Stabilisation of biological samples

Patent metadata

Field	Value
Publication number	US-11525155-B2
Application number	US-201414777902-A
Country	US
Kind code	B2
Filing date	Mar 18, 2014
Priority date	Mar 18, 2013
Publication date	Dec 13, 2022
Grant date	Dec 13, 2022

Abstract

The present invention provides methods and composition suitable for stabilizing cell-containing samples such as blood samples. The stabilizers used are primary or secondary carboxylic acid amides.

First claim

The invention claimed is:

1. A method for stabilizing a cell-containing biological sample, comprising:
a) contacting the sample with
(i) at least one carboxylic acid amide,
wherein the carboxylic acid amide is selected from primary carboxylic acid amides and secondary carboxylic acid amides having formula 1

wherein R1 is hydrogen or C1-05 alkyl, wherein R2 is hydrogen or C1-05 alkyl, wherein R3 is hydrogen, and wherein R4 is oxygen, and
(ii) a caspase inhibitor,

wherein the cell-containing biological sample is selected from the group consisting of body fluids, body secretions, body excretions, swab samples, and animal cell-containing biological samples,
wherein said method is not based on cell lysis, and
wherein the stabilization does not involve the use of a cross-linking agent that induces protein-nucleic acid and/or protein-protein crosslinks, the use of a formaldehyde releaser, or freezing the sample.

2. The method according to claim 1 , wherein the stabilization results in a stabilization of intracellular RNA, and/or wherein the extracellular nucleic acid population comprised in the cell-containing sample is stabilized.

3. The method according to claim 1 , having one or more of the following characteristics:
a) the method comprises contacting the cell-containing sample with at least one primary carboxylic acid amide selected from the group consisting of formamide, acetamide, propanamide and butanamide;
b) the method comprises contacting the cell-containing sample with butanamide;
c) the method comprises contacting the cell-containing sample with at least one secondary carboxylic acid amide selected from the group consisting of N-alkylformamide, N-alkylacetamide and N-alkylpropanamide; or d) the method comprises contacting the cell-containing sample with at least one secondary carboxylic acid amide selected from N-methylformamide, N-methylacetamide and N-methylpropanamide.

4. The method according to claim 1 , wherein the mixture that is obtained when contacting the cell-containing biological sample with the at least one carboxylic acid amide selected from primary carboxylic acid amides and secondary carboxylic acid amides comprises said carboxylic acid amide in a concentration of at least 0.1%, at least 0.25%, at least 0.5%, at least 0.75%, at least 1%, at least 1.25%, at least 1.5% or at least 2%.

5. The method according to claim 1 , wherein the cell-containing sample is a blood sample, and the method comprises additionally contacting the blood sample with an anticoagulant.

6. The method according to claim 1 , having one or more of the following characteristics:
a) performing the method reduces the degradation of nucleic acids present in the cell-containing sample due to the stabilization;
b) performing the method stabilizes intracellular RNA of the cell-containing biological sample;
c) performing the method stabilizes the transcriptome and/or transcript levels in cells contained in the sample; and/or
d) performing the method stabilizes the transcriptome and/or transcript levels in cells contained in the sample, wherein the transcript level of one or more marker genes selected from c-fos, IL-1beta, IL-8 and p53 is stabilized for at least 24h or at least 48h upon stabilization.

7. The method according to claim 1 , wherein the method is suitable for stabilizing an extracellular nucleic acid population comprised in the cell-containing sample and wherein the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample is reduced.

8. The method according to claim 1 , having one or more of the following characteristics:
a) the stabilization allows isolating cells from the stabilized sample;
b) the cell-containing sample is a blood sample and wherein white blood cells are stabilized;
c) the morphology of cells is preserved;
d) the morphology of nucleated cells is preserved;
e) the sample is a blood sample and contained lymphocytes and/or monocytes are stabilized;
f) cell surface epitopes are preserved; and/or
g) cell surface proteins are preserved.

9. The method according to claim 1 , wherein the method comprises additionally contacting the cell-containing sample with at least one polyethylene glycol.

10. The method according to claim 1 , wherein the method has one or more of the following characteristics:
a) the at least one carboxylic acid amide which is a primary or secondary carboxylic acid amide and optionally further additives are comprised in a stabilising composition and wherein the volumetric ratio of the stabilising composition to the specified volume of the cell-containing sample is selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5;
b) the method comprises subjecting the stabilized cell-containing sample to a nucleic acid analysis and/or detection method;
c) the method comprises isolating intra- and/or extracellular nucleic acids from the stabilized sample and analyzing and/or detecting the isolated nucleic acids;
d) the method comprises removing cells comprised in the stabilized sample;
e) the method comprises storing (i) the stabilized cell-containing biological sample, (ii) the stabilized sample from which cells have been removed and/or (iii) cells removed from the sample; and/or
f) the method comprises removing cells from the stabilized sample and i) analyzing the removed cells and/or ii) isolating biomolecules from removed cells.

11. The method according to claim 1 , wherein the method comprises additionally contacting the cell-containing sample with at least one tertiary amide which is a compound according to formula 1

wherein R1 is a hydrogen residue or an alkyl residue, wherein R2 and R3 are identical or different hydrocarbon residues with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, and wherein R4 is an oxygen, sulphur or selenium residue.

12. The method of claim 1 , further comprising:
b) isolating nucleic acids from the stabilized sample.

13. The method according to claim 12 , wherein step b) comprises isolating intracellular RNA.

14. The method according to claim 12 , wherein cells are separated from the stabilized sample, and wherein in step b) extracellular nucleic acids are isolated from the remaining sample and/or intracellular nucleic acids are isolated from the removed cells.

15. The method according to claim 1 , wherein the cell-containing biological sample is a human body fluid, a human body secretion, a human body excretion, a human swab sample, or a human cell-containing biological sample.

16. The method of claim 1 , wherein the cell-containing biological sample is a body fluid.

17. The method of claim 16 , wherein the body fluid is blood or urine.

18. The method according to claim 1 , wherein in formula 1, R1 is either hydrogen or C1-C5alkyl, R2 is C1-C5 alkyl, R3 is hydrogen, and R4 is oxygen.

19. The method according to claim 1 , wherein in formula 1, R1 is either hydrogen or C1-C4 alkyl, R2 is C1-C4 alkyl, R3 is hydrogen, and R4 is oxygen.

20. The method according to claim 1 , wherein the caspase inhibitor is Quinoline-Val-Asp-CH2-OPH or Z-Val-Ala-Asp(OMe)-FMK.

Assignees

• Qiagen Gmbh

Inventors

• Wyrich Ralf
• Voss Thorsten
• Günther Kalle
• Oelmüller Uwe

Classifications

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• C12Q1/6841

  In situ hybridisation · CPC title

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2527/127

  the enzyme inhibitor or activator used · CPC title