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M Zborowski

Publications and source records attributed to M Zborowski.

At least 19 recordsLinked to original sources
PubMed2001

Mobility measurements of immunomagnetically labeled cells allow quantitation of secondary antibody binding amplification.

Magnetic cell separation methods commonly utilize paramagnetic materials conjugated to antibodies that target specific cell surface molecules. The amount of magnetic material bound to a cell is directly proportional to the magnetophoretic mobility of that cell. A mathematical model has been developed which characterizes the fundamental parameters controlling the amount of magnetic material bound, and thus, the magnetophoretic mobility of an immunomagnetically labeled cell. In characterization of the paramagnetic labeling, one of the parameters of interest is the increase in magnetophoretic mobility due to the secondary antibody binding to multiple epitopes on the primary antibody, referred to as the "secondary antibody binding amplification," Psi. Secondary antibody-binding amplification has been investigated and quantitated by comparing the mobilities of lymphocytes directly labeled with anti-CD4 MACS (Miltenyi Biotec, Auburn, CA) magnetic nanoparticle antibody with the mobilities of lymphocytes from the same sample labeled with two different indirect antibody-labeling schemes. Each indirect labeling scheme incorporated a primary mouse anti-CD4 FITC antibody that provides both FITC and mouse-specific binding sites for two different secondary antibody-magnetic nanoparticle conjugates: either anti-FITC MACS magnetic nanoparticle antibody or anti-mouse MACS magnetic nanoparticle antibody. The magnetophoretic mobilities of the immunomagnetically labeled cells were obtained using Cell Tracking Velocimetry (CTV). The results indicate that an average of 3.4 anti-FITC MACS magnetic nanoparticle antibodies bind to each primary CD4 FITC antibody, Psi(1,2f) = 3.4 +/- 0.33, and that approximately one, Psi(1,2m) = 0.98 +/- 0.081, anti-mouse MACS magnetic nanoparticle antibody binds to each primary mouse CD4 FITC antibody on a CD4 positive lymphocyte. These results have provided a better understanding of the antibody-binding mechanisms used in paramagnetic cell labeling for magnetic cell separation.

Antigen-Antibody Reactions↗
PubMed2001

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Antigen-Antibody Reactions↗
PubMed2001

Measurement of CD2 expression levels of IFN-alpha-treated fibrosarcomas using cell tracking velocimetry.

METHODS: A methodology and a mathematical relationship have been developed that allow quantitation of the expression levels of cellular surface antigens, in terms of antibody binding capacities (ABC). This methodology uses immunomagnetically labeled cells and calibration microbeads combined with cell tracking velocimetry (CTV) technology to measure magnetophoretic mobilities corresponding to cellular ABC. The mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads and cells in a nearly constant magnetic energy gradient. RESULTS: Transformed fibrosarcoma cells were given controlled treatments of interferon-alpha in order to manipulate CD2 antigen expression levels. These cells were then immunomagnetically labeled with anti-CD2 FITC antibodies and anti-FITC MACS paramagnetic nanoparticles. Measured magnetophoretic mobilities were used to calculate ABC for these cells, corresponding to CD2 expression levels. CONCLUSION: The results from CTV and flow cytometry (FCM) qualitatively verify that these fibrosarcoma cells express elevated levels of CD2 molecules with increasing interferon-alpha treatment from 0 to 24 h. The mean basal CD2 expression level, in terms of ABC, was calculated to be 27,000 from CTV analysis, whereas FCM indicates a comparable ABC value of 33,000.

CD2 Antigens↗
PubMed2001

Impact of attachment styles on dream recall and dream content: a test of the attachment hypothesis of REM sleep.

We tested the hypothesis (McNamara 1996; Zborowski and McNamara 1998) that dream recall and dream content would pattern with interpersonal attachment styles. In study I, college student volunteers were assessed on measures of attachment, dream recall, dream content and other psychologic measures. Results showed that participants who were classified as 'high' on an 'insecure attachment' scale were significantly more likely to (a) report a dream, (b) dream 'frequently', and (c) evidence more intense images that contextualize strong emotions in their dreams as compared with participants who scored low on the insecure attachment scale. In study II, 76 community dwelling elderly volunteers completed measures of attachment, and dream recall. Participants whose attachment style was classified as 'preoccupied' were significantly more likely to report a dream and to report dreams with higher mean number of words per dream as compared with participants classified as 'securely' attached or as 'avoidant' or as 'dismissing.' Dream recall was lowest for the avoidant subjects and highest for the preoccupied subjects. These data support the view that rapid eye movement (REM) sleep and/or dreaming function, in part, to promote attachment.

Adult↗
PubMed2000

Magnetophoretic mobilities correlate to antibody binding capacities.

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.

Antigens, Surface↗
PubMed2000

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry.

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.

Cells, Cultured↗
PubMed2000

Study of magnetic particles pulse-injected into an annular SPLITT-like channel inside a quadrupole magnetic field.

