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Results for “Flow Cytometry”

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PubMed2003

[Immunophenotyping of 222 children with acute leukemia by multi-color flow cytometry].

OBJECTIVE: Acute leukemia (AL) is one of the most common malignant diseases in children. AL immunophenotypes are known to be benefit to the predictable prognoses and specific therapy. Recently, the accuracy of AL immunophenotyping was dramatically improved with the application of the flow cytometry, the new monoclonal antibodies, the improvement of the gating strategies and the multi-parameter analytic techniques. The aim of this study was to evaluate the value of multi-color flow cytometry in the immunophenotyping of acute leukemia in children. METHODS: Three- or four-color flow cytometry and CD(45)/Side Scatter (SSC) gating were used to analyze the surface and cytoplasmic (Cy) antigen expressions in 222 successive cases of childhood acute leukemia. RESULTS: Cells from 222 cases of children with acute leukemia were analyzed. Based on the diagnostic criterion proposed by European Group for the Immunological Characterization of Leukemia (EGIL), four categories could be identified: the undifferentiated type accounted for 0.9%, acute myeloid leukemia (AML) 35.1%, acute lymphoblastic leukemia (ALL) 55.9%, and mixed lineage AL 8.1%. Of 124 patients with ALL, 94 patients (75.8%) were classified as B lineage and 30 patients (24.2%) T lineage ALL. Antigen aberrant expressions were found in AML (24.4%), B lineage ALL (36.2%) and T lineage ALL (30.0%). CD(7) was the most commonly expressed lymphoid antigen in AML (12.8%), followed by CD(19) (6.4%) and CD(2) (5.1%). CD(13) was the most commonly expressed myeloid antigen in ALL (18.5%), followed by CD(15) (11.3%), CD(11b) (6.5%) and CD(33) (4.3%). CD(117) and CD(56) presented in 73.3% and 38.0% cases of AML, respectively, but were generally absent on blast cells of ALL. CyCD(22), CyCD(3) and CyMPO were specifically expressed in B lineage, T lineage and myeloid lineage leukemia, respectively, and the first two could be more sensitively detected than they were on cell membrane surface. CONCLUSIONS: Multi-color flow cytometry is a reliable technique in the diagnosis, differential diagnosis and classification of acute leukemia in children.

Acute Disease↗

PubMed2003

Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection.

Platelet-derived microparticles (PMPs) formed by vesiculation during platelet activation seem to play a role in blood coagulation and in pathological disease states. Flow cytometry is currently the gold standard to characterize platelets and PMPs. Using this technique we distinguished between platelets and PMPs based on size and the presence of phosphatidyl serine (PS); PMPs were arbitrarily defined to be smaller than one micrometer and capable of forming a stable complex with fluorescently-labeled Annexin V, a protein that forms a calcium-dependent complex with PS. Further confirmation of PMP and platelet identity was done by use of fluorescently-labeled antibodies against CD41a, a glycoprotein found on the surface of both platelets and PMPs. In this report we also introduce the use of capillary electrophoresis with post-column laser-induced fluorescence detection (CE-LIF) for the analysis of fluorescently labeled platelets and PMPs. While both flow cytometry and CE-LIF can measure individual fluorescent events, only CE-LIF allowed us to calculate individual electrophoretic mobilities of activated platelets and PMPs that were then represented as distributions. A comparison between distributions suggests that PMPs have less negative mobilities. The fact that activated platelet preparations include PMPs partially obscure the interpretation of the data. While PMP and platelet number ml(-1) determined by flow cytometry is lower than the same parameter determined by CE-LIF, signal-to-noise ratio was 20 fold better for flow cytometry than for CE-LIF. This is the first time that a direct comparison between these two techniques is reported.

Blood Platelets↗

PubMedDate not supplied

Comparison of beta-thromboglobulin, flow cytometry, and platelet aggregometry to study platelet activation.

This study compares granule membrane protein (GMP)-140 expression measured by flow cytometry, release of beta-thromboglobulin (beta-TG), and platelet aggregometry as markers of platelet activation in vitro. Whole blood was activated with different concentrations of thrombin. There was a significant increase in beta-TG plasma levels after stimulation with 0.01 and 0.04 U thrombin/ml. There was also an increase in GMP-140 expression, but interindividual variability was high. Aggregometry of platelet-rich plasma did not detect platelet activation and formation of platelet aggregates with 0.05 and 0.1 U thrombin/ml, while flow cytometry showed an early and significant increase of GMP-140 expression with these doses. Beta-TG release is a more sensitive marker of platelet activation than GMP-140 while flow cytometry is easier to perform and less susceptible to artifacts.

