Publications by authors named "Jeffrey J Chalmers"

82 Publications

Formation and manipulation of ferrofluid droplets with magnetic fields in a microdevice: a numerical parametric study.

Soft Matter 2020 Oct;16(41):9506-9518

Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Buffalo, New York 14260, USA.

We present a numerical model that describes the microfluidic generation and manipulation of ferrofluid droplets under an external magnetic field. We developed a numerical Computational Fluid Dynamics (CFD) analysis for predicting and optimizing continuous flow generation and processing of ferrofluid droplets with and without the presence of a permanent magnet. More specifically, we explore the dynamics of oil-based ferrofluid droplets within an aqueous continuous phase under an external inhomogeneous magnetic field. The developed model determines the effect of the magnetic field on the droplet generation, which is carried out in a flow-focusing geometry, and its sorting in T-junction channels. Three-channel depths (25 μm, 30 μm, and 40 μm) were investigated to study droplet deformation under magnetic forces. Among the three, the 30 μm channel depth showed the most consistent droplet production for the studied range of flow rates. Ferrofluids with different loadings of magnetic nanoparticles were used to observe the behavior for different ratios of magnetic and hydrodynamic forces. Our results show that the effect of these factors on droplet size and generation rate can be tuned and optimized to produce consistent droplet generation and sorting. This approach involves fully coupled magnetic-fluid mechanics models and can predict critical details of the process including droplet size, shape, trajectory, dispensing rate, and the perturbation of the fluid co-flow for different flow rates. The model enables better understanding of the physical phenomena involved in continuous droplet processing and allows efficient parametric analysis and optimization.
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http://dx.doi.org/10.1039/d0sm01426eDOI Listing
October 2020

Self-Assembly and sedimentation of 5 nm SPIONs using horizontal, high magnetic fields and gradients.

Sep Purif Technol 2020 Oct 4;248. Epub 2020 May 4.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 320 Koffolt Laboratories, 151 West Woodruff Avenue, Columbus, OH 43210, USA.

Superparamagnetic iron oxide nanoparticles (SPIONs) are employed in multiple applications, especially within medical and chemical engineering fields. However, their magnetic separation is very challenging as the magnetophoretic motion is hindered by thermal energy and viscous drag. Recent studies have addressed the recovery of SPIONs by a combination of cooperative magnetophoresis and sedimentation. Nevertheless, the effect of horizontal, high fields and gradients on the vertical sedimentation of SPIONs has not been described. In this work, we report, for the first time, the magnetically facilitated sedimentation of 5 nm particles by applying fields and gradients perpendicular to gravity. The magnetic field was generated by quadrupole magnetic sorters and the process was measured with time by tracking the concentration along the length of a channel contacting the 5 nm SPIONs within the quadrupole field. Our experimental data suggest that aggregates of 60-90 particles are formed in the system; thus, particle agglomeration by dipole-dipole interactions was promoted, and these clusters settled down as a result of gravitational forces. Multiple variables and parameters were evaluated, including the initial SPION concentration, the temperature, the magnetic field and gradient and operation time. It was found that the process was improved by decreasing the initial concentration and the temperature, but the magnitude of the magnetic field and gradient did not significantly affect the sedimentation. Finally, the separation process was rapid, with the systems reaching the equilibrium in approximately 20 minutes, which is a significant advantage in comparison to other systems that require longer times and larger particle sizes.
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http://dx.doi.org/10.1016/j.seppur.2020.117012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7351086PMC
October 2020

Hyperferritinemia in critically ill COVID-19 patients - Is ferritin the product of inflammation or a pathogenic mediator?

Clin Chim Acta 2020 Oct 21;509:249-251. Epub 2020 Jun 21.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 151 West Woodruff Avenue, Columbus, OH 43210, United States. Electronic address:

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http://dx.doi.org/10.1016/j.cca.2020.06.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7306200PMC
October 2020

Continuous-Flow Separation of Magnetic Particles from Biofluids: How Does the Microdevice Geometry Determine the Separation Performance?

Sensors (Basel) 2020 May 27;20(11). Epub 2020 May 27.

Department of Chemical and Biomolecular Engineering, ETSIIT, University of Cantabria, Avda. Los Castros s/n, 39005 Santander, Spain.

The use of functionalized magnetic particles for the detection or separation of multiple chemicals and biomolecules from biofluids continues to attract significant attention. After their incubation with the targeted substances, the beads can be magnetically recovered to perform analysis or diagnostic tests. Particle recovery with permanent magnets in continuous-flow microdevices has gathered great attention in the last decade due to the multiple advantages of microfluidics. As such, great efforts have been made to determine the magnetic and fluidic conditions for achieving complete particle capture; however, less attention has been paid to the effect of the channel geometry on the system performance, although it is key for designing systems that simultaneously provide high particle recovery and flow rates. Herein, we address the optimization of Y-Y-shaped microchannels, where magnetic beads are separated from blood and collected into a buffer stream by applying an external magnetic field. The influence of several geometrical features (namely cross section shape, thickness, length, and volume) on both bead recovery and system throughput is studied. For that purpose, we employ an experimentally validated Computational Fluid Dynamics (CFD) numerical model that considers the dominant forces acting on the beads during separation. Our results indicate that rectangular, long devices display the best performance as they deliver high particle recovery and high throughput. Thus, this methodology could be applied to the rational design of lab-on-a-chip devices for any magnetically driven purification, enrichment or isolation.
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http://dx.doi.org/10.3390/s20113030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308945PMC
May 2020

Phase I Study of Veliparib on an Intermittent and Continuous Schedule in Combination with Carboplatin in Metastatic Breast Cancer: A Safety and [18F]-Fluorothymidine Positron Emission Tomography Biomarker Study.

