US20140227688A1 - Stabilisation and isolation of extracellular nucleic acids - Google Patents

Stabilisation and isolation of extracellular nucleic acids Download PDF

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US20140227688A1
US20140227688A1 US14/347,051 US201214347051A US2014227688A1 US 20140227688 A1 US20140227688 A1 US 20140227688A1 US 201214347051 A US201214347051 A US 201214347051A US 2014227688 A1 US2014227688 A1 US 2014227688A1
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sample
cell
nucleic acids
inhibitor
caspase
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Martin Horlitz
Anabelle Schubert
Markus Sprenger-Haussels
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Qiagen GmbH
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Qiagen GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor

Definitions

  • the technology disclosed herein relates to methods and compositions suitable for stabilizing the extracellular nucleic acid population in a cell-containing sample, in particular a blood sample, and to a method for isolating extracellular nucleic acids from respectively stabilized biological samples.
  • Extracellular nucleic acids have been identified in blood, plasma, serum and other body fluids. Extracellular nucleic acids that are found in respective samples are to a certain extent degradation resistant due to the fact that they are protected from nucleases (e.g. because they are secreted in form of a proteolipid complex, are associated with proteins or are contained in vesicles).
  • nucleases e.g. because they are secreted in form of a proteolipid complex, are associated with proteins or are contained in vesicles.
  • the presence of elevated levels of extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response. Additionally, elevated fetal DNA/RNA in maternal blood is being used to determine e.g.
  • extracellular nucleic acids are in particular useful in non-invasive diagnosis and prognosis and can be used e.g. as diagnostic markers in many fields of application, such as non-invasive prenatal genetic testing, oncology, transplantation medicine or many other diseases and, hence, are of diagnostic relevance (e.g. fetal- or tumor-derived nucleic acids).
  • extracellular nucleic acids are also found in healthy human beings. Common applications and analysis methods of extracellular nucleic acids are e.g. described in WO97/035589, WO97/34015, Swarup et al, FEBS Letters 581 (2007) 795-799, Fleischhacker Ann. N.Y.
  • the first step of isolating extracellular nucleic acids from a cell-containing biological sample such as blood is to obtain an essentially cell-free fraction of said sample, e.g. either serum or plasma in the case of blood.
  • the extracellular nucleic acids are then isolated from said cell-free fraction, commonly plasma, when processing a blood sample.
  • obtaining an essentially cell-free fraction of a sample can be problematic and the separation is frequently a tedious and time consuming multi-step process as it is important to use carefully controlled conditions to prevent cell breakage during centrifugation which could contaminate the extracellular nucleic acids with cellular nucleic acids released during breakage.
  • cell-containing samples may also comprise other nucleic acids of interest that are not comprised in cells.
  • pathogen nucleic acids such as viral nucleic acids.
  • the sample comprises a high amount of cells as is the case e.g. with whole blood samples.
  • the need to directly separate e.g. the plasma from the blood is a major disadvantage because many facilities wherein the blood is drawn (e.g.
  • plasma that is obtained under regular conditions often comprises residual amounts of cells which accordingly, may also become damaged or may die during handling of the sample, thereby releasing intracellular nucleic acids, in particular genomic DNA, as is described above.
  • genomic DNA in particular genomic DNA
  • these remaining cells also pose a risk that they become damaged during the handling so that their nucleic acid content, particularly genomic (nuclear) DNA and cytoplasmic RNA, would merge with and thereby contaminate respectively dilute the extracellular, circulating nucleic acid fraction.
  • Blood samples are presently usually collected in blood collection tubes containing spray-dried or liquid EDTA (e.g. BD Vacutainer K 2 EDTA).
  • EDTA chelates magnesium, calcium and other bivalent metal ions, thereby inhibiting enzymatic reactions, such as e.g. blood clotting or DNA degradation due to DNases.
  • EDTA is an efficient anticoagulant, EDTA does not efficiently prevent the dilution respectively contamination of the extracellular nucleic acid population by released intracellular nucleic acids.
  • the extracellular nucleic acid population that is found in the cell-free portion of the sample changes during the storage.
  • EDTA is not capable of sufficiently stabilising the extracellular nucleic acid population in particular because it can not avoid the contamination of the extracellular nucleic acid population with e.g. genomic DNA fragments which are generated after blood draw by cell degradation and cell instability during sample transportation and storage.
  • sample processing techniques which result in a stabilisation of the extracellular nucleic acid population comprised in a biological sample, in particular a sample containing cells, including samples suspected of containing cells, in particular whole blood, plasma or serum, thereby making the handling, respectively processing of such samples easier (e.g. by avoiding the need to directly separate plasma from whole blood or to cool or even freeze the isolated plasma) thereby also making the isolation and testing of extracellular nucleic acids contained in such samples more reliable and consequently, thereby improving the diagnostic and prognostic capabilities of the extracellular nucleic acids.
  • a solution for preserving extracellular nucleic acids in whole blood samples e.g. for prenatal testing and/or for screening for neoplastic, in particular premalignant or malignant diseases.
  • an object of the present invention to overcome at least one of the drawbacks of the prior art sample stabilization methods.
  • a method that is capable of stabilising a cell-containing sample, in particular whole blood.
  • it is an object of the present invention to stabilise the extracellular nucleic acid population contained in a biological sample and in particular to avoid a contamination of the extracellular nucleic acid population with genomic DNA, in particular fragmented genomic DNA.
  • it is in particular an object of the present invention to provide a method suitable for stabilising a biological sample, preferably a whole blood sample, even at room temperature, preferably for a period of at least two, preferably at least three days.
  • a sample collection container in particular a blood collection tube that is capable of effectively stabilising a biological sample and in particular the extracellular nucleic acid population comprised in the sample.
  • the present invention is based on the finding that certain additives are surprisingly effective in stabilizing cell-containing biological samples comprising extracellular nucleic acids, in particular whole blood samples or samples derived from whole blood such as e.g. blood plasma. It was found that these additives are highly efficient in stabilizing the extracellular nucleic acid population and in particular are capable to avoid or at least significantly reduce contaminations with genomic DNA, in particular fragmented genomic DNA.
  • a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample wherein a sample is contacted with
  • R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, more preferred a methyl 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
  • R4 is an oxygen, sulphur or selenium residue.
  • a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample wherein the sample is contacted with at least one apoptosis inhibitor.
  • the cell-containing sample is selected from whole blood, plasma or serum.
  • the apoptosis inhibitor reduces contaminations of the extracellular nucleic acid population with intracellular nucleic acids, in particular fragmented genomic DNA, that originate from cells contained in the sample, e.g. from damaged or dying cells.
  • the apoptosis inhibitor reduces the degradation of nucleic acids present in the sample.
  • the stabilization according to the present invention using an apoptosis inhibitor has the effect that the extracellular nucleic acid population contained in the sample is substantially preserved in the state it had shown at the time the biological sample was obtained, respectively collected.
  • a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample wherein a sample is contacted with at least one hypertonic agent, which is capable of stabilizing cells comprised in the sample.
  • a hypertonic agent which is capable of stabilizing cells comprised in the sample.
  • cell shrinking that is induced by mild hypertonic effects (osmosis) results in a considerable increase of the cell stability.
  • the hypertonic agent in particular reduces the release of intracellular nucleic acids, in particular genomic DNA, from the contained cells into the extracellular portion or compartment of the sample.
  • the stabilization according to the present invention using a hypertonic agent has the effect that the extracellular nucleic acid population contained in the sample is substantially preserved in the state it had shown at the time the biological sample was obtained, respectively collected.
  • a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample wherein a sample is contacted with at least one compound according to formula 1
  • R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, more preferred a methyl 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
  • R4 is an oxygen, sulphur or selenium residue. It was found that adding a respective compound as an advantageous stabilizing effect on the extracellular nucleic acid population.
  • a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample wherein a sample is contacted with
  • the combination of these stabilizing agents is remarkably effective in inhibiting the release of intracellular nucleic acids, in particular genomic DNA, from the contained cells into the extracellular portion of the sample. Furthermore, it was shown that the degradation of nucleic acids present in the sample is highly efficiently prevented. In particular, less fragmented genomic DNA is found in respectively stabilized samples.
  • the stabilization according to the present invention using this combination of stabilizing additives has the effect that the extracellular nucleic acid population contained in the sample is substantially and effectively preserved in the state it had shown at the time the biological sample was obtained, respectively collected (e.g. drawn in the case of blood) and that in particular contaminations of the extracellular nucleic acid population with fragmented genomic DNA are reduced.
  • a respective combination may comprise at least one apoptosis inhibitor, at least one hypertonic agent and/or at least one compound according to formula 1 as defined above, for example (1) a combination of at least one apoptosis inhibitor and at least one compound according to formula 1 as defined above, (2) a combination of at least one hypertonic agent and at least one compound according to formula 1 or (3) a combination of all three stabilizing agents, i.e. at least one apoptosis inhibitor, at least one hypertonic agent and at least one compound according to formula 1.
  • a respective combination may also comprise additional additives that enhance the stabilizing effect such as e.g. chelating agents.
  • additional additives that enhance the stabilizing effect
  • e.g. chelating agents In case the sample is blood or a sample derived from blood, usually an anticoagulant is also added. Chelating agents such as e.g. EDTA are suitable for this purpose.
