Numerical modeling and in situ small angle X-ray scattering characterization of ultra-small SPION magnetophoresis in a high field and gradient separator.

Magnetic nanoparticles (MNPs) have recently gained significant attention in various fields, including chemical and biomedical applications, due to their exceptional properties. However, separating MNPs from solution via magnetophoresis is challenging when MNPs are smaller than 50 nm as Brownian forces become on the order of the magnetic forces. In this study, we successfully separated small MNPs (5-30 nm) by utilizing high magnetic fields and gradients generated by economical permanent magnets. In situ small angle X-ray scattering (SAXS) was used to investigate the time-dependent concentration changes in the ferrofluid, and the results validated that only the 30 nm particles experienced particle aggregation or agglomeration, indicating that dipole-dipole interactions did not play a discernable role in the separation process for particles smaller than ∼15 nm. However, numerical simulations have provided further validation that in the absence of particle-particle interactions, even MNPs with diameters less than 15 nm exhibited magnetophoresis that effectively counteracted the effects of Brownian motion.

Measuring magnetic force field distributions in microfluidic devices: Experimental and numerical approaches.

Precisely and accurately determining the magnetic force and its spatial distribution in microfluidic devices is challenging. Typically, magnetic microfluidic devices are designed in a way to both maximize the force within the separation region and to minimize the necessity for knowing such details-such as designing magnetic geometries that create regions of nearly constant magnetic force or that dictate the behavior of the magnetic force to be highly predictable in a specified region. In this work, we present a method to determine the spatial distribution of the magnetic force field in a magnetic microfluidic device by particle tracking magnetophoresis. Polystyrene microparticles were suspended in a paramagnetic fluid, gadolinium, and this suspension was exposed to various magnetic field geometries. Polystyrene particle motion was tracked using a microscope and images processed using Fiji (ImageJ). From a sample with a large spatial distribution of particle tracks, the magnetic force field distribution was calculated. The force field distribution was fitted to nonlinear spatial distribution models. These experimental models are compared to and supported by 3D simulations of the magnetic force field in COMSOL.

The Unique Magnetic Signature of Sickle Red Blood Cells: A Comparison Between the Red Blood Cells of Transfused and Non-Transfused Sickle Cell Disease Patients and Healthy Donors.

Sickle cell disease (SCD) is an inherited blood disorder that affects millions of people worldwide, especially in low-resource regions of the world, where a rapid and affordable test to properly diagnose the disease would be highly valued. Magnetophoresis is a technique that could simultaneously analyze, quantify, and potentially separate the patient's sickle red blood cells (RBCs) from healthy RBCs, but the magnetic characteristics of sickle RBCs have yet to be reported. In this work, we present the single cell magnetic characterization of RBCs obtained from SCD patients. Sufficient single cells are analyzed from patient samples undergoing transfusion therapy and not yet having transfusion therapy (TP and NTP, respectively), such that means and distributions of these single RBC mobilities are created in the form of histograms which facilitated comparison to RBCs from healthy donors (HD). The magnetic characterization is obtained using a technique known as Cell Tracking Velocimetry (CTV) that quantitatively characterizes the RBC response to magnetic and gravitational fields. The magnetic properties of RBCs containing oxygenated, deoxygenated hemoglobin (Hb) and methemoglobin (oxyHb-RBCs, deoxyHb-RBCs, and metHb-RBCs) are further determined. The NTP samples reported the highest magnetic character, especially when compared to oxyHb-RBCs from HD, which implies impaired oxygen binding capabilities. Also, the oxygen-Hb equilibrium curves are obtained to estimate the magnetic character of the cells under intermediate oxygen levels. Our results confirm higher magnetic moment of SCD blood (NTP) under intermediate oxygen levels. These data demonstrate the potential feasibility of magnetophoresis to identify, quantify and separate sickle RBCs from healthy RBCs.

Potential of cell tracking velocimetry as an economical and portable hematology analyzer.

