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Results for “Flow Cytometry”

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PubMed1998

A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry.

A whole-blood flow cytometry-based assay was utilized to assess CD4 and CD8 T-lymphocyte activation in response to phytohemagglutinin (PHA) stimulation. T-lymphocyte activation was assessed by qualitative (percent CD69) and semiquantitative (anti-CD69 antibody binding capacity) measurements of CD69 surface expression. Whole-blood samples from 21 healthy and 21 human immunodeficiency virus (HIV)-infected (<500 absolute CD4 counts per mm3) individuals were stimulated with 20 microg of PHA per ml for 18 to 24 h. The proportions of activated CD4 and CD8 T lymphocytes expressing CD69 (percent CD69) and the levels of CD69 expression on each T-lymphocyte subset (anti-CD69 antibody binding capacity) were measured. By using this assay system, T-lymphocyte activation was impaired in both CD4 and CD8 T-lymphocyte subsets of HIV-infected individuals. The proportions of CD69-positive CD4 and CD8 T lymphocytes were 43 and 27% lower, respectively, in samples from HIV-infected individuals compared to samples from healthy individuals. Similarly, the levels of CD69 expression on each activated CD4 and CD8 T-lymphocyte subset were 48 and 51% lower, respectively. These results suggest that both qualitative and semiquantitative measurements of CD69 surface expression by flow cytometry can be used to assess T-lymphocyte activation.

Antigens, CD↗

PubMed1981

Clinical application of flow cytometry: a review.

A survey of the use of flow cytometry for clinical purposes is given. In the last decade the main clinical application of this technique has been measurements of cellular DNA content for estimation of cell cycle distribution and ploidy studies. A large body of data is now available on the presence of aneuploidy in different malignant diseases. By measurements with high resolution, the demonstration of abnormal cellular DNA content in several types of tumors can be of definite diagnostic value when combined with conventional diagnostic procedures. The prognostic significance of different types of DNA aberrations is so far not established. Attempts to monitor cancer treatment by studying altered cell cycle distribution have not been successful, although some applications are of potential value. The main reasons for this are the complexity of tumor tissue as well as difficulties with interpretation of altered cell cycle distribution caused by drug combinations. For further progress in this field more emphasis on other cell constituents than DNA measured by flow cytometry is desirable, either as single or as multiparameter measurements.

Blood Cells↗

PubMed2004

Flow cytometry for high-throughput, high-content screening.

Flow cytometry is a mature platform for quantitative multi-parameter measurement of cell fluorescence. Recent innovations allow up to 30-fold faster serial processing of bulk cell samples. Homogeneous discrimination of free and cell-bound fluorescent probe eliminates wash steps to streamline sample processing. Compound screening throughput may be further enhanced by multiplexing of assays on color-coded bead or cell suspension arrays and by integrating computational techniques to create smaller, focused compound libraries. Novel bead-based assay systems allow studies of real-time interactions between solubilized receptors, ligands and molecular signaling components that recapitulate and extend measurements in intact cells. These new developments, and its broad usage, position flow cytometry as an attractive analysis platform for high-throughput, high-content biological testing and drug discovery.

Automation↗

PubMed1998

The clinical significance of flow cytometry crossmatching in heart transplantation.

