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Results for “Flow Cytometry”

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PubMedDate not supplied

Evaluation of methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as reagents in flow cytometry of hematopoietic progenitor cells.

BACKGROUND: Flow cytometry is used to quantitate CD34+ hematopoietic progenitor cells for transplantation. The present study evaluates methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as flow cytometry reagents. STUDY DESIGN AND METHODS: The human myeloid leukemic cell lines KG 1 a (CD34+) and K562 (CD34-) were grown in culture under standard conditions and then prepared on ficoll gradients of different densities to determine which gave the component that was most reproducible. After ficoll preparation, the cells were frozen in standard cryopreservation media and four methods of thawing were examined. Determination of the method that gave the cell component that was most reproducible was based on viability, percentage of cell recovery, and maintenance of CD34 antigenicity status. RESULTS: Ficoll gradient preparation improved the ease of flow cytometry analysis when original viability was low, and it produced a more uniform cell population. However, it resulted in significant cell loss for both cell lines. While the cell recovery for K562 cells was not significantly different with any of the densities of ficoll, recovery was significantly better for KG 1 a cells with ficoll at a specific gravity of 1.077. Of the thawing methods examined, all three that involved a rapid thaw at 37 degrees C were statistically equivalent to each other and were better than thawing at 4 degrees C. CONCLUSION: With a standardized method of preparing cell lines as reagents for quality control purposes, data comparison among cell processing laboratories may more readily be initiated. Such cell lines could also be useful as a teaching tool for flow cytometry and in proficiency testing.

Acute Disease↗

PubMed1980

Detection of human ADCC activity using automated flow cytometry.

Using automated flow cytometry techniques we have developed a rapid assay to measure human antibody-dependent cell-mediated cytotoxicity (ADCC). By staining cell cultures for DNA content, chick red blood cell targets can be readily distinguished from human effector cells and ADCC can be measured by changes in their relative proportions. The sensitivity and rapidly of the assay is shown by the finding that at an effector to target ratio of only 2:1, 42% killing can be detected after 1 h incubation.

Antibodies↗

PubMed1993

Detection of ploidy in colorectal tumors. A comparison between flow cytometry and cytogenetics.

Parallel investigations of ploidy by flow cytometry and cytogenetics were performed in 20 colorectal tumors. Flow cytometry detected an aneuploidy in 13 tumors with DNA indices ranging from 1.13 to 2.21. The other samples exhibited an apparent diploid DNA content. Cytogenetic analyses revealed an abnormal chromosome count in 14 cases and a frequent implication of numerical or structural changes in chromosomes 1, 7, 12, 17, 18, and 20. DNA content evaluated by both techniques was generally concordant with minor discrepancies not exceeding 10%. In six cases, cytogenetics failed to find the cell populations detected by flow cytometry. These results indicate that flow cytometry and cytogenetics are reliable and complementary techniques, particularly in near-diploid tumors, where flow cytometry has some difficulties in detecting variations from diploidy below to 8%.

Adenocarcinoma↗

PubMed1999

Plug flow cytometry: An automated coupling device for rapid sequential flow cytometric sample analysis.

BACKGROUND: The tools for high throughput flow cytometry have been limited in part because of the requirement that the samples must flow under pressure. We describe a simple system for sampling repetitively from an open vessel. METHODS: Under computer control, the sample is loaded into a sample loop in a reciprocating eight-way valve by the action of a syringe. When the valve position is switched, the plug of sample in the sample loop is transported to the flow cytometer by a pressure-driven fluid line. By coupling the plug-forming capability to a second multi-port valve, samples can be delivered sequentially from separate vessels. RESULTS: The valve is able to deliver samples at rates ranging up to about 9 samples per minute. Each plug of sample has uniform delivery characteristics with a reproducible coefficient of variation (CV). Even at the highest sampling rate, carryover between samples is limited. CONCLUSIONS: Plug-flow flow cytometry has the potential to automate the delivery of small samples from unpressurized sources at rates compatible with many screening and assay applications.