  • Respective stabilizing combinations can be according to a fifth sub-aspect advantageously used in a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample according to the first aspect of the present invention.
  • a method for isolating extracellular nucleic acids from a biological sample comprises the steps of:
  • Stabilization in step a) can be achieved e.g. according to one of the five sub-aspects of the first aspect according to the present invention as described above.
  • the stabilization according to the present invention has the effect that the extracellular nucleic acid population contained in the sample is substantially preserved in the state it had shown at the time the biological sample was obtained, respectively collected. Therefore, extracellular nucleic acids obtained from a respectively stabilized sample comprise less contaminations with intracellular nucleic acids, in particular fragmented genomic DNA, that results e.g. from decaying cells comprised in the sample compared to extracellular nucleic acids that are obtained from an unstabilized sample.
  • the substantial preservation of the extracellular nucleic acid population is an important advantage because this stabilization/preservation enhances the accuracy of any subsequent tests. It allows for standardizing the isolation and subsequent analysis of the extracellular nucleic acid population, thereby making diagnostic or prognostic applications that are based on the extracellular nucleic acid fraction more reliable and more independent from the used storage/handling conditions. Thereby, the diagnostic and prognostic applicability of the respectively isolated extracellular nucleic acids is improved.
  • the teachings of the present invention have the advantage that the ratio of certain extracellular nucleic acid molecules can be kept substantially constant compared to the ratio at the time the sample was collected. The stabilization achieves that intracellular nucleic acids are substantially kept within the cells and that extracellular nucleic acids are substantially stabilized.
  • composition suitable for stabilizing a cell-containing biological sample comprising:
  • a respective stabilizing composition is particularly effective in stabilizing a cell-containing biological sample, in particular whole blood, plasma and/or serum by stabilizing the cells and the extracellular nucleic acid population comprised in said sample.
  • at least two of the stabilizing agents defined in a) to c) more preferred all of the stabilizing agents defined in a) to c) are present in the stabilizing composition.
  • a respective stabilizing composition allows the storage and/or handling, e.g. shipping, of the sample, e.g. whole blood, at room temperature for at least two, or preferably at least three days without substantially compromising the quality of the sample, respectively the extracellular nucleic acid population contained therein.
  • the time between sample collection, e.g. blood collection, and nucleic acid extraction can vary without substantial effect on the extracellular nucleic acid population contained in the sample. This is an important advantage as it reduces the variability in the extracellular nucleic acid population attributable to different handling procedures.
  • a container for collecting a cell-containing biological sample preferably a blood sample
  • the container comprises a composition according to the third aspect of the present invention.
  • a respective container e.g. a sample collection tube comprising the stabilizing composition
  • a respective sample collection container in particular a blood collection tube, is capable of stabilising blood cells and extracellular nucleic acids and optionally, viruses respectively viral nucleic acids contained in a blood sample or a sample derived from blood.
  • a method comprising the step of collecting, preferably withdrawing, a biological sample, preferably blood, from a patient directly into a chamber of a container according to the fourth aspect of the present invention.
  • a method of producing a composition according to the third aspect of the present invention wherein the components of the composition are mixed, preferably are mixed in a solution.
  • solution refers to a liquid composition, preferably an aqueous composition. It may be a homogenous mixture of only one phase but it is also within the scope of the present invention that a solution comprises solid components such as e.g. precipitates.
  • FIG. 1 a shows a gel picture after chip electrophoresis of DNA isolated from samples treated with caspase inhibitors (Example 1).
  • FIG. 1 b is a diagram showing the effect of caspase inhibitors on the increase of ribosomal 18S DNA in plasma (Example 1).
  • FIG. 2 a shows a gel picture after chip electrophoresis of DNA isolated from samples treated with different concentrations of the caspase inhibitor Q-VD-OPH in combination (Example 2).
  • FIG. 2 b is a diagram showing the effects of different concentrations of the caspase-inhibitor Q-VD-OPH in combination with glucose on the increase of ribosomal 18S DNA in the plasma (Example 2).
  • FIG. 4 a shows a gel picture after chip electrophoresis of DNA isolated from samples treated with dihydroxyacetone dissolved in different buffers (Example 3).
  • FIG. 4 b is a diagram showing the effect of dihydroxyacetone on the increase of ribosomal 18S DNA (Example 3).
  • FIG. 5 shows the blood cell integrity measured by flow cytometry for blood cells treated with different concentrations of dihydroxyacetone (Example 4).
  • FIG. 6 a shows a gel picture after chip electrophoresis of DNA isolated from samples treated with different concentrations of dihydroxyacetone (Example 4).
  • FIG. 6 b is a diagram showing the effect of different dihydroxyacetone concentrations on the increase of ribosomal 18S DNA (Example 4).
  • FIG. 7 a shows the blood cell integrity measured by flow cytometry for blood cells treated with a combination of elevated K 2 EDTA, Q-VD-OPH and DHA (Example 5).
  • FIG. 7 b is a diagram showing the effect of the combination of EDTA, DHA and Q-VD-OPH on the increase of 18S DNA (Example 5).
  • FIG. 9 is a diagram showing the effects of different concentrations of DMAA on the increase of ribosomal 18S DNA in the plasma.
  • FIG. 10 is a diagram showing the influence of different sugar alcohols on the increase of 18S rDNA (Example 8)
  • FIG. 11 is a diagram showing the influence of substances on the increase of 18S rDNA (Example 9)
  • FIG. 12 is a diagram showing the influence of substances on the increase of 18S rDNA (Example 10)
  • FIG. 13 is a diagram showing the influence of substances on the increase of 18S rDNA (Example 11)
  • FIG. 14 is a diagram showing the influence of substances on the increase of 18S rDNA (Example 11)
  • FIG. 15 is a diagram showing the influence of substances on the increase of 18S rDNA
  • FIG. 16 is a diagram showing the ccfDNA increase in plasma fraction of whole blood incubated for up to 6 days at 37° C. (Example 13)
  • FIG. 19 is a diagram showing the mean copies (Example 14)
  • FIG. 20 is a diagram showing the percent of 18S compared to BD Vacutainer K2E (Example 14)
  • FIG. 21 is a diagram showing the decrease of HIV, incubated in whole blood at 37° C., purified from plasma (Example 15)
  • FIG. 22 is a diagram showing the decrease of HCV, incubated in whole blood at 37° C., purified from plasma (Example 15)
  • FIG. 23 is a diagram showing the influence of propionamid on 18S rDNA increase Donor 1 (Example 16)
  • FIG. 24 is a diagram showing the influence of propionamid on 18S rDNA increase Donor 2 (Example 16)
  • the present invention is directed to methods, compositions and devices and thus to technologies suitable for stabilizing the extracellular nucleic acid population comprised in a cell-containing biological sample.
  • the stabilization technologies disclosed herein reduce the risk that the extracellular nucleic acid population is contaminated with intracellular nucleic acids, in particular fragmented genomic DNA, which derives from, e.g. is released from damaged and/or dying cells contained in the sample. Therefore, the present invention achieves the stabilization of the sample and hence the stabilization of the extracellular nucleic acid population comprised therein without the lysis of the contained cells. Rather, cells contained in the sample are stabilized thereby substantially preventing or reducing the release of intracellular nucleic acids.
  • the remarkable stabilization that is achieved with the methods and compositions of the present invention allows the storage and/or handling of the stabilized sample for a prolonged period of time at room temperature without jeopardizing the quality of the sample, respectively the extracellular nucleic acids contained therein.
  • the time between sample collection and nucleic acid extraction can vary without significant effect on the composition of the extracellular nucleic acids population. This allows the standardization of e.g.
  • the samples, respectively the extracellular nucleic acids obtained from respectively stabilized samples become more comparable.
  • the teachings of the present invention obviate the necessity to directly separate cells contained in the sample from the cell-free portion of the sample in order to avoid, respectively reduce contaminations of the extracellular nucleic acids with intracellular nucleic acids, in particular fragmented genomic DNA, that is otherwise released from decaying cells. This advantage considerably simplifies the handling of the samples, in particular the handling of whole blood samples. E.g.
  • ⁇ blood samples obtained in a clinic and stabilized according to the teachings of the present invention can be shipped at room temperature and the plasma containing the extracellular nucleic acids can be conveniently separated in the receiving clinical lab.
  • teachings of the invention are also advantageous when processing cell-depleted biological samples, or samples commonly referred to as “cell-free” such as e.g. blood plasma or serum.
  • Respective cell-depleted or “cell-free” biological samples may still (also depending on the used separation process) comprise residual cells, in particular white blood cells which comprise genomic DNA, which accordingly, pose a risk that the extracellular nucleic acid population becomes increasingly contaminated with intracellular nucleic acids, in particular fragmented genomic DNA, if the (potentially) remaining cells are damaged or die during the shipping of storing process.
  • the technology of the present invention allows to efficiently preserve the extracellular nucleic acid population of the sample at the time the sample is collected and contacted with the stabilizing agents, said samples can be properly worked up in the receiving facilities in order to isolate the extracellular nucleic acids from said samples while substantially avoiding respectively reducing contaminations of the extracellular nucleic population with intracellular nucleic acids.
  • the facilities receiving the samples such as e.g. laboratories usually also have the necessary equipment such as e.g. high speed centrifuges (or other means, see also below) to efficiently remove cells comprised in the samples, including residual cells that might be present in cell-depleted samples such as e.g. in blood plasma.