BACKGROUND: Flow cytometry crossmatching is more sensitive than cytotoxic methods in identifying preformed antibodies to donor alloantigens. However, the significance of a positive flow crossmatch remains unknown for a recipient of a heart transplant who has a negative anti-human globulin crossmatch. METHODS: Flow crossmatching was performed retrospectively for 92 recipients of a primary cardiac allograft who underwent transplantation with a negative AHG crossmatch. RESULTS: Forty-six patients were flow crossmatch-positive for alloantibody: 20 were positive on both T and B lymphocytes, 12 were positive only on B lymphocytes, and 13 were positive only on T lymphocytes. Eleven had autoantibody invalidating the flow crossmatch with donor cells. Thirty-six patients had negative flow crossmatch. A significantly higher incidence of graft dysfunction with vascular rejection by 6 months was found for patients who had a positive flow crossmatch on B lymphocytes. This group also had an increased incidence of mortality within this same period. Patients who were flow crossmatch-positive on T and B lymphocytes were more likely to experience greater than two episodes of treated cellular rejection within the first 6 months. Flow crossmatch-positive patients stayed longer in the hospital in comparison to the other two groups, although the increases were not statistically significant. There were no differences between groups with regard to time to first rejection, absence of rejection episodes, episodes of decreased cardiac index (<2.3 L/m2), depressed left and right ventricular ejection fraction, or development of transplant atherosclerosis. CONCLUSION: A positive flow crossmatch identified a subset of patients who are predisposed to development of vascular rejection or are more likely to have frequent cellular rejection.

Female↗

PubMed1998

Basics of flow cytometry.

In summary, a beginner requires fundamental knowledge about flow cytometric instrumentation in order to effectively use this technology. It is important to remember that flow cytometers are very complex instruments that are composed of four closely related systems. The fluidic system transports particles from a suspension through the cytometer for interrogation by an illumination system. The resulting light scattering and fluorescence is collected, filtered, and converted into electrical signals by the optical and electronics system. The data storage and computer control system saves acquired data and is also the user interface for controlling most instrument functions. These four systems provide a very unique and powerful analytical tool for researchers and clinicians. This is because they analyze the properties of individual particles, and thousands of particles can be analyzed in a matter of seconds. Thus, data for a flow cytometric sample are a collection of many measurements instead of a single bulk measurement. Basic knowledge of instrumentation is a tremendous aid to designing experiments that can be successfully analyzed using flow cytometry. For example, it is important to know the emission wavelength of the laser in the instrument that will be used for analysis. This wavelength is critical knowledge for selecting probes. It is also important to understand that a different range of wavelengths is detected for each fluorescent channel. This will aid selection of probes that are compatible with the flow cytometer. Understanding the complication that emission spectra overlap contributes to detection can be used to guide fluorochrome selections for multicolor analysis. All of these experiment design considerations that rely on knowledge of how flow cytometers work are a very practical and effective means of avoiding wasted time, energy, and costly reagents. Data analysis is a paramount issue in flow cytometry. Analysis includes interpreting as well as presenting data that has been stored in list-mode files. Data analysis is very graphically oriented. There are a number of types of graphic representation that are available to visually aid data analysis. Two standard types of displays are used. These data plots are one-parameter histograms and bivariate plots. A user must be familiar with these two fundamental types of display in order to effectively analyze data. Histograms are the most simple modes of data representation. Histograms allow visualization of a single acquired parameter. Mean fluorescence and distributional statistics can be obtained based on markers that the user can graphically set on the plot. Percentages of positively expressing particles relative to a control sample can also obtained in a similar manner. In addition, multiple histograms can be overlayed on one another to depict qualitative and quantitative differences in two or more samples. Two-parameter data plots are somewhat more complicated than histograms; however, they can yield more information. Two-parameter dot plots of FSC vs SSC allow visualization of both light-scattering parameters that are important for identifying populations of interest. Bivariate fluorescent plots allow discrimination of dual-labeled populations that might remain hidden if histograms were used to display fluorescent data. Two-parameter plots that combine one light-scattering parameter and a fluorescent parameter are useful for analyzing control samples to elucidate the origin of nonspecific binding. Data analysis is very graphically oriented. Experience and pattern recognition become important when using two-parameter data plots for qualitative as well as quantitative analysis. The technique of gating or drawing regions on dual parameter light-scatter plots allows one to exclude information and examine the population of interest by disallowing particles that might confound or interfere with analysis. This is one of the fundamental uses for gating. (ABSTRACT TRUNCATED)

Animals↗

PubMedDate not supplied

Relationship between sperm viability as determined by flow cytometry and nonreturn rate of dairy bulls.