Anemia and iron deficiency continue to be the most prevalent nutritional disorders in the world, affecting billions of people in both developed and developing countries. The initial diagnosis of anemia is typically based on several markers, including red blood cell (RBC) count, hematocrit and total hemoglobin. Using modern hematology analyzers, erythrocyte parameters such as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), etc. are also being used. However, most of these commercially available analyzers pose several disadvantages: they are expensive instruments that require significant bench space and are heavy enough to limit their use to a specific lab and lead to a delay in results, making them less practical as a point-of-care instrument that can be used for swift clinical evaluation. Thus, there is a need for a portable and economical hematology analyzer that can be used at the point of need. In this work, we evaluated the performance of a system referred to as the cell tracking velocimetry (CTV) to measure several hematological parameters from fresh human blood obtained from healthy donors and from sickle cell disease subjects. Our system, based on the paramagnetic behavior that deoxyhemoglobin or methemoglobin containing RBCs experience when suspended in water after applying a magnetic field, uses a combination of magnets and microfluidics and has the ability to track the movement of thousands of red cells in a short period of time. This allows us to measure not only traditional RBC indices but also novel parameters that are only available for analyzers that assess erythrocytes on a cell by cell basis. As such, we report, for the first time, the use of our CTV as a hematology analyzer that is able to measure MCV, MCH, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), the percentage of hypochromic cells (which is an indicator of insufficient marrow iron supply that reflects recent iron reduction), and the correlation coefficients between these metrics. Our initial results indicate that most of the parameters measured with CTV are within the normal range for healthy adults. Only the parameters related to the red cell volume (primarily MCV and RDW) were outside the normal range. We observed significant discrepancies between the MCV measured by our technology (and also by an automated cell counter) and the manual method that calculates MCV through the hematocrit obtained by packed cell volume, which are attributed to the artifacts of plasma trapping and cell shrinkage. While there may be limitations for measuring MCV, this device offers a novel point of care instrument to provide rapid RBC parameters such as iron stores that are otherwise not rapidly available to the clinician. Thus, our CTV is a promising technology with the potential to be employed as an accurate, economical, portable and fast hematology analyzer after applying instrument-specific reference ranges or correction factors.

SPIONs self-assembly and magnetic sedimentation in quadrupole magnets: Gaining insight into the separation mechanisms.

Superparamagnetic iron oxide nanoparticles (SPIONs) are currently popular materials experiencing rapid development with potential application value, especially in biomedical and chemical engineering fields. Examples include wastewater management, bio-detection, biological imaging, targeted drug delivery and biosensing. While not exclusive, magnetically driven isolation methods are typically required to separate the desired entity from the media in specific applications and in their manufacture and/or quality control. However, due to the nano-size of SPIONs, their magnetic manipulation is affected by Brownian motion, adding considerable complexities. The two most common methods for SPION magnetic separation are high and low gradient magnetic separation (HGMS and LGMS, respectively). Nevertheless, the effect of specific magnetic energy fields on SPIONs, such as horizontal (perpendicular to gravity), high fields and gradients (higher than LGMS) on the horizontal magnetophoresis and vertical sedimentation of SPIONs has only recently been suggested as a way to separate very small particles (5 nm). In this work, we continue those studies on the magnetic separation of 5-30 nm SPIONs by applying fields and gradients perpendicular to gravity. The magnetic field was generated by permanent magnets arranged in quadrupolar configurations (QMS). Different conditions were studied, and multiple variables were evaluated, including the particle size, the initial SPIONs concentration, the temperature, the magnetic field gradient and the magnetic exposure time. Our experimental data show that particles are subjected to horizontal magnetic forces, to particle agglomeration due to dipole-dipole interactions, and to vertical sedimentation due to gravity. The particle size and the type of separator employed (i.e. different gradient and field distribution acting on the particle suspension) have significant effects on the phenomena involved in the separation, whereas the temperature and particle concentration affect the separation to a lesser extent. Finally, the separation process was observed to occur in less than 3 mins for our experimental conditions, which is encouraging considering the long operation time (up to days) necessary to separate particles of similar sizes in LGMS columns that also employ permanent magnets.

Recovery of Magnetic Catalysts: Advanced Design for Process Intensification.

The design of microdevices in which components with magnetic character must be separated and recovered from reactive media benefits from the advantages of microfluidics and meets the criteria for process intensification; however, there are open questions, such as the design of the most appropriate magnet arrangement, that need further research in order to increase the magnetic gradient exerted on the particles. Herein, we focus on the continuous recovery of magnetic microparticles, that can be used as support to facilitate the recovery of biocatalysts (magnetic microcatalysts, MMCs) from biological fluids. We analyze and compare the performance of two typical magnetophoretic microdevices for addressing bead recovery: (i) annular channels with a quadrupole orientation of the permanent magnets (quadrupole magnetic sorter, QMS) and (ii) the standard design, which consists of rectangular channels with a single permanent magnet to generate the magnetic field. To this end, an experimentally validated computational fluid dynamics (CFD) numerical model has been employed. Our results reveal that for devices with the same width and length, the micro-QMS, in comparison to a rectangular channel, could accomplish the complete particle retrieval while (i) processing more than 4 times higher fluid velocities, treating more than 360 times higher flow rates or (ii) working with smaller particles, thus reducing by 55% the particle mass. Additionally, the parallel performance of ≈300 micro-QMSs fulfills the processing of flow rates as high as 200 L·h-1 while entirely capturing the magnetic beads. Thereby, this work shows the potential of the QMS advanced design in the intensification of the recovery of catalysts supports of magnetic character.

