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Results for “Flow Cytometry”

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PubMed1982

Enrichment of epidermal Langerhans cells: studies with a monolayer technique and flow cytometry sorting.

Langerhans cells were enriched from epidermal cell suspensions by a monolayer technique based on the association of Langerhans cells with solid matrix-bound anti-Ia antibodies or by flow cytometry sorting of fluorescein-isothiocyanate labeled anti-Ia reactive cells. The monolayer technique gave a moderate enrichment (15-37%) whereas considerably higher purity (73-87%) was obtained by flow cytometry sorting. The identity of the enriched anti-Ia reactive cells as Langerhans cells was established by histochemical techniques or electron microscopy. The monolayer-enriched Langerhans cells could function as stimulating cells in the mixed leukocyte culture reaction and as antigen-presenting cells.

Animals↗

PubMed2002

Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls.

BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells.

Animals↗

PubMed1996

Rapid susceptibility testing for nontuberculosis mycobacteria using flow cytometry.

We demonstrated previously that susceptibility testing of Mycobacterium tuberculosis could be accomplished within 24 h after the organisms were incubated with antituberculosis agents by using fluorescein diacetate (FDA) staining and flow cytometry. Continued studies have now shown that assay suspensions containing M. avium, M. fortuitum, M. gordonae, or M. marinum incubated with various concentrations of ciprofloxacin, clarithromycin, erythromycin, kanamycin, rifampin, tobramycin hydrolyzed less FDA than drug-free controls. Suspensions of treated and nontreated mycobacteria could be easily differentiated at 6 and 24 h after the initiation of the susceptibility assays by using FDA staining and flow cytometry. In addition, multiplication of the mycobacteria was not required to discern differences between drug-free suspensions of mycobacteria and those treated with antimycobacterial agents. The flow cytometric assay is simple, reproducible, and rapid.

Drug Resistance, Microbial↗

PubMed1994

[DNA cytometry using paraffin embedded tumor tissues. Comparison of flow cytometry (FCM) and image cytometry (ICM)].

In order to examine the correlation of cytometric DNA ploidy in paraffin-embedded tumour material, a comparison was made of flow (FCM) and image cytometry (ICM); in the case of ICM FEULGEN-stained tissue sections (8-10 microns) and in some cases nuclear suspensions were used. Tumour tissue from seminomas (FCM: n = 50; ICM/section: n = 28), oral squamous carcinomas (FCM: n = 110; ICM/section: n = 50) and adenocarcinomas of the gallbladder (FCM: n = 50; ICM/section: n = 46; ICM/nuclear susp.: n = 40) was studied. The concordance regarding diploid/non-diploid characteristics was 96.5% in the case of seminomas, 87% in oral carcinomas and 100% in gallbladder carcinomas. A statistically significant correlation of DNA indices of FCM with ICM histograms was found in all three organ tumours examined. 6% of oral and up to 18% gallbladder carcinomas, however, could not be evaluated by means of FCM due to poor quality of the histograms (coefficient of variation (CV) > 6%). In the determination of DNA indices and resolution of multiploid stemlines, FCM analysis proved to be superior. Regional intratumoural differences and rare tumour populations were best detected by ICM using sections. Without correction factors being used, these sections did not show any significant deviation of DNA indices from the other methods used. Our study has revealed that in paraffin-embedded tumour material FCM and ICM usually provide well-correlating results, when standardised and controlled preparation and measuring procedures are applied.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗

PubMed1992

Quantitation of biological tumor markers (p53, c-myc, Ki-67 and DNA ploidy) by multiparameter flow cytometry in non-small-cell lung cancer.

Fifteen primary non-small-cell lung carcinomas (8 adenocarcinomas and 7 squamous-cell carcinomas) were analyzed by multiparameter flow cytometry for their expression of p53 and c-myc proteins. In addition, the fraction of cells staining with the proliferation-associated antibody Ki-67 and DNA ploidy was determined. These 4 biological markers were analyzed in parallel samples from a single-cell suspension made from fresh, frozen biopsies. Thus, the internal relationship between these markers within each tumor-cell population was established. Three different anti-p53 antibodies were used: PAb 421, PAb 1801 and PAb 240. All 15 tumors were p53-positive with the antibodies PAb 1801 and PAb 240, whereas only 9 were positive as judged by the antibody PAb 421. This indicates that the choice of p53 antibody is not irrelevant. Ten tumors were c-myc-positive; 7 of these were adenocarcinomas. The c-myc-positive tumors had a significantly higher level of p53 expression, judged by PAb 1801 and PAb 240, than c-myc-negative tumors. For PAb 421, there was no difference. We did not find any correlation between Ki-67 staining and expression of p53 and c-myc proteins, either with DNA ploidy, S-phase fraction or histological type. Our study indicates that there might be an association between accumulation of p53 protein and c-myc over-expression in non-small-cell lung cancer, and that this in particular might apply to adenocarcinomas. Furthermore, we show that multiparameter flow cytometry is a powerful tool in the study of the relationship between different markers in a cell population.

