Stabilisation and isolation of extracellular nucleic acids

1. A container suitable for collecting a biological sample, comprising:

a stabilizing composition suitable for stabilizing an extracellular nucleic acid population comprised in the sample, wherein said stabilizing composition comprises (a) a caspase inhibitor; and

the container further comprising 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 arranged in a linear or branched manner, and R4 is an oxygen, sulphur or selenium residue.

2. The container according to claim 1 , wherein the sample is a blood, plasma or serum sample.

3. The container according to claim 1 , wherein the at least one compound according to formula 1 is comprised in the stabilizing composition.

4. The container according to claim 1 , wherein in formula 1, R1 is a C1-C5 alkyl residue.

5. The container according to claim 1 , wherein the stabilizing composition additionally comprises an anticoagulant.

6. The container according to claim 1 , wherein the container is evacuated.

7. The container according to claim 1 , wherein the stabilizing composition is capable of reducing (1) the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample, and/or (2) the degradation of nucleic acids present in the sample.

8. The container according to claim 1 , wherein

a) the caspase inhibitor has one or more of the following characteristics:

i) it is a pancaspase inhibitor,

ii) it is a caspase-specific peptide,

iii) it is a caspase-specific peptide modified by an aldehyde, nitrile or ketone compound, and/or

iv) it is selected from the group consisting of Q-VD-OPh having the structure:

and Z-Val-Ala-Asp(OMe)-FMK having the structure:

and/or

b) wherein the compound according to formula 1 has one or more of the following characteristics:

i) R1, R2 and R3 comprise 1 to 5 carbon atoms,

ii) R1, R2 and R3 comprise 1 or 2 carbon atoms,

iii) R4 is oxygen,

iv) it is a N,N-dialkyl-carboxylic acid amide,

v) it is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide, and/or

vi) it is N,N-dimethylpropanamide.

9. The container according to claim 1 , wherein the stabilizing composition additionally comprises at least one anticoagulant.

10. The container according to claim 9 , wherein the at least one anticoagulant is a chelating agent.

11. The container according to claim 10 , wherein the chelating agent is EDTA.

12. The container according to claim 1 , wherein the stabilizing composition comprises the caspase inhibitor in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics:

a) it comprises the caspase inhibitor in a concentration of at least 0.01 μM, at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least 1 μM, at least 2.5 μM or at least 3.5 μM;

b) it comprises the caspase inhibitor in a concentration range selected from 0.01 μM to 100 μM, 0.05 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 40 μM, 1 μM to 30 μM, and 2.5 μM to 25 μM.

13. The container according to claim 1 , wherein the stabilizing composition comprises in addition to the caspase inhibitor:

(b) at least one hypertonic agent which stabilizes cells potentially comprised in the sample,

(c) the at least one compound according to formula 1; and

(d) optionally at least one anticoagulant.

14. The container according to claim 1 , wherein the stabilizing composition comprises the compound according to formula 1 in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics:

a) it comprises the compound according to formula 1 in a concentration of at least 0.1%, at least 0.5%, at least 1%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%;

b) it comprises the compound according to formula 1 in a concentration range selected from 0.1% to 50%, 0.5% to 25%, 0.75% to 20%, 1% to 15%, and 1% to 10%.

15. The container according to claim 1 , wherein the stabilizing composition comprises:

(a) at least one pancaspase inhibitor as caspase inhibitor, (b) at least one hypertonic agent,

(c) the at least one compound according to formula 1, and (d) optionally a chelating agent as anticoagulant.

16. The container according to claim 1 , wherein stabilization of the extracellular nucleic acid population is achievable without refrigeration for a time period selected from

a) at least one day;

b) at least two days;

c) at least three days;

d) one day to three days;

e) one day to six days; and/or

f) one day to seven days.

17. The container according to claim 1 , wherein the container has an open top, a bottom, and a sidewall extending therebetween defining a chamber, wherein the stabilization composition is comprised in the chamber.

18. The container according to claim 17 , wherein 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.

19. The container according to claim 18 , wherein the closure is capable of being pierced with a needle or cannula, and wherein the reduced pressure is selected to draw a specified volume of a liquid sample into the chamber.

20. The container of claim 19 , wherein the chamber is at a reduced pressure selected to draw a specified volume of a liquid sample into the chamber, and wherein the stabilizing composition is a liquid and is disposed in the chamber such that the volumetric ratio of the stabilising composition to the specified volume of the 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.

21. The container according to claim 1 , suitable for collecting a blood sample, wherein the stabilizing composition further comprises an anticoagulant, and is capable of reducing (1) the release of genomic DNA from cells in the sample into the cell-free portion of the sample, and (2) the degradation of nucleic acids in the sample.

22. The container according to claim 13 , wherein the stabilizing composition comprises the hypertonic agent in a concentration that when the sample is added to the stabilizing composition, the resulting mixture has one or both of the following characteristics:

a) it comprises the hypertonic agent in a concentration of at least 0.05M or at least 0.1M;

b) it comprises the hypertonic agent in a concentration range selected from 0.05M to 2M, 0.1 to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M, and 0.1M to 0.6M.

23. The container according to claim 1 , wherein the stabilizing composition has one or more of the following characteristics:

a) it is capable of reducing the release of genomic DNA from cells contained in the sample into the cell-free portion of the sample;

b) it is capable of reducing the degradation of nucleic acids present in the sample;

c) it is provided in a solid form;

d) it is provided in a liquid form; and/or

e) it is capable of stabilizing the extracellular nucleic acid population contained in said sample at room temperature for at least 3 days.

24. The container according to claim 23 , wherein in b), the stabilizing composition is capable of reducing the degradation of genomic DNA present in the sample.

25. The container according to claim 1 , wherein the container additionally comprises the sample, and wherein the sample has one or more of the following characteristics:

a) it comprises extracellular nucleic acids;

b) it is selected from the group consisting of whole blood, plasma, serum, lymphatic fluid, urine, liquor, cerebrospinal fluid, ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, semen/seminal fluid, swabs/smears, body fluids, body secretions, nasal secretions, vaginal secretions, wound secretions and excretions and cell culture supernatants;

c) it is a cell-depleted or cell containing body fluid;

d) it is selected from whole blood, plasma and/or serum; and/or

e) it is whole blood.

26. A method for collecting a sample, comprising collecting a sample from a patient into a chamber of the container according to claim 1 .

27. The container according to claim 13 , wherein the hypertonic agent has one or more of the following characteristics:

i) it is uncharged,

ii) it stabilizes the cells comprised in the sample by inducing cell shrinking,

iii) it is cell impermeable,

iv) it is water-soluble,

v) it is a hydroxylated organic compound,

vi) it is a polyol,

vii) it is a hydroxy-carbonyl compound,

viii) it is a carbohydrate or a sugar alcohol, and/or

ix) it is dihydroxyacetone.