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Results for “Flow Cytometry”

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PubMed2001

Immunophenotypic analysis of hematogones (B-lymphocyte precursors) in 662 consecutive bone marrow specimens by 4-color flow cytometry.

Bone marrow hematogones (B-lymphocyte precursors) may cause problems in diagnosis because of their morphologic and immunophenotypic similarities to neoplastic lymphoblasts. The purposes of this prospective, multiparametric flow cytometry study were to quantify hematogones across age groups and a spectrum of clinical conditions, to identify factors that affect the relative quantity of hematogones, and to compare their immunophenotype with that of neoplastic lymphoblasts. A total of 662 consecutive marrow specimens were analyzed for hematogones using one of two 4-color antibody combinations; hematogones were identified in 528 (79.8%). There was a significant decline in hematogones with increasing age (P <.001), but a broad range was found at all ages and many adults had a relatively high number. Specimens processed by density gradient had a higher mean percent hematogones than those processed by erythrocyte lysis (P <.001). There was a direct decline in hematogones with increasing marrow involvement with neoplastic cells. A total of 8% of the 662 specimens contained 5% or more hematogones: 24.6% of specimens from patients aged less than 16 years and 6.3% from those 16 and older (P <.000 01). Increased hematogones were observed most often in patients with lymphoma, marrow regenerative states, immune cytopenias, and acquired immunodeficiency syndrome. Hematogones always exhibited a typical complex spectrum of antigen expression that defines the normal antigenic evolution of B-cell precursors and lacked aberrant expression. In contrast, lymphoblasts in 49 cases of precursor B-ALL showed maturation arrest and exhibited 1 to 11 immunophenotypic aberrancies. Four-color flow cytometry with optimal combinations of antibodies consistently distinguishes between hematogones and neoplastic lymphoblasts.

Adolescent↗

PubMed2001

Flow cytometry of peripheral blood and bone marrow cells from patients with hairy cell leukemia: phenotype of hairy cells, lymphocyte subsets and detection of minimal residual disease after treatment.

By flow cytometry (FC) and an extensive panel of markers we characterized leukemia cells from the peripheral blood (PB) and bone marrow (BM) of 13 symptomatic patients with hairy cell leukemia (HCL). Hairy cells (HCs) identified in the large cell gate always expressed B-cell markers - CD19, CD20, CD22, HLA-DR, and 'HCL-restricted' markers - CD22+CD11c, CD25 and CD103. Other markers, not followed regularly, were occasionally expressed, such as CD34, CD38, CD71, CD15, CD10 and kappa/lambda light chains. Furthermore, in one patient with suspect but not proved HCL in PB or BM, neither morphologically nor immunologically, we confirmed the diagnosis of HCL. Only the immunophenotyping of splenic cells after splenectomy confirmed HCL diagnosis. Flow cytometry was repeated at 3-5 month intervals, after treatment with 2-Chlorodeoxyadenosine (CdA) or less frequently alpha-interferon (IFN). We investigated serially lymphocyte subsets after treatment and we found profound and persistent CD4+ lymphopenia in majority of studied patients after CdA treatment. Simultaneously we investigated the value of FC to detect minimal residual disease (MRD) and to establish, whether MRD+ could predict relapse. Detection of MRD in our series predicted hematological relapse only in one case with persistent MRD+, in majority of cases with occasionally found MRD+ phenotype, did not. Using quantitative immunophenotyping we observed significantly higher values of molecule numbers of hairy cell B-cell markers, comparing to B-cells in nonleukemic gate of the same sample. Our study showed 1) the diagnostic value of FC in management of HCL patients, 2) long-lasting response in the majority of patients after CdA, 3) a profound and persistent CD4+ lymphopenia in CdA treated patients, 4) some correlation between persistent MRD staining and hematological relapse, and 5) further, till now not described activated feature of HCs, given by the increased values of molecular numbers (molecules of equivalent soluble fluoresceine - MESF) in B-cell antigens of HCL.

