Name: detection of rhesus factor in human blood samples using magnetic levitation
Uploaded: 2023-11-30T12:47:36.000+00:00
Duration: 1 min 32 s
Description: All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human ...

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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 1x1&#160;mm squared glass capillary tube. The device was 3-D printed using an AutoCAD design, which is available upon request.
<ol>
	<li>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 K&#246;hler illumination requirements.</li>
	<li>Support and level the stand on a breadboard table using 2 or 3 lab jacks.</li>
	<li>To limit vibrations, support the breadboard table with rubber dampening feet.</li>
	<li>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.</li>
	<li>Attach the magnetic levitation device to the lab jack using two mini-series optical posts.</li>
</ol>
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.
<ol>
	<li>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).</li>
	<li>Add 12 &#181;L 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 &#181;L of 0.3 M Sulfo-N-Hydroxysuccinimide (NHS)&#160; (MW 217.14, 0.0651 g in 1 mL) in ice-cold water.</li>
	<li>Place tubes on an orbital shaker and shake vigorously for 1 h at room temperature to activate the carboxyl groups on beads.</li>
	<li>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).</li>
	<li>Pellet the beads by centrifuging at 20,000 x&#160;g&#160;for 10 min, or, if the beads cannot be pelleted, use a 0.45 &#181;m centrifuge tube filter. Aspirate the supernatant or flow-through.</li>
	<li>Wash the beads with 1 mL of 10x PBS 3 times as in step 2.5.</li>
	<li>Calculate 25 &#181;g 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.</li>
	<li>Place tubes on a tube rocker set on a low speed. Roll tubes gently at room temperature overnight for coupling.</li>
	<li>Repeat step 2.5 and wash the beads twice with 1 mL of 10x PBS.</li>
	<li>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.</li>
	<li>Repeat step 2.5 and wash the beads once in 1 mL of Dulbecco&#39;s phosphate-buffered saline (DPBS). The beads can be stored in 200 &#181;L of &#160;DPBS at 4 &#176;C until needed.&#160;&#160;&#160;&#160; 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Collection and Preparation of Blood for Rh(+) Detection
<ol>
	<li>Using a one-click lancing device, prick the finger of an Rh(+) donor and collect 10 &#181;L of blood into 1 mL of DPBS.</li>
	<li>Stain the Rh+ cells with a fluorescent plasma membrane stain. Optionally, add 1 &#181;L of fluorescent dye to the 1 mL suspension of Rh+ cells (1:1000 dilution).</li>
	<li>Incubate the cells with the fluorescent dye at 37 &#176;C for 15 min.</li>
	<li>Pellet the cells by spinning at 5,600 x&#160;g&#160;for 15 s and wash 3 times using 1 mL of DPBS. Resuspend in 1 mL of Hanks&#39;&#160;balanced salt solution&#160;(HBSS)&#160; with calcium and magnesium (HBSS++).</li>
	<li>Using the one-click lancing device, prick the finger of an Rh(-) donor and collect 2 &#181;L of blood. 
	NOTE: If preparing more than 2 conditions, collect enough blood to add 1 &#181;L of Rh(-) blood to each tube.</li>
	<li>Prepare the necessary experimental tubes: Beads alone, immunoglobulin G (IgG) control, sample.
	<ol>
		<li>Beads alone: add 174 &#181;L of HBSS++, 1 &#181;L of IgG control beads, 1 &#181;L of high-density beads (1.2 g/mL), and 24 &#181;L of 500 mM Gd3+&#160;(60 mM).</li>
		<li>IgG control: add 172 &#181;L of HBSS++, 1 &#181;L of IgG control beads, 1 &#181;L of high-density beads, 1 &#181;L of Rh- blood, 1 &#181;L of stained Rh+ blood suspension, and 24 &#181;L of 500 mM Gd3+.</li>
		<li>Sample tube add 172 &#181;L of HBSS++, 1 &#181;L of anti-RhD coated beads, 1 &#181;L of high-density beads, 1 &#181;L of Rh- blood, 1 &#181;L of stained Rh(+) blood suspension, and 24 &#181;L of 500 mM Gd3+. 
		NOTE: High density beads are added to Rh samples for reference.</li>
	</ol>
	</li>
</ol>
4. Analyzing Cells on the Magnetic Levitation Device
<ol>
	<li>Perform instrument startup according to manufacturer instructions.</li>
	<li>Load 50 &#181;L 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.</li>
	<li>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.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-(N-Morpholino)ethanesulfonic acid hydrate</td>
			<td>Sigma Aldrich</td>
			<td>M-2933</td>
			<td>(MES); component of activation buffer</td>
		</tr>
		<tr>
			<td>50x2.5x1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197&#34;) thickness</td>
			<td>K&#38;J Magnetics</td>
			<td>Custom</td>
			<td>Magnets used for the magnetic levitation device</td>
		</tr>
		<tr>
			<td>Capillary Tube Sealant (Critoseal)</td>
			<td>Leica Microsystems</td>
			<td>267620</td>
			<td>Used to cap the ends of the capillary tubes</td>
		</tr>
		<tr>
			<td>Centrifuge tube filters (Corning Costar Spin-X)</td>
			<td>Sigma Aldrich</td>
			<td>CLS8163</td>
			<td>Used to wash beads</td>
		</tr>
		<tr>
			<td>Compact Lab Jack</td>
			<td>Thorlabs</td>
			<td>LJ750</td>
			<td>Used for adjusting the magnetic levitation device</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Solution for bead suspensions</td>
		</tr>
		<tr>
			<td>Ethanolamine</td>
			<td>Sigma Aldrich</td>
			<td>E9508-100ML</td>
			<td>Used during a wash step for beads</td>
		</tr>
		<tr>
			<td>Fluorescent Plasma Membrane Stain (CellMask Green)</td>
			<td>Invitrogen</td>
			<td>C37608</td>
			<td>Used to stain Rh+ cells</td>
		</tr>
		<tr>
			<td>Gadoteridol Injection</td>
			<td>ProHance</td>
			<td>NDC 0270-1111-03</td>
			<td>Gadolinium (Gd3+); magnetic solution used to suspend cells</td>
		</tr>
		<tr>
			<td>HBSS++</td>
			<td>Gibco</td>
			<td>14025-092</td>
			<td>Solution for sample preparation</td>
		</tr>
		<tr>
			<td>Mini Series Post Collar</td>
			<td>Thorlabs</td>
			<td>MSR2</td>
			<td>Used to secure magnetic levitation device to lab jacks</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N&#8242;-ethylcarbodiimide hydrochloride</td>
			<td>Sigma Aldrich</td>
			<td>E1769-10G</td>
			<td>(EDC); used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Normal Rabbit IgG Control</td>
			<td>R&#38;D Systems</td>
			<td>AB-105-C</td>
			<td>Used to coat beads as a control condition</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (10X Solution, pH 7.4)</td>
			<td>Boston Bioproducts</td>
			<td>BM-220</td>
			<td>Component of coupling buffer, used for washing steps</td>
		</tr>
		<tr>
			<td>Polysorbate 20 (Tween 20)</td>
			<td>Sigma Aldrich</td>
			<td>P7949-500ML</td>
			<td>Component of activation buffer</td>
		</tr>
		<tr>
			<td>Polystyrene Carboxyl Polymer</td>
			<td>Bangs Laboratories</td>
			<td>PC06004</td>
			<td>Top density beads (1.05 g/mL), used for antibody coupling</td>
		</tr>
		<tr>
			<td>Rabbit RhD Polyclonal Antibody</td>
			<td>Invitrogen</td>
			<td>PA5-112694</td>
			<td>Used to coat beads for the dectection of Rh factor in red blood cells</td>
		</tr>
		<tr>
			<td>Research Grade Microscope</td>
			<td>Olympus</td>
			<td>Provis AX-70</td>
			<td>Microscoped used to mount magnetic levitation device and view levitating cells</td>
		</tr>
		<tr>
			<td>Rubber Dampening Feet</td>
			<td>Thorlabs</td>
			<td>RDF1</td>
			<td>Used to support the breadboard table</td>
		</tr>
		<tr>
			<td>Square Boro Tubing</td>
			<td>VitroTubes</td>
			<td>8100-050</td>
			<td>Capillary tube used for loading sample into Maglev</td>
		</tr>
		<tr>
			<td>Sulfo-NHS</td>
			<td>Thermoscientific</td>
			<td>24510</td>
			<td>Used in antibody coupling reaction</td>
		</tr>
		<tr>
			<td>Translational Stage</td>
			<td>Thorlabs</td>
			<td>PT1</td>
			<td>Used for focusing and for scanning capillary tube</td>
		</tr>
	</tbody>
</table>