Oncologist 2020 08 17;25(8):e1158-e1169. Epub 2020 Jun 17.

Stefanie Spielman Comprehensive Breast Center, The Ohio State University, Columbus, Ohio, USA.

Background: Poly(ADP-ribose) polymerase inhibitors (PARPis) are U.S. Food and Drug Administration (FDA) approved for treatment of BRCA-mutated metastatic breast cancer. Furthermore, the BROCADE studies demonstrated benefit of adding an oral PARPi, veliparib, to carboplatin and paclitaxel in patients with metastatic breast cancer harboring BRCA mutation. Given multiple possible dosing schedules and the potential benefit of this regimen for patients with defective DNA repair beyond BRCA, we sought to find the recommended phase II dose (RP2D) and schedule of veliparib in combination with carboplatin in patients with advanced breast cancer, either triple-negative (TNBC) or hormone receptor (HR)-positive, human epidermal growth receptor 2 (HER2) negative with defective Fanconi anemia (FA) DNA-repair pathway based on FA triple staining immunofluorescence assay.

Materials And Methods: Patients received escalating doses of veliparib on a 7-, 14-, or 21-day schedule with carboplatin every 3 weeks. Patients underwent [18]fluoro-3'-deoxythymidine ( FLT) positron emission tomography (PET) imaging.

Results: Forty-four patients (39 TNBC, 5 HR positive/HER2 negative with a defective FA pathway) received a median of 5 cycles (range 1-36). Observed dose-limiting toxicities were grade (G) 4 thrombocytopenia (n = 4), G4 neutropenia (n = 1), and G3 akathisia (n = 1). Common grade 3-4 toxicities included thrombocytopenia, lymphopenia, neutropenia, anemia, and fatigue. Of the 43 patients evaluable for response, 18.6% achieved partial response and 48.8% had stable disease. Median progression-free survival was 18.3 weeks. RP2D of veliparib was established at 250 mg twice daily on days 1-21 along with carboplatin at area under the curve 5. Patients with partial response had a significant drop in maximum standard uptake value (SUV ) of target lesions between baseline and early in cycle 1 based on FLT-PET (day 7-21; p  = .006).

Conclusion: The combination of continuous dosing of veliparib and every-3-week carboplatin demonstrated activity and an acceptable toxicity profile. Decrease in SUV on FLT-PET scan during the first cycle of this therapy can identify patients who are likely to have a response.

Implications For Practice: The BROCADE studies suggest that breast cancer patients with BRCA mutation benefit from addition of veliparib to carboplatin plus paclitaxel. This study demonstrates that a higher dose of veliparib is tolerable and active in combination with carboplatin alone. With growing interest in imaging-based early response assessment, the authors demonstrate that decrease in [18]fluoro-3'-deoxythymidine positron emission tomography (FLT-PET) SUV during cycle 1 of therapy is associated with response. Collectively, this study established a safety profile of veliparib and carboplatin in advanced breast cancer while also providing additional data on the potential for FLT-PET imaging modality in monitoring therapy response.
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http://dx.doi.org/10.1634/theoncologist.2020-0039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418347PMC
August 2020

Quantification of the Mean and Distribution of Hemoglobin Content in Normal Human Blood Using Cell Tracking Velocimetry.

Anal Chem 2020 01 7;92(2):1956-1962. Epub 2020 Jan 7.

William G. Lowrie Department of Chemical and Biomolecular Engineering , The Ohio State University , 315 Koffolt Laboratories, 151 West Woodruff Avenue , Columbus , Ohio 43210 , United States.

The current clinical method for detecting anemia focuses on measuring the concentration of hemoglobin (Hb) in blood. However, recent developments in particle tracking algorithms and the understanding of the relationship between Hb and magnetism has enabled the quantitative measurement of the Hb content in a single red blood cell, RBC, based on magnetophoretic mobility. To further explore this relationship, 22 human blood samples obtained from 17 healthy volunteers were analyzed by the cell tracking velocimetry system, and the calculated Hb concentration from these measurements was compared to the values measured by UV-visible spectrophotometry, the standard method for measuring Hb in clinical laboratories. The results show close correlations between the mean of the spectrophotometric and magnetophoretic methods; however, single cell analysis with the magnetophoretic mobility method allows further elucidation of the distribution of Hb concentration within RBCs from a donor sample to be determined. Histograms of these magnetophoretic mobility distributions indicate that the fraction of RBCs that are below the bulk Hb concentration that defines anemia varies not only from donor to donor but also in the same donor over time. Consistent with a variable fraction below the anemic Hb concentration, the distribution around the mean has a large range. Previous studies have indicated that RBCs lose Hb during ex vivo storage; however, it is not known if this variability in the distribution of Hb content is a function of the age of the RBCs in a donor, suggesting a variable rate in RBC production between donors, or variability in available iron at the time of RBC formation. We suggest our cell tracking velocimetry system can reveal more information regarding this matter.
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http://dx.doi.org/10.1021/acs.analchem.9b04302DOI Listing
January 2020

Biomolecular detection, tracking, and manipulation using a magnetic nanoparticle-quantum dot platform.

J Mater Chem B 2020 04;8(16):3534-3541

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.