  • the present invention has many advantages when stabilizing biological samples which comprise a large amount of cells such as e.g. whole blood samples, but also has important advantages when stabilizing biological samples which comprise only a small amount of cells or which may only be suspected of containing cells such as e.g. plasma, serum, urine, saliva, synovial fluids, amniotic fluid, lachrymal fluid, ichors, lymphatic fluid, liquor, cerebrospinal fluid and the like.
  • a method suitable for stabilizing the extracellular nucleic acid population comprised in a cell-containing sample, preferably a blood sample is provided, by contacting the sample with a cell-containing sample, preferably a blood sample.
  • the risk is reduced that the extracellular nucleic acid population is contaminated with intracellular nucleic acids, in particular fragmented genomic DNA originating from contained cells, e.g. from damaged or dying cells and/or the degradation of nucleic acids present in the sample is reduced, respectively inhibited.
  • This has the effect that the composition of the extracellular nucleic acid population comprised in said sample is substantially preserved, respectively stabilized.
  • extracellular nucleic acids or “extracellular nucleic acid” as used herein, in particular refers to nucleic acids that are not contained in cells. Respective extracellular nucleic acids are also often referred to as cell-free nucleic acids. These terms are used as synonyms herein. Hence, extracellular nucleic acids usually are present exterior of a cell or exterior of a plurality of cells within a sample.
  • extracellular nucleic acids refers e.g. to extracellular RNA as well as to extracellular DNA. Examples of typical extracellular nucleic acids that are found in the cell-free fraction (respectively portion) of biological samples such as body fluids such as e.g.
  • blood plasma include but are not limited to mammalian extracellular nucleic acids such as e.g. extracellular tumor-associated or tumor-derived DNA and/or RNA, other extracellular disease-related DNA and/or RNA, epigenetically modified DNA, fetal DNA and/or RNA, small interfering RNA such as e.g. miRNA and siRNA, and non-mammalian extracellular nucleic acids such as e.g. viral nucleic acids, pathogen nucleic acids released into the extracellular nucleic acid population e.g. from prokaryotes (e.g. bacteria), viruses, eukaryotic parasites or fungi.
  • mammalian extracellular nucleic acids such as e.g. extracellular tumor-associated or tumor-derived DNA and/or RNA, other extracellular disease-related DNA and/or RNA, epigenetically modified DNA, fetal DNA and/or RNA, small interfering RNA such as e.g. miRNA and siRNA, and non-m
  • the extracellular nucleic acid is obtained from respectively is comprised in a body fluid as cell-containing biological sample such as e.g. blood, plasma, serum, saliva, urine, liquor, cerebrospinal fluid, sputum, lachrymal fluid, sweat, amniotic or lymphatic fluid.
  • a body fluid such as e.g. blood, plasma, serum, saliva, urine, liquor, cerebrospinal fluid, sputum, lachrymal fluid, sweat, amniotic or lymphatic fluid.
  • extracellular nucleic acids that are obtained from circulating body fluids as circulating extracellular or circulating cell-free nucleic acids.
  • extracellular nucleic acid in particular refers to mammalian extracellular nucleic acids, preferably disease-associated or disease-derived extracellular nucleic acids such as tumor-associated or tumor-derived extracellular nucleic acids, extracellular nucleic acids released due to inflammations or injuries, in particular traumata, extracellular nucleic acids related to and/or released due to other diseases, or extracellular nucleic acids derived from a fetus.
  • extracellular nucleic acids” or “extracellular nucleic acid” as described herein also refers to extracellular nucleic acids obtained from other samples, in particular biological samples other than body fluids. Usually, more than one extracellular nucleic acid is comprised in a sample.
  • a sample comprises more than one kind or type of extracellular nucleic acids.
  • extracellular nucleic acid population refers to the collective of different extracellular nucleic acids that are comprised in a cell-containing sample.
  • a cell-containing sample usually comprises a characteristic and thus unique extracellular nucleic acid population.
  • the type, kind and/or the amount of one or more extracellular nucleic acids comprised in the extracellular nucleic acid population of a specific sample are important sample characteristics.
  • changes in the extracellular nucleic acid population with respect to the quantity, the quality and/or the composition of the comprised extracellular nucleic acids, in particular changes attributable to an increase of released genomic DNA are over the stabilization period considerably reduced (preferably by at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%) compared to an unstabilized sample or a corresponding sample that is e.g. stabilized by EDTA in case of a blood sample or a sample derived from blood.
  • At least one apoptosis inhibitor is used for stabilizing the sample.
  • the apoptosis inhibitor alone is effective in stabilizing a cell-containing sample and to substantially preserve the extracellular nucleic acid population from changes in its composition in particular arising from contaminations with fragmented genomic DNA.
  • the sample can be contacted with the apoptosis inhibitor, e.g. by adding the apoptosis inhibitor to the sample or vice versa.
  • the at least one apoptosis inhibitor present in the resulting mixture supports the stabilization of cells contained in the sample and inhibits the degradation of nucleic acids comprised in the sample thereby substantially preserving the extracellular nucleic acid population.
  • apoptosis inhibitor refers to a compound whose presence in a cell-containing biological sample provides a reduction, prevention and/or inhibition of apoptotic processes in the cells and/or makes the cells more resistant to apoptotic stimuli.
  • Apoptosis inhibitors include but are not limited to proteins, peptides or protein- or peptide-like molecules, organic and inorganic molecules.
  • Apoptosis inhibitors include compounds that act as metabolic inhibitors, inhibitors of nucleic acid degradation respectively nucleic acid pathways, enzyme inhibitors, in particular caspase inhibitors, calpain inhibitors and inhibitors of other enzymes involved in apoptotic processes. Respective apoptosis inhibitors are listed in Table 1.
  • the at least one apoptosis inhibitor that is used for stabilizing the cell-containing biological sample is selected from the group consisting of metabolic inhibitors, caspase inhibitors and calpain inhibitors. Suitable examples for each class are listed in Table 1 in the respective category.
  • the apoptosis inhibitor is cell-permeable.
  • apoptosis inhibitors either from the same or a different class of apoptosis inhibitors, respectively to use a combination of different apoptosis inhibitors which inhibit apoptosis either by the same or a different working mechanism.
  • the apoptosis inhibitor is a caspase inhibitor.
  • caspase inhibitors Members of the caspase gene family play a significant role in apoptosis.
  • the substrate preferences or specificities of individual caspases have been exploited for the development of peptides that successfully compete caspase binding.
  • caspase-specific peptides e.g. aldehyde, nitrile or ketone compounds.
  • E.g. fluoromethyl ketone (FMK) derivatized peptides such as Z-VAD-FMK act as effective irreversible inhibitors with no added cytotoxic effects.
  • FMK fluoromethyl ketone
  • Further suitable caspase inhibitors are synthesized with a phenoxy group at the C-terminus.
  • An example is Q-VD-OPh which is a cell permeable, irreversible broad-spectrum caspase inhibitor that is even more effective in preventing apoptosis than Z-VAD-FMK.
  • the caspase inhibitor is a pancaspase inhibitor and thus is a broad spectrum caspase inhibitor.
  • the caspase inhibitor comprises a modified caspase-specific peptide.
  • said caspase-specific peptide is modified by an aldehyde, nitrile or ketone compound.
  • the caspase specific peptide is modified preferably at the carboxyl terminus with an O-Phenoxy or a fluoromethyl ketone (FMK) group.
  • the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-VAD(OMe)-FMK.
  • Z-VAD(OMe)-FMK a pancaspase inhibitor
  • Q-VD-OPh which is a broad spectrum inhibitor for caspases
  • Q-VD-OPh is cell permeable and inhibits cell death by apoptosis.
  • Q-VD-OPh is not toxic to cells even at extremely high concentrations and consists of a carboxy terminal phenoxy group conjugated to the amino acids valine and aspartate.
  • caspase inhibitors that is used as apoptosis inhibitor for stabilizing the cell-containing sample is one which acts upon one or more caspases located downstream in the intracellular cell death pathway of the cell, such as caspase-3.
  • the caspase inhibitor is an inhibitor for one or more caspases selected from the group consisting of caspase-3, caspase-8, caspase-9, caspase-10 and caspase-12. It is also within the scope of the present invention to use a combination of caspase inhibitors.
  • the mixture that is obtained after contacting the biological sample with the at least one apoptosis inhibitor may comprise the apoptosis inhibitor (or combination of apoptosis inhibitors) in a concentration selected from the group of at least 0.01 ⁇ M, at least 0.05 ⁇ M, at least 0.1 ⁇ M, at least 0.5 ⁇ M, at least 1 ⁇ M, at least 2.5 ⁇ M or at least 3.5 ⁇ M. Of course, also higher concentrations can be used.
  • Suitable concentration ranges for the apoptosis inhibitor(s) when mixed with the cell-containing biological sample include but are not limited to 0.01 ⁇ M to 100 ⁇ M, 0.05 ⁇ M to 100 ⁇ M, 0.1 ⁇ M to 50 ⁇ M, 0.5 ⁇ M to 50 ⁇ M, 1 ⁇ M to 40 ⁇ M, more preferably 1 ⁇ M to 30 ⁇ M or 2.5 ⁇ M to 25 ⁇ M.
