bluuebunny/crossref_metadata_2025_split huggingface dataset:
A rapid mix flow cytometer with subsecond kinetic resolution
Authors: Nolan John P. , Posner Richard G. , Martin John C. , Habbersett Robert , Sklar Larry A. 
Published: June 2005

AbstractKinetic approaches are valuable tools for mechanistic studies of cell function. Flow cytometry is well suited to make sensitive kinetic measurements, but the time required to deliver mixed samples to the point of measurement (10–20 s in a conventional cytometer) limits analysis of rapidly occurring events. To address this limitation, we adapted a syringe‐based stopped‐flow rapid mixing device to a modified commercial flow cytometer to achieve mixing and measurement of sample in under 1 s. Because such screw‐driven mixers are designed to deliver fluid at rates of microliters per millisecond and cytometers accept samples at microliters per second, the syringe mixer was modified with a screw to allow sample delivery at rates as low as 1.8 μ1/s. A custom‐made nozzle holder featuring a fast‐acting three‐way sample delivery valve and a 1.5‐ μ1 dead volume was designed for a Becton Dickinson FACS stream‐in‐air flow nozzle. Syringe motors and valves are computer controlled, as is the start signal for an adjustable time ramp. A stable sample stream can be established within the sheath stream in less than 1 s, enabling fluorescence measurements of microspheres with coefficients of variation of ∼5%. Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at times of under 300 ms. Efficient mixing of reagents is demonstrated by the iodide quenching of microspheres surface labeled with fluorescein isothiocyanate (FITC). The instrument is capable of quantitatively proportioning cells and reagent, thereby allowing precise control of reagent concentration and dilution. Rapid kinetic measurements of intact cells are demonstrated by FITC‐formyl peptide binding to cell surface receptors. © 1995 Wiley‐Liss, Inc.
Electropermeabilization and fluorescent tracer exchange: The role of whole‐cell capacitance
Authors: Sukhorukov Vladimir L. , Djuzenova Cholpon S. , Frank Hermann , Arnold W. Michael , Zimmermann Ulrich 
Published: June 2005

AbstractTransmembrane crossing of charged fluorescent tracers such as propidium iodide (PI) and carboxyfluorescein (CF) can be used to quantitate membrane permeabilization. Murine myeloma Sp2/0‐Ag14 cells were loaded with CF (0.1 fmol/cell) before electropulsation (0.5–3.0 kV/cm, 40 μs) in medium containing 25–50 μg/ml PI at 21–23°C. Cytograms of PI vs. CF fluorescence showed three readily distinguishable subpopulations: (1) intact living cells with CF but without PI (these form >95% of the prepulsed population), (2) transiently electropermeabilized but resealed cells showing both CF and low‐level PI fluorescence, and (3) permanently permeabilized cells without CF but with very high PI fluorescence. Despite the ready influx of PI, the efflux of CF from transiently permeabilized cells was negligible and was insensitive to pulse parameters; however, electrically killed cells (subpopulation 3) lost all CF fluorescence and probably lost their cytoplasm. This difference in transmembrane passage of the dyes is best explained by binding of intracellular CF to macromolecules (and/or organelles). In isotonic “pulse medium,” the membranes resealed after electropulsing with a time constant (τR) of about 2 min. In 150 mOsm medium, resealing was faster (typically τR ∼ 0.5 min). The population distribution of PI uptake [coefficient of variation (CV) > 40%] was very broad and could not be accounted for by the radius dependence of pulse‐induced voltage (CVradius ∼ 10%). The variability in PI uptake could be explained if the electrical energy of the charged membrane, which depends on the whole‐cell capacitance (Cc), was taken into account. Evaluation of the Cc values with single‐cell resolution was based on measurement of the electrical charging time constant of the plasma membrane by electrorotation. © 1995 Wiley‐Liss, Inc.
Effect of cell density on the expression of adhesion molecules and modulation by cytokines
Authors: Stanley A. J. , Banks R. E. , Southgate J. , Selby P. J. 
Published: June 2005