Biomarkers, Tumor↗

PubMed2003

Detection of minimal residual disease in unselected patients with acute myeloid leukemia using multiparameter flow cytometry for definition of leukemia-associated immunophenotypes and determination of their frequencies in normal bone marrow.

BACKGROUND AND OBJECTIVES: Detection of minimal residual disease (MRD) by multiparameter flow cytometry is an emerging prognostic factor in patients with acute myeloid leukemia (AML). The present analysis aimed at improving the applicability of this approach to more patients with AML. DESIGN AND METHODS: Bone marrow samples from unselected patients with AML at diagnosis and from healthy volunteers were immunophenotyped applying triple-stainings of 31 antigens. Leukemia-associated immunophenotypes were defined by gating on populations displaying an aberrant or infrequent immunophenotype and by applying Boolean algebra. The combination of gates obtained was applied to list mode data files containing measurements of normal bone marrow samples. Dilution experiments of AML samples in normal bone marrow were performed to test the linearity of measurements. RESULTS: At least one aberrant/infrequent immunophenotype was identified (median, 2; range, 1-5) in all of 68 analyzed AML patients. The median frequencies of cells displaying an aberrant/infrequent immunophenotype within normal bone marrow ranged from 0.00% to 1.20% (median, 0.07%). Limiting this analysis to only the most sensitive aberrant/infrequent immunophenotype per patient resulted in frequencies of cells displaying an aberrant/infrequent immunophenotype within normal bone marrow ranging from 0.00% to 0.43% (median, 0.05%). Serial dilution experiments confirmed the linearity of measurements (R>0.90 in all cases analyzed). INTERPRETATION AND CONCLUSIONS: The application of multiparameter flow cytometry to identify cells displaying an aberrant/infrequent immunophenotype and to quantify MRD is feasible in unselected patients with AML.

Acute Disease↗

PubMed1989

Detection of the ras p21 gene product in human leukemias by flow cytometry.

To date, the detection of oncogene products in human neoplasms has relied primarily upon immunoblot analysis of specimen homogenates. Herein are reported the results of a study using flow cytometry to evaluate the expression of the ras p21 gene product in both fresh and cryopreserved specimens of human acute leukemias. Cell lines known to express ras p21 were used as positive controls and normal peripheral blood was used as a negative control. Intensity of staining for ras p21 (Ip21) was expressed as the ratio of the peak channel numbers of the peak generated by staining with anti-ras p21 to the peak obtained by staining with an isotype control. Using this method, 21 out of 32 clinical specimens of acute leukemia were found to express ras p21 in elevated amounts compared to normal peripheral blood. Flow cytometry appears to be a practical method for routine screening of clinical specimens for the expression of oncogene products on individual cells rather than cell homogenates.

Flow Cytometry↗

PubMed1998

Simultaneous cell cycle and phenotypic analysis of primary uveal melanoma by flow cytometry.

BACKGROUND AND PURPOSE: DNA ploidy and cell cycle measurements of uveal melanoma tissue are regarded as having limited prognostic significance. In contrast, dual-parameter (DNA monoclonal antibody) flow cytometry offers a convenient and rapid way to screen tumour samples for a variety of phenotypic markers, whilst simultaneously measuring DNA ploidy and cell cycle, and therefore has the increased potential to identify clinically relevant indicators of disease progression. The aim of the present study was to identify a simple yet robust method for isolating, preserving and staining cells that could be analysed by flow cytometry. METHODS: Using a simple preparation procedure, a panel of membrane-associated antibodies (ICAM-1, W632, HLA-DR) and nuclear or cytoplasmic oncoprotein antibodies (c-erbB-2, c-myc, bcl-2, p53), together with positive (PHM-5) and negative (FITC F(ab')2) controls, were assayed. It was considered important to test the protocol with markers expressed on the cell surface, and in the cytoplasm and nucleus, so as not to be restrictive and thereby exclude an antigen of potential clinical interest. In addition, such panels would also enable the generation of a 'phenotypic profile' for each specimen that may reveal clinically significant trends. RESULTS: Our results indicate that tissue dissociation followed by brief fixation in 1% paraformaldehyde and permeabilisation in 70% methanol produces a stable single cell suspension, which can subsequently be stained with a wide range of antibodies for the accurate identification of cells in a potentially heterogeneous tumour population. CONCLUSION: This technology can rapidly identify sub-populations of cells expressing differing levels of proteins, which may prove to be indicative of disease progression for this aggressive disease.

