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PubMed2003

Biosafety concerns for shared flow cytometry core facilities.

Many researchers who need flow cytometry for their projects have neither sufficient funds nor the work volume to justify the purchase of an analytic cytometer or cell sorter. In shared flow cytometry facilities, costs for instrument purchases, cytometer maintenance, and personnel are pooled to provide economic services for a multitude of users when they are required. Owing to the diverse nature of the samples that are submitted to core facilities, the biohazard potential of the samples can vary dramatically. For the safety of facility personnel and users, it is critical that information about hazards contained in the samples be transmitted to instrument operators before flow cytometry experiments are started. During 1999 the former Biosafety Committee of the International Society for Analytical Cytology formulated a framework biosafety questionnaire for shared facilities designed to request information about the hazard potential of experimental samples from investigators who wish to use the facility. In this report we review safety issues that are pertinent to flow cytometry core facilities by discussing the individual components of this biosafety questionnaire.

Animals↗

PubMed1994

[Quantitative analysis of blood group antigen of transitional cell carcinoma of the urinary tract by flow cytometry].

We propose a new method of quantitative measurement of cell surface blood group antigen (BGA) in superficial transitional cell carcinoma (TCC) of the urinary tract by using flow cytometry. Flow cytometric analysis was performed on 22 cases of superficial urinary tract TCC registered at the department of Urology, Okayama University Hospital during April, 1992-Feb. 1993. Fresh samples were divided into two specimens. One specimen was fixed in 20% formalin and was subjected to immunohistochemical staining of BGA using avidine-biotine complex (ABC) method. The other specimen was dissociated into single cell with mincing. The single cell was further divided into two specimens. One specimen was incubated on ice with anti-ABH mouse monoclonal antibody (DAKO)as a primary antibody. The other specimen was incubated on ice with mouse IgM as a negative control. These two specimens were reacted with FITC-conjugated rabbit anti-mouse IgM and propidium iodide (PI). Next, these cells were subjected to the flow cytometry using FACStar (Becton & Dickinson). By gating on the bivariate display of FSC vs. FL2 (PI), all nucleated cells were obtained separately and the positive rate was measured. The positive rate of FCM was successfully correlated with the degree of immunohistochemical stain. Compared with immunohistochemical staining thus far, this new technique provides a way to standardize the quantitative measurement of BGA expressions.

ABO Blood-Group System↗

PubMed2002

Drug sensitivity assay for leukaemic cells by flow cytometry.

BACKGROUND & OBJECTIVES: Drug sensitivity assays are useful in oncology practice for evaluating the sensitivity of malignant cells to anti-cancer drugs. The usefulness of such assays for the prediction of clinical response to therapy has also been demonstrated. The existing methods used for this purpose are time consuming and labour intensive. Here we report a simplified flow cytometry based assay for evaluating the in vitro drug sensitivity of leukaemic cells. METHODS: The chemo-sensitivity of three human leukaemic cell lines (a lymphoblastoid cell line, Jurkat; an erythroleukaemic cell line, K 562 and a myelomonocytic cell line HL-60) was investigated by flow cytometry. Flow cytometry was used to determine LD50 (50% inhibitory concentration) for prednisolone on Jurkat and daunorubicin on HL 60 and K 562 cell lines respectively. Per cent cell death could directly be assessed on a flow cytometer by measuring the fluorescence after staining with propidium iodide (PI). For comparison MTT assay was also performed using prednisolone on Jurkat and daunorubicin on HL-60. RESULTS: Cytotoxic effect of drugs was found to be dose dependent. Mean LD50 of prednisolone for Jurkat cells by flow cytometry was 0.805 +/- 0.058 mg/ml and by MTT assay 0.866 +/- 0.115 mg/ml. Mean LD50 of daunorubicin for HL-60 was 1.96 +/- 0.05 micrograms/ml by flow cytometry and 1.90 +/- 0.282 micrograms/ml by MTT assay. The mean LD50 of daunorubicin to K 562 was 0.49 +/- 0.049 mg/ml by the flow cytometry method. The inter-assay variation for the LD50 by flow cytometry based assay was found to be 6, 14 and 10 per cent for Jurkat, HL-60 and K 562 respectively. INTERPRETATION & CONCLUSION: We report a flow cytometry based drug-sensitivity assay for leukaemic cells, which uses a single dye staining and is rapid, technically simple and reproducible. The results compare well with the more commonly used MTT assay, which is labour intensive and time consuming. The limitation of our method is that it can only be used for studying cells in suspension and is therefore not suitable for adherent cell lines.

