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PubMed1989

Procedural guidelines for performing immunophenotyping by flow cytometry.

Flow cytometry is a rapidly expanding technology that is moving from the research laboratory into the clinical laboratory. Recent advances in availability and reproducibility of monoclonal antibody reagents specific for a wide range of cell types coupled with lower costs for increasingly automated flow cytometers with powerful and user friendly data analysis capabilities have made flow cytometry the method of choice for immunophenotyping in the clinical laboratory. However, there is great variability in the level and type of quality assurance procedures used from laboratory to laboratory. A subcommittee established by the National Committee for Clinical Laboratory Standards (NCCLS), composed of representatives from industry, academia, professional societies, and regulatory agencies, has drafted consensus procedures which address specific problems and suggested solutions for performance of immunophenotyping by flow cytometry. This paper is based on the authors' discussions with the NCCLS Committee but does not represent an official NCCLS position. The official NCCLS document on this subject (H42) is expected to be published in 1989.

Adult↗
PubMed1983

Analysis of pleural effusions using automated flow cytometry.

Flow cytometry allows rapid and accurate analysis of the deoxyribonucleic acid (DNA) content of a large number of cells. In solid tumors, the presence of aneuploidy has been shown to correlate wall with the presence of neoplastic cells. Both cytologic examination and DNA analysis by flow cytometry were performed on pleural effusions from 33 patients. Results of the two examinations were in agreement in 10 of 12 malignant pleural effusion (two false-negatives) and in 20 of 21 benign effusions. One patient with cirrhosis, ascites and Nocardia pneumonia had hypodiploid cells (false-positive) in the pleural fluid. All patients who had a malignancy, but whose pleural effusion proved to be due to a benign cause, had cells with normal DNA content in their pleural effusion. DNA analysis using flow cytometry can be rapidly performed and is highly specific and sensitive. The finding of hyperdiploid cells is highly suggestive of malignancy.

Adult↗
PubMed1982

Preparation of rat islet B-cell-enriched fractions by light-scatter flow cytometry.

Flow cytometry has been examined as a method to separate islet cells into homogeneous subpopulations. Collagenase-isolated rat islets were dissociated into single cells and these were analyzed and sorted according to their low forward angle light scattering properties by using automated flow cytometry. Light scatter histograms showed two peaks of viable cells. Radioimmunoassay of hormone content in cell fractions collected across the the two peaks showed that glucagon-containing cells were concentrated towards the left side of the left peak and somatostatin-containing cells were concentrated towards the right side of the left peak, whereas insulin-containing cells were clearly enriched in the right peak. The B-cell-enriched fraction (90% B cells, 3% A cells, 2% D cells) exhibited significant insulin secretory responses to glucose (16.7 mM), and 3-isobutyl-1-methylxanthine (0.1 mM), during a 24-h culture period, and these responses were slightly greater than those observed in the original mixed islet cell preparation (66% B cells, 14% A cells, and 4% D cells). These results indicate that flow cytometry can be applied to sort pancreatic islet cells into populations enriched in specific endocrine cell types for further study of the functions of individual cell types.

Animals↗
PubMed1991

Detection of anti-equine neutrophil antibody by use of flow cytometry.

Flow cytometric and conventional fluorescence microscopic methods were compared to detect heterologous (rabbit) neutrophil antibody bound to equine neutrophils. Unfixed and paraformaldehyde-fixed neutrophils were treated with normal rabbit serum or various dilutions of an antineutrophil serum. The cells were then reacted with fluorescein conjugates of goat anti-rabbit IgG, staphylococcal protein A, and streptococcal protein G. Antibody binding was evaluated by use of fluorescence microscopy and flow cytometry. Unfixed neutrophils treated with normal rabbit serum did not fluoresce, whereas many of the fixed neutrophils had distinct cytoplasmic and some membranous (nonspecific) fluorescence. Unfixed cells treated with the antiserum had localized areas (capping) of intense membrane fluorescence, whereas fixed cells had bright uniform membranous fluorescence. The intensity of specific fluorescence varied with the antiserum dilution and the conjugate. On flow cytometry, over 80% of unfixed cells treated with antiserum dilutions up to 1:1,024, 1:2,048, and 1:256 fluoresced, respectively, with anti-IgG, protein-G, and protein-A conjugates. Fixed cells generally had similar percentages of fluorescent cells, but at a higher (1-step) antiserum dilution. It was concluded that flow cytometry is more sensitive than conventional fluorescence microscopy to detect antibodies associated with equine neutrophils.

