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PubMed1994

Survival of Staphylococcus aureus in lakewater monitored by flow cytometry.

The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-IgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.

Bacterial Proteins↗

PubMed2001

Quantification of Cryptosporidium parvum oocysts in mouse fecal specimens using immunomagnetic particles and two-color flow cytometry.

Although single-color flow cytometry has been shown to be more sensitive than fluorescence microscopy for the quantification of Cryptosporidium parvum oocysts, this method has not been optimized. Monoclonal antibody OW50, specific to the cell wall of oocysts, was conjugated to superparamagnetic particles, to fluorescein isothiocyanate, and to r-phycoerythrin. The oocysts were then double stained with the fluorochrome-labeled OW50 and were placed in tubes with known numbers of highly fluorescent polystyrene beads, allowing quantification of the oocysts without dependence on acquired sample volume by flow cytometry. Data from 2-color flow cytometry using logical gating of the oocysts and beads showed a linear relationship between dilutions of a purified oocyst suspension and the mean numbers of oocysts detected (r2 = 1.00). An average of 15 purified oocysts/ml were counted in a dilution with a theoretical concentration of 12 oocysts/ml. Known numbers of purified oocysts were seeded into normal mouse fecal specimens, captured by OW50-labeled immunomagnetic particles, eluted with 5% potassium dichromate at low pH, and double stained with fluorochrome-labeled OW50. By flow cytometry, the mean recovery was 43.1% (+/-8.3%), and as few as 133 oocysts were detected. The captured and eluted oocysts were infective in neonatal BALB/c mice. This 2-color flow cytometry method, used in conjunction with the capture and elution of oocysts by and from immunomagnetic particles, provides a powerful tool for not only the quantification and purification of C. parvum oocysts from different sources but also for the characterization of oocysts in vitro and in vivo.

Animals↗

PubMed1997

Quantitative analysis and isolation of Escherichia coli O157:H7 in a food matrix using flow cytometry and cell sorting.

Flow cytometry is a potentially valuable analytical method in microbiology providing the ability to analyze rapidly large numbers of individual microorganisms by several parameters. With a flow cytometer with enhanced light scatter sensitivity and a conventionally configured sorting cytometer, a series of comparative studies to determine the ability of the two flow systems and the antibody-direct epifluorescent filter technique (Ab-DEFT) to detect and enumerate Escherichia coli O157:H7 were made. Initial experiments used culture-derived mixtures of non-pathogenic E. coli and serial dilutions of E. coli O157:H7. Subsequent studies involved analysis of enrichment cultures from ground beef inoculated with E. coli O157:H7. Comparison of flow cytometry with microscopy and plate counts produced similar results at higher concentrations in both culture mixtures and beef enrichments. At the lowest concentrations Ab-DEFT was more sensitive, however, the time required for analysis was much less with flow cytometry. With a cytometer with enhanced light scatter sensitivity designed for bacterial analysis, O157:H7 could be distinguished from E. coli strain HB101 on the basis of light scatter. This instrument also provided direct count data for selected populations. In experiments using cell sorting to isolate target organisms, the purity of fluorescent-labeled E. coli O157:H7 sorted from beef enrichment cultures and plated was not affected by the level of background organisms, as is often the case in conventional plating procedures.

Animals↗

PubMed1991

Flow cytometry: a diagnostic aid.

Flow cytometry can provide the clinician with a wealth of information on a given disease entity. It uses fluorescence and laser technology, thereby allowing characterization of a host of cell parameters and functions. Flow cytometry can be used to diagnose and prognose immunologic, infectious, and malignant disease, with minimal invasiveness. Any material within the reach of an aspiration needle is amenable to cytometric analysis. A qualified pathologist, well-trained technologist, and well-informed clinician all play crucial roles in the appropriate application of cytometric analysis of the clinical specimen. The wide variety of applications make flow cytometry a useful tool for all clinicians, regardless of practice location.

Clinical Laboratory Techniques↗

PubMed1996

Consensus protocol for the flow cytometric immunophenotyping of hematopoietic malignancies. Working Group on Flow Cytometry and Image Analysis.