Antibiotics, Antineoplastic↗

PubMed2000

Detection of significant bacteriuria by automated urinalysis using flow cytometry.

A new flow cytometry-based automated urine analyzer, the UF-50, was evaluated for its ability to screen urine samples for significant bacteriuria. One hundred eighty-six urine specimens from patients attending an outpatient clinic of a university-based hospital were examined. The results obtained with the UF-50 were compared with those obtained by conventional quantitative urine culture. The UF-50 detected significant bacteriuria with a sensitivity of 83.1%, a specificity of 76.4%, a positive predictive value of 62.0%, a negative predictive value of 90.7%, and an accuracy of 78.5%. These results are comparable to those obtained by previously reported screening procedures. Besides detecting significant bacteriuria, the UF-50 can also perform routine urinalysis, including measurement of concentrations of red blood cells, white blood cells, epithelial cells, and casts, within 70 s. This capability renders this new flow cytometry-based urine analyzer superior to previously reported rapid screening methods.

Bacteria↗

PubMed1983

Combined flow cytometry and image cytometry of the same cytological sample.

Flow cytometry and image cytometry, two measuring techniques in the field of analytical cytology, can be used sequentially on the same cytological sample. Cells stained with a fluorochrome for the determination of for example, DNA or RNA content are first analysed in suspension by flow cytometry. The results of the fluorescence analysis of the individual cells are presented after data processing as frequency histograms of the DNA or RNA content of all the cells of the sample. In these histograms certain cell populations such as those with an increased DNA content are defined and these are then selected for further investigation. This is achieved by sorting cells of interest into centrifugation buckets by means of electrostatic deflection of the droplets containing such cells. Sorted cell populations are then centrifuged on to glass slides and stained according to the acriflavine Feulgen-SITS staining procedure, a quantitative method for DNA and protein. Image cytometry of these stained cells is performed with a computer controlled television based image analysis system (LEYTAS). With this system abnormal cells with elevated DNA content or increased chromatin contrast are automatically detected, thereby eliminating almost all artefacts and normal cells. Subsequently detected objects are stored in grey-value memories after the automated analysis for visual examination by the cytologist. The possibilities of combined flow cytometry and image cytometry are illustrated in typical examples in the field of cervical, bladder and mammary cytology.

Breast↗

PubMed1985

Flow cytometry in body fluid analysis.

Flow cytometry uses state-of-the-art technology to examine quickly and accurately an almost limitless number of characteristics of individual cells. Present and future applications of flow cytometry to the study of the various body fluids are discussed.

Antigens↗

PubMed2005

The role of flow cytometry in companion animal diagnostic medicine.

Flow cytometry is a powerful tool for characterising the composition of complex cell populations. The accuracy and precision of this technology for describing and enumerating cells exceeds traditional methods. The number of diagnostic veterinary laboratories with access to a dedicated machine is increasing, and there is the potential to offer a clinical flow cytometry service. The improved availability of monoclonal antibodies (mAb) to cell markers expressed by the leukocytes of companion animals, permits the implementation of comprehensive mAb panels suitable for diagnosis of lympho- and myeloproliferative disease. Reticulated erythrocyte and platelet quantification, antiglobulin assays for immune-mediated cytopenias, lymphocyte subset analysis, and immunophenotyping of lymphoma and leukemia, have been validated for companion animal samples on the flow cytometer. It is now timely to consider the role of flow cytometry in diagnostic practice, and the requirement for quality assurance and standardization of testing procedures.

