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PubMed1994

DNA or cell kinetics flow cytometry analysis of 33 small gastrointestinal cancers treated by photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) mediated by hematoporphyrin derivative (HPD) is a new treatment for cancers of small volume undergoing Phase II or III clinical trials in various medical fields. However, there is a lack of prognostic criteria of efficacy as in other cancer treatment. METHODS: Cell DNA content or cell kinetics throughout the cell cycle were analyzed by flow cytometry and propidium iodide staining before and after HPD-PDT in 33 patients with Tis or T1 cancers of the gastrointestinal tract. The authors compared results in near-diploid cancers with those obtained in normal corresponding tissue. RESULTS: Complete local tumor destruction and negative histologic findings (complete response [CR]) were observed in 17 of 33 patients during a period averaging 15.7 months. Flow cytometry DNA analysis was feasible in 32 patients. Aneuploidy, found in 15 of the 32 indicated a poor prognosis because 5 of 15 patients with aneuploid tumors were classified as having CR, compared with 12 of 17 patients with near-diploid tumors (P < 0.05). Changes in ploidy after PDT in 11 patients consisted of a reduction in the number of aneuploid peaks in 8 patients and the appearance of one aneuploid peak in 3 patients. Percentages of cells in SG2M phase in near-diploid tumors differed from those observed in control subjects for adenocarcinomas, and there was no significant decrease after HPD-PDT. There was no correlation between the decrease of SG2M cells and the response to HPD-PDT. CONCLUSION: Results obtained with PDT in this series of patients confirm previously published findings. Changes occurring in the ploidy of PDT-treated patients demonstrate that PDT acts directly on cancer cells in humans and not only on tumor vasculature. However, response to PDT varies from one cell population to another. The appearance of aneuploid populations after PDT suggests that destruction of sensitive cell populations allows the growth of aneuploid clones that initially are not detectable by flow cytometry.

Adenocarcinoma↗

PubMed1989

A new membrane permeabilization method for the detection of intracellular antigens by flow cytometry.

This article describes a new cell membrane permeabilization method for the detection of intracellular antigens by immunofluorescence staining and flow cytometry. The number of cells remained unaltered and no cell aggregation or loss of intracellular antigenicity was observed after this permeabilization treatment. A mixed leukocyte population from human peripheral blood was used in this study and the leukocytes were fixed and permeabilized, which permitted monoclonal anti-vimentin antibodies to reach intracellular antigens. The stabilization of cell membranes and preservation of intracellular antigenicity was achieved with paraformaldehyde fixation. This pretreatment prevents cell destruction and subsequent treatment with the detergent n-octyl-beta-D-glucopyranoside results in permeabilization of the cell membrane. The procedure does not alter the expression of cell surface antigens, which is of importance if phenotypic characterization of intracellularly stained cells is to be undertaken. Furthermore, this simple, rapid and reproducible technique makes it possible to detect and distinguish between different human peripheral blood leukocytes without prior purification steps. The leukocyte subpopulations remain well-separated and easily detectable by flow cytometry.

Antibodies, Monoclonal↗

PubMed1995

[The role of flow cytometry in the study of physiopathology of neutrophilic granulocytes].

Flow cytometry represents an interesting methodological approach to human neutrophil function assessment and to the study of neutrophil biology. Several aspects of neutrophil activation can be evaluated by means of such technology: phagocytosis, respiratory burst, surface markers, modifications in intracellular pH, actin polymerization, membrane potential, aggregation. Neutrophil flow cytometric evaluation is suitable for whole blood evaluation and has been shown to be very specific and sensitive. In particular, whole blood methods have been developed in order to investigate phagocytosis, hydrogen peroxide production, and surface markers expression (integrins, receptors for the Fc fragment of IgG, selectins, etc.). Other aspects of neutrophil activation have to be investigated by using purified neutrophils.

Flow Cytometry↗

PubMed2000

Anti-paternal antibodies by flow cytometry in the management of alloimmunization on recurrent miscarriages.

