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Results for “Flow Cytometry”

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PubMed1993

Flow cytometry in comparison with mitotic index in predicting disease outcome in transitional-cell bladder cancer.

The DNA content and S-phase fraction were measured by flow cytometry in 448 tumour biopsy specimens from transitional-cell bladder cancer (TCC). The samples were also analyzed for mitotic index, WHO grade and papillary status, and histological and flow cytometric data were then correlated to clinical behaviour of tumours during a mean follow-up period of 9.9 years. TNM classification, WHO grade, papillary status, mitotic index, DNA ploidy and S phase fraction were significantly interrelated. Twenty-four percent of tumours showed heterogeneous DNA indices when measured from multiple samples (measured in 94 cases). Of the histological parameters, independent predictors of progression in superficial tumours were the S-phase fraction and mitotic index. In superficial tumours, S-phase fraction and the mitotic index included all the available independent prognostic information in survival analysis, whereas in muscle-invasive tumours T category was the most important prognostic factor. The results suggest that DNA ploidy has no independent prognostic value in transitional-cell bladder cancer, whereas proliferation indices (SPF, mitotic index) are important prognostic factors. Accordingly, malignancy classification of papillary bladder tumours can be based on proliferation indices alone. Nodular tumours run an unfavourable course and their malignancy grading by flow cytometry or by mitotic index is not relevant.

Aged↗

PubMed1985

Studies on urinary bladder carcinoma by morphometry, flow cytometry, and light microscopic malignancy grading with special reference to grade II tumours.

Biopsies from 28 patients with urinary bladder carcinoma were investigated by flow cytometry and morphometry. Histopathological grading on 1.5 microns thick glycol methacrylate sections was also performed. Nuclear profile areas, nuclear volume densities and mitotic indices were usually larger in the higher grades of malignancy. All grade I tumours were diploid and all grade III tumours were aneuploid. Out of 13 grade II tumours 8 were diploid and 5 aneuploid. In these latter five cases nuclear profile areas were at the high end of the spectrum. The data show that flow cytometry and morphometry could be a valuable tool in the diagnosis of urinary bladder carcinoma. Our data also suggest that a subdivision of the grade II tumours might be possible and meaningful in the assessment of prognosis.

Adult↗

PubMed1992

Clinical applications of flow cytometry. Immunologic methods for measuring cell membrane and cytoplasmic antigens.

Flow cytometric analysis is the method of choice for classifying leukemia and lymphomas. When combined with other clinical and morphometric data, a definitive diagnosis can be made in the most difficult cases. By using a combination of specific antibodies that are each labeled with a different fluorochrome, panels can be designed to follow residual disease with greater precision than any other method. The detection of intracellular proteins is less well developed. Because antibodies can bind nonspecifically to dead cell components, there may be considerable biological noise. This noise reduces the sensitivity for detecting the desired protein within the cell. Until this problem is solved, it is likely that other methods may provide more reliable data. The major advantages of flow cytometry are rapid cell processing, cell by cell analysis, and the simultaneous evaluation of multiple markers. The latter is especially important because more tests can be performed using less clinical material. When flow cytometric results are combined with histologic results, the confidence in professional interpretation is far greater than either can provide alone. It is likely that the future application of these approaches will significantly improve diagnosis and patient care.

Antigens, Neoplasm↗

PubMedDate not supplied

[DNA ploidy and cell cycle phase analysis with flow cytometry in bladder wash. Preliminary experience].

OBJECTIVE: To analyze the prognostic significance in bladder carcinoma of DNA ploidy and cell phase fractions measured by bladder wash flow cytometry. METHODS: Samples were obtained from 25 patients by bladder irrigation; 16 before surgery and 9 during follow-up cystoscopic examination. Cells were stained with propidium iodide and analyzed with the FacScan flow cytometer and Cellfit 2.01 (Becton-Dickinson). RESULTS: The number of cells obtained was sufficient for flow cytometric analysis in all cases. In 13 tumor samples (8 superficial and 5 invasive tumors), aneuploidy cells were detected in 3 cases that had a worse outcome; the only superficial tumor in which aneuploidy was detected presented a recurrent bladder carcinoma 15 months later. Of the 5 patients with invasive tumors, two patients with aneuploidy died within 6 months from tumor metastases. Of the patients without macroscopic tumor, only one showed an increase in the percentage of the S phase fraction (19.5% of cells in S phase). A recurrent bladder carcinoma was detected in this patient 6 months after the analysis. In patients with macroscopic tumor, analysis of the S phase fraction was not relevant for prognosis. CONCLUSIONS: Analysis of DNA ploidy and cell phase fractions by flow cytometry of bladder washings can increase the prognostic information in bladder carcinoma. Aneuploidy was associated with a worse prognosis and an increase in the S phase fraction predicted a recurrent bladder carcinoma months before it manifested clinically.