detection of rhesus factor in human blood samples using magnetic levitation

Source: Thompson, L. et al., <a href="https://app.jove.com/v/62550">Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates the detection of the Rhesus (Rh) factor in blood samples using a magnetic levitation instrument. The magnetic field aligns and levitates beads and blood cells based on density. Treating the samples with anti-Rh antibodies results in intermediate-density bead-cell complexes&#39; formation, allowing for the differentiation of Rh-positive and Rh-negative red blood cells.

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 ...

Take a paramagnetic gadolinium solution responsive to external magnetic fields.
Introduce fluorescently labeled Rhesus or Rh factor-positive and unlabeled Rh factor-negative blood samples.
Add anti-RhD antibody-coated low-density polymer beads and uncoated high-density beads.
Anti-RhD antibodies bind to RhD-antigens on Rh-positive RBCs, forming bead-RBC complexes.
Now, load the sample into a capillary tube and seal it. Place the tube between magnets in the magnetic levitation device.
In the magnetic field generated by like-poles-facing magnets, the paramagnetic solution exerts magnetic and buoyant forces on sample components, causing them to levitate at heights proportional to their densities.
Low-density and high-density beads levitate at the top and bottom of the capillary, respectively, establishing density height references.
Blood cells, being diamagnetic, are repelled by a strong magnetic field near the magnets and levitate at an intermediate position.
The bead-RBC complexes levitate between low-density beads and RBCs, with fluorescent cells in this layer confirming Rh-positive RBC presence.

detection-rhesus-factor-human-blood-samples-using-magnetic

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

116 Aufrufe. Quelle: Thompson, L. et al., Quantifizierung von zellulären Dichten und antigenen Eigenschaften mittels magnetischer Levitation.J. Vis. Exp.(2021) Dieses Video zeigt den Nachweis des Rhesusfaktors (Rh) in Blutproben mit einem Magnetschwebegerät. Das Magnetfeld richtet Kügelchen und Blutzellen basierend auf der Dichte aus und lässt sie schweben. Die Behandlung der Proben mit Anti-Rh-Antikörpern führt zur Bildung von Kügelchen-Zell-Komplexen mittlerer Dichte, die die Unterscheidung von Rh-...

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The protocol described details an experimental procedure to quantify Red Blood Cell (RBC) aggregates under a controlled and constant shear rate, based on image processing techniques. The goal of this protocol is to relate RBC aggregate sizes to the corresponding shear rate in a controlled microfluidic environment.

Controlled Environment Mikrofluidik für dynamische Untersuchung der Aggregation roter Blutzellen

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JoVE Journal

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Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

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Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

Transkript

Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation