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Results for “Flow Cytometry”

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At least 379 records · Page 21Linked to original sources

PubMed1989

Inhibition of cellular esterases by the antitumour imidazotetrazines mitozolomide and temozolomide: demonstration by flow cytometry and conventional spectrofluorimetry.

Using flow cytometry and conventional spectrofluorimetry we have previously shown that chloroethylnitrosoureas (CNUs) can exhibit marked inhibition of cellular enzymes catalysing hydrolysis of fluorescein diacetate (FDA). More potent inhibition was seen for the carbamoylating CNUs, whereas alkylating agents were largely inactive. We now report results obtained with the developmental imidazotetrazines mitozolomide and temozolomide in comparison with BCNU, the novel alkylating agents clomesome and cyclodisone, and the active mitozolomide metabonate MCTIC. Inhibition of EMT6 mouse mammary-tumour esterases was seen for mitozolomide and temozolomide, and activity against purified porcine carboxylesterase was demonstrated. Flow cytometric analysis showed that inhibition occurred across the entire EMT6 cell population, with no evidence of a subpopulation resistant to enzyme inhibition. Inhibitory potency for the imidazotetrazines was much weaker than for BCNU. With EMT6 cells, I50 values from flow cytometry were 9.7 x 10(-3) M and 1.5 x 10(-3) M for mitozolomide and temozolomide compared with 3.7 x 10(-4) M for BCNU. These were higher than the ID50 values for in vitro antitumour activity (MTT assay), 8.5 x 10(-6) M in the case of mitozolomide and 1.2 x 10(-5) M for BCNU, but similar to that of 5.6 x 10(-4) M for the less toxic temozolomide. MCTIC and cyclodisone showed very low activity, but significant inhibition was seen for clomesome. The results are consistent with the view that the imidazotetrazines do not exhibit major carbamoylating ability, although significant effects are seen at cytotoxic concentrations of temozolomide. In addition, the potential for the generation of carbamoylating species at the enzyme active site cannot be ruled out.

Animals↗

PubMed2003

[Detection of minimal residual disease in childhood B-ALL by flow cytometry].

OBJECTIVE: To establish a flow cytometric method for detecting minimal residual disease (MRD) in children with B-ALL and evaluate its clinical application. METHODS: Fifty-eight childhood B-ALL cases entered this study and 30 MRD analyses were performed after remission induction therapy. Four-color fluorochrome labeled monoclonal antibodies were used to analyze the cell immunophenotypes. Cells from normal bone marrow were used as controls. The leukemic cell populations located in flow cytometry dot plots different from those of normal were considered to be the markers of interest in the first step screening, and then used to monitor MRD step after therapy. RESULTS: Fifty-eight cases of childhood B-ALL were screened for antibodies combinations of interest and were identified in 89.7% (52/58) of these cases. The four-color antibody combinations consisted of CD(10)/CD(34)/CD(19) plus another effective marker such as CD(38), CD(65), CD(66c), CD(21). The sensitivity of this method was 0.01%, much higher than microscopic inspection. In 8 cases whose bone marrow microscopically showed no residual leukemic cells, the percentage of leukemic cells were identified with this method of 0.028%, 1.430%, 3.050%, 0.015%, 5.660%, 2.700%, 0.027%, and 0.069%, respectively. CONCLUSION: The application of flow cytometry in MRD monitoring can significantly improve the detection sensitivity in childhood B-ALL, thus facilitate the further treatment decision and follow-up.

Burkitt Lymphoma↗

PubMedDate not supplied

Clinical relevance of low levels of preformed alloantibodies detected by flow cytometry in the first year post-kidney transplantation.