Anticoagulants↗

PubMed1985

Evaluation of platelet glycoprotein Ib by fluorescence flow cytometry.

Platelet glycoprotein Ib (GpIb), a receptor for von Willebrand's factor (vWF), was studied by way of fluorescence flow cytometry. Using a sandwich staining technique, GpIb was identified by a monoclonal antibody (6D1) directed against an epitope close to the vWF binding site. Platelets from normal individuals were symmetrically distributed with respect to GpIb content. Treatment of washed platelets with plasmin resulted in progressive loss of GpIb as measured by fluorescence flow cytometry and by loss of agglutination response when combined with ristocetin in the presence of vWF. In mixing experiments with GpIb-deficient and normal platelets, it was possible to detect a subpopulation of deficient cells comprising 2% of the total population. Streptokinase treatment of platelet-rich plasma caused loss of the agglutination response to ristocetin and the emergence of a population of GpIb-deficient platelets. Fluorescence flow cytometry appears to be an important new technique by which to study platelet surface receptors.

Blood Platelets↗

PubMed1997

Determination of parameters that characterize effector-target conjugation of human NK and LAK cells by flow cytometry.

Effector-target conjugation between different cell populations of human NK cells and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios. Non-linear and linear regression methods were applied to these isotherms to calculate the binding parameters that characterize the process of conjugation, namely, the maximum effector and target conjugate frequencies, the dissociation constant of the conjugates formed, the binding units and the area under the binding isotherms. The results obtained show that: (1) flow cytometry analysis of effector-target conjugation is faster, unbiased and more suitable than microscopic counting of conjugates, thereby permitting the analysis of larger number of conjugates in shorter times, (2) the binding parameters derived from conjugate frequencies obtained by flow cytometry analysis differ from those obtained by microscopy, (3) the discrepancies between the two methods are due to the presence of several cells engaged in multicellular conjugates that are detected as single particles by flow cytometry and (4) the analysis of population distributions of the conjugates formed at different values of the effector-to-target ratio permit the above discrepancies to be corrected.

Flow Cytometry↗

PubMed1988

Application of flow cytometry and automated image analysis to the study of prostate cancer.

Flow cytometry and image analysis are complementary quantitative cytologic techniques that have demonstrated utility in the assessment and analysis of prostate cancer and other urologic and nonurologic tumors. This review is intended to assess the current state of the art and to project future directions and applications of these modalities in the pathologic assessment of prostate cancer. Special attention is directed toward the topics of prostate cancer detection and diagnosis, determination of patient prognosis, and the monitoring of patient response to therapy. We recommend that 1) flow cytometry and image analysis be used to determine pathologic parameters that will help in predicting poor patient prognosis and 2) quantitative cytologic determinations of DNA content be included in clinical trials so that their ultimate role in monitoring patient response to therapy can be determined. This knowledge will allow the development of protocols designed to test the value of earlier institution of multimodal therapy in high-risk populations.

Flow Cytometry↗

PubMed1981

Characterization of cultured human tumor cell lines by flow cytometry DNA stemline.

The DNA stemlines for 12 human tumor cell lines were determined by flow cytometry, and the determinations were compared with other methods available for characterizing cultured cells. In four of ten cases where karyotyping had been done, there was a reasonably good correlation between DNA and karyotype stemlines, but in six cases they differed. Two distinct stemlines were present in two cases. The measurements by flow cytometry can be carried out rapidly and easily and may be useful indicators of extraneous cell contamination or a special clone-derived cell population that has developed during culture passage. In addition, information is provided on the cell cycle distribution. It is proposed that flow cytometry determinations of the DNA stemline be used to help characterize cultured cell lines and to ensure their identity.

Cell Cycle↗

PubMed1993

Image and flow cytometry: companion techniques for adherent and non-adherent cell analysis and sorting.

