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PubMed1993

Diagnosis of paroxysmal nocturnal haemoglobinuria by phenotypic analysis of erythrocytes using two-colour flow cytometry with monoclonal antibodies to DAF and CD59/MACIF.

We investigated the relationship between the complement lysis sensitivity test and two-colour flow cytometric analysis using monoclonal antibodies to decay accelerating factor (DAF) and CD59/membrane attack complex inhibitory factor (MACIF) in patients with paroxysmal nocturnal haemoglobinuria (PNH) and other haematological diseases. Flow cytometry showed that all 59 PNH patients had two or three erythrocyte populations, while all 74 patients with other haematological diseases and all 31 healthy volunteers had a single erythrocyte population. We compared the percentage of PNH III erythrocytes in the lysis test with the percentage of negative cells shown by flow cytometry in 52 PNH patients, and found a significant correlation (r = 0.960, P < 0.001). However, in 13 patients the erythrocyte phenotypes did not correspond in both tests. This was generally related to difficulty of detecting PNH II erythrocytes in the lysis test. In the PNH patients the ranges of mean fluorescence intensity for the negative, intermediate and positive erythrocyte populations were respectively 1.1-2.5, 2.2-29, and 61-600 for CD59/MACIF positivity and 1.9-7.2, 3.6-22. and 31-350 for DAF positivity. In contrast, the mean intensities in healthy volunteers ranged from 190 to 720 for CD59/MACIF and from 150 to 350 for DAF. These findings suggest that PNH can be diagnosed and phenotypic analysis of PNH erythrocytes can be performed by respectively assessing the fluorescence profiles and mean fluorescence intensities of both proteins using flow cytometry. Flow cytometry may provide a superior diagnostic method to the traditional tests for PNH.

Adult↗

PubMed1992

Immunoglobulin VH usage analysis by fluorescent in situ hybridization and flow cytometry.

We have devised a flow cytometry-based fluorescent in situ hybridization assay that permits analysis of gene expression in a large number of single cells. In this technique, fixed and permeabilized cells are incubated with biotinylated single-stranded RNA probes and by means of a fluorescently labelled second-step reagent, the cells are analyzed by flow cytometry. This is a rapid and simple method that allows all of the steps in the procedure to be performed on cells in suspension. Using this approach, we demonstrate here that immunoglobulin heavy chain variable region (VH) gene expression can be analyzed among individual cells using particular VH family-specific probes. This technique has a high degree of accuracy (greater than 97%) in detecting the fraction of cells expressing a specific message in a population and is sensitive enough to detect immunoglobulin message in LPS activated B cells. The technique has been applied successfully to monitor gene expression in homogeneous and heterogeneous populations. It also allows concurrent analysis of cell surface proteins and gene expression through two-color flow cytometry. This method of monitoring gene expression in individual cells may have a number of applications in immunology and cell biology.

Animals↗

PubMed1998

[In vitro detection of prostate cancer circulating cells by immunocytochemistry, flow cytometry and RT-PCR PSA].

OBJECTIVE: To evaluate 3 in vitro methods detection (immunocytochemistry, flow cytometry and RT-PCR PSA) of circulating prostate cancer cells from a model of uncap dilution in immortalised lymphocytes. METHODS: In vitro comparison of 3 techniques (immunocytochemistry, flow cytometry, RT-PCR PSA) was performed from a range of dilutions of LbCap cells in immortalised human lymphocytes (concentration range: 1 LnCap cell per 100 lymphocytes to 1 LnCap cell per 100 million lymphocytes). Cells were detected by anti-PSA (prostate specific antigen) and PAP (prostatic acid phosphatase) antibody by immunochemistry, by fluorescent linked antipancytokeratin antibody by flow cytometry and RT-PCR PSA. RESULTS: The limit of detection was 1 LnCap cell per 200,000 lymphocytes (1/2.10(5)) for immunochemistry, 1 LnCap cell per 1,000 lymphocytes (1/1.10(3)) for flow cytometry and 1 LnCap cell per 10 million lymphocytes (1/10(7)) for RT-PCR PSA. CONCLUSION: RT-PCR, due to its most perceptible limit of detection, appears to be the method of choice for the detection of prostatic epithelial cells. Immunocytochemistry has the advantage of providing a quantitative approach. Flow cytometry is limited by the limit of detection of the apparatus used. The prognostic significance of detection of circulating prostate cancer cells remains to be clarified, but the detection of these cells and their correlation with the primary tumour will provide a better understanding of metastatic phenomena.

