Cell Analysis Method Using Divalent Carboxylic Acid Heating
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Solution Overview
Problem
Conventional cell analysis methods face challenges in preserving the three-dimensional structure of cells from tissue specimens, leading to difficulties in quality control and changes in cellular characteristics when isolating individual cells from living tissues, particularly in FFPE samples, resulting in incomplete separation and analysis errors.
Innovation Solution
A method involving aldehyde-fixed cells embedded in a water-insoluble medium, where the embedding medium is removed, and the sample is heated in the presence of a divalent carboxylic acid compound, followed by enzyme treatment to disperse cells efficiently, allowing for high-precision analysis using a flow cytometer.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cells are isolated from living tissue for analysis, then individual cell analysis becomes possible, but the original three-dimensional structural characteristics are lost and quality control becomes difficult
Solution Approach 1:
The tissue specimen is fixed with aldehyde and embedded in water-insoluble embedding medium before analysis to preserve the three-dimensional structure. This preliminary preservation action allows subsequent processing to recover individual cells while maintaining structural integrity, resolving the contradiction between individual cell analysis and structural preservation
2Productivity
If FFPE tissue specimens are heated in citric acid solution to separate cells, then cell separation is achieved, but the dispersion efficiency is insufficient and cell lumps remain
Solution Approach 1:
The patent changes the chemical parameters of the heating solution from citric acid to a divalent carboxylic acid compound (such as oxalic acid, malic acid, succinic acid, or maleic acid). This parameter change in the chemical composition significantly improves cell dispersion efficiency by preventing cell lump formation, thereby enabling accurate flow cytometry analysis
Solution Approach 2:
The patent replaces the mechanical/enzymatic cell separation approach with a chemical solution-based approach using divalent carboxylic acid compounds. This substitution achieves superior cell dispersion by dissolving cellular aggregates through chemical action rather than relying on mechanical separation or enzyme digestion alone
3Ease of operation
If embedding medium is removed and enzyme treatment is applied to disperse cells, then cell separation is improved, but incomplete separation and cell lumps still occur
Solution Approach 1:
The divalent carboxylic acid compound acts as an intermediary substance during the heating process, facilitating complete cell separation by preventing aggregate formation. This intermediary chemical agent bridges the gap between embedding medium removal and enzyme treatment, ensuring reliable and complete cell dispersion without lumps
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 enhances cell dispersion efficiency, minimizing errors due to cell lumps and maintaining the original characteristics of the three-dimensional structure, enabling high-precision analysis of cells from tumor, non-tumor, and normal tissues.
Implementation Method 1
heating the sample obtained in step (a) in the presence of a divalent carboxylic acid compound, thereby obtaining the heat-treated sample containing the tissue
Implementation Method 2
bringing the sample obtained in step (b) into contact with an enzyme with a cell dispersion activity to separate, into individual cells, cells contained in the sample obtained in step (b)
Implementation Method 3
obtaining, using the flow cytometer, optical information on the cells in the sample obtained in step (c)
Data Source
Figure 1
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Figure 3A~3D
AI summary
A cell analysis method for analyzing, using a flow cytometer, cells obtained from a tissue specimen which is aldehyde-fixed and embedded in a water-insoluble embedding medium. The method comprises: (a) removing embedding medium from a sample obtained from the tissue specimen which is aldehyde-fixed and embedded in the water-insoluble embedding medium, thereby obtaining the sample comprising the tissue from which the embedding medium is removed; (b) heating the sample obtained from step (a) in the presence of a divalent carboxylic acid compound to obtain a heat-treated sample comprising the tissue; (c) contacting the heat-treated sample from step (b) with an enzyme having a cell dispersion activity to separate the sample into individual cells, thereby obtaining the sample comprising the individual cells dispersed in a solvent; (d) subjecting the separated cells from step (c) to flow cytometry to obtain optical information ; and (e) analyzing the cells based on the optical information obtained in the step (d).