Flow cytometry (FMC) is an analytical and preparative technique whereas image analysis is only applied to cell analysis. Recently, image analysis has been adapted as a preparative method using a new technique: image cytometry for analysis and sorting (ICAS). FCM and ICAS are complementary. Flow cytometry allows rapid, quantitative and precise study of fluorescence and light scattering in a large number of cells in suspension, while ICAS analyses fewer cells (adherent cells or tissue) on the basis of fluorescence, morphology and size. ICAS can use these criteria to destroy unwanted cells and hence sort selected cells. ICAS can also be used for confocal microscopy and laser surgery.

Cell Adhesion↗

PubMed1987

[A new hypothesis on the natural history of bladder cancer based on the study of tumor DNA levels by flow cytometry].

72 bladder tumors were studied for nuclear DNA content with flow cytometry. A bimodal DNA profile was present in 33 of them (45%). The following findings concerning the aneuploid second peak of these 33 tumors are remarkable. Aneuploid peak DNA index distribution is discontinuous: there is no peak below 1.5 nor between 2.3 and 2.7. Aneuploid peak importance (second peak cell percentage versus all tumor cells in the same sample) increases when its DNA index decreases from 2.0 to 1.5 = this percentage is on average 45% for a DNA index of 2.0 and increases to 75% when DNA index decreases to 1.5. Aneuploid peak mitotic activity increases when DNA index decreases from 2.0 to 1.5 = the percentage of S G2 M cells of the aneuploid peak is in the range of 15% for a DNA index of 2.0 and in the range of 22% for a DNA index of 1.5. These findings are in favor of a dynamics in bladder cancer natural history. Tumors are supposed to share the same clonal evolution, in 3 stages. First stage: transformed tumor cell DNA profile is unimodal with a DNA index in the region of 1; second stage: due to chromosomic non-dysjunction during mitosis, a second peak appears with a DNA index of 2. Third stage: DNA index of this aneuploid second peak progressively decreases from 2 to 1.5 as a consequence of non vital chromosomes loss by tumor cells. It is suggested that DNA index as defined by flow cytometry does not have an absolute prognostic value per se, but in combination with tumors mitotic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗

PubMed2002

Ploidy and imprinting in hydatidiform moles. Complementary use of flow cytometry and immunohistochemistry of the imprinted gene product p57KIP2 to assist molar classification.

OBJECTIVE: To determine whether the combination of flow cytometry and immunohistochemistry for p57 expression is useful for the pathologic classification of diagnostically challenging hydatidiform moles. STUDY DESIGN: Six probable hydatidiform moles that were difficult to classify pathologically were reevaluated by histology, flow cytometry and immunohistochemistry for p57. RESULTS: In all cases, the pathologic diagnoses were easily resolved: two cases were partial moles (triploid, p57 positive), two cases were early complete moles (diploid, p57 negative), and two cases were twin gestations with normal villi (diploid, p57 positive) admixed with villi from a complete hydatidiform mole (diploid, p57 negative). CONCLUSION: Immunostaining for the paternally imprinted, maternally expressed p57 gene product is a practical and accurate adjunct to flow cytometry in the pathologic classification of hydatidiform moles.

Cyclin-Dependent Kinase Inhibitor p57↗

PubMed1992

Sensitive detection of RNAs in single cells by flow cytometry.

A rapid and sensitive fluorescent in situ hybridization method has been developed to probe RNA contents of individual cells by flow cytometry. Fixed cells in suspension were hybridized with 5' end-fluorophore-labeled oligodeoxynucleotides complementary to defined regions of the RNA of interest and analyzed by flow cytometry. With this method, we monitored combinations of histone H4 mRNA, 18S rRNA and 28S rRNA levels in synchronized HeLa S3 cells by multicolor analysis. A fluorescence signal equivalent to 1800 copies of histone H4 mRNA per cell was detected with signal-to-background ratio of 5.4. If non-specific binding of the fluorophore-labeled probe can be reduced, as few as 100 copies of mRNA of the size of H4 could be detected in individual cells by flow cytometry.

Base Sequence↗

PubMed2002

CD4 lymphocyte enumeration in patients with human immunodeficiency virus infection using three-colour and four-colour dual-platform flow cytometry: an inter-laboratory comparative evaluation.