  • concentrations were found to be more effective, however, good stabilizing results were also achieved at lower concentrations. Hence, an efficient stabilization is also achieved at lower concentrations e.g.
  • concentrations apply to the use of a single apoptosis inhibitor as well as to the use of a combination of caspase inhibitors. If a combination of caspase inhibitors is used, the concentration of an individual apoptosis inhibitor that is used in said mixture of apoptosis inhibitors may also lie below the above mentioned concentrations, if the overall concentration of the combination of apoptosis inhibitors fulfils the above mentioned features.
  • a lower concentration that still efficiently stabilizes the cells and/or reduce the degradation of nucleic acids in present in the sample has the advantage that the costs for stabilisation can be lowered.
  • Lower concentrations can be used e.g. if the apoptosis inhibitor is used in combination with one or more stabilizers as described herein.
  • the aforementioned concentrations are in particular suitable when using a caspase inhibitor, in particular a modified caspase specific peptide such as Q-VD-OPh and/or Z-VAD(OMe)-FMK as apoptosis inhibitor.
  • the above mentioned concentrations are e.g. very suitable for stabilizing whole blood, in particular 10 ml blood.
  • Suitable concentration ranges for other apoptosis inhibitors and/or for other cell-containing biological samples can be determined by the skilled person using routine experiments, e.g. by testing the apoptosis inhibitors, respectively the different concentrations in the test assays described in the examples.
  • the apoptosis inhibitor will, in an effective amount, decrease or reduce apoptosis in a cell-containing biological sample by at least 25 percent, at least 30 percent, at least 40 percent, at least 50 percent, preferably, by at least 75 percent, more preferably, by at least 85 percent as compared to a control sample which does not contain a respective apoptosis inhibitor.
  • At least one hypertonic agent is used for stabilizing the sample, wherein the used hypertonic agent stabilizes cells comprised in the sample.
  • the hypertonic agent alone is effective in stabilizing a cell-containing sample and substantially preserving the composition of the extracellular nucleic acid population comprised therein.
  • the hypertonic agent induces cell shrinking by mild hypertonic effects (osmosis), thereby increasing the cell stability. Therefore, the cells are less prone to e.g. mechanically induced cell damage.
  • the sample can be contacted with the hypertonic agent, e.g. by adding the hypertonic agent to the sample or vice versa.
  • the hypertonic agent present in the resulting mixture in particular is suitable for stabilizing cells contained in the sample, thereby reducing the amount of intracellular nucleic acids, in particular genomic DNA that is released from damaged cells.
  • the extracellular nucleic acid population is substantially preserved and the risk of contaminating respectively diluting the extracellular nucleic acids with intracellular nucleic acids, in particular genomic DNA, is reduced.
  • the hypertonic agent is sufficiently osmotically active to induce cell shrinking (the cells release water), however, without damaging the cells i.e. without inducing or promoting cell lysis, respectively cell rupture.
  • the hypertonic agent preferably has a mild osmotic effect.
  • it is desirous that interactions between the hypertonic agent and the sample are predominantly limited to the cell stabilization effect basically in order to avoid unwanted side effects.
  • an uncharged hypertonic agent is used.
  • Using an uncharged hypertonic agent has the advantage that even though the cells shrink respectively are stabilized due to the osmotic effect of the hypertonic agent, interactions between the hypertonic agent and other compounds comprised in the sample are limited compared to the use of a charged hypertonic agent.
  • the hypertonic agent is a hydroxylated organic compound and accordingly, carries at least one hydroxyl group.
  • the hydroxylated organic compound comprises at least two hydroxyl groups.
  • the hydroxylated organic compound is a polyol.
  • the polyol comprises 2 to 10 hydroxyl groups, preferably 3 to 8 hydroxyl groups.
  • the hydroxylated organic compound may comprise 2 to 12 carbon atoms, preferably 3 to 8 and can be a cyclic or linear molecule, branched or un-branched; it can be saturated or unsaturated; aromatic or non-aromatic.
  • the hydroxylated organic compound is a hydroxy-carbonyl compound.
  • a hydroxy-carbonyl compound is a compound possessing one or more hydroxy (OH) groups and one or more carbonyl groups.
  • Hydroxylated organic compounds may include but are not limited to hydroxylated ketone compounds and carbohydrates, or compounds derived therefrom.
  • the hydroxylated organic compound is a polyalcohol, in particular a sugar alcohol.
  • hydroxylated organic compounds include but are not limited to carbohydrates such as glucose, raffinose, succrose, fructose, alpha-d-lactose monohydrate, inositol, maltitol, mannitol, dihydroxyacetone, alcohols such as glycerol, erythritol, mannitol, sorbitol, volemitol, or sugar alcohols. Suitable examples are also listed in the table below. It is also within the scope of the present invention to use combinations of respective hydroxylated organic compounds.
  • the polyols and sugar alcohols listed above may be replaced by alcohols with less hydroxyl groups (e.g., hexane-1,2,3,4,5-pentol, pentane-1,2,3,4-tetraol).
  • the hydroxylated organic compound is no alcohol having 1 to carbon atoms and carrying only one hydroxyl group.
  • alcohols with only one hydroxyl group are excluded as hydroxylated organic compound.
  • the hydroxylated organic compound that can be used as stabilizer according to the present invention preferably is water-soluble and non-toxic to the cells comprised in the biological sample to be stabilized.
  • the hydroxylated organic compound does not induce or support the lysis of the cells contained in the biological sample and accordingly, preferably does not function as a detergent or as cell membrane dissolving agent.
  • a suitable hydroxylated organic compound according to the present invention achieves a stabilizing effect of the cell-containing sample by improving the preservation of the composition of the extracellular nucleic acid population as can be e.g. tested by the assays described in the example section.
  • Adding a hydroxylated organic compound to a cell-containing biological sample such as e.g. whole blood increases the concentration of said hydroxylated organic compound in the cell-free portion respectively fraction (e.g. the blood plasma) and thus forces blood cells to release water into the plasma as a result of an osmotic (hypertonic) effect.
  • a hydroxylated organic compound is used which is closely related to a product of the cell metabolism but preferably can not be utilized by the cells.
  • cells contained in the biological sample are essentially impermeable for the hypertonic agent that is used for stabilization.
  • the hypertonic agent which preferably is a hydroxylated organic compound as described in detail above, is essentially cell impermeable.
  • Essentially cell impermeable in this respect in particular means that the concentration of the hypertonic agent, which preferably is a hydroxylated organic compound, is substantially higher in the extracellular portion of the sample than inside the cells contained in the biological sample that is stabilized according to the teachings of the present invention.
  • the hypertonic agent which preferably is a hydroxylated organic compound, is non-toxic, so that the cell viability is not compromised. This is preferred to avoid disturbing influences on the cell metabolism.
  • the hypertonic agent is dihydroxyacetone (DHA).
  • DHA is a carbohydrate and usually serves as tanning substance in self-tanning lotions.
  • DHA surprisingly has a remarkable stabilizing effect on cell-containing biological samples, in particular whole blood samples and samples derived from whole blood such as blood plasma or serum.
  • DHA does naturally not occur in mammalian cells except for the phosphoric acid ester of DHA, dihydroxyacetone-phosphat, an intermediate product of glycolysis. Thus DHA is not expected to be actively transported or to diffuse into blood cells.
  • the hypertonic agent is not dihydroxyaceton-phosphate.
  • the mixture that is obtained when contacting the cell-containing biological sample with the at least one hypertonic agent may comprise the hypertonic agent or mixture of hypertonic agents in a concentration of at least 0.05M, preferably 0.1M, preferably at least 0.2M, more preferred at least 0.25M. Of course, also higher concentrations can be used. Suitable concentration ranges for the hypertonic agent can be selected from 0.05M to 2M, 0.1M to 1.5M, 0.15M to 0.8M, 0.2M to 0.7M or 0.1M to 0.6M. Respective concentrations are particularly suitable when using a hydroxylated organic compound, e.g. a carbohydrate such as dihydroxyacetone as hypertonic agent. The above mentioned concentrations are e.g.
  • Suitable concentration ranges for other hypertonic agents and/or other cell-containing biological samples can also be determined by the skilled person using routine experiments, e.g. by testing the hypertonic agents, respectively different concentrations thereof in the test assays described in the examples.
  • R1 is a hydrogen residue or an alkyl residue, preferably a C1-C5 alkyl residue, more preferred a methyl 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
  • R4 is an oxygen, sulphur or selenium residue.
  • a compound according to formula 1 described above is effective in achieving a remarkable stabilizing effect and in substantially preserving the composition of the extracellular nucleic acid population in the stabilized sample. Also a mixture of one or more compounds according to formula 1 can be used for stabilization.
  • the hydrocarbon residues R2 and/or R3 can be selected independently of one another from the group comprising alkyl, including short chain alkyl and long-chain alkyl, alkenyl, alkoxy, long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl, alkylsilyloxy, alkylene, alkenediyl, arylene, carboxylates and carbonyl.
  • General groups, for instance alkyl, alkoxy, aryl etc. are claimed and described in the description and the claims.
  • the following groups are used within the generally described groups within the scope of the present invention:
  • the chain length n of R2 and/or R3 can in particular have the values 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20.
  • R2 and R3 have a length of the carbon chain of 1-10.
