Process and composition for the stabilization of cell-free nucleic acids and cells

Abstract

A method for stabilizing cell-free nucleic acids. The method includes providing a composition and applying the composition to a biological sample as a stabilizing agent for the cell-free nucleic acids contained in the biological sample. The composition includes at least one buffering compound that buffers to a pH value of 7 or below, at least one anticoagulant and urotropin in aqueous solution.

Claims

1. A method for stabilizing cell-free nucleic acids, the method comprising providing a composition and applying the composition to a biological sample as a stabilizing agent for the cell-free nucleic acids contained in the biological sample, wherein the composition comprises at least one buffering compound that buffers to a pH value of 7 or below, at least one anticoagulant and urotropin in aqueous solution.

2. The method according to claim 1, wherein the composition consists of the at least one buffering compound, the at least one anticoagulant which is a chelating agent, and the urotropin.

3. A method for stabilizing cell-free nucleic acids, the method comprising providing a composition and applying the composition to a biological sample as a stabilizing agent for the cell-free nucleic acids contained within the biological sample, wherein the composition comprises, as a dry mixture, at least one buffering compound that is configured to buffer the biological sample to a pH value of 7 or below, at least one anticoagulant and urotropine.

4. The method according to claim 1, wherein the composition contains no quenching compound for free or for dissolved formaldehyde.

5. The method according to claim 1, wherein the at least one buffering compound buffers the aqueous solution to a pH value in the range of 3.5 to 7 and has a buffer capacity that is equal to a concentration of 0.3 to 1 M citrate, and in that 1 to 30 wt./vol.-% urotropin is dissolved in the solution.

6. The method according to claim 1, wherein the at least one buffering compound and the at least one anticoagulant are formed by citrate buffer.

7. The method according to claim 1, wherein the at least one buffering compound is selected from citrate buffer, acetate buffer, MES (2-N-morpholinoethanolsulfonic acid), PIPES (piperazine-N,N′-bis-2-ethanolsulfonic acid), MOPS (3-N-morpholinopropanesulfonic acid), phosphate buffer and mixtures of at least two thereof, and in that the at least one anticoagulant is a chelating agent selected from citrate and EDTA and mixtures thereof.

8. The method according to claim 1, wherein the biological sample is whole blood and the composition is contained in a blood collection tube in a volume fraction of up to 20% of the maximum sample volume to be drawn, for which the blood collection tube is configured.

9. The method according to claim 1, wherein the biological sample is urine.

10. The method according to claim 1, wherein the biological sample in a mixture with the composition is stable at 0 to 37° C. for 1 h to 14 d.

11. The method according to claim 1, wherein the applying comprises contacting the sample with the composition to produce a mixture of the biological sample and the composition and storing the mixture of the biological sample and the composition at 0 to 37° C. for 1 hour to 14 days.

12. The method according to claim 11, wherein the composition is contacted with the sample in a volume fraction of at maximum 20% of the sample volume.

13. The method according to claim 11, comprising, separating the mixture into a cell-free fraction, and adding proteinase to the mixture prior to the storing.

14. The method according to claim 13, comprising isolating nucleic acids from the cell-free fraction and analyzing the cell-free fraction.

15. The method according to claim 1, wherein the biological sample is whole blood or urine.

16. The method according to claim 1, wherein the anticoagulant is a chelating agent and the composition comprises PEG.

17. The method according to claim 3, wherein the anticoagulant is a chelating agent and the composition comprises PEG.

Description

(1) The invention is now described in more detail by way of an example with reference to the figures, which show in

(2) FIG. 1 the result of a PCR amplification of cell-free DNA from a sample following different storage periods and in

(3) FIG. 2 the result of PCR amplifications of two DNA segments as a quotient of the concentrations of the amplificates following different storage periods.

Example 1: Isolating and Analysing Cell-Free DNA from Whole Blood

(4) The composition was produced by mixing 0.5 M citric acid (monohydrate) in water and 0.5 M trisodium citrate (dihydrate) in water until pH 4.2 was reached and subsequently adding and dissolving of 18 wt./vol.-% urotropin.

(5) As an example for a biological sample, whole blood from 3 donors was drawn into 4 identical blood collection tubes to 4.9 mL each that each contained 0.49 mL of the composition (10 vol.-% of the whole blood). From these blood collection tubes that contained the mixture of whole blood and of the composition and that were stored at 22.5° C., one each was processed on day 0 (T0), day 3 (T3), day 7 (T7) and day 14 (T14), respectively, and cell-free DNA was isolated therefrom and analyzed. For this, a cell-free fraction was generated from each blood collection tube via a two-step centrifugation process (10 min, 2,000×g; 15 min, 15,000×g).

