with Ki-67 and propidium iodide: analysis by flow cytometry].

Growth fractions in the cell cycle were demonstrated by flow cytometry with monoclonal antibody Ki-67 for gastric cancer. By setting the cut off line at the lowest channel number of S-phase, the Ki-67 labeling rate was calculated by Ki-DNA dual fluorescence analysis. In addition to 32 gastric cancers, we examined three cell lines (Colo 320, NUGC4 and MKN28) and compared the result with the BrdUrd labeling rate. The G0G1 ratio obtained with BrdUrd was generally correlated with the G0 + G1 ratio obtained with Ki-67. The S-phase fractions obtained with Ki-67, however were a little different from those obtained with BrdUrd because of the existence of S0. The mean Ki-67 labeling rate of gastric cancer was 45.1% (16.2-66.3%). Fifteen cases received Ki-67 immunohistochemical study in the same samples. The results of flow cytometric analysis were parallel to those of microscopic study, and a correlation line: y = 0.626x + 15.9145, r = 0.8031, (p less than 0.001) was obtained. Ki-67 antibodies may provide useful information on cell kinetics.

Antibodies, Monoclonal↗

PubMed1998

Novel approach for simultaneous evaluation of cell phenotype, apoptosis, and cell cycle using multiparameter flow cytometry.

Apoptosis is a vital process for organism development and, when disrupted, can lead to abnormalities including cancer and autoimmune diseases. We demonstrate a novel multicolor flow cytometry approach for quantifying apoptosis and cell cycle information of phenotypically distinct populations, using less than 2 x 10(5) cells per sample. We used incorporation of Cy5-dUTP into DNA strand breaks by the terminal dUTP nucleotide end labeling (TUNEL) method to determine apoptosis, while cell cycle information was assessed with an ultraviolet DNA binding dye, DAPI. To simultaneously determine surface phenotype, we used paraformaldehyde fixation and a gentle permeabilization protocol combined with FITC- and PE-labeled surface antibodies. Using these fluorochromes, and three-laser instrumentation, we quantified apoptosis and cell cycle phase in lymphocyte subpopulations from heterogeneous human and murine cell sources, subjected to various culture conditions. Further, we used this method to detect divergent rates of apoptosis in a human, heterogeneous lymphocyte tumor population, demonstrating a potential application for clinical and/or research settings. Thus, we describe a six-parameter, four-color flow cytometry approach for evaluating apoptosis and cell cycle with dual surface labels. This method may also be useful as a generalized scheme to assess simultaneously two intracellular targets in a mixed cell population.

Animals↗

PubMed1994

Flow cytometry of blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells and lymphocyte subsets after treatment with 2-chlorodeoxyadenosine.

By flow cytometry and an extensive set of markers, we characterized leukemic cells from the blood and bone marrow of 68 symptomatic patients with hairy cell leukemia (HCL). Hairy cells identified in the large cell gate always expressed CD19, CD20, HLA-DR, CD45RA, and B-ly 7. Other markers were occasionally expressed, such as CD38, CD45RO, CD23, CD15, CD4, CD5, and CD10 (expressed on more than 20% of the hairy cells in 44%, 25%, 21%, 18%, 12%, 10%, and 5% of evaluated cases, respectively). During treatment with 2-chlorodeoxyadenosine (CdA), the median lymphocyte counts decreased from 2,000/microL to 300/microL. Flow cytometry was repeated at the nadir (n = 24) of lymphocyte counts, at 3 months (n = 46), at 6 months (n = 50), at 1 year (n = 39), and at 2 years (n = 12) after treatment. The initial decrease of CD8+ and CD20+ cells was greater than that of CD4+ and natural killer (NK) cells, leading to an increasing CD4/CD8 ratio. Median nadir values of CD4+, CD8+, CD20+, and NK cells were 128/microL, 78/microL, 10/microL, and 13/microL, respectively. The subsequent recovery was quicker for CD8+ and NK cells, leading to a normalization within 3 months, whereas CD20+ and CD4+ cells required 1 or 2 years to enter the normal range. The CD4/CD8 ratio thus decreased after the nadir and remained less than 1. CD45RA+ CD4 cells and CD45RA+/CD45RO+ double-positive cells were less affected by CdA. Activated T cells, ie, HLA-DR+ cells, rarely decreased below the normal range and often recovered with an overshoot. CD10+ cells increased in the bone marrow posttreatment as an indication of normal B-cell regeneration in 16 of 36 (44%) patients. The quick regeneration of certain lymphoid subsets might explain the lack of late infections in CdA-treated HCL patients.