Advantages of the continuous magnetic flow sorting for biomedical applications over current, batch-wise magnetic separations include high throughput and a potential for scale-up operations. A continuous magnetic sorting process has been developed based on the quadrupole magnetic field centered on an annular flow channel. The performance of the sorter has been described using the conceptual framework of split-flow thin (SPLITT) fractionation, a derivative of field-flow fractionation (FFF). To eliminate the variability inherent in working with a heterogenous cell population, we developed a set of monodisperse magnetic microspheres of a characteristic magnetization, and a magnetophoretic mobility, similar to those of the cells labeled with a magnetic colloid. The theory of the magnetic sorting process has been tested by injecting a suspension of the magnetic beads into the carrier fluid flowing through the sorter and by comparing the theoretical and experimental recovery versus total flow-rate profiles. The position of the recovery maxima along the total flow-rate axis was a function of the average bead magnetophoretic mobility and the magnetic field intensity. The theory has correctly predicted the position of the peak maxima on the total flow-rate axis and the dependence on the bead mobility and the field intensity, but has not correctly predicted the peak heights. The differences between the calculated and the measured peak heights were a function of the total flow-rate through the system, indicating a fluid-mechanical origin of the deviations from the theory (such as expected of the lift force effects in the system). The well-controlled elution studies using the monodisperse magnetic beads, and the SPLITT theory, provided us with a firm basis for the future sorter evaluation using cell mixtures.

Magnetics↗
PubMed1999

Quantification of cellular properties from external fields and resulting induced velocity: cellular hydrodynamic diameter.

An experimental technique is discussed in which the size distribution of a population of cells is determined by calculating each cell's settling velocity. The settling velocity is determined from microscopically obtained images which were recorded on SVHS tape. These images are then computer imaged and processed, and the cell's location and velocity are determined using a computer algorithm referred to as cell tracking velocimetry (CTV). Experimental data is presented comparing the distribution of human lymphocytes and a human breast cancer cell line, MCF-7, determined using a Coulter counter and the CTV approach.

Algorithms↗
PubMed1999

Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.

Cells, Cultured↗
PubMed1999

Flow rate optimization for the quadrupole magnetic cell sorter.

The quadrupole magnetic cell sorter is a form of split-flow thin-channel (SPLITT) separation device. It employs a quadrupole magnetic field and annular channel geometry. Immunomagnetic labels are used to bind to specific receptors on the surface of the cells of interest. It is the interaction of these labels with the magnetic field that brings about the selective isolation of these cells. The SPLITT separation devices have generally been based on parallel-plate geometry, usually with effectively constant field strength applied across the channel thickness. The nonconstant field strength and annular channel geometry of the magnetic cell sorter require that a new strategy be developed for optimization of inlet and outlet flow rates. We present such a strategy here based on a consideration of certain specific cell trajectories within the system.

Cell Separation↗
PubMed1999

Detection of rare MCF-7 breast carcinoma cells from mixtures of human peripheral leukocytes by magnetic deposition analysis.

BACKGROUND: The presence of malignant breast cancer cells in bone marrow or peripheral blood is a prognostic factor. We tested the capacity of a novel magnetic cell analyzer to detect rare cancer cells in mixtures with human peripheral leukocytes. METHODS: Human peripheral leukocytes were spiked with cells of the MCF-7 line, and the cell mixture was labeled with anti-epithelial membrane antigen antibody and a magnetic colloid. The MCF-7 cells were selectively captured on a magnetic deposition substrate from the flowing leukocyte and MCF-7 cell mixture. RESULTS: The recovery of the MCF-7 cells from the original mixture ranged from 20% to 60%. The limit of detection of the MCF-7 cells was 10(-6) (n = 9). The morphology of the captured cancer cells was well preserved and comparable to that observed in cytospin smears. All deposited cells were located in a small area of 1.4 mm x 6 mm and could be quickly identified with an optical microscope following Wright's staining. CONCLUSIONS: This is a proof-of-principle study using a simplified model of rare cancer cells in a leukocyte mixture. The clinical relevance of the method will be tested in the future by extension to patient bone marrow samples and using antibody cocktails to increase specificity against the breast carcinoma cells.

Antibodies, Monoclonal↗
PubMed1998

Continuous, flow-through immunomagnetic cell sorting in a quadrupole field.

