SEARCH · PubMed Health

Results for “Flow Cytometry”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,027 records · Page 57Linked to original sources

PubMed1996

An assessment of the effects of cadaver donor bone marrow on kidney allograft recipient blood cell chimerism by a novel technique combining PCR and flow cytometry.

A new technique, the PCR-flow assay is described that has allowed for the serial identification and quantitation of discrete mononuclear cell subsets of donor (or recipient) bone marrow derived cells in cadaver kidney transplant recipients infused postoperatively with donor vertebral body bone marrow cells. With fixed permeabilized cells in flow cytometry the amplification power of the polymerase chain reaction (PCR), using fluorescent-labeled primers to identify single copy HLA class II DRbeta1 genes of either donor or recipient origin, is combined with multi-color fluorochrome-labeled CD epitope-specific monoclonal antibodies. The details of the methodology are described; these support the utility of the assay. Initial observations were made on the chimeric makeup of the peripheral blood as well as iliac crest bone marrow between six months and one year posttransplantation in recipients serially followed weekly and then monthly, concomitantly compared with a control group of stable kidney transplant recipients using similar therapeutic protocols, who did not receive cadaver bone marrow. Several findings are of note. In 14 recipients of two bone marrow infusions totalling a mean of 6.29+/-2.18x10(10) cells, donor CD34 positive (+) (immature) cells were fourteen times as numerous in peripheral blood six months postoperatively as in six recipients given half as many bone marrow cells in one infusion (averaging 3.02+/-0.5x10(10)). These donor CD34+ cells unexpectedly averaged 36+/-7% of the total (donor plus recipient) CD34+ subset counted. Moreover, iliac crest bone marrow aspirates contained an average of thirteen times this number of CD34+ cells than in the peripheral blood, supporting the notion of engraftment. Of additional interest, between six months and one year posttransplant although no donor cells could be detected in peripheral blood of the controls there was an identifiable presence of donor CD34+ cells in their iliac crest bone marrow, albeit 10-fold less than the marrow-infused patients. In the clinical follow-up, although there were three unrelated mortalities, there were no additional kidney losses with current serum creatinine concentrations averaging 1.3+/-0.06 mg/dl. In conclusion, the PCR-flow assay presents the possibility of identifying discrete subsets of donor or recipient cells that may have an immunoregulatory function.

Adoptive Transfer↗

PubMed1995

The role of flow cytometry in non-resected cervical carcinoma.

Flow cytometry (FCM) has a prognostic value for many malignant neoplasms in terms of treatment response rate and survival. However, its role in non-resected cervical carcinoma remains uncertain. We have collected 96 paraffin-embedded specimens taken from non-resected cervical cancer patients treated by radiotherapy (RT) alone between 1984 and 1986. Our data revealed that FCM has little correlation with patients' age, pathological grade and clinical stage. Ploidy pattern and clinical stage correlate significantly with complete remission (CR) rate (p = 0.001 and 0.03). Most diploid or low-stage tumours (IB to IIA) obtained CR after RT alone. The application of an intravaginal extension electron cone (IVEC) (p = 0.019) and CR status (p = 0.0001) yield significant better overall survival (OS) rates than their alternative groups. The Cox regression model has confirmed these two variables as having an independent influence on OS. We thus conclude that both ploidy pattern and S-phase fraction (SPF) predict neither pre-treatment biological behaviour of the tumours nor overall survival. However, ploidy pattern has an independent influence on CR rate.

Adult↗

PubMed1983

Flow cytometry of keratinocytes.

A prerequisite for using flow cytometry (FCM) is the availability of isolated single cells. Procedures for separation and isolation of keratinocytes from animals and man are available, and the resulting single cell suspensions have been subjected to FCM measurements. The major advantage of the method is the accuracy and speed with which a variety of cellular constituents can be quantified. FCM of keratinocytes has, hitherto, been mainly confined to measurements of nuclear DNA for estimation of cell-cycle distributions and for ploidy studies. In mouse epidermis, cell-cycle distributions were estimated from sequentially obtained DNA histograms and evaluated with other cell kinetic measurements, resulting in new information about epidermal cell-cycle progression, not achievable by any of the methods alone. The best way, therefore, to increase our knowledge of keratinocyte proliferation, is the combined use of DNA FCM and other cell kinetic methods. DNA FCM has also been applied to healthy and diseased human epidermis, and may add valuable information to the classification of skin disease in selected cases. It is believed that further progress in the characterization of keratinocyte growth and development will depend on parameters other than DNA alone.

