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.

Inverted Linear Halbach Array for Separation of Magnetic Nanoparticles.

A linear array of Nd-Fe-B magnets has been designed and constructed in an inverted Halbach configuration for use in separating magnetic nanoparticles. The array provides a large region of relatively low magnetic field, yet high magnetic field gradient in agreement with finite element modeling calculations. The magnet assembly has been combined with a flow channel for magnetic nanoparticle suspensions, such that for an appropriate distance away from the assembly, nanoparticles of higher moment aggregate and accumulate against the channel wall, with lower moment nanoparticles flowing unaffected. The device is demonstrated for iron oxide nanoparticles with diameters of ~ 5 and 20 nm. In comparison to other approaches, the inverted Halbach array is more amenable to modeling and to scaling up to preparative quantities of particles.

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 field application or mechanical stimulation via magnetic microparticles does not enhance chondrogenesis in mesenchymal stem cell sheets.

Using a novel magnetic field bioreactor, this work evaluated the chondrogenesis of scaffold-free human mesenchymal stem cell sheets in response to static and variable magnetic fields, as well as mechanical stimulation via 4.4 µm magnetic particles. Neither static nor variable magnetic fields generated by 1.44-1.45 T permanent magnets affected cartilage formation. Notably, magnetic field-induced mechanical stimulation by magnetic particles, which applied forces to the cells and ECM statically (4.39 pN) or cyclically (1.06-63.6 pN; 16.7 mHz), also did not affect cartilage formation.

Interpersonal deviance and consequent social impact in hypothetically schizophrenia-prone men.

Interpersonal deviance is central to the theory of and research on schizotypal psychopathology. The present study investigated interpersonal deviance and its corresponding impact among hypothetically schizotypic, or schizophrenia-prone, men, defined by high scores on the Perceptual Aberration-Magical Ideation (Per-Mag) Scale. In a videotaped interview, high-scoring Ss relative to control Ss were rated as more odd (p < .001) and more avoidant (p < .05) in their interview behavior and made the interviewers feel more anxious (p < .05), more angry (p < .05), and less interested (p < .05). Other analyses revealed that oddness was the strongest discriminating variable and that this behavior could not be accounted for by social anxiety or lack of interest. These results provide further construct validation for the Per-Mag scale and suggest that interpersonal factors may influence the eventual adjustment of high-scoring individuals.

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 through, immunomagnetic cell separation.

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

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.

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.

A comparison of efficacy of Tolpa Torf Preparation (TTP) in the treatment of cervicitis with or without surgery.

Tolpa Torf Preparation (TTP) is an immunomodulating drug produced by Torf Corporation, Wroclaw and registered for human use in Poland. TTP enhances the process of tissue regeneration. Authors evaluate TTP effectiveness in the treatment of inflammatory states of the cervix, especially cervical erosions and the influence of this preparation of the macroscopic, cytological and bacteriological state of the cervix. TTP was used in 31 patients with the diagnosis of cervical erosion. All patients treated as yet were classified into 3 groups, depending on the treatment of cervical erosion used previously. TTP was administered orally in the dose of 5 mg (in 10 ml of water) daily during 10 days and locally in the form of tampons soaked with 1% TTP solution in the volume of 5 ml also during 10 days. TTP administered this way has beneficial therapeutic effects on the healing of cervical erosion accelerating the process of epithelialization and bringing normalization of the cytological picture. Especially beneficial in the treatment of cervical erosion is combined use of TTP and electrocoagulation or curettage--the healing time can be shortened by half.

Lymphocyte fractionation using immunomagnetic colloid and a dipole magnet flow cell sorter.

The relationship between cell function and surface marker expression is a subject of active investigation in biology and medicine. These investigations require separating cells of a homogeneous subset into multiple fractions of varying marker expression. We have developed a novel cell sorter, the dipole magnet flow sorter (DMFS), which separates selected T lymphocyte subpopulations, targeted by immunomagnetic colloid, into multiple fractions according to cell surface marker expression, as determined by flow cytometry. A narrow stream of cells is introduced into a sheath of carrier fluid in a rectangular channel while subjected to a perpendicular magnetic force. The special design of the pole pieces ensures a constant magnetic force acting on the magnetically labeled cells in the separation area. Cells are spread across the flow in relation to their magnetophoretic mobility. Separation is achieved by control of the positions of the effluent stream boundaries, which separate fluid volumes with cells of different magnetophoretic mobility. CD4 and CD8 T lymphocytes labeled with primary antibody-fluorescein isothiocyanate (FITC) conjugate and anti-FITC-magnetic colloid are the chosen cell systems. Flow cytometry analysis shows that, for CD4 cells, a three-fold increase in total marker number per cell is observed when comparing the highest to the lowest fluorescence fractions. Similarly, a four-fold increase in total marker number is observed for CD8 cells. We also observed the separation of two dissimilar cell types that differed in expression of the CD4 marker, monocytes and T helper lymphocytes. We believe that this type of separation is applicable to any cells in suspension for which a suitable antibody exists and, due to the comparatively gentle nature of the process, is particularly suitable for the sorting of fragile cells.

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.

A comprehensive investigation of putative correlates of bulimia among college-age women: object relations, dependency, ego defenses, trait anxiety, and depression.

This study investigated putative correlates of bulimia, which included measures of object relations impairment, dependency, ego defenses, trait anxiety and depression. Subjects were college-age women drawn from a larger sample (n = 417) and selected on the basis of their scores on the revised version of the Bulimia Test. The bulimia group was composed of those scoring in the top 5% (n = 23), concordant with the base rate of bulimia in this population, whereas controls (n = 23) were conservatively selected from those scoring around the mean (56th-61st percentile). A multivariate analysis of variance revealed a significant group difference across dependent variables (p < .05), and univariate Fs uniformly demonstrate predicted group differences. A stepwise discriminant analysis was performed to assess the relative contribution of these variables toward the differentiation between psychometrically defined, bulimic and nonbulimic women, revealing that trait anxiety, dependency, and depression reliably differentiated these groups, accounting for 37% of between-groups variance. In replicating and extending prior research, the results have implications for elucidating the phenomenology and potential etiologic factors associated with bulimia and for informing treatment and future research.

Experimental study of extracorporeal perfusion for septic shock.

The authors evaluated the efficacy of treatment by extracorporeal perfusion on experimental canine septic shock. Canine septic shock was produced by intravenous infusion of Escherichia coli endotoxin and treated by three techniques: no treatment (Sham), hemoperfusion over Polymyxin B immobilized fiber (PMX), and plasma perfusion over anion exchange resin (Resin). The 24 hr survival rates of the Sham, PMX, and Resin groups were 0%, 80%, and 40%, respectively. In the PMX group, blood pressure was significantly better over 6 hr than that recorded in the Sham group. In the PMX group, phagocytic function evaluated by neutrophil function, opsonic index, and complement were better than that of the Sham group. In addition, blood endotoxin levels in the PMX group were significantly lower, resulting in a significant suppression of TNF release. In the Resin group, some parameters were significantly better than those of the Sham group, but the efficacy of this treatment was less than that of the PMX treatment. Hemoperfusion over Polymyxin B immobilized fibers can detoxify circulatory endotoxin, resulting in improvement of systemic and organic disorders caused by sepsis.

Rapid cell isolation by magnetic flow sorting for applications in tissue engineering.

Rapid and efficient cell sorting methods are important for tissue progenitor cell isolation. We built and evaluated a laboratory prototype of a continuous flow, quadrupole magnetic cell sorter. The sorter was tested on a model cell system of human peripheral lymphocytes. The helper T cell subpopulation was targeted by primary, mouse anti-CD4 monoclonal antibody conjugated to a fluorochrome (FITC), and magnetized by secondary, anti-FITC antibody magnetic colloid. The purities and recoveries of the cell fractions were measured by flow cytometry and an automated cell counter. Cells were spread across the flow according to their magnetophoretic mobilities. The purity of the CD4 cell enriched fraction was 99.6%, and the purity of the CD4 cell depleted fraction was 2% for an initial CD4 cell purity of 36%; the corresponding recovery of the enriched CD4 cell fraction was 59% at a sorting speed of 4,200 cells/s (four experiments). The recovery could be increased to 90% with a concomitant decrease in the purity of CD4 cell enriched fraction to 66%. This type of sorting should be applicable to any cells in suspension for which a suitable antibody exists, in particular, to large, fragile cells.

Removal of trypsin complexed alpha-2 macroglobulin by plasma fractionation.

The aim of this study is to assess in vitro the efficacy of a plasma fractionator to remove trypsin complexed alpha 2MG (alpha-2 macroglobulin) from human plasma in a manner analogous to the reticuloendothelial system (RES). Eval filter type "4A" (Kuraray Co., Osaka, Japan) was chosen as a plasma fractionator. Two and one half liters of bovine trypsin spiked human plasma was perfused in vitro through the fractionator in a single pass mode (n = 5). The concentrations of complexed alpha 2MG, total alpha 2MG, albumin, and IgM were measured before and after fractionation, and the concentration of free alpha 2MG and the sieving coefficients of each solute were calculated. The concentration of the trypsin complexed alpha 2MG measured by ELISA was significantly decreased by fractionation with Eval "4A" from 103.7 +/- 16.7 to 13.8 +/- 8.2 mg/L (reduction of 86.7%). Mean sieving coefficients of each solute were 0.133 +/- 0.079 in complexed alpha 2MG, 0.203 +/- 0.065 in free alpha 2MG, 0.203 +/- 0.065 in total alpha 2MG, 0.770 +/- 0.130 in albumin, and 0.070 +/- 0.010 in IgM. Although in vivo study will be required in patients with acute pancreatitis, in vitro study shows the feasibility of membrane plasma fractionation in eliminating trypsin complexed alpha 2MG.

Extracorporeal endotoxin removal in a canine model of septic shock.

The authors induced endotoxic shock in an animal model and attempted to treat this state by direct hemoperfusion over a modified anion sorbent column. It has been shown that the reversal of septic shock correlates with the efficiency of extracorporeal endotoxin removal. In this experiment, there were five control animals (sham) and five test animals (hemoperfusion over sorbent column). The efficacy of treatment was evaluated by survival at 24 hr, changes in mean arterial pressure, blood-acid base balance, and plasma endotoxin levels. There was 0% survival in the control group and 100% survival in the test group. The control dogs never recovered from shock or metabolic acidosis, but the test animals were at their initial values for these parameters by 6 hr. The endotoxin levels measured at 6 hr were higher in the control group (265 +/- 88 ng/ml) as compared with the test group (7.0 +/- 6.2 ng/ml). Direct hemoperfusion over a modified sorbent column effectively removed endotoxin and reversed the course of fatal septic shock.

Clinical trials of a cryoglobulin filter.

The authors report the results of clinical trials of a high capacity cryoglobulin filter (Cryofilter) in seven patients with cryoglobulinemia unresponsive to high doses of prednisone or immunosuppressive drugs who required plasmapheresis. The objective of this study was to test the safety and efficacy of the cryofilter in a limited patient population according to the investigational Device Exemption guidelines of the FDA. The cryoglobulins were selectively filtered from plasma at 4 degrees C by a cryofilter characterized by a membrane surface area of 0.135 m2 and an average pore size of 4.3 microns. Safety was evaluated by patients vital signs, complement activation, and clinical score of symptoms in the course of 10 treatments. Efficacy of cryofiltration was evaluated by comparing sieving of the cryoglobulins to that of albumin; immunoglobulins G, A, and M; and fibrinogen. All seven patients completed the series of 10 treatments without notable complement activation or any signs of discomfort. The cryofilter was particularly selective in patients with high cryoglobulin concentrations. Improvement in clinical symptoms was observed in all patients.

Magnetic flow sorting using a model system of human lymphocytes and a colloidal magnetic label.

Cells of identical physical properties that differ in the expression of surface proteins can be sorted conveniently using immunospecific stains conjugated to fluorescent, or magnetic, labels. Immunomagnetic cell sorting using commercial batch sorters offers advantages of high sorting capacity, high viability of sorted fractions, and high depletion rates; its disadvantages are low enrichment rate and batch processing. The authors developed and tested a continuous, flow-through magnetic cell sorter for small volume, experimental cell enrichment. Freshly isolated human peripheral lymphocytes were labeled using an immunofluoromagnetic sandwich consisting of mouse anti human CD8 monoclonal antibody-fluorescein conjugate and rat anti mouse polyclonal antibody-colloidal iron-dextran conjugate. A total of 2-3 min lymphocytes were sorted per hour using a saturation magnetic field of 1.334 T and a five channel sorter. The fluorescent cells were distributed among the channels in relation to their fluorescence intensity and magnetic susceptibility. The purity (68-85%) and enrichment rates (16-34x) were comparable to those of commercial batch magnetic separators; sorting capacity and recovery of the enriched fractions (up to 32%) were limited by the small scale of the sorter. Future direction is focused on increasing the resolution, recovery, and sorting capacity of the enriched fractions, and testing the sorter on other cell systems.

Cryofiltration apheresis for treatment of cryoglobulinemia associated with hepatitis C.

Cryofiltration apheresis (CA) is a specific therapy for treatment of patients with cryoglobulinemia. We evaluated the safety and efficacy of CA in patients with mixed cryoglobulinemia associated with hepatitis C. As reported previously, the Cryoglobulin Filter comprises a membrane module inside a refrigeration unit on-line with a Spectra Apheresis System (COBE, Denver, CO). The efficacy of cryofiltration was measured by comparing the sieving coefficient of cryoprecipitable proteins (CPP) to that of albumin and comparing the systemic CPP concentration ratio post to pre treatment. Five patients were enrolled in this study, and a minimum of 10 procedures were performed for each patient. The risk for hepatitis C was multiple blood transfusions, intravenous drug abuse, immunosuppressive therapy, or renal transplantation. Four patients had Type II mixed cryoglobulinemia, and one patient had Type III. Four patients had chronic renal failure; one with liver cirrhosis received alpha interferon along with CA. One patient had no response to conventional plasma exchange and immunosuppressive therapy secondary to repeated infections and sepsis; CA was the only viable therapy for this patient. The maximum CPP concentration before therapy ranged from 1,440 to 7,440 micrograms/ml. The plasma CPP sieving coefficient at 1 L filtrate ranged from 0.25 to 0.74 (average +/- SE, 0.51 +/- 0.19; n = 39). The sieving coefficient for albumin was 1 (n = 50). The systemic CPP ratio post to pre treatment ranged from 0.28 to 0.83 (average +/- SE, 0.59 +/- 0.20; n = 37). No adverse effects specific to CA were observed. The CA was safe and effective and possibly the only choice of therapy in patients with cryoglobulinemic hepatitis C who have no response to plasma exchange and immunosuppressive therapy.