Acid Phosphatase↗

PubMed1993

Detection of antisperm antibodies on the surface of living spermatozoa using flow cytometry: preliminary study.

OBJECTIVE: To assess the use of flow cytometry (FCM) analysis of living spermatozoa subjected to indirect immunofluorescence staining with the mouse anti-human immunoglobulin (Ig)G monoclonal antibodies (FCM test) as an objective method for the detection of antisperm antibodies in semen and serum. DESIGN: The comparative studies were conducted with the direct and indirect mixed antiglobulin reaction (MAR) and FCM test performed by FCM analyzer FACSCAN (Becton Dickinson Immunocytometry Systems, Mountain View, CA). SETTING: Clinical infertile service. PATIENTS: Seventy-six semen and 20 sera samples of infertile patients were analyzed. RESULTS: For IgG in semen there was a highly significant correlation between the direct MAR and FCM test. Six of 12 IgG-positive samples revealed also IgA, and just one sample, which showed negative MAR, was positive for IgM in FCM test. Flow cytometry test and indirect MAR showed also a highly significant correlation of the results of IgG detection in sera. CONCLUSIONS: Flow cytometry test may be used to verify that IgG antisperm antibodies are on the surface of living spermatozoa either retrieved directly from an ejaculate or after exposure to serum and to determine proportion of antisperm antibodies-positive spermatozoa, titer, and quantity of antibodies bound to the cell surface.

Animals↗

PubMed1992

[Evaluation of platelet intracellular calcium ion concentrations with flow cytometry].

Platelet intracellular Ca++ ([Ca++] i) was measured by flow cytometry, using a new Ca(++)-sensitive fluorescent dye, fluo 3. The acetoxymethyl derivative of fluo 3, fluo 3 AM, was incorporated into platelets most efficiently in the presence of 1.5 micrograms/ml pluronic F-127, a surfactant often used to facilitate intracellular incorporation of lipophilic agents. [Ca++] i measurement with flow cytometry proved to be more sensitive than that with ordinary fluorescence spectrophotometers, detecting cells with elevated [Ca++] i at much lower agonist concentrations. Two-dimensional analysis using forward scatter intensities and [Ca++] i more clearly defined a subset of platelets responsive to low concentrations of agonists. In normal subjects [Ca++] i hardly changed in response to low-dose thrombin (0.002 U/ml), while it invariably showed a marked increase in response to high-dose thrombin (0.02 U/ml). Thus, these dose of thrombin were used to evaluate clinically whether platelets was hyper-reactive or hypo-reactive. Of 44 patients with diabetes mellitus, 6 patients had hypo-responsive platelets, while platelets were sensitive to low concentrations of thrombin in 3 patients. Since such differences cannot be detected by conventional spectrophotometric methods, [Ca++] i measurement by flow cytometry may prove useful tool for clinical evaluation of platelet function.

Blood Platelets↗

PubMed1994

Abnormal flow cytometry profiles in patients with interstitial cystitis.

Flow cytometry was performed on bladder cells from patients with interstitial cystitis and control patients. Cells were processed in standard fashion for flow cytometry with propidium iodide staining and analysis was restricted to samples with sufficient cells for cytokeratin gating and acceptable coefficients of variation. Of 14 interstitial cystitis patients 4 (29%) demonstrated aneuploid deoxyribonucleic acid (DNA) profiles as evidenced by a discrete peak with a DNA index of 1.2 or greater in the cytokeratin positive population. The aneuploid peak accounted for up to 54% of the cytokeratin positive population in these samples. No such aneuploid DNA profiles were evident in specimens obtained from control patients. A significant DNA tetraploid population, as evidenced by a 4C (G2) peak greater than 20%, was observed in 6 of 14 interstitial cystitis patients (43%) and 8 of 11 controls (72%). Manual counting of the per cent of binucleated cytokeratin positive cells in the cytokeratin stained population and nuclear preparations of several samples for flow analysis indicate that apparent DNA tetraploidy in the interstitial cystitis and control patients is due to an abundance of binucleated cells. Aneuploid DNA profiles on barbotage specimens from interstitial cystitis patients may reflect a real karyotypic abnormality or altered chromosome complement (true aneuploidy), abnormal chromatin structure or abnormal cytoplasmic binding of the propidium iodide stain. This finding may signal an underlying abnormality of the epithelial cell population in some patients with the clinical diagnosis of interstitial cystitis.