AbstractLittle is known about the regulation of integrin expression. Using flow cytometry, we have characterized the expression of integrin subunits and other adhesion molecules in bladder and colonic cell lines and studied their modulation by the cytokines tumor necrosis factor‐α (TNF‐α) and interferon‐γ (IFN‐γ). We show that although TNF‐α and IFN‐γ may modulate integrin expression, these effects are, in the case of some integrin subunits, secondary to cytokine‐induced changes in cell density. Cell density‐dependent changes in the expression of α2‐, α3‐, α6‐, β1‐, and β4‐integrin subunits were seen. IFN‐γ produced density‐independent decreases in expression of α2‐ and β1‐integrins and increases in expression of intercellular adhesion molecule‐1 (ICAM‐1), α3‐, α6‐, and β4‐integrins. TNF‐α produced increases in β1‐integrin and ICAM‐1 expression which were also density‐independent. The expression of some integrin subunits is clearly density‐dependent and the alterations seen are dependent on the cell line studied. This study illustrates the importance of including appropriate density‐related controls in all investigations examining adhesion molecule expression. © 1995 Wiley‐Liss, Inc.
Rapid detection of antimicrobial activity using flow cytometry
Authors: Durodie J. , Coleman K. , Simpson I. N. , Loughborough S. H. , Winstanley D. W. 
Published: June 2005

AbstractDifferent antibiotic classes vary considerably in their modes of action and hence in their effects on the bacterial cell. Flow cytometry was used to analyse E. coli cells treated with five antibiotics differing a their modes of action. The ratio of protein content, as measured by fluorescence, to forward light scatter (i.e., FL1:FSC) provided a simple and reliable way of detecting within 2 h of treatment antimicrobial activity at a 0.5 minimum inhibitory concentration. This ability to detect antimicrobial activity rapidly offers considerable advantages in drug research for the rapid detection of novel antimicrobials and may, with further development, find a use in the clinic for rapid susceptibility testing as an aid to the selection of therapy. © 1995 Wiley‐Liss, Inc.
DNA aneuploidy in acute myeloblastic leukemia is associated with a high expression of lymphoid markers
Authors: Vidriales M. B. , Orfao A. , López‐Berges M. C. , González M. , López‐Macedo A. , Ciudad J. , López A. , Cañizo M. C. , Garcia M. A. , Miguel J. F. San 
Published: June 2005

AbstractIn the present study the DNA cell content of 205 de novo acute myeloblastic leukemia (AML) patients is analyzed at flow cytometry in order to determine both the incidence of DNA aneuploidy in AML patients and the clinical and biological characteristics of AML aneuploid cases. All technical procedures were performed in accordance with the proposed guidelines of the DNA Cytometric Consensus Conference. Our results show that the incidence of DNA aneuploidy is quite low (4.8%), with most cases (n = 8) hyperdiploid and only a small proportion (n = 2) hypodiploid. No major differences were detected between the aneuploid and diploid cases with respect to the clinical and prognostic disease characteristics. Regarding the immunophenotype of the blast cells, the aneuploid cases displayed both a higher incidence of immature myeloblastic phenotype and a greater expression of lymphoid‐associated antigens. In summary, our results show that AML patients display a quite low incidence of DNA aneuploidy and that despite the fact that these cases do not display particular clinical characteristics, they show an association with the expression of lymphoid‐related markers. © 1995 Wiley‐Liss, Inc.
Variability in absolute lymphocyte counts obtained by automated cell counters
Authors: Simson E. , Groner W. 
Published: June 2005

AbstractThere is increasing interest in the absolute lymphocyte count. This is partly driven by the need to obtain absolute values for lymphocyte subsets such as absolute CD4+ counts in human immunodeficiency virus (HIV)‐infected persons. The absolute total lymphocyte count is usually determined in the routine hematology laboratory on a separate sample from the same patient specimen and then combined with percentage results from flow cytometry to obtain the absolute value of the lymphocyte subsets.We have studied analytic variability in the absolute lymphocyte determination and compared it to the variability of the total white blood count (WBC). In a series of 524 specimens, four different automated methods were compared to each other and to the traditional eye count differential. The automated methods were four widely used automated cell counters (Technicon H*1, TOA NE8000, Coulter STKS, and Abbott CD3000). The results indicate that analytic variability in the absolute lymphocyte counts, due, primarily, to method variability, is significant and is larger than the variability typically observed on interlaboratory trials of relative CD4 counts. These method biases cannot easily be reduced by calibration, since the cell classification algorithms are built‐in features of the various cell counters. Analytic variability of the absolute lymphocyte counts was found to be 12.4% compared with analytic variability of only 4.9% for total WBC counts on the same samples.Our data suggest that more precise results would be obtained if flow cytometry results expressed each phenotype as a fraction of the leukocytes as well as total lymphocytes. Conversion to absolute values could then be accomplished through determination of the total WBC in the routine hematology laboratory. © 1995 Wiley‐Liss, Inc.
Immunophenotyping of congenital leukemia
Authors: McCoy J. Philip , Overton W. Roy 
Published: June 2005