Adult↗

PubMed1993

Analysis of DNA aneuploidy and c-myc oncoprotein content of canine plasma cell tumors using flow cytometry.

To derive a method for determining malignant potential of plasma cell tumors, a retrospective analysis of the DNA ploidy and relative p62c-myc oncoprotein content using bivariate flow cytometry was performed on 23 formalin-fixed, paraffin-embedded tissues from 23 dogs. The samples included one tissue each from 17 males and six females 2 to 16 years of age (mean = 7.5 years). Twelve breeds were represented, including three Cocker Spaniels, three Golden Retrievers, and five of mixed breed. Ten of the samples were histologically classified as malignant-plasma cell tumors, and ten specimens were classified as benign. Three samples of plasmacytic inflammation, from two Cocker Spaniels and one Shih Tsu, were included as controls. The ploidy and relative c-myc content data obtained were compared with the histologic grade. A significant difference in ploidy was found between benign and malignant tumors (P < or = 0.05). Five of nine malignant plasma cell tumors were aneuploid; the remainder were diploid (4/9) or tetraploid (1/9). Only one of the benign plasmacytomas was aneuploid (1/10), whereas six were diploid (6/10), and three were tetraploid (3/10). All of the controls were diploid (3/32). When relative amounts of p62c-myc from malignant and benign tumors were compared by flow cytometry, a greater significant difference was established (P < or = 0.01) than bu using aneuploidy alone. Relative values of p62c-myc content ranged from 219 to 553 units in 8/10 malignant plasma cell tumors and from 86 to 392 units in 3/10 benign plasmacytomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2005

Standard and quantitative analysis of cyclin E threshold by cyclin E/DNA multiparameter flow cytometry.

The threshold of cyclin E expression at G1/S boundary is a characteristic feature of cell cycle progressing. In this study, we tried to develop a quantitative approach to analyze cyclin E threshold by multiparameter flow cytometry. The expression of cyclin E in exponentially growing MOLT-4 cells was detected under different photomultiplier tube (PMT) voltages by cyclin E/DNA multiparameter flow cytometry. Additionally, cyclin E was detected in cells which were treated with caffeine and cycloheximide (CHX) under the same PMT voltage. Moreover, the expression of cyclin E in MOLT-4 cells was compared with that in JURKAT cells. Cyclin E threshold was quantified by formula B2/AxC (A, B, C indicates the minimum, threshold, and maximum of cyclin E fluorescence intensity, respectively). Results showed that in MOLT-4 cells, cyclin E threshold calculated by formula B2/AxC was invariable under different PMT settings. It was decreased in cells treated with caffeine and remained changeless in cells treated with cycloheximide. Cyclin E threshold in JURKAT cells was much lower than that in MOLT-4 cells. It was suggested that Formula B2/AxC we firstly set up could be used to analyze cyclin E expression threshold quantitatively.

Caffeine↗

PubMed1991

DNA analysis of cardiac myxomas: flow cytometry and image analysis.

Cardiac myxoma is the most common primary tumor of heart, but there is a longstanding controversy over whether it is a true neoplasm or a reactive lesion. We analyzed 24 cardiac myxomas from 22 patients: 22 by DNA flow cytometry and five by image analysis. Two myxomas were aneuploid; one of those analyzed by flow cytometry, and the other by image analysis. Proliferative fractions (S + G2/M) were high in three tumors from patients with multiple myxomas (mean, 15.9%; SD, 4.0%) as compared with 12 solitary uncomplicated myxomas (mean, 7.7%; SD, 6.0%). S-phase and proliferative fractions were low in embolic, recurrent, and solitary myxomas. The presence of aneuploidy in some myxomas supports a neoplastic origin for this tumor.

Adult↗

PubMed1985

Application of flow cytometry to analyses of cultured human glioma and fetal brain cells.