Animals↗

PubMed1989

Deoxyribonucleic acid flow cytometry of benign prostatic disease.

Deoxyribonucleic acid flow cytometry was performed on aspirated prostatic cells from 198 patients who had benign cytological or histological findings. Unsatisfactory acellular histograms were obtained from 10.6 per cent of the cases. Three-fourths of the satisfactory samples (more than 5,000 cells after subtracting debris) showed the expected single peak deoxyribonucleic acid diploid to near diploid histograms. Unexpectedly, the remaining samples were deoxyribonucleic acid aneuploid, most having 2 peridiploid peaks (deoxyribonucleic acid index 0.82 to 1.31). Usually, proliferation was low with less than 20 per cent hyperdiploid cells and with 2.5 +/- 1.5 per cent G2 cells. In 10 per cent of the single peak histograms there was evidence of inflammation, identified as an increase in hyperdiploid cells without an increased percentage of G2 cells but with a tail of high channel values. The aforementioned histogram features were considered to be benign findings. Seven per cent of the samples had deoxyribonucleic acid histograms suggestive of prostate cancer. Of these samples 7 had diploid or peridiploid aneuploid histograms with high proliferation (more than 20 per cent hyperdiploid cells with 8.5 +/- 3.8 per cent G2 cells), while 5 had histograms with deoxyribonucleic acid aneuploidy other than peridiploidy.

DNA↗

PubMed1993

The use of flow cytometry for the investigation of cell death.

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented.

Animals↗

PubMed1987

Centromeric index versus DNA content flow karyotypes of human chromosomes measured by means of slit-scan flow cytometry.

We have applied slit-scan flow cytometry (SSFCM) to classify human chromosomes according to their centromeric index (CI) and relative DNA content. The resulting bivariate--CI vs. DNA content--distributions shows 14 peaks for normal human chromosomes. Distinct peaks are produced by chromosomes 1, 2, 3, 4 + 5, 6 + 7 + X, 8, 13 + 14 + 15, 16, 17 + 18, 19 + 20, and 21 + 22 + Y. In addition, chromosomes 9 through 12 are resolved into three peaks. The identity of the chromosomes comprising each peak was determined by comparing CI vs. DNA content distributions measured for normal human chromosomes by means of SSFCM with CI and DNA content values measured for human chromosomes with image analysis. The accuracy of CI measurement by SSFCM was verified by measuring CIs for human chromosomes isolated from human/rodent hybrid cell lines containing only a few known human chromosomes. These studies showed CIs measured for human chromosomes 1-19 and 21 to be in close agreement with the CIs calculated by means of image analysis. We further confirmed the chromosome assignments for each peak by showing that the relative volumes of the peaks in the CI vs. DNA content distributions for chromosomes from normal cells are similar to the relative frequencies of chromosomes expected for these peaks based on the peak assignments.

Animals↗

PubMedDate not supplied

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry.

We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.

Calcium↗

PubMed1983

T-cell subpopulations in the peripheral blood of patients with connective tissue diseases as determined by flow cytometry using monoclonal antibodies.

We studied by flow cytometry using monoclonal antibodies the T3, T4, and T8 subpopulations of T cells in the peripheral blood of 109 patients with various connective tissue diseases who were not receiving any treatment. Comparison of the results was made with those obtained with normal controls matched for age and sex with each connective tissue disease group. When compared as disease groups, patients with systemic lupus erythematosus (n = 41) had decreased T8 cells, but patients with active disease (n = 17) had all three T-cell subpopulations lower than their controls, whereas those with inactive disease (n = 24) showed no differences. Patients with rheumatoid arthritis (n = 23) had decreased T3 and T8 cells, whereas patients with scleroderma (n = 22) only had decreased T3 cells, and patients with primary Sjögren's syndrome (n = 15) had lower proportions of all three T-cell subpopulations than their matched controls. Patients with mixed connective tissue disease (n = 8) had proportions of all three T-cell subpopulations akin to those of their matched controls, but showed a tendency to have decreased T8 cells that reached statistical significance when compared to the entire control group. Although our findings tend to support the notion that the abnormalities in immunoregulatory T-cell circuits leading to autoimmunity are different in each connective tissue disease, the great variability found in both patients and controls seems to preclude the use of these determinations in individual patients for clinical purposes.