Adult↗

PubMed2000

Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry.

BACKGROUND: An analysis was carried out to determine the sources and extent of errors encountered in the quantitation of the volume of fetomaternal hemorrhage (FMH) by flow cytometry. Different assay conditions were compared, to define the simplest, most accurate protocol. STUDY DESIGN AND METHODS: D-, D+, and artificial FMH (mixtures of D+ and D- RBCs) were stained either by a direct method (using FITC-conjugated IgG3 D MoAb [BRAD-3]), with or without dual labeling with PE-conjugated anti-GPA, or by indirect methods (using polyclonal anti-D followed by FITC- or biotin-conjugated anti-IgG reagents). Cells were selected for flow cytometric analysis on the basis of either forward or side scatter (log FSC/log SSC) characteristics or of GPA+ labeling or were unselected. The numbers of events labeled with anti-D were determined from histograms. For some samples, 10 replicates of 500,000 events each were analyzed. RESULTS: Background fluorescent events in 10 directly labeled gated D- samples ranged from 0.007 to 0.023 percent, equivalent to 0.15- to 0.51-mL FMH. Both the use of a gate on log FSC/SSC or the selection of GPA+ events only resulted in a reduction in FMH of 0.3 mL or less. The intra-assay variation in FMH, or sampling error, was found to be approximately 10 percent at low artificial FMH (<10 mL) but greater (< or =50% with a CV of 15%) with D- samples. Direct staining was quicker and produced a lower background than indirect staining. CONCLUSION: The inherent sampling error that is due to the random distribution of rare events throughout the blood sample contributed greatly to the variation in the volume of FMH calculated by flow cytometry. The FMH should not be underestimated. For a routine assay, a simplified protocol and calculation will be sufficiently accurate to determine the dose of prophylactic anti-D that should be given to the patient.

Adult↗

PubMed1983

Flow cytometry of reticulocytes applied to clinical hematology.

Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5-8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

Anemia, Aplastic↗

PubMed1986

Flow cytometry of colorectal carcinoma with three-year follow-up.

Flow cytometry DNA measurements were carried out on colorectal carcinomas from a series of 33 patients who have since been followed for three to four years. The tumors could be subclassified by this technique into 15 that had near-diploid DNA and 18 that were aneuploid. Prior reports had suggested that the near-diploid tumors carry a better prognosis than aneuploid tumors, but in this study the distribution by Dukes' stage and survival were the same in both groups.

Adult↗

PubMed1984

Endosome pH measured in single cells by dual fluorescence flow cytometry: rapid acidification of insulin to pH 6.

The acidification of various ligands was measured on a cell by cell basis for cell suspensions by correlated dual fluorescence flow cytometry. Mouse 3T3 cells were incubated with a mixture of fluorescein- and rhodamine-conjugated ligands, and the ratio of fluorescein and rhodamine fluorescence was used as a measure of endosome pH. The calibration of this ratio by both fluorometry and flow cytometry is described. Dual parameter histograms of average endosome pH per cell versus amount of internalization were calculated from this data, for samples in the absence and presence of chloroquine added to neutralize acidic cellular vesicles. The kinetics of acidification of insulin were measured and compared with previous results obtained with the chloroquine ratio technique. Rapid acidification of internalized ligand was observed both for insulin, which was mostly internalized via nonspecific pathways, and for alpha 2-macroglobulin, which was mainly internalized by specific receptor-mediated endocytosis. The average pH observed for internalized insulin was less than pH 6 within 10 min after addition of insulin. At 30 min, the average pH began to decrease to approximately pH 5, presumably because of fusion of endosomes with lysosomes.

Animals↗

PubMed1988

Flow cytometry in cytopathology. Overview and perspectives.

