Adaptive Immune Cell Quantification via Synthetic Template Normalization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods cannot accurately quantify the absolute number of adaptive immune cells in complex biological samples, such as blood or tissues, and do not allow for the determination of their absolute number in diseased tissues, which is crucial for diagnostic and prognostic purposes in cancer and other conditions.
Innovation Solution
A method involving multiplex PCR and sequencing is used to amplify and quantify rearranged CDR3 oligonucleotide sequences from T cell receptor or Immunoglobulin loci, employing synthetic templates with unique barcodes to determine the amplification factor and calculate the total number of T or B cells relative to the total number of genomes in the sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR methods are used to amplify immune cell DNA, then amplification of target sequences is achieved, but accurate quantitation of absolute cell numbers cannot be obtained due to amplification bias and lack of normalization controls
Solution Approach 1:
The patent introduces synthetic template DNA as an intermediary substance that serves as a normalization control. These synthetic templates are added at known concentrations to the PCR reaction mixture, allowing comparison between expected and actual amplification outcomes. This intermediary enables the calculation of amplification factors that correct for bias, thereby resolving the contradiction between achieving amplification and maintaining quantitation accuracy.
Solution Approach 2:
The patent implements a feedback mechanism by measuring the actual amplification of synthetic templates and using this information to adjust and normalize the quantitation of target immune cell DNA. The amplification factor derived from synthetic template performance feeds back into the calculation process, allowing correction of amplification bias and enabling accurate absolute quantitation despite variations in PCR efficiency.
2Productivity
If multiplex PCR is used to amplify multiple immune cell targets simultaneously, then productivity increases, but measurement precision decreases due to competition for reagents and amplification variability
Solution Approach 1:
The patent uses synthetic template DNA as an intermediary normalization control that is co-amplified with target sequences in the multiplex PCR reaction. This synthetic template experiences the same reagent competition and amplification conditions as the target DNA, allowing accurate measurement of amplification efficiency despite multiplex complexity. The synthetic template serves as an internal reference that enables precise quantitation even when multiple targets are amplified simultaneously.
3Measurement precision
If synthetic templates with unique barcodes are used to normalize amplification, then measurement precision improves, but device complexity increases due to additional reagents and computational steps
Solution Approach 1:
The patent uses synthetic template DNA sequences that copy or mimic the structure and amplification characteristics of genuine immune cell DNA templates. These synthetic copies contain unique barcodes for identification but otherwise replicate the amplification behavior of real targets, allowing normalization without requiring complex additional equipment or procedures. The copying approach simplifies the system by using informationally equivalent substitutes rather than complex physical measurement devices.
Solution Approach 2:
The patent changes the parameter of template origin from natural to synthetic, and adds barcode sequences as a distinguishing feature. This parameter change allows the synthetic templates to be easily identified and differentiated from genuine immune cell DNA through sequence analysis, enabling automated computational distinction without complex physical separation methods. The barcode parameter provides a simple molecular tag that simplifies data processing despite adding reagent complexity.
4Reliability
If absolute quantitation of adaptive immune cells is achieved, then diagnostic and prognostic value increases, but current methods fail due to inability to distinguish immune cells from other cell types in complex mixtures
Solution Approach 1:
The patent extracts or isolates the specific genetic marker (rearranged CDR3 sequences of TCR or Ig loci) that is unique to adaptive immune cells from the complex mixture of all cellular DNA in the sample. By designing PCR primers that specifically amplify only these immune cell-specific sequences, the method effectively extracts the signal of interest from the background of non-immune cell DNA, enabling specific detection and quantitation of adaptive immune cells in heterogeneous tissue 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 enables accurate absolute quantitation and relative representation of adaptive immune cells in biological samples, providing valuable information for diagnostic and prognostic purposes, particularly in cancer and infectious diseases.
Implementation Method 1
amplifying by multiplex PCR and sequencing: i) rearranged CDR3 oligonucleotide sequences from T cell receptor (TCR) loci from T cells or Immunoglobulin (Ig) loci from B cells
Implementation Method 2
amplifying by multiplex PCR and sequencing: i) rearranged CDR3 oligonucleotide sequences from T cell receptor (TCR) loci from T cells or Immunoglobulin (Ig) loci from B cells
Data Source
Figure 1(A)~1(B)
Figure 2
Figure 3
AI summary
A relative representation of adaptive immune cells in a biological sample is quantified using multiplex PCR and sequencing of adaptive immune cells, control genes, and synthetic template molecules. Disclosed herein are methods for quantifying a number of adaptive immune cells in a biological sample, and methods for quantifying a relative representation of adaptive immune cells in a biological sample that comprises a mixture of cells comprising adaptive immune cells and cells that are not adaptive immune cells. Methods are provided for amplifying by multiplex PCR and sequencing a first set of synthetic templates each comprising one TCR or IgV segment and one TCR of Ig J or C segment and a unique bar code.