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PubMed2003

Metabolic activity in filamentous fungi can be analysed by flow cytometry.

The use of flow cytometry in combination with fluorescent dyes as a technique to rapidly differentiate and enumerate bacterial and yeast cells is well established. We have shown that through the judicial choice of stains, the nondestructive screening and sorting of fungal material is possible. The early stages of growth, from germination through hyphal development of three filamentous fungal species, Penicillium, Phoma and Trichoderma, have been followed using forward- and side-angle scatter on a Becton Dickinson FACSCalibur flow cytometer. By staining isolates with the permeant fluorogenic substrates, dihydroethidium and hexidium iodide metabolic activity in the developing hyphae has been measured. We have been able to demonstrate that there is a 12-13 h window of opportunity during which germination and the early stages of hyphal development of filamentous fungi can be analysed by flow cytometry.

Antarctic Regions↗

PubMed1997

The combined effects of temperature, pH and NaCl on growth of Debaryomyces hansenii analyzed by flow cytometry and predictive microbiology.

Flow cytometry was applied to determine growth of Debaryomyces hansenii in a laboratory medium. Viable yeasts were enumerated after staining with the fluorogenic ester fluorescein diacetate (FDA). Initial studies showed that the flow cytometric determinations correlated well with viable yeast populations determined as colony forming units (CFU) whereas the relationship between CFU and optical density was only linear over a narrow range of cell concentrations, 10(5.5)-10(7.5) cells/ml. The flow cytometric measurements could reliably detect D. hansenii at concentrations as low as 10(2) cells/ml whereas the lower detection limit using optical density measurements was 10(5)-10(6) cells/ml. Growth was determined by flow cytometry at different combinations of temperatures (10-30 degrees C), pH (4.7-6.0) and NaCl concentrations (1-12% w/v). Growth curves were generated by fitting a modified Gompertz equation to the growth data using non-linear regression analysis. Lag phase duration and maximum specific growth rates were derived and quadratic polynomial models were developed describing the effects of environmental conditions on the growth parameters. Model validation based upon repetition of experiments and use of another laboratory medium showed good agreement between observed and predicted maximum specific growth rates whereas predicted lag phases were shorter than the observed lag phases.

Flow Cytometry↗

PubMed1983

Analysis of malaria parasite-infected blood by flow cytometry.

The use of flow cytometry in the quantitative analysis of blood from mice infected with Plasmodium vinckei has been studied. Several fluorescent dyes responsive to cell membrane potential were screened and one dye, 3,3'-dimethyloxacarbocyanine (DiOC1(3) ), was chosen for further study. Mature red blood cells (mRBC), immature RBC (imRBC), and parasitized RBC (pRBC) could be recognized and counted in the flow cytometer. When infected blood was separated on a Percoll gradient and fractions analyzed by flow cytometry using DiOC1(3), distinct populations of pRBC were recognized, the frequency of which varied with density. These subpopulations could not be correlated with distinct morphologic stages but varied with the size or age of the growing parasite. Methods combining the use of DiOC1(3) with a DNA specific-dye, Hoechst 33342, are discussed as an approach to more complete analysis of the blood of malaria-infected animals.

Animals↗

PubMed1994

Rapid assay for pathogenic Salmonella organisms by immunofluorescence flow cytometry.

Multi-parameter flow cytometry was investigated for the rapid detection of specific serotypes of salmonellas (S. typhimurium and S. montevideo) labelled with fluorescent monoclonal antibodies, both in pure culture and in a typical food matrix (full-fat milk). In all cases, the method was accurate to levels of below 10(4) target cells per ml for a total assay time of about 30 min. After 6 h non-selective enrichment in the presence of a 10,000-fold excess of competing micro-organisms (Escherichia coli) the corresponding detection limit was about 20 cells ml-1. These results suggest that flow cytometry has significant potential for the detection of pathogenic micro-organisms in the food industry.

Animals↗

PubMedDate not supplied

Oncology applications of flow cytometry.

The application of flow cytometry in evaluation of neoplasms is reviewed, with emphasis on DNA content analysis and surface-membrane antigen analysis. By measuring total nuclear DNA content one can determine the ploidy status and S-phase fraction of tumor cells. Aneuploidy, an abnormal DNA content, is a marker of malignancy and often a predictor of poor prognosis in a number of tumor categories. S-phase fraction reflects the proliferative activity of the tumor and is therefore related to the aggressiveness of the tumor. Surface-membrane antigen analysis of hematologic malignancies using monoclonal antibodies is useful in determining lineage and differentiation of tumor cells. This information is helpful in diagnosis and monitoring of patients and also has prognostic values. The flow cytometry is a new tool for the management of cancer patients because of its role in diagnosis, monitoring, and prognosis of tumors.

