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PubMed1980

Characterization of hematologic malignancies by flow cytometry.

Flow cytometry of cellular DNA content revealed ploidy abnormalities in 15% of 170 patients with various leukemias, in 50% of 26 patients with malignant lymphomas, and in 68% of 110 patients with multiple myeloma, for an overall incidence of DNA content abnormality of 37% in 306 patients with hematologic malignancies. Since the incidence of ploidy abnormality in over 100 solid tumors exceeded 90%. DNA flow cytometry is also ideally suited to screen for bone marrow metastases. Cell separation by centrifugal elutriation was shown to permit enrichment of aneuploid cells, including one example where cells with abnormal DNA content were not recognized in the unfractionated sample. Biparametric measurements of acridine orange-stained cells for DNA and RNA content analysis were suitable to enhance the discriminatory power of flow cytometric detection of lymphoma and myeloma tumor cells in heterogeneous cell populations of bone marrow and lymph nodes. DNA/RNA measurements in leukemia, (the disease category with the lowest incidence of abnormal DNA mode) revealed a markedly higher mean RNA content in acute myeloid leukemia compared with normal or acute lymphoblastic leukemia bone marrow. While these different RNA content patterns were found in whole marrow, cell separation by centrifugal elutriation of normal marrow disclosed cell subpopulations of myeloid precursor cells with a RNA content pattern similar to that of unseparated AML marrow. Hence, the described differences in RNA content between normal and AML marrow seem to be related to the greater heterogeneity of differentiated cells in normal marrow and per se do not appear to be a unique feature of the leukemia disease process.

Bone Marrow↗

PubMed1988

Principles of flow cytometry.

Flow cytometers can analyze a populations of cells, one at a time, at rates of 1,000 to 10,000 cells per second. They can provide distributions of parameters, not just the MCV. A broad variety of measurement parameters is available, ranging from simple sizing to esoteric measures of membrane fluidity or epitope density. The resulting analysis gives low resolution compared to imaging of cells but gives excellent statistical samples of the parameters that it can detect. The parameter values for individual cells can be correlated, and displayed in a variety of multi-dimensional formats. When the analysis reveals subsets of cells, it is possible to sort the detected subsets of cells into separate chambers for further culture or test. Flow cytometers are already in clinical laboratories, performing size analysis in multiparameter hematology instruments. As you have seen, this usage barely scratches the surface of the capabilities of this technology. It now remains for the research clinician to adapt flow cytometry to a broader range of useful clinical applications.

Calibration↗

PubMed1984

Cellular DNA of human neoplastic B-cells measured by flow cytometry.

Flow cytometric analysis of DNA of tumor cells rapidly provides information on cell kinetics and tumor ploidy. Human B-cell lymphomas, however, often contain high numbers of nonneoplastic cells, mainly T-lymphocytes, which may hamper the accurate measurement of cell cycle phases and ploidy level of these tumors. The neoplastic cells in each B-cell lymphoma express a single immunoglobulin light chain. Therefore, we labeled surface immunoglobulin light chains to discriminate between predominantly neoplastic B-cells and nonneoplastic cells in the same tissues. Using this label as well as antibodies against nonneoplastic T-cells, we performed multiparameter correlated flow cytometric analysis of 52 human B-cell lymphomas measuring cellular DNA in neoplastic and nonneoplastic populations from the same tissues without physical separation of cells. Comparison of cellular DNA of immunoglobulin light chain-bearing neoplastic cells with that of nonneoplastic cells from the same tumor enabled us to detect DNA changes (aneuploidy) in almost 80% of the lymphomas, an incidence higher than observed previously by conventional DNA analysis of unseparated cells. These ploidy changes were confirmed by comparing in the same tumor the DNA of normal T-cells with that of predominantly neoplastic cells. The proportion of neoplastic cells in the synthetic phase of the cell cycle (S-fraction) varied widely from tumor to tumor. Lymphomas with high neoplastic S-fractions (higher than 10%) were mostly hyperdiploid tumors and histologically corresponded to intermediate- and high-grade unfavorable lymphomas. Tumors with low neoplastic S-fractions (less than 5%) were predominantly diploid and near diploid, histologically low-grade lymphomas. Six lymphomas showed two discrete cell populations bearing the same immunoglobulin light chain but containing different amounts of DNA suggesting the presence of two neoplastic clones in the same tumor (biploidy). In two patients in whom the lymphoma relapsed at 17 and 34 months, respectively, after the initial biopsies, repeat tumor samples were obtained. Despite an increase in the neoplastic S-fraction, no change in ploidy level was observed in either case. Light scatter analysis suggested a relationship between cell size and genomic size; large cells in these tumors were mostly presynthetic aneuploid cells. The ability to measure DNA, antigens, and cell size in individual cells in a rapid, correlated manner is a unique attribute of flow cytometry.(ABSTRACT TRUNCATED AT 400 WORDS)

B-Lymphocytes↗

PubMed1989

Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry.

