Frozen Biological Sample Preparation with Non-Crosslinking Fixation
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Solution Overview
Problem
Existing methods for preserving biological samples for analysis face challenges in maintaining morphology and integrity of biological components, as they often require laborious paraffin-embedding and immediate freezing, and introduce chemical modifications or degradation due to cross-linking agents.
Innovation Solution
A method involving fixation with a non-crosslinking fixative solution followed by incubation in an aqueous cryoprotectant solution and freezing, which preserves morphology and integrity of biological components without the need for paraffin-embedding or immediate freezing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If fixation with cross-linking agents such as formaldehyde is used, then sample morphology is well preserved, but severe chemical modifications and degradation of biomolecules occur
Solution Approach 1:
The patent removes cross-linking agents from the fixation process and replaces them with non-crosslinking fixatives such as alcohols (methanol, ethanol), acids (acetic acid, formic acid), or their combinations. This extraction of the harmful cross-linking step while retaining the morphology-preserving function resolves the contradiction between morphology preservation and biomolecule integrity
Solution Approach 2:
The patent changes the chemical parameters of the fixation process by using different fixative compositions (e.g., Carnoy's solution, Methacarn, ethanol-acetic acid mixtures) with varying ratios of alcohols and acids. These parameter changes allow optimization of both morphology preservation and biomolecule protection without relying on cross-linking agents
2Shape
If paraffin-embedding is used for preservation of tissue morphology, then morphology is well retained, but the method is laborious and takes up to two days
Solution Approach 1:
The patent removes the paraffin-embedding step entirely from the sample preparation workflow. By using non-crosslinking fixatives that are compatible with frozen sectioning, the method eliminates the time-consuming paraffin infiltration, embedding, and subsequent deparaffinization steps, reducing processing time from up to two days to a matter of hours
Solution Approach 2:
The patent performs preliminary fixation with non-crosslinking fixatives that prepare the tissue for direct freezing and sectioning. This preliminary action eliminates the need for subsequent paraffin processing steps, as the tissue is already adequately fixed and preserved for frozen section analysis
3Loss of time
If immediate freezing of the sample is performed, then processing time is reduced, but morphology preservation is compromised
Solution Approach 1:
The patent applies preliminary fixation with non-crosslinking fixatives before freezing to stabilize the tissue morphology. This preliminary action ensures that when immediate freezing is performed, the tissue structure is already preserved and resistant to freezing artifacts, achieving both rapid processing and morphology preservation
4Stability of the object's composition
If cross-linking agents are used for fixation, then tissue structure is stabilized, but purification of biological components suitable for molecular analysis is prevented
Solution Approach 1:
The patent removes cross-linking agents from the fixation process, extracting the harmful chemical modifications that prevent biomolecule purification. By using non-crosslinking fixatives such as alcohols and acids, the tissue structure is stabilized through precipitation and coagulation rather than cross-linking, allowing subsequent purification of DNA, RNA, and proteins for molecular analysis
Solution Approach 2:
The patent changes the chemical mechanism of fixation from cross-linking to precipitation and coagulation by using different fixative compositions. These parameter changes in fixation chemistry stabilize tissue structure through alternative mechanisms that do not interfere with biomolecule purification and molecular analysis
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 allows for high-quality preservation of DNA, RNA, and proteins, enabling excellent performance in molecular analysis and diagnostics, while avoiding chemical modifications and sample degradation.
Implementation Method 1
Non-crosslinking fixative solutions advantageously do not introduce crosslinks and achieve tissue fixation by different mechanisms, one important mechanism being the precipitation of proteins
Implementation Method 2
They may also disrupt the hydrophobic interactions that give many proteins their tertiary structure
Implementation Method 3
incubating the fixed biological sample in an aqueous solution comprising a cryoprotectant
Implementation Method 4
Cryoprotectants are thought to decrease the mobility of water molecules thereby preventing the formation of ice crystals
Implementation Method 5
freezing the cryoprotected biological sample
Data Source
AI summary
The present invention pertains to a method of preparing a frozen biological sample, comprising the steps of fixing the biological sample with a non-crosslinking fixative solution, incubating the fixed biological sample in an aqueous solution comprising a cryoprotectant, and freezing the cryoprotected biological sample. The method advantageously allows to preserve both, morphology of the sample as well as biological components such as nucleic acids and proteins, in high quality for subsequent analysis. The method is robust, simple and neither requires laborious steps associated with paraffin-embedding nor immediate freezing of the sample. Also provided are advantageous uses and kits.