DNA, Neoplasm↗

PubMed2006

Rapid assessment of the physiological status of Streptococcus macedonicus by flow cytometry and fluorescence probes.

Flow cytometry in combination with fluorescence probes was applied to rapidly assess the physiological status of Streptococcus macedonicus ACA-DC 198, a newly described member of the lactic acid bacteria group with technologically important features (e.g. lantibiotic production). A sonication procedure was developed for disaggregating typical streptococci chains in order to optimize cell preparations for single cell analysis. Single stained live and dead populations of S. macedonicus cells were clearly resolved based on membrane potential by bis-oxonol [DiBAC(4)(3)], membrane integrity by Propidium Iodide (PI) and enzymatic activity as well as membrane integrity by Carboxyfluorescein Diacetate (cFDA). Further, estimation of both live and dead cells by a cFDA/PI two-colour flow cytometric assay showed excellent correlation with the dead cells in the samples (dead(FCM)=0.9945 dead(S)-0.806, R(2)=0.9986 and live(FCM)=-0.978 dead(S)+98.895, R(2)=0.9992). Finally, the assay was applied to study the physiology of S. macedonicus after acid stress. Interestingly, in situ assessment of the physiological status of stressed S. macedonicus cells by flow cytometry and single cell sorting revealed the coexistence of three distinct subpopulations according to their fluorescence labelling behaviour and culturability, representing intact/culturable, permeabilized/dead and potentially injured cells with the latter exhibiting both metabolic activity and membrane permeabilization as well as decreased culturability.

Cell Membrane Permeability↗

PubMed1996

DNA ploidy and protein content in bronchioloalveolar carcinoma multi-variable flow cytometry.

Multi-variable flow cytometry studies were done on paraffin-embedded surgical specimens from 97 patients with bronchiolalveolar carcinoma (BAC) and on 10 normal lung tissue specimens to assess the value of nuclear protein content besides DNA ploidy. Tumor tissues were stained for DNA content with propidium iodide and for protein content with fluorescein isothiocyanate. DNA ploidy measurements were successful in 87 of the 97 specimens. Twenty-four (28%) specimens were DNA diploid, and 63 (72%) were DNA nondiploid (DNA tetraploid or DNA aneuploid). There was no significant difference in survival between patients with DNA diploid and with DNA nondiploid tumors (P = 0.69). The protein histogram pattern was bimodal in 26/87 (30%) of patients; these had significantly shorter survival than patients with either normal or right-shift protein histogram patterns (61/87) (P = 0.0033). The nuclear protein measurement was an independent prognostic indicator when corrected for tumor grade and tumor size in a Cox model analysis (P = 0.04). The nuclear protein measurement correlated with nuclear size as determined by nuclear volume measurements. The combination of DNA ploidy, protein, and cell size measurements by flow cytometry provide a useful biologic basis for the variable prognosis seen with BAC tumors.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed1999

Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry.

The use of flow cytometry for quantifying fetomaternal haemorrhage is increasing, and has been shown to be more accurate than the Kleihauer-Betke test for evaluating larger bleeds of over 4 mL in volume. Red cells are stained with fluorescently labelled monoclonal anti-D. Cells for analysis are normally gated manually on the basis of forward and side scatter. We investigated whether the use of an antiglycophorin A monoclonal antibody conjugate (red cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R1r RBC were prepared to simulate 1, 0.5, 0.25, 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse monoclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoclonal anti-D was used to determine the proportion of D-positive cells. Results from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and ungated data. The results showed that single labelling with manual gating gave falsely low FMH estimates. We conclude that use of a fluorescently labelled antiglycophorin A antibody improves the accuracy of the FMH measurement by flow cytometry, as manual subjective gating of RBC excludes a higher proportion of fetal than of adult RBC.

Animals↗

PubMed2006

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy.

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).

Animals↗

PubMed1999

Variable selection and multivariate methods for the identification of microorganisms by flow cytometry.

BACKGROUND: When exploited fully, flow cytometry can be used to provide multiparametric data for each cell in the sample of interest. While this makes flow cytometry a powerful technique for discriminating between different cell types, the data can be difficult to interpret. Traditionally, dual-parameter plots are used to visualize flow cytometric data, and for a data set consisting of seven parameters, one should examine 21 of these plots. A more efficient method is to reduce the dimensionality of the data (e.g., using unsupervised methods such as principal components analysis) so that fewer graphs need to be examined, or to use supervised multivariate data analysis methods to give a prediction of the identity of the analyzed particles. MATERIALS AND METHODS: We collected multiparametric data sets for microbiological samples stained with six cocktails of fluorescent stains. Multivariate data analysis methods were explored as a means of microbial detection and identification. RESULTS: We show that while all cocktails and all methods gave good accuracy of predictions (>94%), careful selection of both the stains and the analysis method could improve this figure (to > 99% accuracy), even in a data set that was not used in the formation of the supervised multivariate calibration model. CONCLUSIONS: Flow cytometry provides a rapid method of obtaining multiparametric data for distinguishing between microorganisms. Multivariate data analysis methods have an important role to play in extracting the information from the data obtained. Artificial neural networks proved to be the most suitable method of data analysis.

