Biomedical subjects

Jeffrey J Chalmers

Publications and source records attributed to Jeffrey J Chalmers.

At least 19 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↗

PubMed2007

Application of immunomagnetic cell enrichment in combination with RT-PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood.

Detection of rare, circulating tumor cells (CTC's) in human peripheral blood is a potential indicator of prognosis and diagnosis in oncology. Typical methods to detect these CTC's are either by immunocytochemistry (ICCS) or RT-PCR. However without accurate, rapid, and reproducible enrichment processes, these detection techniques are labor intensive and/or unreliable. In this article, a repeatable enrichment process that included a flow-through immunomagnetic cell separation system, the quadrupole magnetic sorter (QMS) was optimized with the aid of a statistical analysis software package. The QMS was operated in a negative mode of operation by immunomagnetically targeting normal human peripheral blood lymphocytes (PBL) through the CD45 surface marker. Three head and neck squamous carcinoma cell lines (HNSCC), Detroit-562, SCC-4, and CAL-27, were used to determine the sensitivity of RT-PCR for the epidermal growth factor receptor (EGFR) in spiked PBL. The detection purity needed for detection was found to be one cell in 10(4), one cell in 10(3), and one cell in 10(5) for the Detroit-562, SCC-4, and CAL-27, respectively. The actual number of cancer cells needed for RT-PCR detection ranged from 30 to 1 cell. To mimic the potential concentration of rare CTC present in peripheral blood of cancer patients, the spiking concentration was chosen to be one cancer cell per 10(5) total leukocytes from healthy donors. Using a single step immunomagnetic labeling, the final, optimized enrichment process produced a 57.6 +/- 30.3-fold (n = 6) enrichment of the rare cancer cells with a final cancer cell recovery of (77.8 +/- 6.6)%.

Biomarkers, Tumor↗

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

Comparison of two immunomagnetic separation technologies to deplete T cells from human blood samples.

The objective of this study was to compare the performance of two immunomagnetic separation technologies to deplete T cells from buffy coats of human blood. Specifically, two versions of the commercial MACS(R) Technology: MiniMACS and SuperMACS, and a prototype, flow-through system, the QMS, were evaluated. Peripheral blood mononuclear leukocytes (PBL) were isolated from buffy coats and an immunomagnetic separation of CD3(+) cells was conducted using company and optimized labeling protocols. To mimic peripheral blood containing bone marrow purged hematopoietic stem cells, HSC, CD34 expressing-cells (KG1a) were spiked into PBL prior to T-cell depletion once optimized depletion conditions were determined. Once the labeling protocol was optimized, the MiniMACS system performed well by producing a highly enriched CD3(+) fraction, and a respectable level of depletion of T cells and recovery of KG1a cells in the depleted fraction; an average log(10) depletion of T cells of 2.88 +/- 0.17 and an average recovery of the KG1a cells of 60.8 +/- 5.94% (n = 14). The performance of the SuperMACS system was very similar with an average log(10) depletion of T cells of 2.89 +/- 0.22 and an average recovery of KG1a of 63.1 +/- 8.55% (n = 10). In contrast, the QMS system produced an average log(10) depletion of T cells of 3.98 +/- 0.33 (n = 16) with a corresponding average recovery of 57.9 +/- 16.6% of the spiked CD34+ cells. The aforementioned QMS performance values were obtained using sorting speeds ranging from 2.5 x 10(4) to 1.7 x 10(5) cells per second. It is suggested that the lack of a 100% recovery of the unlabeled KG1a cells is the result of a previously reported "drafting" phenomena which pulls unlabeled cells in the direction of the magnetically labeled cells thereby resulting in loss of the unlabeled cells.

Antigens, CD34↗

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

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↗

PubMed2005

Cell tracking velocimetry as a tool for defining saturation binding of magnetically conjugated antibodies.

