Femtogram Resolution of Iron Content on a Per Cell Basis: Ex Vivo Storage of Human Red Blood Cells Leads to Loss of Hemoglobin.

The magnetic characteristics of hemoglobin (Hb) changes with the binding of dioxygen (O2) to the heme prosthetic groups of the globin chains: from paramagnetic ferrous Hb to diamagnetic ferrous oxyhemoglobin (oxyHb) with reversibly bound O2, or paramagnetic ferric methemoglobin (metHb). When multiplied over the number of Hb molecules in a red blood cell (RBC), the effect is detectable through motion analysis of RBCs in a high magnetic field and gradient. This motion is referred to as magnetophoretic mobility, which can be conveniently expressed as a fraction of the cell sedimentation velocity. In this Article, using a previously developed and reported instrument, cell tracking velocimetry (CTV), we are able to detect difference in Hb concentration in two RBC populations to a resolution of 1 × 107 Hb molecules per cell (4 × 107 atoms of Fe per cell or 4-5 femtograms of Fe). Similar resolution achieved with inductively coupled plasma-mass spectrometry requires on the order of 105-106 cells and provides an average, whereas CTV provides a measurement for each cell. CTV analysis revealed that RBCs lose, on average, 17% of their Hb after 42 days of storage, the maximum FDA-approved length of time for the cold storage of RBCs in additive solution. This difference in Hb concentration was the result of routine RBC storage; clinical implications are discussed.

Steering microtubule shuttle transport with dynamically controlled magnetic fields.

Nanoscale control of matter is critical to the design of integrated nanosystems. Here, we describe a method to dynamically control directionality of microtubule (MT) motion using programmable magnetic fields. MTs are combined with magnetic quantum dots (i.e., MagDots) that are manipulated by external magnetic fields provided by magnetic nanowires. MT shuttles thus undergo both ATP-driven and externally-directed motion with a fluorescence component that permits simultaneous visualization of shuttle motion. This technology is used to alter the trajectory of MTs in motion and to pin MT motion. Such an approach could be used to evaluate the MT-kinesin transport system and could serve as the basis for improved lab-on-a-chip technologies based on MT transport.

New insights into shear-sensitivity in dinoflagellate microalgae.

A modification of a flow contraction device was used to subject shear-sensitive microalgae to well-defined hydrodynamic forces. The aim of the study was to elucidate if the inhibition of shear-induced growth commonly observed in dinoflagellate microalgae is in effect due to cell fragility that results in cell breakage even at low levels of turbulence. The microalgae assayed did not show any cell breakage even at energy dissipation rates (EDR) around 10(12)Wm(-3), implausible in culture devices. Conversely, animal cells, tested for comparison purposes, showed high physical cell damage at average EDR levels of 10(7)Wm(-3). Besides, very short exposures to high levels of EDR promoted variations in the membrane fluidity of the microalgae assayed, which might trigger mechanosensory cellular mechanisms. Average EDR values of only about 4·10(5)Wm(-3) increased cell membrane fluidity in microalgae whereas, in animal cells, they did not.

Occupational heat-related illness emergency department visits and inpatient hospitalizations in the southeast region, 2007-2011.

BACKGROUND: Heat-related illness (HRI) is an occupational health risk for many outdoor, and some indoor, workers. METHODS: Emergency department (ED) and inpatient hospitalization (IH) data for 2007-2011 from nine southeast states were analyzed to identify occupational HRI numbers and rates, demographic characteristics, and co-morbid conditions. RESULTS: There were 8,315 occupational HRI ED visits (6.5/100,000 workers) and 1,051 IHs (0.61/100,000) in the southeast over the study period. Out-of-state residents comprised 8% of ED visits and 12% of IHs. Rates for both, ED visits and IHs were significantly elevated in males and blacks. Younger workers had elevated rates for ED visits, while older workers had higher IH rates. CONCLUSIONS: This is the first study to evaluate occupational HRI ED visits and IHs in the southeast region and indicates the need for enhanced heat-stress prevention policies in the southeast. Findings from this study can be used to direct state health department tracking and evaluation of occupational HRI.

Manipulation of magnetically labeled and unlabeled cells with mobile magnetic traps.

A platform of discrete microscopic magnetic elements patterned on a surface offers dynamic control over the motion of fluid-borne cells by reprogramming the magnetization within the magnetic bits. T-lymphocyte cells tethered to magnetic microspheres and untethered leukemia cells are remotely manipulated and guided along desired trajectories on a silicon surface by directed forces with average speeds up to 20 microm/s. In addition to navigating cells, the microspheres can be operated from a distance to push biological and inert entities and act as local probes in fluidic environments.