Fluorescent and magnetic materials play a significant role in biosensor technology, enabling sensitive quantification and separations with applications in diagnostics, purification, quality control, and therapeutics. Here, we present a magneto-fluorescent biosensor/separations platform consisting of quantum dots (QDs) and superparamagnetic iron oxide nanoparticles (SPIONs) that are separately encapsulated in amphiphilic block co-polymer micelles conjugated to DNA or protein (i.e., single-stranded (ss) DNA derived from the mRNA of the tumor suppressor protein p53 or avidin protein). Analytes were detected via an aggregation sandwich assay upon binding of at least 1 QD and 1 SPION-containing micelle to result in a fluorescent/magnetic composite. Multiplexed isolation of protein and DNA biomolecules was demonstrated by using QDs of varying emission wavelength; QD fluorescence intensity could be correlated with analyte concentration. Sequential or parallel biomolecule separation was achieved by adding appropriately functionalized SPION-containing micelles and applying user-controlled magnetic fields via patterned magnetic disks and wires. QD fluorescence was used to continuously visualize analyte separation during this process. This QD/SPION platform is simple to use, demonstrates ∼10-16 M sensitivity in analyte detection (comparable to competing QD biosensors based on energy transfer) with specificity against 1 and 2 basepair mismatches in DNA detection, molecular separations capability in solutions of ∼10-10 M, and permits simultaneous or parallel, multiplexed separation of protein and DNA. Thus, this versatile platform enables self-assembly-based rapid, sensitive, and specific detection and separation of biomolecules, simultaneously and with real-time visualization. This technology demonstrates potential for nanoscale assembly, biosensing, and bioseparations.
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http://dx.doi.org/10.1039/c9tb02481fDOI Listing
April 2020

A Subpopulation of Monocytes in Normal Human Blood Has Significant Magnetic Susceptibility: Quantification and Potential Implications.

Cytometry A 2019 05 8;95(5):478-487. Epub 2019 Apr 8.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio.

The presence of iron in circulating monocytes is well known as they play essential roles in iron recycling. Also, the storage of this metal as well as its incorrect uptake and/or release are important data to diagnose different pathologies. It has been demonstrated that iron storage in human blood cells can be measured through their magnetic behavior with high accuracy; however, the magnetic characteristics of monocytes have not been reported so far to the best of our knowledge. Therefore, in this work, we report, for the first time, the physical and magnetic properties of human monocytes, along with plasma platelets, oxyhemoglobin red blood cells (oxyHb-RBCs), and methemoglobin red blood cells (metHb-RBCs). The different cell populations were separated by Ficoll-density gradient centrifugation, followed by a flow sorting step to isolate monocytes from peripheral blood mononuclear cells. The different fractions were analyzed by Coulter Counter (for determining the size distribution and concentration) and the sorted monocytes were qualitatively analyzed on ImageStream, a state-of-the-art imaging cytometer. The analysis of the Coulter Counter and ImageStream data suggests that although there exists contamination in the monocyte fraction, the integrity of the sorted monocytes appears to be intact and the concentration was high enough to precisely measure their magnetic velocity by Cell Tracking Velocimetry. Surprisingly, monocytes reported the highest magnetic mobility from the four fractions under analysis, with an average magnetic velocity 7.8 times higher than MetHb-RBCs, which is the only type of cells with positive magnetic velocities. This value is equivalent to a susceptibility 2.5 times higher than the value reported by fresh MetHb-RBCs. It should be noted that this is the first study that reports that a subpopulation of human monocytes is much more magnetic than MetHb-RBCs, opening the door to the possible isolation of human monocytes by label-free magnetic techniques. Further, it is suggested that these magnetic monocytes could "contaminate" positively selected, immunomagnetically labeled blood cells (i.e., during a process using magnetically conjugated antibodies targeting cells, such as CD34 positive cells). Conversely, these magnetic monocytes could be inadvertently removed from a desired blood population when one is using a negative magnetic isolation technique to target cells for removal. © 2019 International Society for Advancement of Cytometry.
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http://dx.doi.org/10.1002/cyto.a.23755DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6985909PMC
May 2019

Quantitative characterization of the regulation of iron metabolism in glioblastoma stem-like cells using magnetophoresis.

Biotechnol Bioeng 2019 07 24;116(7):1644-1655. Epub 2019 Apr 24.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, Ohio.

This study focuses on different iron regulation mechanisms of glioblastoma (GBM) cancer stem-like cells (CSCs) and non-stem tumor cells (NSTCs) using multiple approaches: cell viability, density, and magnetophoresis. GBM CSCs and NSTCs were exposed to elevated iron concentration, and their magnetic susceptibility was measured using single cell magnetophoresis (SCM), which tracks the magnetic and settling velocities of thousands of individual cells passing through the magnetic field with a constant energy gradient. Our results consistently demonstrate that GBM NSTCs have higher magnetic susceptibility distribution at increased iron concentration compared with CSCs, and we speculate that it is because CSCs have the ability to store a high amount of iron in ferritin, whereas the free iron ions inside the NSTCs lead to higher magnetic susceptibility and reduced cell viability and growth. Further, their difference in magnetic susceptibility has led us to pursue a separate experiment using a quadrupole magnetic separator (QMS), a novel microfluidic device that uses a concentric channel and permanent magnets in a special configuration to separate samples based on their magnetic susceptibilities. GBM CSCs and NSTCs were exposed to elevated iron concentration, stained with two different trackers, mixed and introduced into QMS; subsequently, the separated fractions were analyzed by fluorescent microscopy. The separation results portray a successful label-less magnetic separation of the two populations.
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http://dx.doi.org/10.1002/bit.26973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6693654PMC
July 2019

Single cell magnetometry by magnetophoresis vs. bulk cell suspension magnetometry by SQUID-MPMS - a comparison.

J Magn Magn Mater 2019 Mar 28;474:152-160. Epub 2018 Oct 28.

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, OH 44195, United States.

