Detection of Rhesus Factor in Human Blood Samples using Magnetic Levitation

Abstract

Source: Thompson, L. et al., Quantification of Cellular Densities and Antigenic Properties using Magnetic Levitation. J. Vis. Exp. (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' formation, allowing for the differentiation of Rh-positive and Rh-negative red blood cells.

Protocol

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 betw…

Materials

2-(N-Morpholino)ethanesulfonic acid hydrate	Sigma Aldrich	M-2933	(MES); component of activation buffer
50×2.5×1 mm magnets, Nickel (Ni-Cu-Ni) plated, grade N52, magnetized through 5mm (0.197") thickness	K&J Magnetics	Custom	Magnets used for the magnetic levitation device
Capillary Tube Sealant (Critoseal)	Leica Microsystems	267620	Used to cap the ends of the capillary tubes
Centrifuge tube filters (Corning Costar Spin-X)	Sigma Aldrich	CLS8163	Used to wash beads
Compact Lab Jack	Thorlabs	LJ750	Used for adjusting the magnetic levitation device
DPBS, no calcium, no magnesium	Gibco	14190-144	Solution for bead suspensions
Ethanolamine	Sigma Aldrich	E9508-100ML	Used during a wash step for beads
Fluorescent Plasma Membrane Stain (CellMask Green)	Invitrogen	C37608	Used to stain Rh+ cells
Gadoteridol Injection	ProHance	NDC 0270-1111-03	Gadolinium (Gd3+); magnetic solution used to suspend cells
HBSS++	Gibco	14025-092	Solution for sample preparation
Mini Series Post Collar	Thorlabs	MSR2	Used to secure magnetic levitation device to lab jacks
N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride	Sigma Aldrich	E1769-10G	(EDC); used in antibody coupling reaction
Normal Rabbit IgG Control	R&D Systems	AB-105-C	Used to coat beads as a control condition
Phosphate Buffered Saline (10X Solution, pH 7.4)	Boston Bioproducts	BM-220	Component of coupling buffer, used for washing steps
Polysorbate 20 (Tween 20)	Sigma Aldrich	P7949-500ML	Component of activation buffer
Polystyrene Carboxyl Polymer	Bangs Laboratories	PC06004	Top density beads (1.05 g/mL), used for antibody coupling
Rabbit RhD Polyclonal Antibody	Invitrogen	PA5-112694	Used to coat beads for the dectection of Rh factor in red blood cells
Research Grade Microscope	Olympus	Provis AX-70	Microscoped used to mount magnetic levitation device and view levitating cells
Rubber Dampening Feet	Thorlabs	RDF1	Used to support the breadboard table
Square Boro Tubing	VitroTubes	8100-050	Capillary tube used for loading sample into Maglev
Sulfo-NHS	Thermoscientific	24510	Used in antibody coupling reaction
Translational Stage	Thorlabs	PT1	Used for focusing and for scanning capillary tube