Adaptive Immune Cell Quantification via Multiplex dPCR
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Solution Overview
Problem
Current methods for quantifying adaptive immune cells in solid tissues are limited, requiring labor-intensive isolation and resulting in incomplete recovery and poor quality of data, especially when detecting lymphocytes in complex biological samples.
Innovation Solution
A method using multiplex digital polymerase chain reaction (dPCR) with V-segment and J-segment primers to amplify and quantify rearranged T cell receptor (TCR) or immunoglobulin (Ig) CDR3-encoding regions in DNA samples, allowing for the relative representation of adaptive immune cells to be determined without isolating them from the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If labor-intensive isolation methods are used to quantify adaptive immune cells, then cell separation is achieved, but recovery is incomplete and data quality is poor
Solution Approach 1:
The patent extracts and amplifies specific DNA sequences (TCR and Ig CDR3 regions) from total DNA extracted from complex tissue samples. By using highly specific primers that target only adaptive immune cell receptor genes, the method isolates the signal of interest without requiring physical cell separation, thereby achieving complete recovery while maintaining high quantification accuracy
Solution Approach 2:
The patent introduces PCR amplification as an intermediary step between DNA extraction and quantification. The exponential amplification of target sequences serves as a mediator that converts trace amounts of adaptive immune cell DNA into detectable signals, enabling accurate quantification without loss of rare cells during isolation procedures
2Reliability
If current quantification methods are applied to complex biological samples, then adaptive immune cells can be detected, but the process is labor-intensive and recovery is incomplete
Solution Approach 1:
The patent develops a universal PCR-based approach that can quantify adaptive immune cells in any complex biological sample type (fresh tissue, frozen tissue, fixed tissue, liquid biopsies) using the same methodology. The method simultaneously achieves detection, quantification, and sample type agnosticism, eliminating the need for different isolation protocols for different sample types
Solution Approach 2:
The patent replaces mechanical cell isolation and separation systems with a molecular biology-based PCR system. Instead of using flow cytometry, magnetic separation, or manual isolation techniques, the method uses sequence-specific DNA amplification to detect and quantify adaptive immune cells, dramatically simplifying the procedure while improving reliability
3Ease of operation
If adaptive immune cells are isolated from complex samples, then cell separation is achieved, but quality of data is poor
Solution Approach 1:
The patent creates amplified copies of target DNA sequences through PCR. By generating millions of copies of the CDR3 regions from even a single adaptive immune cell, the method produces high-quality detectable signals without requiring physical isolation of cells, thereby maintaining both operational simplicity and measurement precision
Solution Approach 2:
The patent extracts only the relevant genetic information (TCR and Ig CDR3 sequences) from total DNA while leaving the rest of the genomic material behind. This selective extraction of diagnostic sequences enables accurate quantification without the need to isolate entire cells, preserving data quality while simplifying the procedure
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 highly sensitive and accurate quantification of adaptive immune cells in complex mixtures, detecting as few as ten adaptive immune cells per 10,000, and is applicable to fresh, frozen, or fixed tissues, providing valuable diagnostic and prognostic information.
Implementation Method 1
amplifying test sample template DNA extracted from the test biological sample in a multiplex digital polymerase chain reaction (dPCR) that comprises: (i) a plurality of V-segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide
Implementation Method 2
V-segment oligonucleotide primers that are each independently capable of specifically hybridizing to at least one polynucleotide encoding a T cell receptor (TCR) V-region polypeptide or an immunoglobulin (Ig) V-region polypeptide
Data Source
AI summary
Compositions and methods are described for highly sensitive quantification of the relative representation of DNA from adaptive immune cells (e.g., T and/or B lymphocytes) in DNA extracted from complex mixtures of cells that include cells which are not adaptive immune cells. Included are methods for determining the relative presence in a tumor of tumor infiltrating lymphocytes (TIL), the relative presence of lymphocytes infiltrating a somatic tissue that is the target of an autoimmune disease, and the relative presence of lymphocytes infiltrating a transplanted organ.