Animals↗
PubMed2006

Comparison of sample fixation and the use of LDS-751 or anti-CD45 for leukocyte identification in mouse whole blood for flow cytometry.

Flow cytometry methods used to measure leukocyte function often entail sample preparation procedures that cause artifactual cell activation. To avoid leukocyte activation by isolation techniques, some preparation methods use fluorescent markers to discriminate leukocytes from erythrocytes in whole blood. One of these markers, laser dye styryl-751(LDS-751), has been used to distinguish leukocytes by staining nucleic acid, but has been found to stain other blood cells and dead cells indiscriminately. Thus, LDS-751 may not be an appropriate reagent for leukocyte identification in whole blood. Fixing samples with formaldehydes increases cell permeability and causes surface protein cross-linking that may alter staining of both intra- and extracellular markers. The degree of this sample alteration by formaldehyde fixation, however, remains in question. In addition, little is known about flow cytometry and sample preparation methods in mouse whole blood. The purpose of this study was to determine if labeling leukocytes with a monoclonal antibody specific to leukocyte common antigen (CD45) was superior to labeling with LDS-751 and to determine the effect of sample fixation on a mouse whole blood preparation for flow cytometry. Samples were incubated with CD16/CD32 Fc receptor blocker, and either 10 microg/ml LDS-751 or phosphate buffered saline (PBS). The samples were then fixed with paraformaldehyde or diluted with PBS followed by incubation with 5 microg/ml PerCP-conjugated anti-CD45, 5 microg/ml FITC-conjugated anti-CD11b, or 80 microM dichlorofluorescein diacetate. We found that samples labeled with LDS-751 demonstrated decreased fluorescence intensity for granulocyte CD11b expression and ROS production compared to samples labeled with anti-CD45. In addition, sample fixation decreased mean fluorescence intensity in samples labeled with either LDS-751 or anti-CD45. We conclude that labeling leukocytes with monoclonal antibody CD45 in a mouse whole blood preparation is preferable, as it provides improved measurement of leukocyte indices compared to LDS-751. Also, while sample fixation prior to antibody staining caused a decrease in overall fluorescence; it can be used to successfully identify extra-cellular markers.

Animals↗
PubMedDate not supplied

Attributes and applications of flow cytometry.

Flow cytometry (FCM) permits high speed measurement of fluorescence from individual cells flowing in a liquid medium. Cells stained with fluorescent dyes that bind specifically and proportionally to desoxyribonucleic acid (DNA) emit fluorescence that reflects their DNA content. Since neoplastic cells often contain abnormal amounts of DNA, FCM can be used as a rapid means to detect neoplasia. Flow cytometry can also provide cell kinetic information by quantitating cells in the various phases of the cell cycle. These capabilities make FCM a useful tool in the study of patients with neoplasia.

Aneuploidy↗
PubMed2006

A general method for bead-enhanced quantitation by flow cytometry.

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry.

Adoptive Transfer↗
PubMed1990

Chlorophyll a fluorescence measurements of isolated spinach thylakoids obtained by using single-laser-based flow cytometry.

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that chlorophyll a fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax-Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: 1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p-benzoquinone, phenyl-benzoquinone, and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. 2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70 degrees C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. 3) Addition of an inhibitor diuron up to 100 microM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.

Chlorophyll↗
PubMed1996

Analysis of pleural effusions using flow cytometry.