A newly developed flow cytometric method for determination of sperm concentration and viability was tested in an insemination trial with cryopreserved bull sperm to establish the relationship between sperm viability and nonreturn rates. Semen for experimental inseminations was produced from 157 young sires (114 Holstein and 43 Jersey), each contributing 4 experimental semen collections. Straws containing approximately 15 x 10(6) motile sperm before freezing were used in 118,680 experimental inseminations performed by 254 artificial insemination technicians in 6352 Danish herds. Statistical analysis based on 44,946 experimental first inseminations showed that the major part (95.4%) of variation in the 56-day nonreturn rate (NRR56) was residual. Only 0.38% of the total variation in NRR56 was due to bulls and differences between ejaculate within bull. However, bulls were preselected, and a relatively high insemination dose was used. Correlations between sperm viability as assessed by flow cytometry and NRR56 was slightly lower than observed for microscopic assessment of sperm motility. However, flow cytometry makes it possible to achieve an objective and precise determination of sperm viability. It was therefore possible to calculate the effect on NRR56 provided selection of semen is based on the flow cytometric method. Three freezing extenders were used in this experiment, but a significant difference in NRR56 was not observed. Flow cytometric results for 1 extender (Biociphos Plus) indicated poorer sperm survival during postthaw incubation compared with Triladyl extender with whole and with clarified egg yolk.

Animals↗

PubMed2001

Application of flow cytometry for ecological monitoring of the rumen microbial ecosystem.

Flow cytometry in combination with fluorescently labeled ribosomal RNA oligonucleotide probes was used for enumeration and monitoring of ruminal bacteria. The polyanionic azo dye Trypan Blue was used for discrimination between live bacterial cells and inorganic particles and the separation was further improved by lysozyme treatment and sonication. Cy3-labeled universally conserved probe EUB338 and FITC-labeled Prevotella bryantii specific probe PBB14 were used for in situ hybridization in mixed culture experiments and in samples of crude rumen fluid. The results were analyzed by flow cytometry. The separation of P. bryantii and Butyrivibrio fibrisolvens, another ruminal bacterium, in mixed culture experiments was satisfactory and enabled monitoring of these bacteria in a test system. P. bryantii cells were detected in crude rumen fluid samples only after supplementation with pure culture cells; this implicates a low concentration of P. bryantii cells in vivo (less than 100/nL, i.e. 10(5) per mL).

Animals↗

PubMedDate not supplied

Flow cytometry for detection and evaluation of urinary bladder carcinoma.

Flow Cytometry (FCM) DNA assays of bladder irrigation specimens are now recognized as a clinically useful and reliable means of detecting and monitoring carcinoma of the bladder. This technique, which identifies carcinoma by the presence of an aneuploid population of cells, can be carried out on specimens obtained in an outpatient or hospital setting and is easily performed in any medium-sized laboratory. It is most sensitive to superficial and high grade tumors. Overall, nearly 80% of superficial carcinomas of bladder will have positive flow cytometry, comparing very favorably with conventional cytology. Until now, the widest clinical application of FCM has been in monitoring the conservative treatment of stage 0-1 flat and papillary carcinomas, but newly developed dual parameter measurements are capable of quantifying proliferative activity, oncogene expression, growth factor receptors, and other cellular features that may better characterize the biologic potential of these tumors and can be expected to aid in the selection and timing of treatment.

Aneuploidy↗

PubMed1989

Predictive value of DNA measurements in bladder washings. Comparison of flow cytometry, image cytophotometry, and cytology in patients with a past history of urothelial tumors.

Comparative DNA ploidy measurements were carried out by flow cytometry and by image analysis on cells in 71 bladder washing specimens from 50 patients with past histories of bladder tumors. Among the specimens classified as diploid or questionable by flow cytometry, 14 showed the presence of aneuploid DNA values documented by image analysis. In 18 of the 50 patients, recurrent tumors were observed during a relatively brief period of follow-up. In 15 of them the DNA pattern was aneuploid and in three it was questionable. In nine of the 15 patients, both methods of DNA analysis disclosed aneuploidy, but in six patients aneuploidy was detected by image analysis only. A combination of DNA aneuploidy, whether observed by flow cytometry, image analysis, or both, and of positive or suspicious urine cytology is highly predictive of recurrence of high grade bladder tumors. Image analysis of DNA content in bladder washings adds information of clinical value above and beyond that obtained by flow cytometry.