DNA, Neoplasm↗

PubMed2000

Measurement of estrogen receptors in intact cells by flow cytometry.

BACKGROUND: Estrogen receptor (ER) levels in tumor cells are important for determining the outcome of treatment and the prognosis of breast cancer patients. Flow cytometry is a convenient tool for quantifying the ER in cells, but a more sensitive, reproducible method for immunostaining the ER with anti-ER antibody is needed. Materials and Methods ER-positive human breast cancer cells MCF-7 and T47D, and ER-negative MDA-MBA-321 cells, were fixed and permeabilized by three different protocols. The cells were then stained by indirect immunofluorescence, using two commercial antibodies to ER (MA1-310 and DAKO 1D5), or by direct immunofluorescence using FITC-labeled anti-idiotypic antibody clone 1D(5). The stained cells were analyzed by flow cytometry. RESULTS: The fixation of cells with a mixture of 0.25% paraformaldehyde and 70% methanol, permeabilization with 0.05% Triton X-100, and increasing antibody and antigen reaction time led to 80-99% of cells being stained with anti-ER antibodies. The relative brightness of ER immunostaining was as follows: anti-idiotypic antibody ID5 > MA1-310 > DAKO 1D5. CONCLUSIONS: Direct immunofluorescence with the FITC-labeled anti-idiotypic antibody of permeabilized cells resulted in improved specific staining of the ER, as compared to indirect immunofluorescence with anti-ER antibodies of fixed and permeabilized cells. Increasing the length of staining, and treatment of cells with Triton X-100, are both necessary to improve the staining of intracellular antigen for flow cytometric analysis.

Antibodies, Anti-Idiotypic↗

PubMedDate not supplied

Assessment of viability of yeast by flow cytometry using Light Green as vital dye exclusion.

A comparative study of two different staining methods for estimation of yeast's viability is assessed by microbial flow cytometry. Propidium iodide (PI) a well known nucleic-acid binding vital dye exclusion is compared with light green (LG), a cytoplasmic function related vital dye widely used in histological, histochemical and immunocytochemical staining. The yeast model used was heat-killed Candida guilliermondii cells. The stained heat-killed bacterial population showed on the dot-plot flow cytometer analysis, a clear separation towards the green-orange (350-600 nm) for LG, and orange-red (550-725 nm) for PI on the fluorescence spectrum-zone channels. Both dyes showed by non-parametric Kolmcgorov-Smirnov, 99% of probability of difference between heat-killed and live microorganism's histogram-population, with a D = ranking from 0.50-0.64 for LG to 0.91-0.98 for PI. Higher cost and carcinogenic risk when handling PI, make LG a better choice for flow cytometry yeast viability tests.

Candida↗

PubMedDate not supplied

[A comparative assessment of diagnostic value of the study of leukocytes and lymphocytes by microscopy and laser flow cytometry in patients with HIV infection].

A total of 135 subjects, 54 of them HIV infected and 81 donors (controls) were examined by microscopy and laser flow cytometry. A certain reduction of the total leukocyte count and of absolute lymphocyte count in the peripheral blood and a comparatively higher percent content of these cells were observed in HIV infected subjects examined for the first time. Laser flow cytometry proved to be a more informative method both for the assessment of the total count of leukocytes (p < 0.05) and for a complex assessment of cell counts (total leukocyte count, absolute and relative counts of lymphocytes) in the peripheral blood (p < 0.01) of patients with HIV infection.

Flow Cytometry↗

PubMed1997

Rapid susceptibility testing of fungi by flow cytometry using vital staining.