Flow cytometry allows a rapid and accurate analysis of the cells in serous fluids. The aim of this study was to evaluate the use of flow cytometric analysis in malignant pleural effusions. 26 patients (13 females, 13 males; mean age 52 +/- 19 years; range 16-82) were included in the study. 15 had malignant pleural effusions (7 adenocarcinoma, 2 lymphoma, 2 chronic myeloid leukemia, 1 ovarian carcinoma, 1 small cell lung carcinoma, 1 squamous cell lung carcinoma and empyema, and 1 malignant mesothelioma) with positive cytology. 2 had benign effusions associated with malignancy (1 squamous cell lung carcinoma and congestive heart failure, and 1 neuroblastoma and hypoproteinemia). 9 had benign effusions (3 tuberculosis, 1 congestive heart failure, 3 parapneumonic pleural effusion, 1 benign mesothelioma, and 1 pulmonary embolism). Flow cytometric analysis of pleural effusions revealed an increased DNA index in malignant effusions: 1.32 +/- 0.44 versus 0.88 +/- 0.23 in benign effusions (p < 0.04). The cell cycle distribution of cells such as G1/G0 and S in malignant effusions did not differ from that of benign pleural effusions; however G2+M increased significantly in malignant effusions (p < 0.03). Using analysis of mononuclear immunophenotyping, CD3+, CD4+, and CD8+ cells did not show any significant difference between the two groups. The lymphocyte activation marker CD38 was positive in 57.6 +/- 11.5% of malignant fluid cells and 38.5 +/- 6.2% of benign fluid cells (p < 0.04). The mean carcinoembryonic antigen levels in malignant and benign pleural effusions were 98.7 +/- 157.3 and 0.9 +/- 1.2 ng/ml, respectively (p < 0.03). In conclusion, the results of our study indicate that finding cells with an abnormal DNA content strongly supports the diagnosis of malignant pleural effusions. Additionally, mononuclear cell phenotypes have to be taken into consideration for malignant pleural effusions, particularly activated T cells. We recommend that flow cytometry should be performed if the cytology is equivocal.

Adolescent↗
PubMed1994

Spectra of fluorescent dyes used in flow cytometry.

Flow cytometry uses a relatively small set of dyes for immunochemistry and nucleic acid detection, most of which have been known and used reliably in flow cytometry for several years. These can usually be combined to make simultaneous two-color measurements of multiple cell-surface antigens and nucleic acid content. Because of the overlap of dye spectra and difficulties in finding dyes with substantial Stokes shifts that can be excited by the argon laser, simultaneous three-color or more detection can be more difficult. A basic knowledge of the factors that go into producing the fluorescent signal, including the spectra of dyes and their overlap, is necessary in planning multicolor experiments. By contrast, there have been a number of new fluorescent probes developed for detecting ions, membrane potential, metabolism, organelles, and other properties of living cells as well as for determining cell viability, proliferation, and cell tracking. So far most of these physiological probes are being used only for fundamental research rather than for cell classification. However, as research activity expands in this area, its diagnostic potential is likely to be increasingly appreciated.

Animals↗
PubMed2002

Immunophenotypic variability of B-cell non-Hodgkin lymphoma: a retrospective study of cases analyzed by flow cytometry.

Flow cytometric analysis is important in the diagnosis, classification, and follow-up of non-Hodgkin lymphoma. It is assumed that the lymphoma phenotype for each patient remains unchanged over time and is consistent from one specimen to another. To determine the variability in expression of lymphoid antigens, we reviewed 211 flow cytometry specimens of malignant lymphoma from 81 patients. Some antigens showed a stable pattern of expression such as CD5, CD10, CD19, CD20, and HLA-DR. In contrast, CD21, CD22, CD23, and CD25 showed more variability from one specimen to another. We believe several factors affect the stability of antigen expression. True differences in expression most probably are related to the biology and function of the different antigens. For instance, CD19 and CD20 are essential in cell maturation and function and, therefore, are present on the majority of cells. In contrast, CD22 has a role during B-cell activation and, therefore, is more variable. Lack of standardization inflow cytometry procedures also is responsible for some variability. Instrument settings for adequate compensation and the criteria used to determine when an antigen is reported as positive are important considerations when evaluating flow cytometry histograms.

Antibodies, Monoclonal↗
PubMedDate not supplied

Analysis of coccidian oocyst populations by means of flow cytometry.

