Epigenetic Markers for T-Lymphocyte Identification
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Solution Overview
Problem
Current methods for identifying CD4 and CD8 T-lymphocytes are challenging due to the need for freshly isolated or immediately fixated cell samples, making routine applications difficult.
Innovation Solution
A method based on DNA methylation analysis that involves analyzing the methylation status of specific CpG positions in the CD3δ/γ/ε genes and other markers such as SLA2, CHRNA3, and LCK to identify CD3+ T-lymphocytes, including CD4+ and CD8+ subsets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional methods (flow cytometry with surface markers) are used to identify T-lymphocytes, then cell identification can be performed, but the method requires freshly isolated or immediately fixated cell samples, making routine applications difficult
Solution Approach 1:
The patent uses DNA methylation patterns as a molecular copy of cell identity that persists in archived samples. Instead of requiring live cells with surface markers, the method analyzes epigenetic marks in DNA that remain stable in frozen or stored samples, effectively copying the cellular identity information into a stable molecular form that can be read from archived material
Solution Approach 2:
The patent shifts the detection parameter from dynamic surface marker expression (which changes with cell state and requires live cells) to static DNA methylation patterns (which are stable and persist in archived samples). This parameter change enables the use of frozen or stored samples while maintaining identification reliability
2Ease of operation
If DNA methylation analysis is used to identify T-lymphocytes from archived samples, then ease of operation is improved, but measurement precision and reliability may be compromised due to sample quality degradation
Solution Approach 1:
The patent focuses analysis on specific local regions of the genome (CpG sites within the CD3 gene locus) that have distinctive methylation patterns for T-cell identification. By concentrating on these specific loci rather than analyzing the entire genome, the method maintains high measurement precision even when overall sample quality is degraded, as these specific regions preserve the epigenetic signature
Solution Approach 2:
The patent performs preliminary characterization of the DNA sample quality and methylation pattern profile before final analysis. By assessing sample suitability in advance and selecting appropriate CpG sites based on preliminary data, the method ensures that only high-quality measurements are performed, maintaining precision while working with archived samples
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 allows for the reliable and convenient identification of T-lymphocytes even from sub-optimal quality samples, providing a robust tool for clinical applications and enabling the measurement of T-lymphocyte subsets in various contexts.
Implementation Method 1
analysing the methylation status of at least one CpG position in the CD3δ/γ/ε genes
Data Source
AI summary
The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD3a/b/c/d/g genes, in particular their “upstream” regulatory regions, and in particular the promoter and other conserved regions of the gene cd3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4 or/and memory and/or naive T lymphocytes. Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3a/b/c/d for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.