Biomedical subjects

P Stephen Williams

Publications and source records attributed to P Stephen Williams.

11 recordsLinked to original sources

PubMed2007

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis.

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.

Antigens, CD34↗

PubMed2006

Binding affinities/avidities of antibody-antigen interactions: quantification and scale-up implications.

Bioaffinity interactions have been, and continue to be, successfully adapted from nature for use in separation and detection applications. It has been previously reported that the magnetophoretic mobility of labeled cells show a saturation type phenomenon as a function of the concentration of the free antibody-magnetic nanoparticle conjugate which is consistent with other reports of antibody-fluorophore binding. Starting with the standard antibody-antigen relationship, a model was developed which takes into consideration multi-valence interactions, and various attributes of flow cytometry (FCM) and cell tracking velocimetry (CTV) measurements to determine both the apparent dissociation constant and the antibody-binding capacity (ABC) of a cell. This model was then evaluated on peripheral blood lymphocytes (PBLs) labeled with anti CD3 antibodies conjugated to FITC, PE, or DM (magnetic nanoparticles). Reasonable agreements between the model and the experiments were obtained. In addition, estimates of the limitation of the number of magnetic nanoparticles that can bind to a cell as a result of steric hinderance was consistent with measured values of magnetophoretic mobility. Finally, a scale-up model was proposed and tested which predicts the amount of antibody conjugates needed to achieve a given level of saturation as the total number of cells reaches 10(10), the number of cells needed for certain clinical applications, such as T-cell depletions for mismatched bone marrow transplants.

Antibody Affinity↗

PubMed2006

Continuous flow magnetic cell fractionation based on antigen expression level.

Cell separation is important in medical and biological research and plays an increasingly important role in clinical therapy and diagnostics, such as rare cancer cell detection in blood. The immunomagnetic labeling of cells with antibodies conjugated to magnetic nanospheres gives rise to a proportional relationship between the number of magnetic nanospheres attached to the cell and the cell surface marker number. This enables the potential fractionation of cell populations by magnetophoretic mobility (MM). We exploit this feature with our apparatus, the Dipole Magnet Flow Fractionator (DMFF), which consists of an isodynamic magnetic field, an orthogonally-oriented thin ribbon of cell suspension in continuous sheath flow, and ten outlet flows. From a sample containing a 1:1 mixture of immunomagnetically labeled (label+) and unlabeled (label-) cells, we achieved an increase in enrichment of the label+ cell fraction with increasing outlet numbers in the direction of the magnetic field gradient (up to 10-fold). The total recovery of the ten outlet fractions was 90.0+/-7.7%. The mean MM of label+ cells increased with increasing outlet number by up to a factor of 2.3. The postulated proportionality between the number of attached magnetic beads and the number of cell surface markers was validated by comparison of MM measured by cell tracking velocimetry (CTV) with cell florescence intensity measured by flow cytometry.

Antigens↗

PubMed2006

Hemoglobin degradation in malaria-infected erythrocytes determined from live cell magnetophoresis.

During intra-erythrocytic development, malaria trophozoites digest hemoglobin, which leads to parasite growth and asexual replication while accumulating toxic heme. To avoid death, the parasite synthesizes insoluble hemozoin crystals in the digestive vacuole through polymerization of beta-hematin dimers. In the process, the heme is converted to a high-spin ferriheme whose magnetic properties were studied as early as 1936 by Pauling et al. Here, by magnetophoretic cell motion analysis, we provide evidence for a graduated increase of live cell magnetic susceptibility with developing blood-stage parasites, compatible with the increase in hemozoin content and the mechanism used by P. falciparum to avoid heme toxicity. The measured magnetophoretic mobility of the erythrocyte infected with a late-stage schizont form was m = 2.94 x 10(-6) mm3 s/kg, corresponding to the net volume magnetic susceptibility (relative to water) of Deltachi = 1.80 x 10(-6), significantly higher than that of the oxygenated erythrocyte (-0.18x10(-6)) but lower than that of the fully deoxygenated erythrocyte (3.33x10(-6)). The corresponding fraction of hemoglobin converted to hemozoin, calculated based on the known magnetic susceptibilities of hemoglobin heme and hemozoin ferriheme, was 0.50, in agreement with the published biochemical and crystallography data. Magnetophoretic analysis of live erythrocytes could become significant for antimalarial drug susceptibility and resistance determination.

Animals↗

PubMed2005

Cell sorting by one gravity SPLITT fractionation.

