Stabilisation of biological samples

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.

1 . A container for collecting a cell-containing biological sample, comprising
 c) at least one carboxylic acid amide, wherein the carboxylic acid amide is selected from primary carboxylic acid amides and secondary carboxylic acid amides; and   d) at least one anticoagulant.   
     
     
         2 . The container according to  claim 1 , wherein the container additionally comprises
 (i) at least one apoptosis inhibitor; and/or   (ii) at least one poly(oxyethylene) polymer, preferably at least one polyethylene glycol.   
     
     
         3 . The container according to  claim 2 , wherein the container comprises a polyethylene glycol as poly(oxyethylene) polymer. 
     
     
         4 . The container according to  claim 1 , having one or more of the following features:
 (i) the container is evacuated;   (ii) the container is a tube, the bottom is a closed bottom, the container further comprises a closure in the open top, and the chamber is at a reduced pressure; and/or   (iii) the container is for drawing blood from a patient.   
     
     
         5 . The container according to  claim 1 , wherein the at least one carboxylic acid amide and the at least one anticoagulant are provided as a stabilizing composition. 
     
     
         6 . The container according to  claim 5 , wherein the at least one carboxylic acid amide is in a concentration of at least 1% in the stabilizing composition. 
     
     
         7 . The container according to  claim 1 , wherein the at least one carboxylic acid amide has formula 1 
       
         
           
           
               
               
           
         
         wherein R1 is a hydrogen residue or an alkyl residue, wherein R2 is selected from a hydrogen residue and a hydrocarbon residue with a length of the carbon chain of 1-20 atoms arranged in a linear or branched manner, wherein R3 is a hydrogen residue, and wherein R4 is oxygen. 
       
     
     
         8 . The container according to  claim 1 , wherein the at least one carboxylic acid amide has one or more of the following characteristics:
 a) it is selected from the group consisting of formamide, acetamide, propanamide and butanamide,   b) it is selected from butanamide and formamide, and/or   c) it is butanamide.   
     
     
         9 . The container according to  claim 1 , wherein the at least one carboxylic acid amide is selected from the group consisting of N-alkylformamide, N-alkylacetamide and N-alkylpropanamide or is selected from N-methylformamide, N-methylacetamide and N-methylpropanamide. 
     
     
         10 . The container according to  claim 1 , wherein the anticoagulant is a chelating agent, preferably EDTA. 
     
     
         11 . The container according to  claim 1 , further comprising at least one tertiary amide. 
     
     
         12 . The container according to  claim 11 , wherein the tertiary amide 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. 
       
     
     
         13 . The container according to  claim 12 , having one or more of the following features:
 a) the tertiary amide according to formula 1 is a carboxylic acid amide;   b) the tertiary amide according to formula 1 is a N,N-dialkylpropanamide; and/or   c) the tertiary amide according to formula 1 is selected from the group consisting of N, N-dimethylformamide, N, N-dimethylacetamide, N, N-dimethylpropanamide, N, N-dimethylbutanamide.   
     
     
         14 . The container according to  claim 6 , wherein the composition further comprises at least one apoptosis inhibitor, preferably a caspase inhibitor. 
     
     
         15 . The container according to  claim 14 , wherein the apoptosis inhibitor is a caspase inhibitor which has one or more of the following characteristics:
 a) the caspase inhibitor is a pancaspase inhibitor,   b) the caspase inhibitor is a caspase specific peptide modified by an aldehyde, nitrile or ketone compound,   c) the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-Val-Ala-Asp(OMe)-FMK, and/or   d) the caspase inhibitor is Q-VD-OPh.   
     
     
         16 . The container according to  claim 6 , wherein the composition is capable of stabilizing the gene transcription profile of contained cells and/or is capable of stabilizing an extracellular nucleic acid population comprised in a cell-containing sample. 
     
     
         17 . The container according to  claim 6 , wherein the stabilization composition does not comprise additives in a concentration that induce or promote cell lysis, and wherein the stabilization composition does not comprise a cross-linking agent that induces protein-DNA and/or protein-protein crosslinks or involve the use of a formaldehyde releaser. 
     
     
         18 . The container according to  claim 6 , wherein the stabilization composition has one or more of the following characteristics:
 a) it is capable of stabilizing cells and reducing the release of genomic DNA from cells contained in the cell-containing biological sample into the cell-free portion of the sample;   b) it is capable of reducing the dilution of the extracellular DNA population comprised in the biological sample with genomic DNA originating from cells contained in the stabilized sample;   c) it is capable of reducing the dilution of the extracellular nucleic acid population comprised in the biological sample with intracellular nucleic acids originating from cells contained in the stabilized sample;   d) the stabilization composition does not comprise additives in a concentration wherein said additives would induce or promote cell lysis;   e) the stabilization composition does not comprise a cross-linking agent that induces protein-DNA and/or protein-protein crosslinks;   f) the stabilization composition does not comprise formaldehyde, formaline, paraformaldehyde or a formaldehyde releaser;   g) the stabilization composition does not comprise a toxic agent;   h) the stabilization composition is capable of stabilizing extracellular nucleic acid population comprised in the cell-containing biological sample without refrigeration, preferably at room temperature, for a time period selected from at least two days, at least three days, at least two days to three days, at least two days to six days and/or at least two days to seven days; and/or   i) the stabilization composition comprises at least one poly(oxyethylene) polymer, preferably a polyethylene glycol.   
     
     
         19 . The container according to  claim 1 , wherein the cell-containing sample is a blood sample. 
     
     
         20 . The container according to  claim 19 , wherein upon contact of the stabilizing composition with a blood sample, the transcript level of one or more marker genes selected from c-fos, IL-1beta, IL-8 and p53 is stabilized for at least 48 h upon stabilization. 
     
     
         21 . The container according to  claim 5 , wherein the container is pre-filled with a defined amount of the stabilizing composition either in solid or liquid form and is provided with a defined vacuum and sealed with a septum. 
     
     
         22 . The container according to  claim 5 , comprising the stabilizing composition mixed with a cell-containing sample, wherein preferably, the volumetric ratio of the stabilizing composition to 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. 
     
     
         23 . The container according to  claim 6 , wherein the sample is a blood sample, and wherein the morphology of and/or cell surface epitopes on white blood cells, preferably lymphocytes, is preserved. 
     
     
         24 . The container according to  claim 6 , wherein the stabilizing composition is provided as a mixture with a biological sample, and wherein said sample has one or both of the following characteristics:
 a) it comprises extracellular nucleic acids;   b) it is whole blood;   c) it is a body fluid; and/or   d) it is urine.   
     
     
         25 . A method comprising the step of collecting a sample from a patient into a chamber of a container according to  claim 1 . 
     
     
         26 . A system comprising a container according to  claim 1  configured to collecting a sample from a patient into a chamber of the container.