Adult↗

PubMedDate not supplied

Integrated photothermal flow cytometry in vivo.

The capability of integrated flow cytometry to detect, in real time, moving cells in their natural states in vivo is demonstrated in a study of circulating red and white blood cells in lymph and blood flow of rat mesentery. This system combines dual pump-probe photothermal (PT) techniques, such as PT imaging, the PT thermolens method, and PT velocimetry, with high-resolution (up to 0.3 microm), high-speed (up to 1000 fps) transmission digital microscopy (TDM) and fluorescence imaging. All PT techniques are based on irradiation of cells in rat mesenteric microvessels with a spectrally tunable laser pulse (420 to 570 nm, 8 ns, 0.1 to 300 microJ) and on detection of temperature-dependent variations of the refractive index with a second continuous probe laser beam (633 nm, 1.4 mW). We focus on intravital monitoring of the integral PT response from single, moving, unlabeled cells (from 100 to 500 cells in one measurement). Potential in vivo applications of this new optical tool, called PT flow cytometry (PTFC), are discussed, including identification of selected cells with differences in natural absorptive properties and sizes, determination of laser-induced cell damage, estimation of flow velocity, and monitoring of circulating cells labeled with PT probes.

Animals↗

PubMed1999

[Flow cytometry: diagnostic, therapeutic and prognostic usefulness in solid tumors].

Flow cytometry is a new technique measuring several optical parameters of particular cells in cell suspension. These parameters are: optical density, light scatter and fluorescence emission. Optical assay conjugated with digital data processing permit the precise characterisation of chosen cellular subpopulations. The optical signs transformed by photomultipliers as analogue signals are presented as descriptive cytograms. Digitally converted signals analysed by multichannel analyser are stored as histigrams. Flow cytometry is widely used in the diagnostics of blood neoplastic diseases and, in a less routine manner, in the diagnostics of solid tumours. The latter requires accurate disaggregation of tumour tissue to obtain homogenous cell suspension. Different, more or less efficient, mechanical or enzymatic disaggregation techniques are used to prepare appropriate cell suspensions. Flow cytometry DNA analysis with simultaneous determination of several protein proliferation factors are heavily used in research and clinical laboratories to follow tumour cell proliferation, efficacy of cancer therapy and finally as prognostic factors. Although these assays yield promising results in some specific tumours, their value and accuracy seems still equivocal.

Cell Movement↗

PubMed1994

Demonstration of cytoplasmic and nuclear antigens in acute leukaemia using flow cytometry.

AIMS: To detect cytoplasmic and nuclear antigens using flow cytometry in acute leukaemia and to use this technique for double marker combinations. METHODS: Cytoplasmic staining was carried out in samples from 40 cases of acute leukaemia with monoclonal antibodies against the myeloid antigen CD13, the lymphoid antigens CD3, CD22, mu chain and the enzymes terminal deoxynucleotidyl transferase (TdT) and myeloperoxidase (MPO). The cells were fixed with paraformaldehyde and permeabilised with Tween 20 and Becton Dickinson's FACS lysing solution. Flow cytometry results were compared in the same cases with immunocytochemistry results using the alkaline phosphatase anti-alkaline phosphatase method. RESULTS: The gentle permeabilisation induced by this method permitted preservation of the membrane antigens and the size and morphology of the cells. The results using flow cytometry were comparable with those obtained using immunocytochemistry, with nearly complete concordance in most cases. CONCLUSIONS: This technique is simple, rapid, sensitive and reproducible and it is suitable for double staining procedures, such as nuclear and cytoplasmic, nuclear and membrane, or cytoplasmic and membrane. It therefore provides a powerful tool for extending the use of immunophenotyping for the diagnosis and follow up of acute leukaemia. It could also be used for the investigation of minimal residual disease.

Acute Disease↗

PubMed2003

Modified flow cytometry and cell-ELISA methodology to detect HLA class I antigen processing machinery components in cytoplasm and endoplasmic reticulum.