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.

Bacteria, Anaerobic↗

PubMed1994

An optimized method for routine HLA-B27 screening using flow cytometry.

Flow cytometry and monoclonal antibodies are promising tools for HLA-antigen detection. Previous approaches have been hampered by the lack of a carefully standardized system for calibration and sample analysis. A new system for HLA-B27 screening was developed using a FACScan flow cytometer, software for automated calibration and analysis, calibration beads, and the anti-HLA-B27-FITC/anti-Leu4-PE (CD3) monoclonal antibodies. The median fluorescence channel result for the HLA-B27-FITC signal of CD3+ T lymphocytes is compared to a decision marker. Values lower than this threshold are read as HLA-B27 negative and those above are recommended for retesting with the classic microcytotoxicity assay on the presumption of HLA-B27 positivity. The anti-HLA-B27 antibody reacts with all six HLA-B27 subtypes and shows a weaker binding to HLA-B7. The screening test results were compared with those from the microcytotoxicity assay for HLA-typing in studies involving several European centers. The observed sensitivity was 100% (95% Cl:98.6-100) and the specificity was 97.4% (95% Cl: 96.4-98.3). Other performance studies verified the reproducibility and reliability of results obtained with the screening system.

Anticoagulants↗

PubMed2003

Analysis of RNA-protein interactions by flow cytometry.

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Flow Cytometry↗

PubMed2001

Quantitative flow cytometry.

Flow cytometers are instruments that can quantify fluorescence intensity data and provide unique information about cell populations. Significant advances have been made in terms of calibration reagents, evaluation of the quality and limitations of the monoclonal antibodies, standardized sample preparation, and data analysis to ensure interlaboratory comparability and reproducibility. Efforts to standardize quantitative fluorescence intensity measurements impact current clinical flow cytometry applications (i.e., immunophenotyping), but also emerging technologies, such as microarray assays, which also require calibration of fluorescence intensity across different platforms.

Data Collection↗

PubMed1988

Stage D1 prostatic adenocarcinoma: significance of nuclear DNA ploidy patterns studied by flow cytometry.

Flow cytometric analysis of nuclear DNA ploidy pattern was performed on 91 samples of prostatic adenocarcinoma from patients with stage D1 disease (metastatic deposits in pelvic lymph nodes). All patients had undergone radical retropubic prostatectomy and bilateral pelvic lymphadenectomy. Clinical follow-up ranged from 5 to 19 years. Nuclei were extracted from paraffin-embedded archival material. Isolated nuclei were stained with propidium iodide. The DNA ploidy pattern was diploid (normal) in 42% of tumors, tetraploid in 45%, and distinctly aneuploid in 13%. Only 15% of DNA diploid tumors progressed locally or systemically, whereas 75% of tumors with an abnormal DNA ploidy pattern (tetraploid or aneuploid) subsequently progressed (P less than 0.0001). Among low-grade tumors, ploidy analysis detected a subgroup associated with a poor prognosis; among high-grade tumors, a subgroup associated with a favorable prognosis was detected. None of the patients with a DNA diploid tumor died of prostatic cancer during the period of observation. In contrast, 43% of patients with DNA tetraploid tumors and 44% of those with DNA aneuploid tumors had died of prostatic cancer 10 years after surgical treatment (P less than 0.001). Determination of nuclear DNA ploidy pattern by flow cytometry provides objective, highly significant, prognostic information for patients with stage D1 prostatic carcinoma.

Adenocarcinoma↗

PubMedDate not supplied

Aneuploid and polyploid cellular DNA heterogeneity in insect cell material of diptera species analyzed by flow cytometry.

Flow cytometric 1-parameter DNA analysis and 2-parameter DNA/protein analysis have been performed with cell material of the diptera species Chironomus thummi, Drosophila melanogaster, Calliphora vicina and Musca domestica using an impulse cytophotometer with a new quartz objective, that was especially manufactured for cytofluorometric investigations. The occurrence of heterogenous cell populations with aneuploid and polyploid DNA content within the cell material of different developmental stages of diptera species have been determined, whereby in larvae polyploid cell populations and in imagos aneuploid cell populations predominate. Partially separation of 2 C cells from other cell populations with higher DNA content can be done by Ficoll-Hypaque centrifugation as demonstrated with cell material from Chironomus larvae. For flow cytometric DNA analysis of insect cell material a simple and rapid cell preparation and staining technique is presented by using the DNA-specific fluorochrome DAPI in combination with the protein fluorochrome sulforhodamine 101. Employment of flow cytometry in diptera genetics might be a new tool for cytological and cytogenetic investigations as shown with the classical genetic objects Chironomus and Drosophila.