The future applications of flow cytometry (FCM) are briefly discussed. Whereas the early goals of its clinical applications were cancer diagnosis and classification, it has now become apparent that FCM can more successfully provide information on a variety of cell constituents of crucial importance in the evaluation of tumor prognosis and treatment sensitivity. It is suggested that (1) the data being provided by FCM will probably lead to a more clinically relevant classification of tumors, based more on the biochemical/molecular markers of individual cells and an assessment of tumor heterogeneity and less on classical cell morphology and tumor progression, and (2) FCM seems likely to become established as a routine methodology in cytopathology.

Cytological Techniques↗

PubMed1997

Rare-event sorting by fixed-time flow cytometry based on changes in intracellular free calcium.

Neurotransmitter induced intracellular free calcium, [Ca2+]i , increases were used to sort rare responding cells by fixed-time flow cytometry. Non-transfected and transfected NIH/3T3 mouse fibroblasts were stimulated with a cocktail of the neurotransmitters oxytocin, serotonin, substance P, noradrenalin, vasopressin, and neurotensin. In both cultures no detectable response to this cocktail was found. Non-transfected cells were stimulated with the cocktail and sorted for rare responders. After two subsequent aseptic sorts, each followed by subsequent cultivation, cell cultures with more than 60% serotonin responsive cells were obtained. The initial frequency of these responders was less than 3 x 10(-4). NIH/3T3 cells transfected with total genomic DNA from the rat pituitary-gland cell line GH3 were sorted with a cocktail without serotonin. After two sorts and subsequent cloning two clones were obtained. Each of these clones was sensitive to one component of the cocktail (oxytocin or substance P). The initial frequency of one responder type was estimated to be less than 2 x 10(-5). These results demonstrate that sorting by fixed-time flow cytometry is a sensitive tool to enrich very rare cells from heterogeneous cultures.

3T3 Cells↗

PubMed1996

Flow cytometry of mesophyll and bundle sheath chloroplast thylakoids of maize (Zea mays L.).

Chlorophyll fluorescence at short and long wavelengths was used to sort thylakoid membranes of maize, a plant with the C4 dicarboxylic acid pathway of photosynthesis, in a flow cytometer. The method yielded two distinct particle populations that were identified as mesophyll and bundle sheath thylakoids by low-temperature fluorescence spectroscopy and by the pigment ratio of chlorophyll a/b. Mesophyll and bundle sheath thylakoids were essentially pure after sorting by flow cytometry. Fluorescence data and chlorophyll a/b pigment ratios of thylakoids separated by flow cytometry were compared with earlier data of preparations obtained by conventional isolation procedures. Our results indicate that impure mesophyll and bundle sheath membranes were used in most previous investigations. We were unable to detect the major light-harvesting complex of PS II (LHC II) in our pure bundle sheath thylakoids using fluorescence excitation spectroscopy. Therefore, we believe that the previously reported presence of LHC II in bundle sheath chloroplasts of maize can be attributed to mesophyll contamination.

Chloroplasts↗

PubMed2001

Diagnostic performance of the urinary bladder carcinoma antigen ELISA test and multiparametric DNA/cytokeratin flow cytometry in urine voided samples from patients with bladder carcinoma.