Antibodies, Monoclonal↗

PubMed1994

The crossmatch in renal transplantation. Evaluation of flow cytometry as a replacement for standard cytotoxicity.

Flow cytometry (FC) is increasingly being used as a crossmatch procedure in addition to the standard complement-dependent cytotoxicity (CDC) test. In fact, FC offers a number of advantages over CDC and has the potential to become the primary crossmatch technique for cadaveric donor renal transplantation. We evaluated this possibility in 230 patients crossmatched by both CDC and FC. The results showed that when the T cell crossmatch was negative by FC it was always negative by CDC, and that when the T cell results were positive by CDC (IgM antibodies excluded) they were also positive by FC. As expected, a number of tests were T cell-positive by FC but negative by CDC. A T cell CDC crossmatch was more likely to be positive when FC was positive for both T and B cells and when FC results were quantitatively higher. However, FC was unable to consistently predict a positive, dithiothreitol-resistant B cell CDC crossmatch. A policy to transplant patients with negative FC results (70% of the patients evaluated) and not to transplant sensitized patients with FC+ T cell results (10%) would allow us to make a final decision with only FC in 80% of the cases. Actual graft survival was similar for nonsensitized first-transplant candidates with positive (83%) or all patients with negative (86%) FC results. We conclude that FC is sufficient to make a final decision in most cases. Wider utilization will require improvements in the ability of FC to measure B cell antibodies and to quantitate antibodies to T cells.

B-Lymphocytes↗

PubMed1990

New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy.

16 fluorochromes were examined for their ability to label viable lymphocytes in vitro and yield fluorescence detectable by fluorescence microscopy and flow cytometry. Of these fluorochromes, four intracellular dyes were found to be suitable for in vivo migration studies. They were H33342, the well known DNA-binding dye which excites and emits in the UV range, and three fluorescein based cytoplasmic dyes, namely BCECF-AM, Calcein-AM and CFSE which excite and emit in the visible range. Lymphocytes labelled with H33342, BCECF-AM and Calcein-AM were suitable for short term in vivo migration experiments with detection by flow cytometry 2-3 days post injection. In contrast lymphocytes labelled with CFSE, a fluorochrome which can covalently couple with intracellular macromolecules, were detected by flow cytometry up to 8 weeks post injection and thus this fluorochrome is ideal for long term migration experiments. Due to marked differences in fluorescence profiles, BCECF-AM and Calcein-AM could be used for short term double labelling experiments using the flow cytometer in which entry of injected lymphocytes into lymphoid organs was quantified. Similarly, in vivo localization of lymphocyte subpopulations could be examined by fluorescence microscopy utilizing differences in fluorescence excitation and emission spectra of lymphocytes labelled with H33342 and one of the fluorescein based dyes.

Animals↗

PubMed1995

CD4 Lymphocyte enumeration: comparison between flow cytometry and enzyme immunoassay.

Assessment of the CD4 lymphocyte number, currently performed by flow cytometry, is one of the main laboratory tests for establishing progression to acquired immunodeficiency syndrome (AIDS). An enzyme immunoassay has recently been commercialized which can be very useful for counting CD4 cells in laboratories where flow cytometers are not available. In the present study, a comparative evaluation of CD4 positive lymphocytes with flow cytometry and the enzyme assay was made in healthy subjects (N = 30, R = 0.88, P < 0.0001), human immunodeficiency virus (HIV)-infected individuals (N = 80, R = 0.91, P < 0.0001), and patients with autoimmune diseases (N = 28, R = 0.82, P < 0.001) or psoriasis (N = 18, R = 0.76, P = 0.01). A correlation between the two methodologies was not found in psoriatic patients under treatment with cyclosporin A (N = 7, R = 0.05, not significant). Some differences could be found at low CD4 lymphocyte levels since the influence of CD4 antigen eluted from monocytes or soluble CD4 in the whole blood sample could cause overestimation of CD4 cell numbers by the enzyme assay.

Acquired Immunodeficiency Syndrome↗

PubMed1979

"Cytogenetic" studies of spermatids of mice carrying Cattanach's translocation by flow cytometry.

The DNA content of spermatids of mice carrying Cattanach's translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X- and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2 +/- 0.1% of the total haploid genome as compared to 3.4 +/- 0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanach's translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.

