Flow Cytometry Lymphocyte Detection with PBS-EDTA Background Control
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Solution Overview
Problem
Existing methods for detecting lymphocyte subpopulations in immune cells are inefficient, leading to waste of reagents, inaccurate results due to unclean backgrounds and debris, and inability to detect small populations accurately, with incomplete definition of DC cell subtypes and imbalance in lymphocyte subpopulation analysis.
Innovation Solution
A method involving pre-cooling, use of PBS-EDTA solution, controlled flow speed, and lymphocyte separation to enrich lymphocytes, ensuring accurate and comprehensive detection of lymphocyte subpopulations, including DC cells, by reducing antibody and reagent usage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If red blood cells are lysed by using red blood cell lysis solution after dyeing immunofluorescent antibody, then red blood cells are removed, but the background becomes unclean and debris increases, causing data errors
Solution Approach 1:
The patent applies preliminary action by performing red blood cell lysis before immunofluorescent antibody staining. This sequence prevents the antibody from binding to red blood cells that would later be lysed, avoiding the generation of debris and maintaining a clean background. The method specifies adding red blood cell lysis solution to the sample, incubating for 10-15 minutes, then adding the fluorescently labeled antibody, thereby resolving the contradiction between removing red blood cells and maintaining data accuracy.
2Measurement precision
If traditional flow cytometry methods are used to detect lymphocyte subpopulations, then detection can be performed, but reagent and antibody consumption increases and small lymphocyte populations cannot be detected accurately
Solution Approach 1:
The patent applies the extraction principle by isolating and enriching lymphocytes from whole blood through density gradient centrifugation before detection. This extraction of the target cell population from the complete blood sample reduces the volume of reagents and antibodies needed for staining, as the antibody only needs to cover the enriched lymphocyte population rather than the entire blood sample. This resolves the contradiction between detection accuracy and reagent consumption.
Solution Approach 2:
The patent performs preliminary lymphocyte enrichment through density gradient centrifugation before immunofluorescent staining. This preliminary separation concentrates the lymphocytes of interest, allowing for more accurate detection of small subpopulations while reducing the total number of cells that require antibody labeling, thereby decreasing reagent consumption.
3Device complexity
If lymphocyte subpopulations are defined incompletely without considering immune system function, then detection is simpler, but the division of subpopulations is not comprehensive and balanced
Solution Approach 1:
The patent applies local quality by defining lymphocyte subpopulations based on their specific functional roles within the immune system. Different subsets are classified according to their distinct functions (e.g., cytotoxic, helper, regulatory, memory functions), with each subset defined by specific marker combinations that reflect their functional characteristics. This functional-based classification approach achieves comprehensive and balanced subpopulation division while maintaining detection feasibility.
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
Enables reliable and efficient detection of lymphocyte subpopulations, including DC cells, with reduced reagent consumption and improved accuracy, providing a comprehensive understanding of the immune system's status.
Implementation Method 1
CN107063982A and US2018/246105 A1 disclose relevant methods for detecting immune cells using lymphocyte separation solutions
Implementation Method 2
the PBS-EDTA solution in step a) or step b) is a mixed solution containing 0.005-0.05M PBS and a final concentration of 2-3mM EDTA with pH of 7.2-7.4
Implementation Method 3
the pre-cooling temperature in step a) is a temperature commonly used for flow cytometric detection in the art, including but not limited to 2-8°C
Data Source
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AI summary
The present application relates to the field of medicines, and in particular to a flow cytometric detection method for lymphocyte in immune cells. The method includes steps of: a) lymphocyte samples stained with immunofluorescent antibodies were added into the pre-cooled PBS-EDTA solution, and the cells were mixed and prepared for flow cytometry detection; b) starting up and warming up a flow cytometer system, adjusting the flow speed, then adding PBS-EDTA solution into a sample tube, and flushing a nozzle system of liquid stream; c) the sample of homogenous cells obtained in step a) was added to the sample tube and then tested. The present detection method can obviously separate cell subpopulations, so that the analysis and detection are more accurate, especially suitable for detecting cell subpopulations with small number of cells, and saving antibodies and reagents.