PROBLEM: There is no reliable laboratory test available to diagnose immunologically mediated miscarriages. We investigated the clinical significance of maternal anti-paternal leukocyte antibodies by flow cytometry after alloimmunization. METHOD OF STUDY: The flow cytometry crossmatch (FCXM) was performed in 158 patients with a history of three or more unexplained first-trimester miscarriages without live birth. After negative FCXM patients were immunized, subsequent pregnancy outcomes and FCXM results were followed. RESULTS: Of 112 subsequent pregnancies, 83 of 100 (83.0%) FCXM-positive patients after immunotherapy had successful pregnancy outcomes, whereas seven of 10 (70.0%) FCXM-negative patients had miscarriages (P = 0.0001). The percent live birth ratio was 2.77 (CI, 1.07-7.16; P=0.0001) for FCXM-positive patients compared to FCXM-negative patients. The calculated predictive value showed that 75.6% of FCXM-negative patients would have subsequent miscarriages. CONCLUSIONS: Positive FCXM is closely associated with successful pregnancy outcome following immunotherapy. We propose that FCXM might be included in the routine laboratory tests for the management of recurrent miscarriages.

Abortion, Habitual↗

PubMed1999

Characterization of binding of adenylate cyclase toxin to target cells by flow cytometry.

Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K(+) and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K(+) efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 micrograms/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37 degrees C; however, the total binding at 0 degrees C is less than that at 37 degrees C. In human erythrocytes, AC toxin does not cause an increase in K(+) efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K(+) efflux, and hemolysis produced by AC toxin.

Adenylate Cyclase Toxin↗

PubMed2004

The use of flow cytometry in the evaluation of cell viability of cryopreserved sperm of the marine shrimp (Litopenaeus vannamei).

Although the cryopreservation of penaeid prawn sperm or embryos has definite applications in the aquaculture industry, there is no protocol routinely used for this procedure. One of the main problems relies on the limitations for the determination of sperm cell viability. In this study, we evaluated the toxicity and cryoprotectant effect of four agents, at three different concentrations, in sperm suspension, spermatic mass, and complete spermatophore of the marine shrimp Litopenaeus vannamei. Cells were frozen by fast and slow cooling rates. After thawing, they were analyzed by optical microscopy and flow cytometry, which was also utilized to determine spermatic viability by DNA staining with propidium iodine. Considering viability by morphotype analysis, the best result was obtained when the spermatic mass was frozen by slow cooling rate in the presence of methanol (61.6%). There was a positive correlation between morphotype analysis and flow cytometry, although the percentage of viable cells was always lower when determined by the later. These results show that flow cytometry is a valuable tool to evaluate sperm cell viability in decapod species and it is more sensitive technique than optical microscopy.

Animals↗

PubMedDate not supplied

Slit scan flow cytometry of isolated chromosomes following fluorescence hybridization: an approach of online screening for specific chromosomes and chromosome translocations.

The recently developed methods of non radioactive in situ hybridization of chromosomes offer new aspects for chromosome analysis. Fluorescent labelling of hybridized chromosomes or chromosomal subregions allows to facilitate considerably the detection of specific chromosomal abnormalities. For many biomedical applications (e.g. biological dosimetry in the low dose range), a fast scoring for aberrations (e.g. dicentrics or translocations) in required. Here, we present an approach depending on fluorescence in situ hybridization of isolated suspension chromosomes that indicates the feasibility of a rapid screening for specific chromosomes or translocations by slit scan flow cytometry. Chromosomes of a Chinese hamster x human hybrid cell line were hybridized in suspension with biotinylated human genomic DNA. This DNA was decorated with FITC by a double antibody system against biotin. For flow cytometry the chromosomes were stabilized with ethanol and counterstained with DAPI or propidium iodide (PI). An experimental data set of several hundred double profiles was obtained by two parameter slit scan flow cytometry and evaluated automatically. The evaluation algorithm developed allowed a classification of chromosomes according to the number of centromeres and their chromosomal positions in less than 1 msec per individual profile. Approximately 20% of the measured DAPI profiles showed a bimodal distribution with a significant centromeric dip indicating a "normal" chromosomal morphology and a correct alignment in the flow system. In many cases, profiles of a "normal" bimodal fluorescence distribution of the DNA stain (DAPI, PI) were correlated with a "normal" FITC profile. Due to their centromeric indices these profiles agreed well to the expected human chromosomes of the cell line. In some cases of "normal" DAPI (PI) profiles, "aberrant" FITC profiles were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