A 1-h assay for antifungal susceptibility testing measuring the impairment of fungal metabolic activity was developed. Yeast viability was analyzed by flow cytometry with a novel fluorescent probe, FUN-1, which emits a red fluorescence when the yeast is metabolically active. For nine Candida albicans strains tested, this method yielded results comparable to those obtained by the standard M27 procedure for amphotericin B, flucytosine, fluconazole, and ketoconazole. Whether the flow cytometry antifungal susceptibility test results correlate with the in vivo activities of the drugs remains to determined.

Candida albicans↗

PubMed1983

Centromeric index measurement by slit-scan flow cytometry.

We report here the application of slit-scan flow cytometry (SSFCM) in the classification of muntjac, Chinese hamster, and human chromosomes according to centromeric index (CI) and total fluorescence. Chromosomes were isolated from mitotic cells, stained with propidium iodide and processed through the SSFCM where fluorescence profiles were measured. The centromere for each profile was taken as the point of maximum difference between the measured profile and a standard profile having no centromeric dip. The areas under the profile on either side of the centromere were then calculated and the CI was calculated as the ratio of the larger area to the total area under the profile. Relative DNA contents for each chromosome were taken to be proportional to the total fluorescence. Mean CI's for muntjac chromosomes 1, 2, and X + 3 were 0.52, 0.88, and 0.73, respectively; CI's for Chinese hamster M3-1 chromosomes 1, 2, 5, 8, and M2 were 0.53, 0.55, 0.57, 0.77, and 0.86, respectively; and average CI's for chromosome groups 4 + t (X;5), 6 + 7 + Y, 9 + M1, and 10 + 11 were 0.56, 0.82, 0.58, and 0.60, respectively. These results were, on average, within 4.4% of CI measurements made by image cytometry. CI's measured for human chromosomes 9 through 12, were, on average, within 2.0% of those made by image cytometry.

Animals↗

PubMed1994

[Flow cytometry in diagnosis and treatment of bladder tumor].

The cellular DNA contents of 139 bladder irrigations from 52 cases of bladder tumors were determined by flow cytometry. The results were compared with those of pathological grading and exfoliative cytology. It is suggested that cytological results were less accurate than DNA contents. High aneuploid DNA contents in tumor cells were marked by low grade and deep penetration of the bladder tumor. Total cystectomy was needed for bladder tumor with high aneuploid DNA, and partial cystectomy and TURBT for that tumor showed high recurrence with instillation of BCG. The results showed that DNA content of tumor is correlated with the nuclear grade, especially in poorly differentiated neoplasms, but flow cytometry can not replace cystoscopy examination.

Adult↗

PubMed1990

[Purification kinetics of human tumors measured by flow cytometry].

We report on the use one-parameter DNA flow cytometry in the evaluation of the proliferation kinetics of malignant tumors. We emphasize the need of a background correction of the histograms in cases where the S-phase portion is used as a prognostic parameter for patients with malignant tumors. Flow cytometric investigations during the last 12 years with many thousand samples of human malignant tumors have shown that the described methodology can be used routinely as "assistant diagnosis" for a distinct group of tumors. These tumors comprise carcinomas of the breast and the ovaries and probably also of the endometrium. Regarding the relevance of the method for oropharyngeal squamous carcinomas, gliomas and meningiomas we have to await longterm results. In contrast, at present the method does not seem to be applicable for colorectal carcinomas and it is apparently not suitable for stomach carcinomas. It is also questionable whether data on proliferation kinetics gained in human tumors transplanted to nude mice can be as a basis in chemotherapeutic treatment of patients from whom the tumor originated.

Animals↗

PubMed2005

Detecting protein-protein interaction in live yeast by flow cytometry.

BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.

Bacterial Proteins↗

PubMed2003

Center for Disease Control (CDC) flow cytometry panel for human immunodeficiency virus infection allows recognition of infectious mononucleosis caused by Epstein-Barr virus or cytomegalovirus.