Paramagnetic constituents of a cell have strong effect on cell's volume magnetic susceptibility even at low volume fraction because of their high susceptibility relative to that of the diamagnetic cell constituents. The effect can be measured at a single cell level by measuring cell terminal velocity in viscous media using a microscope equipped with a well-defined field and gradient magnet configuration (referred to as magnetophoretic analysis by cell tracking velocimetry, CTV). The sensitivity of such a microscopic-scale magnetometry was compared to that of a reference method of superconducting quantum interference-magnetic properties measurement system (SQUID-MPMS) using a red blood cell (RBC) suspension model. The RBC hemoglobin oxygen saturation determines the hemoglobin molecular magnetic susceptibility (diamagnetic when fully oxygenated, paramagnetic when fully deoxygenated or converted to methemoglobin). The SQUID-MPMS measurements were performed on an average of 5,000 RBCs in 20 μL physiological phosphate buffer at room temperature, those by CTV on a single cell track in a mean magnetic field of 1.6 T and mean gradient of 240 T/m, repeated for an average of 1,000 tracks per sample. This suggests 5,000× higher sensitivity of cell susceptometry by magnetophoretic analysis than by SQUID-MPMS. The magnetophoretic mean RBC magnetic susceptibilities were in the range determined by SQUID-MPMS (lower limit) and theory (upper limit). The ability of magnetophoretic analysis to resolve susceptibility peaks in a mixed cell populations was confirmed for an oxy RBC and met RBC mixture. Magnetophoretic analysis by CTV provides new tool for studies of emergence of paramagnetic reaction products in the cell.
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http://dx.doi.org/10.1016/j.jmmm.2018.10.108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453790PMC
March 2019

Single cell analysis of aged RBCs: quantitative analysis of the aged cells and byproducts.

Analyst 2019 Jan;144(3):935-942

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, 320 Koffolt Laboratories, 151 West Woodruff Avenue, Columbus, OH 43210, USA.

This study initially focused on characterizing the aging process of red blood cells by correlating the loss of hemoglobin and the translocation of phosphatidylserine (PS) in expired human red blood cells, hRBCs. Five pre-storage, leukoreduced hRBC units in AS-5 solution were stored between 1 and 6 °C for 42 days. Aliquots from each of these units were stained with Annexin-V FLUOS, which binds to externalized PS, and the hemoglobin within the cells was placed in a methemoglobin state with sodium nitrite, metHb. These aliquots were subsequently sorted into four sub-populations, ranging from no PS expression to high PS expression using a BD FACS ARIAIII. Each of these sub-fractions were introduced into the cell tracking velocimetry apparatus which measured both the magnetically-induced and the gravity-induced velocity. Subsequently, the samples were removed from the cell tracking velocimetry instrument and characterized using the Multisizer 4e Coulter Counter. From the magnetically-induced velocity, the amount of hemoglobin, in pg Hb per cell can be determined, and using an average value of the density of RBCs, the size can be determined. For the PS negative sub-fraction of RBCs, the size of the RBC was as expected but the average hemoglobin, Hb, content was below the threshold which defines anemia. In contrast, unexpected results were observed with the various levels of expression of PS. First, virtually all of the PS expressing cells were significantly smaller, on the order of 1 micron, than a normal RBC after 42 days of storage; yet the density of these small cells/microvesicles was such that they had settling velocities similar to normal-sized RBCs. Further, while the total amount of Hb per small cell/microvesicle was only approximately 25% of the full-sized RBCs, the volume of these small cells/microvesicles is only 1/200 of the PS negative RBCs. This suggests that these PS expressing cells are shrunken RBCs, or shrunken microvesicles from RBCs that concentrated the Hb internally. These results suggest not only a relationship between the loss of hemoglobin and the amount of PS exposed on the cellular outer wall, but also a mechanism by which these aged RBCs break down. It is not known at this time whether this is an artifact of storage or similar mechanisms occur in circulation within the human body.
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http://dx.doi.org/10.1039/c8an01904eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6506859PMC
January 2019

Implementing Liquid Biopsies in Clinical Trials: State of Affairs, Opportunities, and Challenges.

Cancer J 2018 Mar/Apr;24(2):61-64

A primary goal of personalized medicine is to develop tumor-specific biomarkers to aid in treatment selection and to better evaluate response to targeted therapies. The assessment of circulating blood markers as surrogate real-time biopsies of disease status, termed liquid biopsies, has been under investigation. There are many different types of liquid biopsies each with different functionalities and limitations. These include tumor markers, circulating tumor cells, cell-free DNA, and extracellular vesicles including exosomes. Multiple clinical trials have evaluated liquid biopsies as prognostic biomarkers with positive results. Additional studies are underway to evaluate liquid biopsies as predictive biomarkers, pharmacodynamic biomarkers, and surrogate efficacy endpoints for treatment response evaluation. There are several challenges in and barriers to implementation of liquid biopsies into clinical trials and subsequently into routine clinical practice, which are addressed in this review.
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http://dx.doi.org/10.1097/PPO.0000000000000309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5880324PMC
March 2019

Continuous, intrinsic magnetic depletion of erythrocytes from whole blood with a quadrupole magnet and annular flow channel; pilot scale study.

Biotechnol Bioeng 2018 06 13;115(6):1521-1530. Epub 2018 Mar 13.

William G. Lowrie Department of Chemica, The Ohio State University, Columbus, Ohio.