A method for simultaneous nuclear immunofluorescence and DNA content quantitation using monoclonal antibodies and flow cytometry.

A preparative technique for the two-parameter flow cytometric study of nuclear antigen expression is reported. This method employs a brief sequential treatment of cells at 4 degrees C first with 0.5% paraformaldehyde and second with 0.1% Triton X-100 in phosphate-buffered saline followed by cellular staining with indirect immunofluorescence and propidium iodide. Using this technique, cellular morphology is preserved, cell clumping is minimized, and high-quality indirect immunofluorescence and DNA staining are obtained with a minimum of nonspecific labeling. Utilizing nuclear antigen-specific monoclonal antibodies in conjunction with this technique, the cell-cycle phase-dependent expression of such antigens is examined. From these data, the utility of two-parameter flow cytometry in the identification and quantification of cell-cycle-dependent modulation of nuclear antigens is discussed.

Antibodies, Monoclonal↗

PubMed2005

Flow cytometry susceptibility testing for the antifungal caspofungin.

Rapid antifungal susceptibility testing for the antifungal agent caspofungin can be performed using flow cytometry (FC). An FC procedure using acridine orange provided minimum inhibitory concentration (MIC) results within 7 to 9 h which were compared with results obtained using the NCCLS M27-A2 protocol. To evaluate the consistency of this method, susceptibility testing using caspofungin was performed using 73 isolates of eight different species of Candida from various clinical samples in Central California. Macrotiter or microdilution tests were performed according to the NCCLS M27-A2 protocol, and the MICs were compared to those provided by our flow cytometry method. All isolates tested had results within the sensitive interpretive category, and 90% of the results compared within 1 dilution, showing excellent agreement between the methods. The MIC at which 50% of the isolates tested were inhibited (MIC50) and the MIC90 of caspofungin for all eight Candida species were within 1 dilution. This flow cytometer 7-h protocol for testing the antifungal susceptibility of Candida species to caspofungin provided results equivalent to those obtained with the M27-A2 protocol.

Antifungal Agents↗

PubMed1989

Issues for quality assurance in clinical flow cytometry.

An increasing number of clinical laboratories are using flow cytometry to analyze cells stained with fluorescent antibodies or dyes. Many articles discuss the analytical performance of immunofluorescent assays and DNA staining procedures. However, quality assurance transcends the performance of analytical methods and includes preanalytic, analytic, and postanalytic phases of the test procedure. This review focuses on preanalytic and analytic factors that affect the results of flow cytometric analysis of cells stained with fluorescent antibody or dyes specific for nucleic acid. The article reviews the controls used to assess instrument performance followed by a discussion of biological variables, and reagent, methodological, and instrumental factors that may affect the interpretation of these results.

Antigens↗

PubMed2007

Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening.

Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cellos functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed.

Animals↗

PubMed2001

Application of gel microdroplet and flow cytometry techniques to selective enrichment of non-growing bacterial cells.