BACKGROUND: Continuous flow immunomagnetic separation is an attractive alternative to current batch mode immunomagnetic separation methods because it is capable of high sorting speeds at mild cell conditions, and grants the operator better control of separation process. The control of the separation is dependent on knowledge of the amount of magnetic label attached to the cell (magnetic labeling intensity), however. Determination of the magnetic labeling is accomplished by measuring cell magnetophoretic mobility using a newly developed technique of Cell Tracking Velocimetry (CTV). METHODS: Flow cytometry was used to define the antibody binding characteristics of a fluorescently tagged primary antibody. Subsequently, CTV was used to measure antibody-binding characteristics of a magnetically tagged secondary antibody. RESULTS: The results of this study show that CTV is capable of providing valuable information concerning the cell labeling by magnetically tagged antibodies. It was demonstrated that the magnetically conjugated antibody binding curve exhibits the same exponential increase to saturation characteristics as that seen with the fluorescently tagged antibody. Further, it was shown that the intensity of the secondary magnetic labeling is directly proportional to the intensity of the primary fluorescent label. CONCLUSIONS: CTV is an accurate tool for evaluation of magnetically conjugated antibodies. The ability to determine the intensity of magnetic labeling is necessary for the development of continuous flow immunomagnetic separations based on cell magnetophoresis.

Antibodies, Monoclonal↗

PubMed2005

Magnetic cell sorting.

Antibodies reacting to specific cell surface markers can be bound to magnetic beads and used to specifically capture cells exhibiting the marker. This approach allows the selective enrichment of specific cell subpopulations and as the methods are amenable to use in blood, tissue fluids and culture medium living cells can be recovered that can subsequently be subcultured. Negative cell separation can also be used where undesirable cell types are tagged using magnetic beads and removed from the desirable cell population.

Antibodies↗

PubMed2005

Cell enrichment and immunochemical staining.

Cell populations can be enriched using a number of physicochemical methods based on characteristics, such as density, size, and volume. Centrifugation using density gradients is typically applied to isolate populations with similar characteristics. Cells obtained using these methods can be further authenticated using specific antibodies that react to markers on the cell surface, allowing the identification of cell subtypes.

Animals↗

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↗

PubMed2004

Evaluation of a contraction flow field on hydrodynamic damage to entomopathogenic nematodes-A biological pest control agent.

Mechanized production and delivery of biological pesticides presents challenges because the biological agents must remain viable during these processes. This study evaluates the effect of flow through an abrupt contraction, where flow characteristics similar to that found within bioprocesses and spray equipment are developed, on damage to a benchmark biological pest control agent, entomopathogenic nematodes (EPNs). An opposed-pistons, contraction flow device generated volumetric flow rates ranging between 8.26 cm(3)/s and 41.3 cm(3)/s. Four EPN species were evaluated: Heterorhabditis bacteriophora, Heterorhabditis megidis, Steinernema carpocapsae, and Steinernema glaseri. Damage was quantified by counting living and dead EPNs. Optical and cold field emission scanning electron microscope (CFE-SEM) images provided qualitative information to describe how the damage occurred. The experimental flow field was completely described using FLUENT, a computational fluid dynamics program. Local flow parameters computed in FLUENT were compared to EPN damage. The type and extent of damage varied between EPN species. Damaged Heterorhabditis spp. generally remained whole with an internal rupture located near the center of the body, while Steinernema spp. most often broke into several pieces. The fast-transient stress field generated at the entrance to the contraction caused a momentary tensile loading and then relaxation that damaged the EPNs. At high flow rates, the tensile stresses became large enough to cause failure of the EPN structural membrane. The relative elasticity of the EPN structural membrane may explain the differences in damage observed between the species. It is speculated that the internal rupture of the Heterorhabditis spp. occurred during the processes of stretching and relaxing at the contraction entrance. Appreciable damage was observed at lower average energy dissipation rates for H. bacteriophora (1.23E + 8 W/m(3)), H. megidis (1.72E + 8 W/m(3)), and S. glaseri (2.89E + 8 W/m(3)) compared to S. carpocapsae (3.70E + 8 W/m(3)). Energy dissipation rates within an equipment component should be kept below 1E + 8 W/m(3) to avoid hydrodynamic damage to EPNs. The relationship between average energy dissipation and EPN damage provides important information for future simulation efforts of actual spray equipment components.

Animals↗

PubMed2004

Enrichment of rare cancer cells through depletion of normal cells using density and flow-through, immunomagnetic cell separation.

