Amplicon Region Methylation Analysis for Non-Classical Monocyte Identification
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for identifying and quantifying non-classical monocytes are not robust enough, particularly in complex samples, and often require cell purification or enrichment, which can be cumbersome and inefficient.
Innovation Solution
Analyzing the methylation status of specific CpG positions within the 2213 amplicon region, specifically CpG positions 39 and 169, to differentiate non-classical monocytes from classical monocytes and other cells, using bisulfite convertibility as an indicator, without the need for cell purification or enrichment, and employing techniques like PCR or bisulfite sequencing for quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell purification or enrichment is performed to identify non-classical monocytes, then measurement precision is improved, but device complexity and time consumption increase
Solution Approach 1:
The invention extracts the key distinguishing feature (DNA methylation pattern at specific CpG positions) from the complex cell population, allowing direct identification of non-classical monocytes through methylation analysis without physical separation. This extracts the essential identification marker while eliminating the need for complex purification devices and procedures.
Solution Approach 2:
The invention changes the identification parameter from cell surface markers (requiring flow cytometry and purification) to DNA methylation status (detectable by bisulfite sequencing). This parameter change enables direct analysis of whole blood samples, eliminating the need for cell enrichment while maintaining high identification accuracy.
2Measurement precision
If cell purification or enrichment is performed to identify non-classical monocytes, then measurement precision is improved, but time consumption increases
Solution Approach 1:
The invention performs preliminary action by targeting specific CpG positions (39 and 169) in the 2213 amplicon region that are already known to differ between monocyte subsets. By pre-selecting these informative positions, the method eliminates time-consuming exploratory purification steps while ensuring accurate identification from the start.
Solution Approach 2:
The method extracts only the essential methylation information from specific CpG positions relevant to non-classical monocyte identification, bypassing the time-consuming process of physical cell separation while obtaining the same identification accuracy directly from whole blood DNA.
3Ease of operation
If whole blood samples are analyzed directly without purification, then ease of operation is improved, but measurement precision deteriorates
Solution Approach 1:
The invention applies local quality by focusing analysis on specific local features (CpG positions 39 and 169 within the 2213 amplicon) that have distinct methylation patterns in non-classical monocytes. This localized approach maintains simplicity of whole blood analysis while achieving high precision through targeted examination of discriminative regions.
Solution Approach 2:
The method changes the detection parameter from cell surface protein expression (requiring complex immunophenotyping in mixed samples) to DNA methylation status at specific CpG sites. This parameter change enables direct analysis of whole blood with high precision, as methylation patterns are cell-type specific and can be detected without physical separation.
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 reliable and quantitative identification and measurement of non-classical monocytes in whole blood or tissue samples, providing a robust tool for diagnosing autoimmune diseases, infections, and cancers, with high specificity and sensitivity, and can be applied in clinical routine without the need for purification or staining procedures.
Implementation Method 1
analyzing the methylation status of at least one CpG position... using bisulfite convertibility as an indicator
Data Source
Figure 1
Figure 2a~2b
AI summary
The present invention relates to a method, in particular an in vitro method, for identifying non-classical monocytes, comprising analyzing the methylation status of at least one CpG position in the mammalian genomic region comprising an amplicon, wherein a demethylation or lack of methylation of said region is indicative for a non-classical monocyte, when compared to a classical monocyte or a non-monocyte cell. The analyses according to the invention can identify non-classical monocytes on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The present invention furthermore provides an improved method for quantifying non-classical monocytes, in particular in complex samples. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.