Flow cytometry has become the preferred method for the lineage assignment and maturational analysis of malignant cells in acute leukemias and lymphomas. Multiparametric immunophenotyping allows the detection of aberrant antigen coexpression and the analysis of heterogeneity and clonality of malignant cells in leukemias and lymphomas. The complexity of multiparameter analysis techniques and the multitude of available monoclonal antibodies demand a standardization of protocols for the use of flow cytometry in clinical laboratories in order to achieve interlaboratory reproducibility. Therefore, the Working Group on Flow Cytometry and Image Analysis has started an initiative in order to establish a consensus protocol on the current methods of the phenotyping of hematological neoplasias as a basis for quality assurance and support for upcoming technologies such as quantitative analysis of antigen densities and automated knowledge-based analysis software. In addition to general recommendations on assay procedures and quality control specific recommendations are given for the selection of two-color reagent panels and data interpretation in an attempt to define a basis for cross-evaluation against the different currently established laboratory protocols.

Acute Disease↗

PubMed1997

Prognostic significance of DNA ploidy and proliferation in 309 colorectal carcinomas as determined by two-color multiparametric DNA flow cytometry.

BACKGROUND: Although DNA flow cytometry has been shown to be of independent value in determining the prognosis of colorectal carcinoma, a number of well-designed studies with contradictory findings have left unresolved the clinical significance of DNA ploidy and proliferation in biologically meaningful subsets of colorectal carcinoma cases. METHODS: To evaluate the prognostic significance of DNA ploidy and proliferation as determined by flow cytometry in a prospective series of 309 human colorectal carcinomas with 4-6 years of follow-up, fresh tumors were mechanically dissociated into whole cell suspensions and dual fluorescence-labeled to allow gated analysis of subpopulations with phenotypic markers. Software programs with histogram-dependent algorithms employing background, aggregate, and debris correction were used in DNA and cell cycle quantitation. Data were analyzed according to recommendations of the 1992 DNA Flow Cytometry Consensus Conference. RESULTS: None of the clinical, site, or staging parameters, including TNM stage variables, correlated with any flow cytometric DNA ploidy or proliferation measurement. Tumors classified as DNA aneuploid or tetraploid, by any definition, did not differ in prognosis or correlate with stage or any pathologic parameter. Univariate Kaplan-Meier survival analysis showed prognostic significance of the following: Dukes staging, individual components of TNM stage (tumor depth, lymph node status, and metastasis), vascular invasion, histologic pattern of tumor infiltration, and peritumoral lymphocytic inflammation. DNA ploidy status and proliferation measurements were not predictive of survival for the overall group or within any particular stage. Only Dukes Stage D (metastasis), vascular invasion, and depth of invasion (T classification) were significant independent predictors of survival in multivariate Cox regression models. CONCLUSIONS: In this analysis, DNA ploidy and proliferation measurements were not predictive of survival in any stage of colorectal carcinoma. However, clinical and pathologic documentation of staging and select histopathologic observations were significant predictors of survival in univariate and multivariate analyses.

Adult↗

PubMed2006

A quantitative exploration of surface antigen expression in common B-cell malignancies using flow cytometry.

The use of flow cytometry to diagnose hematological malignancies has become routine due to its ability to often differentiate between morphologically similar diseases based on antigens expressed on the surface of malignant cells. In an attempt to expand on the utility of flow cytometry in the study of B-cell malignancies we have used the most reliable quantitative methodology, QIFI (quantitative indirect immunofluorescence assay), to study the expression of CD5, CD10, CD11c, CD19, CD20, CD22, CD23, and CD79b in 384 cases of several common B-lineage malignancies, including: B-ALL, CLL, SLL, hairy cell leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. The impetus behind this extensive, single institution study of surface antigens was two-fold: evaluating similarities and differences of antigen expression between B-cell neoplasms and finding additional clinical utility for the quantitative flow cytometric data generated. Our results show that each distinct malignant histology has its own quantitative pattern of surface antigen expression. In most cases, these quantitative patterns do not increase the ability of flow cytometry to distinguish between them. However, a high expression of specific antigens on a given B-cell malignancy may potentially identify optimal therapeutic targets for current and/or future monoclonal antibody-based therapies.

