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PubMed2004

Comparative study of monocyte enumeration by flow cytometry: improved detection by combining monocyte-related antibodies with anti-CD163.

Monocyte-counting performance by hematological cell counters often lacks satisfactory accuracy and precision. Flow cytometry, the alternative, is a reliable reference method for monocyte enumeration in evaluation of the performance of automated analyzers. The present study was focused on evaluation and optimization of monocyte enumeration by flow cytometry. Fifty blood samples were stained with same-color CD163, CD64, and CD14 monoclonal antibodies (MoAbs), single or in mixtures, along with different colors of anti-CD45 and anti-CD16. The percentage positive monocytes and their median fluorescence intensity (MFI) were compared between the resulting MoAb combinations. CD163(+)14 increased monocyte detection by 5% compared with CD14 alone. The CD64(+)CD163 mixture resulted in a 6% detection increase relative to CD64 alone. CD64(+)CD14 labeled 2% more monocytes than CD14 and 0.03% more compared with CD64. Combining CD14 with CD163 or CD64 increased MFI 24% and 14% relative to CD14. CD163(+)CD64 increased monocyte MFI 115% compared with CD64. We found that combining same-color antibodies improved monocyte detection, especially when anti-CD163 was added to CD64 or CD14 antibodies. The use of CD163 antibody is the recommended flow cytometric reference method for monocyte counting.

Antibodies, Monoclonal↗

PubMed1995

Comparative polyclonal antithymocyte globulin and antilymphocyte/antilymphoblast globulin anti-CD antigen analysis by flow cytometry.

Polyclonal antithymocyte globulin (ATG)/antilymphocyte and antilymphoblast globulins (ALG) antibodies have been used successfully in transplantation, aplastic anemia and graft-versus-host disease. Flow cytometry has been used to analyze peripheral blood lymphocyte populations in transplant patients receiving polyclonal ATG/ALG preparations for immunosuppression. Recent reports have indicated clinical dose adjustment based on levels of patient's cells expressing various CD antigens. In vitro analysis of individual polyclonal ATG/ALG CD antigen specificity could identify appropriate antigens for clinical monitoring as well as provide useful in vitro activity data. Therefore, a flow cytometry based assay to characterize and compare activities to specific CD antigens found on the surface of peripheral blood lymphocytes has been developed. Activities found in four lots each of horse ATG (ATGAM, Upjohn), rabbit and horse ATG (thymoglobulin and lymphoglobulin, Merieux), horse ALG (Minnesota), and rabbit ATG (Fresenius) have been compared for CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD18, CD28, CD44, CD45, and TCR-alpha/beta antigens. Quantitation is achieved by measuring the concentration of ATG/ALG required to give 50% inhibition of antigen specific fluorescent-labeled monoclonal antibody relative to buffer controls. The three horse products tested have similar activity to most antigens tested. However, Fresenius rabbit ATG has the lowest activity for almost all antigens tested whereas the Merieux rabbit ATG has activities closer to the horse products. This technique allows for rapid in vitro comparison of reactivities to individual lymphocyte antigens as well as in vitro analysis of consistency.

Animals↗

PubMed1988

Nasal-secretion leukocyte populations determined by flow cytometry during acute rhinovirus infection.

Leukocyte populations in the secretions of volunteers challenged by intranasal inoculation with rhinovirus serotype 25 were evaluated by means of flow cytometry. With the light-scatter properties of peripheral blood leukocytes as the standard of reference, significant increases (P less than .05) of both lymphocytes and phagocytes (polymorphonuclear leukocytes plus monocytes) were detected in the nasal secretions of persons infected by the viral challenge. There was a direct correlation of nasopharyngeal symptom severity with both the percentage of lymphocytes (P less than .05) and the percentage of phagocytes (P less than .001). Monoclonal antibodies for specific cell-membrane antigens identified the lymphocytes and phagocytes as leukocytes and also demonstrated the presence of a population of monocytic cells during the phase of maximal symptoms. The panel of monoclonals chosen did not unequivocally identify a lymphocyte population except in the presence of nosebleed. However, the results show that flow cytometry can be used to investigate nasal-secretion cell populations during the rhinovirus common cold.

Adult↗

PubMed1995

Increase in total protein following infection of CV-1 cells with SV40 virus as assayed by flow cytometry.