Aneuploidy↗

PubMed1993

Development of clinical standards for flow cytometry.

Flow cytometers are instruments that can determine multiparameter data simultaneously and have a great potential in providing unique information about cells. To transfer this potential to the clinical laboratory, the development and the proper use of standards and calibrators are required to ensure comparability and reproducibility of data from different flow cytometers. Although there are a number of types of standards for flow cytometry, each has its specifically designed purpose to ensure that data from this complex instrument are accurate, precise, and reproducible among instruments over time.

CD3 Complex↗

PubMed1987

Detection of human immunodeficiency virus-infected lymphoid cells at low frequency by flow cytometry.

Flow cytometric detection of human immunodeficiency virus (HIV)-infected lymphoid cells at low frequencies is described. Infected cells from human T lymphoid cell lines H9 and A3.01 were detected at frequencies as low as 10(-4) following indirect immunofluorescence labeling. For labeling, cells were treated with an HIV-inactivating, permeabilizing fixative followed by binding of a monoclonal antibody specific for the HIV major core protein p24, and then by binding of fluorescein isothiocyanate-conjugated F(ab')2 fragments of goat anti-mouse immunoglobulin antibody. We compared two fixation procedures, one using a mixture of methanol and acetone, the other a three-step fixation using methanol, paraformaldehyde and Triton X-100. The latter fixation protocol was found to be superior in its ability to resolve mixtures of infected and uninfected cells. The method allowed determination of the percentage of the cell population that was infected and the relative amount of p24 antigen per cell. At analysis rates of several thousand cells/s, detection of HIV-infected cells as rare events was possible. Excellent agreement was obtained between flow cytometric evaluation and reverse transcriptase (RT) assay of infected H9 cells cocultured with uninfected H9 cells in various proportions for 7 days. In time course of infection experiments, cultures infected by small numbers of viral particles were positive by flow cytometry up to 3 days earlier than by RT assay.

Acquired Immunodeficiency Syndrome↗

PubMed2000

Immunohistochemistry can be used to subtype acute myeloid leukemia in routinely processed bone marrow biopsy specimens. Comparison with flow cytometry.

Flow cytometry (FC) is the preferred method of immunophenotyping acute myeloid leukemia (AML). However, there are situations in which FC is unavailable and in which immunohistologic staining of bone marrow biopsy specimens can be used to provide immunophenotypic information. To evaluate immunohistologic staining and to confirm its value, we selected 80 newly diagnosed cases of AML that were classified according to French-American-British (FAB) criteria and confirmed by flow cytometric analysis for this study. Paraffin-embedded bone marrow specimens were stained using a panel of antibodies that included CD34 (QBEND10), antimyeloperoxidase (anti-MPO), antihemoglobin, factor VIII-related antigen, and 3 epitopes of CD68 (HAM56, KP1, and PG-M1). Our findings suggest that with the use of the paraffin-reactive antibodies CD34 (QBEND10), MPO, CD68 (PG-M1), antihemoglobin, and factor VIII-related antigen, immunohistochemistry can be used to subclassify AML. Comparison of immunohistochemical results with FC immunophenotyping suggests that there is significant concordance in the results for markers that can be used with both techniques, indicating that the sensitivity and specificity of both methods is comparable (P > .53 in all cases).

Acute Disease↗

PubMed2004

Techniques: GPCR assembly, pharmacology and screening by flow cytometry.

Flow cytometers are well known for their ability to analyze and sort cells at high rates based on physiological responses and expression of protein markers. The potential for flow cytometry in G-protein-coupled receptor (GPCR) research, however, is less well appreciated. Potential applications include: (i) the homogenous discrimination of free and bound ligands or proteins in both cellular and microsphere-based assays; and (ii) multiplexed ('suspension array') analysis of cell responses and protein-protein interactions. Innovative sample-handling systems also provide sub-second resolution of interaction kinetics and 1 second per well throughput of microliter-sized samples from multiwell plates. Flow cytometric methods using microspheres for analysis of GPCRs that interact with intracellular and extracellular binding partners such as ligands, G proteins and kinases have been established. These analyses can produce quantitative pharmacological data analogous to radioligand assays, and, in some cases, the probes can be integrated into the assembly as fluorescent fusion proteins.