The need for innovative separative techniques suitable for the fractionation of biomaterials prompted this investigation into the performance of the gravitational split-flow thin channel (G-SPLITT) system as a cell sorter. The rigorous mathematical description of the separation mechanism allows achievement of fast separation of several million myeloma cells from healthy splenocytes using flow conditions calculated from theory. Separation in G-SPLITT is based on differences in sedimentation rate. For accurate prediction of the optimal working conditions, this parameter was directly measured by cell tracking velocimetry rather than relying on a measure of diameter (by Multisizer) and an assumed density for each cell population. We also discuss the influence of different flow conditions on the effectiveness of separation.

Cell Separation↗

PubMed2005

Establishment and implications of a characterization method for magnetic nanoparticle using cell tracking velocimetry and magnetic susceptibility modified solutions.

Magnetic micro and nanoparticles conjugated to affinity labels have become a significant, commercial reagent. It has been demonstrated that the performance of cell separation systems using magnetic labels is a function of the magnitude of the magnetic force that can be generated through labeling. This magnetic force is proportional to the number of magnetic particles bound to the cell, the magnetic energy gradient, and the particle-field interaction parameter. This particle-field interaction parameter, which is the product of the relative volumetric, magnetic susceptibility and the volume of the micro or nanoparticle, is a fundamental parameter which can be used to characterize the magnetic particles. An experimental technique is presented which measures the volumetric magnetic susceptibility of particles through the use of susceptibility modified solutions and an experimental instrument, Cell Tracking Velocimetry, CTV. Experimental studies were conducted on polystyrene microspheres alone and those bound to four different magnetic nanoparticles. The experimentally determined values of the magnetic susceptibility of the polystyrene microspheres are consistent with values found from literature. Consequently, magnetic susceptibility measurements of these polystyrene microspheres bound with the magnetic nanoparticles combined with particle size measurements using commercial dynamic light scattering instrument allowed estimates of the particle-field interaction parameter to be made for four commercial, magnetic nanoparticles. The value found for MACS beads is close to what is reported from an independent study. The values for MACS beads and Imag beads are found to agree with what is observed from experiments. Finally, an experimental demonstration of the impact that differences in this field interaction parameter has on the labeling of human lymphocytes is presented.

Cell Movement↗

PubMed2004

Control of magnetophoretic mobility by susceptibility-modified solutions as evaluated by cell tracking velocimetry and continuous magnetic sorting.

With the analytical expression for the magnetophoretic mobility of an ideal, linearly polarizable sphere undergoing creeping motion in viscous medium, we have shown that both attractive and repulsive motions are possible in the magnetic field. We have validated theoretical predictions using magnetic monodisperse microspheres of 5.2-microm diameter and nonmagnetic polystyrene microspheres of 6.99-microm diameter suspended in solutions of paramagnetic ions. The microsphere magnetophoretic mobility was measured using a modified particle tracking velocimetry system, developed in-house and called a cell tracking velocimeter. The product of measured mobility and viscosity agrees well with the theoretical prediction, differing only by approximately 11%. Further, a 26% increase in resolution between magnetic and nonmagnetic particle distributions was evaluated when paramagnetic ion carrier was used instead of water. Continuous particle sorting based on differences in magnetophoretic mobility was performed with another device developed by us, the quadrupole magnetic flow sorter (QMS). In the QMS, the introduction of paramagnetic ions into the carrier was effective in suppressing nonspecific crossover (i.e., the transport of low-mobility particles into the magnetic particle fraction) in particles and in biologically relevant red blood cells and thus showed promise as a means of increasing the purity of the magnetic separation.

Journal Article↗

PubMed2003

Splitter imperfections in annular split-flow thin separation channels: experimental study of nonspecific crossover.

The separation performance of a split-flow thin (SPLITT) separation device depends on uniformity of channel thickness and the precise placement of the flow splitters at fixed distances between the channel walls. The observation of nonspecific crossover, that is, the transport of sample materials across the channel thickness without the influence of an applied field, has routinely been taken to indicate the presence of irregularities in splitter shape or placement. Computational fluid dynamics software may be used to predict the influence of splitter imperfections on nonspecific crossover, where it is assumed that sample transport is by convection alone. A previous study has shown how small inlet splitter imperfections can account for the relatively low levels of nonspecific crossover observed with typical annular SPLITT devices. This study, however, could not distinguish between the possible sources of nonspecific crossover; hydrodynamic lift or shear-induced diffusion could have contributed. To confirm the validity of the computational approach, a series of experiments has been carried out on a channel having a deliberately and severely bent splitter. Nonspecific crossover was measured for a range of inlet and outlet flow rate ratios, with the bent splitter placed at both the channel inlet and outlet. The severity of the splitter distortion was sufficient to produce significant nonspecific crossover over a wide range of flow conditions. Good agreement was found between experiment and prediction based on computational fluid dynamics, with experiment generally showing only slightly higher crossover than prediction. The quantitative agreement for this extreme case suggests that the contribution to nonspecific crossover due to geometrical imperfections can be well described using computational fluid dynamics.

Chemical Fractionation↗

PubMed2003

Magnitude and direction of thermal diffusion of colloidal particles measured by thermal field-flow fractionation.

In this paper we provide experimental evidence showing that various types of submicrometer-sized particles (latexes, inorganic, and metallic), suspended in either aqueous or nonaqueous carrier liquids to which a temperature gradient dT/dx is applied, experience a force in the direction opposite to that of dT/dx. This behavior is similar to that of small particles such as soot, aerosols, and small bubbles suspended in stagnant gases across which temperature gradients are applied, a phenomenon known as "thermophoresis in gases." We report the use of a thermal field-flow fractionation (ThFFF) apparatus in two different configurations to establish the direction of particle motion subject to a temperature gradient. The first approach employed the conventional horizontal ThFFF channel orientation. In this case, small electrical potentials were applied across the narrow channel thickness either to augment or to act in opposition to the applied thermal gradient, depending on whether the accumulation wall was maintained at a positive or negative potential relative to the depletion wall. Thus, by observing the changes in the retention behavior of surface-charged latices or silica particles with changes in potential difference across the channel thickness, we were able to ascertain the direction of migration of the particles in the thermal gradient. The second approach involved the use of a ThFFF column oriented vertically in an implementation of a technique known as thermogravitational FFF. In this approach, the convective flow along the channel length (due to density gradients associated with the temperature gradient) couples with the thermal diffusion effect across the channel thickness to result in a combined particle retention mechanism. A retarded upward migration rate is indicative of accumulation of particles at the cold wall, while enhanced upward migration would indicate a hot-wall accumulation. From the results of our investigations, we conclude that submicrometer-sized particles suspended in either aqueous or nonaqueous carrier liquids and subjected to a temperature gradient migrate from the hot wall toward the cold wall of a ThFFF channel.

Journal Article↗

PubMed2003

Splitter imperfections in annular split-flow thin separation channels: effect on nonspecific crossover.

The separation performance of split-flow thin (SPLITT) separation channels generally falls short of ideal behavior. There are many possible contributing factors to the loss of separation resolution, and these are discussed in the text. The possibility that small imperfections in the splitters play a significant role is examined in this study. Computational fluid dynamics is used to determine the flow pattern within an annular SPLITT channel having small imperfections in the inlet splitter. These results are used to calculate the nonspecific crossover of particles from the inner annular inlet to the outer annular outlet under various flow rate regimes. Nonspecific crossover, obtained through convective transport alone, and not the result of field-induced transport, is often used as a check of channel behavior. The results of a typical experimental determination of nonspecific crossover are included for comparison. It is concluded that geometrical imperfections can indeed play a significant role in the loss of resolution observed for these systems.

Cell Separation↗

PubMed2002

Field and flow programming in frit-inlet asymmetrical flow field-flow fractionation.

The separation of wide molecular mass (Mr) ranges of macromolecules using frit inlet asymmetrical flow field-flow fractionation (FI-AFlFFF) has been improved by implementing a combination of field and flow programming. In this first implementation, field strength (governed by the cross flow-rate through the membrane-covered accumulation wall) is decreased with time to obtain faster elution and improved detection of the more strongly retained (high Mr) materials. The channel outlet flow-rate is optionally held constant, increased, or decreased with time. With circulation of the flow exiting the accumulation wall to the inlet frit, the dual programming of cross flow and channel outlet flow could be implemented using just two pumps. With this flow configuration, the channel outlet flow-rate is always equal to the channel inlet flow-rate, and these may be programmed independently of the cross flow-rate through the membrane. FI-AFlFFF retains its operational advantage over conventional asymmetrical flow FFF (AFlFFF). Unlike conventional AFlFFF, FI-AFlFFF does not require time consuming, and experimentally inconvenient, sample focusing and relaxation steps involving valve switching and interruption of sample migration. The advantages of employing dual programming with FI-AFlFFF are demonstrated for sets of polystyrene sulfonate standards in the molecular mass range of 4 to 1000 kDa. It is shown that programmed FI-AFlFFF successfully expands the dynamic separation range of molecular mass.

Chromatography, High Pressure Liquid↗