Quantification of non-specific binding of magnetic micro- and nanoparticles using cell tracking velocimetry: Implication for magnetic cell separation and detection.

The maturation of magnetic cell separation technology places increasing demands on magnetic cell separation performance. While a number of factors can cause sub-optimal performance, one of the major challenges can be non-specific binding of magnetic nano- or microparticles to non-targeted cells. Depending on the type of separation, this non-specific binding can have a negative effect on the final purity, the recovery of the targeted cells, or both. In this work, we quantitatively demonstrate that non-specific binding of magnetic nanoparticles can impart a magnetization to cells such that these cells can be retained in a separation column and thus negatively impact the purity of the final product and the recovery of the desired cells. Through experimental data and theoretical arguments, we demonstrate that the number of MACS magnetic particles needed to impart a magnetization that is sufficient to cause non-targeted cells to be retained in the column to be on the order of 500-1,000 nanoparticles. This number of non-specifically bound particles was demonstrated experimentally with an instrument, cell tracking velocimeter, CTV, and it is demonstrated that the sensitivity of the CTV instrument for Fe atoms contained in magnetic nanoparticles on the order of 1 x 10(-15) g/mL of Fe.

Magnetic wire traps and programmable manipulation of biological cells.

We present a multiplex method, based on microscopic programmable magnetic traps in zigzag wires patterned on a platform, to simultaneously apply directed forces on multiple fluid-borne cells or biologically inert magnetic microparticles or nanoparticles. The gentle tunable forces do not produce damage and retain cell viability. The technique is demonstrated with T-lymphocyte cells remotely manipulated (by a joystick) along desired trajectories on a silicon surface with average speeds up to 20 microm/s.

Mobility measurements of immunomagnetically labeled cells allow quantitation of secondary antibody binding amplification.

Magnetic cell separation methods commonly utilize paramagnetic materials conjugated to antibodies that target specific cell surface molecules. The amount of magnetic material bound to a cell is directly proportional to the magnetophoretic mobility of that cell. A mathematical model has been developed which characterizes the fundamental parameters controlling the amount of magnetic material bound, and thus, the magnetophoretic mobility of an immunomagnetically labeled cell. In characterization of the paramagnetic labeling, one of the parameters of interest is the increase in magnetophoretic mobility due to the secondary antibody binding to multiple epitopes on the primary antibody, referred to as the "secondary antibody binding amplification," Psi. Secondary antibody-binding amplification has been investigated and quantitated by comparing the mobilities of lymphocytes directly labeled with anti-CD4 MACS (Miltenyi Biotec, Auburn, CA) magnetic nanoparticle antibody with the mobilities of lymphocytes from the same sample labeled with two different indirect antibody-labeling schemes. Each indirect labeling scheme incorporated a primary mouse anti-CD4 FITC antibody that provides both FITC and mouse-specific binding sites for two different secondary antibody-magnetic nanoparticle conjugates: either anti-FITC MACS magnetic nanoparticle antibody or anti-mouse MACS magnetic nanoparticle antibody. The magnetophoretic mobilities of the immunomagnetically labeled cells were obtained using Cell Tracking Velocimetry (CTV). The results indicate that an average of 3.4 anti-FITC MACS magnetic nanoparticle antibodies bind to each primary CD4 FITC antibody, Psi(1,2f) = 3.4 +/- 0.33, and that approximately one, Psi(1,2m) = 0.98 +/- 0.081, anti-mouse MACS magnetic nanoparticle antibody binds to each primary mouse CD4 FITC antibody on a CD4 positive lymphocyte. These results have provided a better understanding of the antibody-binding mechanisms used in paramagnetic cell labeling for magnetic cell separation.

Effects of antibody concentration on the separation of human natural killer cells in a commercial immunomagnetic separation system.