Flow cytometry and cell-enzyme linked immunosorbent assay (ELISA) are useful techniques for the quantitative analysis of cell surface antigen expression. Furthermore, flow cytometry can detect intracellular markers in cells permeabilized to facilitate the intracellular penetration of antibodies. However, to the best of our knowledge, neither method has been used to detect antigens located in the endoplasmic reticulum (ER) of cells. This limitation has a negative impact on the analysis of the expression of HLA class I antigen processing machinery components in cells. Therefore in this study, we show that markers located in cytoplasm and ER can be detected by flow cytometry and cell-ELISA in cells sequentially fixed with paraformaldehyde, heated in a microwave oven, permeabilized with saponin and reacted with monoclonal antibodies (mAb). Utilizing LMP10 as an intracytoplasmic marker and calreticulin and tapasin as ER luminal markers, we show that the modified flow cytometry and cell-ELISA are sensitive, simple and reproducible methods to detect HLA class I antigen processing machinery components in cells. Furthermore, testing of 10 human cell lines with HLA class I antigen processing machinery component-specific mAb has shown that the results obtained with the modified flow cytometry and cell-ELISA are significantly correlated. These results altogether indicate that the modified flow cytometry and cell-ELISA methods we have described will facilitate the analysis of the expression of HLA class I antigen processing machinery components in cells under physiological and pathological conditions. The resulting information will contribute to the characterization of the effect of changes in the expression of antigen processing machinery components on the recognition of cells by the host's immune system.

Animals↗

PubMed1993

Flow cytometry. From research to clinical laboratory applications.

Flow cytometry is a technology that has made a successful transition from the research laboratory to the clinical laboratory. There are numerous clinical applications of flow cytometry. In hematology, the major applications have been in leukocyte phenotyping for the diagnosis and classification of leukemias and lymphomas and enumeration of peripheral blood lymphocytes in immune disorders. Applications in the phenotyping and functional characterization of hematopoietic and lymphoid cells such as erythroid cells, including reticulocytes, platelets and megakaryocytes, monocytes/macrophages, and granulocytes, are likely to increase and demonstrate additional diagnostic and prognostic clinical utility.

Antibodies, Monoclonal↗

PubMed2004

Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications.

Analysis of protein phosphorylation with flow cytometric techniques has emerged as a powerful tool in the field of immunological signaling, allowing cellular subsets in complex populations to be analyzed accurately and rapidly. In this review, we examine the development of phospho-epitope, or phospho-specific, flow cytometry and the premises upon which the technique is based. Phospho-specific flow cytometry is compared to traditional biochemical methods, and its advantages, such as single cell analysis, multiparameter data acquisition, rapid protocols, and the ability to analyze rare cell subsets, are detailed. We also discuss the many technical considerations that must be addressed when developing new antibodies or analyzing new epitopes including antigen accessibility, stability of the phospho-epitope, fluorophore selection, surface phenotype integrity, and antibody suitability for staining epitopes inside fixed and permeabilized cells. The methods that have been used to date are described in light of these technical considerations. The importance of developing bioinformatic platforms in parallel with these techniques is emphasized due to the large, multiparameter datasets that are rapidly accumulated and which require more efficient data viewing and complex clustering methods than currently available for flow cytometric data. Finally, we discuss the potential clinical applications of phospho-specific flow cytometry in analyzing immune cell development and antigen-specific immune responses, as well as pharmacodynamic profiling of disease states or drug efficacy and specificity against particular signaling proteins.

Animals↗

PubMed1997

Proposed new data file standard for flow cytometry, version FCS 3.0.

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http://nucleus.immunol.washington.edu/ISAC.ht ml.

Database Management Systems↗

PubMed1995

Clinical significance of quantitative analysis of carcinoembryonic antigen assessed by flow cytometry in fresh human gastric cancer cells.

The expression of carcinoembryonic antigen(CEA) on tumor cells freshly excised from 51 patients with gastric cancer was studied using flow cytometry. The expression of CEA by flow cytometry was more quantitative than that by immunohistochemical staining. There was no relationship between the fluorescence intensity assessed by flow cytometry and serum CEA levels, except for patients with a high titer of serum CEA. The patients with high grade CEA expression on tumor cells by flow cytometry had poor prognoses, compared to patients with low CEA expression in undifferentiated gastric cancer. Thus, it is suggested that the quantitative CEA expression on tumor cells by flow cytometry could be a useful prognostic marker in postoperative gastric cancer patients.

