Particle Enumeration in Cell Compositions via Osmotic Lysis
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Solution Overview
Problem
Current methods for detecting and enumerating particles, such as bead particles, in a cell composition are inaccurate due to difficulties in distinguishing particles from cells and often damage the particles or leave residual debris, leading to incomplete removal and false positives/negatives.
Innovation Solution
The method involves osmotic cell lysis using hypotonic or hypertonic solutions to separate cells from particles, followed by rinsing and washing to remove debris, preserving the integrity of the particle surface for accurate enumeration using techniques like flow cytometry or microscopy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current methods for detecting particles in cell composition are used, then the detection process is simple, but the accuracy and precision of particle enumeration is poor due to inability to distinguish particles from cells and presence of residual debris
Solution Approach 1:
The method segments the detection process into distinct phases: cell lysis phase (using hypotonic solution to burst cells), debris removal phase (using centrifugation and filtration), and particle enumeration phase (using flow cytometry or microscopy). This segmentation allows each phase to be optimized independently, achieving accurate particle counting while managing overall process complexity through structured steps.
Solution Approach 2:
The method performs preliminary actions before particle detection by first lysing cells with hypotonic solution and then removing debris through centrifugation and filtration. These preliminary steps prepare the sample by separating particles from cellular materials, enabling accurate subsequent enumeration without interference from intact cells or residual debris.
2Reliability
If harsh methods are used to remove cells and debris, then complete removal is achieved, but particle integrity is damaged leading to false results
Solution Approach 1:
The method uses parameter changes in osmotic conditions (hypotonic to isotonic transition) to achieve selective cell lysis without particle damage. By controlling the osmotic gradient and incubation conditions, cells undergo controlled swelling and lysis while particles, being osmotically resistant, maintain their structural integrity and surface properties for reliable detection.
Solution Approach 2:
The hypotonic solution acts as an intermediary agent that selectively targets cells for lysis while leaving particles unaffected. This intermediary mechanism allows indirect removal of cells and debris without direct harsh treatment of particles, preserving particle integrity for accurate subsequent enumeration.
3Measurement precision
If multiple washing steps are performed to remove debris, then particle purity increases, but particle loss and enumeration errors increase
Solution Approach 1:
The method employs hydraulic principles through centrifugation to separate particles from debris based on density differences. The centrifugal force creates a density gradient that allows efficient separation in a single step, reducing the need for multiple washing operations and minimizing particle loss while maintaining high particle purity.
Solution Approach 2:
The method uses filtration through porous materials to remove residual debris from the particle suspension. The porous filter allows particles to pass through while trapping smaller debris molecules, achieving effective purification without mechanical stress that would damage particles or cause loss.
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
This approach allows for reliable and precise detection of particle presence, absence, and concentration in cell compositions, reducing false positives and negatives, and maintaining the integrity of the particle surface for effective enumeration.
Implementation Method 1
incubating a sample comprising at least a portion of a cell composition or a sample derived from the cell composition under a condition sufficient to induce osmotic lysis of one or more cells in the sample
Implementation Method 2
contacting the sample with a hypotonic solution
Implementation Method 3
incubating the lysed cell composition or a composition derived from the lysed cell composition with a hypertonic solution
Data Source
AI summary
Provided herein are methods of assessing or determining the presence or absence of particles, such as bead particles, present in a cell composition. Also provided are articles of manufacture and kits for use in the methods.