OBJECTIVE: To determine the prevalence of transplants performed with a false-negative cytotoxicity cross-match and to analyze the clinical relevance of alloantibodies (Ab) detected only by flow cytometry (flow). METHODS: We studied 66 patients undergoing kidney transplantation from a cadaveric donor. All patients had a simultaneous negative T+AHG+DTT and B+DTT. Pretransplant sera were retrospectively analyzed by flow cytometry according to an Emory University protocol: (1) T+ and B-: Ab anti-class I; (2) T- and B+: anti-class II; (3) T+B+: anti-class I + II. Chi-square, Fisher exact, Student t test, and Kaplan Meier analyses were employed with significance assigned at P < or = .05. RESULTS: The overall incidence of false-negative cytotoxicity was 33.3% (22/66), namely, 6.1% (n = 4) anti-class I; 9.1% (n = 6) anti-class II; and 18.2% (n = 12) anti-class I + II. Primary nonfunctioning grafts occurred in 6.8% (3/44) and 13.6% (3/22) negative and positive flow patients (two anti-class I + II and one class II; P = .39). The incidence of graft loss in the first year was respectively, 13.6% (6/44) and 18.2% (4/22; two anti-class II and two anti-class I + II; P = .72). Compared to flow-negative grafts, creatinine levels were significantly higher among flow-positive patients at 8 and 12 weeks. One-year graft survivals were 86.4% among negative versus 81.8% for the positive group (P = .67). CONCLUSIONS: We observed that 33% of kidney transplant recipients had low levels of alloantibodies detected only by flow. This single factor was associated with the worst graft function in the first trimester with a suggestion of a higher risk for non-functioning graft.

Cadaver↗

PubMed1989

[Value of cell cycle analysis by flow cytometry in gynecology and obstetrics].

Flow cytometry is a new technique that makes it possible to study the cell cycle and the DNA content of thousands of cells per minute. Its value in oncology is prognostic, there being a correlation between the level of DNA and mitotic activity with the power to invade tissues. After a description of the cell cycle, a review of the literature demonstrates its value in gynaecology and obstetrics. Aneuploidy and marked mitotic activity correlate well with a poor prognosis for: CIN: aneuploid lesions are less likely to regress spontaneously and are found more frequently in higher grades. This is a reason for treating aneuploid CIN. Adenocarcinoma of the endometrium: aneuploidy is the best prognostic measure when analysing cells in a multifactorial way to include the stage, the grade, the degree of myometrial invasion and the receptors. Sarcoma of the uterus: in a small series aneuploidy occurred frequently and was of poor prognosis. Molar pregnancy: flow cytometry is a diagnostic aid because the hydatidiform mole is diploid with a marked degree of mitotic activity. The prognostic value has still to be determined. Ovary: borderline tumours are always diploid. In cancers of the ovary aneuploidy is a sign of aggression and predictive of survival.

Aneuploidy↗

PubMed2002

Flow cytometry in human reproductive biology.

Flow cytometry (FC) is an analytical cytology technique which has been extensively used for decades. It has many advantages compared with other similar methods for the study of cell biology, even on a molecular basis. FC allows the cell-by-cell analysis of many optical or immunological features in the same sample, at the same time, and at a rate of thousands of cells per second, generating immense quantities of data and thus providing almost limitless information which is statistically robust due to the number of units studied. The aim of this review is to describe the contribution of FC to the study of physiological and pathological processes related to human reproduction, and to discuss how this technique has been used in research, as well as its clinical applications in this field. We have used some practical examples selected from the most relevant studies within a wide range of investigations published in the literature, and we have also drawn on our own experience of using flow cytometry to study different phenomena related to reproduction. It is conclued that FC is a useful instrument for basic investigation of gynecological issues, as well as for the study of male reproductive characteristics, either in research applications or directly for clinical diagnostic purposes. Future development of these techniques will permit further advances both in our knowledge and in the improvement of assisted reproduction techniques.

Acrosome Reaction↗

PubMed2001

Use of overlapping peptide mixtures as antigens for cytokine flow cytometry.

Intracellular cytokine staining and flow cytometry can be used to measure T-cell responses to defined antigens. Although CD8+ T-cell responses to soluble proteins are inefficiently detected by this approach, peptides can be used as antigens. Using overlapping peptides spanning an entire protein sequence, CD8+ T-cell responses can be detected to multiple epitopes, regardless of HLA type. In this study, overlapping peptide mixes of various lengths were compared and 15 amino acid peptides with 11 amino acid overlaps were found to stimulate both CD4+ and CD8+ T-cell responses. Such peptide mixes stimulated CD4+ T-cell responses equivalent to those observed with whole recombinant protein, while simultaneously stimulating CD8+ T-cell responses much higher than those observed with whole protein. Although 8-12 amino acid peptides produced the highest level of CD8+ T-cell responses, 15 amino acid peptides were still very effective. Peptides that were 20 amino acids in length, however, did not stimulate strong CD8+ T-cell responses at the same peptide dose. The cytokine responses to individual epitopes added up approximately to the response to the entire mix, demonstrating that large mixes can detect responses in a quantitative fashion. Unlike whole protein antigens, peptide mixes were effective at stimulating responses in both cryopreserved PBMC and blood stored for 24 h at room temperature. Thus, overlapping 15 amino acid peptide mixes may facilitate the analysis of antigen-specific CD4+ and CD8+ T-cell responses by cytokine flow cytometry, using clinical specimens that include shipped blood or cryopreserved PBMC.