AbstractCongenital leukemia is a rare but well‐documented disease in which a leukemic process is detected at birth or very shortly thereafter. An estimated 175–200 reports of congenital leukemia have appeared in the literature. The majority of the cases reported have not undergone thorough immunophenotyping, but rather have been assigned lineage based on cytochemical and morphological studies. Historically, a large proportion of congenital leukemias have been thought to be of the myeloid lineage, in contrast to pediatric leukemias in general, which are primarily lymphoid. The precise proportions of the lineage assignments may be distorted by the inclusion of cases of transient myeloproliferative disorders (TMD) as congenital leukemia. The immunophenotyping data available to date suggest that congenital leukemias are phenotypically heterogeneous, lacking any common distinguishing markers. The prognosis for congenital leukemias is usually poor if leukemoid reactive processes, such as TMD, are carefully excluded. © 1995 Wiley‐Liss, Inc.
Congenital leukemia: Report of two cases
Authors: McCoy J. Philip , Travis Susan F. , Blumstein Linda , Birdsall Peggy B. , Schroeder Kathy , Overton W. Roy , McMorrow Lydia E. , Campbell Tamberly , Wineburg Marissa , Greenbaum Barbara H. , Hirsch Kenneth H. , Donaldson Milton H. 
Published: June 2005

AbstractCongenital leukemia is a rare disease in which a leukemic process is present at birth or immediately thereafter. The majority of cases presented in the literature were reported prior to the availability of contemporary immunophenotyping methods, and lineage assignment was often made on the basis of morphology alone. Congenital leukemias may be of various lineages, although, historically, monocytic and myelomonocytic congenital leukemias appear to be the most prevalent. We present two cases of congenital leukemia with detailed immunophenotypic and cytochemical characterization. One case is of the lymphoid lineage, and the second is of myelomonocytic lineage. Neither patient displayed trisomy 21. © 1995 Wiley‐Liss, Inc.
Alterations in phenotype and cell‐surface antigen expression levels of human monocytes: Differential response to in vivo administration of rhm‐csf or rhgm‐csf
Authors: Schmid Ingrid , Baldwin Gayle C. , Jacobs Edwin L. , Isacescu Valentin , Neagos Negoita , Giorgi Janis V. , Glaspy John A. 
Published: June 2005

AbstractWe investigated, via multicolor flow cytometry, the in vivo effects of colony‐stimulating factors (CSFs) on cell size, frequencies, and expression of surface antigens on peripheral blood monocytes from melanoma patients treated concurrently with CSFs and tumor‐specific monoclonal antibody (mAb) R24. Recombinant human macrophage colony‐stimulating factor (rhM‐CSF) increased cell size, relative percentages of monocytes, percentages of CD14+, HLA‐DQ+, CD11b+, and CD16+ monocytes, and cell‐surface expressions of HLA‐DR and CD11b; rhM‐CSF also up‐regulated cell‐surface expression of CD14 on CD14brightCD16− monocytes. Recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGM‐CSF) increased cell size, percentages of CD14+, HLA‐DQ+, and CD11b+ monocytes, and cell‐surface expressions of HLA‐DR, HLA‐DQ, CD11b, and CD58. Relative percentages of monocytes and CD16+ cells and cell‐surface expression of CD14 on CD14brightCD16− monocytes decreased. In addition, monocytes derived from patients treated with rhM‐CSF showed functional activity when assayed in vitro for antibody‐dependent cellular cytotoxicity (ADCC). During treatment and coincident with increased CD16 expression, monocytes derived from rhM‐CSF patients had enhanced levels of cytotoxicity towards melanoma target cells compared to healthy controls and to patients treated with rhGM‐CSF. © 1995 Wiley‐Liss, Inc.
Peripheral blood cell preparation influences the level of expression of leukocyte cell surface markers as assessed with quantitative multicolor flow cytometry
Authors: Islam Dilara , Lindberg Alf A. , Christensson Birger 
Published: June 2005

