Stabilization method for biological samples by combination of heating and chemical fixation

a biological sample and chemical fixation technology, applied in the direction of sampling, material testing goods, measurement devices, etc., can solve the problems of inability difficult to detect the presence, and difficult to achieve effective heating, so as to avoid degradation of other constituents of the sample, shorten the time needed to obtain blockage of enzymatic processes, and facilitate effective heating

Inactive Publication Date: 2015-07-09
DENATOR
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This method effectively minimizes sample degradation, allowing for reliable and reproducible preservation of proteins and peptides, enabling accurate analysis and comparison of protein content across samples.

Problems solved by technology

Sample degradation, however, is both hard to impede, and hard to detect.
The result is that many analyses are unable to detect the presence of species that have degraded long before the analysis is carried out; correspondingly, such analyses may in fact identify degradation products of critical components in place of the original components.
Despite their ubiquity, proteins are extremely sensitive to their environments and thus are not always easy to detect and to identify because they can degrade very quickly.
Once an organism dies, or once a sample of tissue is extracted from a living organism, the regulatory balance of the organism or in the sample is lost and key proteins start to break down.
For example, some proteins whose natural role is to digest other proteins (a “proteolytic” function), and whose natural levels and activities are regulated while an organism is alive, may go out of control after death.
However, the study of tissue samples from patients or model organisms usually exposes the samples to a certain period of oxygen and nutrient depletion before homogenization and protease inactivation occurs.
Use of aldehyde solutions is problematic because it penetrates tissue slowly, ˜0.5-2 mm / h, allowing for degradation of macromolecules prior to complete fixation and consequently does not arrest natural degradation of proteins (Fox et al., “Formaldehyde fixation” J. Histochem. Cytochem. 33:845-853, 1985).
Microwave irradiation is problematic because it is generally non-uniform, that is, some parts of the sample reach a temperature that is high enough to cause sample breakdown.
Furthermore, parts of the sample can reach temperatures above 100° C. creating small holes due to steam eruptions.
Furthermore, microwave irradiation has formerly been applied to living (non-human) subjects as part of a sacrificial protocol and thus has yet to be established as a tool for analyzing samples that have been extracted from subjects, both human and non-human.
This method does not address the problems related to rapid post-sampling degradation of the sample.
However, full benefit of the application of this technology e.g. in immunohistochemical studies is limited by the rapid loss of phosphorylation in samples before complete stabilization of the sample can be achieved (Mandell, “Phosphorylation state-specific antibodies.

Method used

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  • Stabilization method for biological samples by combination of heating and chemical fixation
  • Stabilization method for biological samples by combination of heating and chemical fixation

Examples

Experimental program
Comparison scheme
Effect test

example 1

tochemical Visualization of the Phosphorylated Form of Creb in Samples of Mice Brain After Different Treatments

[0080]Experimental

[0081]Biological Samples and Treatments

[0082]Mice were sacrificed by cervical dislocation and sample collected immediately. Whole brains were excised immediately following sacrifice and were subjected whole to either of the following treatments: A: Directly submerged in 10% neutral buffered formalin (NBF), B: Kept at room temperature for 15 min prior to being submerged in NBF. C: Kept at room temperature for 2 h prior to being submerged in NBF. D: Stabilized by heat treatment in the Stabilizor T1 instrument (Denator AB, Sweden) at auto settings for fresh tissue and directly after treatment submerged in NBF.

[0083]All samples were kept for 24 h in formalin solution at room temperature prior to paraffin imbedding. All samples were incubated without further sub dissection in the NFB. Prior to paraffin embedding samples were cut into halves and embedded such th...

example 2

tion of Samples from Mice Brain

[0092]Experimental

[0093]Biological Samples and Treatments

[0094]Mice were sacrificed by cervical dislocation and sample collected immediately. Whole brains were excised immediately following sacrifice and stabilized by heat treatment in the Stabilizor T1 instrument (Denator AB, Sweden) at auto settings for fresh tissue and subsequently incubated in room temperature for the following periods of time, A: 0 min, B: 15 min, C: 6 h, and D: 24 h prior to being submerged in NBF.

[0095]All samples were kept for 24 h in formalin solution at room temperature prior to paraffin imbedding. All samples were incubated without further sub dissection in the NFB. Prior to paraffin embedding samples were cut into halves and embedded such that sections could be cut through the centre of the original sample.

[0096]Histochemistry, Immunohistochemistry, and Image capture and processing were performed as described in Example 1.

[0097]Results

[0098]FIG. 2 describes the results of I...

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Abstract

The present invention provides methods for stabilizing a biological sample for analysis. The invention more particularly provides methods combining heat treatment and chemical fixation of biological samples in order to maintain protein primary structure and post-translational modifications, such as protein phosphorylations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This present application is a Continuation of U.S. Nonprovisional application Ser. No. 13 / 377,944 filed on 17 Jan. 2012; which claims the benefit of PCT International Application Serial No. PCT / SE2010 / 050633 filed on 8 Jun. 2010; which claims the benefit of SE Application No. 0950572-8 filed 30 Jul. 2009; each of which is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION[0002]It is key to any analysis of a biological sample that the integrity of its constituents is conserved between the time that the sample is extracted from a living organism and the time that the analysis is carried out. Sample degradation, however, is both hard to impede, and hard to detect. The result is that many analyses are unable to detect the presence of species that have degraded long before the analysis is carried out; correspondingly, such analyses may in fact identify degradation products of critical components in place of the origin...

Claims

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Application Information

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Patent Type & AuthorityApplications(United States)
IPC IPC(8): G01N1/30G01N1/44
CPCG01N1/30G01N2001/305G01N1/44
InventorBOREN, MATSSKOLD, KARLSODERQUIST, MARCUS
OwnerDENATOR