Bacillus subtilis↗

PubMed2005

Neoplasia detection in Macoma balthica from the Gulf of Gdansk: comparison of flow cytometry, histology and chromosome analysis.

A flow cytometry protocol was applied for the detection of neoplasia in Macoma balthica L. from the Gulf of Gdansk (Baltic Sea, Poland). A simple method, based on an osmotic shock, was used to permeabilise gill cells. The cytometric pattern of normal clams consisted of 2 peaks, a major peak B and a smaller peak C. The cytometric pattern of affected clams consisted of 2 peaks named B' and C'. Two parameters were used to define the stages of abnormalities in M. balthica clams based on the percentage of cells in peaks B, C, B' and C' and on the ratio between the fluorescence value of peaks B, C, B' and C' in all individuals. Three stages of neoplasia were clearly distinguished by flow cytometry considering peak C'. Stage 1 was characterised by a major population of cells in peak B' and more than 10% of cells in the C' peak. Stage 2 consisted of a lower percentage of cells in peak B' and more than 25% of cells in peak C'. Stage 3 of the neoplasia was characterised by a further reduction in peak B' and more than 40% of cells in peak C'. Flow cytometry allowed for objective detection of neoplasia and provided a rapid method for measuring the DNA content of thousands of cells per individual. The accuracy of flow cytometry was assessed by comparing with standard histological techniques, used here as a reference technique for the detection of neoplasia, and with chromosome analysis. All individuals were analysed in parallel using the 3 techniques. The proportion of normal and affected individuals diagnosed using flow cytometry was comparable to the proportion determined by histology and chromosome analysis.

Analysis of Variance↗

PubMed2003

Using flow cytometry to quantify microbial heterogeneity.

Flow cytometry is a powerful technique for the study of single cells, and thus it is of particular utility in the study of heterogeneity in microbial populations. This review seeks to highlight the role of flow cytometric analyses in studies of microbial heterogeneity, drawing wherever possible on recently published research articles. Whilst microbial heterogeneity is well documented in both natural and laboratory environments, the underlying causes are less well understood. Possible sources for the heterogeneity that is observed in microbial systems are discussed, together with the flow cytometric tools that aid its study. The role of flow cytometry in molecular biology is discussed with reference to gene reporter systems, which enable heterogeneity of gene expression to be monitored. With the recent sequencing of a variety of microbial genomes, it is anticipated that flow cytometry will have an increasing role to play in studying the effects of gene expression and mutation on heterogeneity, and in resolving the interactions of genetics and physiology.

Bacteria↗

PubMed2002

Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry.

AIMS: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). METHODS: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1-120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. RESULTS: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. CONCLUSION: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.

B-Lymphocytes↗

PubMed2005

Plant DNA flow cytometry and estimation of nuclear genome size.

BACKGROUND: DNA flow cytometry describes the use of flow cytometry for estimation of DNA quantity in cell nuclei. The method involves preparation of aqueous suspensions of intact nuclei whose DNA is stained using a DNA fluorochrome. The nuclei are classified according to their relative fluorescence intensity or DNA content. Because the sample preparation and analysis is convenient and rapid, DNA flow cytometry has become a popular method for ploidy screening, detection of mixoploidy and aneuploidy, cell cycle analysis, assessment of the degree of polysomaty, determination of reproductive pathway, and estimation of absolute DNA amount or genome size. While the former applications are relatively straightforward, estimation of absolute DNA amount requires special attention to possible errors in sample preparation and analysis. SCOPE: The article reviews current procedures for estimation of absolute DNA amounts in plants using flow cytometry, with special emphasis on preparation of nuclei suspensions, stoichiometric DNA staining and the use of DNA reference standards. In addition, methodological pitfalls encountered in estimation of intraspecific variation in genome size are discussed as well as problems linked to the use of DNA flow cytometry for fieldwork. CONCLUSIONS: Reliable estimation of absolute DNA amounts in plants using flow cytometry is not a trivial task. Although several well-proven protocols are available and some factors controlling the precision and reproducibility have been identified, several problems persist: (1) the need for fresh tissues complicates the transfer of samples from field to the laboratory and/or their storage; (2) the role of cytosolic compounds interfering with quantitative DNA staining is not well understood; and (3) the use of a set of internationally agreed DNA reference standards still remains an unrealized goal.