AbstractWe have compared the influence of sample preparation upon the level of surface expression of T, B, and NK cell‐related antigens as assessed by flow cytometry. Lysed whole blood (WBL), Ficoll‐Paque separated peripheral blood lymphocyte (F‐PBL), and frozen peripheral blood lymphocyte (Fr‐PBL) were analyzed via single‐ and multicolor flow cytometry. The percentage of positive cells expressing the individual cell surface markers was not affected by the procedure for preparation of WBL, F‐PBL, and Fr‐PBL. In contrast, the fluorescence intensity level of individual cell surface markers varied depending on cell preparation. By using Quantum Simply Cellular (QSC) microbeads, the antibody binding capacity (ABC) of single‐color stained cells was quantified and compared. The amount of monoclonal antibody (MAb) anti‐CD3‐FITC bound to Fr‐PBL (mean ABC = 137,040) was significantly higher (P < 0.001) that the amounts bound to WBL (mean ABC = 112,410) and F‐PBL (mean ABC = 107,738). In multicolor analysis, the fluorescence intensity of CD3‐FITC and CD4‐FITC was significantly higher on Fr‐PBL than on WBL and F‐PBL; CD8‐PE and CD20‐PerCP was significantly higher on WBL. Furthermore, the intensity of CD3 and CD4 was different on T‐cell subsets. The intensity of CD3 staining in three‐color analysis was lower than with single‐color staining using the same fluorochrome.We conclude that particularly the method of cell preparation but also the selection of MAb combinations may influence the level of staining of certain lymphocyte antigens. This may be of relevance in the analysis of cellular activation and regulation of differentiation. © 1995 Wiley‐Liss, Inc.
HLA‐B27 screening by flow cytometry
Authors: Lingenfelter Beth , Fuller Thomas C. , Hartung Leah , Hunter Jay , Wittwer Carl 
Published: June 2005

AbstractA flow cytometric assay for lymphocyte HLA‐B27 expression using a two‐color direct immunofluorescent assay was compared to traditional microlymphocytotoxicity testing on 209 clinical samples. For the flow cytometric assay, whole blood was mixed with a monoclonal anti‐B27 conjugated to fluorescein‐isothiocyanate (FITC) and anti‐CD3 conjugated to phycoerythrin (PE). The samples were analyzed with flow cytometry by gating on CD3 positive events and anti‐B27 staining intensity was evaluated as median channel fluorescence of the histogram peak. The median channel fluorescence was least with B27 negative and B7 negative samples (84 ± 17), intermediate with samples that were B27 negative but B7 positive (118 ± 13), and greatest with samples that were B27 positive (155 ± 13). In addition to cross‐reactivity with the B7 antigen (n = 38), the monoclonal anti‐B27 cross‐reacted with HLA‐B37 positive samples (n = 3) and HLA‐B39 positive samples (n = 3). Using a median channel fluorescence cutoff of 136, 39 of the 40 B27 positive samples gave positive results in the flow cytometric assay for a sensitivity of 97.6%. The specificity was 95.9% with 7 false positives of 169 B27 negative samples. The flow cytometric HLA‐B27 assay is a convenient, useful screening test. For greatest specificity, samples positive by flow cytometry should be confirmed by conventional microlymphocytotoxicity or by use of other monoclonal antibodies directed against B27. © 1995 Wiley‐Liss, Inc.
Lymphocyte subset determination using a hematology analyzer
Authors: Hudson James C. , Brunhouse Robert F. , Garrison Connie , Rodriguez Carlos M. , Zwerner Robert , Russell Thomas R. 
Published: June 2005

AbstractAnti‐CD4 antibody (T4)‐coated microspheres were used to label CD4 cells in whole blood. The mixture was lysed and analyzed by a modified Coulter VCS hematology analyzer, which differentiated microspherelabeled cells by a change in Coulter volume, conductance, and light scatter. %CD3+/CD4+ fluorescent values from a profile were compared to %CD4 values using the VCS‐microsphere method. CD3 gating was used to exclude CD4+ monocytes from the 9OLS‐FALS lymphocyte gate. The results correlated well (R = 0.996). The percentage of CD4+ lymphocytes from profile scatterplots and VCS scatterplots showed a line of regression close to the equivalence line (n = 76, slope = 0.96) when CD3 gating was used for the profile. These results suggest that CD3 gating, though necessary for 9OLS‐FALS scatterplots, may not be necessary for volume‐conductance‐light scatterplots. © 1995 Wiley‐Liss, Inc.
Contribution of ploidy and cell kinetics from fine‐needle aspirates for the diagnosis of breast lesions: Study of 606 consecutive cases
Authors: Daidone Maria Grazia , Martelli Gabriele , Pilotti Silvana , Luisi Antonella , Fariselli Giuseppe , De Palo Giuseppe , De Yoldi Gianfranco Coopmans , Silvestrini Rosella 
Published: June 2005