INTRODUCTION: This study aims to evaluate the correlation and agreement between 2 methodologies of CD4 lymphocyte enumeration. MATERIALS AND METHODS: Fifty-two blood samples from patients with human immunodeficiency virus (HIV) infection were sent for CD4 lymphocyte enumeration at 2 major hospitals using dual-platform flow cytometry where the absolute lymphocyte counts were determined on separate haematology analyzers. CD4 cell enumeration was accomplished using 3-colour flow cytometry on the FACSCalibur cytometer (Becton Dickinson) in Hospital A and 4-colour flow cytometry on the Coulter Epic XL cytometer (Beckman Coulter) in Hospital B. The percentages and absolute counts of CD4 lymphocytes obtained were analysed using both linear regression and Bland-Altman plots. RESULTS: On linear regression plots, the total white cell counts, absolute lymphocyte counts, CD4 lymphocyte percentages and absolute CD4 counts from the 2 hospitals correlated well with correlation coefficients, r > 0.95. On the Bland-Altman plots, there was a mean difference of 0.13 x 10(9)/L and 0.06 x 10(9)/L in the total white cell and absolute lymphocyte counts from the 2 hospitals respectively. The CD4 lymphocyte percentages revealed a mean difference of only 0.05% (95% limits of agreement, -3.6 to 3.7%) but there was a mean difference of 14/uL in the absolute CD4 lymphocyte counts (95% limits of agreement, -113 to 142/uL). CONCLUSIONS: The CD4 lymphocyte percentages obtained using the 3-colour and 4-colour flow cytometry correlated and agreed well. However, the absolute CD4 lymphocyte counts obtained using the dual-platform technique in both the hospitals did not agree well. Hence, absolute CD4 lymphocyte counts from the 2 hospitals cannot be used interchangeably in clinical practice due to poor inter-laboratory comparability.

CD4 Lymphocyte Count↗

PubMed1996

Astrocyte differentiation in primary culture followed by flow cytometry.

Astrocyte proliferation and differentiation in primary culture were followed by flow cytometry. The time-courses of the percentages of astrocytes in the different cell-cycle phases suggest that astrocytes proliferate during the first 10 days in culture thereafter reaching confluence. Two types of astrocytes are identified immunocytochemically: one growing in the bed monolayer, identified as type-1 astrocytes, and another growing on the top of the monolayer, identified as type-2 astrocytes. In addition, three populations identified as being formed of type-1, type-2 and putative progenitor cells, respectively, were followed by flow cytometry. Progenitor cells were the major type 2 h after plating (89%) but their percentage decreases sharply (to 16%) during the first 5 days in culture, with no ensuing changes. In contrast, the percentage of type-1 cells (11%) rapidly increased after plating reaching a maximum 5 days later (73%). Later, it decreased (to 47%) and was maintained thereafter. The percentage of type-2 cells was undetectable immediately after plating but increased from the 3rd to the 10th day with no further changes. Our results suggest that progenitor cells differentiate into type-1 astrocytes triggered by the culture medium but the differentiation of progenitor cells into type-2 astrocytes is brought about by some type-1-secreted factor. In this work we report a rapid and simple method for following the growth and differentiation of rat brain astrocytes in primary culture.

Animals↗

PubMed1994

Rapid determination of antifungal activity by flow cytometry.

We have developed a rapid assay of antifungal activity which utilizes flow cytometry to detect accumulation of a vital dye in drug-damaged fungal cells. Results of these studies suggest that flow cytometry may provide an improved, rapid method for determining and comparing the antifungal activities of compounds with differing modes of action.

Antifungal Agents↗

PubMed1990

Quality control in flow cytometry for diagnostic pathology. I. Cell surface phenotyping and general laboratory procedures.

Flow cytometric analyses have become commonplace in the clinical laboratory for determining cell lineage and for quantitation of cells bearing a given phenotype. Because these assays are being conducted to support diagnoses or assist in determining therapy, it is crucial to ensure that these tests are highly accurate and reproducible within a laboratory and among laboratories involved in similar endeavors. This quality assurance has been slow evolving in clinical flow cytometry for a variety of reasons: the exquisite sensitivity and delicacy of the instrumentation that recognize previously undetectable variations in staining; the constant improvement of the hardware and software; the rapid development of new techniques and reagents of clinical interest; and the failure of any existing specialty or subspecialty to encompass all aspects of flow cytometry. This article provides an overview of quality assurance necessary for the flow cytometric analysis of cell surface markers. Practical experience, published studies, and suggested guidelines from accreditation agencies have been combined to develop the text.

Cell Separation↗

PubMed2002

Comparison of urine cell characteristics by flow cytometry and cytology in patients suspected of having bladder cancer.

