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Results for “Flow Cytometry”

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PubMed1988

[Cytogenetic study of cell sub-populations in human leukemias (AML, CML) sorted by flow cytometry].

Multivariate analysis, flow cytometry and sorting were used to distinguish and enrich subpopulations of bone marrow cells in cases of acute myeloblastic leukemia and blast crisis of chronic myelocytic leukemia: blast cells with a high rate of S/G2-M phase of the cell cycle; non-blast cells or blasts with a low rate of S/G2-M. These populations were separated on the basis of their forward angle and large angle light scatter of the laser beam, and of their Hoechst 33342 fluorescence intensity. About one million cells of each population were sorted and sorting purity was controlled by cytometry and microscope examination. Cell viability was good. Karyotypes of sorted cell populations were carried out using a cell synchronisation technique and showed different chromosomal markers.

Bone Marrow Cells↗

PubMed1996

Clinical application of flow cytometry to urological malignancies.

Flow cytometric DNA analysis provides rapid, quantitative objective information regarding the biological behavior of urological malignancies. Moreover, clinical applications of the many recent advances made the flow cytometry are expected to materialize soon. For instance, flow cytometric DNA ploidy analysis for human bladder cancers may provide a significant diagnostic and prognostic potential. Also, flow cytometric DNA analysis of irrigation specimens produces a higher sensitivity than conventional cytology for detecting bladder cancer. However, there are obvious pitfalls with this approach since diploid or near-diploid tumors cannot always be recognized by DNA analysis alone. One of the most significant advantages possible with flow cytometry is its capability of analyzing simultaneously multiple parameter on single cells. The integration of the DNA content with proliferative activity should yield important information significant to the biological behavior of individual tumors. Flow cytometric DNA/bromodeoxyuridine bivariate analysis can be used as an effective adjunct to histological examination for prognostication and decision-making in treatment of bladder cancer patients. Therefore, multiparameteric flow cytometric analysis can be used to isolate specific tumor cells from mixed cell populations, and should receive even increased attention as a valuable diagnostic technique and prognostic factor. In the present review, the efficacy of flow cytometric DNA ploidy analysis integrated with cell proliferation markers is discussed.

Cell Division↗

PubMed1985

Detection of distinct subpopulations of Langerhans cells by flow cytometry and sorting.

Flow cytometry was found to be a very appropriate tool for the study of Langerhans cells (LC), which represent a minor cell population (2-3%) of human epidermis, and allowed us to obtain new phenotypic, functional, and cell cycle data on these rare cells. The phenotypic analysis of cell surface antigens demonstrates the existence of two subpopulations of LC: the former is HLA-DR+ and OKT 6+ (about 90% of total HLA-DR+ cells) and the latter is HLA-DR+ and OKT 6- (about 10% of total HLA-DR+ cells). These subpopulations of LC are both able to stimulate the proliferation of peripheral blood lymphocytes (PBL) in the presence of keratinocytes i.e., in mixed skin lymphocyte reaction (MSLR). Analysis of the cell cycle could be performed on OKT 6+ LC. Results show that they can be found in the various phases of the cell cycle, suggesting that the large majority of Langerhans cells are able to proliferate in situ in normal human epidermis.

Antibodies, Monoclonal↗

PubMed1984

Increased DNA and/or RNA content of synovial fluid cells in rheumatoid arthritis: a flow-cytometry study.

Flow-cytometry studies of DNA and RNA content were carried out in acridine orange-stained synovial fluid lymphocytes from 11 patients presenting with classical or definite rheumatoid arthritis. Monoclonal antibodies were used to detect specific T cell surface antigens (OKT3, OKT4, OKT8) and antigens associated with lymphocyte activation (OKIa 1, OKT10). T3 positive cell percentages were comparable to those of normal blood, although T4/T8 ratios were decreased in 4 out of 5 cases, and HLA-DR positive cells increased. Six out of 11 patients showed percentages of dividing cells varying from 2.2 to 7.2% as compared with less than 1% in the other patients and in normal blood. Nondividing cells were characterised by an increase in their RNA content compared with normal blood. A greater increase of RNA content was observed in patients with lower percentages of dividing cells, suggesting a G1/S block. Changes in cellular DNA and/or RNA contents provide a valuable parameter of lymphocyte activation, not necessarily linked to the expression of differentiation antigens by activated cells.