Aged↗

PubMed2005

A novel flow cytometry based assay for quantification of corneal angiogenesis in the mouse model of herpetic stromal keratitis.

In this study a novel flow cytometry based quantitative assay for measuring corneal angiogenesis is demonstrated. Corneas of Balb/c mice were lightly scarified and infected with 5 x 10(5), 5 x 10(4) and 5 x 10(3) pfu Herpes simplex virus 1 (HSV-1) RE virus. Development of corneal angiogenesis was studied on day 15 post infection by direct visualisation of infected cornea with a slit-lamp biomicroscope. Endothelial cells constituting the newly developed blood vessels in cornea were stained with murine anti CD31 antibody on frozen corneal sections and corneal whole mounts at day 15 post infection. Total number of endothelial cells was quantified at day 15 post infection by flow cytometry. Mice infected with different doses of HSV-1 RE developed severe to mild corneal angiogenesis at day 15 post infection. Endothelial cells constituting the newly formed blood vessels expressed CD31 at day 15 post infection. Flow cytometry revealed that, the number of CD31 positive cells isolated from diseased corneas increased with the increase in Neovascularisation index. The flow cytometry analysis used in this present study is a useful, accurate and cost effective method for quantifying corneal angiogenesis.

Animals↗

PubMed2000

Immunohistochemical detection of CD10 in paraffin sections of hematopoietic neoplasms: a comparison with flow cytometry detection in 56 cases.

Paraffin-section immunohistochemistry with heat-induced epitope retrieval using a newly characterized monoclonal antibody (clone 56C6) against the CD10 antigen was performed on 56 hematopoietic tumors previously studied for CD10 expression by flow cytometry. The cases included 33 precursor B-lymphoblastic leukemias, 10 acute myeloid leukemias, five precursor T-lymphoblastic leukemias, five follicular lymphomas, and three Burkitt cell leukemias. Forty of the 56 cases were CD10 positive by flow cytometry studies, including all five follicular lymphomas (100%); 30 of 33 (91%) cases of precursor B-lymphoblastic leukemias, two of three (66%) cases of Burkitt cell leukemias, two of five (40%) cases of precursor T-lymphoblastic leukemias, and none of the 10 cases of acute myeloid leukemia. Thirty-nine of the 40 (97%) flow cytometric CD10-positive cases also expressed CD10 by immunohistochemistry in formalin- or B5-fixed, paraffin-embedded tissue, with only one case of precursor B-lymphoblastic leukemia being positive by flow cytometry and negative by immunohistochemistry. The 16 CD10-negative flow cytometry specimens were all also negative by immunohistochemistry. Thirty-seven CD10 immunohistochemistry positive cases showed a diffuse membranous staining pattern and two cases demonstrated a Golgi staining pattern. The fixation methods (10% neutral buffered formalin versus B5) and decalcification did not affect the CD10 immunostaining results. This study demonstrates that the new CD10 monoclonal antibody clone 56C6 is a reliable marker for detection of CD10 antigen expression in formalin-and B5-fixed paraffin-embedded tissue after heat-induced epitope retrieval when compared with flow cytometry detection of fresh tissue samples.

Antibodies, Monoclonal↗

PubMed1990

Clinical cancer progression in urinary bladder tumors evaluated by multiparameter flow cytometry with monoclonal antibodies. Laval University Urology Group.

