Epigenetic Immune Cell Counting Using qPCR Methylation Markers
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Solution Overview
Problem
Current methods for immune cell counting, such as flow cytometry, require fresh blood samples and are limited in applicability, especially in resource-poor settings, and lack standardized measurements, leading to suboptimal treatment decisions and diagnostic challenges.
Innovation Solution
A quantitative methylation assay using epigenetic qPCR-based methods that allow for the counting of immune cells in fresh, frozen, or dried blood samples by analyzing DNA methylation markers specific to T cells, CD4+ T cells, CD8+ T cells, B-cells, and NK cells, utilizing a panel of recombinant nucleic acids and plasmids to normalize and correct for amplification efficiencies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If conventional flow cytometry or mass cytometry is used for immune cell counting, then cell counting can be performed, but the process is complex, requires multiple reagents and instruments, and is not suitable for point-of-care settings
Solution Approach 1:
The patent extracts the epigenetic marking information from complex cellular analysis and isolates it as a specific detectable signal. By focusing only on epigenetic markers rather than requiring full flow cytometry analysis, the method simplifies the measurement process while maintaining immune cell counting capability
Solution Approach 2:
The patent introduces epigenetic markers as intermediary substances that mediate between the cell and the detection system. These markers serve as simplified intermediaries that carry specific signals detectable by standard PCR equipment, eliminating the need for complex flow cytometry instrumentation
2Ease of manufacture
If epigenetic markers are used for cell counting, then simpler detection is possible, but the method must be validated to ensure accuracy comparable to conventional methods
Solution Approach 1:
The patent employs feedback through validation experiments that compare epigenetic marker-based counting results against conventional flow cytometry and mass cytometry results. This feedback loop ensures the simplified method achieves comparable measurement precision by verifying its performance against established standards
Solution Approach 2:
The patent changes the detection parameter from physical cell properties (measured by flow cytometry) to epigenetic molecular markers. This parameter transformation enables use of simpler PCR-based detection while maintaining accuracy through the specific and reliable nature of epigenetic markings on immune cell DNA
3Adaptability or versatility
If standard PCR equipment is used instead of flow cytometry, then instrument availability improves, but the method must overcome limitations in detecting cellular heterogeneity
Solution Approach 1:
The patent replaces the mechanical and optical complexity of flow cytometry with biochemical detection using PCR. By substituting physical cell sorting and optical detection with molecular amplification and detection, the method achieves comparable heterogeneity detection using widely available standard PCR equipment
Data Source
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AI summary
The present invention relates to improved methods for epigenetic blood and immune cell counting, and respective uses and kits.