We describe an application of gel microdroplet (GMD) and flow cytometry techniques to selective enrichment of non-growing Leuconostoc mesenteroides cells, which are well culturable on other media, from a mixture with Bacillus subtilis cells in nutrient broth. After encapsulating cells of the mixed population within GMDs and a brief incubation in nutrient broth, the inability of L. mesenteroides cells to form microcolonies within GMDs allowed their discrimination from B. subtilis cells. After staining the GMD mixture with 6-carboxyfluorescein diacetate, which showed no influence on cell viability, the GMDs containing single cells of L. mesenteroides were selectively collected using flow cytometry sorting based on differences in fluorescence intensity. The cells of L. mesenteroides retained viability during the process.

Bacillus subtilis↗

PubMed1989

Chromosome banding analysis by slit-scan flow cytometry.

We have investigated the use of fluorescence banding patterns for the resolution of metaphase chromosomes by slit-scan flow cytometry. Fluorescence scans of R-banded chromosomes have been obtained for the entire human karyotype. Metaphase chromosomes were R-banded in suspension by staining with chromomycin A3 after hypotonic treatment in Ohnuki's buffer. Specific fluorescent landmark bands were detected for human chromosomes 1-12. Scans obtained for chromosomes 13-22 did not contain sufficient information for classification. Characteristic fluorescence patterns for human chromosomes 1 and 3 provided the clearest evidence for the detection of R-bands by slit-scan flow cytometry. Specific patterns were detected for human chromosomes 9-12 in which the number and placement of the fluorescent bands served as classifiers.

Cell Line↗

PubMed1987

Purification and identification of murine epidermal dendritic cells: flow cytometry and immunogold labeling.

We report on application of flow cytometric and immunogold labeling techniques to purify and identify two types of murine epidermal dendritic cells: Langerhans cells (LC) and Thy-1-positive dendritic epidermal cells (Thy 1+-dEC). After density centrifugation of epidermal cell (EC) suspensions through Ficoll gradients. IA-positive LC and Thy 1+-dEC are labeled with monoclonal antibodies (fluorescein-conjugated anti-IAd for LC and anti-Thy 1.2-biotin, followed by avidin-phycoerythrin, for Thy 1+-dEC). The fluorescence-activated cell sorter (FACS) is then used to obtain 95-98% pure populations of these dendritic cells with a yield of 2-4 X 10(6) cells and a viability of 80-90%. A post-fixation, pre-embedding immunogold labeling technique using 15 nm and 40 nm colloidal gold particles is employed to identify LC and Thy 1+-dEC, respectively, to confirm the purity of the sorting and to estimate the number of IA antigenic sites per LC. With transmission electron microscopy, ultrastructural morphology of sorted LC is preserved; however, Birbeck granules are markedly diminished compared to the pre-sorted population of LC. In contrast, characteristic dense-core granules are readily visualized in sorted Thy 1+-dEC. Purification of epidermal dendritic cells by flow cytometry may be a useful technique to employ in functional studies of epidermal dendritic cells.

Animals↗

PubMed2004

Detection of residual neuroblastoma cells in bone marrow: comparison of flow cytometry with immunocytochemistry.

BACKGROUND: Because the cytomorphologic examination of bone marrow (BM) aspirates appears not sensitive enough to detect residual neuroblastoma cells, two four-color flow cytometric assays using different combinations of CD9, CD81, CD56, CD45, and anti-GD2 were evaluated. METHODS: The sensitivity of the flow cytometric assays was assessed by spiking experiments in normal peripheral blood samples. Twenty-eight BM samples, 12 biopsies, and 3 peripheral blood stem cell (PBSC) preparations from 22 patients with neuroblastoma were analyzed. The results were compared with those of an anti-GD2 immunocytochemical reference assay. RESULTS: Flow cytometric and immunocytochemical analyses showed residual neuroblastoma cells in four BM samples. One PBSC preparation and 20 BM samples were negative for both assays. Four BM and two PBSC samples scored positive for the immunocytochemical assay but were negative for the flow cytometric tests. This was due to the limited number of cells that were flow cytometrically analyzed. A strong correlation between the flow cytometric and immunocytochemical tests was found (chi2 = 6.4, P = 0.011). CONCLUSIONS: When an equal amount of cells is analyzed, the sensitivity of the flow cytometric assays is to be about 10 times lower than that of the immunocytochemical test. However, the flow cytometric assays can be used to screen for residual cells in clinical samples with a sensitivity of one neuroblastoma cell in 10(4) to 10(5) normal mononuclear cells. Flow cytometry is simple, quick, and cost effective compared with immunocytochemistry. In addition, the flow cytometric assays can be used to screen for residual neuroblastoma cells in case of a GD2-negative primary tumor. Therefore we recommend flow cytometry for the detection of residual neuroblastoma cells.