Magnetophoretic and spectral characterization of oxyhemoglobin and deoxyhemoglobin: Chemical versus enzymatic processes.

A new method for hemoglobin (Hb) deoxygenation, in suspension or within red blood cells (RBCs) is described using the commercial enzyme product, EC-Oxyrase®. The enzymatic deoxygenation method has several advantages over established deoxygenation methodologies, such as avoiding side reactions that produce methemoglobin (metHb), thus eliminating the need for an inert deoxygenation gas and airtight vessel, and facilitates easy re-oxygenation of Hb/RBCs by washing with a buffer that contains dissolved oxygen (DO). The UV-visible spectra of deoxyHb and metHb purified from human RBCs using three different preparation methods (sodium dithionite [to produce deoxyHb], sodium nitrite [to produce metHb], and EC-Oxyrase® [to produce deoxyHb]) show the high purity of deoxyHb prepared using EC-Oxyrase® (with little to no metHb or hemichrome production from side reactions). The oxyHb deoxygenation time course of EC-Oxyrase® follows first order reaction kinetics. The paramagnetic characteristics of intracellular Hb in RBCs were compared using Cell Tracking Velocimetry (CTV) for healthy and sickle cell disease (SCD) donors and oxygen equilibrium curves show that the function of healthy RBCs is unchanged after EC-Oxyrase® treatment. The results confirm that this enzymatic approach to deoxygenation produces pure deoxyHb, can be re-oxygenated easily, prepared aerobically and has similar paramagnetic mobility to existing methods of producing deoxyHb and metHb.

Intrinsically magnetic susceptibility in human blood and its potential impact on cell separation: Non-classical and intermediate monocytes have the strongest magnetic behavior in fresh human blood.

The presence of iron in circulating monocytes is well known as they play an essential role in iron recycling. It has been demonstrated that the iron content of blood cells can be measured through their magnetic behavior; however, the magnetic properties of different monocyte subtypes remain unknown. In this study we report, for the first time, the magnetic behavior of classical, intermediate and non-classical monocytes, which may be related to their iron storage capacity. The magnetic properties of monocytes were compared with those of other blood cells, such as lymphocytes and red blood cells in the oxyhemoglobin and methemoglobin states, and a cancer cell type. For this analysis, we used an instrument referred to as a Cell Tracking Velocimetry (CTV), which quantitatively characterizes the magnetic behavior of biological entities. Our results revealed that significant fractions of the intermediate and non-classical monocytes (up to 59% and 65% depending on the sample, respectively) have paramagnetic properties, suggesting their higher iron storage capacities. Moreover, our findings have implications for the immunomagnetic separation industry; we propose that negative magnetic isolation techniques for recovering monocytes from blood should be used with caution, as it is possible to lose magnetic monocytes when using this technique.

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding.

The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.

A comparison of alendronate to varying magnitude PEMF in mitigating bone loss and altering bone remodeling in skeletally mature osteoporotic rats.

Pulsed electromagnetic field (PEMF) treatments stimulate bone formation activities though further work is needed to optimize its therapeutic benefit. PEMF can generate local potential gradients and electric currents that have been suggested to mimic bone electrochemical responses to load. In line with this reasoning, a recent publication reported that PEMF application on isolated bone tissue induced detectable micro-vibrations (doi:https://doi.org/10.1109/TMAG.2016.2515069). To determine the ability of PEMF to intervene in a rat model of osteoporosis, we tested its effect on trabecular and cortical bone following ovariectomy. Four PEMF treatments, with increasing sinusoidal amplitude rise with time (3850 Hz pulse frequency and 15 Hz repetition rate at 10 tesla/sec (T/s), 30 T/s, 100 T/s, or 300 T/s), were compared to the efficacy of an osteoporosis drug, alendronate, in reducing levels of trabecular bone loss in the proximal tibia. Herein, the novel findings from our study are: (1) 30 T/s PEMF treatment approached the efficacy of alendronate in reducing trabecular bone loss, but differed from it by not reducing bone formation rates; and (2) 30 T/s and 100 T/s PEMF treatments imparted measurable alterations in lacunocanalicular features in cortical bone, consistent with osteocyte sensitivity to PEMF in vivo. The efficacy of specific PEMF doses may relate to their ability to modulate osteocyte function such that the 30 T/s, and to a lesser extent 100 T/s, doses preferentially antagonize trabecular bone resorption while stimulating bone formation. Thus, PEMF treatments of specific magnetic field magnitudes exert a range of measurable biological effects in trabecular and cortical bone tissue in osteoporotic rats.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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 B0 = 1.36 T at the pole tips and a constant field gradient of B0 /r0 = 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 × 106 -9 × 106 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 mm2 (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.

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

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 T2 /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.