Animals↗

PubMed2000

Use of flow cytometry to quantify mouse gastric epithelial cell populations.

Flow cytometry provides the opportunity to quantify cell populations within a total cell suspension. The quality of flow cytometry is strongly dependent on the isolation of intact viable cells. However, techniques to isolate mouse gastric cells for flow cytometry have not been evaluated. The objective of this study was to develop an effective method for isolating intact viable cells from mouse gastric tissue for flow cytometry. Cells were isolated from mouse stomach and spleen by either enzymatic separation or mechanical dissociation. A Percoll density gradient was used to separate viable cells from cellular debris. Cells were labeled with fluorescently tagged ligand or antibody and analyzed by flow cytometry. According to propidium iodide staining, there was a higher percentage of viable cells after mechanical dissociation (10-20%) compared to enzymatic separation (1%). After Percoll centrifugation there was a further increase in the percent of viable cells (50-80%). Gastrin (G), somatostatin (D), and parietal cells represented 0.6%, 3%, and 8% of the total epithelial cell population, respectively. T and B lymphocytes made up 4% and 2% in the gastric mucosa. Dissociated splenocytes were comprised of 20% T cells and 14% B cells. The ability to reliably resolve a cellular fraction that comprises only 0.6% of the input marks a substantial improvement over morphometric methods. Therefore, mechanical dissociation of the stomach followed by use of a Percoll gradient is the preferred method for isolating viable intact gastric epithelial cells for flow cytometry.

Animals↗

PubMed2004

Flow cytometry: a new method to investigate the properties of water-in-oil-in-water emulsions.

We present a new and facile method to evaluate w/o/w emulsions containing fluorescent markers by flow cytometry. Flow cytometry allows simultaneous measurement of w/o/w emulsion droplets "marked" with a fluorescent marker or "blank" without the need for complicated sample preparation. The yield of preparation of the w/o/w emulsion and the release rate of the fluorescent marker FITC-BSA were investigated by this new method. The release fraction (after 24 h) of FITC-BSA from the w/o/w emulsion decreased with increasing concentration of FITC-BSA inside the internal phase, just like the release fraction of NaCl as marker from the w/o/w emulsion. Flow cytometry results show that the yield and release behavior in w/o/w emulsions are in agreement with results reported by more complicated methods.

Journal Article↗

PubMed1986

Detection of central nervous system relapse in acute leukemia by multiparameter flow cytometry of DNA, RNA, and CALLA.

DNA/RNA flow cytometry studies were performed on the spinal fluid samples of thirty patients with acute leukemia or lymphoma at the time of clinical central nervous system relapse, and compared with similar studies of 56 patients (98 specimens) who had leukemia in remission with no evidence of CNS disease. Twelve of the 30 patients with CNS involvement had cells with abnormal DNA content in the spinal fluid (40%); the remaining eighteen had cells with diploid DNA content. In the group of 18 with diploid DNA, 10 had other abnormalities detected by flow cytometry. These included eight patients with acute leukemia who had cells with high RNA content, and two patients with non-Hodgkin's lymphoma who had markedly increased proliferation. Of the 22 patients studied by conventional cytology nine were negative for malignant cells, and in eight of these patients flow cytometry studies of DNA/RNA demonstrated abnormalities. Common ALL antigen was demonstrated by flow cytometry in three out of five cases studied. Thus, abnormal DNA content, increased RNA content, increased proliferation and/or expression of the cell surface antigen CALLA identified CNS relapse by flow cytometry in 22 of 30 patients with acute leukemia or lymphoma. The technique appears to be at least as sensitive as conventional cytology and identifies CNS relapse in some patients with negative cytology.

Antigens, Neoplasm↗

PubMed1981

Responses of synchronized HeLa cells to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate as evaluated by flow cytometry.