Multiparameter flow cytometry studies were performed on clinical samples of human bladder tumors to simultaneously analyse DNA content and the expression of surface glycoproteins defined by monoclonal antibodies T16, Om5, T43, and T138. The results of tests performed on 80 samples of bladder irrigations and tumors from 68 patients were correlated with clinical findings at the time of sampling and with disease outcome prospectively (mean follow-up 2 years). Measuring the level of the panurothelial antigen T16 provided more precision in DNA analysis and served as an internal standard to measure the relative expression of the other cell surface antigens studied. The panel of monoclonal antibodies improved the analytical capacity to study the heterogeneity of antigenic phenotypes within individual samples. Aneuploidy frequently correlated with high stage cancers and with a high rate of clinical cancer progression defined as metastasis or death by cancer. However ploidy was not an entirely reliable prognostic indicator since a significant proportion of Ta and T1 nonprogressing tumors were aneuploid, while in 6/20 cases of cancer progression, the samples were near diploid. Contrary to Om5, T43 and T138 antigens were expressed significantly more often on aneuploid samples, although they appear to provide additional information. T138 was positive on 17/18 samples from patients with high stage cancers of which five were near diploid. It was also positive on 4/5 samples from patients with Ta and T1 tumors in whom disease progressed to metastasis and death. Overall, the expression of T138 antigen was a better single indicator of clinical cancer progression than was ploidy. Further stratification was obtained with combined results of DNA and T138 antigen studies. Within the near diploid group, the incidence of bladder cancer death was 0/26 for T138 negative and 5/13 (38%) for T138-positive patients (P less than 0.01). In the aneuploid group incidence of bladder cancer death was 2/10 (20%) for T138-negative and 12/19 (63%) for T138-positive patients (P less than 0.05). These results suggest that simultaneous flow cytometry measurements of DNA and surface antigens may better assess the prognostic behavior of human bladder tumors.

Antibodies, Monoclonal↗

PubMed1989

Limitations in the measurement of c-myc oncoprotein and other nuclear antigens by flow cytometry.

Recent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation-associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c-myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa-S3 cells. The cells were stained with monoclonal antibodies either to the c-myc oncoprotein or to p105, a prolifera-tion-associated nuclear antigen. Protein-levels were measured on the basis of anti-c-myc or anti-p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c-myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.

Antibodies, Monoclonal↗

PubMed2005

Discrimination of resident and infiltrated alveolar macrophages by flow cytometry in influenza A virus-infected mice.

Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC18(5)-DS to discriminate resident alveolar macrophages from newly infiltrating monocytes/macrophages in mice with and without pulmonary influenza A virus infection. Leukocytes in bronchoalveolar lavage (BAL) and peripheral blood were analyzed by 2-color flow cytometry as a function of time following intravenous injection of DiIC18(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC18(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC18(5)-DS positive, and almost all of these were monocytes/macrophages based on labeling with fluorescein isothiocyanate (FITC)-conjugated antibody to the Mac-3 marker. Only 3.2% +/- 0.3% of peripheral blood monocytes (approximately 0.16% of total peripheral blood leukocytes) were DiIC18(5)-DS positive at 6 days after injection, whereas > 95% of BAL leukocytes were strongly dye-positive on days 6 to 28. When DiIC18(5)-DS was injected in mice 6 days prior to intranasal challenge with influenza A, flow cytometry indicated that 57.8% 5.6% and 60.7% +/- 8.5% of macrophages/monocytes in BAL were newly infiltrated (i.e., DiIC18(5)-DS negative, Mac-3 positive) at 4 and 7 days, respectively, post viral infection. The discrimination of subpopulations of resident and newly recruited macrophages in BAL should facilitate future mechanistic studies on pulmonary infection and inflammatory lung injury.

Animals↗

PubMed2003

Quick detection of Leishmania in peripheral blood by flow cytometry. Is prestorage leucodepletion necessary for leishmaniasis prevention in endemic areas?

