Maciej Zborowski

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.

Continuous cell separation using novel magnetic quadrupole flow sorter

Abstract A laboratory prototype of a flow cell sorter based on magnetic quadrupole field was built and evaluated. The magnetic force acting on magnetically labeled cells in such a field has a `centrifugal’ character which provides a basis for the design of a continuous separation process. The sorter was tested on a model cell system of human peripheral lymphocytes labeled with magnetic colloid.

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patients blood from 4.05 × 109 to 8.04 × 103 cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patients blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log10) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534.

Flow Through, Immunomagnetic Cell Separation

A brief, process‐oriented overview of immunologically based cell separation technology is presented. In addition, the design and preliminary experimental data of two unique flow‐through immunomagnetic cell separation devices are presented. The first design is based on a dipole magnetic field, while the second design is basis on a quadrupole magnetic field. The dipole design can “fractionate” an inlet, magnetically labeled, cell stream into different outlet streams on the basis of the degree to which the cell is immunomagnetically labeled. The quadrupole separator splits an inlet, immunomagnetically labeled, cell stream into two outlet streams in which the purity, recovery, and potentially the degree to which the cells are immunomagnetically labeled is controlled by the flow rates in the inlet and outlet flows. A 99% purity and 86% recovery have been achieved with this system. Some distinct advantages of these two systems are the potential of high purity, recovery, and throughput at a cost which is potentially significantly lower than current, comparable technologies.

Rare cell separation and analysis by magnetic sorting.

The separation and or isolation of rare cells using magnetic forces are commonly used and growing in use ranging from simple sample prep for further studies to a FDA approved, clinical diagnostic test. This growth is the result of both the demand to obtain homogeneous rare cells for molecular analysis and the dramatic increases in the power of permanent magnets that even allow the separation of some unlabeled cells based on intrinsic magnetic moments, such as malaria parasite-infected red blood cells.

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.

An instrument to determine the magnetophoretic mobility of labeled, biological cells and paramagnetic particles

Abstract An instrument is described and discussed which can determine the magnetophoretic mobility of immunomagnetically labeled cells and paramagnetic particles. Through the use of a well-characterized magnetic energy gradient and a computer algorithm, cell tracking velocimetry, it is possible to obtain the mean and distribution of the magnetophoretic mobility for samples with greater than 10 3 individuals.

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.

Determination of the magnetic susceptibility of labeled particles by video imaging

Magnetic cell sorting is gaining in popularity as a method to separate and recover viable cells which differ in functionality, but not in physical characteristics. We are developing a continuous cell sorter to overcome the restrictions of current batch methods, which are limited in their throughput and separation efficiency and which can also subject cells to potentially detrimental physical stresses. Of primary importance in the design and operation of a continuous magnetic cell sorter is the degree of cell magnetization, which is characterized by a property called the magnetic susceptibility. Current techniques for measuring susceptibility give only population average values. We describe here a video-based technique for quickly measuring the susceptibility of large numbers of individual particles. Paramagnetic particles are pumped across the interpolar gap of a permanent magnet and the magnetic field-induced deflections are recorded by video microscopy. Susceptibility is computed from force balances and the measured velocities for individual particles. Our results show that this method can provide susceptibility values which agree well with values obtained from other established methods, while additionally providing population statistics not available by the other methods. The versatility of this method is also demonstrated.

Multiparameter Analysis, including EMT Markers, on Negatively Enriched Blood Samples from Patients with Squamous Cell Carcinoma of the Head and Neck

Epithelial to mesenchymal transition (EMT) has been hypothesized as a mechanism by which cells change phenotype during carcinogenesis, as well as tumor metastasis. Whether EMT is involved in cancer metastasis has a specific, practical impact on the field of circulating tumor cells (CTCs). Since the generally accepted definition of a CTC includes the expression of epithelial surface markers, such as EpCAM, if a cancer cell loses its epithelial surface markers (which is suggested in EMT), it will not be separated and/or identified as a CTC. We have developed, and previously reported on the use of, a purely negative enrichment technology enriching for CTCs in the blood of squamous cell carcinoma of the head and neck (SCCHN). This methodology does not depend on the expression of surface epithelial markers. Using this technology, our initial data on SCCHN patient blood indicates that the presence of CTCs correlates with worse disease-free survival. Since our enrichment is not dependent on epithelial markers, we have initiated investigation of the presence of mesenchymal markers in these CTC cells to include analysis of: vimentin, epidermal growth factor receptor, N-cadherin, and CD44. With the aid of confocal microscopy, we have demonstrated not only presumed CTCs that express and/or contain: a nucleus, cytokeratins, vimentin, and either EGFR, CD44, or N-cadherin, but also cells that contain all of the aforementioned proteins except cytokeratins, suggesting that the cells have undergone the EMT process. We suggest that our negative depletion enrichment methodology provides a more objective approach in identifying and evaluating CTCs, as opposed to positive selection approaches, as it is not subjective to a selection bias and can be tailored to accommodate a variety of cytoplasmic and surface markers which can be evaluated to identify a multitude of phenotypic patterns within CTCs from individual patients, including so-called EMT as presented here.