Adolescent↗

PubMed2000

[Determination, by in situ hybridization on interphasic nucleus, a cytogenetic DNA index; application to breast cancer; comparison of this DNA index to DNA indexes determined by imaging and flow cytometry].

In oncology, flow cytometry (FCM) and image cytometry (ICM) are commonly used to detect DNA aneuploid cell populations in solid tumors. Agreement between these two approaches is good. The use of both techniques in association minimizes the rate of FCM and ICM false negatives and gives better DNA pattern characterization, particularly for detection of any tumoral component in the FCM DNA diploid peak. Nevertheless, discrepancies exist between the FCM and the ICM DNA index values: the ICM DNA index is often greater than the FCM DNA index. The aim of the present study was to establish a cytogenetic DNA index by determining the chromosomal ploidy using a molecular cytogenetic approach and to compare it to the FCM and ICM DNA indexes. We present here the fluorescence in situ hybridization (FISH) technique we have adapted to the study of breast cancer in order to count the number of copies of the 22 + X human chromosomes in interphasic nuclei. This was achieved using a panel of 21 indirect FITC labeled probes which recognize specific chromosomic DNA sequences. Preliminary results obtained from DNA diploid and DNA aneuploid tumors are discussed.

Breast Neoplasms↗

PubMed2002

Nonparametric discriminant analysis of phytoplankton species using data from analytical flow cytometry.

BACKGROUND: Analytical flow cytometry (AFC) provides rapid and accurate measurement of particles from heterogeneous populations. AFC has been used to classify and identify phytoplankton species, but most methods of discriminant analysis of resulting data have depended on normality assumptions and outcomes have been disappointing. METHODS AND RESULTS: In this study, we consider nonparametric methods based on density estimation. In addition to the familiar kernel method, methods based on wavelets are also implemented. Full five-dimensional wavelet estimation proves to be computationally prohibitive with current workstation power, so we employ projection pursuit for reduction of dimensionality. AFC typically produces very large samples, so we also investigate data simplification through binning. Further modifications to the discrimination strategy are suggested by specific features of phytoplankton data, namely, a hierarchical group structure, the possible presence of many groups, and the likelihood of encountering an aberrant group in a test sample. CONCLUSIONS: We apply all the resultant procedures to appropriate subsets of a very large data set, demonstrate their efficacy, and compare their error rates with those of more conventional methods. We further show that incorporation of the specific features of phytoplankton data into the analysis leads to improved results and provides a general framework for analysis of such data.

Animals↗

PubMed2003

How to optimize multiparameter flow cytometry for leukaemia/lymphoma diagnosis.

Multiparameter flow cytometry can allow for accurate lineage assignment of leukaemia cell populations in approximately 99% of cases, whereby the emphasis lies in the word 'can'. Despite the fact that the very few markers that are lineage-specific are localized inside the cell (e.g. myeloperoxidase, lactoferrin, cytoplasmic CD3, cytoplasmic CD22), several investigators still shy away from including these essential test elements in their routine panels. Of course, the staining of intracellular antigens requires the added effort of determining optimal conditions. Published suggestions often need to be revised, and cell lines must be used as positive and negative controls. The same holds true for other new and exciting applications of flow cytometry, such as the monitoring of minimal residual disease (MRD) or the establishment of physiological assays (e.g. measuring the activity of drug-efflux pumps). The beauty of-and unfortunately for some, the problem with-multiparameter flow cytometry is that although immunophenotyping by flow cytometry has become a routine approach to the diagnosis of haematological malignancies it is a discipline that is still in development. New antibodies are continually being introduced and new diagnostic and prognostically relevant subtypes are being published in almost every issue of the major scientific journals. It is therefore very important for the flow cytometrist not only to strive for optimal performance of all tests employed but also to keep up with new knowledge and to incorporate it into the interpretation of routine specimens. We owe it to our patients to diagnose their disease accurately and in accordance with accepted standards of interpretation so that the treating physicians can trust immunophenotyping results and act accordingly in the management of their patients.

