Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

Optimization of procedures for counting viruses by flow cytometry

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.     

Fine affinity discrimination by yeast surface display and flow cytometry

Yeast surface display is a eucaryotic system for the directed evolution of protein binding and stability. For antibody affinity maturation, achievable single-pass enrichment factors are a critical variable. Both reliable recovery of rare clones (yield) and effective differentiation between clones of only slightly improved affinity (purity) are paramount. To validate yeast display's purification potential, trial sorting experiments were performed. The D1.3 (anti-hen egg lysozyme) single chain variable fragment antibody and a 2-fold higher affinity mutant (M3) were each displayed on the surface of Saccharomyces cerevisiae. M3-displaying cells were mixed into the D1.3-displaying cells at a ratio of 1:1000. Cells were fluorescently labeled according to antigen equilibrium binding and then sorted using a flow cytometer. Single-pass enrichment of M3-displaying cells was 125-fold (+/- 65-fold). This level of performance is achievable because of the precision and reproducibility of optimal labeling conditions. This work further demonstrates the capability of yeast display for very fine discrimination between mutant clones of similar affinity. Because large improvements in affinity typically result from combinations of small changes, this capability to identify subtle improvements is essential for rapid affinity maturation.     

Detection of intracytoplasmic cytokines by flow cytometry

Flow cytometry has been used as a powerful technique for studying cell surface antigen expression as well as intracellular molecules. Its capability of analyzing multiple parameters simultaneously on a single cell has allowed identification and studies of functional cell subsets within heterogeneous populations. In this respect, several techniques have been developed during the past few years to study cytokine-producing cells by flow cytometry in humans and several animal models.     

Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis.     

Detection of anti-phosphatidylethanolamine antibodies using flow cytometry.

BACKGROUND: The finding that lupus anticoagulant (LA) is significantly associated with anti-phosphatidylethanolamine (PE) activity has led to great interest in its relation to the clinical features of the antiphospholipid syndrome (APS). Considerable variability has, however, been reported in the prevalence of anti-PE antibodies in APS patients using enzyme-linked immunosorbent assay (ELISA) methodology. The lack of standardization and differences in technique may in part explain these discrepancies. PE binds variably to different types of microtiter wells, reflected in the consequent detection, or lack of detection, of anti-PE antibodies. This study describes the use of flow cytometry as an alternative method for the detection of anti-PE antibodies. METHODS: Six LA-positive plasma samples were used in this original study. Polystyrene beads were coated with PE overnight. These were subsequently incubated with patient plasma. Both IgG and IgM binding were detected by flow cytometry using a cocktail of fluorescently labelled anti-human Ig isotypes. RESULTS: When these results were compared with those from ELISA, flow cytometric analysis provided an apparent enhanced detection of anti-PE antibodies. It was found that 6/6 were IgM anti-PE positive by flow cytometry, whereas 5/6 were IgM by ELISA; 2/6 negative for anti-cardiolipin antibodies by ELISA were positive by flow cytometry; and 2/6 positive for antiphosphatidylcholine antibodies in cytometry were negative by ELISA. CONCLUSIONS: With appropriate quantification, this method may be more sensitive than ELISA in detecting anti-PE antibodies in plasma samples of patients with APS.     

Detection of minimal residual disease in acute leukemia by flow cytometry.

Patients with acute leukemia in clinical remission may still have up to 10(10) residual malignant cells (the upper limit of detection by standard morphologic techniques). Sensitive techniques to detect minimal residual disease (MRD) may allow better estimates of the leukemia burden and help the selection of appropriate therapeutic strategies. Flow cytometry and polymerase chain reaction have emerged as the most promising methods for detecting submicrospopic levels of leukemia. Flowcytometric detection of MRD is based on the identification of immunophenotypic combinations expressed on leukemic cells but not on normal hematopoietic cells. It affords the detection of one leukemic cell among 10,000 normal bone marrow cells, and can be currently applied to at least two thirds of all patients with acute leukemia. Prospective studies in large series of patients have demonstrated a strong correlation between MRD levels during clinical remission and treatment outcome. Therefore, MRD assays can be reliably used to assess early response to treatment and predict relapse. In this review, we discuss methodologic aspects and clinical results of flowcytometric detection of MRD in patients with acute leukemia.     

Detection of endometrial cancer by flow cytometry using two monoclonal antibodies.