Methods are described to study cell surface and cytoplasmic antigens of cultured human glioma, fetal brain cells and fibroblasts using flow cytometry. This required harvesting the cultured cells with Versene or mild trypsin treatment and fixation in 4% paraformaldehyde prior to staining for glial fibrillary acidic protein (GFAP) and fibronectin using indirect immunofluorescence. At passage 10, 38% of fetal brain cells [CHII] were GFAP-positive but at passage 14 only 3.5% expressed GFAP. Two glioblastomas and an anaplastic astrocytoma had 38.8%, 6.7% and 81.3% GFAP-positive cells, respectively. Of the 10(4) cells studied, 91.6%, 79.1% and 40.8% were fibronectin-positive for glioblastoma multiforme [12-18], oligodendroglioma [12-10] and fetal brain [CHII] cells, respectively. Two fibroblast lines had 33.5% and 43.1% of the cells expressing fibronectin. The validity of these results was confirmed by staining for GFAP and fibronectin using peroxidase-antiperoxidase and immunofluorescence microscopy. Using low angle forward light scatter to estimate cell size and gating techniques it was found that GFAP-positive CHII and anaplastic astrocytoma cells were generally larger than GFAP-negative cells of the same type. No correlation between cell size and fibronectin expression was found for glioblastoma [12-18] cells. These results demonstrate the validity of the described methods and illustrate some specific applications and the potential value of flow cytometry to neurooncology.

Brain Neoplasms↗

PubMedDate not supplied

[Fine-needle puncture-aspiration in the measurement of the DNA content by flow cytometry in head and neck tumors. Methodology and applications].

Fine needle aspiration was used to obtain samples from 15 patients with head and neck tumors for posterior analysis of their DNA content by flow cytometry. This technique afforded rapid and readily reproducible high quality histograms. A review was made of the malignancy criteria according to variations in DNA content. Good patient tolerance made it possible to apply the technique repeatedly to obtain samples for DNA analysis by flow cytometry, a method of great interest in monitoring response to different treatment protocols (chemotherapy, radiotherapy).

Biopsy, Needle↗

PubMed2005

[Quantification of minimal residual disease by multiparameter flow cytometry in acute myeloid leukemia. From diagnosis to prognosis].

Today, multiparameter flow cytometry allows the simultaneous assessment of up to five surface and cytoplasmic antigens in different cell populations from peripheral blood and bone marrow samples. Due to the high efficiency of modern flow cytometers a comprehensive characterization is achieved within a short time. In the setting of diagnosing acute myeloid leukemias (AML) this method is used not only to subclassify AML and separate it from acute lymphoblastic and biphenotypic leukemias but also for the identification of leukemia-associated aberrant immunophenotypes (LAIPs). Since these LAIPs are defined individually for each patient, leukemic bone marrow cells can be detected during the course of treatment using the LAIP allowing the quantification of minimal residual disease (MRD) which is not detectable by cytomorphology. Due to its close correlation with the course of the disease and with the risk of relapse the MRD represents an important prognostic parameter which is increasingly used for stratification of therapy in clinical trials.

Bone Marrow Cells↗

PubMed1990

Comparison of morphometry and DNA flow cytometry with standard prognostic factors in bladder cancer.

In 83 bladder cancer patients with adequate follow-up (mean 13 years, range 9-22) the prognostic value of morphometric, DNA flow cytometric and clinical parameters was assessed. Paraffin embedded material was used in flow cytometry. Univariate life-table analysis showed the statistically significant relation of clinical stage, histological grade, mean nuclear area, the Standard Deviation (SD) of nuclear area, mean maximal nuclear diameter, mean nuclear perimeter and the volume corrected mitotic index (M/V index) to survival when bladder cancer deaths alone were used in the analysis. The recurrence of bladder cancer could be predicted with the M/V index. Survival analysis with Cox's regression model pointed to primary tumour clinical stage as the most important prognostic factor of crude survival. Histologically, grade and the SD of nuclear area were the best prognostic factors. Primary tumour stage and histological grade were the best predictors of death from bladder cancer. In Cox's model, histoquantitative methods are almost as good as clinical staging in predicting prognosis. DNA flow cytometry of paraffin embedded material offered no advantage over clinical stage, histological grade or morphology in assessing prognosis.