The changes in cell size and total protein were determined for G1-arrested, contact-inhibited CV-1 cells infected with Simian virus 40 (SV40). The assays used were the Biorad total protein assays (Bradford and DC protein assays) on a standard number of cells, total protein as assayed by fluorescein isothiocyanate (FITC) and SR101 by flow cytometry, orthoganol (90 degrees) light scatter by flow cytometry, and direct microscopic measurement with an ocular micrometer. Uninfected CV-1 cells and two cell lines with variations in DNA content (diploid vs. tetraploid) were used as controls for the studies presented. The results demonstrated a 40-60% increase in total protein at 32 to 42 h postinfection. These increases were similar to values obtained due to cellular changes resulting from viral replication and cell death.

Animals↗

PubMedDate not supplied

Detection and quantitation of sperm-bound antibodies by flow cytometry of human semen.

Detection of sperm surface antibodies is important for the diagnosis and treatment of infertility. The authors have investigated the methodologic aspects of flow cytometry (FCM) to detect sperm-bound antibodies and to quantitate the sperm antibody load (antibody molecules/spermatozoa). To obtain reliable results, dead spermatozoa must be excluded from analysis because they can bind antibody nonspecifically, and comprise 10% to 58% (n = 28) of the ejaculate in subfertile men. Flow cytometry estimation of dead cells correlates (r = 0.83) significantly with the manual Eosin Y method. After staining washed sperm samples (n = 26) with fluorescein-isothiocyanate-conjugated F(ab')2 fragments of anti-IgG and IgA antibodies, the sperm load was 11,500 +/- 8,600 IgG molecules and 13,200 +/- 9,500 IgA molecules per spermatozoa. The sperm antibody load measured on different occasions could be compared between patients or in the same patient by the use of calibration standards. Since the inter- and intra-assay variation of the FCM assays was less than 10%, FCM has the potential reliably and objectively to monitor the sperm antibody load during corticosteroid treatment.

Antibodies↗

PubMed1989

Shipboard analytical flow cytometry of oceanic ultraphytoplankton.

Shipboard analysis of marine ultraphytoplankton by flow cytometry is a powerful method to classify these cells according to in vivo fluorescence characteristics and size. At present, this ataxonomic-allometric approach allows recognition of phycoerythrin-containing cyanobacteria, cryptomonads, very small red-fluorescing cells (presumably prochlorophytes), and eukaryotic algae of various sizes in many open ocean samples. The speed at which flow cytometric analysis can be performed on freshly collected samples permits a high degree of sampling resolution in both space and time. A flow cytometric view is presented of the vertical distribution of ultraphytoplankton at various sites in the north Atlantic and of experiments wherein phytoplankton were incubated in an artificial light gradient and under simulated in situ conditions.

Atlantic Ocean↗

PubMed1989

Flow cytometry of DNA content and other indicators of proliferative activity.

Flow cytometric DNA content analysis, employing fluorescent stains that bind stoichiometrically to DNA, is widely used by researchers and clinicians to monitor cell growth kinetics and to detect abnormalities in nuclear DNA content in tumor cells. Measurement precision is critical because small differences in DNA content may be significant; plastic spheres and cell nuclei are used for instrument calibration and standardization. Preparative techniques must provide a uniformly stained sample representative of the cells or tissue under investigation; multiparameter gating may facilitate cell selection within samples. Flow cytometry of light scattering, surface or intracellular antigens, RNA, chromatin structure, and/or DNA synthesis may provide additional useful information about neoplastic cell behavior.

Calibration↗

PubMed1990

Direct estimation of serological H-Y antigen by flow cytometry.

This study was undertaken to evaluate the efficiency of flow cytometry in the detection of the serological H-Y antigen, and to survey expression of H-Y in the normal human population. Peripheral blood leukocytes (granulocytes) were reacted with monoclonal H-Y antibody, gw-16, and with FITC-conjugated goat anti-mouse IgG, and then were scored for fluorescence in the flow cytometer. Assigned H-Y phenotype was correlated accurately with sex phenotype in 33 out of 38 women and 62 out of 65 men. Data were evaluated by exploratory data analysis (EDA).

Antibodies, Monoclonal↗

PubMed2003

Flow cytometry analysis of proliferative lesions at the gastrocystoplasty anastomosis.