Adolescent↗

PubMed1995

Comparison of CD4 cell count by a simple enzyme-linked immunosorbent assay using the TRAx CD4 test kit and by flow cytometry and hematology.

Measurement of CD4 T-lymphocyte levels is clinically useful in monitoring immune status in a number of conditions, including human immunodeficiency virus (HIV) infection, in which the absolute CD4 count is used to guide therapy. The absolute CD4 count is obtained by multiplying the results of the leukocyte count and the differential with a hematology cell counter and the percentage of CD4+ T lymphocytes determined by flow cytometry. These techniques require expensive, complex instrumentation, and interlaboratory results are difficult to standardize and reproduce. The rapid growth of HIV infection worldwide has increased the need for more-reproducible and cost-effective methods for CD4 T-cell monitoring. The TRAx CD4 test kit is based on a novel adaptation of conventional enzyme-linked immunosorbent assay (ELISA) and permits the simple quantitation of total CD4 protein from whole-blood lysates. In this study, the relationship between total CD4 protein measured in units per milliliter (TRAx) and in cells per microliter (flow cytometry and hematology) was defined in a multisite clinical study using linear regression analysis. Data from 230 HIV-seronegative and 321 HIV-seropositive specimens were used to calibrate the TRAx assay recombinant CD4 standards and controls in equivalent CD4 T lymphocytes per microliter (cells per microliter). The calibration of the TRAx CD4 assay in cells per microliter was validated with a second group of specimens from 17 healthy volunteers and 20 HIV-seropositive patients which were collected and tested under strictly controlled conditions intended to minimize the effects of specimen aging on the results of the reference method. These data were also used to estimate the variability of absolute CD4 count by cytometric methods as well as the precision of the TRAx CD4 result after it was calibrated in cells per microliter. Overall, correlations between the two methods ranged from 0.87 to 0.95. Additional studies demonstrated that the contribution of CD4 protein from monocytes and any soluble CD4 in sera are negligible in the TRAx assay and do not significantly affect results. This new method represents a promising alternative to absolute CD4 T-cell enumeration by flow cytometry and hematology.

Adolescent↗

PubMed1986

Isolation of viable type II alveolar epithelial cells by flow cytometry.

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.

Acridine Orange↗

PubMed1996

Characterization of camel leukocytes by flow cytometry and microscopic evaluation of granulocyte phagocytosis of fluorescent bacteria.

Peripheral blood was obtained from four lactating camels (Camelus bactrianus) and flow cytometry was used to characterize cell populations. The ability of the granulocytes to engulf fluorescent bacteria was studied in vitro using fluorescence microscopy. Three clusters of blood cells (mononuclear cells, neutrophils and eosinophils) were identified by flow cytometry and fluorescent microscopy. Milk, on the other hand, was dominated by cell fragments and no distinct cluster formation was found. The mean yield of blood granulocytes and monocytes isolated on Ficoll gradient was 92.2 +/- 5.4% and 7.9 +/- 5.7%, respectively. Cell viability was 95%. The mean percentage of phagocytic cells was 71.8 +/- 5.9% at 10 min and increased to 97.3 +/- 0.5% at 60 min when observation was terminated. The average number of bacteria per phagocyte was 8.7 +/- 2.1 and 13.1 +/- 0.9 at 10 and 60 min incubation time.

Animals↗