Clinical Trials as Topic↗

PubMed2000

Flow cytometry: principles and clinical applications in hematology.

The use of flow cytometry in the clinical laboratory has grown substantially in the past decade. This is attributable in part to the development of smaller, user-friendly, less-expensive instruments and a continuous increase in the number of clinical applications. Flow cytometry measures multiple characteristics of individual particles flowing in single file in a stream of fluid. Light scattering at different angles can distinguish differences in size and internal complexity, whereas light emitted from fluorescently labeled antibodies can identify a wide array of cell surface and cytoplasmic antigens. This approach makes flow cytometry a powerful tool for detailed analysis of complex populations in a short period of time. This report reviews the general principles in flow cytometry and selected applications of flow cytometry in the clinical hematology laboratory.

Blood Cells↗

PubMed2000

[Flow cytometry--use in cell biology and benefits in clinical medicine].

Flow cytometry is an automated method for measuring features of single cells in suspension. Many parameters may be measured simultaneously at high speed, objectively and with good statistics. The method has been increasingly used in the context of medical diagnosis and prognosis over the last ten years. Clinical-chemical departments are major users of flow cytometry for differential cell counts of leukocyte populations in blood by light scatter measurements. For immunophenotyping of acute leukaemias, flow cytometry is used routinely in all university hospitals in Norway. The method is also used to classify chronic lymphoproliferative disease, and for quantification of CD34-positive stem cells by high-dose chemotherapy with autologous stem cell support. Flow cytometry is routinely used for classifying non-malignant haematological disease like congenital and acquired immunodeficiencies. In diagnostic pathology, DNA flow cytometry is used for analysis of DNA ploidy and S-phase fractions in malignant tumours. DNA aneuploidy is a strong prognostic factor in some childhood tumours and in gynaecological malignant tumours, and flow cytometry is used in tumour biology for measurements of growth and apoptotic cell death. Furthermore, the method is an important research tool in many areas of biomedical research for detection of structural and functional cellular features.

Animals↗

PubMed1994

Effects of environmental factors present in the St. Lawrence Estuary (Quebec, Canada) on experimental survival of Salmonella salamae as determined by flow cytometry.

Survival of Salmonella salamae in the St. Lawrence Estuary was studied experimentally during an oceanographic cruise using in situ exposure diffusion chambers. The abundance distribution (colony-forming units) of culturable S. salamae on media was compared with the distribution of cells enumerated by flow cytometry. Flow cytometry was also used to characterize the size distribution and DNA content of cells exposed to various environmental factors. Solar radiation, starvation, and a gradual increase in salinity led to an abrupt loss of the ability of S. salamae cells to form cultures and to a gradual reduction in the cell size and DNA content. Conversely, starvation combined with a gradual increase in salinity in the absence of sunlight led to a gradual loss of the cells' ability to form cultures and an abrupt reduction in cell size and DNA content (i.e., a rapid increase in cell damage). Mortality (i.e., a decrease in total cell count) of S. salamae placed in darkness began at a lower salinity (11.4/1000) than did the mortality of cells exposed to sunlight (23.1/1000). Therefore, the S. salamae cells exposed to sunlight seemed to be more resistant to gradual salinity stress than the cells that were not subjected to sunlight.

DNA, Bacterial↗

PubMed1995

Laboratory practices in reporting flow cytometry phenotyping results for leukemia/lymphoma specimens: results of a survey.