AIM: To analyze the distribution of lymphocyte subsets in the peripheral blood of patients with infectious mononucleosis caused by Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and to investigate the possible diagnostic usefulness of flow cytometry panel recommended by the Center for Disease Control and Prevention (CDC) for HIV-1 infection. METHODS: The study included 130 immunocompetent adults with infectious mononucleosis caused by EBV (n=103) and CMV (n=27) and 50 controls. EBV-infected patients were divided into two groups based on typical (n=92) or atypical (n=11) clinical presentation of the disease. Lymphocyte subpopulations were determined by flow cytometry and a panel of monoclonal antibodies recommended by the CDC for the immunophenotyping of patients infected with human immunodeficiency virus (HIV). RESULTS: Patients with typical and atypical presentation of EBV-induced infectious mononucleosis showed increased percentages of total T-cells, cytotoxic-suppressor CD8(+) T cells and activated HLA-DR(+) T cells compared to healthy controls. Percentages of CD4(+) T cells, as well as CD4/CD8 ratio, were significantly decreased. Absolute counts of CD4(+) T cells and percentages of B cells did not differ from healthy controls. Pattern of changes in CMV-infected patients was completely identical to that in healthy controls, although less pronounced. CONCLUSION: Lymphocyte subpopulations represented in the CDC panel for HIV are sufficient for the recognition of patients with infectious mononucleosis caused by EBV and CMV. Flow cytometry can be useful support for reaching diagnosis in patients with atypical clinical presentation of EBV-induced infectious mononucleosis.

Adult↗

PubMed1995

Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry.

We have compared the influence of sample preparation upon the level of surface expression of T, B, and NK cell-related antigens as assessed by flow cytometry. Lysed whole blood (WBL), Ficoll-Paque separated peripheral blood lymphocyte (F-PBL), and frozen peripheral blood lymphocyte (Fr-PBL) were analyzed via single- and multicolor flow cytometry. The percentage of positive cells expressing the individual cell surface markers was not affected by the procedure for preparation of WBL, F-PBL, and Fr-PBL. In contrast, the fluorescence intensity level of individual cell surface markers varied depending on cell preparation. By using Quantum Simply Cellular (QSC) microbeads, the antibody binding capacity (ABC) of single-color stained cells was quantified and compared. The amount of monoclonal antibody (MAb) anti-CD3-FITC bound to Fr-PBL (mean ABC = 137,040) was significantly higher (P < 0.001) that the amounts bound to WBL (mean ABC = 112,410) and F-PBL (mean ABC = 107,738). In multicolor analysis, the fluorescence intensity of CD3-FITC and CD4-FITC was significantly higher on Fr-PBL than on WBL and F-PBL; CD8-PE and CD20-PerCP was significantly higher on WBL. Furthermore, the intensity of CD3 and CD4 was different on T-cell subsets. The intensity of CD3 staining in three-color analysis was lower than with single-color staining using the same fluorochrome. We conclude that particularly the method of cell preparation but also the selection of MAb combinations may influence the level of staining of certain lymphocyte antigens. This may be of relevance in the analysis of cellular activation and regulation of differentiation.

Adult↗

PubMed2001

Effects of mismatched refractive indices in aquatic flow cytometry.

BACKGROUND: Forward-angle light scatter, as measured by flow cytometry, can be used to estimate the size spectra of cell assemblages from natural waters. The refractive index of water samples from aquatic environments can differ because of a variety of factors such as dissolved organic content, aldehyde preservative, sample salinity, and temperature. In flow cytometric analyses, mismatch between the refractive indices of the sheath fluid and the sample causes distortion of the forward-angle light scatter signal. We measured the effect of this mismatch on cell size measurements. METHODS: We examined the error by measuring the scatter signal of a variety of particle types and sizes and changing the sheath-to-sample salinity ratio. The effects were characterized for standard microspheres, cultured phytoplankton cells of different sizes, and natural populations from an estuarine river. RESULTS: We found that the distorted scatter signals resulted in an increase in the apparent size of small cells (1--2 microm) by a factor of 4.5 times. Cells in the size range of 3--5 microm were less affected by the salinity differences, and cells larger than 5 microm were not affected. Chlorophyll and phycoerythrin fluorescences and 90 degrees light scatter signals were not changed by sheath and sample salinity differences. CONCLUSIONS: Care must be taken to ensure that the sheath and sample refractive index are matched when using forward light scatter to measure cell size spectra, especially in estuarine studies, where salinity can vary greatly. Of the factors considered that can change the sample refractive index, salinity gradients in an estuary cause the largest index mismatch and, consequently, the largest error in scatter.

