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Results for “Flow Cytometry”

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PubMed2002

Ability of bivariate cytokeratin and deoxyribonucleic acid flow cytometry to determine the biologic aggressiveness of resectable non-small cell lung cancer.

OBJECTIVE: The purpose of this study was to clarify the significance of bivariate cytokeratin and DNA flow cytometry for analysis of the biologic aggressiveness of resectable non-small cell lung cancer. METHODS: In 92 patients who underwent curative operations, the DNA ploidy status and S-phase fractions of the cancer cell populations inside the tumors were analyzed by a cytokeratin gating technique with paraffin-embedded specimens and were correlated with the surgical results. RESULTS: Ninety tumors yielded assessable DNA histograms. DNA diploidy was detected in 25 tumors with a mean S-phase fraction of 14.3% +/- 4.7%, and DNA aneuploidy was detected in 65 tumors with a mean S-phase fraction of 15.1% +/- 7.1%. The 5-year overall and recurrence-free survivals were 73.3% and 70.3%, respectively. Multivariate analysis showed that only TNM staging was a prognostic factor after surgery. There was a negative correlation between the logarithms of S-phase fraction and the disease-free interval for 22 patients with proven recurrence (P =.006). The tumors with high S-phase fractions recurred more rapidly than did those with low S-phase fractions. CONCLUSION: In a bivariate analysis of cytokeratin and DNA flow cytometry in resectable non-small cell lung cancer, the S-phase fraction appeared to be correlated with the disease-free interval. However, DNA ploidy and S-phase fraction were not predictive of either recurrence or survival after operation. Thus DNA flow cytometry may be of limited use for the analysis of the biologic aggressiveness of lung cancer.

Adult↗

PubMed1995

Cytogenetic and flow cytometry DNA analysis of regional heterogeneity in a low grade human glioma.

This study combined flow cytometry with standard cytogenetic analysis of first division cells to evaluate regional heterogeneity in 38 spatially mapped regions of a low grade human oligoastrocytoma. Histologically, the tumor was relatively homogeneous. In contrast, flow cytometry and cytogenetic analyses identified variable percentages of near-diploid (ND; 35 to 57 chromosomes/metaphase) and near-tetraploid (81-103 chromosomes/metaphase) populations. The largest proportion of cells in the ND population was 46,XY with normal Giemsa bands; however, four karyotypically unrelated ND clones also were identified. The development of these clonal populations centered around a region in which more than 50% of the cells contained nonclonal abnormalities and which demonstrated more histological pleomorphism than any other region. The frequency of the nonclonal karyotypes suggested that this region was genetically unstable. Three of the clonal ND populations resided in small, spatially discreet areas of the tumor. The largest and the most widely distributed clonal population, 47,XY,+7, underwent further evolution to give rise to seven additional sidelines. This investigation demonstrates that low grade gliomas have areas of genetic instability capable of generating mutant cells with the capacity to proliferate and to form cellular foci. As a result, multiple, spatially distinct clonal populations can exist in low grade gliomas, some of which are capable of further cytogenetic evolution and clonal expansion, resulting in tumor progression.

Adult↗

PubMed1989

[Simultaneous double fluorescence flow cytometry of lysed whole blood for prenatal diagnosis of combined immunodeficiency].

Simultaneous two-color flow cytometry of lysed whole blood allows the collection of maximal information from minimal blood volumes. This method was used for prenatal diagnosis of severe combined immunodeficiency by analysis of fetal blood. The data demonstrate that flow cytometry of lysed whole blood provides a reliable tool for prenatal diagnosis of certain immunodeficiencies. Moreover, the method described seems highly suitable for immunological monitoring of preterm infants and newborns. To enable the diagnosis of more subtle immunodeficiency states in these patients, however, valid normal values for all investigated parameters need to be defined first for the respective age/weight groups.

Female↗

PubMed1999

[Detection of multiple myeloma cells using multicolor immunofluorescence and flow cytometry].

The advent of new therapeutic approaches to multiple myeloma made necessary the introduction of novel methods for detection of minimal residual disease. Among others approaches residual disease can be detected by the immunofluorescence using flow cytometry. We have examined the co-expression of CD19, CD38, CD45, CD54, CD56, and CD138 molecules in cells of peripheral blood and bone marrow aspirates in patients with multiple myeloma by 3-color flow cytometry. For the detection and characterization of multiple myeloma cells, combinations of following antibodies were used: anti-CD19 FITC, anti-CD38 FITC, anti-CD38 PE, anti-CD54 FITC, anti-CD56 PE-Cy5, anti-CD45 PE, anti-CD45 PE-Cy5 (Immunotech) and anti CD138 PE (Serotec). The samples were analyzed using EPICS XL (Coulter) flow cytometer, and the analysis was based on at least 10,000 events. Samples from 17 patients were analyzed. The percentage of multiple myeloma cells ranged between 0.3% and 54.2% in bone marrow aspirates and between 0.0 and 11.8% in periferal blood. The expression of CD138, CD38, CD54 and CD56 molecules was found in 100%, 100%, 85% and 68% of examined cases, respectively. In our opinion, multiple myeloma cells are best characterized by following combinations of antibodies: CD38 FITC/CD138 PE/CD45 PE-Cy5, CD54 FITC/CD138 PE/CD56 PE-Cy5 or CD54 FITC/CD38 PE/CD 56 PE-Cy5. The identification of a malignant clone is the first and the most important step in the characterization of the disease, determination of its prognosis and the detection of residual disease after treatment. Three-color flow cytometry represents a method which can meet these goals.

Antigens, CD↗

PubMed1991

[Use of flow cytometry in the study of a non-specific immunodeficiency in children. A case].

Using flow cytometry, we explored a case of nonspecific immunodeficiency in a seven month old girl with repeated infections. This method showed evidence of granulocyte phagocytosis and oxidative metabolism abnormalities suggesting the diagnosis of a variant form of chronic granulomatous disease (CGD). Findings also showed that flow cytometry can be useful to study phagocytic cells during the neonatal period as it allows rapid multiparametric analysis with a very small amount of blood.

Complement C3b Inactivator Proteins↗

PubMed2002

Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA.

To establish standard methods for measuring anti-ErbB-2 antibody, a flow cytometric and an enzyme-linked immunosorbent assay (ELISA) were developed and compared. In the flow cytometric assay, the antibody was measured by binding to human breast cancer cell line SKBR3 and the result expressed as mean channel fluorescence (MCF). In ELISA, the antibody was measured by binding to a recombinant, secreted human ErbB-2 containing the N-terminal 505 amino acids of ErbB-2 fused to myc and His tags (secE2/myc/His or secE2) and the result expressed as O.D.(405). A mouse anti-human ErbB-2 mAb 9G6 was used as the standard. Using flow cytometry, MCF of 9G6 binding increased with linearity between 0.6 and 10 microg/ml. Using ELISA, O.D.(405) increased with linearity between 0.015 and 1 microg/ml, indicating greater sensitivity of ELISA. The titer of an immune mouse serum was determined to be 400 and 8000 with flow cytometric and ELISA assay, respectively, consistent with the assay sensitivity. Based on standard curves generated with 9G6, antibody concentration in the same serum sample was calculated to be approximately 230 microg/ml by ELISA and approximately 270 microg/ml by flow cytometry. Therefore, excellent concordance in antibody concentration was obtained with different assays using linear regression and properly diluted samples. This concordance was verified with multiple serum samples.

Animals↗

PubMed1989

[Measurement of DNA content and analysis of chromosome using flow cytometry].

This include review of recent paper about sensitivity and effective of radiation therapy using flow cytometry. This paper discuss the prediction of tumor response. As predictor of response in cancer. It is DNA aneuploidy, growth fraction (% S fraction), chromosome changes, and monoclonal antibody by oncogene product protein. It is possible that the protocol of cancer treatment will be determined based on prediction data from an individual cancer. Flow cytometry would be a useful tool to get a predictor of radiation, drugs response on each cancer.

Aneuploidy↗

PubMed1994

Comparison of fluorescence in situ hybridization with flow cytometry and static image analysis in ploidy analysis of paraffin-embedded prostate adenocarcinoma.