The ability to separate RBCs from the other components of whole blood has a number of useful clinical and research applications ranging from removing RBCs from typical clinical blood draw, bone marrow transplants to transfusions of these RBCs to patients after significant blood loss. Viewed from a mechanistic/process perspective, there are three routine methodologies to remove RBCs: 1) RBCs lysis, 2) separation of the RBCs from the nucleated cells (i.e., stem cells) based on density differences typically facilitated through centrifugation or sedimentation agents, and 3) antibody based separation in which a targeted RBC is bound with an affinity ligand that facilitates its removal. More recently, several microfluidic based techniques have also been reported. In this report, we describe the performance of continuous RBC separation achieved by the deflection of intrinsically magnetic, deoxygenated RBCs as they flow through a magnetic energy gradient created by quadrupole magnet. This quadrupole magnetic, with aperture of 9.65 mm, has a maximum field of B  = 1.36 T at the pole tips and a constant field gradient of B /r  = 286 T/m. The annular flow channel, contained within this quadrupole magnet, is 203 mm long, has an inner radius of 3.98 mm, and an inner, outer radius of 4.36 mm, which corresponds to an annulus radius of 380 micrometer. At the entrance and exit to this annular channel, a manifold was designed which allows a cell suspension and sheath fluid to be injected, and a RBC enriched exit flow (containing the magnetically deflected RBCs) and a RBC depleted exit flow to be collected. Guided by theoretical models previously published, a limited number of operating parameters; total flow rate, flow rate ratios of flows in and flow out, and ratios of RBC to polystyrene control beads was tested. The overall performance of this system is consistent with our previously presented, theoretical models and our intuition. As expected, the normalized recovery of RBCs in the RBC exit fraction ranged from approximately 95% down to 60%, as the total flow rate through the system increased from 0.1 to 0.6 ml/min. At the cell concentrations studied, this corresponds to a flow rate of 1.5 × 10 -9 × 10  cells/min. While the throughput of these pilot scale studies are slow for practical applications, the general agreement with theory, and the small cross-sectional area in which the actual separation is achieved, 77 mm (annulus radius times the length), and corresponding volume of approximately 2 mls, suggests the potential to scale-up a system for practical applications exists and is actively being pursued.
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http://dx.doi.org/10.1002/bit.26581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311700PMC
June 2018

Correlation of simulation/finite element analysis to the separation of intrinsically magnetic spores and red blood cells using a microfluidic magnetic deposition system.

Biotechnol Bioeng 2018 05 9;115(5):1288-1300. Epub 2018 Feb 9.

William G. Lowrie Department of Chemical and Biomolecular Engineering Director, Analytical Cytometry Shared Resource, The OSU Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio.

Magnetic separation of cells has been, and continues to be, widely used in a variety of applications, ranging from healthcare diagnostics to detection of food contamination. Typically, these technologies require cells labeled with antibody magnetic particle conjugate and a high magnetic energy gradient created in the flow containing the labeled cells (i.e., a column packed with magnetically inducible material), or dense packing of magnetic particles next to the flow cell. Such designs, while creating high magnetic energy gradients, are not amenable to easy, highly detailed, mathematic characterization. Our laboratories have been characterizing and developing analysis and separation technology that can be used on intrinsically magnetic cells or spores which are typically orders of magnitude weaker than typically immunomagnetically labeled cells. One such separation system is magnetic deposition microscopy (MDM) which not only separates cells, but deposits them in specific locations on slides for further microscopic analysis. In this study, the MDM system has been further characterized, using finite element and computational fluid mechanics software, and separation performance predicted, using a model which combines: 1) the distribution of the intrinsic magnetophoretic mobility of the cells (spores); 2) the fluid flow within the separation device; and 3) accurate maps of the values of the magnetic field (max 2.27 T), and magnetic energy gradient (max of 4.41 T /mm) within the system. Guided by this model, experimental studies indicated that greater than 95% of the intrinsically magnetic Bacillus spores can be separated with the MDM system. Further, this model allows analysis of cell trajectories which can assist in the design of higher throughput systems.
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http://dx.doi.org/10.1002/bit.26550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338348PMC
May 2018

Tessellated permanent magnet circuits for flow-through, open gradient separations of weakly magnetic materials.

J Magn Magn Mater 2017 Apr 15;427:325-330. Epub 2016 Nov 15.

Department of Biomedical Engineering, Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195.

Emerging microfluidic-based cell assays favor label-free red blood cell (RBC) depletion. Magnetic separation of RBC is possible because of the paramagnetism of deoxygenated hemoglobin but the process is slow for open-gradient field configurations. In order to increase the throughput, periodic arrangements of the unit magnets were considered, consisting of commercially available Nd-Fe-B permanent magnets and soft steel flux return pieces. The magnet design is uniquely suitable for multiplexing by magnet tessellation, here meaning the tiling of the magnet assembly cross-sectional plane by periodic repetition of the magnet and the flow channel shapes. The periodic pattern of magnet magnetizations allows a reduction of the magnetic material per channel with minimal distortion of the field cylindrical symmetry inside the magnet apertures. A number of such magnet patterns are investigated for separator performance, size and economy with the goal of designing an open-gradient magnetic separator capable of reducing the RBC number concentration a hundred-fold in 1 mL whole blood per hour.
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http://dx.doi.org/10.1016/j.jmmm.2016.11.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5667671PMC
April 2017

Magnetic Quantum Dots Steer and Detach Microtubules From Kinesin-Coated Surfaces.

Biotechnol J 2018 Jan 9;13(1). Epub 2017 Oct 9.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH, USA.

The microtubule (MT)-kinesin system has been extensively studied because of its role in cellular processes, as well as its potential use for controllably transporting objects at the nanoscale. Thus, there is substantial interest in methods to evaluate MT properties, including bending radius and the binding energy of kinesin motor proteins to MT tracks. Current methods to identify these properties include optical tweezers, microfluidic devices, and magnetic fields. Here, the use of magnetic quantum dots (i.e., MagDots) is evaluated as a method to study MT-kinesin interactions via applied magnetic forces. Magnetic fields are generated using a magnetic needle whose field gradient is quantified by finite element modeling (FEM). Magnetic force is applied to MagDot-labeled MTs and demonstrated sufficient to steer and detach MTs from kinesin-coated surfaces. Taking advantage of the dual-functionality of MagDots, the magnetic force experienced by a single MagDot and the number of MagDots on MTs are determined. The total force exerted on MTs by MagDots is estimated to be ≈0.94-2.47 pN. This approach could potentially be used to interrogate MT properties and MT-kinesin interactions, enhancing our biological understanding of this system and enabling further development of MT shuttles for nanotransport.
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http://dx.doi.org/10.1002/biot.201700402DOI Listing
January 2018

Multispectral Imaging Analysis of Circulating Tumor Cells in Negatively Enriched Peripheral Blood Samples.