  • the chain length n can in particular have the values 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
  • R2 and R3 have a length of the carbon chain of 1-5 and in this case the chain length can in particular have the values 1, 2, 3, 4 and 5.
  • Particularly preferred is a chain length of 1 or 2 for R2 and R3.
  • the chain length n of R1 preferably has the value 1,2,3,4 or 5. Particularly preferred is a chain length of 1 or 2 for R1.
  • R4 preferably is oxygen
  • the compound according to formula 1 is a N,N-dialkyl-carboxylic acid amide.
  • Preferred R1, R2, R3 and R4 groups are described above.
  • the compound is selected from the group consisting of N,N-dimethylacetamide; N,N-diethylacetamide; N,N-dimethylformamide and N,N-diethylformamide.
  • N,N-dialkylpropanamides such as N,N-dimethylpropanamide as is shown in the examples.
  • the substance according to formula 1 is N,N-dimethlylacetamide (DMAA).
  • DMAA N,N-dimethlylacetamide
  • the mixture that is obtained when contacting the cell-containing biological sample with a compound according to formula 1 or a mixture of respective compounds may comprise said compound or mixture of compounds in a final concentration of at least 0.1%, at least 0.5%, at least 0.75%, at least 1%, at least 1.25% or at least 1.5%.
  • a suitable concentration range includes but is not limited to 0.1% up to 50%.
  • Preferred concentration ranges can be selected from the group consisting of 0.1% to 30%, 0.1% to 20%, 0.1% to 15%, 0.1% to 10%, 0.1% to 7.5%, 0.1% to 5%, 1% to 30%, 1% to 20%, 1% to 15%, 1% to 10%, 1% to 7.5%, 1% to 5%; 1.25% to 30%, 1.25% to 20%, 1.25% to 15%, 1.25% to 10%, 1.25% to 7.5%, 1.25% to 5%; 1.5% to 30%, 1.5% to 20%, 1.5% to 15%, 1.5% to 10%, 1.5% to 7.5% and 1.5% to 5%.
  • Respective concentrations are particularly suitable when using a N,N-dialkyl-carboxylic acid amide, e.g.
  • N,N-dimethylacetamide, N,N-diethylacetamide, N,N-diethylformamide or N,N-diemethylformamide or N,N-dimethylpropanamide as stabilizing agent.
  • concentrations are e.g. very suitable for stabilizing whole blood or blood products such as plasma.
  • Suitable concentration ranges for other compounds according to formula 1 and/or other cell-containing biological samples can also be determined by the skilled person using routine experiments, e.g. by testing the compound, respectively different concentrations thereof in the test assays described in the examples.
  • the compound according to formula 1 is used in combination with a chelating agent for stabilizing the cell containing sample.
  • a chelating agent can be used as anticoagulant when stabilizing a blood sample or a sample derived from blood such as e.g. plasma or serum. Suitable chelating agents and concentration ranges are provided below.
  • a method suitable for stabilizing a cell-containing sample preferably a blood sample is provided, wherein said method comprises contacting the sample with
  • the apoptosis inhibitor and the hypertonic agent which both alone are already effective in stabilizing a cell-containing sample (see above and examples), are used in combination.
  • the stabilization effect can be increased and/or the concentration of the individual components (the apoptosis inhibitor and/or the hypertonic agent) may also be reduced while still efficiently preserving the extracellular nucleic acid population in the sample, and in particular avoiding, respectively reducing the contamination by intracellular nucleic acids in particular fragmented genomic DNA that is released from damaged or decaying cells contained in the sample.
  • using a respective combination is particularly effective in stabilizing a cell-containing sample, even very complex samples such as a whole blood sample.
  • apoptosis inhibitors in combination with different hypertonic agents.
  • Suitable and preferred embodiments of the apoptosis inhibitor and the hypertonic agent as well as suitable and preferred concentrations of the respective agents suitable for achieving an efficient stabilization of the sample are described in detail above in conjunction with the embodiments, wherein either an apoptosis inhibitor or a hypertonic agent is used to stabilize the cell-containing biological sample. It is referred to the above disclosure which also applies to the embodiment, wherein an apoptosis inhibitor is used in combination with a hypertonic agent.
  • At least one caspase inhibitor preferably a modified caspase specific peptide, preferably modified at the C-terminus with an O-phenoxy group such as Q-VD-OPh
  • at least one hydroxylated organic compound e.g. a carbohydrate, such as dihydroxyacetone or a polyol, as hypertonic agent.
  • a respective combination is remarkably effective in stabilizing a cell-containing biological sample, in particular a whole blood sample, at room temperature for more than 3 days and even for 6 days.
  • a combination of stabilizing agents which comprises at least one apoptosis inhibitor, at least one hypertonic agent and/or at least one compound according to formula 1 as defined above.
  • respective combinations include (1) a combination of at least one apoptosis inhibitor and at least one compound according to formula 1 as defined above, (2) a combination of at least one hypertonic agent and at least one compound according to formula 1 as defined above or (3) a combination of all three stabilizing agents, i.e. at least one apoptosis inhibitor, at least one hypertonic agent and at least one compound according to formula 1 as defined above.
  • a respective combination may also comprise additional additives that enhance the stabilizing effect such as e.g. anticoagulants and chelating agents.
  • the combination of stabilizing agents comprises a caspase inhibitor and an anticoagulant, preferably a chelating agent such as EDTA.
  • a chelating agent such as EDTA.
  • Respective combinations can be according to a fifth sub-aspect advantageously used in a method suitable for stabilizing an extracellular nucleic acid population comprised in a cell-containing sample according to the first aspect of the present invention.
  • the stabilizing effect observed with combinations of stabilizing agents is stronger than the effect observed for any of the individual stabilizing agents when used alone and/or allows to use lower concentrations, thereby making combinatorial use of stabilizing agents an attractive option.
  • apoptosis inhibitor Suitable and preferred embodiments of the apoptosis inhibitor, the hypertonic agent and the compound according to formula 1 defines above as well as suitable and preferred concentrations of the respective agents suitable for achieving an efficient stabilization of the sample are described in detail above in conjunction with the embodiments, wherein either an apoptosis inhibitor, a hypertonic agent or a compound according to formula 1 is used to stabilize the cell-containing biological sample.
  • extracellular nucleic acids are usually not present “naked” in the sample but are e.g. stabilized to a certain extent by being released protected in complexes or by being contained in vesicles and the like. This has the effect that extracellular nucleic acids are already to a certain extent stabilized by nature and thus, are usually not degraded rapidly by nucleases in cell-containing samples such as whole blood, plasma or serum.
  • one of the primary problems is the dilution, respectively the contamination of the extracellular nucleic acid population by intracellular nucleic acids, in particular fragmented genomic DNA, that originates from damaged or dying cells that are contained in the sample.
  • the stabilization technology according to the present invention is of particular advantage in this respect because it not only substantially preserves the extracellular nucleic acids present in the sample and e.g. inhibits degradation of the comprised extracellular nucleic acids (preferably at least by 60%, at least by 70%, at least by 75%, at least by 80%, at least by 85%, at least by 90% or most preferably at least by 95% over the stabilization period compared to an unstabilized sample or an EDTA stabilized sample) but furthermore, efficiently reduces the release of genomic DNA from cells contained in the sample and/or reduces the fragmentation of respective genomic DNA.
  • using the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 for stabilizing the cell-containing sample according to the teachings of the present invention has the effect that the increase of DNA that results from a release of DNA from cells contained in the sample is reduced compared to a non-stabilized sample.
  • said release of genomic DNA is reduced by at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 10-fold, at least 12-fold, at least 15-fold, at least 17-fold or at least 20-fold over the stabilization period compared to the non-stabilized sample or a corresponding sample that is stabilized with EDTA (in particular in case of a blood sample or a sample derived from blood such as plasma or serum).
  • said release of genomic DNA is reduced by at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% over the stabilization period compared to the non-stabilized sample or a corresponding sample that is stabilized with EDTA (in particular in case of a blood sample or a sample derived from blood such as plasma or serum).
  • the release of DNA can be determined e.g. by quantifying the ribosomal 18S DNA as is described herein in the example section.
  • standard EDTA stabilized blood samples show a 40-fold increase of DNA determined e.g. at day 6 of storage at room temperature in a respective assay (see FIG. 2 b ).
  • the stabilization achievable with the teachings of the present invention remarkably reduces this release of DNA even down to e.g. a maximum of 4-fold.
  • the extracellular nucleic acid population contained in the sample is considerably stabilized compared to samples stabilized in standard EDTA tubes.
  • the stabilization effect that is achieved with the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as taught by the present invention results in that the release of DNA from cells contained in the sample is at least reduced to a maximum of 10-fold, preferably 7-fold, more preferably 5-fold and most preferably is at least reduced to a maximum of 4-fold, as is e.g. determinable in the 18S DNA assay described in the examples.
  • an effective stabilization of the extracellular nucleic acid population is achievable for a period of at least up to 6 days.
  • the DNA release can be reduced at least to a maximum of two-fold as e.g. determinable in the 18S DNA assay described in the examples.
  • the DNA release can be reduced to 2fold or less up to three days of storage when using the stabilizing methods according to the present invention.
  • This is a remarkable improvement in the stabilization of the extracellular nucleic acid population compared to the prior art methods. This significantly enhances the accuracy of any subsequent tests. In certain cases, for example if the sample material has to be transported for long distances or stored for longer periods e.g.