Antigens, CD↗

PubMed1990

Flow cytometry to evaluate acrosome-reacted sperm.

Flow cytometry was used in the scoring of acrosome-reacted human sperm. Propidium iodide was used for detection of the nonviability of the sperm. Fluoresceinated pea lectin was used to detect acrosome-reacted sperm. The results obtained by flow cytometry and those obtained by fluorescence microscopy were compared to determine if flow cytometry can serve as a more accurate, faster, and simpler method. It was possible to detect human sperm by flow cytometry. The percentage of propidium iodide labeled sperm determined by flow cytometry was close to that obtained by fluorescence microscopy. Comparison of the percentage of acrosome-reacted sperm determined by flow cytometry and fluorescence microscopy showed that these methods gave very similar results (r = 0.98, p less than 0.001). Objective scoring of more sperm was possible by flow cytometry than by fluorescence microscopy, and flow cytometry was useful as a simple method for evaluation of acrosome-reacted human sperm.

Acrosome↗

PubMed2000

Analysis of free intracellular calcium by flow cytometry: multiparameter and pharmacologic applications.

Flow cytometry offers numerous advantages over traditional techniques for measuring intracellular Ca(2+) in lymphoid and nonlymphoid cells. In particular, the heterogeneity of cell responses can be defined by flow cytometry, and multiparameter analyses permit the determination of intracellular Ca(2+) in surface-marker-defined target cells as well as correlation of changes in Ca(2+) with other biochemical markers, including ligand binding. This article presents several established methods for measuring intracellular Ca(2+) by flow cytometry in lymphoid and nonlymphoid cells. Examples are provided for determination of Ca(2+) in human peripheral blood leukocytes and two human epithelial cell lines grown in monolayer. In addition, applications are reviewed or presented for correlating changes in intracellular Ca(2+) with other cell parameters, including cell cycle analysis, changes in cell membrane integrity, and the induction of apoptosis markers. Finally, a number of novel sample handling capabilities useful for performing kinetic analyses of Ca(2+) changes by flow cytometry are now available and one application is presented which is finding utility in pharmacologic studies.

Aniline Compounds↗

PubMed1998

Flow cytometry in diagnostic cytology.

Flow cytometry (FCM) is a useful adjunct to cytologic examination, because the quantitative biochemical information it provides complements the morphologic information gained during visual examination. It aids in the interpretation of bladder washings, and is particularly useful for the assessment of lymphoid lesions, whether they originate from fine-needle aspiration, cerebrospinal fluid, or effusions. Optimal use of FCM frequently requires assessment of more than one parameter; simultaneous use of cell differentiation markers and nuclear DNA quantitation is often significantly more useful than either alone. Despite the utility of FCM, however, the potential for future development appears to be limited. Improvements in image cytometry allow reasonable assessment of ploidy and S-fraction to be made from specimens prepared on glass slides. Multiparameter measurements may also be accomplished with imaging techniques, which allow the further advantage of visual identification of cells with equivocal morphologic changes. The development of artificial intelligence methods for use with imaging technology has also significantly exceeded that of FCM. Finally, image cytometry is often more useful for samples with few cells. Other challenges are posed by immunocytochemical methods which compete with flow cytometry as tools for assessment of proliferation. Given the relatively high cost of FCM instrumentation, survival of FCM as an ancillary technique in cytopathology will require further technical refinements to offset the advantages currently associated with image cytometry and immunocytochemistry.

Biopsy, Needle↗

PubMed1991

Flow cytometry of breast cancer.