Methods Mol Biol 2017 ;1634:219-234

William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, 151 W. Woodruff Ave., Columbus, OH, 43210, USA.

A variety of biomarkers are present on cells in peripheral blood of patients with a variety of disorders, including solid tumor malignancies. While rare, characterization of these cells for specific protein levels with the advanced technology proposed, will lead to future validation studies of blood samples as "liquid biopsies" for the evaluation of disease status and therapeutic response. While circulating tumor cells (CTCs) have been isolated in the blood samples of patients with solid tumors, the exact role of CTCs as clinically useful predictive markers is still debated. Current commercial technology has significant bias in that a positive selection technology is used that preassumes specific cell surface markers (such as EpCAM) are present on CTCs. However, CTCs with low EpCAM expression have been experimentally demonstrated to be more likely to be missed by this method. In contrast, this application uses a previously developed, technology that performs a purely negative enrichment methodology on peripheral blood, yielding highly enriched blood samples that contain CTCs as well as other, undefined cell types. The focus of this contribution is the use of multispectral imaging of epifluorescent, microscopic images of these enriched cells in order to help develop clinically relevant liquid biopsies from peripheral blood samples.
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http://dx.doi.org/10.1007/978-1-4939-7144-2_19DOI Listing
April 2018

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

Biotechnol J 2017 Sep 14;12(9). Epub 2017 Aug 14.

Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, USA.

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs.
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http://dx.doi.org/10.1002/biot.201600750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633046PMC
September 2017

Circulating tumor cells in head and neck cancer: A review.

World J Otorhinolaryngol Head Neck Surg 2016 Jun 21;2(2):109-116. Epub 2016 Jul 21.

Department of Otolaryngology-Head and Neck Surgery, Wexner Medical Center at Ohio State University, Columbus, OH, USA.

Carcinoma of the head and neck represents 3.5% of all cancers, and the vast majority of these tumors are squamous cell carcinoma (HNSCC). With a stable overall survival rate of 50% among all stages, there is continued interested in developing measures for early detection and disease aggressiveness. Circulating tumor cells (CTCs) have been identified as a potential marker for early metastatic disease, response to treatment, and surveillance in head and neck squamous cell carcinoma. In this article, techniques of CTC detection, applications of CTC technology, and outcomes of HNSCC patients will be discussed.
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http://dx.doi.org/10.1016/j.wjorl.2016.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5698518PMC
June 2016

Effect of surgical intervention on circulating tumor cells in patients with squamous cell carcinoma of the head and neck using a negative enrichment technology.

Head Neck 2016 12 5;38(12):1799-1803. Epub 2016 Jun 5.

William G. Lowrie Department of Chemical and Biomolecular Engineering, Ohio State University, Columbus, Ohio.

Background: The purpose of this study was to investigate the impact of surgical intervention on detection of circulating tumor cells (CTCs) in patients with squamous cell carcinoma of the head and neck (SCCHN.) METHODS: We utilized a negative depletion technique to identify cytokeratin (CK)-positive CTCs. The numbers of CTCs immediately before and after surgical resection were compared.

Results: Seventy-six blood samples from 38 patients with SCCHN were examined. Seventy-nine percent of the patients had CTCs detected before and after surgery. A total of 7.89% had no CTCs before surgery, yet had CTCs identified after surgery. Overall, 60.5% of patients had an increased number of CTCs/mL after surgery with a mean increase of 6.63-fold. A statistically significant increase in CTCs was seen after surgery (p = .02).

Conclusion: The timing of sample collection in patients with SCCHN who have surgical intervention can potentially impact the number of CTCs identified. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1799-1803, 2016.
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http://dx.doi.org/10.1002/hed.24519DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5118182PMC
December 2016

Tailoring the surface charge of dextran-based polymer coated SPIONs for modulated stem cell uptake and MRI contrast.

Biomater Sci 2015 Apr 26;3(4):608-16. Epub 2015 Feb 26.

Department of Chemistry, University of Liverpool, Liverpool, UK.

Tracking stem cells in vivo using non-invasive techniques is critical to evaluate the efficacy and safety of stem cell therapies. Superparamagnetic iron oxide nanoparticles (SPIONs) enable cells to be tracked using magnetic resonance imaging (MRI), but to obtain detectable signal cells need to be labelled with a sufficient amount of iron oxide. For the majority of SPIONs, this can only be obtained with the use of transfection agents, which can adversely affect cell health. Here, we have synthesised a library of dextran-based polymer coated SPIONs with varying surface charge from -1.5 mV to +18.2 mV via a co-precipitation approach and investigated their ability to be directly internalised by stem cells without the need for transfection agents. The SPIONs were colloidally stable in physiological solutions. The crystalline phase of the particles was confirmed with powder X-ray diffraction and their magnetic properties were characterised using SQUID magnetometry and magnetic resonance. Increased surface charge led to six-fold increase in uptake of particles into stem cells and higher MRI contrast, with negligible change in cell viability. Cell tracking velocimetry was shown to be a more accurate method for predicting MRI contrast of stem cells compared to measuring iron oxide uptake through conventional bulk iron quantification.
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http://dx.doi.org/10.1039/c5bm00011dDOI Listing
April 2015

Gene expression patterns through oral squamous cell carcinoma development: PD-L1 expression in primary tumor and circulating tumor cells.