Flow cytometry was employed as a tool to analyze and characterize batches of oocysts from laboratory and field isolates of Eimeria spp. from chickens and to propagate sub-populations of batches of oocysts. Oocyst batches were cleaned of debris by a combination of salt flotation, washing and treatment with dilute sodium hypochlorite (1.5% aqueous). Oocyst size and shape were registered by forward-angle light scatter with the argon laser excitation set at 488 nm at 300 mW. Sub-populations of oocysts were collected by map gating and used for microscopy or for propagation. The profile of particle size was characteristic for each species. Propagation of sub-populations of oocysts of specified sizes resulted in cultures of coccidia that were pure species or nearly pure species. The small size of E. mitis caused difficulty in separation from the remaining fine debris. This technique was useful for studying the mixed isolates by bit-map gating had the same limitations as micromanipulation because of the overlapping size of Eimeria spp. Characterization is further limited by the lack of suitable size/shape standards for flow cytometry.

Animals↗
PubMed1996

A novel method of microwave treatment for detection of cytoplasmic and nuclear antigens by flow cytometry.

Flow cytometry has recently become a useful technique for the quantitative analysis of cytoplasmic and nuclear antigens. We report here a rapid, simple, reproducible, and sensitive method for the simultaneous detection of cytoplasmic and nuclear antigens by flow cytometry. This technique involves the treatment of cell suspensions with 60 s of microwave oven heating after fixation with 2% paraformaldehyde. Following this treatment a number of cytoplasmic and nuclear antigens were detected on the human myelomonocytic cell line U937 (CD68, PCNA and Ki-67), peripheral blood leukocytes from both normal donors and leukemia patients (CD68, lipocortin-1 and PCNA) and a rat mesangial cell line 1097 (desmin, alpha-smooth muscle actin) using a standard indirect immunofluorescent staining with mouse monoclonal antibodies (mAbs). There are several advantages of this technique over the routinely used methods currently available. Firstly, microwave treatment is a rapid, simple, and reproducible method, which largely reduces both time and cost expenditure, and makes this technique widely available for flow cytometric analysis in many areas of diagnostic and research purposes. Secondly, microwave treatment produces optimal results for simultaneous detection of both cytoplasmic (CD68, lipocortin-1, desmin, alpha-smooth actin) and nuclear (PCNA, Ki67) antigens. Thirdly, microwave treatment also produces a discrete profile for DNA content analysis. Finally, microwaving retains a clear discrimination between cells and debris as measured by light scatter. This study demonstrates that microwave treatment is a powerful technique which will be particularly applicable to flow cytometric analysis in the detection of many cytoplasmic and nuclear antigens.

Animals↗
PubMed2000

Detection of massive transplacental haemorrhage by flow cytometry.

Flow cytometry has been shown to be a more accurate and sensitive method than the Kleihauer-Betke test for the measurement of feto-maternal haemorrhage in Rh(D) incompatibility. This report describes the successful use of flow cytometry to detect and monitor the management of a massive transplacental haemorrhage (105 ml) of fetal Rh(D) positive cells in a Rh(D) negative woman. The report highlights the accuracy and reproducibility of the test and the stability of a blood sample when transferred 596 kilometres to a central testing facility.

Adolescent↗
PubMed1990

Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry.

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.

Animals↗
PubMed2000

Recent developments in flow cytometry.

Flow cytometry has experienced considerable expansion of available parameters during the last years. Various new techniques that allow the direct analysis of antigen-specific lymphocytes by direct labeling with antigen or according to functional parameters following in vitro antigen stimulation will have enormous impact on immunological research, diagnosis, and therapy. This article concentrates on the basic principles and applications of these important technological developments in flow cytometry and focuses on the analysis of specific lymphocytes.

Cytokines↗
PubMedDate not supplied

Detection of intracytoplasmic cytokines by flow cytometry.

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.

Animals↗
PubMed1992

Detection of borreliacidal antibodies in hamsters by using flow cytometry.

Flow cytometry can be used to detect antibody that kills Borrelia burgdorferi. Borreliacidal activity was detected within 3 h of incubating B. burgdorferi with immune serum and complement. Right-angle light scatter and propidium iodide fluorescence were the cytometric parameters which correlated best with in vitro killing of B. burgdorferi. Flow cytometry is a rapid method for determining the presence of borreliacidal activity and may lead to a better serodiagnostic test for the detection of Lyme disease.

Animals↗