Antibodies, Monoclonal↗

PubMed1998

CD34+ subset and tumor cell quantitation by flow cytometry--step toward quality assessment of autografts in B cell malignancies.

To day it is possible to predict the probability of fast engraftment based on a very simple flow cytometry standard analysis of CD34+ cells as documented by the 28 laboratories within the NSCL-G. However, the risk for delayed platelet engraftment still needs to be predicted in clinical practice for patients receiving less than 10 x 10(6) CD34+ cells/kg. Here we present data from our center supporting that identification by double staining of uncommitted (CD34+/CD38-) and lineage specific (CD34+/CD61+) progenitors may allow us to predict patients at high risk for prolonged platelet recovery. Following high dose therapy more than 30% of patients with haematological malignancies do suffer from disease recurrence within the first 3-6 months following high dose therapy. Today there are strong indications that such patients may have been transplanted with an autograft contaminated with a high number of potentially malignant B cells. Here we present a novel methodology for quantitation of blood circulating tumor cells by combining flow cytometry, cell sorting, limiting dilution and single cell RT-PCR. Such methodology has documented mobilization of clonal B cells following priming of the peripheral blood stem cell harvest and it can be used to identify minor populations and predict the efficacy of patient specific purging strategy. Consequently, quality assessment of autografts may include techniques which can predict fast three lineage engraftment as well as the risk for prolonged platelet recovery and can identify the group of patients/autografts with a strong contamination of potential tumor cells with a risk of early relapse. The future supportive cell therapy may depend upon improvements of such technologies and strategies including the selective administration of lineage specific growth factors e.g., trombopoietin as well as patient specific controlled purging strategies.

Antigens, CD19↗

PubMed2001

Correlation of hematopoietic progenitor cell count determined by the SE-automated hematology analyzer with CD34(+) cell count by flow cytometry in leukapheresis products.

The yield of stem cell collection after mobilization is crucial for autologous peripheral blood stem cell (PBSC) transplantation. Quantitative determinations of CD34(+) cells using flow cytometry or stem cell culture have been used, but these methods require much time, technical experience, and expensive reagents. The automated hematology analyzer (Sysmex SE-9000trade mark, TOA, Japan) equipped with the Immature Information (IMI) channel for immature myeloid cells can detect IMI(+) cells within 90 sec. Detection is made possible by the combination of a special reagent system and direct current/radiofrequency biosensors. We studied the relation of IMI(+) cells and variable cell counts with CD34(+) cell yield in autologous stem cell harvest. In a series of 32 patients (median age, 44 years; M:F = 11:21), 184 leukaphereses were performed after mobilization regimens with chemotherapy and G-CSF or G-CSF alone. Full blood cell counts were enumerated on peripheral blood (PB) samples taken prior to each leukapheresis. Mononuclear cell (MNC) and IMI(+) cell counts by automated hematology analyzer and flow cytometry based CD34(+) cell yield were measured on the harvested product. The relationship among PB white blood cells (WBC), PB monocytes, IMI(+) cells, MNC, and CD34(+) cell yield in a single leukapheresis was estimated by Pearson correlation analysis. PB WBC count showed no correlation with CD34(+) cell yield in a single leukapheresis (r = 0.02, P = 0.81). PB monocyte count showed a weak correlation (r = 0.21, P = 0.01) and MNC in harvest also showed a weak correlation (r = 0.36, P = 0.0001) with CD34(+) cell yield. In contrast, CD34(+) cell yield correlated well with IMI(+) cell count (r = 0.68, P = 0.0001), and data could be fitted by a linear regression equation, y = 0.330 + 0.974x. IMI(+) cell assay by the automated hematology analyzer correlated well with the CD34(+) cell yield in a mobilized autologous stem cell harvest. The IMI(+) cell count might be used as a simple and efficient indicator of blood stem cell mobilization and collection.

Adolescent↗

PubMed1998

[Measurement of Candida-specific lymphocyte proliferation by flow cytometry in children with atopic dermatitis].