Adult↗

PubMed1986

DNA ploidy and proliferation characteristics of bowel polyps analysed by flow cytometry compared with cytology and histology.

56 human adenomatous bowel polyps and 44 samples of macroscopically unchanged bowel mucosa taken at a distance of 2-10 cm from the polyp were investigated by histology, cytology, and flow cytometry in respect to their dysplasia grade and DNA distributions. 30 human adenomatous polyps of the same localization were investigated by histology and flow cytometry; in 14 cases in addition to the DNA-patterns per se their changes mediated by heparin, a polyanion, were recorded. Histological and cytological investigations revealed mild dysplasia in 25 cases, moderate dysplasia in 43 cases and severe dysplasia in 18 cases. DNA aneuploidy was correlated with the histological finding of mild dysplasia three times, of moderate dysplasia twice and severe dysplasia five times. All other cases, also macroscopically unchanged bowel mucosa revealed DNA diploidy. In these diploid cases, cell cycle stage analysis yielded a higher proportion of cells in S-phase and G2M phase in adenomas with severe dysplasia in comparison to adenomas with moderate and mild dysplasia (P less than 0.05). Macroscopically unchanged bowel mucosa showed about the same values as adenomas with mild dysplasia. The heparin mediated increase in fluorescence intensity is different for adenomas with mild and moderate dysplasia, and attributed to the latter a higher proliferative activity (P less than 0.05).

Adenoma↗

PubMed1979

A system for storage and retrieval of individual cells following flow cytometry.

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.

Cytological Techniques↗

PubMed2001

Prognostic value of DNA flow cytometry in sympathoadrenal paragangliomas.

OBJECTIVE: To determine whether ploidy patterns are related to prognosis in sympathoadrenal paragangliomas (SAP) using flow cytometry. STUDY DESIGN: DNA flow cytometric analysis of formalin-fixed, paraffin-embedded tumor samples from 36 patients with SAP was performed. Eight cases fulfilled at least one of the following malignancy criteria: (1) extensive invasion of adjacent structures (5 cases), (2) local recurrence (3 cases), or (3) metastases (4 cases). RESULTS: Of the 36 tumors, 22 (61%) showed nondiploid patterns (12 aneuploid, 10 tetraploid). All diploid tumors were benign, while all malignant cases showed nondiploid patterns (P = .0131). The differences between diploid and aneuploid tumors and between diploid and tetraploid tumors, with regard to the malignancy of the disease, were statistically significant (P = .03311 and .01976, respectively). Only one malignant tumor had a DNA index < 1.75 (P = .00259). CONCLUSION: Anomalous DNA ploidy patterns are frequent in SAP, without necessarily implying malignancy. However, diploid DNA content may be a marker of a good prognosis. The likelihood of malignancy is greater in the tetraploid and peritetraploid range.

Adolescent↗

PubMed1994

Detection and quantitation of cell-cell electrofusion products by flow cytometry.

A cytometric method for detecting and quantitating hybrid cells that resulted from cell-cell electrofusion was developed. Cells from two different lines and two vital fluorescent dyes were used in conjunction with a flow cytometer to demonstrate the method. Each dye was used to stain one cell type prior to electrofusion. Hybrid cells exhibited dual fluorescence while unfused cells retained their single fluorescence. Flow cytometry was used to detect dual fluorescing hybrid cells that resulted from electrofusion. Fluorescent microscopy was used to confirm that 92% of the cytometrically detected cells were hybrids. Flow cytometry was also used to quantitatively show differences in hybrid yields for samples fused using different electrical conditions. These results demonstrate that the method can be used for detection and quantitation of electrofusion products. The methodologies presented are not specific to the cell types used; they can be adapted for use with other cell types.

Animals↗

PubMed1995

Comparison of DNA content in non-Hodgkin's lymphoma as measured by flow cytometry and cytogenetics.

Specific cytogenetic changes such as t(14;18) and t(8;14) are associated with specific histologic subtypes of non-Hodgkin's lymphoma (NHL) and may predict disease outcome. Nonspecific cytogenetic changes include other structural rearrangements or numerical changes such as monosomies and trisomies, which may cause changes in total cellular DNA content. In many solid tumors, the presence of abnormal DNA content may be predictive of clinical behavior. NHL biopsies, however, contain normal (diploid) as well as abnormal cells, and DNA changes in the peridiploid range are detectable by cytogenetic analysis, but not consistently by flow cytometry. In the present study, we performed flow cytometric and cytogenetic analysis of DNA on biopsies from 129 patients with non-Hodgkin's lymphoma (NHL). Cytogenetic studies were successful on 88 (68%) of the samples. There was 55% concordance between flow cytometric and cytogenetic techniques in detecting aneuploid DNA content, with the majority of discrepancies occurring in the peridiploid range. We also detected six samples which were aneuploid by flow cytometry, but diploid by cytogenetics. We suggest that a reasonable approach to determine DNA content, as it relates to prediction of outcome in NHL, would be to combine data from both of these techniques and analyze the results in terms of ranges of DNA rather than by categorizing as diploid versus aneuploid.

Aneuploidy↗

PubMed1992

Adhesion of subsets of human blood mononuclear cells to endothelial cells in vitro, as quantified by flow cytometry.

Binding of leucocytes to endothelial cells (EC) is essential as an initial step in inflammatory responses. We present a rapid, non-radioactive method to measure adhesion of human lymphoid cells to EC using flow cytometry. Freshly isolated peripheral blood mononuclear cells (PBMC) were allowed to adhere to EC grown in 24-well plates. Non-adhering cells were removed, after which adhering cells and EC were dissociated using trypsin/EDTA. These samples were subsequently analysed by flow cytometry, using scatter properties to distinguish between adhering cells and EC. The ratio of the number of adhering leucocytes and EC was calculated to quantify adhesion. Results of the flow cytometric adhesion assay were comparable to those obtained with a conventional adhesion assay using chromium-labelled cells. We additionally show that by using the flow cytometric adhesion assay, adhesion of lymphocytes and monocytes present within the adhering PBMC can be quantified simultaneously. As a model, the contribution of LFA-1 (CD11a/CD18) and ICAM-1 (CD54) in adhesion of PBMC to EC was studied. It was found that adhesion of lymphocytes and monocytes is regulated differently by phorbol ester and that the relative contribution of LFA-1 and ICAM-1 differs for both cell types.

Antigens, CD↗

PubMedDate not supplied

Population dynamics of a continuous fermentation of recombinant Saccharomyces cerevisiae using flow cytometry.

The plasmid instability of genetically modified microorganisms during prolonged bioreactor operations is one of the major problems to be overcome in the production of recombinant proteins. The use of flow cytometry to monitor a fermentation process with recombinant cells in a CSTR is reported here. This technique has been applied to determine the fraction of plasmid-bearing cells (P+) of a recombinant Saccharomyces cerevisiae strain harboring the EXG1 gene in a continuous stirred tank bioreactor with a working volume of 2 L. The different levels in the expression of the EXG1 gene, which encodes the enzyme exo-beta-glucanase, were used to determine the P+ fraction. Other parameters such as viability, cellular protein, cell size and structure were also monitored using flow cytometry. This technique has two main advantages over the conventional method of determining the P+ fraction (plating in selective and non-selective solid media): (a) it takes a very short period of time to obtain a measurement that provides multiple parametric information; and (b) it is more representative of the bioreactor cell population since it can analyze thousands of cells in the same sample. A continuous operation (432 h) with the recombinant strain in a CSTR was carried out to test the application of this technique. Measurements of cellular exo-beta-glucanase activity and cellular protein content closely correlates to the measured fraction of plasmid-containing cells in the population. Moreover, the standard deviation of the fraction of P+ cells determined using this technique was very low (about 2%). Recombinant protein production also increased the size of the yeast cells, whereas the recombinant cells also had a more complex internal structure than the non-recombinant host strain.

Bioreactors↗

PubMed1997

Multiparameter flow cytometry as a tool to evaluate antipsoriatic therapy.

Objective comparison of different antipsoriatic therapies requires quantitative assessment of disease severity. However, clinical assessment with the widely used Psoriasis Area and Severity Index (PASI) introduces inaccuracy. An alternative is the quantitative analysis of different epidermal cell parameters using multiparameter flow cytometry. Our aim in the present study was to compare the clinical and flow cytometric approach to monitor disease activity and to evaluate antipsoriatic efficacy. Clinical scores for erythema, induration and scaling were assessed and biopsies for flow cytometric analysis were obtained from the psoriatic plaques of 89 patients before and after treatment with different therapeutic regimens consisting of vitamin D3 analogues and corticosteroids. In total, 219 epidermal cell suspensions were analysed using triple-labelling, with the simultaneous staining of markers for epidermal proliferation (DNA dye TO-PRO-3), differentiation (antikeratin 10), and inflammation (antivimentin). Correlation analysis was performed on 166 paired values obtained from 83 patients. A highly significant correlation was observed between erythema and the percentage of vimentin-positive cells, between scaling and the percentage of keratin 10 positive keratinocytes, and between induration and the number of basal keratinocytes in the S- and G2M phase, when all 166 biopsies were assessed. The correlation remained in the same range if the analysis was restricted to the 83 pretreatment biopsies. In contrast to the clinical scores, the flow cytometric analysis permitted a clear separation between the antiproliferative and anti-inflammatory or keratinization-enhancing effects of antipsoriatic treatment. The vitamin D3 analogues proved to exert a mainly antiproliferative effect. The combination of calcipotriol and a topical corticosteroid improved all cell biological markers substantially, and clobetasol monotherapy had a powerful effect on these markers. In conclusion, multiparameter flow cytometry has been shown to be a sensitive tool to evaluate the growth inhibiting, anti-inflammatory and keratinization-enhancing effects of antipsoriatic therapies.

Biomarkers↗

PubMed1997

Comparison of DNA flow cytometry and fluorescence in situ hybridization with a set of 10 chromosome-specific DNA probes in four head and neck carcinomas.

Four squamous cell carcinomas of the oral cavity were simultaneously analysed by DNA flow cytometry (FCM) and fluorescence in situ hybridization (FISH) with alpha-telomeric DNA probes specific for chromosomes 1, 2, 3, 6, 7, 8, 10, 12, 17, and X. Nuclei with 2 hybridization signals for most chromosomes were in agreement with flow cytometric DNA content in a diploid tumor, but a monosomy 8 for half of the cells indicated a tumor clone with an aberrant chromosome 8. Aberrations of virtually all chromosomes were found in 3 aneuploid tumors. Comparing the relative numbers of disomal and aneusomal nuclei with the corresponding proportions of DNA diploid and aneuploid sample cells provided evidence that the flow cytometrically diploid cell population of an aneuploid tumor consisted of both karyotypically normal and abnormal cells. Although there was a high degree of concordance between aberrant DNA content and magnitude of chromosomal aberration, discrepancies between both methods suggested a notable clonal heterogeneity in aneuploid tumor cell populations. These results demonstrate that a simultaneous application of DNA flow cytometry contributes to broaden the utility of interphase cytogenetics.

Aneuploidy↗