PURPOSE: Proliferative epithelial metaplasia that develops in the anastomotic line after gastrocystoplasty has unknown malignant potential. Flow cytometry analysis of cell cycle profiles is used to predict the neoplastic progression of metaplastic lesions in other proliferative epithelium. We used this technique to evaluate transitional cell metaplasia in rat gastrocystoplasty specimens. MATERIALS AND METHODS: A total of 50 prepubescent female Long-Evans rats were randomly assigned to an experimental group (gastrocystoplasty) or a control group (sham operation). At 21 to 27 months (mean 24.9) after operation 12 rats per group survived to sacrifice. Metaplastic lesions were microdissected to yield a minimum of 10(4) cells for DNA flow cytometry and cell cycle analysis. Transitional cell epithelium from sham specimens and gastric epithelium from experimental animals served as controls. RESULTS: Transitional cell hyperplasia and metaplasia with cyst formation were found in the anastomotic line in all 12 augmented bladders (100%). No proliferative lesions developed in control animals. No nuclear pleomorphism or mitotic changes were identified on routine histological examination. The epithelial cell turnover rate was 10 times higher in the gastrocystoplasty junctional zone than in control bladders (mean 2.2% versus 0.1% S phase) but lower than in native stomach epithelium (mean 3.3% S phase). Of 12 experimental specimens 1 showed near diploid DNA aneuploidy. No DNA abnormalities were detected in control bladder or stomach specimens. CONCLUSIONS: In this animal model histologically benign appearing proliferative lesions that develop in the anastomotic zone after long-term gastrocystoplasty harbor cell cycle and DNA ploidy abnormalities.

Anastomosis, Surgical↗

PubMed1988

Enumeration of IFN-gamma-producing cells by flow cytometry. Comparison with fluorescence microscopy.

A new intracytoplasmic immunofluorescence staining to detect and quantify human interferon-gamma (IFN-gamma)-producing cells by means of flow cytometry is described. Mononuclear leukocytes, stimulated in vitro to produce IFN-gamma, were fixed and made permeable to antibodies by sequential exposure to paraformaldehyde and the detergent n-octyl-glucoside. Cytoplasmic IFN-gamma was demonstrated by indirect immunofluorescence using IFN-gamma-specific mouse monoclonal antibodies. The staining exhibited a very characteristic morphology and was localized in the Golgi apparatus. An excellent agreement between the enumeration of cytoplasmic IFN-gamma-positive cells by immunofluorescence microscopy and flow cytometry was noted. However, the latter has the advantage of a standardized control, is less labor consuming and is observer independent.

Adult↗

PubMed1986

Flow cytometry of bacteria: cell cycle kinetics and effects of antibiotics.

Flow cytometric determination of the DNA and protein content of E. coli has been carried out by means of a microscope-based flow cytophotometer with a high pressure arc lamp excitation ligh source. Fluorescence (DNA)/light scatter (total cell protein) dual parameter histograms with a resolution cv of 5% were obtained for cells labeled with a combination of mithramycin and ethidium bromide. Histograms of E. coli in rapid and slow exponential growth are presented to exemplify how the cell cycle kinetics of bacteria can be studied in much more detail than has been possible by other methods. Significant effects of chloramphenicol and penicillin on the cell cycle distribution and cell numbers of E. coli cultures were evident after one hour of culture. The data provided information on which parts of the cell cycle and what types of processes were affected by the drug. It appears that flow cytometry may become a valuable tool in studies of the cell cycle of bacteria as well as in clinical drug testing.

Anti-Bacterial Agents↗

PubMed1987

A two-color flow cytometry assay for detection of hairy cells using monoclonal antibodies.

We have developed a simple two-color immunofluorescence assay equally suited for microscopy and flow cytometry detecting hairy cells (HCs) in single cell suspensions, based on the concomitant reactivities with the B cell-specific monoclonal antibody B1 (CD20) and the monocyte/HC-associated antibody SHCL-3 (CD11c). Thus, HCs can be demonstrated in peripheral blood, bone marrow, and spleen specimens from hairy cell leukemia (HCL) patients even when they constitute less than 1% of the cell suspension. Likewise, admixture experiments with normal mononuclear cells and the MOLT-4 T-acute lymphocytic leukemia (ALL) cell line demonstrated that HCs could be detected in amounts as low as 1%. The validity of this assay has been ascertained by the lack of double marker positivity in cell suspensions from B-chronic lymphocytic leukemia (CLL) and acute myelogenous leukemia (AML) patients that only expressed B1 or SHCL-3, respectively. Furthermore, other malignant blood diseases, including malignant lymphomas, acute leukemias, and chronic leukemias disclosed no double marker positive cells. In a clinical setting, this assay was used for purifying HCs (by flow cytometry) from the peripheral blood from patients with no apparent morphological evidence of circulating HC infiltration and for monitoring the effect of interferon therapy. In conclusion, this assay should be of value for both diagnosis and monitoring patients with HCL.