  • the process according to the invention makes it possible for the first time for these tests to be carried out after such a period of time.
  • the samples may also be further processed earlier, if desired. It is not necessary to make use of the full achievable stabilization period.
  • the stabilization that is achieved with the present invention reduces variations in the extracellular nucleic acid population that may result from a different handling/processing of the samples (e.g. storage conditions and periods) after they were collected. This greatly improves the standardization of handling and molecular analysis.
  • additives may be used in addition to the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as defined above in order to further stabilize the cell-containing sample.
  • suitable additives may also contribute to the stabilization effect may also depend on the type of cell-containing sample to be stabilized.
  • an anticoagulant e.g. selected from the group consisting of heparin, ethylenediamine tetraacetic acid, citrate, oxalate, and any combination thereof.
  • the anticoagulant is a chelating agent.
  • a chelating agent is an organic compound that is capable of forming coordinate bonds with metals through two or more atoms of the organic compound.
  • Chelating agents according to the present invention include, but are not limited to diethylenetriaminepentaacetic acid (DTPA), ethylenedinitrilotetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA) and N,N-bis(carboxymethyl)glycine (NTA).
  • EDTA is used.
  • the term “EDTA” indicates inter alia the EDTA portion of an EDTA compound such as, for example, K 2 EDTA, K 3 EDTA or Na 2 EDTA.
  • a chelating agent such as EDTA also has the advantageous effect that nucleases such as DNases are inhibited, thereby e.g. preventing a degradation of extracellular DNA by DNases.
  • EDTA used/added in higher concentrations is capable of reducing the release of intracellular nucleic acids, in particular genomic DNA from the cells thereby supporting the stabilizing effect that is achieved by the apoptosis inhibitor, the hypertonic agent and/or the at least one compound according to formula 1.
  • EDTA alone is not capable of efficiently inhibiting the fragmentation of e.g. genomic DNA that is released from the cells contained in the sample. Thus, EDTA does not achieve a sufficient stabilization effect.
  • the concentration of the chelating agent, preferably EDTA, in the biological sample that is mixed with one or more of the stabilizing compounds described above is in the range selected from the group consisting of 0.05 mM to 100 mM, 0.05 mM to 50 mM, 0.1 mM to 30 mM, 1 mM to 20 mM and 2 mM to 15 mM after the contacting step.
  • Respective concentrations are particularly effective when stabilising blood, plasma and/or serum samples, in particular 10 ml blood samples.
  • Additional additives can also be used in order to further support the stabilization of the cell-containing sample, respectively support the preservation of the extracellular nucleic acid population.
  • respective additives include but are not limited to nuclease inhibitors, in particular RNase and DNase inhibiting compounds.
  • RNase inhibitors include but are not limited to anti-nuclease antibodies or ribonucleoside-vanadyl-complexes.
  • the cell-containing biological sample which preferably is a blood sample or a sample derived from blood such as plasma or serum, is contacted with:
  • the components of the stabilizing composition can be comprised, respectively dissolved in a buffer, e.g. a biological buffer such as MOPS, TRIS, PBS and the like.
  • a buffer e.g. a biological buffer such as MOPS, TRIS, PBS and the like.
  • the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as defined above as well as the optionally present further additives can be e.g. present in a device, preferably a container, for collecting the sample or can be added to a respective collection device immediately prior to collection of the biological sample; or can be added to the collection device immediately after the sample was collected therein. It is also within the scope of the present invention to add the stabilizing agent(s) and optionally, the further additive(s) separately to the cell containing biological sample. However, for the ease of handling, it is preferred that the one or more stabilizing agents and optionally the further additives are provided in one composition.
  • the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as described above and optionally the further additive(s) are present in the collection device prior to adding the sample. This ensures that the cell-containing biological sample is immediately stabilized upon contact with the stabilizing agent(s).
  • the stabilisation agent(s) are present in the container in an amount effective to provide the stabilisation of the amount of cell containing sample to be collected, respectively comprised in said container.
  • the sample can be mixed with the stabilization agent(s) directly after and/or during collection of the sample thereby providing a stabilized sample.
  • the sample is mixed with the stabilization agent(s) directly after and/or during the collection of the sample. Therefore, preferably, the stabilization agent(s) and additives described above are provided in form of a stabilizing composition.
  • said stabilizing composition is provided in liquid form. It can be e.g. pre-filled in the sample collection device so that the sample is immediately stabilized during collection.
  • the stabilizing composition is contacted with the cell-containing sample in a volumetric ratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. It is a particular advantage of the teachings of the present invention that stabilization of a large sample volume can be achieved with a small volume of the stabilizing composition. Therefore, preferably, the ratio of stabilizing composition to sample lies in a range from 1:2 to 1:7, more preferred 1:3 to 1:5.
  • cell-containing sample refers to a sample which comprises at least one cell.
  • the cell-containing sample may comprise at least two, at least 10, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 1500, at least 2000 or at least 5000 cells.
  • cell-containing samples comprising considerably more cells are encompassed by said term and can be stabilized with the teachings according to the present invention.
  • the term “cell-containing sample” also refers to and thus encompasses cell-depleted samples, including cell-depleted samples that are commonly referred to as “cell-free” such as e.g. blood plasma as respective samples often include residual cells.
  • cell-free samples such as blood plasma comprise residual amounts of cells which accordingly, pose a risk that the extracellular nucleic acid population becomes contaminated with intracellular nucleic acids released from said residual cells. Therefore, respective cell-depleted and “cell-free” samples are according to one embodiment also encompassed by the term “cell-containing sample”.
  • the “cell-containing sample” may comprise large amounts of cells, as is the case e.g. with whole blood, but may also only comprise merely minor amounts of cells.
  • the term “cell containing sample” also encompasses samples that may only be suspected of or pose a risk of containing cells.
  • the method according to the present invention has considerable advantages as these residual cells may also result in a undesired contamination of the comprised extracellular nucleic acids.
  • Using the stabilizing technology of the present invention also ensures that respective samples which only comprise residual amounts of cells or are merely suspected of or pose a risk of residual amounts of cells, are efficiently stabilized as is also described in detail above.
  • Using the stabilizing method according to the present invention has the advantage that irrespective of the composition of the sample and the number of cells contained therein, the extracellular nucleic acid population contained therein is substantially preserved, respectively stabilized, thereby allowing for standardizing the subsequent isolation and/or analysis of the contained extracellular nucleic acids.
  • the cell-containing biological sample is selected from the group consisting of whole blood, samples derived from blood, plasma, serum, sputum, lachrymal fluid, lymphatic fluid, urine, sweat, liquor, cerebrospinal fluid, ascites, milk, stool, bronchial lavage, saliva, amniotic fluid, nasal secretions, vaginal secretions, semen/seminal fluid, wound secretions, and cell culture supernatants and supernatants obtained from other swab samples.
  • the cell-containing biological sample is a body fluid, a body secretion or body excretion, preferably a body fluid, most preferably whole blood, plasma or serum.
  • the cell-containing biological sample comprises extracellular nucleic acids.
  • the cell-containing biological sample is a non-fluid sample derived from a human or animal, such as e.g. stool, tissue or a biopsy sample.
  • a human or animal such as e.g. stool, tissue or a biopsy sample.
  • Other examples of cell-containing biological samples that can be stabilized with the method according to the present invention include but are not limited to biological samples cell suspensions, cell cultures, supernatant of cell cultures and the like, which comprise extracellular nucleic acids.
  • the samples can be kept at room temperature or even at elevated temperatures e.g. up to 30° C. or up to 40° C.
  • a stabilization effect is achieved for at least two days, preferably at least three days; more preferred at least one day to six days, most preferred for at least one day to at least seven days at room temperature.
  • the samples that were stabilized according to the method of the present invention were not substantially compromised when stored for 3 days at room temperature.
  • the stabilisation efficiency is particularly good during this time period.
  • the extraordinary long stabilisation times and stabilisation efficiencies that are achievable with the method according to the present invention provides an important safety factor.
  • compositions according to the present invention allow the stabilization also of large volumes of biological samples with small volumes of added substances because the additives that are used according to the teachings of the present invention are highly active.
  • This is an important advantage because the size/volume of the sample poses considerable restrains on the subsequent isolation procedure in particular when intending to use automated processes for isolating the extracellular nucleic acids contained in the samples.
  • extracellular nucleic acids are often only comprised in small amounts in the contained sample.
  • processing larger volumes of a cell-containing sample such as e.g. a blood sample has the advantage that more circulating nucleic acids can be isolated from the sample and thus are available for a subsequent analysis.
  • the stabilization of the biological sample may either be followed directly by techniques for analysing nucleic acids, or the nucleic acids may be purified from the sample.
  • the sample that was stabilized according to the method of the present invention can be analysed in a nucleic acid analytic and/or detection method and or may be further processed.
  • extracellular nucleic acid can be isolated from the stabilized sample and can then be analysed in a nucleic acid analytic and/or detection method or may be further processed.
  • a method for isolating extracellular nucleic acids from a cell-containing biological sample comprises the steps of:
  • the stabilization according to the present invention has the effect that the extracellular nucleic acid population contained in the sample is substantially preserved in the state it had shown at the time the biological sample was obtained, respectively drawn.