Laboratory directors who routinely utilize flow cytometry for at least part of their diagnostic evaluations in leukemias or lymphomas were surveyed by mail. The survey consisted of 12 questions about the flow cytometry procedures used by the laboratory in evaluating leukemias and lymphomas and on the format and content of their official report. It also requested an example of a typical leukemia/lymphoma report and solicited write-in comments about additional important aspects of using flow cytometry to evaluate leukemia and lymphomas not covered by the questionnaire. The goal of the survey, which was sponsored by the Clinical Cytometry Society (CCS), was to document what directors of flow cytometry laboratories currently consider to be the appropriate contents of a clinical leukemia/lymphoma phenotyping analysis and in what manner and detail they report such flow cytometry results to clinicians. The survey indicated that a large number of markers are routinely evaluated to phenotype leukemias (mean = 19) and lymphomas (mean = 16). Light scatter gating, using CD45/14 to monitor the gate selected, is currently employed by a 2:1 ratio over the next most population gating strategy (CD45 vs. 90 degrees LS). Peripheral blood, bone marrow, and lymphoid tissue constitute the majority of clinical specimens evaluated for leukemia and/or lymphoma. Two-color analysis, primarily for surface markers, is currently the standard method for flow cytometry measurements in routine diagnostic studies of leukemia and lymphoma. The official flow cytometry laboratory report is most commonly an individual-lab-generated, paper report form. A discussion of the potential benefits that might result from the development of improved computerized reporting software and from the increased use of antibody-defined, lineage gating is offered. A composite report format is presented that demonstrates the flow measurements and quality control data included in the best of the example clinical reports submitted as part of the survey and considered important by a majority of our survey respondents. The example report is intended to be a basis for further discussion within the flow cytometry community on whether minimum reporting standards for leukemia and/or lymphoma flow cytometry results can and should be developed.

Antigens, CD↗

PubMed2001

The relevance of flow cytometry for biochemical analysis.

Flow cytometry (FCM) allows the simultaneous measurement of multiple fluorescences and light scatter induced by illumination of single cells or microscopic particles in suspension, as they flow rapidly through a sensing area. In some systems, individual cells or particles may be sorted according to the properties exhibited. By using appropriate fluorescent markers, FCM is unique in that multiple structural and functional parameters can be quantified simultaneously on a single-particle basis, whereas up to thousands of biological particles per second may be examined. FCM is increasingly used for basic, clinical, biotechnological, and environmental studies of biochemical relevance. In this critical review, we summarize the main advantages and limitations of FCM for biochemical studies and discuss briefly the most relevant parameters and analytical strategies. Graphical examples of the biological information provided by multiparametric FCM are presented. Also, this review contains specific sections on flow cytoenzymology, FCM analysis of isolated subcellular organelles, and cell-free FCM.

Animals↗

PubMed1982

Flow cytometry followup of patients with low stage bladder tumors.

A total of 98 urologic outpatients with a history of conservatively treated low stage bladder tumors underwent 325 examinations during a 2-year interval, each comprising conventional cytology, cystoscopy and automated flow cytometry. The flow cytometry results agreed with cystoscopic findings and conventional cytology in 80 per cent of the examinations, while cystoscopy and cytology agreed in 75 per cent. Assuming cystoscopic findings were entirely accurate, the false negative rate for flow cytometry was 8.3 per cent and the false positive rate was 5.8 per cent. On occasion, flow cytometry diagnosis of bladder tumors antedated the development of visible tumor by up to 12 months. Flow cytometry provided an objective and quantitative assay of epithelial abnormalities and, in the absence of skilled, conventional cytology, it appears to be a valuable new tool for the urologist in the diagnosis, management and followup of patients with bladder tumors or a history of bladder tumor.

Carcinoma, Transitional Cell↗

PubMed2002

Emerging technology and future developments in flow cytometry.

The authors' view of the future of flow cytometry is based on a belief that the single most important aspect flow cytometry offers to the investigator is high-speed interrogation of correlated measurements on a cell-by-cell basis. Over the next several years, an enormous increase in the capabilities of cytometry in general, and flow cytometry in particular, is likely to occur. A brief description of some of those capabilities is the subject of this article.

Antibodies, Monoclonal↗

PubMed1998

Flow cytometry of bacterial cells: comparison between different flow cytometers and different DNA stains.