Antibodies, Monoclonal↗

PubMedDate not supplied

Flow cytometry and pigment analyses as tools to investigate the toxicity of herbicides to natural phytoplankton communities.

Characterisation of natural phytoplanktonic communities is currently being advanced through flow cytometry and high resolution pigment analyses. To date, toxicological methods to assess impacts of herbicides on natural phytoplankton populations are lacking. Here, we report the novel use of these techniques in combination to study changes in phytoplankton populations exposed to 2-methylthio-4-tertiary-butylamino-6-cyclopropylamino-s-triazine (Irgarol 1051), a herbicide used in antifouling paints. Flow cytometry results revealed that following a 72-h exposure to approximately 100 ngL(-1), eukaryote abundance was less than half that in the controls. High performance liquid chromatographic analyses of pigments demonstrated that 19'-hexanoyloxyfucoxanthin was selectively lost relative to the control. This carotenoid is specific to the prymnesiophytes which are key constituents of phytoplanktonic communities within temperate marine waters. Values of EC(50) (72 h) as low as 70 ngL(-1) were calculated from the selective reduction in this compound. Concentrations substantially exceeding this level have been reported in UK and other European coastal waters.

Chromatography, High Pressure Liquid↗

PubMed1988

Cell kinetics in human malignancies studied with in vivo administration of bromodeoxyuridine and flow cytometry.

Bromodeoxyuridine (BrdUrd) is a pyrimidine analogue which is incorporated into the DNA of proliferating cells. When in vivo BrdUrd infusion is coupled with bivariate flow cytometry to measure cell BrdUrd incorporation and DNA content, both the percentage of DNA-synthesizing cells [BrdUrd-labeling index (LI)] and the DNA synthesis time (TS) can be determined on the same tissue sample. From experimentally determined LI and TS, the potential doubling time of the population and its cell production rate are calculated. To ascertain whether the BrdUrd infusion method is clinically feasible and if data are reliable, we studied patients with leukemia, refractory anemia, multiple myeloma, and brain and gastric tumors. The BrdUrd incorporation data were compared with those determined on duplicate samples with the techniques conventionally used for LI and TS values, i.e., 3H- and 14C-labeled thymidine autoradiography, respectively. The complete BrdUrd procedure takes 6-9 h, and no immediate toxicity from BrdUrd administration has been observed. In an 8-month period, 154 patients were studied. Successful LI and TS determinations were obtained in 78.9 and 59.7% of cases, respectively, more often in hematological than in solid tumors. The values for LI and TS assessed with the BrdUrd technique were very close to those found with 3H- and 14C-labeled thymidine autoradiography (r = 0.88, P less than 0.005, and r = 0.89; P less than 0.005, respectively). The potential doubling time and production rate were accordingly similar. These data indicate that in vivo BrdUrd infusion coupled with flow cytometry measurements can be performed in clinical settings and that this method is reliable. It could be used for kinetic studies in clinical trials aimed at evaluating the prognostic relevance of proliferative parameters and for planning radio- and/or chemotherapy.

Autoradiography↗

PubMed1993

Rapid, automated separation of specific bacteria from lake water and sewage by flow cytometry and cell sorting.

The use of fluorescence-activated flow cytometric cell sorting to obtain highly enriched populations of viable target bacteria was investigated. Preliminary studies employed mixtures of Staphylococcus aureus and Escherichia coli. Cells of S. aureus, when mixed in different proportions with E. coli, could be selectively recovered at a purity in excess of 90%. This was possible even when S. aureus composed only approximately 0.4% of the total cells. Cell sorting was also tested for the ability to recover E. coli from natural lake water populations and sewage. The environmental samples were challenged with fluorescently labelled antibodies specific for E. coli prior to cell sorting. Final sample purities of greater than 70% were routinely achieved, as determined by CFU. Populations of E. coli released into environmental samples were recovered at greater than 90% purity. The use of flow cytometry and cell sorting to detect and recover viable target bacteria present at levels of less than 1% within an indigenous microflora was also demonstrated.