BACKGROUND: The magnetic separation of a cell population based on cell surface markers is a critical step in many biological and clinical laboratories. In this study, the effect of antibody concentration on the separation of human natural killer cells in a commercial, immunomagnetic cell separation system was investigated. METHODS: Specifically, the degree of saturation of antibody binding sites using a two-step antibody sandwich was quantified. The quantification of the first step, a primary anti-CD56-PE antibody, was achieved through fluorescence intensity measurements using a flow cytometer. The quantification of the second step, an anti-PE-microbeads antibody reagent, was achieved through magnetophoretic mobility measurements using cell tracking velocimetry. RESULTS: From the results of these studies, two different labeling protocols were used to separate CD56+ cells from human, peripheral blood by a Miltenyi Biotech MiniMACS cell separation system. The first of these two labeling protocols was based on company recommendations, whereas the second was based on the results of the saturation studies. The results from these studies demonstrate that the magnetophoretic mobility is a function of both primary and secondary antibody concentrations and that mobility does have an effect on the performance of the separation system. CONCLUSIONS: As the mobility increased due to an increase in bound antibodies, the positive cells were almost completely eliminated from the negative eluent. However, with an increase in bound antibodies, and thus mobility, the total amount of positive cells recovered decreases. It is speculated that these cells are irreversibly retained in the column. These results demonstrate the complexity of immunomagnetic cell separation and the need to further optimize the cell separation process.

Separation of a breast cancer cell line from human blood using a quadrupole magnetic flow sorter.

We have developed a quadrupole magnetic flow sorter (QMS) to facilitate high-throughput binary cell separation. Optimized QMS operation requires the adjustment of three flow parameters based on the immunomagnetic characteristics of the target cell sample. To overcome the inefficiency of semiempirical operation/optimization of QMS flow parameters, a theoretical model of the QMS sorting process was developed. Application of this model requires measurement of the magnetophoretic mobility distribution of the cell sample by the cell tracking velocimetry (CTV) technique developed in our laboratory. In this work, the theoretical model was experimentally tested using breast carcinoma cells (HCC1954) overexpressing the HER-2/neu gene, and peripheral blood leukocytes (PBLs). The magnetophoretic mobility distribution of immunomagnetically labeled HCC1954 cells was measured using the CTV technique, and then theoretical predictions of sorting recoveries were calculated. Mean magnetophoretic mobilities of (1-3) x 10(-4) mm(3)/(T A s) were obtained depending on the labeling conditions. Labeled HCC1954 cells were mixed with unlabeled PBLs to form a "spiked" sample to be separated by the QMS. Fractional recoveries of cells for different flow parameters were examined and compared with theoretical predictions. Experimental results showed that the theoretical model accurately predicted fractional recoveries of HCC1954 cells. High-throughput (3.29 x 10(5) cells/s) separations with high recovery (0.89) of HCC1954 cells were achieved.

Evaluation of eluents from separations of CD34+ cells from human cord blood using a commercial, immunomagnetic cell separation system.

Human CD34+ cells from cord blood were separated in a two-step process using a commercial, immunomagnetic cell retention system. The performance of the system was evaluated by analyzing a number of eluents from the separations with a number of analytical techniques. In addition to cell counts and flow cytometry analysis, a new experimental technique that is undergoing development, cell tracking velocimetry (CTV), was used. CTV measures the degree to which a cell is immunomagnetically labeled, known as the magnetophoretic mobility, of a population of cells on a cell-by-cell basis and presents the results in the form of a histogram similar to flow cytometry data. The average recovery and purity of CD34+ cells from 10 separations was 52% and 60%, respectively. CTV analysis indicated that the mean magnetophoretic mobility of the positively enriched CD34 cells was 9.64 x 10(-5) mm3/T-A-s, while the mean mobility from negative eluents was -2.02 x 10(-6) mm3/T-A-s, very similar to the mobility of unlabeled cells. Within the positive eluents, the range of magnetophoretic mobility was approximately 50-fold, representing a plausible 50-fold range in surface CD34 antigen expression. CTV analysis also indicated that in some separations, positive cells were not retained by the immunomagnetic cell retention system. Finally, preliminary studies indicate that monocytes might be a primary cause in the lower purities and recoveries seen in this study. It is suggested that the monocytes phagocytose the magnetic nanobeads and become sufficiently magnetized to be retained within the Miltenyi column, reducing the purity of the positive eluent.

Measurement of CD2 expression levels of IFN-alpha-treated fibrosarcomas using cell tracking velocimetry.