BACKGROUND: The objective of the current study was to comparatively analyze the sensitivity and specificity of flow cytometric DNA/cytokeratin 8/18 measurements and the urinary bladder carcinoma antigen (UBC) enzyme linked immunoabsorbent assay (ELISA) test for the detection of bladder carcinoma in voided urine samples. METHODS: Eighty-one fresh urine voided samples, preserved frozen for a maximum period of 3 months, belonging to patients with an active bladder carcinoma (n = 37), patients who were free of disease as confirmed by cystoscopy (n = 19), patients receiving intravesical therapy (n = 17), and individuals with other benign and malignant conditions (n = 8), were collected. Flow cytometry measurements of thawed samples were based on the detection of cytokeratin (CK) 8+ and CK18+ cells using the 3F3 and 6D7 monoclonal antibodies alone or in combination with the measurement of cell DNA contents, after propidium iodide staining. Urinary bladder carcinoma antigen test was measured by ELISA. RESULTS: Patients were grouped according to the presence (n = 44) or absence (n = 29) of bladder carcinoma as confirmed by cystoscopy, and taking cutoffs of 9.7 microg/L for UBC-ELISA, 75% for the percentage of 3F3 (+) and 6D7 (+) cells, and 10.6% for the proportion of hyperdiplod cells that suggested a specificity of 83%, the individual sensitivity obtained for each parameter was 77%, 5%, 9%, and 77%, respectively. The presence of DNA aneuploid populations showed a relatively low sensitivity (36%) although it was the most specific parameter (93%). Combining UBC antigen test with the proportion of cells showing DNA content higher than 2n increased to 89% the sensitivity of the UBC antigen alone. However, false-positive results for both techniques were found in individuals with urologic diseases other than bladder carcinoma and in patients receiving intravesical therapy. CONCLUSIONS: The authors' results suggest that the combined use of the UBC antigen test and DNA/cytokeratin flow cytometry double stainings for the analysis of freshly obtained urine voided samples, cryopreserved to assure cellular integrity, is of great clinical utility for the detection of tumor recurrence in patients with bladder carcinoma.

Aged↗

PubMed1992

[DNA ploidy pattern of flow cytometry as indicator of malignancy and prognosis in bladder cancer].

Flow cytometry (FCM) of bladder cancer has unique characteristics. First, bladder washing is a suitable material to measure in terms of single cell suspension, while the urine appears inadequate because of the low sensitivity for detecting bladder cancer. Second, the role of FCM depends on the different features of superficial, in situ or invasive bladder cancer. In superficial cancers, FCM can detect the presence of cancer, predict intravesical recurrence and evaluate the response of intravesical instillation therapy. In carcinomas in situ, frequent occurrence of polyclonal aneuploid may indicate the aggressive nature of high grade malignancy. Early removal of bladder may be endorsed unless BCG instillation is effective. For invasive cancers, FCM does not seem to be so useful, but FCM relates well to other prognostic parameters.

Carcinoma, Transitional Cell↗

PubMed1983

Complement-mediated lysis of pigeon erythrocyte ghosts analysed by flow cytometry. Evidence for the involvement of a 'threshold' phenomenon.

Flow-cytometric analysis of complement-mediated lysis of antibody-coated pigeon erythrocyte ghosts containing fluorescein was carried out to determine whether lysis involved a gradual release of fluorescein or a 'threshold' release from individual cells. Antibody-coated ghosts were comprised of three subpopulations identified by fluorescence and scatter (size). These were: (a) highly fluorescent, medium scatter, (b) medium fluorescence, high scatter, and (c) low (or zero) fluorescence, low scatter. Lysed ghosts and isolated nuclei were identified by fluorescence microscopy and scanning electron microscopy. Fluorescence distributions analysed by flow cytometry indicated that, after complement attack, those ghosts remaining intact retained all their fluorescent label. A time course of changes in ratios of the three subpopulations indicated that once lysis of an individual ghost was initiated, release of label was complete within 1 min; no stages of intermediary fluorescence appeared, and those ghosts remaining at the end of the experiment retained the same fluorescence intensity as control ghosts. The results supported the hypothesis that complement-mediated cell lysis is a 'threshold' phenomenon; a submaximal response by a cell population representing a complete response by only some of the cells rather than a partial response by all of the cells.

Animals↗

PubMed1990

Validation of flow cytometry for rapid enumeration of bacterial concentrations in pure cultures.

Flow cytometry was investigated as a rapid detection and counting method for bacteria in pure cultures. A simple two-parameter detection scheme was employed: particle size was measured by forward angle light scatter and nucleic acid content by fluorescence of the DNA/RNA-binding dye ethidium bromide. The technique gave results that correlated exceptionally well with conventional plate counting for four species of bacteria, and concentrations in the range 10(2) to 10(7) cfu/ml. Cytometric counts were obtained in a few minutes, as compared with 2 d required for the plate counts. Under ideal conditions, each bacterial species examined exhibited a characteristic 'signature' on the cytometer, which could be explained by its known properties and morphology.

Aeromonas↗