Adenocarcinoma↗

PubMed1991

Quantitative DNA analysis in breast carcinomas: a comparison between image analysis and flow cytometry.

We compared the results of quantitative DNA analysis of fresh tumor tissue from 50 invasive breast carcinomas by image analysis and flow cytometry. For image analysis, Feulgen-stained slides of tumor imprints and of disaggregated tumor cytospin preparations were evaluated with the CAS-200 image analyzer. For flow cytometry, propidium iodide-stained disaggregated tumor cells were analyzed with the Coulter EPICS-C flow cytometer. The two methods yielded comparable results. The DNA indices obtained by the two methods showed close correlation by linear regression analysis (r = 0.86, P less than 0.001). There were 26 diploid (52%) and 24 nondiploid (48%) carcinomas. The ploidy pattern between the two methods showed agreement in 41 carcinomas (82%) and discordance in two (4%). Three tumors (6%) were equivocal by flow cytometry and four (8%) by image analysis. The equivocal cases presented potential sources of error in the evaluation of histograms in the near-tetraploid region by flow cytometry and in the near-diploid region by image analysis. Image analysis required smaller tissue samples and permitted direct visualization and selection of tumor cells. It also detected more tetraploid carcinomas. In contrast, flow cytometry analyzed larger cell samples and provided histograms with better resolution. It more readily detected the presence of multiple aneuploid peaks and also the presence of aneuploid peaks in the near-diploid range. The presence of aneuploidy was significantly related to the loss of hormone receptor expression, high mitotic rate, and high histologic and nuclear grades. Our study indicates that image analysis and flow cytometry provide comparable results in a majority of breast carcinomas.(ABSTRACT TRUNCATED AT 250 WORDS)

Breast Neoplasms↗

PubMed2000

Rapid DNA fingerprinting of pathogens by flow cytometry.

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.

DNA Fingerprinting↗

PubMed2002

Hyphenation of sedimentation field flow fractionation with flow cytometry.

Interest in the development of field flow fractionation (FFF) systems for cell sorting recently increased with the possibility of collecting and characterizing viable cellular materials. There are various tools for the analysis of cell characteristics, but the reference is small- and large-angle light scattering often coupled with fluorimetric measurements. The well-known flow cytometry (FC) cell analysis techniques can be associated with FFF leading to the possibility of collecting information provided by a remarkable separation technique for micron-sized particles (cells) operating in the steric-hyperlayer elution mode with multiparametric detection provided by flow cytometry. Moreover FFF derived cell characteristics can be correlated with FC characteristics to describe in a unique way the nature of the eluted materials. Experimental demonstrations are described herein using nucleated cells (HL-60 cell lineage) and human red blood cells (HRBC).

Chemical Fractionation↗

PubMed2003

The use of flow cytometry to assess neutrophil infiltration in the injured murine spinal cord.

Inflammatory cells, including neutrophils, are likely candidates in promoting early cell death after spinal cord injury. We describe a simple and reliable method for obtaining neutrophils from the injured murine spinal cord for flow cytometric quantification. Mice were subjected to either a moderate or severe spinal cord contusion injury and euthanized 24 h later. The area of maximal damage, designated the epicenter, was prepared for assessment of myeloperoxidase (MPO) activity, quantitative immunocytochemistry, or quantification of immunolabeled neutrophils by flow cytometry. For flow cytometry, a cell suspension was prepared from the epicenter by gentle mechanical disruption. After centrifugation, the pellet was resuspended, immunolabeled for neutrophils, and analyzed. There was no detectable MPO activity in the injured spinal cord. In contrast, neutrophil infiltration was confirmed by immunocytochemistry and found to be significantly greater in the more severely injured group. Flow cytometry, using a standard neutrophil marker, revealed a similar significant increase in immunolabeled cells in the more severely injured group. However, when cell viability was determined in the neutrophil labeled population, no significant difference in the numbers of live neutrophils were noted between the two injured groups. Together, these findings demonstrate an effective method for the detection and quantification of viable neutrophils in the injured murine spinal cord.

Animals↗