Flow cytometry (FCM) has become a generally accepted analytical method of worldwide application. Considerably achievements in flow cytometry hardware, staining techniques, especially immunoflow cytometry, together with progress in software programmes have significantly contributed to persistent expansion of the method. Some of the inbuilt rapidity of FCM has been lost because of additional preparatory steps and incubations. Mammary carcinoma is one of those malignant tumours which have been most extensively investigated by FCM. Evidence has been repeatedly produced to the existence of correlations among the most important FCM data, prognostic factors, and follow-up findings. Further improvement of the method and search for the best possible clinical application actually are the main challenges to FCM of breast cancer, today.

Aneuploidy↗

PubMed2000

Immunophenotypes in adult acute lymphocytic leukemia. Role of flow cytometry in diagnosis and monitoring of disease.

Flow cytometry has revolutionized the study of hematopoietic cells. Immunophenotyping by multiparameter flow cytometry supplements conventional morphologic diagnosis by providing information on cell lineage and differentiation in ALL and helps monitor disease by improving sensitivity in detecting minimal residual disease. The use of multiple MoAbs and multicolor study by flow cytometry has revealed heterogeneity among ALL and mixed-lineage acute leukemia, which are assigned to the same diagnostic categories by morphology. As technology has improved, clinical and research applications of flow cytometry have expanded to include evaluation of nuclear markers, oncogene proteins, apoptosis, cytokine receptors, and drug resistance. Expanded identification of MoAbs against leukemia-specific markers and the use of QFCM be a significant in managing patients with ALL in the future. In addition, flow cytometry and flow cytometric sorting will be combined more and more with other technologies, such as molecular probing or fluorescence in situ hybridization (FISH). The sorting of rare malignant cells based on immunophenotype and subsequent confirmation by PCR or FISH has already been proven feasible. Ultimately, it is hoped that further definition of subgroups of ALL by immunophenotyping using prognostically significant markers and the use of hybrid technologies of flow cytometry and molecular analysis or cytogenetics will improve treatment strategies for patients with ALL.

Adult↗

PubMed1994

[Flow cytometry and digestive pathologies].

Flow cytometry provides rapid evaluation of nuclear DNA content and cell cycle analysis. The major applications of flow cytometry in gastroenterology are the evaluation of DNA content and proliferative indices as prognostic indicators of gastrointestinal malignancies, and the screening of premalignant conditions of the digestive tract.

Digestive System Diseases↗

PubMed2006

Hyperspectral imaging system using acousto-optic tunable filter for flow cytometry applications.

A major advantage of flow cytometry is its flexible and open instrument configuration, which is highly suitable for systems integration. This flexibility permits the coupling of auxiliary instrumentation that may offer the measurement of parameters other than those typically measured by this multiparameter measurement technique. On the basis of this advantage, we explore the principle and application of hyperspectral imaging (HSI), which has the potential to be a useful add-on feature to flow cytometry applications. Application of HSI to flow cytometry involves the acquisition of spatial information and rendering it in spectral form. In this work, we describe the development and application of an HSI system which provides both spectral and spatial information. Spectral information was generated by obtaining an entire spectrum of a single sample site within a wavelength region of interest, while spatial information was generated by recording a two-dimensional (2D) image of an area of the sample of interest at one specific wavelength. HSI is a promising additional feature to flow cytometry since it can provide both spatial (image format) and spectral information in addition to the multiparameter information already available from flow cytometry measurements.

Acoustics↗

PubMedDate not supplied

Impact of standardization on clinical cell analysis by flow cytometry.

The evolution of flow cytometry from a research tool to a pivotal technology for clinical diagnostic purposes has required significant efforts to standardize methods. The great advantage of flow cytometry is that it's applications are highly amenable to standardization. Here, we review the efforts that have been made for flow cytometric applications in four major fields of clinical cell analysis: CD4+ T-cell enumeration, CD34+ hematopoietic stem and progenitor cell enumeration, screening for the HLA-B27 antigen and leukemia/lymphoma immunophenotyping. These standardization efforts have been parallelled by the establishment of external quality assessment (EQA) schemes in many countries worldwide. The goal of these EQA exercises has been primarily educa-tional, but their results will increasingly serve as a basis for laboratory accreditation. This important development requires that the EQA schemes, in particular the quality of the distributed samples and the procedures for evaluating the results, meet the highest standards.