Animals↗

PubMed1995

[Principles of flow cytometry].

Flow cytometry (FCM) allows the simultaneous measurement of different parameters in a population of cell or sub-cellular particles, which are individually analysed at a rate of 500 to 5,000 objects/second. Results are displayed as univariate or bivariate histograms, giving the distribution of the population in relation to the studied parameters. Moreover, FCM offers the possibility to physically sort the sub populations defined by the analysis.

Cell Cycle↗

PubMed1995

Immune functions in beluga whales (Delphinapterus leucas): evaluation of phagocytosis and respiratory burst with peripheral blood leukocytes using flow cytometry.

Flow cytometric assays using peripheral blood were developed to study phagocytosis and respiratory burst, the two major functions of neutrophils and among the most important non-specific defense mechanisms, in beluga whales. The use of flow cytometry avoids the problems associated with the isolation and purification of different cell types, and allows the measurement of a large number of cells (10,000) in a very short period of time. The methods described will be used to compare these functions in blood samples from highly contaminated beluga whales from the St. Lawrence and from relatively clean arctic beluga whales.

Animals↗

PubMed1995

Influence of incubation on the chromatin condensation and nuclear stability of human spermatozoa by flow cytometry.

Flow cytometry analysis was used for the accurate and objective evaluation of sperm chromatin condensation and chromatin stability of sperm nuclei. It was also possible to determine the influence of incubation on sperm chromatin. Different types of spermatozoa were studied: unprocessed spermatozoa at 1 and 45 min after ejaculation, after swim-up (migrated), spermatozoa incubated for 6 h in non-capacitating conditions (aged), or in B2 medium (capacitated) or B2 medium followed 1 h later with A23187 (reacted). All types of spermatozoa were analysed before and after treatment with various decondensation agents: sodium dodecyl sulphate (SDS), SDS plus EDTA and SDS plus disulphide-reducing agent [dithiotreitol (DTT)]. Sperm nuclei were enzymatically isolated and stained with propidium iodide. Three flow cytometric parameters were then measured: forward light scatter (cellular size), side light scatter (cellular complexity) and fluorescence (uptake of propidium iodide). Fluorescence was the most suitable parameter to study the degree of condensation and resistance to decondensation of DNA in the spermatozoa. Unprocessed spermatozoa 1 min after ejaculation underwent decondensation by all assessed treatments (anionic detergent, chelating or disulphide-reducing agents). Unprocessed spermatozoa 45 min after ejaculation and migrated spermatozoa did not undergo decondensation with SDS treatment, but decondensation occurred after treatment with SDS+EDTA or SDS+DTT. Spermatozoa incubated for 6 h under both non-capacitating (aged spermatozoa) and capacitating conditions (capacitated spermatozoa) and reacted spermatozoa were decondensed only after treatment with SDS+DTT.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcimycin↗

PubMed1983

Flow cytometry.

Flow cytometry (FCM) allows the rapid measurement of a variety of physical and biochemical properties of individual cells suspended in a liquid medium. Recent developments now allow FCM to assess cellular properties with a speed and precision unobtainable by any other means. Because of its multiple capabilities, FCM is being used for ever greater numbers of applications, in both basic and applied biology, and its expected soon to be widely used in the clinical laboratory.

Antigens, Surface↗

PubMed1993

Enzyme activities of six different dehydrogenases in Ehrlich ascites cells measured by flow cytometry.

Flow cytometric measurements of the activities of lactate dehydrogenase, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase in single Ehrlich ascites tumour cells are described using a tetrazolium salt/fluorescent formazan reaction. Applying cyano-ditolyl-tetrazolium chloride (CTC) as redox dye indicating enzyme reaction, and DAPI as a fluorochrome for nuclear DNA staining, the bivariate flow cytometric assay of enzyme activity and cell cycle analysis was established. Furthermore, adopting the calibration procedure reported formerly, consisting of biochemical determination and flow cytometry of the same sample performed parallelly, the enzyme activities were expressed in biochemical units. The dehydrogenase activities found in Ehrlich ascites cells were 97.5 fmol H2 per average positive cell during 5 min for lactate dehydrogenase, 69.0, 10.6, 25.3, 29.7, and 19.0 fmol H2 per average positive cell during 20 min for glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, glycerol-3-phosphate dehydrogenase, succinate dehydrogenase and glutamate dehydrogenase, respectively. This quantitative procedure can offer an alternative analytic tool for enzyme cytology.

Animals↗