Nuclear DNA ploidy has been shown to have an important prognostic association for patients with adenocarcinoma of the prostate. Flow cytometry and static image analysis are ploidy methods that have been used in prostate carcinoma. Fluorescence in situ hybridization (FISH) using chromosome-specific probes can be used to evaluate the ploidy of interphase nuclei. In this study FISH was compared with flow cytometry and static image analysis in determining ploidy in paraffin-embedded tissue from 34 prostatic adenocarcinomas. Ploidy status using FISH was determined by enumerating centromeres of two chromosomes (8 and 12) by use of directly-labeled alpha-satellite DNA probes in isolated whole nuclei obtained by the Hedley technique. All three methods identified 11 of 34 cases as diploid and 17 of 34 cases as nondiploid (82% concordance). Six cases were discordant; two cases had discrepant results by each method. Ploidy classification as determined by FISH had an 88% concordance with ploidy classification by either flow cytometry or static image analysis. In conclusion, FISH was found to be a sensitive method of ploidy analysis in isolated paraffin-embedded nuclei from prostate adenocarcinomas. When the chromosomes commonly involved in aneuploidy have been identified in prostate adenocarcinoma, FISH has the potential to provide greater sensitivity for aneuploidy detection compared with currently available methods.

Adenocarcinoma↗

PubMed1992

[Development of a new supplementary diagnostic method for endometrial cancer by flow cytometry employing anti-endometrial cancer antibody, MSN-1 and MSN-3].

In cytology, it is often difficult to make a definite diagnosis of endometrial cancer. In order to devise a new supplementary method, using flow cytometry we analyzed the reactivities of endometrial cells with two monoclonal antibodies (MSN-1 and MSN-3), both of which are strongly reactive to endometrial cancer cells. Employing either or both antibodies in flow cytometry, we investigated the usefulness of this analysis for distinguishing endometrial cancer cells from normal endometrial cells. Results revealed a low positive rate for normal endometrium: 9.2% with MSN-1 and 5.0% with MSN-3. In contrast, for endometrial cancer the positive rate was high: 81.2% with MSN-1 and 43.8% with MSN-3. Combined analysis with both antibodies increased the positive rate for endometrial cancer to 93.7%. These results suggest that analysis by flow cytometry employing MSN-1 and MSN-3 may have a diagnostic value for endometrial cancer. We are also investigating the usefulness of two-color analysis using these antibodies.

Antibodies, Monoclonal↗

PubMedDate not supplied

[Flow cytometry estimate of s phase cell cycle and prognosis of osteosarcoma in children and adolescents].

Flow cytometry analysis has been used to measure the S phase in paraffin embedded osteosarcoma tissues of 22 children and adolescents who were surgically treated. The aim of this study was the assessment of S phase cell cycle in relation to survival, localization of tumour and histopathological grading. Our findings demonstrate the usefulness of flow cytometry determination of S phase cell cycle as a prognostic factor. Higher percentage of S phase cells predict worse prognosis.

Adolescent↗

PubMed1992

DNA ploidy and other results of DNA flow cytometry as prognostic factors in operable breast cancer: 10 year results of a randomised study.

We evaluated breast cancer specimens from 241 patients of a controlled clinical trial by means of DNA flow cytometry. We report the correlations between DNA index (DNI) and fraction of cells in S-phase (SPF) and other prognostic parameters. Both univariately and in a Cox model, the predictive power of these factors is evaluated after a follow-up of more than 10 years. There are strong correlations between DNI and SPF (P = 0.0001) and between flow cytometry parameters and clinical and histopathological factors such as axillary lymph node involvement, tumour size and histological grade. In univariate analysis DNI fails to provide prognostic information, whereas SPF turns out to be able to differentiate between patients at high and low risk for relapse and death (P = 0.002). In the multivariate Cox model, too, SPF is an important prognostic parameter with respect to patient survival (relative risk: +86%), only surpassed by nodal involvement. DNI, however, turns out to be an independent predictor of relapse free survival and distant recurrence free survival. By combination of DNI and SPF, patients can be divided into three prognostic subgroups. We conclude that data from DNA flow cytometry can be of great importance for the decision on the level of aggressiveness of adjuvant therapy for an individual patient and therefore may help to avoid overtreatment and toxicity.

Adult↗

PubMed1989

The clinical application of aspiration deoxyribonucleic acid flow cytometry to neurologically impaired men entering an electroejaculation program.