To study a role of Candida albicans in the development of atopic dermatitis (AD) from the viewpoint of cellular responses, we measured Candida-specific lymphocyte proliferation by flow cytometry in children with AD. There was no apparent age-dependent change in the level of Candida-SIF (stimulation index measured by flow cytometry) in either AD or non-atopic control subjects. The level of Candida-SIF was significantly higher in AD patients than in non-atopic controls (178.0 +/- 89.3 vs 137.9 +/- 37.6, p < 0.02), and the incidence of subjects with the elevated Candida-SIF level (> or = 200) was significantly higher in AD patients than in non-atopic controls (27.9% (17/61) vs 2.6% (1/38), p < 0.005). There was no correlation between the levels of Candida-SIF and Candida-specific IgE antibody. These results suggest that Candida albicans contributes to the development of AD in some patients not only by Type I, but also by Type IV hypersensitivity reactions.

Adolescent↗

PubMed1987

DNA flow cytometry and histopathological grading of paraffin-embedded prostate biopsy specimens in a survival study.

Methods to disintegrate old paraffin-embedded tissue blocks for the application of DNA flow cytometry open up new possibilities for retrospective studies on the correlation between tumor cell nuclear DNA pattern and prognosis of the neoplastic disease. In the present work we used such a method to study the relationship between DNA ploidy, histopathological grade, and survival for 50 patients with prostate carcinomas diagnosed 1958-1974. Plugs of histologically identified tissue from benign and tumor areas were sampled from paraffin blocks of prostate biopsy specimens by using a 4-mm skin biopsy punch. Thirty-micron sections were cut from each plug for dewaxing and disintegration. The cell suspensions obtained were stained with 4',6-diamidino-2-phenylindole dihydrochloride and analyzed by flow cytometry. In about one-half of the cases where two or more plugs were analyzed we found a heterogeneous tumor cell nuclear DNA pattern. No apparent correlation was found between the histopathological grade and the DNA ploidy. Using Cox's multiple regression analysis, we found a significant correlation between DNA ploidy and survival of these patients (P = 0.043) when we controlled for histopathological grade (Dhom grade), acid phosphatase level, occurrence of metastases, age, year of diagnosis, and type of biopsy. The correlation between DNA ploidy and survival was just above the level of significance (P = 0.059) when Gleason grade was substituted for Dhom grade in the regression model.

Acid Phosphatase↗

PubMed1984

Flow cytometry isolation and improved visualization of sorted mouse chromosomes. Purification of chromosomes X and ISO-1 from cell lines with Robertsonian translocations.

While analysis and sorting of human chromosomes by flow cytometry has been widely used, isolation of a pure mouse chromosome remains very difficult, since most murine chromosomes are quite similar in size. To overcome this problem, we have analysed mouse cell lines having either Robertsonian translocations or isochromosomes. The resulting metacentric chromosomes are very different in size and in morphology from normal mouse acrocentric chromosomes. These characteristics have been analysed by computer-monitored flow cytometry, facilitated by improvements in the chromosome extraction procedure. Signals characteristic of the iso-lq chromosome in cell line PCC4 azaR1, and of the normal X chromosome in the mouse strain 22CD have thus been obtained. These chromosomes have been sorted and can be easily recognized by fluorescence microscopy when collected onto serum-albumin-coated microscope slides. The technical modifications made, coupled with the existence of a great diversity of metacentric chromosomes resulting from Robertsonian translocations, should allow the purification of a number of different mouse chromosomes.

Animals↗

PubMed1994

[Breast cancer and flow cytometry: comparative study of DNA ploidy pattern with clinicopathological parameters].

There is evidence that DNA quantization and histopathological classification of breast cancer may be useful for its therapeutic management. DNA flow cytometry, clinical and anatomopathological features of 60 paraffin embedded primary breast cancer tissue samples were studied. The aneuploidy percentage was 67%. There was a correlation between DNA index and degree of cellular pleomorphism, degree of differentiation and the fraction of cells in S phase. Likewise a correlation was found between the degree of cellular pleomorphism and the mitotic index. DNA cytophotometry was useful to solve cases of difficult diagnosis with flow cytometry ("near" diploid and tetraploid tumors). No correlation was found between aneuploidy, percentage of cells in phase S, degree of cellular atypia and mitotic degree with clinical stage, degree of lymph node involvement, tumoral size or age. It is suggested that these variables may have an independent behavior.

Adult↗

PubMed1992

[Analysis of mononuclear cells in urine using flow cytometry: a useful tool for monitoring disease activity of proliferative glomerulonephritis].