Animal Diseases↗

PubMed1983

DNA cytometry of primary breast cancer. Comparison of microspectrophotometry and flow cytometry, and different preparation methods for flow cytometric measurements.

In 18 biopsy specimens from human breast carcinoma a comparison was made between DNA measurements obtained by microspectrophotometry (MSP) of Feulgen-stained nuclei in imprints and flow cytometry (FCM) of nuclei stained with ethidium bromide. For each specimen FCM was performed both with ethanol-fixed cells and unfixed cells. In addition, single cell suspensions were made from other 11 fresh mammary cancer biopsies. Parts of these suspensions were analysed both by MSP (Feulgen-stained smears) and FCM (ethanol-fixed, mitramycin-stained cells). The MSP histograms show selected tumour cells and tumour-like cells. This explains the higher proportion of cells with DNA content above the 2 c level. A good agreement was found between the results obtained by MSP and FCM with regard to the ploidy of the DNA stemline(s). FCM of fixed cells (multiple-step procedure) yielded a slightly lower proportion of diploid cells than FCM of the unfixed cells (one-step procedure), probably owing to loss of small cells during the different preparation steps. It is concluded that the results from DNA-histograms obtained from MSP and FCM can be compared as to DNA-stemline ploidy of the cell population but not as to the proportion of cells with non-diploid DNA-content.

Aneuploidy↗

PubMed1994

A knowledge-based system for the interpretation of flow cytometry data in leukemias and lymphomas.

Flow cytometry immunophenotyping and analysis of DNA ploidy and proliferative activity have become extremely helpful techniques for diagnosing and subclassifying hematopoietic cell populations in a modern, full-service hematopathology laboratory. The number of physicians with special training in the interpretation of these studies is limited. A knowledge-based computer system has been designed to aid in the interpretation of immunophenotyping and DNA flow cytometry results in hematopoietic disease. The system, known as "Professor Fidelio," is a heuristic classification system that reasons on the basis of defined diagnostic patterns. In this study, Fidelio was tested as a stand-alone system on 366 specimens from two large tertiary medical centers. Fidelio's interpretation was considered to be appropriate in all cases. In 300 of 366 (82%) specimens, the system's interpretation agreed with the diagnosis of record. Many of the disagreements could be traced to errors in the recording of the original diagnosis and minor differences in diagnostic criteria between Fidelio's knowledge base and the criteria in use at the medical centers. When used in a stand-alone mode, Fidelio's interpretation was less specific than the diagnosis of record in certain lymphoproliferative disorders that require morphologic information for subclassification. Professor Fidelio is one module in a workstation for the diagnostic hematology laboratory. This workstation is designed for interpretive reporting, education, and database functions for clinical research. Clinical and morphologic information are shared between Fidelio and the other modules for peripheral blood analysis, bone marrow morphology, and lymph node interpretation by means of a relational database. The system will be useful in hospitals that lack individuals specially trained in flow cytometry.(ABSTRACT TRUNCATED AT 250 WORDS)

Artificial Intelligence↗

PubMed2001

Molecular identification using flow cytometry histograms and information theory.

Flow cytometry is a common technique for quantitatively measuring the expression of individual molecules on cells. The molecular expression is represented by a frequency histogram of fluorescence intensity. For flow cytometry to be used as a knowledge discovery tool to identify unknown molecules, histogram comparison is a major limitation. Many traditional comparison methods do not provide adequate assessment of histogram similarity and molecular relatedness. We have explored a new approach applying information theory to histogram comparison, and tested it with histograms from 14 antibodies over 3 cell types. The information theory approach was able to improve over traditional methods by recognizing various non-random correlations between histograms in addition to similarity and providing a quantitative assessment of similarity beyond hypothesis testing of identity.

Animals↗

PubMed1985

The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry.

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry with tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd. The use of BrdUrd allowed the visualization of a population of S-phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.

Animals↗

PubMed1986

Flow cytometry evaluation of cell-mediated cytotoxicity.