METHODS: A methodology and a mathematical relationship have been developed that allow quantitation of the expression levels of cellular surface antigens, in terms of antibody binding capacities (ABC). This methodology uses immunomagnetically labeled cells and calibration microbeads combined with cell tracking velocimetry (CTV) technology to measure magnetophoretic mobilities corresponding to cellular ABC. The mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads and cells in a nearly constant magnetic energy gradient. RESULTS: Transformed fibrosarcoma cells were given controlled treatments of interferon-alpha in order to manipulate CD2 antigen expression levels. These cells were then immunomagnetically labeled with anti-CD2 FITC antibodies and anti-FITC MACS paramagnetic nanoparticles. Measured magnetophoretic mobilities were used to calculate ABC for these cells, corresponding to CD2 expression levels. CONCLUSION: The results from CTV and flow cytometry (FCM) qualitatively verify that these fibrosarcoma cells express elevated levels of CD2 molecules with increasing interferon-alpha treatment from 0 to 24 h. The mean basal CD2 expression level, in terms of ABC, was calculated to be 27,000 from CTV analysis, whereas FCM indicates a comparable ABC value of 33,000.

Magnetophoretic mobilities correlate to antibody binding capacities.

METHODS: A methodology and a mathematical theory have been developed, which allow quantitation of the expression levels of cellular surface antigens using immunomagnetic labels and cell tracking velocimetry (CTV) technology. RESULTS: Quantum Simply Cellular (QSC) microbeads were immunomagnetically labeled with anti-CD2 fluorescein isothiocyanate (FITC) antibodies and anti-FITC MACS paramagnetic nanoparticles. Magnetophoretic mobility has been defined as the magnetically induced velocity of the labeled cell or microbead divided by the magnetophoretic driving force, proportional to the magnetic energy density gradient. DISCUSSION: Using computer imaging and processing technology, the mobility measurements were accomplished by microscopically recording and calculating the velocity of immunomagnetically labeled QSC microbeads in a nearly constant magnetic energy gradient. A calibration curve correlating the measured magnetophoretic mobility of the immunomagnetically labeled microbeads to their antibody binding capacities (ABC) has been obtained. CONCLUSION: The results, in agreement with theory, indicate a linear relationship between magnetophoretic mobility and ABC for microbeads with less than 30,000 ABC. The mathematical relationships and QSC standardization curve obtained allow determination of the number of surface antigens on similarly immunomagnetically labeled cells.

The use of magnetite-doped polymeric microspheres in calibrating cell tracking velocimetry.

Continuous magnetic separation, in which there is no accumulation of mass in the system, is an inherently dynamic process, requiring advanced knowledge of the separable species for optimal instrument operation. By determining cell magnetization in a well-defined field, we may predict the cell trajectory behavior in the well-characterized field environments of our continuous separators. Magnetization is determined by tracking the migration of particles with a technique known as cell tracking velocimetry (CTV). The validation of CTV requires calibration against an external standard. Furthermore, such a standard, devoid of the variations and instabilities of biological systems, is needed to reference the method against day-to-day shifts or trends. To this end, a method of synthesizing monodisperse, magnetite-doped polymeric microspheres has been developed. Five sets of microspheres differing in their content of magnetite, and each of approximately 2.7 microm diameter, are investigated. An average gradient of 0.18 T/mm induces magnetic microsphere velocities ranging from 0.45 to 420 microns/s in the CTV device. The velocities enable calculation of the microsphere magnetization. Magnetometer measurements permit the determination of magnetization at a flux density comparable to that of the CTV magnet's analysis region, 1.57 T. A comparison of the results of the CTV and magnetometer measurements shows good agreement.

Cell damage of microcarrier cultures as a function of local energy dissipation created by a rapid extensional flow.

Microcarrier cultures of Chinese hamster ovary cells were subjected to a range of energy dissipations created by an abrupt contraction. These flow conditions can be characterized as a rapidly transient, extensional, and shear flow. Cell damage was measured using a lactate dehydrogenase assay. The laminar flow in the device was modeled using two commercial, computation fluid-dynamic codes: POLYFLOW and FLUENT. Cell damage was correlated to numerical values of energy dissipation. The magnitude of energy dissipation at which cell damage began to be detected, 10(4) ergs cm(-3) s(-1) (10(4) cm(2) s(-3)), is consistent with values of energy dissipation estimated in bioreactors operated under conditions which result in cell damage. This magnitude of energy dissipation is orders of magnitude lower than those values reported to cause damage to suspended animals cells which is also consistent with generally accepted experimental observations. Finally, an analysis and discussion of the presence and relative importance with re- spect to cell damage of shear vs. extensional flow is included.

Cell-microcarrier adhesion to gas-liquid interfaces and foam.