For the neurologically impaired patient electroejaculation with a rectal probe can furnish sperm for artificial insemination. However, documentation of pregnancies in this patient group has been limited. Deoxyribonucleic acid flow cytometry has been shown to be a reproducible quantitative method with which to evaluate spermatogenesis. We assessed the ability of deoxyribonucleic acid flow cytometry on testicular aspirates to predict the quality of sperm production in 13 men undergoing electroejaculation. Semen analysis was performed on antegrade and retrograde specimens. Deoxyribonucleic acid histograms from testicular aspirates were evaluated for the relative proportions of haploid, diploid and tetraploid cells. These ratios were compared to the means reported for our own series of 25 patients who at vasovasostomy had sperm intraoperatively and to the similar figures from a group of 10 accident victims. Five patients had normal testicular ploidy compartments defined as within 2 standard deviations from control means. Of the 5 patients 4 had normal sperm counts and motilities (30 per cent or greater). Two patients have contributed sperm for artificial insemination; 1 has resulted in pregnancy. Three patients had moderately abnormal (2 to 4 standard deviations) testicular ploidy classes. These patients had adequate sperm counts but low motilities (5 per cent or less). Of the 5 patients with markedly abnormal (greater than 4 standard deviations) histograms 4 remained azoospermic despite repeated attempts at electroejaculation. Our data support the use of deoxyribonucleic acid flow cytometry on testicular aspirates as a predictor of which patients might succeed and those who are unlikely to succeed in an electroejaculation program.

Adult↗

PubMed2004

Description of a flow cytometry approach based on SYBR-14 staining for the assessment of DNA content, cell cycle analysis, and sorting of living normal and neoplastic cells.

RATIONALE: This study aimed to expand the utilization of a simplified flow cytometric approach that employing SYBR-14/PI staining into broader flow cytometry applications, including (i) measurement of the DNA content; (ii) performing cell cycle analysis on mammalian cells; and (iii) sorting of live SYBR-14-stained mammalian cells based on DNA content. MATERIAL AND METHODS: Cell lines of human origin were stained with SYBR-14 and propidium iodide (PI) and assessed by a dual-color flow cytometry. Finally, sorting of living SYBR-14-stained human cell lines was performed. RESULTS: Dual staining with SYBR-14 and PI of human cells followed by flow cytometry analysis demonstrates that in addition to quality assessment, this staining could be utilized to determinate the DNA content on mammal cells. In addition, it resolves the diploid, tetraploid, and aneuploid DNA content. Furthermore, the SYBR-14-stained mammal cells were efficiently sorted based on DNA content and live cells were obtained. All these features have not been previously described with the utilization of this staining approach. CONCLUSIONS: Results of this study demonstrate that this flow cytometric approach not only allows assessment of the viability of cells, but also the DNA content of mammal cells. In addition, this approach allows one to sort viable cells stained with SYBR-14. These findings open-up unexpected and unrestricted avenues for sorting of living mammal cells and provide significant advantages over the traditionally cumbersome sorting approaches for living cells, which demand very specialized and expensive UV light sources as well as sophisticated sorting procedures.

Cell Cycle↗

PubMed1979

Identification of polymorphonuclear leukocytes in cytologic samples for flow cytometry.

Inflammatory cells are commonly present in cytologic specimens obtained for flow cytometry, and may interfere with the analysis of epithelial cells. We have found that detergent (Triton X-100) pretreatment in the two-step acridine orange staining procedure disrupts granulocyte cell membranes to yield bare nuclei; bladder epithelial and squamous cells on the other hand are quite resistant to the detergent treatment. Being deprived of their cytoplasmic RNA, the granulocytes lose red fluorescence. Moreover, the shearing forces in the cytometer extend the multisegmented granulocyte nuclei and align them in the direction of flow. Thus, they present as elongated objects in the measuring system, giving a large DNA fluorescence pulsewidth (nuclear size). These two phenomena make it possible to identify granulocytes in the recorded data, where they are discernible from the mononucleated leukocytes and from epithelial cells. By data selection the granulocytes can be excluded, rendering epithelial cell populations more amenable to analysis. This method may make it unnecessary to remove physically leukocytes from the specimen before flow cytometry; it may also provide a way to analyze the morphology of granulocyte nuclei and to assess methods to manipulate their membrane stability. Full protection from membrane disruption is accomplished by alcohol fixation, and partial protection by 20-30% serum.

Cytological Techniques↗

PubMed1996

Karyotyping of individual cells with flow cytometry.