Leishmaniasis is a serious health problem in various endemic areas. There are reports that the parasite can be transmitted via blood transfusions. We studied the clinical utility of flow cytometry for the screening of blood donors in an endemic area in Greece. Samples from 2000 blood donors from the area of Lasithi, Crete, Greece were examined by flow cytometry after labelling with a polyclonal anti-leishmania antibody conjugated with fluorescein-isothiocyanate derived from infected canines in the area. The same blood samples were simultaneously examined for serum anti-leishmania antibodies, May-Grünwald staining of peripheral blood smears and polymerase chain reaction (PCR) in buffy coat to the minicircle of kinetoplastic DNA. Direct sequencing of the PCR-amplified area helped discriminate leishmania species. Flow cytometry detected 33 cases with parasites in the peripheral blood leucocytes (1.65%), which were confirmed by PCR. One PCR-positive case was negative by flow cytometry. After prestorage leucodepletion, no sample was positive by PCR. Anti-leishmania antibodies were positive in 304 (15%) cases. Flow cytometry was found to be a sensitive and rapid method of detecting leishmania in peripheral blood samples. Leucodepletion effectively reduces the detection of the parasite, thus minimizing the potential risk of leishmania transmission through blood transfusions in endemic areas.

Adolescent↗

PubMed1994

Flow cytometry of liver fine needle aspirates.

Fine needle aspiration analysis of intraabdominal masses is increasing in frequency. This study was designed to evaluate whether flow cytometry could be of value in the cytopathology interpretation of such specimens. Over a 4-year time span, 129 consecutive liver fine needle aspirates were evaluated by flow cytometry for DNA content, ploidy, and proliferation rate. Only excess cells remaining in the needle after cytology samples were prepared were included in this study. Overall sensitivity was 75% and specificity was 94%. In addition, flow cytometry results were pivotal for at least two specimens in achieving the appropriate diagnosis. For these reasons, it was concluded that flow cytometry could be a valuable adjunctive technology, to the cytopathologist in the interpretation of liver fine needle aspirates.

Biopsy, Needle↗

PubMed1998

Immunomagnetic separation and flow cytometry for rapid detection of Escherichia coli O157:H7.

A rapid method for detecting Escherichia coli O157:H7 combining immunomagnetic beads (IMB) and flow cytometry was developed. Labeling antigens separated by IMB with fluorescent antibody enabled the detection of < 10(3) CFU bacteria per ml in pure culture. The optimum concentration of magnetic beads for flow cytometry was lower (ca. 10(5) particles per ml) than that reported for conventional IMB assay (more than 6 x 10(6) to 8 x 10(6) particles per ml). Immunomagnetic separation and flow cytometry (IMFC) were evaluated for detecting E. coli O157:H7 in the presence of a competing microorganism and for detecting antibodies in potassium phosphate buffer. The total assay time from separating antigens with IMB to analyzing with flow cytometry was about 1 h. IMFC detected 10(3) to 10(4) CFU of E. coli O157:H7 per ml in ground beef enrichment broth and could effectively discriminate between E. coli O157:H7 and competing natural flora. The new assay system provides another approach to separation and detection of low populations of pathogens and shows potential for detecting low concentrations of toxins and other soluble antigens directly from food in a short time.

Animals↗

PubMed1994

Detection of borreliacidal antibody by using acridine orange and flow cytometry.

Borreliacidal antibody has been shown to be important for the serodiagnosis of Lyme disease and determination of immune status. Our results show that borreliacidal antibody can be rapidly and accurately detected by flow cytometry. Acridine orange was added to normal and immune sera containing Borrelia burgdorferi organisms in the presence and absence of complement prior to data acquisition by flow cytometry. The flow cytometric parameters of side scatter and detection of acridine orange fluorescence were used to determine events per minute (number of labeled spirochetes), percent shift in fluorescence (number of dead spirochetes), and mean channel fluorescence (intensity of fluorescence-labeled spirochetes) of acridine orange-labeled spirochetes. Borreliacidal antibody was detected as early as 4 h, with optimal detection 16 to 24 h after incubation of B. burgdorferi organisms with immune serum and complement. Our results also showed that complement was necessary for detection of borreliacidal antibody. Flow cytometry with acridine orange-labeled spirochetes provides a rapid means for detection of borreliacidal antibody.

Acridine Orange↗