BACKGROUND: To develop a supplementary diagnostic method for endometrial cancer by measuring the reactivity of various endometrial lesions with two monoclonal antibodies. METHODS: We investigated the reactivity of various endometrial lesions with two monoclonal antibodies (MSN-1 and MSN-3) by flow cytometry (one-color and two-color methods). RESULTS: The two-color method appeared to be suitable for use in place of simultaneous performance of the one-color methods with MSN-1 and MSN-3. The positivity rate for normal endometrium was 16.0% with the two-color method, which was lower than the rate of 30.0% obtained with concomitant used of the one-color methods. The positivity rate for endometrial cancer was high, 84.0%, with the two-color method. The positivity rate was 85.7% for well-differentiated endometrial cancer, 71.4% for moderately differentiated cancer, and 100.0% for poorly differentiated cancer; thus, the rate was high irrespective of the cellular differentiation. CONCLUSIONS: The two-color method is more useful than the one-color method as a supplementary diagnostic procedure for endometrial cancer.     

Detection of a Plasmodium vivax erythrocyte binding protein by flow cytometry.

BACKGROUND: The malaria parasite Plasmodium vivax preferentially invades reticulocytes. It is therefore relevant for vaccine development purposes to identify and characterize P. vivax proteins that bind specifically to the surface of reticulocytes. We have developed a two-color flow cytometric erythrocyte binding assay (F-EBA) that has several advantages over traditional erythrocyte binding assays (T-EBAs) used in malaria research. We demonstrate the use of F-EBA using the P. vivax Duffy binding protein region II (PvDBP-RII) recombinant protein as a model. This protein binds to all erythrocytes that express the Duffy receptor (Fy) and discriminates binding between normocytes and reticulocytes. METHODS: F-EBAs were performed by incubating freshly isolated Aotus nancymai, Macaca mulatta, Saimiri boliviensis, and human erythrocytes with PvDBP-RII, a fluorescent anti-His tag detection antibody, and thiazole orange before flow cytometric analysis. T-EBAs employing immunoblot detection with an anti-His antibody were performed concomitantly. RESULTS: PvDBP-RII bound to A. nancymai, M. mulatta, and human Fy+ erythrocytes, but not human Fy- erythrocytes, by F-EBAs and T-EBAs. However, F-EBAs exhibited higher sensitivity and better concordance between experiments compared with T-EBAs. CONCLUSIONS: F-EBA is a rapid, simple, and reliable method for quantifying the ability of malaria proteins to bind to the surface of erythrocytes. F-EBA can discriminate binding between erythrocyte subpopulations without enrichment protocols and may be more reliable and sensitive than T-EBAs in identifying novel erythrocyte binding proteins.     

Narrow-angle forward light scattering from individual algal cells: implications for size and shape discrimination in flow cytometry

Single-cell forward light scattering patterns have been examinedfor four algal species (one pennate diatom, two green flagellatesand one filamentous cyanobacterium), mounted statically in afocused laser beam. In all cases, the distribution of lightintensity at narrow angles (within the first scattering lobe)is well described by diffraction theory. Narrow-angle forwardscattering measurements can therefore be used in principle todeduce the size and approximate shape of algal cells. The feasibilityof using this technique in flow cytometry has been tested usingan instrument which orientates elongated cells uniformly inthe flow stream, and uses fibre optics to make azimuthally resolvedforward scatter measurements at sub-degree polar angles. Withthis instrument it is possible to discriminate between specieswith similar volume and fluorescence characteristics using forwardlight scattering as a shape-sensitive parameter.     

Detection of hemoglobin variants in erythrocytes by flow cytometry

BACKGROUND: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F. METHODS: Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells. RESULTS: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols. CONCLUSIONS: This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.     

Analysis and discrimination of necrosis and apoptosis (programmed cell death) by multiparameter flow cytometry.

Necrosis and apoptosis are two distinct modes of cell death which differ in morphology, mechanism and incidence. Membrane disruptants, respiratory poisons and hypoxia cause ATP depletion, metabolic collapse, cell swelling and rupture leading to inflammation. These are typical features of necrosis. Apoptosis plays a crucial role in embryogenesis and development and is also prevalent in tumours. It is characterised by cell shrinkage, chromatin condensation and systematic DNA cleavage. Apoptotic cells are rapidly engulfed by phagocytes, thus preventing inflammatory reaction to degradative cell contents. In vivo, apoptosis is almost impossible to quantify due to problems of heterogeneity and the short half-life of an apoptotic cell. In vitro, mechanistic studies are further complicated by a late phase of apoptosis where the cell membrane becomes permeable to vital dyes and which occurs in the absence of phagocytes. Here we describe a novel and rapid multiparameter flow cytometric assay which discriminates and quantifies viable, apoptotic and necrotic cells via measurement of forward and side light scatter (proportional to cell diameter and internal granularity, respectively) and the DNA-binding fluorophores Hoechst 33342 and propidium. It is anticipated that mechanistic studies of apoptosis in a variety of cell types will greatly benefit from this mode of analysis.     

A system for storage and retrieval of individual cells following flow cytometry.

