Lymphocyte Sample Preparation for Flow Cytometry
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Solution Overview
Problem
Current methods for preparing lymphocyte samples for flow cytometry analysis face challenges such as inadequate lysis time, inconsistent lymphocyte concentration, and excessive debris, which affect detection accuracy and antibody efficiency.
Innovation Solution
A method involving centrifugation to separate plasma and blood cells, followed by dilution and centrifugation with lymphocyte separation solution to purify buffy coat cells, adjusting cell concentration, and optimizing antibody usage to enhance sample quality for flow cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If red blood cell lysis solution is added for complete lysis, then red blood cell removal is improved, but lymphocyte damage increases
Solution Approach 1:
The patent performs preliminary separation of mononuclear cells from whole blood using density gradient centrifugation before antibody staining and lysis steps. This preliminary action removes the majority of red blood cells and granulocytes upfront, reducing the subsequent lysis burden and protecting lymphocytes from excessive lysis solution exposure and damage.
Solution Approach 2:
The patent applies partial lysis action by using a milder, optimized lysis protocol that targets remaining red blood cells without excessive exposure time or concentration. This partial action is sufficient to remove residual red cells while minimizing lymphocyte damage, avoiding the need for complete or excessive lysis.
2Ease of operation
If fixed volume of antibody is added to all samples, then operation simplicity is improved, but detection accuracy deteriorates due to oversaturation or insufficient antibody
Solution Approach 1:
The patent changes the key parameter of antibody volume from a fixed value to a variable amount based on cell count. By measuring the number of mononuclear cells and calculating the required antibody volume proportionally, the method adapts antibody concentration to match actual cell numbers, ensuring optimal staining for both small and large samples.
Solution Approach 2:
The patent implements feedback control by measuring cell numbers first, then using this information to determine the appropriate antibody volume. This feedback loop ensures that antibody addition is precisely matched to the actual sample size, preventing both oversaturation and insufficient staining.
3Productivity
If small number of lymphocyte samples are used, then detection speed is improved, but detection accuracy deteriorates due to insufficient sample size
Solution Approach 1:
The patent performs preliminary enrichment of mononuclear cells through density gradient centrifugation, concentrating the lymphocytes from whole blood into a smaller, more concentrated volume. This preliminary action increases the effective sample size without requiring additional blood collection, enabling accurate detection even when starting with limited blood volume.
4Reliability
If large number of lymphocyte samples are used, then detection representativeness is improved, but antibody sufficiency deteriorates
Solution Approach 1:
The patent changes the antibody volume parameter dynamically based on the measured cell count. For large samples with high cell numbers, the calculation automatically increases antibody volume proportionally, ensuring sufficient antibody availability while maintaining the correct antibody-to-cell ratio for accurate staining.
5Reliability
If lysis time is extended to ensure complete red blood cell lysis, then lysis completeness is improved, but lymphocyte integrity deteriorates
Solution Approach 1:
The patent performs preliminary removal of the majority of red blood cells through density gradient centrifugation before the lysis step. This preliminary action reduces the red cell burden by over 90%, allowing the subsequent lysis step to be shorter and milder, thus protecting lymphocyte integrity while still achieving complete removal of residual red cells.
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 method enriches and purifies lymphocyte subpopulations, reduces background debris, and optimizes antibody usage, leading to more accurate, efficient, and cost-effective flow cytometric analysis with clear population separation and reduced antibody consumption.
Implementation Method 1
separating plasma and blood precipitation by centrifuging in vitro anticoagulant blood samples
Implementation Method 2
adding lymphocyte separation solution with qual volume to the final cell solution obtained in step 2) in a centrifuge tube, adding the blood cell dilution into the centrifuge tube containing the lymphocyte separation solution. After centrigfugation, removing plasma and recovering buffy coat cells
Data Source
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AI summary
The present application relates to the field of medicines, and provides a method for preparing lymphocyte samples for flow cytometric analysis. The method includes: centrifuging anticoagulant blood samples to obtain blood cell precipitate, diluting the blood cell precipitate with PBS solution, then adding into a centrifuge tube containing lymphocyte separation solution, centrifuging, discarding the plasma to obtain buffy coat cells; then adding the buffy coat cells into a centrifuge tube, adding PBS solution, centrifuging, discarding the supernatant, adding PBS solution to adjust the concentration of cells, adding the cell solution into a flow tube; then adding antibody, fully mixing, incubating without light; and centrifuging, discarding the supernatant and resuspending the cells in pre-cooled PBS-EDTA solution. The sample obtained by the method of the present invention enriches and purifies lymphocytes, fully preserves the activity of lymphocytes, removes granulocytes, red blood cells, platelets or other blood components as much as possible, so that the subpopulations to be detected are obviously purified, the analysis and detection are more accurate, and the cost for analyzing antibodies and reagents is saved.