Animals↗

PubMed1998

Determination of the biomasses of small bacteria at low concentrations in a mixture of species with forward light scatter measurements by flow cytometry

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4', 6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 &mgr;g/liter. The lowest value for mean dry mass, 0.027 +/- 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

Journal Article↗

PubMed2001

Cell cycle analysis by cyclin E + A/DNA multiparameter flow cytometry in exponential growth MOLT-4 cells.

OBJECTIVE: To establish a new method for analyzing the cell cycle in tumor which is a kind of cell cycle disease. METHODS: Mixtures of cyclin E and cyclin A antibodies were incubated with fixed MOLT-4 cells, and measured by flow cytometry. RESULTS: We developed a cyclin E + A/DNA flow cytometry analysis method, which may distinguish G0, early G1, late G1, S, G2 and M phase cells, rather than three phases in the DNA content histogram. CONCLUSION: Cyclin E + A/DNA multiparameter flow cytometry can simultaneously differentiate in the same sample six cell groups: G0, early G1, late G1, S, G2 and M phase cells. It performed better than any other cell cycle analysis methods that we have used and has a definite cell biology foundation.

Cell Cycle↗

PubMed1985

Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry.

This report describes the fluorescence hybridization of DNA sequence probes to interphase nuclei in suspension and the quantification of bound probe by dual beam flow cytometry. Nuclear proteins were first cross-linked with dimethylsuberimidate to prevent disintegration of the nuclei during denaturation and hybridization. To demonstrate that in situ hybridization can be performed in suspension, stabilized mouse thymocyte nuclei were hybridized with a probe for mouse satellite DNA sequences. The DNA probes were labeled with 2-acetylaminofluorene. After hybridization, an indirect immunofluorescent labeling procedure was used to visualize the target sequences. With dual beam flow cytometry, both the amount of hybridized probe and the DNA content of individual nuclei were determined. Thus, the specificity of DNA hybridization can be combined with the speed and quantitative analysis provided by flow cytometry.

2-Acetylaminofluorene↗

PubMed1991

DNA flow cytometry in uterine endometrial carcinoma.

The DNA content was investigated by means of flow cytometry in frozen tumor specimens from 33 patients with uterine endometrial carcinoma. The association was tested between ploidy and S-phase fraction on one hand and clinico-pathological parameters on the other. Aneuploidy appeared in 15 (45%) of the patients, and the average S-phase fraction was 0.18 (0.04-0.34). There was a highly significant correlation between DNA-Index (DI) and histologic differentiation (p = 0.01) and a less significant correlation between DI and estrogen receptor content (p = 0.04). DNA ploidy measured by flow cytometry is a highly reproducible parameter that in addition to routine histopathological examination may provide useful information. Further studies are needed to establish the prognostic importance of the flow cytometric parameters.

Carcinoma↗

PubMed2003

Monitoring of acute myeloid leukemia by flow cytometry.

Monitoring of minimal residual disease (MRD) becomes increasingly important in the risk-adapted management of patients with acute myeloid leukemia (AML). In selected patients with AML, multiparameter flow cytometry has shown accuracy and sensitivity in the quantification of MRD levels with independent prognostic impact. The applicability of this approach is superior to that of other methods such as quantitative polymerase chain reaction: Up to 80% of all patients can be monitored by flow cytometry. Nonetheless, significant technical advances are anticipated to extend the applicability of flow cytometry to 100% and to improve its sensitivity. Large clinical trials will determine the role of immunologic monitoring in the prognostic stratification of patients with AML.

Acute Disease↗

PubMed1990

Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry.

Random samples of cryopreserved, milk-extended semen, collected from 20 Holstein bulls at about 14 mo of age (young) and again at about 4 yr of age (mature), were evaluated at thawing and during 3-h incubation to compare semen quality of young versus mature bulls. Evaluation by differential interference contrast microscopy showed greater proportions of cytoplasmic droplets in semen from young versus mature bulls. Mature bulls exhibited greater proportions of intact acrosomes in freshly thawed semen than did young bulls. Evaluation of sperm chromatin structure by flow cytometry after staining with acridine orange showed lower values for mature versus young bulls, indicating resistance of DNA in nuclear chromatin to acid denaturation increased with age. Correlations between ages for most sperm morphology, acrosome integrity, and flow cytometry variables were high and positive. Nonreturn rate for young bulls was positively related to morphologically normal sperm and acrosomal integrity and negatively related to flow cytometry traits. Results suggest semen quality of young bulls was related to subsequent quality as mature bulls. With flow cytometry, differences were detected between semen samples that were not evident with light microscopy.

Acrosome↗