Antibodies, Bacterial↗

PubMed1998

Occult B cell malignancies can be detected by three-color flow cytometry in patients with cytopenias.

Patients with unexplained cytopenias often present a diagnostic dilemma with minimal morphologic or cytogenetic changes to identify the underlying disease process. We have used multidimensional flow cytometry in a study of patients with cytopenias and found that this technology established, changed, or refined the diagnosis in 17/121 patients. Using the flow cytometric technique of CD45 and right angle light scatter (SSC) gating with two additional markers in a three-color analysis, eight of 121 patients were found to have hairy cell leukemia (HCL), in the absence of definitive morphologic findings of HCL. Two additional patients were found to have non-Hodgkin's lymphoma (NHL). Myeloid abnormalities, myelodysplasia (MDS) or acute leukemia was detected in seven of 56 patients with unexplained pancytopenia. Six of 65 patients identified with cytopenias resulting from lymphoid neoplasms had been referred for bone marrow transplantation (BMT) with a presumptive diagnosis of MDS, with subsequent deferral of BMT upon correct diagnosis. The screening technique is incorporated into an extensive immunophenotyping scheme to identify hematopoietic abnormalities using multidimensional flow cytometry (MDF). HCL cells (detected as low as 1.3%) reside in the same position as normal monocytes in the CD45 and SSC plots but could be distinguished from monocytes based on the expression of HLA-DR without CD11b, and expression of CD19. Further phenotyping of the abnormal population confirmed immunoglobulin light chain restriction, CD11c, and CD25 expression. Non-Hodgkin's lymphoma was detected as aberrant mature lymphocytes expressing B lymphoid markers, CD5 and light chain restriction. Myeloid abnormalities were identified in the myeloblast or maturing myeloid cell fractions. The flow cytometric scheme described can be used in primary diagnosis. The technique is definitive, sensitive, and stresses the importance of distinguishing lymphoid from myeloid etiology of cytopenias.

Acute Disease↗

PubMed1989

Quantitation of conjugate formation between human polymorphonuclear leukocytes and antibody-coated target cells by flow cytometry: the role of Fc receptor and LFA-1 antigen.

The specific binding of human polymorphonuclear leukocytes (PMN) to antibody-coated target cells was characterized by flow cytometry. PMN were labeled with phycoerythrin-E (PE) via a granulocyte-specific monoclonal antibody (leu-M1) and mixed with fluorescein isothiocyanate-labeled K562 tumor cells sensitized with rabbit antiserum. Specific conjugates were formed as analyzed by two-color fluorescence in a flow cytometer. The formation of stable conjugates was dependent on initiation of contact, temperature, time, and antiserum concentration. Studies with inhibitors implicate that microfilaments, but not microtubules, Ca2+, Mg2+, or energy-dependent processes were a prerequisite for binding of PMN to the antibody-coated target cells. No conjugates were formed when uncoated target cells were used or when the experiment was performed in the presence of protein A, indicating that binding was specifically mediated through Fc receptors (FcR). Monoclonal antibodies against the FcRII and FcRIII were used to address the role of these receptors in conjugation. One of the two anti-FcRIII antibodies and an anti-FcRII antibody effectively prevented conjugation. A monoclonal antibody directed against the common beta-chain of the adhesion molecule family and a combination of antibodies against the alpha-chain of LFA-1 and Mo-1 also blocked conjugation when target cells were sensitized under suboptimal conditions. The antibody against the beta-chain also diminished killing of antibody-coated K562, as measured by chromium release when included in the cytotoxicity assay. These results indicate that flow cytometry permits accurate quantitation and characterization of the binding between PMN and antibody-coated target cells, which in principle, can be prevented by monoclonal antibodies against surface receptors. Binding is primarily established by both the FcRII and FcRIII. Adhesion-associated molecules on the PMN surface contribute to optimal binding.

Antibodies, Monoclonal↗

PubMed1998

Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia.

We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

Amino Acid Substitution↗