OBJECTIVE: To develop a reliable technique to enrich for rare cells in blood suspensions using only negative selection steps including a flow-through immunomagnetic cell separations system and by optimizing variables normally encountered during such enrichment processes. METHODS: A human breast cancer cell line was cultivated and spiked at a ratio of 1 cancer cell to 10(5) total leukocytes in buffy coat or 1 cancer cell to 10(8) total cells in whole blood samples. The final, optimized process consisted of: a red cell lysis step, immunomagnetically staining leukocytes with an anti-CD45 PE, anti- MACS sandwich, immunomagnetic sorting using a flow-through system (QMS), and a final cell analysis step using either an automated cell counter, filtration, and visual counting or a cytospin analysis. RESULTS: The final, optimized process produced a final enrichment of the rare cancer cells of 5.17 log(10) and an average, final recovery of 46%. It should be noted that a negative depletion protocol was used (i.e., no labeling of the rare cancer cells was used). CONCLUSIONS: To the authors' knowledge, no examples in the literature exist of a 5.17 log(10) enrichment of cancer cells in human blood using a negative depletion protocol. The closest example is a 4 log(10) enrichment in which two positive magnetic cell separation steps were used (none were used in this study). Ongoing studies are investigating further modifications of the precommercial, prototype flow-through immunmagnetic separation system to increase both the enrichment and recovery rate. However, even at current performance levels, the presented process could significantly improve visual and molecular analysis of rare cells in blood.

Antibodies, Monoclonal↗

PubMed2003

Magnetic cell separation: characterization of magnetophoretic mobility.

Magnetic cell separation has become a popular technique to enrich or deplete cells of interest from a heterogeneous cell population. One important aspect of magnetic cell separation is the degree to which a cell binds paramagnetic material. It is this paramagnetic material that imparts a positive magnetophoretic mobility to the target cell, thus allowing effective cell separation. A mathematical relationship has been developed to correlate magnetic labeling to the magnetophoretic mobility of an immunomagnetically labeled cell. Four parameters have been identified that significantly affect magnetophoretic mobility of an immunomagnetically labeled cell: the antibody binding capacity (ABC) of a cell population, the secondary antibody amplification (psi), the particle-magnetic field interaction parameter (DeltachiV(m)), and the cell diameter (D(c)). The ranges of these parameters are calculated and presented along with how the parameters affect the minimum and maximum range of magnetophoretic mobility. A detailed understanding of these parameters allows predictions of cellular magnetophoretic mobilities and provides control of cell mobility through selection of antibodies and magnetic particle conjugates.

Antigen-Antibody Reactions↗

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

Characterization of antibody binding to three cancer-related antigens using flow cytometry and cell tracking velocimetry.

Proper antibody labeling is a fundamental step in the positive selection/isolation of rare cancer cells using immunomagnetic cell separation technology. Using either a two-step or single-step labeling protocol, we examined a combination of six different antibodies specific for three different antigens (epithelial specific antigen, epithelial membrane antigen, and HER-2/Neu) on two different breast cancer cell lines (HCC1954 and MCF-7). When a two-step labeling protocol was used (i.e., anti-surface marker-fluoroscein-isothiocyanate [FITC] [primary Ab], anti-FITC magnetic colloid [secondary Ab]) saturation of the primary antibody was determined using fluorescence intensity measurements from flow cytometry (FCM). The saturation of the secondary antibody (or saturation of a single-step labeling) was determined using magnetophoretic mobility measurements from cell tracking velocimetry (CTV). When the maximum magnetophoretic mobility was the primary objective, our results demonstrate that the quantities necessary for antibody saturation with respect to fluorescence intensity were generally higher than those recommended by the manufacturer. The results demonstrate that magnetophoretic mobility varies depending on the types of cell lines, primary antibodies, and concentration of secondary magnetic colloid-conjugated antibody. It is concluded that saturation studies are a vital preparatory step in any separation method involving antibody labeling, especially those that require the specificity of rare cell detection.

Antigen-Antibody Reactions↗

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↗

PubMed2003

Red blood cell magnetophoresis.

The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.

Cell Movement↗