A flow cytometric study was carried out on the effects of tumor-promoting 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-8) M) on HeLa cells synchronized by amethopterin for DNA synthesis. Cells treated with TPA at the time of release from the amethopterin block showed a delayed passage through S phase and partially through G2 in their immediate life span as measured 24 hr after release. This late G2 delay was not observed when TPA was added to cells during late S or G2 phase. In this case, however, a direct inhibition of cells in G2 became evident as observed about 15 hr after release from block. None of these effects was caused by the nonpromoting 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate (10(-6) M). These data support observations obtained with asynchronous cultures. The TPA effects resemble those reported after X-irradiation of cell cultures.

Cell Cycle↗

PubMed2002

Flow cytometry and its application in small animal oncology.

Flow cytometry measures multiple characteristics of single cells. The use of flow cytometry in the veterinary clinical laboratory has increased considerably during the past decade. The most common applications of flow cytometry in small animal oncology are measurement of DNA content in tumours and immunophenotyping of haematopoietic malignancies. DNA ploidy and S-phase rate of various tumours in dogs have been found to be independent predictor of patient outcome. In dogs with lymphomas immunophenotyping is recommended as a part of the patient work-up. Flow cytometry has shown to be a suitable method for immunophenotyping of canine lymphomas. However, it has not become a routine technique in small animal oncology yet. This report reviews the applications of flow cytometry in small animal oncology. Besides basic principles and technical aspects, the clinical relevance of DNA-analysis and immunophenotyping are discussed.

Animals↗

PubMed2000

Migration of mononuclear cells in the modified Boyden chamber as evaluated by DNA quantification and flow cytometry.

In vitro migration of mononuclear cells in the modified Boyden chamber was evaluated using flow cytometry and DNA quantification (Hoechst 33258) of all adherent and nonadherent cells. The effects of different membrane pore sizes, cell concentrations and incubation times were studied. Pore sizes of 3 and 5 microm resulted in a reduction in the number of nonadherent cells compared with a pore size of 8 microm. Reducing the incubation time from 60 to 40 and 20 min resulted in too few migrating monocytes for analysis by flow cytometry. Flow cytometry showed that both monocytes and lymphocytes migrated and adhered to the membrane when using peripheral blood mononuclear cells (PBMC) to study monocyte migration. Migration of lymphocytes under these conditions is a novel observation. A substantial number of migrated cells could be identified by flow cytometry and quantified by DNA measurement as nonadherent below the membrane. Samples of synovial fluid (n = 49) and plasma (n = 133) as chemoattractants analysed in triplicate resulted in mean coefficient of variation (CV) values of 11 and 9%, respectively. Variation from assay to assay on the same day, using N-formyl-methionyl-leucyl-phenylanine (fMLP) 10(-7) M as chemoattractant resulted in a CV of 13%. Day-to-day variation, using fMLP 10(-7) M as chemoattractant and the same well on three different days, resulted in a CV of 21%. These results were obtained using a pore size of 5 microm, a PBMC concentration of 3 x 10(6)/ml and 60 min of incubation. The combination of DNA quantification and flow cytometry thus allowed characterization and quantification of subsets of migrating adherent as well as nonadherent mononuclear cells.

Antigens, CD↗

PubMed1999

[Flow cytometry in hematologic diagnosis].

The paper presents flow cytometry method in haematology diagnostics. Beside to the estimation of blast cells phenotype in acute leukaemia, flow cytometry allows to evaluate the presence of minimal residual disease (MRD) and multi drug resistance protein (MDR). Flow cytometry plays a crucial role in the estimation of DNA profile in cancer cells and evaluation of stem cells. Repeatedly flow cytometry is used to estimate platelets and reticulocytes.

DNA, Neoplasm↗

PubMed2005

Telomere length on bladder washing samples from patients with bladder cancer correlates with tumor characteristics flow cytometry method for quantitative fluorescence in situ hybridization (flow-FISH technique).