As the study of metaphase chromosomes with flow cytometry presupposes mixing all chromosomes from many cells before analysis, important information is lost. To overcome this limitation we have developed a novel cell-oriented flow cytometric method for chromosome analysis. A flow cytometer supplied with a special device for disruption of metaphase chromosomes is the heart of the method. Cells with stained chromosomes are pressed into the disruption device and then converted in batches of chromosomes. Chromosome fluorescence and time intervals between fluorescence pulses are registered. There are large time intervals between neighboring batches because a sparse cell suspension is used and, in turn, there are small time intervals inside batches. Taking account of time intervals, it is possible to identify individual cell flow karyotypes. This highly productive method for individual cell analysis (100-200 cells/min) ensures detecting the changes in cell flow karyotypes, i.e., under- and overrepresentation of chromosomes, aberrations, and amplification of DNA.

Animals↗

PubMed2003

Studies on murine thyroiditis: new insights from organ flow cytometry.

The pathogenesis of autoimmune diseases is frequently studied in murine models, in which disease outcome is traditionally assessed by light microscopy. To determine whether digital imaging improves reliability of the histopathologic assessment, and whether flow cytometry is applicable directly on the murine thyroid, we studied 395 CBA/J mice 3 weeks after thyroglobulin immunization, and 192 nonimmunized CBA/J mice. Digital imaging significantly improved reliability of the histopathological assessment (r = 0.988, 95% confidence interval: 0.980-0.992, p < 0.0001), and flow cytometry on the murine thyroid could be performed successfully. We also found that normal thyroids contained a higher than expected number of hematopoietic cells in the interstitium. We suggest that digital imaging offers a better means of estimating disease outcome, and that flow cytometry performed at the target organ levels reflects the autoimmune pathogenesis more closely than when performed on peripheral lymphoid organs. These methods should also be applicable to other organ systems targeted by autoimmune attack, such as heart, exocrine, and other endocrine glands.

Animals↗

PubMed1977

Chromomycin A3 as a fluorescent probe for flow cytometry of human gynecologic samples.

Chemical, physical and optical properties of chromomycin A3 are examined so as to ascertain appropriate staining and analysis procedures for flow cytometry of human gynecologic samples. Fluorescence excitation and emission spectra of chromomycin A3-stained cervical cells are compared with those of chromomycin A3-stained deoxyribonucleic acid. Conditions for deoxyribonucleic acid-specific staining of cervical cells are presented, and staining specificity of cervical cells with chromomycin A3 is compared to that obtained with ethidium bromide, propidium iodide and Hoechst 33258. Also presented is a brief review of two parameter flow cytometry as a prescreening procedure for detection of cervical neoplasia. Results of flow cytometry and cell sorting are interpreted based on the deoxyribonucleic acid-specificity of chromomycin A3 staining.

Cell Nucleus↗

PubMed1985

The insulin receptor: characterization and regulation using insulin-antiinsulin antibody complexes as a probe for flow cytometry.

The primary approach for the characterization of the insulin receptor has been through the study of its interaction with 125I-labeled insulin. Recently, we demonstrated that insulin receptors can also be identified by flow cytometry using antibodies to the receptor. In the present study, we characterized the insulin receptor on human lymphoblastoid cells (IM-9) and studied its regulation using insulin and antiinsulin antibodies as a probe for flow cytometry. The mean peak fluorescence of the cells treated with insulin followed by antiinsulin serum was 30-50 U above the control value. There was a close correlation between [125I]insulin binding and peak fluorescence. Fish insulin, which has about 50% the affinity of porcine insulin for the insulin receptor but does not bind to antiinsulin antibodies, did not enhance antiinsulin antibody binding, but competed for the pork insulin-antiinsulin antibody complexes in a dose-dependent manner. Exposure of IM-9 cells to insulin or antireceptor antibodies resulted in reduction in the number of insulin receptors. Cells down-regulated with 10(-6) M insulin or a monoclonal antibody to the insulin receptor had 40% of the [125I]insulin binding of the control cells and 40-50% of the peak fluorescence when insulin-antiinsulin was the probe for the immunofluorescence studies. Cells down-regulated with human autoantibodies to the receptor had 4% [125I]insulin binding and 10% peak fluorescence. In all cases, receptors were lost proportionally from all cells, yielding a single symmetrical fluorescent peak. These date indicate that flow cytometry with insulin-antiinsulin antibody complexes provides a new approach to the measurement of insulin receptors, since it provides direct measurement of the occupied receptor.

Antibodies↗