Antigens, CD34↗

PubMed1999

Antifungal susceptibility testing of Candida species by flow cytometry.

The feasibility of flow cytometric antifungal susceptibility testing has been studied using the fluorescent anionic membrane potential probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. The in vitro antifungal susceptibility testing of amphotericin B was performed on 8 Candida isolates from clinical specimens and 2 ATCC strains by flow cytometry with the results compared to those of the National Committee of Clinical Laboratory Standards (NCCLS) M27-T, broth macrodilution method. The flow cytometric method is based on an increase of fluorescence given out by DiBAC4(3) in fungi when they are killed by antifungal agents. Minimum inhibitory concentration (MIC) of amphotericin B ranged from 0.25 to 1 microg/mL. All results agreed within +/-2 dilution between the flow cytometric method and the M27-T method. MIC with ATCC strains were within recommended ranges of M27-T. The new flow cytometric method revealed a clear and distinct reproducible test end point. A four hr of incubation was sufficient for the test. In conclusion, flow cytometry using DiBAC4(3) is a rapid and accurate in vitro antifungal susceptibility testing method.

Amphotericin B↗

PubMed1990

Analysis of sperm cell viability, acrosomal integrity, and mitochondrial function using flow cytometry.

A triple staining procedure was developed to evaluate bull spermatozoa using flow cytometry. Flow cytometric estimates of cell viability, measured by propidium iodide (PI) exclusion, and acrosomal integrity, measured by Pisum sativum agglutinin (PSA) binding acrosomal contents, were equivalent to estimates made by using standard laboratory assays. Mitochondrial function, measured by rhodamine 123 (R123) fluorescence, was depressed by the mitochondrial inhibitors rotenone (64%) or monensin (52%), establishing that mitochondrial damage can be detected. Dilauroylphosphatidylcholine (PC12) or lysophosphatidylcholine (LPC) was used to destabilize sperm membranes. When challenged with 15-30 microM PC12, selective exposure of PSA binding sites occurred without induction of PI uptake or loss of R123 staining. However, PC12 concentrations greater than 60 microM resulted in a loss of R123 fluorescence intensity. In contrast, greater than 1200 microM LPC was required to expose PSA binding sites, which also resulted in PI uptake. By using flow cytometry, these three stains in combination can be used to correlate three different features simultaneously on individual spermatozoa and assay thousands of cells per sample without extensive preparation.

Acrosome↗

PubMed1995

Flow cytometry in the diagnosis of cancer.

Flow cytometry has rapidly expanded from basic research to clinical laboratories mainly due to its unique characteristics regarding cell analysis. Among the clinical uses of flow cytometry cancer represents one of the most relevant. Several applications of flow cytometry can currently be applied to the study of cancer, including the detection of tumour cell DNA aneuploidy, the analysis of tumour cell proliferation and the immunophenotyping of leukemias. Although standardized flow cytometry protocols for these applications are scanty, the clinical value has been clearly established. The presence of DNA aneuploidy and a high proportion of S-phase tumour cells have been associated with tumour malignancy and a poor prognosis. The immunophenotype of leukaemia is of great help both for the diagnosis and classification of chronic lymphoproliferative disorders and acute leukaemias, especially in acute lymphoblastic leukemia cases and the M0, M3-variant, M6 and M7 acute myeloblastic leukaemia subtypes. In addition, it allows the identification of relatively rare leukemia cases such as the biphenotypic and the Nk-cell lineage leukemias. The development of flow cytometry is continuously bringing new applications into the clinical laboratory in the area of cancer diagnosis.

Flow Cytometry↗