OBJECTIVE: The purpose of the present study was to evaluate the length of telomeres in patients with bladder cancer using a quantitative flow cytometry (flow-FISH) technique. METHODS: Bladder washing samples from 51 patients with bladder cancer were obtained immediately before transurethral resection. The average length of telomere repeats was measured by flow-FISH, as previously reported. Results were expressed in molecular equivalents of soluble fluorochrome (MESF) units. RESULTS: Bladder washing specimens provided adequate cell numbers for flow-FISH in 49 cases. The TEL means were 1014.71, 2343.36, 5567 and 18267.57 for Ta, T1, T2 and T3/4 tumors, respectively. Regarding grade it was obtained a mean MESF value of 1379.46, 3391.29 and 15925.11 for G1, G2 and G3, respectively. ANOVA demonstrated statistically significant differences in stage (p: 0.014) and tumor grades (p: 0.012). In relation to ploidy, we found a mean MESF value of 2701.37 and 16085.44 MESF units for diploid and aneuploid cells, respectively. Significant difference (p: 0.003) was observed between both groups. CONCLUSION: To date, this is the first report wherein telomere length was measured using flow-FISH method in exfoliated cells in urine from patients with bladder cancer. Further investigations are required to demonstrate whether flow-FISH technique might be considered as a tumor marker of bladder cancer.

Analysis of Variance↗

PubMedDate not supplied

The role of flow cytometry in the diagnosis of lymphoma: a critical analysis.

Flow cytometry, now used routinely to aid in the classification of leukemias, is increasingly being evaluated as a rapid technique for determination of surface antigens on the cells teased from lymph nodes and other masses with suspected lymphoma. The present study reviews biopsy specimens from patients examined during a two year period which were sent for flow cytometry with a diagnosis of suspected lymphoma. Sixteen of 25 samples (64 percent) produced cell suspensions of sufficient quantity and quality to be diagnostically helpful. Results showed that in 9/16 (56 percent) the diagnosis of lymphoma or cancer could be suspected by flow cytometry alone, while 4/16 were consistent with the final tissue diagnosis of normal or reactive hyperplasia. Three samples that came from patients who had morphologic evidence of malignant disease on biopsy (two Hodgkin's disease and one large cell lymphoma) had flow cytometry results that were interpreted as normal. Flow cytometry is rapid and appears to be virtually diagnostic of non-Hodgkin's lymphoma when a majority of cells are B cells with an abnormal kappa/lambda ratio (> 4.0 or < 0.25). Nonhematologic malignancy can be suspected if less than 75 percent of the cells show CD45 (common leukocyte antigen). Hodgkin's disease cannot be detected by flow cytometry as it is currently used, and as many as 15 percent (1/6 in this study) of lymphomas may show normal results. It is extremely helpful when the biopsy sample actually contains the cells of interest in large proportion. Loss of architectural relationships in the course of processing specimens for flow cytometry is a major disadvantage when small foci of lymphoma or tumor cells exist together with large amounts of stroma or normal lymphocytes.

Antigens, CD↗

PubMed2006

[Scanning aneugen and clastogen by micronuclei analysis using flow cytometry].

OBJECTIVE: To explore a flow cytometry (FCM)-based method for discriminating aneugen- or clastogen-induced micronuclei. METHODS: Cells were stained with anti-CD71-FITC and PI, and the PI fluorescent signal intensity of micronucleated reticulocyte (MN-RET) in the peripheral blood of NIH mouse treated with COL or CP was detected by flow cytometry. RESULTS: The ratio of the median of the intensity of MN-RET fluorescent signals to that of nucleated cell was low in the cyclophosphamide treated mouse, while the median was high in the colchicine treated mouse. CONCLUSION: The flow cytometry-based micronucleus assay can be used to discriminate primarily smaller MN induced by the clastogen exposure from the larger MN induced by an aneugen.

