Blood Cell Counting via DNA Methylation Analysis
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Solution Overview
Problem
Conventional methods for determining the cellular composition of blood samples, such as leukocyte differential counts, are labor-intensive, difficult to standardize, and limited by the need for fresh samples, while existing epigenetic approaches like DNA methylation analysis face challenges in clinical routine applicability and precision.
Innovation Solution
A method involving the isolation of genomic DNA from blood samples to identify specific DNA methylation levels at CpG-dinucleotides, using sequences like those in SEQ ID NO: 1-13, to predict the relative distribution of blood cell types, enabling precise estimation of cellular composition even from frozen or sub-optimal samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If conventional flow cytometric technologies are used for leukocyte differential counts, then automated analysis and standardization are improved, but the requirement for fresh blood samples and inability to freeze samples for later analysis worsens
Solution Approach 1:
The patent changes the measurement parameter from flow cytometric properties (electrical impedance, light scattering, fluorescence) to DNA methylation status. This parameter change enables the analysis to be performed on frozen samples because DNA methylation patterns are stable during freezing and storage, whereas flow cytometric properties degrade in frozen samples. The method uses bisulfite conversion to detect methylated vs unmethylated CpG sites, providing a storage-independent measurement approach.
2Measurement precision
If immunophenotypic analysis with fluorescent staining is used, then definition of lymphocyte subsets is improved, but labor intensity and difficulty of standardization worsen
Solution Approach 1:
The patent replaces the mechanical/manual process of immunophenotypic analysis with an automated biochemical assay. Instead of manual fluorescent staining and flow cytometric analysis requiring skilled operators, the method uses automated DNA extraction, bisulfite conversion, and methylation-specific PCR or sequencing. This substitution maintains high precision in lymphocyte subset definition while eliminating labor intensity and improving standardization across different laboratories.
3Adaptability or versatility
If DNA methylation analysis is used for cellular composition determination, then sample storage flexibility is improved, but precision and clinical routine applicability worsen
Solution Approach 1:
The patent extracts and analyzes only the specific DNA methylation markers that are most informative for cellular composition determination. Rather than analyzing the entire genome or using complex epigenetic profiles, the method identifies and measures methylation status at specific CpG sites in genes known to be differentially methylated between cell types (e.g., CD3, CD4, CD8, B-cell, monocyte-specific markers). This extraction of key informative markers maintains precision while simplifying the assay for clinical routine applicability.
4Measurement precision
If manual microscopic evaluation is used for leukocyte differential counts, then sample analysis is improved, but productivity and time consumption worsen
Solution Approach 1:
The patent replaces manual microscopic evaluation with automated molecular biology techniques. The workflow involves automated DNA extraction from blood samples, followed by bisulfite conversion and methylation-specific amplification or sequencing. This automated biochemical approach processes samples much faster than manual microscopy while maintaining or improving precision through objective, quantifiable methylation measurements that are not subject to inter-observer variability.
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 provides robust and precise estimation of blood cell types, overcoming limitations of conventional methods by allowing analysis of frozen samples and small volumes, achieving similar precision to established techniques with improved standardization and cost-effectiveness.
Implementation Method 1
DNA-methylation (DNAm) represents the best understood epigenetic modification. Methyl groups can be added to the 5 th carbon of the pyrimidine ring of cytosine
Data Source
Figure 1A~1E
Figure 2A~3B
Figure 4A~4H
AI summary
The invention relates to a method for determining at least a part of the cellular composition of a blood sample, with which the relative distribution of at least some blood cell types in the sample can be predicted. According to the invention even DNAm levels at individual CG-dinucleotides (CpGs) reflect fractions of granulocytes (including neutrophil and eosinophil granulocytes), CD4+ T-cells, CD8+ T-cells, B-cells, NK-cells, or monocytes. It could be demonstrated that all tested CpG-dinucleotides that are indicative of a specific cell type have lower methylation (DNAm) levels in this specific cell type as in the other cell types.