  • the usually observed high increase in nucleic acids that results from intracellular nucleic acids, in particular genomic DNA, more specifically fragmented genomic DNA, released from damaged or dying cells is efficiently reduced as is demonstrated in the examples. Therefore, the extracellular nucleic acids obtained from a respectively stabilized sample comprise fewer contaminations with intracellular nucleic acids originating from degraded or dying cells comprised in the sample and in particular comprise less amounts of fragmented genomic DNA compared to non-stabilized samples. Furthermore, the unique stabilization step allows to increase the amount of recoverable extracellular nucleic acids.
  • the stabilization method according to the present invention can be performed without the crosslinking of the sample. This is an important advantage over the use of cross-linking agents such as formaldehyde or formaldehyde releasers, as these reagents might reduce the recoverable amount of extracellular nucleic acids due to cross-linking.
  • the method according to the present invention improves the diagnostic and prognostic capability of the extracellular nucleic acids.
  • said stabilization allows the sample to be stored and/or handled, e.g. transported,—even at room temperature—for a prolonged period of time prior to separating the cells contained in the sample and/or prior to isolating the extracellular nucleic acids comprised therein in step b). With respect to the details of the stabilization, it is referred to the above disclosure which also applies here.
  • the cell-containing biological sample such as e.g. a whole blood sample is stabilized in step a) as is described in detail above using at least one apoptosis inhibitor, at least one hypertonic agent and/or at least one compound according to formula 1 as described above, preferably using at least two of these stabilizing agents and optionally, further additives.
  • apoptosis inhibitor e.g. a hypertonic agent
  • at least one compound according to formula 1 e.g. a whole blood sample
  • a caspaseinhibitor in combination with an anticoagulant, preferably a chelating agent as described above, for stabilizing whole blood samples.
  • the cells are separated from the remaining sample in order to obtain a cell-free, respectively cell-reduced or cell-depleted fraction of the sample which comprises the extracellular nucleic acids.
  • cells are removed from the cell-containing sample between step a) and step b).
  • This intermediate step is only optional and e.g. may be obsolete if samples are processed which merely comprise minor amounts of residual cells such as e.g. plasma or serum.
  • respective remaining cells or potentially remaining cells are removed as they might contaminate the extracellular nucleic acid population during isolation.
  • cells including residual cells, can be separated and removed e.g. by centrifugation, preferably high speed centrifugation, or by using means other than centrifugation, such as e.g. filtration, sedimentation or binding to surfaces on (optionally magnetic) particles if a centrifugation step is to be avoided.
  • centrifugation preferably high speed centrifugation
  • means other than centrifugation such as e.g. filtration, sedimentation or binding to surfaces on (optionally magnetic) particles if a centrifugation step is to be avoided.
  • Respective cell removal steps can also be easily included into an automated sample preparation protocol.
  • Respectively removed cells may also be processed further.
  • the cells can e.g. be stored and/or biomolecules such as e.g. nucleic acids or proteins can be isolated from the removed cells.
  • Extracellular nucleic acids are then isolated in step b), e.g. from the cell-free, respectively cell-depleted fraction, e.g. from supernatants, plasma and/or serum.
  • any known nucleic acid isolation method can be used that is suitable for isolating nucleic acids from the respective sample, respectively the cell-depleted sample.
  • Examples for respective purification methods include but are not limited to extraction, solid-phase extraction, silica-based purification, magnetic particle-based purification, phenol-chloroform extraction, chromatography, anion-exchange chromatography (using anion-exchange surfaces), electrophoresis, filtration, precipitation, chromatin immunoprecipitation and combinations thereof.
  • nucleic acids are isolated using a chaotropic agent and/or alcohol.
  • the nucleic acids are isolated by binding them to a solid phase, preferably a solid phase comprising silica or anion exchange functional groups.
  • Suitable methods and kits are also commercially available such as the QIAamp® Circulating Nucleic Acid Kit (QIAGEN), the Chemagic Circulating NA Kit (Chemagen), the NucleoSpin Plasma XS Kit (Macherey-Nagel), the Plasma/Serum Circulating DNA Purification Kit (Norgen Biotek), the Plasma/Serum Circulating RNA Purification Kit (Norgen Biotek), the High Pure Viral Nucleic Acid Large Volume Kit (Roche) and other commercially available kits suitable for extracting and purifying circulating nucleic acids.
  • QIAamp® Circulating Nucleic Acid Kit QIAGEN
  • Chemagic Circulating NA Kit Chemagen
  • the NucleoSpin Plasma XS Kit Macherey-Nagel
  • the Plasma/Serum Circulating DNA Purification Kit Norgen Biotek
  • the Plasma/Serum Circulating RNA Purification Kit Norgen Biotek
  • the High Pure Viral Nucleic Acid Large Volume Kit
  • nucleic acids that are comprised in the sample that is obtained after step a) or optionally obtained after the cells have been removed in the intermediate step are isolated, e.g. are isolated from the cell-free, respectively cell-depleted fraction.
  • total nucleic acids can be isolated from plasma or serum and the extracellular nucleic acids will be comprised as a portion in these extracted nucleic acids. If the cells are efficiently removed, the total nucleic acids isolated will predominantly comprise or even consist of extracellular nucleic acids. It is also within the scope of the present invention to isolate at least predominantly a specific target nucleic acid.
  • a target nucleic acid can be e.g. a certain type of nucleic acid, e.g.
  • RNA or DNA including mRNA, microRNA, other non-coding nucleic acids, epigenetically modified nucleic acids, and other nucleic acids. It is also within the scope of the present invention to e.g. digest the non-target nucleic acid using nucleases after isolation.
  • target nucleic acid also refers to a specific kind of nucleic acid, e.g. a specific extracellular nucleic acid that is known to be a certain disease marker.
  • the isolation of extracellular nucleic acids may also comprise the specific isolation of a respective target nucleic acid e.g. by using appropriate capture probes.
  • the term a target nucleic acid also refers to a nucleic acid having a certain length, e.g.
  • nucleic acid having a length of 2000 nt or less, 1000 nt or less or 500 nt or less. Isolating respective smaller target nucleic acids can be advantageous because it is known that extracellular nucleic acids usually have a smaller size of less than 2000 nt, usually less than 1000 nt and often even less than 500 nt.
  • the sizes, respectively size ranges indicated herein refer to the chain length. I.e. in case of DNA it refers to bp. Focusing the isolation, respectively purification, on respective small nucleic acids can increase the portion of extracellular nucleic acids obtained in the isolated nucleic acids.
  • the stabilization methods according to the present invention allow, in particular due to the inhibition of fragmentation of genomic, intracellular DNA, for a more efficient separation of such high molecular weight genomic DNA from the fragmented extracellular nucleic acid population, e.g., during the nucleic acid extraction procedure.
  • genomic DNA can be removed e.g. by size-selective recovery of DNA more efficiently than without the respective stabilization.
  • Suitable methods to achieve a respective selective isolation of the extracellular nucleic acid population e.g. by depleting the high molecular weight genomic DNA are well-known in the prior art and thus, need no further description here. E.g.
  • a size-selection method that depletes a sample of any nucleic acid larger than 1,000-10,000 nucleotides or base pairs.
  • genomic usually larger than >10,000 bp
  • extracellular nucleic acids usually ⁇ 1000 bp
  • known methods for selectively isolating extracellular nucleic acid from a biological sample could be applied. This also provides further opportunities in order to reduce the amount of intracellular nucleic acids in the isolated extracellular nucleic acid population.
  • the removal of genomic DNA during the nucleic acid extraction protocol could also supplement or even replace a separate high g-force centrifugation of a plasma sample before starting the nucleic acid extraction in order to remove residual cells.
  • Genomic DNA that is released from said residual cells is prevented from becoming massively degraded due to the stabilization according to the present invention, and accordingly, can be removed by size-selective isolation protocols.
  • This option is of particular advantage, as many clinical laboratories do not have a centrifuge capable of performing such a high g-force centrifugation or other means for removing in particular trace amounts of residual cells.
  • the isolated nucleic acids can then be analysed and/or further processed in a step c) using suitable assay and/or analytical methods.
  • they can be identified, modified, contacted with at least one enzyme, amplified, reverse transcribed, cloned, sequenced, contacted with a probe, be detected (their presence or absence) and/or be quantified.
  • Respective methods are well-known in the prior art and are commonly applied in the medical, diagnostic and/or prognostic field in order to analyse extracellular nucleic acids (see also the detailed description in the background of the present invention).
  • extracellular nucleic acids are isolated, optionally as part of total nucleic acid, total RNA and/or total DNA (see above), they can be analysed to identify the presence, absence or severity of a disease state including but not being limited to a multitude of neoplastic diseases, in particular premalignancies and malignancies such as different forms of cancers.
  • neoplastic diseases in particular premalignancies and malignancies such as different forms of cancers.
  • the isolated extracellular nucleic acids can be analysed in order to detect diagnostic and/or prognostic markers (e.g., fetal- or tumor-derived extracellular nucleic acids) in many fields of application, including but not limited to non-invasive prenatal genetic testing respectively screening, disease screening, pathogen screening, oncology, cancer screening, early stage cancer screening, cancer therapy monitoring, genetic testing (genotyping), infectious disease testing, injury diagnostics, trauma diagnostics, transplantation medicine or many other diseases and, hence, are of diagnostic and/or prognostic relevance.
  • the isolated extracellular nucleic acids are analyzed to identify and/or characterize a disease or a fetal characteristic.