Aldehydes↗

PubMed1995

Assessment and significance of diploid-range epithelial populations in DNA aneuploid breast carcinomas using multi-parametric flow cytometry.

A 2-color (PI, cytokeratin--FITC) multi-parametric analysis of intact cells was used to reveal diploid-range epithelial populations by flow cytometry in 108 consecutive DNA aneuploid breast carcinomas. Thirty-eight tumors (35%) contained a significant diploid range epithelial population, defined as cytokeratin-positive cells having a DNA content indistinguishable from that of endogenous lymphocytes and comprising at least 20% of all cytokeratin-positive cells. An additional 23 cases (21%) contained a minor diploid range epithelial population having a normal DNA content and comprising only 5-20% of all cytokeratin-positive cells. Multiple DNA aneuploid stemlines were present in 24 cases (22%). Diploid-range populations were more frequent (91%) in tetraploid cases than in hyperdiploid (32%), hypodiploid (17%) or hypertetraploid cases (20%). The presence of diploid epithelial populations and/or multiple aneuploid stemlines correlated with histologic parameters, including an extensive intraductal component (unimodal--4% vs. multi-modal--57%, P = 0.001), heterogeneous differentiation (unimodal--0% vs. multi-modal--52%, P = 0.001), and multi-focal growth with residual interspersed benign tissue (unimodal--8% vs. multimodal--57%, P = 0.01). These data show that diploid-range epithelial cells are frequent in aneuploid breast carcinomas analyzed by flow cytometry. In some tumors, these populations undoubtedly reflect the presence of residual benign epithelium. The numerical dominance of other histograms by near-diploid measurements suggests the presence of diploid-range neoplastic stemlines which would be 'hidden' by contaminating host-derived cells in single parameter DNA histograms. Finally, the correlation of DNA content heterogeneity with distinctive histologic patterns of breast neoplasia implies that co-existing stemlines may have biological significance in the progression of some tumors.

Aneuploidy↗

PubMed1990

Long-term culture of human bone marrow. I. characterization of adherent cells in flow cytometry.

Using long-term culture techniques, we were able to characterize the different adherent cell elements of the in vitro bone marrow microenvironment by flow cytometry. After 2 weeks of culture, four adherent cell subsets, L, MM, F, and M, were distinguished according to their intrinsic cellular characteristics of volume and spontaneous fluorescence. Using four immunological markers, we identified each population as a hematopoietic lymphocytic group (L), CD45+, VIM2-, CD14-; a hematopoietic myelomonocytic group (MM), CD45+, VIM2+, CD14-; a hematopoietic macrophage group (M), brightly autofluorescent, CD45+, VIM2-, CD14+; and a group of fibroblastoid cells (F) with a very high volume, CD45-, VIM2-, CD14-, and collagen III+. Isolation of the different hematopoietic and nonhematopoietic components of long-term bone marrow culture is thus possible using flow cytometry analysis according to the cell characteristic of volume and spontaneous fluorescence alone.

Antigens, CD↗

PubMed1997

HLA-B27 phenotyping with flow cytometry: further improvement by multiple monoclonal antibodies.

To establish the optimal flow cytometric method for HLA-B27 phenotyping, we compared several strategies, using three monoclonal anti-B27 antibodies (from the HLA-ABC-m3, GS145.2, and FD705 clones). We used a triple-color direct immunofluorescence assay, including a T-lymphocyte-specific antibody as an internal control and an anti-HLA-Bw4 antibody. Blood samples from >400 subjects were tested. From ROC curve analysis none of the three antibodies appeared to be suitable for use as a single typing reagent. The efficiency of the test was affected by cross-reactions with other HLA antigens, notably the HLA-B7 antigen. Preincubation with anti-B7 serum efficiently inhibited this cross-reaction and raised the test efficiency considerably. We concluded that none of the anti-B27 antibodies investigated is suitable for use as a single typing reagent. Additional typing of Bw4 is not valuable, whereas inhibition of cross-reactions due to the B7 antigen will considerably improve the performance of the test. We recommend that two different monoclonal anti-B27 antibodies be used for accurate and reliable HLA-B27 phenotyping with flow cytometry.

Antibodies, Monoclonal↗