Extracellular Nucleic Acid Stabilization Using Apoptosis Inhibitors
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Solution Overview
Problem
Current methods for stabilizing extracellular nucleic acids in cell-containing samples, particularly whole blood, are inefficient, leading to contamination with genomic DNA and degradation, which complicates diagnostic and prognostic applications.
Innovation Solution
A method involving the use of apoptosis inhibitors, hypertonic agents, and specific compounds to stabilize cell-containing samples, preventing the release of intracellular nucleic acids and maintaining the integrity of extracellular nucleic acids, allowing for storage at room temperature for extended periods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to isolate extracellular nucleic acids from cell-containing samples, then the isolation process can be completed, but the extracellular nucleic acids become contaminated with genomic DNA released from broken cells
Solution Approach 1:
The patent applies preliminary action by adding stabilizing agents (apoptosis inhibitors, hypertonic agents, and specific compounds) to the sample immediately upon collection. This prevents cell breakdown and genomic DNA release before the isolation process begins, thereby maintaining the purity of extracellular nucleic acids throughout subsequent handling and storage
Solution Approach 2:
The patent employs preliminary anti-action by using apoptosis inhibitors to prevent programmed cell death and hypertonic agents to prevent osmotic lysis of cells. These agents counteract the harmful processes that would otherwise release genomic DNA into the sample, thereby protecting the integrity of the extracellular nucleic acid population before isolation occurs
2Ease of operation
If samples are stored at room temperature for extended periods, then ease of handling and transport is improved, but extracellular nucleic acids degrade
Solution Approach 1:
The patent introduces intermediary substances (apoptosis inhibitors, hypertonic agents, and specific compounds with formula (1)) that act as mediators between the extracellular nucleic acids and the environmental conditions. These intermediaries protect the nucleic acids from degradation during room temperature storage by preventing cell lysis and stabilizing the nucleic acid population, thereby enabling convenient handling without compromising integrity
Solution Approach 2:
The patent applies parameter changes by modifying the chemical environment of the sample through the addition of stabilizing agents. These agents change the physical-chemical parameters (osmotic pressure, enzymatic activity, cellular stability) to create conditions that allow room temperature storage while maintaining nucleic acid integrity, thus resolving the contradiction between storage convenience and stability
3Reliability
If multiple centrifugation steps are used to remove cells, then cell removal efficiency is improved, but the process becomes time-consuming and complex
Solution Approach 1:
The patent applies preliminary action by stabilizing cells and preventing their breakdown before the isolation process. This preliminary stabilization reduces the need for multiple centrifugation steps to remove cell debris, as cells remain intact and do not release genomic DNA, thereby simplifying the overall process and reducing time requirements while maintaining complete cell removal
Solution Approach 2:
The patent extracts or removes the need for multiple centrifugation steps by using stabilizing agents that prevent cell lysis in the first place. This eliminates the generation of cell debris that would require extensive centrifugation to remove, thereby simplifying the isolation protocol and reducing processing time while maintaining high purity
4Quantity of substance
If cells are allowed to break down during sample processing, then release of intracellular contents occurs, but extracellular nucleic acids become contaminated with released genomic DNA
Solution Approach 1:
The patent employs preliminary anti-action by using apoptosis inhibitors to prevent programmed cell death and membrane-stabilizing agents to prevent osmotic lysis. These agents counteract the cell breakdown process before it can occur, thereby preventing the release of genomic DNA that would contaminate the extracellular nucleic acid population and compromising purity
Solution Approach 2:
The patent converts the potential harm of cell breakdown into a benefit by using controlled stabilization strategies. Instead of allowing uncontrolled lysis that releases genomic DNA, the stabilizing agents maintain cellular integrity or control the breakdown process in a way that preserves the distinction between intracellular and extracellular nucleic acids, thereby maintaining purity
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
The method effectively reduces contamination and degradation of extracellular nucleic acids, ensuring their stability and integrity for diagnostic and prognostic applications, enabling reliable analysis and standardization of sample handling.
Implementation Method 1
contacting the sample with a) at least one apoptosis inhibitor
Implementation Method 2
b) at least one hypertonic agent, which stabilizes the cells comprised in the sample
Implementation Method 3
c) at least one compound according to formula (1) wherein R1 is a hydrogen residue or an alkyl residue, R2 and R3 are identical or different hydrocarbon residues with a length of the carbon chain of 1-20 atoms, and R4 is an oxygen, sulphur or selenium residue
Data Source
AI summary
The present invention provides methods, compositions and devices for stabilizing the extracellular nucleic acid population in a cell-containing biological sample using an apoptosis inhibitor, preferably a caspase inhibitor, a hypertonic agent and/or a compound according to formula 1 as defined in the claims.