(6) From the cell-free plasma generated thereby that contained the composition for stabilization, cell-free DNA was isolated using the NucleoSnap DNA Plasma Kit (available from Macherey-Nagel). In contrast to the Kit instruction, 1.7 mL of the cell-free plasma instead of 3.0 mL were used, and the lysis buffer VL and ethanol were reduced from 3.0 mL to 1.7 mL accordingly. Additionally, the incubation time for the cell-free plasma with proteinase K at room temperature and at 56° C. was each increased from 5 min to 15 min.

(7) The analysis of the cell-free DNA was done by amplifying the 115 bp segment ALU115 and the 247 bp segment ALU247 by means of quantitative PCR as described by Umetani et al. (2006).

(8) FIG. 1 shows the average of the quotients of the concentration of ALU115 at the given timepoint at day 0 (T0), day 3 (T3=Tx), day 7 (T7=Tx) and day 14 (T14=Tx) of the aliquots and the concentration of ALU115 at day 0 (T0). Therein, a quotient (Tx/T0) of 1 showed that the concentration of cell-free DNA in the mixture stays unchanged respectively stable, and also shows that the cell-free DNA in the mixture is amplifiable through PCR without change. This result shows that the cell-free DNA in the mixture of whole blood and of the composition is stabilized in concentration and structure over a storage duration of at least 14 d at 22.5° C. for the subsequent analysis.

(9) FIG. 2 shows the quotients of the absolute amounts of cell-free DNA (cfDNA) on the example of ALU247 and ALU115 at the given timepoints of storing at 25° C. (day 0 (0), day 3 (3), day 7 (7), day 10 (10) and day 14 (14) for the composition according to the invention (cfDNA Exact) and for EDTA samples (EDTA) on day 0 (0) and day 7 (7)). The result shows the stabilization by the composition according to the invention (Exact) by means of the quotient remaining constant over the incubation time. In non-stabilized, EDTA-anticoagulated samples, the ALU247/ALU115 quotient increases with increasing storage duration. An insufficient stabilization of blood samples/biological samples leads to the lysis of cells and thus to the release of the genomic DNA (gDNA). An ALU247/ALU115 quotient of 1, meaning equal amounts of ALU247 and ALU115, is characteristic for gDNA.

(10) These results also show that the composition is suitable for the later isolation of cell-free nucleic acids from a cell-free mixture of the composition with plasma with adsorbing of the nucleic acids to an adsorption agent, and respectively does not impair this isolation.

Example 2: Isolating and Analysing Cells from Whole Blood

(11) According to Example 1, a mixture of the composition and of whole blood was produced and stored at 22.5° C. A second whole blood sample instead of the composition contained physiological saline solution and EDTA (1.6 mg per mL blood) to prevent coagulation. Out of both kinds of whole blood sample, 5.0 mL each underwent an erythrocyte lysis on day 0, day 1, day 3 and day 5, respectively, and the circulating endothelial cells (cEC) were enriched and analyzed by means of the cEC Enrichment and Enumeration Kit of the company Miltenyi Biotech. In healthy humans, the number of circulating endothelial cells lies at ca. 1-20 cells per mL. In various diseases, the number of cECs is in some cases substantially increased. Stabilizing compounds have to ensure that the number of cEC in stored blood samples is stable. Following the analysis described for the Kit, it has shown that over the storage duration the number of evidenced cEC stayed constant only in the samples stabilized according to the invention.

Example 3: Isolating and Analysing Cell-Free DNA from Urine

(12) The composition was produced by mixing 0.5 M citric acid (monohydrate) in water and 0.5 M trisodium citrate (dihydrate) in water until pH 4.2 was reached and subsequently adding and dissolving 15 wt.-%/vol.-% urotropin.

(13) As an example for a biological sample, 90 mL urine were mixed with 1/10 vol. of the composition immediately after sampling. From this mixture that was stored at 22.5° C., one aliquot was each processed on day 0, day 3, day 7 and day 14 and from this cell-free DNA was extracted and analyzed. For this, one cell-free fraction each was generated through centrifugation at 15,000×g for 15 min and separation of the cell-free fraction from one aliquot. From the thus generated cell-free fraction that contained the composition for stabilization, cell-free DNA was isolated by means of the NucleoSnap Plasma Kit (available from Macherey-Nagel).

(14) Compared to urine samples that were identically stored without treatment or with addition of an equal volume of physiological saline solution or of dissolved buffering compound only, it has shown that cell-free DNA was stabilized by the composition.

Example 4: Analysing Stabilized Cells in Urine

(15) According to Example 3, urine was mixed with prostate carcinoma cells (LnCap) and was mixed and stored with the composition or, for comparison, with physiological saline solution.

(16) The analysis ensued on cells sedimented through centrifugation after identical storage period of the comparative samples. It showed that only the composition according to the invention allowed for the flow cytometric detection of the added cells.

(17) Compared to urine samples that were identically stored without treatment or with addition of an equal volume of physiological saline solution or of dissolved buffering compound only, it has shown that the composition stabilized surface markers of the cells during storage.