AbstractA total of 606 consecutive patients with palpable breast lesions underwent physical examination, mammography, and fine‐needle aspiration for cytologic and flow‐cytometric (FCM) DNA analyses. FCM determinations included DNA ploidy and the fraction of cells in S + G2 + M phases. Aneuploid clones were considered indicative of malignancy; diploid, rapidly proliferating (S + G2 + M > 12%) clones were considered suggestive of malignancy; and diploid, slowly proliferating clones were considered indicative of benignancy. Sensitivity, specificity, and the predictive accuracy for positive or negative results of FCM information were 82%, 90%, 93.1% and 69.8%, respectively. These values were lower than those observed for the three conventional diagnostic assays owing to the presence of false‐positive results (in 23 cases) or unassessable DNA plots (in 90 cases). FCM information alone detected five cases in which the other tests gave inconclusive results and in association to the conventional diagnostic triple test, increased the incidence of positive conclusive cases from 97.9% to 99.2%. However, further refinements are needed before this approach can be used as a routine diagnostic tool. © 1995 Wiley‐Liss, Inc.
B‐cell prolymphocytic leukemia expressing discordant myeloid‐associated antigens in simultaneous specimens from bone marrow and peripheral blood
Authors: Emery Christopher L. , Cleveland Ronald P. 
Published: June 2005

AbstractThe case of a 73‐year‐old man with B‐cell prolymphocytic leukemia (PLL) and rapid clinical demise is reported. Flow cytometric immunophenotyping results of specimens obtained from the patient demonstrated a monoclonal CD5 positive B‐cell population with myeloid‐associated marker expression, which was discordant: CD15 and CDllb were expressed in bone marrow leukemic cells, whereas peripheral blood leukemic cells showed virtually no expression of these markers. Discordant immunophenotyping results between bone marrow and peripheral blood cells have been reported recently. Additionally, investigators have associated expression of CD13 and CD11b by chronic B‐cell lymphoid leukemias with a more aggressive clinical course and shorter survival. Expression of these myeloid‐associated antigens by B‐cell prolymphocytes in PLL has not been widely reported. Cytogenetic analysis revealed a karyotype of 46,XY/?44,XYdel(1q),del (3p), whereas molecular genetic studies demonstrated immunoglobulin gene rearrangements in both heavy and light chain regions. Cytochemical staining for PAS (periodic acid‐Schiff), nonspecific esterase and methyl‐green‐pyronin was positive in leukemic cells. © 1995 Wiley‐Liss, Inc.
Laboratory practices in reporting flow cytometry phenotyping results for leukemia/lymphoma specimens: Results of a survey
Authors: Hassett Joseph , Parker John 
Published: June 2005

AbstractLaboratory directors who routinely utilize flow cytometry for at least part of their diagnostic evaluations in leukemias or lymphomas were surveyed by mail. The survey consisted of 12 questions about the flow cytometry procedures used by the laboratory in evaluating leukemias and lymphomas and on the format and content of their official report. It also requested an example of a typical leukemia/lymphoma report and solicited write‐in comments about additional important aspects of using flow cytometry to evaluate leukemias and lymphomas not covered by the questionnaire. The goal of the survey, which was sponsored by the Clinical Cytometry Society (CCS), was to document what directors of flow cytometry laboratories currently consider to be the appropriate contents of a clinical leukemia/lymphoma phenotyping analysis and in what manner and detail they report such flow cytometry results to clinicians. The survey indicated that a large number of markers are routinely evaluated to phenotype leukemias (mean = 19) and lymphomas (mean = 16). Light scatter gating, using CD45/14 to monitor the gate selected, is currently employed by a 2:1 ratio over the next most population gating strategy (CD45 vs. 90° LS). Peripheral blood, bone marrow, and lymphoid tissue constitute the majority of clinical specimens evaluated for leukemia and/or lymphoma. Two‐Color analysis, primarily for surface markers, is currently the standard method for flow cytometry measurements in routine diagnostic studies of leukemia and lymphoma. The official flow cytometry laboratory report is most commonly an individual‐lab‐generated, paper report form. A discussion of the potential benefits that might result from the development of improved computerized reporting software and from the increased use of antibody‐defined, lineage gating is offered. A composite report format is presented that demonstrates the flow measurements and quality control data included in the best of the example clinical reports submitted as part of the survey and considered important by a majority of our survey respondents. The example report is intended to be a basis for further discussion within the flow cytometry community on whether minimum reporting standards for leukemia and/or lymphoma flow cytometry results can and should be developed. © 1995 Wiley‐Liss, Inc.