Animals↗

PubMedDate not supplied

A web-based database for diagnosis of haematologic neoplasms using immunophenotyping by flow cytometry.

The interpretation of immunophenotyping results by flow cytometry involves pattern recognition of different haematologic neoplasms that may have similar immunologic marker patterns. The numerous markers available in the flow cytometry laboratory make these patterns difficult to remember, especially for those of uncommon neoplasms. We describe the design and implementation of a Web-based database for diagnosis of haematologic neoplasms using results of immunophenotyping by flow cytometry. This database aims to assist pathology and haematology residents in interpreting flow cytometry data, and is designed to reach a wide base of users who use a variety of browsers on different computer platforms. Five modules are developed in this comprehensive program: (a) differential diagnosis: to generate a list of differential diagnoses that closely match the marker results in a given case; (b) display of disorders: typical results of markers for each disorder; (c) display of markers: relevant information of each immunologic marker; (d) display of archived cases for a disorder: marker results of cases previously diagnosed for a disorder; and (e) display of summary for archived cases: summary of marker results of all the archived cases for each disorder. Our experience with this Web-based database in teaching pathology residents has been very encouraging. Since the World Wide Web is increasingly more accessible to computer users, it has become an ideal medium for distribution of clinical decision-support software.

Antigens, CD↗

PubMed2003

Flow cytometry in lymphoma diagnosis and prognosis: useful?

Flow cytometry has become an important tool in the diagnosis of mature lymphoid neoplasms and the determination of prognosis in selected cases. The advantages of flow cytometry are based largely on its ability to analyse, rapidly and simultaneously, multiple cell properties in a quantitative manner. Flow cytometric immunophenotyping is useful in diagnosing lymphoma under the WHO classification system, where lymphoid neoplasms are separated into distinct clinical entities based upon morphology, immunophenotype, genetic abnormalities and clinical features. Flow cytometry can quantify the expression of proteins associated with a good or poor prognosis, detect multidrug resistance, and measure cell proliferation, making it useful in measuring prognostic indicators in lymphoid neoplasia. The unique attributes of flow cytometry therefore make it a valuable technique in the diagnosis and classification of lymphomas as well as the assessment of prognostic markers in lymphoma patients.

Flow Cytometry↗

PubMed1989

Measurement variability in DNA flow cytometry of replicate samples.

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.

Cell Line↗

PubMed1994

A comparative study of frozen-section immunoperoxidase and flow cytometry for immunophenotypic analysis of lymph node biopsies.

Immunophenotyping by flow cytometry and frozen-section immunoperoxidase was compared on 21 consecutive lymph node biopsy specimens, of which a diagnosis of lymphoma was made for 11 specimens. Samples for flow cytometry were obtained by a fine-needle aspiration technique. Concordance between frozen-section immunoperoxidase and flow cytometry for all routine markers on all specimens ranged from 76 to 100%. In general, B-cell markers showed poorer concordance than T-cell markers, with kappa and lambda light chains having the poorest concordance, at 76% each. Flow cytometry was significantly more sensitive (90 versus 30%; P < 0.006) and had a significantly higher negative predictive value (100 versus 63%; P < 0.006) than frozen-section immunoperoxidase for demonstrating light-chain restriction. There was no significant difference in the specificities (100 versus 91%) or positive predictive values (100% each) between the two methods. Both methods demonstrated characteristic immunophenotypes for intermediate cell lymphomas, small lymphocytic lymphomas, and T-cell lymphoblastic lymphomas. Frozen-section immunoperoxidase and flow cytometry appear to be significantly concordant methods for immunophenotypic analysis of lymph node biopsies. Light-chain restriction is more readily demonstrated by flow cytometry than frozen-section immunoperoxidase. We believe that ex vivo fine-needle aspiration is a simple and reliable method of obtaining cell suspensions of lymph nodes for flow cytometry.

Flow Cytometry↗