A novel flow cytometry method for the evaluation of cell-mediated cytotoxicity is described. This method uses flow cytometry analysis to distinguish target cells from effector cells by differences in volume and light scatter characteristics. Non-viable target cells, following their interaction with effector cells, are determined via propidium iodide (PI) dye exclusion and then expressed as a percentage of the total target cell population. This assay is suitable both for analysis of systems which allow recycling of cytotoxic effector cells (total cell cytotoxicity assays, TCCA), and of systems in which recycling does not occur (single cell cytotoxicity assays, SCCA). Natural killer (NK) cell-mediated cytotoxicity evaluated by flow cytometry is significantly correlated with the standard 51Cr release assay. Flow cytometry can also be used to evaluate the competitive inhibition that certain cell types exert on the cell-mediated killing of NK-sensitive targets. A prerequisite for this assay is that competitor cells and target cells are distinguishable through their volume and light scatter characteristics. Advantages and pitfalls of the flow cytometry method are discussed, in comparison with the 51Cr-release assay.

Adult↗

PubMed1988

Flow cytometry deoxyribonucleic acid determinations and cytology of bladder washings: practical experience.

Flow cytometry deoxyribonucleic acid measurements of bladder washings, routine bladder irrigation cytology and cystoscopic results were compared in a prospective fashion during 33 months of clinical urology practice. Of the 204 patients (286 specimens) studied 74 had bladder tumors or a history of bladder tumors and 130 had other genitourinary pathological conditions, including hematuria, cystitis, benign prostatic hypertrophy or prostatic carcinoma. Flow cytometry and cytology agreed with the cystoscopic findings in 93 per cent of the cases, whereas flow cytometry agreed with cytology in 84.6 per cent. When combined, flow cytometry and cytology were negative in only 1 case when several papillomas or grade I papillary carcinomas were present on cystoscopy, yielding a diagnostic accuracy of 99 per cent. The false negative rates for flow cytometry and cytology were 4.3 and 5.2 per cent, respectively, with false positive rates of 4.2 and 3.1 per cent, respectively. As expected the specimens that were missed by cytology were low grade tumors and those missed by flow cytometry were usually ulcerated invasive tumors. We believe that flow cytometry is a valuable adjunct to cytology and cystoscopy in the diagnosis, management and followup of patients with known or suspected bladder cancer. In addition, criteria for the diagnosis of cancer are changed such that specimens with a deoxyribonucleic acid tail and a large amount of hyperdiploid cells without a distinct stem line are considered suspicious and indicative of bladder pathology but they are not synonymous with the presence of carcinoma.

Cystoscopy↗

PubMed2004

Role of flow cytometry in pediatric hematopathology.

Multiparameter flow cytometry with optimally selected antibody combinations has expanded the use of this technique beyond traditional applications in hematopathology. By analyzing qualitative patterns of antigen expression on discrete populations or "clusters," one can detect immunophenotypic aberrancy in specific cell populations relative to normal and reactive populations. Evaluation of patterns of antigen expression can also be used to supplement conventional methodologies in the diagnosis and subclassification of certain types of hematologic neoplasia. Finally, the diagnosis of some congenital disorders affecting the hematolymphoid system can be facilitated by the detection of characteristic immunophenotypic changes. Examples of applications of multiparameter flow cytometry with particular relevance to pediatric hematopathology are discussed.

Burkitt Lymphoma↗

PubMed1999

Energy transfer between fluorescein isothiocyanate and propidium iodide--a problem in the estimation of Tpot with the bromodeoxyuridine-DNA flow cytometry technique?

Energy transfer in flow cytometry can occur when two fluorochromes are bound in close proximity (generally within 100 A) and the emission spectrum of one fluorochrome overlaps significantly with the excitation spectrum of the other. The latter criterium is fulfilled for the fluorochromes fluorescein isothiocyanate and propidium iodide and also the former when they, e.g., are used in bromodeoxyuridine - DNA flow cytometry methods. In the present growth kinetic study using this method, we show that energy transfer does take place between fluorescein isothiocyanate and propidium iodide which results in a detected increase in DNA content with 2-3%. Despite the erroneous increase in the obtained DNA content values, this does not seem to have any influence on the calculation of DNA synthesis time and potential doubling time where the DNA content, based on the relative movement principle of the labelled cells, is used.