Oncotarget 2015 Aug;6(25):20902-20

Department of Pathology, Ribeirao Preto School of Medicine, University of Sao Paulo, Ribeirao Preto, Brazil.

Oral squamous cell carcinoma (OSCC) is the most common tumor of the oral cavity and has been associated with poor prognosis. Scarce prognostic markers are available for guiding treatment and/or sub-classifying patients. This study aims to identify biomarkers by searching for genes whose expression is increased or decreased during tumor progression (through T1 to T4 stages). Thirty-six samples from all tumor size stages (from T1 to T4) were analyzed using cDNA microarrays. Selected targets were analyzed by immunohistochemistry and in circulating tumor cells by immunofluorescence and Nanostring. Correlation was shown between PD-L1 and tumor size and lymph node metastasis, HOXB9 and tumor size, BLNK and perineural invasion, and between ZNF813 and perineural invasion. PD-L1 positivity was an independent prognostic factor in this cohort (p = 0.044, HH = 0.426). In CTCs from patients with locally advanced OSCC, we found a strong cytoplasmatic expression of PD-L1. PD-L1 is a ligand of PD-1 and is believed to limit T cell activity in inflammatory responses and limit autoimmune diseases. We demonstrated an important role for PD-L1 in primary tumors according to tumor size, and in disease specific survival. Therefore, we could further determine individuals with PD-L1+ CTCs, and possibly follow treatment using CTCs.
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http://dx.doi.org/10.18632/oncotarget.3939DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4673238PMC
August 2015

Magnetic separation of algae genetically modified for increased intracellular iron uptake.

J Magn Magn Mater 2015 Apr 10;380:201-204. Epub 2014 Sep 10.

Cleveland Clinic, Cleveland, Ohio, U.S.A.

Algae were investigated in the past as a potential source of biofuel and other useful chemical derivatives. Magnetic separation of algae by iron oxide nanoparticle binding to cells has been proposed by others for dewatering of cellular mass prior to lipid extraction. We have investigated feasibility of magnetic separation based on the presence of natural iron stores in the cell, such as the ferritin in () strains. The cell constructs were tested for inserted genes and for increased intracellular iron concentration by inductively coupled plasma atomic absorption (ICP-AA). They were grown in Sueoka's modified high salt media with added vitamin B1 and increasing concentration of soluble iron compound (FeCl EDTA, from 1× to 8× compared to baseline). The cell magnetic separation conditions were tested using a thin rectangular flow channel pressed against interpolar gaps of a permanent magnet forming a separation system of a well-defined fluid flow and magnetic fringing field geometry (up to 2.2 T and 1,000 T/m) dubbed "magnetic deposition microscopy", or MDM. The presence of magnetic cells in suspension was detected by formation of characteristic deposition bands at the edges of the magnet interpolar gaps, amenable to optical scanning and microscopic examination. The results demonstrated increasing cellular Fe uptake with increasing Fe concentration in the culture media in wild type strain and in selected genetically-modified constructs, leading to magnetic separation without magnetic particle binding. The throughput in this study is not sufficient for an economical scale harvest.
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http://dx.doi.org/10.1016/j.jmmm.2014.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774641PMC
April 2015

Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients.

Breast Cancer Res 2014 Mar 6;16(2):R23. Epub 2014 Mar 6.

Introduction: Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.

Methods: A total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).

Results: CD45-negative/CK-positive (CD45- CK+) populations with EpCAM + and EpCAM - expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM - populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM - events/ml than CK + EpCAM + events/ml in both the CD45- and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45- and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM - were associated with worse overall survival (P = 0.0292).

Conclusions: Metastatic breast cancer patients have atypical cells that are CK + EpCAM - circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM - circulating cells as a prognostic and predictive marker.
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http://dx.doi.org/10.1186/bcr3622DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4053256PMC
March 2014

Feasibility study of red blood cell debulking by magnetic field-flow fractionation with step-programmed flow.

Anal Bioanal Chem 2014 Feb 19;406(6):1661-70. Epub 2013 Oct 19.

Department of Biomedical Engineering, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, 44195, USA.

Emerging applications of rare cell separation and analysis, such as separation of mature red blood cells from hematopoietic cell cultures, require efficient methods of red blood cell (RBC) debulking. We have tested the feasibility of magnetic RBC separation as an alternative to centrifugal separation using an approach based on the mechanism of magnetic field-flow fractionation (MgFFF). A specially designed permanent magnet assembly generated a quadrupole field having a maximum field of 1.68 T at the magnet pole tips, zero field at the aperture axis, and a nearly constant radial field gradient of 1.75 T/mm (with a negligible angular component) inside a cylindrical aperture of 1.9 mm (diameter) and 76 mm (length). The cell samples included high-spin hemoglobin RBCs obtained by chemical conversion of hemoglobin to methemoglobin (met RBC) or by exposure to anoxic conditions (deoxy RBC), low-spin hemoglobin obtained by exposure of RBC suspension to ambient air (oxy RBC), and mixtures of deoxy RBC and cells from a KG-1a white blood cell (WBC) line. The observation that met RBCs did not elute from the channel at the lower flow rate of 0.05 mL/min applied for 15 min but quickly eluted at the subsequent higher flow rate of 2.0 mL/min was in agreement with FFF theory. The well-defined experimental conditions (precise field and flow characteristics) and a well-established FFF theory verified by studies with model cell systems provided us with a strong basis for making predictions about potential practical applications of the magnetic RBC separation.
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http://dx.doi.org/10.1007/s00216-013-7394-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943756PMC
February 2014

Isolation and analysis of rare cells in the blood of cancer patients using a negative depletion methodology.

Methods 2013 Dec 20;64(2):169-82. Epub 2013 Sep 20.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University, Columbus, OH 43210, United States.