Antigens, Differentiation, B-Lymphocyte↗

PubMed2003

Lineage-specific identification of nonhematopoietic neoplasms by flow cytometry.

To extend flow cytometry (FC) to the diagnosis of nonhematopoietic neoplasms, we have developed new flow cytometric assays to identify expression of cytokeratin, epithelial cell adhesion molecule (EpCAM)/epithelial glycoprotein-2, myogenin, and CD99. To validate these assays, we correlated the flow cytometric results with the histologic and immunohistochemical results on paraffin-embedded tissue in a series of 21 cases, including 17 carcinomas, 1 atypical carcinoid, 2 rhabdomyosarcomas, and 1 Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET). Six of 7 assayed carcinomas and the carcinoid were positive for cytoplasmic cytokeratin by the flow cytometric assay. EpCAM was expressed by 11 of 12 carcinomas that were assayed by FC. Both rhabdomyosarcomas expressed myogenin by FC, and the ES/PNET case expressed CD99. Interestingly, the blast-associated antigen CD90 was expressed uniformly on the ES/PNET case and on subsets of cells in the rhabdomyosarcoma and carcinoma cases. Potential applications of the flow cytometric assay to nonhematopoietic neoplasms will include evaluating samples with limited material, monitoring disease persistence and recurrence in patients with previous diagnoses, and making rapid diagnoses in urgent cases.

12E7 Antigen↗

PubMed2001

[Flow cytometry].

Recently, flow-cytometric analysis has become the most popular type of cell-surface marker examination and it is particularly indispensable in diagnosing hematological disorders such as leukemia. Since diagnosis becomes more reliable using monoclonal antibodies, cell-surface marker analysis contributes greatly to the diagnosis of hematological disorders. In particular in lymphocytic leukemias, immunological classification becomes possible and since this classification has a close relation to pathophysiology and prognosis, it is very useful in diagnosing and treating patients with lymphocytic leukemia. Attempts at quantitative analysis of cell-surface antigens and expected benefits such as improved detail are also described. Additionally the standardization of cell-surface analysis using flow-cytometry is discussed.

Antibodies, Monoclonal↗

PubMed1990

[DNA cytometry in gastric cancer--a comparison between static cytometry on cytologic and histologic preparations and flow cytometry].

Static cytometry on cytologic and histologic preparations and flow cytometry are the basic techniques of DNA cytometry with certain advantages and drawbacks. An exact and reliable assessment of DNA ploidy and stem line heterogeneity of paraffin-embedded specimens is best performed with static cytometry on smears. The high resolution of DNA histograms in flow cytometry is reduced by the often strong portion of detritus and therefore the internal reference peak or small tumor stem lines might not be identified. The use of histologic preparations is advantageous for the analysis of correlations between histomorphologic tumor heterogeneity and stem line heterogeneity.

DNA, Neoplasm↗

PubMed1982

Quantitative karyotyping of human chromosomes by dual beam flow cytometry.

Dual beam flow cytometry of chromosomes stained with Hoechst 33258 and chromomycin A3 has been proposed as a method for quantitative classification of human chromosomes (bivariate flow karyotyping). In this paper we investigate the sources and magnitudes of variability in the mean fluorescence intensities of each chromosome group resolved in bivariate flow karyotypes and study the impact of this variability on chromosome classification. Replicate bivariate flow karyotypes of chromosomes isolated from lymphocytes from 10 individuals demonstrated that person-to-person variability was significantly greater than run-to-run variability. The total variability was sufficiently small that it did not interfere with classification of normal chromosome types except chromosomes 9 through 12 and chromosomes 14 and 15. Furthermore, the variability was generally smaller than 1/600th of the mitotic genome, so that one-band rearrangements should be detectable in bivariate flow karyotypes.

Flow Cytometry↗

PubMed2003

Laser impact on marine planktonic diatoms: an experimental study using a flow cytometry system.

A flow cytometry system was used to evaluate the impact of pulsed laser irradiations from an Nd:YAG laser on two marine coastal water diatoms, Chaetoceros gracilis and Skeletonema costatum. Three flow speeds, i.e. 9, 18 and 27 ml min-1 and three laser fluences, i.e. 0.025, 0.05 and 0.1 J cm-2 pulse-1 were tested during this study. The reduction in cell density and chlorophyll a (chl a) concentrations were monitored by reference to non-irradiated samples as controls. Upon irradiation, the cell density and the chl a concentrations became reduced significantly compared to the control (one way ANOVA p < 0.001 for the cell density in both the species and p < 0.05 for chl a concentrations in both species). A maximum mortality of 0.77 log10 (about 83%) for C. gracilis and 0.68 log10 (about 78%) for S. costatum was observed at 9 ml min-1 flow speed and 0.1 J cm-2 laser fluence. The maximum reduction observed in the chl a concentration was about 26% (control 0.413 and sample 0.306 mg ml-1) for C. gracilis and 27% (control 0.222 and sample 0.16 mg ml-1) for S. costatum, when the flow rate was 9 ml min-1 and the fluence 0.1 J cm-2. In general, mortality increased with an increase in the laser fluence. The results thus show if the cooling water is laser-irradiated to mitigate biofouling, this could result in significant damage to the planktonic flora of the flowing seawater system, which in turn might reduce algal biofilm formation on industrially important structures. The reduction in the chl a concentration showed that the laser irradiations also could result in a significant reduction in the primary productivity of the cooling water.

Analysis of Variance↗

PubMed1989

Flow cytometry and phytoplankton.

Flow cytometry and sorting are now an important technology in aquatic research. Simultaneous measurements of individual particle cell size, fluorescence, and light scatter properties are directly applicable to current topics in aquatic research. Flow sorting may be employed to obtain subsets of cells for analysis by conventional methods. The manner in which rapid, precise measurements of single cells are made is complex, and the application of this technology to aquatic samples is subject to many analytical constraints. Flow cytometric measurements of algal cell size and pigment autofluorescence are relative and are therefore dependent on the optical configuration and variability of the instrument. Specific types of reference materials are used to establish the validity of analyses: 1) instrument standards, 2) fluorescence controls, and 3) internal stain standards. The selection and application of standards and controls are discussed in the context of allometric (cell size versus pigment fluorescence) and ataxonomic (pigment color groups) methods. The widespread acceptance of particular reference materials among research groups will result in comparable data sets describing aquatic particle distributions.

Cell Separation↗

PubMed1993

Analysis and sorting of chromosomes by flow cytometry: new trends.

Flow cytogenetic is widely used since 1975, and essentially contributes to karyotype analysis and chromosome sorting. The principles of experimentation and its possibilities and limitations are now well known. Recently several new technologies have appeared. What attitude should the cytometrist adopt regarding PCR, microdissection of chromosomes, in situ hybridization, slit-scan flow cytometry or image analysis?

Animals↗

PubMed1990

Flow cytometry: its applications to immunohaematology.

Flow cytometry, using fluorescent probes, has only recently been applied to practical aspects of immunohaematology. It has been used for the detection and quantitation of cell-bound immunoglobulins and complement, minor cell populations, cellular antigens, and platelet and granulocyte antibodies. Practical applications of detecting minor cell populations have been successful in determining the extent of fetal maternal haemorrhage; determining the survival of transfused RBCs (small and large volumes); demonstrating RBC chimaerism (e.g. following bone marrow transplantation) and RBC mosaicism (e.g. demonstration of the McLeod phenotype carrier status). A large variety of cellular antigens have been demonstrated on RBCs, platelets and white cells using flow cytometry. Quantitation of RBC antigens by flow cytometry has been found to be much more objective and accurate than the routinely used serological approaches for establishing zygosity in paternity studies. Flow cytometry has been found to be a sensitive, objective and reproducible method for detecting platelet auto-and alloantibodies, and has also been used to detect granulocyte antibodies. More recently, it has been used to detect antibodies to monocytes and lymphocytes. As flow cytometers become more commonly available, many of these applications may become routine in clinical pathology laboratories.

Antigens, Surface↗

PubMedDate not supplied

Identification of Mycoplasma gallisepticum and M. synoviae by flow cytometry.

Immunofluorescence and flow cytometric methods were examined to detect and distinguish Mycoplasma gallisepticum and M. synoviae. The procedure employed 24-hr broth cultures of each organism, direct immunofluorescence staining with either homologous or heterologous antiserum, and analyses by flow cytometry. The organisms were distinguishable on the basis of fluorescent profiles when stained with the appropriate antiserum.

Cells, Cultured↗

PubMed1990

Reticulocyte count using thiazole orange. A flow cytometry method.

Recently flow cytometry techniques have been developed to replace the microscope reticulocyte count. We used thiazole orange, a RNA binding fluorochrome, to discriminate reticulocytes from mature erythrocytes. Thiazole orange and the Retic-COUNT software package were evaluated for performance of routine analysis on different flow instruments. The applied methodology analysed 10(4) cells semi-automatically in an easily performed manner. Consistent results were obtained with dipotassium EDTA anticoagulated blood (stable for 30 h after venesection), with incubation times in thiazole orange solution ranging from 2 to 7 h at 25 degrees C. This allowed flexibility in specimen collection and storage and assay performance with no change in results. Changes of incubation temperature up to 30 degrees C had no measurable effect. The values obtained showed good linearity, precision and accuracy for normal, low and high reticulocyte counts. However interferences were observed: RBC autofluorescence, nucleated RBC, Howell-Jolly bodies, high leucocyte count, high platelet count and giant platelets, all falsely increased the number of reticulocytes. These artifacts were eliminated by software gate corrections, thus leaving less than 5% of the specimen to be reanalysed by the microscopic method. The thiazole orange flow cytometric method was determined to be a fast, reliable method for the routine clinical quantitation of reticulocytes.

Benzothiazoles↗

PubMed1992

The detection of intracytoplasmic interleukin-1 alpha, interleukin-1 beta and tumour necrosis factor alpha expression in human monocytes using two colour immunofluorescence flow cytometry.

Two colour flow cytometry was used to analyse in situ cytokine expression by human monocytes. Whole blood was cultured in siliconised glass bottles, with or without E. coli lipopolysaccharide (LPS), for various times, and the mononuclear cells (MNCs) then exposed to a variety of permeabilisation procedures prior to flow cytometric analysis. Paraformaldehyde (PF)/saponin fixation preserved cellular morphology, and caused a reproducible degree of permeabilisation (estimated by propidium iodide inclusion: mean 94%, range 86-99% (n = 33)). After fixation with 4% PF and permeabilisation with 1% saponin at 0 degrees C in PBS containing 20% human serum, MNCs were incubated with phycoerythrin(PE)-conjugated mouse anti-CD14 (monocyte phenotype) and polyclonal rabbit anti-human interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumour necrosis factor alpha (TNF-alpha), or control rabbit IgG. Binding of rabbit antibodies was detected using goat anti-rabbit IgG fluorescein isothiocyanate (FITC). FITC fluorescence was increased in CD14 PE positive cells with the three anti-cytokine antibodies following LPS stimulation, compared with controls. There was a reproducible dose related response in monocyte IL-1 beta and TNF-alpha expression following LPS stimulation, with early peaks in TNF-alpha (2 h), compared with IL-1 beta (4 h), and IL-1 alpha (12 h). Specificity of this cytokine detection system was confirmed by inhibition studies using the corresponding recombinant human cytokines, by an absence of staining in CD14 negative or unpermeabilised MNCs, and by the characteristic cytoplasmic localisation of the different cytokines visualised with UV immunochemistry. Hence, the methods described here provide a reproducible, semiquantitative and specific assay for the detection of cell associated monokines. The technique may be applicable to the analysis of a variety of different cytokines in other phenotypically defined cell populations.

Antibodies, Monoclonal↗