Adenocarcinoma↗

PubMed2001

Rapid detection of viable yeasts and bacteria in wine by flow cytometry.

The potential of using flow cytometry (FCM) in combination with fluorescent dyes for rapidly estimating counts of yeasts and malolactic bacteria in laboratory media and wines was examined. In general, there was a good correlation (regression coefficient, 0.94) between viable counts of yeasts determined by FCM and by standard plate assay. The FCM detection limit of yeasts in YPDE medium and in Pinot noir must was 10(3) cells/ml. The lowest bacterial concentration detected by FCM was 10(4) cells/ml. When yeast and malolactic bacteria populations were simultaneously analysed in wine by FCM without any previous sample treatment, difficulties were encountered in the count of bacterial cells due to their size, which is similar to natural debries present in wine. However, after the optimisation of the sample preparation, the technique appeared promising in determining the presence of such microorganisms in wine with one single measurement. Because it is rapid and easy to use, flow cytometry can be considered a useful method for microbiological quality control in wineries and for the investigation of the growth dynamics of microorganisms in wine.

Colony Count, Microbial↗

PubMed1992

Biphenotypic hybrid acute leukaemia detected by two colour flow cytometry.

Immunophenotypic studies using immunofluorescent flow cytometry were performed on the blast cells of 36 patients with acute leukaemia using a panel of eight monoclonal antibodies. Six patients had blasts which co-expressed markers for lymphoid and myeloid differentiation, and which were therefore defined as biphenotypic hybrid acute leukaemia. Of the six, three patients were in the paediatric age group (below 12 years old) while the other three were more than 12 years old. Peripheral blood counts were variable; however, bone marrow infiltration was extensive (blasts > or = 75% in all). At the time of study, remission was achieved in only two patients. The authors' data show that biphenotypic hybrid acute leukaemia is not rare in Malaysia. This represents a subgroup of acute leukaemia identifiable by immunophenotyping but not by the French-American-British classification based on morphological and basic cytochemical studies alone. The recognition of this subgroup is important for both practical and theoretical reasons. There are implications for treatment of the individual patient because treatment directed at a single lineage may not be effective. The two colour flow cytometry proved to be a useful tool for diagnosis and classification of acute leukaemia.

Adolescent↗

PubMed1991

Prognostic value of analysis of DNA in pancreatic adenocarcinoma by flow cytometry.

DNA was analysed by flow cytometry in paraffin-embedded tumour specimens from 58 patients who had undergone resection of pancreatic ductal adenocarcinoma, to see if there was any correlation between DNA analysis and survival. Because of the poor quality of the DNA histogram five cases were excluded from the analysis of DNA-ploidy and 10 from the analysis of the synthesis phase fraction. Aneuploidy was detected in 11 (21%) of the 53 tumours. Neither aneuploidy nor synthesis phase fraction correlated significantly with size, stage or differentiation of the tumours. The survival of the patients with DNA-aneuploid tumours (median 9 months) did not differ significantly from that of the patients with diploid tumours (median 14 months). Four patients survived for 5 years or longer, two of whom had aneuploid tumours. Synthesis phase fraction did not correlate with survival. Using univariate survival analyses, location of the tumour in the head of the pancreas (p = 0.02), a tumour diameter of less than 3.8 cm (p = 0.003), tumour stages I and II (p = 0.03), and a well differentiated tumour (p = 0.04) correlated with favourable survival. In a Cox's regression analysis, however, only the diameter of the tumour had independent prognostic value. We conclude that DNA analysis by flow cytometry is not helpful in selecting patients with resectable pancreatic carcinoma who will benefit from resection.

Adenocarcinoma↗