A system has been developed to deposit cells in indexed locations on a gelatin-coated film following flow cytometry, allowing the measurements made of individual cells to be correlated with observed morphology or with subsequent microspectrophotometric measurements. Samples are deposited in a continuous track on the film by a deposition nib attached to the flow system below the observation point; laminar flow is preserved by adjusting the tape speed and the flow velocity. Locations of individual cells are indicated by etching the film with a spark triggered by the detection of a cell in the flow cytometer. After deposition, the film is dried by forced warm air. Cells on gelatin may be washed and restained with Papanicolaou and other stains with reasonable preservation of morphology. The system may be used for validation of automated cytodiagnostic procedures based on flow cytometry and for biomedical research.     

Detection of bladder carcinoma in females by flow cytometry and cytology

The sensitivity of bladder wash flow cytometry (BWFCM), voided urinary cytology (VUC), and cytology of catheterized urine obtained at the time of cystoscopy (CUC) were reviewed on all women evaluated for bladder cancer at Memorial Sloan-Kettering Cancer Center between June 1985 and December 1986. This comprised sixty-four episodes of pathologically proven bladder cancer in 48 women. Considering positive and suspicious results jointly the sensitivities of BWFCM, CUC and 3 VUC were 75%, 64% and 56%, respectively. If only positive results were considered (i.e., suspicious results considered as negative), the sensitivities of BWFCM, CUC and 3 VUC were 64%, 31% and 32%, respectively. The sensitivities of these tests are less than for a predominantly male population, presumably related to the presence of squamous epithelium and greater frequency of pyuria. However, bladder wash flow cytometry and conventional cytology are still a very valuable addition to cystoscopic examination, and the combination of BWFCM with conventional cytology is more sensitive than either procedure alone.     

Detection of JNK and p38 activation by flow cytometry analysis

JNK and p38 protein kinases are involved in the signal transduction of apoptotic stimulus. JNK and p38 are activated by dual phosphorylation on threonine and tyrosine residues. Different techniques such as Western blotting (WB) and confocal microscopy analysis have been developed to detect the activation by using antibodies that recognize the phosphorylated forms of both enzymes. However, these techniques are time consuming, not quantitative, and dependent on subjective interpretation. Herein, we describe a flow cytometry-based analysis to detect JNK and p38 activation. Using human primary lymphocytes and Jurkat CD4(+) T cells stimulated with PMA/ionomycin, we demonstrate activation (phosphorylation) of JNK and p38, which is further confirmed by two additional established techniques (WB and confocal microscopy). Flow cytometry analysis is shown to be more sensitive than WB to detect JNK and p38 activation, which can be quantitated and enables us to study their activation within cell populations.     

Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry.

The intracellular distribution of important chemotherapeutic antibiotics belonging to the anthracycline group (e.g. adriamycin) can be detected by laser flow cytometry. The indirect method is based on the interference of these compounds with the binding of propidium iodide to the nuclear DNA. While in the direct method, the intracellular fluorescence of these antibiotics is excited and detected with a laser beam in a flow system. The present report demonstrates the use of these two methods for intracellular detection and quantitation of a number of important anthracyclines.     

Microbial determinations by flow cytometry.

Recent improvements in the optics and electronics of flow cytometry systems, as well as in staining techniques, permit the assay of such minute cellular constituents as the DNA and protein contents of micro-organisms. To assess the usefulness of this technique, DNA and protein content distributions were determined in Escherichia coli, Lactobacillus brevis, Lactobacillus casei, Chlorella kessleri 8k, Saccharomyces cerevisiae, Candida utilis, Schizosaccharomyces pombe and Euglena gracilis. Investigations of the DNA content distributions of polyploid strains of Saccharomyces cerevisiae indicated that the method can be used to determine ploidy. The rapidity of flow cytometry measurements allows accurate determinations in large populations.     

Detection of intracytoplasmic immunoglobulin by flow cytometry in B-cell malignancies

The aim of this study was to compare the results of flow cytometric (FCM) determination of heavy and light chain cytoplasmic immunoglobulin (cIg) with those obtained by the peroxidase-antiperoxidase (PAP) method. Fifty-one patients, including five non-T-acute lymphoblastic leukemias, 16 B-chronic lymphocytic leukemias (CLL), 13 non-Hodgkin's lymphomas, seven hairy cell leukemias, four multiple myeloma/plasma cell leukemias, and six T-cell leukemia/lymphomas, as well as 12 normal controls, were studied. Saponin-permeabilized cell suspensions were indirectly stained with monoclonal antibodies and analyzed by flow cytometry. Acetone-fixed cytocentrifuge smears were stained for cIg by the PAP method. The results obtained indicate that: (a) detection of cIg by FCM is a feasible and useful technique to confirm the B-cell lineage of leukemias and lymphomas, particularly those characterized by low-density surface immunoglobulin, such as CLL; and (b) cIg detection by FCM and PAP staining are complementary methods to recognize with certainty the monoclonality of B-cell malignancies.