A variety of enrichment/isolation technologies exist for the characterization of rare cells in the blood of cancer patients. In this article, a negative depletion process is presented and discussed which consists of red blood cell (RBC) lysis and the subsequent removal of CD45 expressing cells through immunomagnetic depletion. Using this optimized assembly on 120 whole blood specimens, from 71 metastatic breast cancer patients, after RBC lysis, the average nucleated cell log depletion was 2.56 with a 77% recovery of the nucleated cells. The necessity of exploring different anti-CD45 antibody clones to label CD45 expressing cells in this enrichment scheme is also presented and discussed. An optimized, four-color immunofluorescence staining is conducted on the cells retained after the CD45-based immunomagnetic depletion process. Different types of rare non-hematopoietic cells are found in these enriched peripheral blood samples and a wide range of external and internal markers have been characterized, which demonstrates the range and heterogeneity of the rare cells.
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http://dx.doi.org/10.1016/j.ymeth.2013.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874448PMC
December 2013

On-chip magnetic separation and encapsulation of cells in droplets.

Lab Chip 2013 Mar;13(6):1172-81

Department of Physics, The Ohio State University, Columbus, Ohio 43210, USA.

Single cell study is gaining importance because of the cell-to-cell variation that exists within cell population, even after significant initial sorting. Analysis of such variation at the gene expression level could impact single cell functional genomics, cancer, stem-cell research, and drug screening. The on-chip monitoring of individual cells in an isolated environment would prevent cross-contamination, provide high recovery yield, and enable study of biological traits at a single cell level. These advantages of on-chip biological experiments is a significant improvement for a myriad of cell analyses methods, compared to conventional methods, which require bulk samples and provide only averaged information on cell structure and function. We report on a device that integrates a mobile magnetic trap array with microfluidic technology to provide the possibility of separation of immunomagnetically labeled cells and their encapsulation with reagents into picoliter droplets for single cell analysis. The simultaneous reagent delivery and compartmentalization of the cells immediately following sorting are all performed seamlessly within the same chip. These steps offer unique advantages such as the ability to capture cell traits as originated from its native environment, reduced chance of contamination, minimal use of the reagents, and tunable encapsulation characteristics independent of the input flow. Preliminary assay on cell viability demonstrates the potential for the device to be integrated with other up- or downstream on-chip modules to become a powerful single-cell analysis tool.
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http://dx.doi.org/10.1039/c2lc41201bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176703PMC
March 2013

Open Gradient Magnetic Red Blood Cell Sorter Evaluation on Model Cell Mixtures.

IEEE Trans Magn 2013 Feb;49(1):309-315

Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195 USA.

The emerging applications of biological cell separation to rare circulating tumor cell (CTC) detection and separation from blood rely on efficient methods of red blood cell (RBC) debulking. The two most widely used methods of centrifugation and RBC lysis have been associated with the concomitant significant losses of the cells of interest (such as progenitor cells or circulating tumor cells). Moreover, RBC centrifugation and lysis are not well adapted to the emerging diagnostic applications, relying on microfluidics and micro-scale total analytical systems. Therefore, magnetic RBC separation appears a logical alternative considering the high iron content of the RBC (normal mean 105 fg) as compared to the white blood cell iron content (normal mean 1.6 fg). The typical magnetic forces acting on a RBC are small, however, as compared to typical forces associated with centrifugation or the forces acting on synthetic magnetic nanoparticles used in current magnetic cell separations. This requires a significant effort in designing and fabricating a practical magnetic RBC separator. Applying advanced designs to the low cost, high power permanent magnets currently available, and building on the accumulated knowledge of the immunomagnetic cell separation methods and devices, an open gradient magnetic red blood cell (RBC) sorter was designed, fabricated and tested on label-free cell mixtures, with potential applications to RBC debulking from whole blood samples intended for diagnostic tests.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047673PMC
http://dx.doi.org/10.1109/tmag.2012.2225098DOI Listing
February 2013

A MagDot-Nanoconveyor Assay Detects and Isolates Molecular Biomarkers.

Chem Eng Prog 2012 Dec;108:41-46

The Ohio State Univ.

The ability to quickly analyze, separate, and manipulate multiple types of biomarkers from small sample volumes is a significant step toward personalized medicine.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286893PMC
December 2012

Assessment of γ-H2AX levels in circulating tumor cells from patients receiving chemotherapy.

Front Oncol 2012 25;2:128. Epub 2012 Oct 25.

William G. Lowrie Department of Chemical and Biomolecular Engineering, The Ohio State University Columbus, OH, USA.

Circulating tumor cells (CTCs) are prognostic markers in a variety of solid tumor malignancies. The potential of CTCs to be used as a "liquid biopsy" to monitor a patient's condition and predict drug response and resistance is currently under investigation. Using a negative depletion, enrichment methodology, CTCs isolated from the peripheral blood of breast cancer patients with stage IV breast cancer undergoing DNA damaging therapy with platinum-based therapy were enriched. The enriched cell suspensions were stained with an optimized labeling protocol targeting: nuclei, cytokeratins 8, 18, and 19, the surface marker CD45, and the presence of the protein γ-H2AX. As a direct or indirect result of platinum therapy, double-strand break of DNA initiates phosphorylation of the histone H2AX, at serine 139; this phosphorylated form is referred to as γ-H2AX. In addition to γ-H2AX staining in specific locations with the cell nuclei, consistent with previous reports and referred to as foci, more general staining in the cell cytoplasm was also observed in some cells suggesting the potential of cell apoptosis. Our study underscores the utility and the complexity of investigating CTCs as predictive markers of response to various therapies. Additional studies are ongoing to evaluate the diverse γ-H2AX staining patterns we report here which needs to be further correlated with patient outcomes.
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http://dx.doi.org/10.3389/fonc.2012.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3480704PMC
November 2012