Flow Cytometry Calibration with Standardized Particles
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Solution Overview
Problem
Current flow cytometry methods face challenges in accurately quantifying target structures on cells due to variability in reagents, marking molecules, and environmental conditions, leading to inconsistent measurements across different flow cytometers.
Innovation Solution
The method involves using standardized calibrators with varying target structure densities and arrangements to normalize flow cytometry measurements, compensating for the influences of reagent tolerances and environmental factors, allowing for precise comparison of fluorescence signals across different instruments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If flow cytometry measurements are performed using different flow cytometers and reagents, then the ability to measure target structures on cells is maintained, but the comparability and precision of measurement values deteriorates due to variability in laser intensity, optical filters, fluidics, and reagent properties
Solution Approach 1:
The patent introduces standardized calibrators as intermediary objects that mediate between the flow cytometer and the cells being measured. These calibrators with known target structure densities serve as reference standards to normalize measurements across different instruments and reagent batches, enabling comparable quantification without requiring direct comparison between different flow cytometers
Solution Approach 2:
The patent changes the measurement parameter from absolute fluorescence intensity to normalized binding events per cell. By using calibrators to establish reference values and expressing results as normalized units rather than raw intensity values, the system compensates for variations in laser intensity, optical filters, and detector settings across different flow cytometers
2Manufacturing precision
If calibration is performed using fluorescent microparticles, then some standardization is achieved, but accurate comparison of fluorescence measurements across different flow cytometers remains insufficient due to variations in particle properties and measurement conditions
Solution Approach 1:
The patent applies local quality by creating calibrators with specific, controlled target structure densities on their surfaces. Different calibrator types have different densities (e.g., 10, 100, 1000 target structures per particle), allowing the system to account for non-linear response at different signal levels and achieve accurate normalization across the full dynamic range of measurements
Solution Approach 2:
The patent segments the calibration process into multiple discrete calibrator types with known, distinct target structure densities. This segmentation allows the system to create multiple reference points that define the relationship between fluorescence signal and binding events across different intensity ranges, enabling more precise normalization than single-point calibration
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 enables precise quantification and comparison of binding events per cell, independent of the flow cytometer used, reagents, and environmental conditions, facilitating the identification of cell subpopulations and improving diagnostic accuracy in diseases like leukemia.
Implementation Method 1
at least the first parameter being a fluorescent radiation which emits the at least one marking molecule upon excitation with the energy beam
Data Source
Figure 1~3
Figure 4
AI summary
In a flow cytometry measurement method, an analysis medium is provided, which contains a fluid and biological cells. A labeling molecule is provided and brought into contact with the analysis medium in such a way that the labeling molecule can bind specifically to a target structure on the cell surface, provided the cell possesses this target structure. Flow cytometry measurements are acquired for each cell for a first and a second physical parameter. The first parameter is the fluorescence emitted by the labeling molecule upon excitation. The cells are classified based on the flow cytometry measurements. A first and a second calibrator are provided, which contain solid particles identical in shape, size, and material.The surface of the first calibrator has an immobilized target structure that matches the target structure of the cells. The second calibrator does not have this target structure. The calibrators are mixed with the analytical medium before the flow cytometry measurements are recorded. First and second flow cytometry measurements are recorded for the calibrators, just as for the cells. A normalized first flow cytometry measurement for the cell is then calculated from the first flow cytometry measurement of the first calibrator, the first flow cytometry measurement of the second calibrator, and the first flow cytometry measurement of the cell.