Cell Nucleus↗

PubMed2003

Analysis and sorting of rye (Secale cereale L.) chromosomes using flow cytometry.

Procedures for chromosome analysis and sorting using flow cytometry (flow cytogenetics) were developed for rye (Secale cereale L.). Suspensions of intact chromosomes were prepared by mechanical homogenization of synchronized root tips after mild fixation with formaldehyde. Histograms of relative fluorescence intensity obtained after the analysis of DAPI-stained chromosomes (flow karyotypes) were characterized and the chromosome content of the DNA peaks was determined. Chromosome 1R could be discriminated on a flow karyotype of S. cereale 'Imperial'. The remaining rye chromosomes (2R-7R) could be discriminated and sorted from individual wheat-rye addition lines. The analysis of lines with reconstructed karyotypes demonstrated a possibility of sorting translocation chromosomes. Supernumerary B chromosomes could be sorted from an experimental rye population and from S. cereale 'Adams'. Flow-sorted chromosomes were identified by fluorescence in situ hybridization (FISH) with probes for various DNA repeats. Large numbers of chromosomes of a single type sorted onto microscopic slides facilitated detection of rarely occurring chromosome variants by FISH with specific probes. PCR with chromosome-specific primers confirmed the identity of sorted fractions and indicated suitability of sorted chromosomes for physical mapping. The possibility to sort large numbers of chromosomes opens a way for the construction of large-insert chromosome-specific DNA libraries in rye.

Cell Separation↗

PubMed2006

Prenatal exclusion/confirmation of Fanconi anemia via flow cytometry: a pilot study.

OBJECTIVE: To explore the potential of flow cytometry in the prenatal exclusion or confirmation of Fanconi anemia (FA). METHODS: Indications for prenatal diagnosis were (1) FA-negative family history, but suspicious ultrasound findings such as radial ray aplasia, (2) FA-positive family history, but without knowledge of the affected gene and/or mutation. Amniotic fluid (AF) cell cultures and umbilical cord (UC) blood cultures were assayed for typical cell cycle changes (G2-phase accumulations) without and with mitomycin C (MMC) treatments using single- and dual-parameter (BrdU-Hoechst) flow cytometry. RESULTS: Single-parameter flow cytometry correctly identified 2 positive and 9 negative cases on the basis of MMC sensitivity of cultivated AF cells. Likewise, 8 negative and 2 positive cases were correctly predicted using bivariate flow cytometry of 72-hour UC blood cultures. In contrast, bivariate flow cytometry applied to AF cells grown in the presence of bromodeoxyuridine (BrdU) yielded false-positive and false-negative results. CONCLUSIONS: Single-parameter flow cytometry of AF cell cultures and bivariate flow cytometry of UC cell cultures have the potential to correctly predict the affected status in cases at risk for FA, whereas bivariate flow cytometry proved unreliable when applied to BrdU-substituted AF cell cultures. Cases with a low a priori risk (e.g. sonographic finding of radial ray abnormalities and negative family history) would benefit most from flow cytometry as a rapid and economical prenatal screening procedure.

Amniocentesis↗

PubMed2002

Basic principles of flow cytometry.

Although the basic principles of flow cytometry have changed little in the past quarter century, the applications of this technology have evolved substantially. As in the past, cytometers interrogate individual cells or particles in a stream with a laser as the cells move past a set of stationary detectors. Increasingly, more colors of fluorescence are being detected by cytometers, faster analysis and sorting rates are becoming possible, cytometers capable of multidirectional sorting are being marketed, and more reagents are becoming available for a wide variety of applications. Furthermore, flow cytometry has not stopped evolving. The development of narrow spectrum flourescent probes, the integration of molecular biologic techniques with flow cytometry, and the evaluation of cell-free markers such as cytokines will be key components in the continuing evolution of flow cytometry.

Analog-Digital Conversion↗

PubMed1980

Flow cytometry and cell sorting.

Flow cytometry has become an important research tool in cytology, genetics, immunology, and microbiology. The information gained from cytometric instruments is quantitative and of high statistical precision, enabling resolution of cell subpopulations. Although increasing, application to cytology is hindered by inadequate appreciation of the nature of flow cytometry and the information obtained. Many cytologic questions can be reexamined from the perspective of this technology to obtain knowledge not accessible with conventional techniques. A flow cytometer and cell sorter are described. The physical, biochemical, and functional properties measurable by these systems are discussed.

Absorption↗