We investigated CD56+ cells (natural killer cells), CD14+ cells (macrophage) and CD3+ cells (pan T cells) in urine using flow cytometry in various renal diseases including idiopathic crescentic glomerulonephritis, IgA nephropathy, membranoproliferative glomerulonephritis membranous nephropathy, pyelonephritis and idiopathic renal hematuria. A remarkable increase of CD56 cells in urine was observed in glomerulonephritis with marked necrotizing/and or crescentic (NC) lesions. The ratio of CD56+ cells/CD3+ cells was correlated with the severity of NC lesions. The ratio of CD14+ cells/CD3+ cells was increased in glomerulonephritis with endocapillary proliferation. Using immunohistochemical methods, CD56+ cells were also observed to be present in cellular crescents in biopsy specimens. These observations suggest that an analysis of these cells in urine using flow cytometry may be a useful tool for monitoring disease activity in proliferative glomerulonephritis.

Antigens, CD↗

PubMed1991

Identification by flow cytometry of the prostaglandin-producing cell populations of term human decidua.

Human term decidua produces prostaglandins (PGs) which have been implicated in the initiation of human parturition. Using flow cytometry to isolate pure cell populations, we have investigated the cell types responsible for decidual PG production. Cell dispersions were prepared enzymatically from decidua vera isolated from term placentae, and were incubated in Dulbecco's Modified Eagle's Medium containing 0.25% bovine serum albumin at 37 degrees C. PGF2 alpha and PGE2 output were measured by radioimmunoassay of the conditioned medium. Production of PGF2 alpha (fmol/10(6) cells per 3 h) exceeded that of PGE2 at 273 (108-322) versus 97 (38-127) respectively (median (range]. The decidual cell dispersions were then incubated with monoclonal antibodies (anti-CD45 which labels the leukocyte common antigen or anti-human leukocyte antigen class II (HLA-DR) which is specific for macrophages in this tissue) and sorted by flow cytometry. The resultant antibody-positive and -negative cell populations were incubated and PG production was measured. Controls showed that antibody labelling and sorting did not alter PG production. PGF2 alpha and PGE2 output by bone marrow-derived (CD45-positive) cell populations exceeded that of non-bone marrow-derived (CD45-negative) cells. Furthermore, we were able to demonstrate that the HLA-DR-positive macrophage population had the highest PGF2 alpha and PGE2 production rates in human term decidua in vitro.

Antibodies, Monoclonal↗

PubMed2005

Flow cytometry analysis of the activity of disinfecting agents tested with Staphylococcus aureus and Escherichia coli.

The efficacy of five disinfecting agents was tested by a new microbiological method, i.e. the flow cytometry. The method is based on kinetic measurements of the effects of a disinfecting agent on the percent of live/dead cells detected with propidium iodide. E. coli ATCC52922 strain and S. aureus ATCC 29213 strain were used in the experiment. From the measurements the killing time 50 (KT50) was estimated as the period of time needed to kill 50% of microorganisms in the disinfected volume. KT50 for ethanol 70% was 11s and it was the most efficient disinfecting agent among all examined. Other commercial preparations were compared with ethanol 70% and were traced throughout the period of 5 min. The results were obtained rapidly, frequently in less than 10 min. In conclusion, the effectiveness of a particular antibacterial disinfectant preparation may be estimated quantitatively within a few minutes by the flow cytometry. The method proved to be very useful for a fast comparison of the effectiveness of various disinfectant preparation against pathological microorganisms.

Disinfectants↗

PubMed2000

A method to study apoptosis in eosinophils by flow cytometry.

The aim of this study was to develop a simple flow cytometric procedure to study eosinophil apoptosis. Eosinophils were isolated from the peripheral blood of healthy, non-allergic individuals and then cultured in basal culture medium. The cells were examined after 24, 48 and 72 h for forward- and side scatter (FS-SSC) pattern, staining with FDA, PI, and anti-CD95, and light microscopic appearance. After culture for >24 h, two populations with different FS-SSC-patterns appeared, referred to as A and B. Population A consisted of living, FDA-positive eosinophils. The eosinophils in population B showed a lower FS scatter than those in population A and a staining pattern with PI indicating the presence of hypodiploid DNA. Anti-CD95 demonstrated a significant staining of the eosinophils in population B, which increased after 2 days in culture. The cells were sorted using a FACS-Scan cell sorter and by Annexin V-coated magnetic beads to permit separate analyses of PI-staining pattern, DNA electrophoresis, and light microscopic examination of the cells in population B. The present study suggest that it is possible to discriminate between apoptotic and living eosinophils using the FS-SSC pattern and the PI-staining pattern obtained by flow cytometry.

Annexin A5↗