The DNA content and light scatter from individual Escherichia coli cells were measured in two flow cytometers with different configurations. The DNA content could be measured with similar resolution either in an Argus flow cytometer equipped with a mercury lamp, or in a FACStar flow cytometer with two argon lasers as light sources. In contrast, light scatter measurements appeared to be a good measure of cell mass only in the Argus instrument. Three DNA stains were compared:, DAPI, Hoechst 33258, and mithramycin A together with ethidium bromide. All three stains yielded DNA histograms of similar quality in both flow cytometers. Optimal results required that the stain and cell concentrations were kept similar, that a fixed rate of sample introduction was used, and that a period of equilibration was allowed during running of each sample. The results demonstrate that conventional, laser-based flow cytometers may be used for high-resolution measurements of bacterial DNA content, thereby making flow cytometry available to an increased number of research groups working with prokaryotes.

Cell Separation↗

PubMed1994

Enumeration of IFN-gamma producing lymphocytes by flow cytometry and correlation with quantitative measurement of IFN-gamma.

We describe a flow cytometry method to simultaneously analyze IFN-gamma production and T cell surface phenotype in freshly isolated lymphocytes without requiring prior in vitro stimulation. We show that enumeration of intracytoplasmic IFN-gamma positive T cells correlates with quantitative measurement of IFN-gamma in culture supernatant fluids. This suggests that cytokines can be reliably measured using flow cytometry. Flow cytometry has the added advantage of simultaneous detection of cell surface markers. Furthermore, we suggest that analysis of ex vivo IFN-gamma production at the single cell level may reflect more accurately T cell IFN-gamma production, by avoiding the polyclonal stimulation of IFN-gamma production observed after short term in vitro stimulation.

Animals↗

PubMed1997

[Flow cytometry in hematology. Methods and technique of flow cytometric analysis].

Reviews the history of flow cytometry, the philosophy of the method, main methods of cell investigation (conductometry, registration of light absorption and diffusion, cytofluorometry, study of fluorescence polarization, and analysis of a wave-shaped signal). Describes the equipment and methods for flow cytofluorometric analysis of cells, used in hematology. Characterizes the hematological analyzers with differentiated estimation of leukocytes.

Animals↗

PubMed1992

Prognostic relevance of DNA flow cytometry in oligodendroglioma.

In a retrospective study of 85 cases, the prognostic value of DNA flow cytometry in oligodendrogliomas was evaluated. Paraffin-embedded material was processed for flow cytometry, and the survival rates of the patients with DNA diploid, aneuploid, and tetraploid tumors were compared using analysis of variance. In addition, the mitotic index was correlated with the results of flow cytometry. Finally, the results of flow cytometry, histopathologic grading, and counting mitoses were tested for dependency. Thirty-one percent of the tumors were diploid, 39% were tetraploid, and 31% were aneuploid. The results of the DNA flow cytometry did not correlate with the survival times (P = 0.798) or with tumor degree. In contrast, the number of mitoses (P less than 0.05), and the grades of the grading system of Smith (P less than 0.003) had relevance for the prognosis. No correlation between flow cytometry, histopathologic grading, and mitotic index was found. It is concluded that flow cytometry has no value in predicting the biologic behavior of oligodendrogliomas, whereas the number of mitoses is a valuable prognostic parameter and thus is considered to be incorporated into the grading system for oligodendrogliomas.

Adolescent↗

PubMed2000

Screening for microorganisms with specific characteristics by flow cytometry and single-cell sorting.

Flow cytometry used in combination with single-cell sorting is a powerful technique for the identification and isolation of microbial cells with particular characteristics, especially when such cells grow more slowly than other cells in a large heterogeneous population. Many applications of flow cytometry with cell sorting, originally used by specialists studying mammalian cells, have been modified so that microorganisms also can be evaluated. The methods can now be used more widely because of the increasing availability of the expensive equipment. There are means for the fluorescence detection of a wide variety of properties, such as amounts of various cell components, specific sequences of peptides and nucleotides, cell functions, and enzyme activities. From the extensive literature, representative reports of an assortment of uses of flow cytometry with cell sorting are reviewed in this article, intended to introduce the technique and its many advantages to microbiologists.

Journal Article↗