A flow-through quadrupole magnetic cell separator has been designed, built, and evaluated by using a cell model system of human peripheral T lymphocytes (CD4+, CD8+, and CD45+ cells). The immunomagnetic labeling was accomplished by using a sandwich of mouse anti-human monoclonal antibody conjugated to fluorescein isothiocyanate and rat anti-mouse polyclonal antibody conjugated to a colloidal magnetic nanoparticle. The feed and sorted fractions were analyzed by FACScan flow cytometry. The magnetically labeled cells were separated from nonlabeled ones in a flow-through cylindrical column within a quadrupole field, which exerted a radial, outward force on the magnetic cells. The flow rate of the cell samples was 0.1-0.75 ml/min, and the flow rate of sheath fluid was 1.5-33.3 times that of the sample flow rate. The maximum shear stress exerted on the cell was less than 1 dyne/cm2, which was well below the level that would threaten cell integrity and membrane disruption. The maximum magnetic field was 0.765 T at the channel wall, and the gradient was 0.174 T/mm. The highest purity of selected cells was 99.6% (CD8 cells, initial purity of 26%), and the highest recovery of selected cells was 79% (CD4 cells, initial purity of 20%). The maximum throughput of the quadrupole magnetic cell separator was 7,040 cells/s (CD45 cells, initial purity of 5%). Theoretical calculations showed that the throughput can be increased to 10(6) cells/s by a scale-up of the current prototype.

Animals↗
PubMed1998

Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter.

The relationship between cell function and surface marker expression is a subject of active investigation in biology and medicine. These investigations require separating cells of a homogeneous subset into multiple fractions of varying marker expression. We have developed a novel cell sorter, the dipole magnet flow sorter (DMFS), which separates selected T lymphocyte subpopulations, targeted by immunomagnetic colloid, into multiple fractions according to cell surface marker expression, as determined by flow cytometry. A narrow stream of cells is introduced into a sheath of carrier fluid in a rectangular channel while subjected to a perpendicular magnetic force. The special design of the pole pieces ensures a constant magnetic force acting on the magnetically labeled cells in the separation area. Cells are spread across the flow in relation to their magnetophoretic mobility. Separation is achieved by control of the positions of the effluent stream boundaries, which separate fluid volumes with cells of different magnetophoretic mobility. CD4 and CD8 T lymphocytes labeled with primary antibody-fluorescein isothiocyanate (FITC) conjugate and anti-FITC-magnetic colloid are the chosen cell systems. Flow cytometry analysis shows that, for CD4 cells, a three-fold increase in total marker number per cell is observed when comparing the highest to the lowest fluorescence fractions. Similarly, a four-fold increase in total marker number is observed for CD8 cells. We also observed the separation of two dissimilar cell types that differed in expression of the CD4 marker, monocytes and T helper lymphocytes. We believe that this type of separation is applicable to any cells in suspension for which a suitable antibody exists and, due to the comparatively gentle nature of the process, is particularly suitable for the sorting of fragile cells.

Biomedical Engineering↗
PubMed1998

Theoretical analysis of cell separation based on cell surface marker density.

A theoretical analysis was performed to determine the number of fractions a multidisperse, immunomagnetically labeled cell population can be separated into based on the surface marker (antigen) density. A number of assumptions were made in this analysis: that there is a proportionality between the number of surface markers on the cell surface and the number of immunomagnetic labels bound; that this surface marker density is independent of the cell diameter; and that there is only the presence of magnetic and drag forces acting on the cell. Due to the normal distribution of cell diameters, a "randomizing" effect enters into the analysis, and an analogy between the "theoretical plate" analysis of distillation, adsorption, and chromatography can be made. Using the experimentally determined, normal distribution of cell diameters for human lymphocytes and a breast cancer cell line, and fluorescent activated cell screening data of specific surface marker distributions, examples of theoretical plate calculations were made and discussed.

Antigens, Surface↗
PubMed1998

A mechanistic model of plasma filtration.

A model describing the sieving and transmembrane pressure behavior of plasma filtration is developed and numerically simulated. The model assumes a mechanistic criteria for particle passage through a membrane with cylindrical pores. The initial pore diameter distribution and porosity are assumed to be known. Model inputs include the particle diameter distribution, concentration and total flow rate of the permeate plasma solution. Outputs of the model include transmembrane pressure, the time-averaged sieving coefficients, and size distributions of the deposited particles and accumulated filtrate particles. Optimal filtration is characterized by high, stable sieving coefficients for desired particles, high retention of larger particles and relatively small increases in transmembrane pressure. These characteristics are realized for membranes with mean pore diameters equal to or slightly larger than mean permeate particle diameters. Simulations demonstrate that the incorporation of membrane properties into models of plasma filtration is both significant and readily possible.

Biophysical Phenomena↗
PubMed1996

Immunomagnetic isolation of magnetoferritin-labeled cells in a modified ferrograph.

Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mononuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two distinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magnetoferritin. Magnetoferritin was obtained by reconstituting the horse spleen ferritin iron core with superparamagnetic magnetite instead of the usual paramagnetic ferrihydrite crystal. The cell deposition on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition analyzer, the ferrograph. The identity of cells in the magnetic deposits was confirmed by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The immunostaining protocol amplified the number of ferritin molecules per cell surface antigen 20-70 times. Magnetoferritin, but not native ferritin, imparted a sufficient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 ml/min. This study showed that the magnetic moment of magnetoferritin was sufficient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modified ferrograph.

Animals↗