Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.

1. Instrument setup

NOTE: Imaging levitating cells requires two rare earth neodymium magnets magnetized on the z-axis to be placed with the same pole facing each other to generate a magnetic field. The distance between the magnets can be customized depending on the intensity of the magnetic field and the density of the targets. In this case the magnets are separated by a 1mm space sufficient for insertion of a 50 mm long 1×1 mm squared glass capillary tube. The device was 3-D printed using an AutoCAD design, which is available upon request.

1. Lay microscope on its side, perfectly horizontal.
   NOTE: A microscope placed in a standard upright position is not directly suitable for imaging levitating objects due to the positions of the magnets with respect to condenser and objective. This limitation can be bypassed by laying the microscope on its side, perfectly horizontal allowing the condenser to focus the light into the capillary, and the objective lens to image the cell levitating in between the magnets, while maintaining Köhler illumination requirements.
2. Support and level the stand on a breadboard table using 2 or 3 lab jacks.
3. To limit vibrations, support the breadboard table with rubber dampening feet.
4. Remove the stage and replace it with a compact lab jack for adjusting the height of the levitation device (y-axis), and two single-axis translational stages; one for adjusting the focus (z-axis), and the second one for scanning the capillary tube.
5. Attach the magnetic levitation device to the lab jack using two mini-series optical posts.

2. Binding of Antibody to Carboxy-Microparticles/Beads (Modified from a protocol by PolyAn)

NOTE: Only low-density beads (1.05 g/mL) need to be coated for Rhesus, Rh(+) detection, but both high- and low-density beads are coated for the detection of extracellular vesicles.

1. Take out 1 mg equivalent of bead suspension and add into 0.5 mL of Activation Buffer (50 mM 2-(N-morpholino)ethanesulfonic acid, MES (MW195.2, 9.72 mg in 1 mL)) pH 5.0 and 0.001% Polysorbate-20).
2. Add 12 µL of freshly made 1.5 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (MW 191.7, 0.28755 g in 1 mL) and 12 µL of 0.3 M Sulfo-N-Hydroxysuccinimide (NHS)  (MW 217.14, 0.0651 g in 1 mL) in ice-cold water.
3. Place tubes on an orbital shaker and shake vigorously for 1 h at room temperature to activate the carboxyl groups on beads.
4. Stop the activation after 1 h by adding 0.5 mL of Coupling Buffer (10x phosphate-buffered saline, PBS, or 0.1 M phosphate pH 7-9).
5. Pellet the beads by centrifuging at 20,000 x g for 10 min, or, if the beads cannot be pelleted, use a 0.45 µm centrifuge tube filter. Aspirate the supernatant or flow-through.
6. Wash the beads with 1 mL of 10x PBS 3 times as in step 2.5.
7. Calculate 25 µg of antibody per mg beads and mix desired antibody with activated beads to a 0.7-1.0 mg/mL final antibody concentration in 10X PBS.
8. Place tubes on a tube rocker set on a low speed. Roll tubes gently at room temperature overnight for coupling.
9. Repeat step 2.5 and wash the beads twice with 1 mL of 10x PBS.
10. Wash with 0.5 mL of 1 M ethanolamine (98% stock = 16.2 M) in buffer pH 8.0 with 0.02% Polysorbate-20 for 1 h at room temperature while gently shaking.
11. Repeat step 2.5 and wash the beads once in 1 mL of Dulbecco's phosphate-buffered saline (DPBS). The beads can be stored in 200 µL of  DPBS at 4 °C until needed.    
    NOTE: The protocol can be paused here.

3. Collection and Preparation of Blood for Rh(+) Detection

1. Using a one-click lancing device, prick the finger of an Rh(+) donor and collect 10 µL of blood into 1 mL of DPBS.
2. Stain the Rh+ cells with a fluorescent plasma membrane stain. Optionally, add 1 µL of fluorescent dye to the 1 mL suspension of Rh+ cells (1:1000 dilution).
3. Incubate the cells with the fluorescent dye at 37 °C for 15 min.
4. Pellet the cells by spinning at 5,600 x g for 15 s and wash 3 times using 1 mL of DPBS. Resuspend in 1 mL of Hanks' balanced salt solution (HBSS)  with calcium and magnesium (HBSS++).
5. Using the one-click lancing device, prick the finger of an Rh(-) donor and collect 2 µL of blood.
   NOTE: If preparing more than 2 conditions, collect enough blood to add 1 µL of Rh(-) blood to each tube.
6. Prepare the necessary experimental tubes: Beads alone, immunoglobulin G (IgG) control, sample.
   1. Beads alone: add 174 µL of HBSS++, 1 µL of IgG control beads, 1 µL of high-density beads (1.2 g/mL), and 24 µL of 500 mM Gd³⁺ (60 mM).
   2. IgG control: add 172 µL of HBSS++, 1 µL of IgG control beads, 1 µL of high-density beads, 1 µL of Rh- blood, 1 µL of stained Rh+ blood suspension, and 24 µL of 500 mM Gd³⁺.
   3. Sample tube add 172 µL of HBSS++, 1 µL of anti-RhD coated beads, 1 µL of high-density beads, 1 µL of Rh- blood, 1 µL of stained Rh(+) blood suspension, and 24 µL of 500 mM Gd³⁺.
      NOTE: High density beads are added to Rh samples for reference.

4. Analyzing Cells on the Magnetic Levitation Device

1. Perform instrument startup according to manufacturer instructions.
2. Load 50 µL of sample into a capillary tube until the tube is filled. Seal the ends of the capillary tube with capillary sealant making sure there are no air bubbles present.
3. Load the capillary tube into the holder between the top and bottom magnets. Adjust the stage and focus for optimal viewing.
   NOTE: Cells/beads can take anywhere from 5-20 min to reach their magnetic equilibrium position based on their density and the concentration of Gd³⁺. The higher the concentration of Gd³⁺, the shorter the time.