The diagnosis of bladder cancer is confirmed by histological analysis of tissue biopsies. Cytology of urine samples is a noninvasive alternative. The aim of this work was to find out whether flow cytometry of urine samples is more sensitive than cytology. For this purpose we studied 115 patients suspected of having bladder cancer. Cells isolated from urine samples were analyzed by cytometry for the expression of cytokeratin and CD 45 and for DNA measurements such as: DNA index, synthesis phase fraction and proliferative index (SPF + G2/M phase). At the same time we carried out cytological analysis. All positive cases were confirmed by histology (21/115), 18 were diagnosed by flow cytometry and 16 by cytology, with a sensitivity of 85.7% and 76.1%, respectively. Two cases were found to be positive by flow cytometry, which were not confirmed by histology, while no false positives were detected by cytology. We found that both techniques gave almost identical results for the diagnosis of bladder cancer, although there were differences in non-malignant samples. In conclusion, flow cytometry is slightly more sensitive than cytology but the combination of the two techniques improves the diagnosis.

Aged↗

PubMed2006

Characterization of antigen-presenting cells in human apical periodontitis lesions by flow cytometry and immunocytochemistry.

AIM: To analyse phenotypic characteristics of antigen-presenting cells (APC), isolated from human periapical lesions by flow cytometry and immunocytochemistry. METHODOLOGY: Sixteen periapical lesions were digested for 15 min with 0.05% collagenase. Mononuclear cells, separated from other inflammatory cells by density centrifugation, were processed for flow cytometry and/or immunocytochemistry. Single and double immunostainings were performed using monoclonal antibodies specific for human CD45, CD3, CD19, CD14, HLA-DR, CD1a, CD83 and CD123. RESULTS: Antigen-presenting cells (HLA-DR(+) cells) represented 32.9 +/- 17.8% of total mononuclear cells. Amongst them, B cells (HLA-DR(+) CD19(+)) were the predominant APC population, followed by activated macrophages (HLA-DR(+) CD14(+)), dendritic cells (DC) (HLA-DR(+) CD14(-) CD19(-) CD3(-)) and activated T cells (HLA-DR(+) CD3(+)). Based on the predominance of T cells (CD3(+)) or B cells and plasma cells (CD19(+) and CD19(lo), respectively) amongst mononuclear cell infiltrates, lesions were divided into T- and B-types. The percentage of DC in T-type lesions (27.1 +/- 6.8% of total HLA-DR(+) cells) was higher, compared with B-type lesions (10.3 +/- 5.2%) (P < 0.01). Within the DC population, the percentages of CD1a (Langerhans cell type) and CD123 (probably plasmacytoid DC type) did not differ significantly between the groups (P > 0.05). However, the percentage of mature DC (CD83(+)) was significantly higher in T-type periapical lesions (P < 0.05). CONCLUSIONS: Flow cytometry and immunocytochemistry are suitable methods for phenotypic analysis of APC after their isolation from human periapical lesions. APC, that were phenotypically heterogeneous, constituted a significant component of infiltrating cells. Lesions with the predominance of T cells were characterized by a higher proportion of mature DC (HLA-DR(+)CD83(+) cells) than lesions with predominance of B cells/plasma cells.

Adolescent↗

PubMed1980

Percentage of cells in the S phase of the cell cycle in human lymphoma determined by flow cytometry.

The percentage of cells with S phase DNA content (S cells) was determined by flow cytometry in cell suspensions obtained from 27 patients with non-Hodgkin's lymphomas. DNA was measured in ethanol-fixed cells stained with propidium iodide after RNase treatment. Cells from 17 samples were also exposed to [3H]-thymidine and labeling indices were determined. An excellent linear correlation was observed between the percentage of S cells calculated by flow cytometry and the labeling index (r = 0.95). To determine the relationship between the percentage of S cells and the clinical behavior of the tumors, the survival of the 27 patients was analyzed. None of the patients was undergoing antineoplastic therapy at the time of the study. The patients were separated into 2 groups according to the percentage of S cells calculated by flow cytometry: those with less than 5% S cells and those with higher than 5% S cells. Clinical follow-up ranged from 5 to 20 months. While 7 of 12 patients with tumors containing more than 5% S cells have died of lymphoma (median survival, 9 months), there has been only one death from lymphoma among 15 patients with tumors containing less than 5% S cells. The difference in survival between these two groups is statistically significant (P = 0.01).

Autoradiography↗