Particle magnetic susceptibility determination using analytical split-flow thin fractionation.

Magnetic split-flow thin (SPLITT) fractionation is a newly developed S

Analytical magnetapheresis of magnetically susceptible particles.

Analytical magnetapheresis is a newly developed technique for analyzing magnetic particles. The magnetically susceptible particles form deposition patterns after flowing through a separation channel in a magnetic field. The separation channel requirements for analytical magnetapheresis are an excellent seal for the carrier flow and ease of disassembly after magnetapheresis. Previously used separation channels often exhibit variable channel leakage and unstable flow velocities. We improved the separation channel assembly to ensure stable, high flow velocities and characterized the system with various magnetically susceptible and labeled particles. Our new separation channel featured silicone sealant with embedded nylon wires and met analytical magnetapheresis requirements. Characterization of this system was performed using several magnetically susceptible particles, and we studied a variety of diamagnetic sample labels with paramagnetic ions and magnetically susceptible particles at different flow-rates and solution pH values. The minimal labeling concentration for complete deposition was determined to be approximately 2.50 x 10(10) ions per particle for test samples at a flow velocity of 0.67 mm s(-1) and a magnetic field gradient of 2.8 T mm(-1). Silicas, yeasts and blood cells were used for these studies. We determined that the minimal difference in magnetic susceptibility (delta(chi)) for successful separation was approximately 2.00 x 10(-6) [SI]. The magnetic susceptibilities of Dynabeads M-450 at several separation distances and flow-rates were determined to be 0.25 [SI], within 2% of values published by other workers. The magnetic susceptibilities of various ion-labeled yeasts and cells were determined and most varied by less than 5% at different flow-rates. The results of this study provide very important references for analytical magnetapheresis applications.

Magnetic split-flow thin fractionation of magnetically susceptible particles.

We recently built a magnetic separation system to extend the applications of split-flow thin (SPLITT) fractionation to magnetically susceptible particles. Here, we characterize the magnetic SPLITT system using magnetically susceptible particles and ion-labeled particles. The flow axis of separation channel was orientated parallel and perpendicular to gravitational forces to exclude and include, respectively, gravitational effects on separation. Both operating modes were used to test the theory experimentally, with emphasis on the parallel mode. The magnetic susceptibilities of carrier and ion-labeled particles were varied, and various ion-labeled and unlabeled particles were studied experimentally, resulting in successful separation of labeled particles, yeasts, and cells from unlabeled ones. The minimal difference in magnetic susceptibility (delta(chi)) required for complete particle separation was about 1.75 x 10(-5) [cgs], corresponding to about 10(9) labeling ions per particle in this study. The throughput was around 7.2 x 10(8) particles/h using the present setup. Magnetic SPLITT fractionation shows good potential for use in obtaining particles magnetic susceptibilities from a simple theoretical treatment.

Immunomagnetic isolation of magnetoferritin-labeled cells in a modified ferrograph.

Pan T, helper, and cytotoxic lymphocytes were isolated from the human peripheral blood mononuclear cell fraction by antibody staining, ferritin labeling, and deposition on glass slides. Two distinct forms of ferritin were used: one was native horse spleen ferritin, and the other was magnetoferritin. Magnetoferritin was obtained by reconstituting the horse spleen ferritin iron core with superparamagnetic magnetite instead of the usual paramagnetic ferrihydrite crystal. The cell deposition on microscopic glass slides in the magnetic field was obtained by an instrument that was adapted from an industrial magnetic deposition analyzer, the ferrograph. The identity of cells in the magnetic deposits was confirmed by comparing the cell fractions in the feed and in the eluate with the use of flow cytometry. The immunostaining protocol amplified the number of ferritin molecules per cell surface antigen 20-70 times. Magnetoferritin, but not native ferritin, imparted a sufficient magnetic moment to cells to deplete the labeled cell population between 67 and 88% of its initial concentration in a magnetic field of 1.67 Tesla (T), a field gradient of 2.57 T/mm, and a flow rate of 0.01 ml/min. This study showed that the magnetic moment of magnetoferritin was sufficient for immunomagnetic isolation of lymphocytes from mononuclear cell preparations in the modified ferrograph.

Analytical magnetapheresis of ferritin-labeled lymphocytes.

Analytical magnetapheresis is a technique for analyzing magnetic particles in suspension. The magnetically susceptible particles form a deposition pattern from the suspending medium under carefully controlled flow and magnetic field conditions. This technique was used to determine the effective magnetic volumetric susceptibility, delta chi, of human lymphocytes labeled with an iron-rich protein, ferritin. Dynabeads M450, monodisperse polymeric beads doped with magnetite, of a diameter 4.5 microns, close to that of human lymphocytes, were used as a reference. The experiment showed an almost complete deposition of ferritin-labeled lymphocytes at an average flow velocity of 0.28 mm/s, a representative magnetic field of 1.67 T, and a magnetic field gradient of 2.57 T/mm. The calculated delta chi was (2.92 +/- 0.24) x 10(-6)[SI] (ferritin-labeled lymphocytes), and the corresponding number of ferritin molecules per lymphocyte was (1.75 +/- 0.44) x 10(7). In comparison, an almost complete deposition of the Dynabeads was observed at a much higher average flow velocity, 15 mm/s, a much lower field, 0.164 T, and a much lower field gradient, 0.025 T/mm. These results corresponded to a much higher delta chi = 0.245[SI] (Dynabeads M450). These results offer important guidelines in evaluating the use of ferritin as a soluble magnetic cell label.

Isolation of human blood cells, platelets, and plasma proteins by centrifugal SPLITT fractionation.

Centrifugal SPLITT fractionation, a technique designed for the continuous high-resolution separation of colloids and low-density particles, is applied here to fresh human blood, producing six purified fractions consisting of proteins and lipoproteins, platelets, red blood cells, lymphocytes, monocytes, and neutrophils. Production of the six fractions requires five steps, each yielding two fractions. These five steps are carried out in sequence using a single apparatus, with conditions varying from step to step in accordance with theoretical guidelines in order to achieve the desired cut points. In the first step, a stream of diluted blood is separated into one fraction consisting of platelets and plasma and another containing blood cells. The throughput of diluted blood is 162 mL/h and that of whole blood is about 2 mL/h or approximately 10(10) cells/h; guidelines are given for significantly increasing throughput. The purity of the blood cell fractions ranged from 92 to 98%, and the viability fell in the range 97-99%.