Microwell Immune Cell Sampling Using Magnetic RBC Separation
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Solution Overview
Problem
Existing methods for sampling immune cells from whole blood in microwell plates face challenges such as clogging due to red blood cells (RBCs), difficulty in distinguishing WBCs and RBCs, and labor-intensive processes like RBC lysis that can affect cell activity and data integrity.
Innovation Solution
A method involving magnetic beads conjugated to antibodies to bind RBCs, a shaker with a magnetic adapter to separate RBCs to the well wall, and a controlled shaking process to isolate immune cells, followed by a sample probe to withdraw a pure immune cell sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If traditional RBC lysis method is used to remove red blood cells from whole blood sample, then RBCs can be removed from the sample, but the method is labor intensive, creates RBC debris that clogs the cytometer flow cell, makes the cytometer very dirty, significantly increases carryover from one sample to another, and may cause loss of data integrity since the hypotonic buffer used in the lysis is not physiological and may affect normal immune cell activity
Solution Approach 1:
The invention extracts and removes RBCs from the whole blood sample using magnetic beads conjugated to anti-human RBC antibodies. The magnetic beads specifically bind to RBCs and are then separated from the sample using a magnetic field, physically extracting the harmful RBC component without lysing them, thereby avoiding debris generation and cytometer contamination
Solution Approach 2:
The invention introduces magnetic beads as an intermediary substance that mediates the separation process. These beads conjugated to anti-RBC antibodies act as a bridge to selectively capture and remove RBCs from the sample, enabling clean separation without direct contact between RBCs and the cytometer system
2Quantity of substance
If gradient centrifugation is used for purification of WBCs, then WBCs can be purified from the sample, but the method is not applicable for microplate format
Solution Approach 1:
The invention replaces the mechanical centrifugation system with a magnetic field-based separation system. Instead of using centrifugal force to separate cells by density, the invention uses magnetic fields to attract and hold magnetic bead-RBC complexes against the well wall, enabling the same purification function in a microplate format without requiring centrifugation equipment
3Ease of operation
If whole blood is sampled directly with a cytometer for WBCs analysis, then sampling can be performed, but whole blood tends to clog small flow paths and populations of WBCs and RBCs are difficult to distinguish
Solution Approach 1:
The invention performs preliminary separation of RBCs from WBCs before cytometer analysis. By using magnetic beads to capture and remove RBCs in advance, the sample is pre-prepared to contain primarily WBCs, eliminating the clogging and discrimination problems that would occur during direct cytometer sampling of whole blood
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Efficient isolation of immune cells from RBCs, preventing clogging and maintaining cell integrity, enabling automated and accurate sampling suitable for flow cytometry.
Implementation Method 1
magnetic beads which are conjugated to antibodies or otherwise designed to bind to RBCs in the sample. The method includes steps of: a) placing the microwell plate on a shaker having a magnetic adapter including a magnet, wherein the magnet causes the RBCs bound to the magnetic beads to be attracted to and migrate to the wall of the well and be held against the wall
Implementation Method 2
b) shaking with the shaker the microwell plate in a manner and for a time period so as to suspend substantially evenly or homogeneously the immune cells in the biological fluid sample within a region of the well
Data Source
AI summary
The present disclosure provides a method for automatic sampling of immune cells from a biological fluid sample deposited in a well of a microwell plate. The microwell plate is placed on a shaker having a magnetic adapter including at least one magnet. The magnet causes red blood cells (RBCs) bound to magnetic beads to be attracted to and migrate to a wall of the well. The shaker is then operated to shake the microwell plate such that the immune cells are substantially isolated from the RBCs in a region of the well. A sample probe is then lowered into the region of the well to withdraw a portion of the sample containing the immune cells.