The interaction of microcarriers, both with and without cells attached, with gas bubbles was studied. These studies consisted of qualitative microscopic observations of microcarriers with bubbles, quantitative measurements of microcarrier entrapment in foam, and quantitative measurements of the effect of bubble rupture at gas-medium interfaces. Ten different "protective additives" were evaluated for their ability to change the dynamic surface tension of the culture media and to prevent microcarrier adhesion to air bubbles during gas sparging and to prevent entrapment in the foam layer. These studies indicate that microcarriers, with and without cells, readily attach to gas-medium interfaces; yet unlike suspended cells, cells attached to microcarriers are not damaged by bubble ruptures at gas-medium interfaces. Only one surfactant was found to substantially prevent microcarrier entrapment in the foam layer; however, this surfactant was toxic to cells. No correlation was observed between surface tension and the prevention of microcarrier adhesion to gas-liquid interfaces. It is suggested that cell damage as a result of sparging in microcarrier cultures is the result of cells, attached to microcarriers, attaching to rising bubbles and then detaching from the microcarrier as this combination rises through the medium. It is further suggested that the hydrodynamic drag force of the rising microcarrier is sufficiently high to remove the bubble-attached cell from the microcarrier.

Study of magnetic particles pulse-injected into an annular SPLITT-like channel inside a quadrupole magnetic field.

Advantages of the continuous magnetic flow sorting for biomedical applications over current, batch-wise magnetic separations include high throughput and a potential for scale-up operations. A continuous magnetic sorting process has been developed based on the quadrupole magnetic field centered on an annular flow channel. The performance of the sorter has been described using the conceptual framework of split-flow thin (SPLITT) fractionation, a derivative of field-flow fractionation (FFF). To eliminate the variability inherent in working with a heterogenous cell population, we developed a set of monodisperse magnetic microspheres of a characteristic magnetization, and a magnetophoretic mobility, similar to those of the cells labeled with a magnetic colloid. The theory of the magnetic sorting process has been tested by injecting a suspension of the magnetic beads into the carrier fluid flowing through the sorter and by comparing the theoretical and experimental recovery versus total flow-rate profiles. The position of the recovery maxima along the total flow-rate axis was a function of the average bead magnetophoretic mobility and the magnetic field intensity. The theory has correctly predicted the position of the peak maxima on the total flow-rate axis and the dependence on the bead mobility and the field intensity, but has not correctly predicted the peak heights. The differences between the calculated and the measured peak heights were a function of the total flow-rate through the system, indicating a fluid-mechanical origin of the deviations from the theory (such as expected of the lift force effects in the system). The well-controlled elution studies using the monodisperse magnetic beads, and the SPLITT theory, provided us with a firm basis for the future sorter evaluation using cell mixtures.

Flow rate optimization for the quadrupole magnetic cell sorter.

The quadrupole magnetic cell sorter is a form of split-flow thin-channel (SPLITT) separation device. It employs a quadrupole magnetic field and annular channel geometry. Immunomagnetic labels are used to bind to specific receptors on the surface of the cells of interest. It is the interaction of these labels with the magnetic field that brings about the selective isolation of these cells. The SPLITT separation devices have generally been based on parallel-plate geometry, usually with effectively constant field strength applied across the channel thickness. The nonconstant field strength and annular channel geometry of the magnetic cell sorter require that a new strategy be developed for optimization of inlet and outlet flow rates. We present such a strategy here based on a consideration of certain specific cell trajectories within the system.

Quantification of cellular properties from external fields and resulting induced velocity: cellular hydrodynamic diameter.

An experimental technique is discussed in which the size distribution of a population of cells is determined by calculating each cell's settling velocity. The settling velocity is determined from microscopically obtained images which were recorded on SVHS tape. These images are then computer imaged and processed, and the cell's location and velocity are determined using a computer algorithm referred to as cell tracking velocimetry (CTV). Experimental data is presented comparing the distribution of human lymphocytes and a human breast cancer cell line, MCF-7, determined using a Coulter counter and the CTV approach.

Quantification of cellular properties from external fields and resulting induced velocity: magnetic susceptibility.

An experimental technique is discussed in which the magnetic susceptibility of immunomagnetically labeled cells can be determined on a cell-by-cell basis. This technique is based on determining the magnetically induced velocity that an immunomagnetically labeled cell has in a well-defined magnetic energy gradient. This velocity is determined through the use of video recordings of microscopic images of cells moving in the magnetic energy gradient. These video images are then computer digitized and processed using a computer algorithm, cell tracking velocimetry, which allows larger numbers (>10(3)) of cells to be analyzed.