  • the isolation method described herein may further comprise a step c) of nucleic acid analysis and/or processing. Therefore, according to one embodiment, the isolated extracellular nucleic acids are analysed in step c) to identify, detect, screen for, monitor or exclude a disease and/or at least one fetal characteristic.
  • the analysis/further processing of the nucleic acids can be performed using any nucleic acid analysis/processing method including, but not limited to amplification technologies, polymerase chain reaction (PCR), isothermal amplification, reverse transcription polymerase chain reaction (RT-PCR), quantitative real time polymerase chain reaction (Q-PCR), digital PCR, gel electrophoresis, capillary electrophoresis, mass spectrometry, fluorescence detection, ultraviolet spectrometry, hybridization assays, DNA or RNA sequencing, restriction analysis, reverse transcription, NASBA, allele specific polymerase chain reaction, polymerase cycling assembly (PCA), asymmetric polymerase chain reaction, linear after the exponential polymerase chain reaction (LATE-PCR), helicase-dependent amplification (HDA), hot-start polymerase chain reaction, intersequence-specific polymerase chain reaction (ISSR), inverse polymerase chain reaction, ligation mediated polymerase chain reaction, methylation specific polymerase chain reaction (MSP), multiplex polymerase chain
  • either or both of the isolating or analyzing steps b) and c) occurs at least one day up to 7 days after the sample has been collected, respectively stabilized according to the teachings of the present invention. Suitable time periods for which the sample, in particular a blood sample, respectively the extracellular nucleic acid population contained therein can be stabilized using the method according to the present invention are also described above and also apply here.
  • the isolation step is performed at least one day, at least 2 days, at least 3 days, at least 4 days, at least 5 days or at least 6 days after the sample was collected and stabilized according to the method according to the present invention.
  • either or both of the isolating or analyzing steps occur without freezing the sample and/or without the use of formaldehyde for preserving the cell-containing biological sample.
  • the biological sample is stabilized after the contact with the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as defined above, preferably in combination with a further additive such as an anticoagulant like EDTA.
  • An anticoagulant is preferably used when stabilizing blood or a sample derived from blood.
  • the respectively stabilized samples can be handled, e.g. stored and/or shipped at room temperature.
  • composition suitable for stabilizing the extracellular nucleic acid population in a biological sample comprising:
  • a respective stabilizing composition is particularly effective in stabilizing a cell-containing biological sample, in particular whole blood, plasma and/or serum by stabilizing the comprised cells and the comprised extracellular nucleic acids thereby substantially preserving, respectively stabilizing the extracellular nucleic acid population.
  • a respective stabilizing composition allows the storage and/or handling, e.g. shipping, of the sample, which preferably is whole blood, at room temperature for at least two, preferably at least three days without substantially compromising the quality of the sample, respectively the extracellular nucleic acid population contained therein. Of course, it is not mandatory to make use of the full possible stabilization period; the samples may also be processed earlier if desired. Contacting the biological sample with the stabilizing composition allows the sample to be stored, and or handled, e.g.
  • the time between the collection or stabilization of the sample and the nucleic acid extraction can vary without substantially affecting the population, respectively the composition of the extracellular nucleic acid population contained therein.
  • the stabilization composition is contacted with the sample immediately after or during collection of the sample.
  • the composition comprises at least one caspase inhibitor and at least one anticoagulant, preferably a chelating agent as described above. It may also comprise further stabilizing agents as described herein.
  • apoptosis inhibitor Suitable and preferred embodiments of the apoptosis inhibitor, the hypertonic agent and/or the compound according to formula 1 as well as suitable and preferred concentrations of the respective compounds are described in detail above in conjunction with the stabilization method. It is referred to the above disclosure which also applies with respect to the stabilization composition.
  • at least one caspase inhibitor preferably a modified caspase specific peptide, preferably modified at the C-terminus with an O-phenoxy group such as Q-VD-OPh
  • at least one hypertonic agent preferably a hydroxylated organic compound such as dihydroxyacetone.
  • Other suitable hydroxylated organic compounds are also described above, it is referred to the respective disclosure.
  • a respective combination is remarkably effective in stabilizing a cell-containing biological sample, in particular a blood sample.
  • the at least one compound according to formula 1 is a N,N-dialkyl-carboxylic acid amide.
  • Preferred R1, R2, R3 and R4 groups are described above.
  • the compound is selected from the group consisting of N,N-dimethylacetamide; N,N-diethylacetamide; N,N-dimethylformamide, N,N-diethylformamide and N,N-dimethylpropanamid.
  • Said compound can also be used in combination with an apoptosis inhibitor, preferably a caspase inhibitor (preferred embodiments are described above, it is referred to the above disclosure) and/or a hypertonic agent, preferably a hydroxycarbon compound (preferred embodiments are described above, it is referred to the above disclosure).
  • an apoptosis inhibitor preferably a caspase inhibitor
  • a hypertonic agent preferably a hydroxycarbon compound
  • the stabilization composition comprises further additives, e.g. an anticoagulant such as a chelating agent in particular if the composition is used for stabilizing whole blood, plasma or serum.
  • an anticoagulant such as a chelating agent in particular if the composition is used for stabilizing whole blood, plasma or serum.
  • the stabilizing composition consists essentially of the mentioned stabilizers and optional additives and optionally, buffering agents.
  • the stabilizing composition stabilizes the sample and thus, does not promote the lysis and/or disruption of the cells contained in the sample.
  • the stabilizing composition may reduce the damage of the cells comprised in the sample as can be e.g. determined by the assay methods described in the example section.
  • the composition may be provided in a solid form. This is e.g. a suitable option if the biological sample to be stabilized contains liquid to dissolve the solid (such as for example cell-containing body fluids, cells in medium, urine) or if liquid, e.g. water is added thereto to dissolve the solid.
  • liquid to dissolve the solid such as for example cell-containing body fluids, cells in medium, urine
  • liquid composition e.g. water is added thereto to dissolve the solid.
  • the advantage of using a solid stabilizing composition is that solids are usually chemically more stable.
  • a liquid composition may be used. Liquid compositions often have the advantage that the mixture with the sample to be stabilised can be quickly achieved, thereby basically providing an immediate stabilising effect as soon as the sample comes into contact with the liquid stabilizing composition.
  • stabilising agent(s) present in the liquid stabilizing composition remain stable in solution and require no pre-treatment-such as for example the dissolving of precipitates of limited solubility-by the user because pre-treatments of this kind pose a risk of variations in the stabilising efficiency.
  • the stabilizing composition is pre-filled in a sample collection device so that the sample is immediately stabilized during collection.
  • the stabilizing composition is contacted with the biological sample in a volumetric ratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. It is a particular advantage of the stabilizing composition of the present invention that stabilization of a large sample volume can be achieved with a small volume of the stabilizing composition. Therefore, preferably, the ratio of stabilizing composition to sample lies in a range from 1:2 to 1:7, more preferred 1:3 to 1:5.
  • the stabilizing composition according to the third aspect of the present invention can be used to stabilize the extracellular nucleic acid population comprised in a cell-containing sample. Furthermore, the stabilizing composition according to the third aspect of the present invention may also be used for stabilizing cells contained in a sample. As described above, the stabilizing composition inter alia reduces the release of genomic DNA from cells that results from decaying cells. Thus, a respective use is also an advantageous and provided by the teachings according to the present invention.
  • compositions according to the third aspect of the present invention are provided, wherein the components of the composition are mixed, preferably in an aqueous solution.
  • composition of the present invention may also be incorporated into a sample collection device, in particular blood collection assembly, thereby providing for a new and useful version of such a device.
  • sample collection devices typically include a container having an open and a closed end.
  • the container is preferably a blood collection tube.
  • the container type also depends on the sample to be collected, other suitable formats are described below.
  • the present invention provides a container for collecting a cell-containing biological sample, preferably a blood sample, wherein the container comprises a stabilizing composition according to the present invention.
  • a respective container e.g. a sample collection tube, which comprises the stabilizing composition according to the present invention, has the advantage that the sample is quickly stabilized when the sample is collected in the respective container. Details with respect to the stabilizing composition were described above, it is referred to the above disclosure which also applies here.
  • a collection container for receiving and collecting a biological sample comprising:
  • the pre-filled components a), b), c) and/or d) can be provided in a liquid or in a dry form.
  • the stabilizing components are provided as a stabilizing composition.
  • a dry form is e.g. a suitable option if the biological sample to be stabilized contains liquid to dissolve the solid (such as for example cell-containing body fluids, cells in medium, urine) or if liquid, e.g. water is added thereto to dissolve the solid.
  • the advantage of using a solid stabilizing composition is that solids are usually chemically more stable than liquids.
  • the inner wall of the container is treated/covered with a stabilizing composition according to the present invention.
  • Said composition can be applied to the inner walls using e.g. a spray-dry-method.
  • Liquid removal techniques can be performed on the stabilising composition in order to obtain a substantially solid state protective composition.
  • Liquid removal conditions may be such that they result in removal of at least about 50% by weight, at least about 75% by weight, or at least about 85% by weight of the original amount of the dispensed liquid stabilising composition.
  • Liquid removal conditions may be such that they result in removal of sufficient liquid so that the resulting composition is in the form of a film, gel or other substantially solid or highly viscous layer. For example it may result in a substantially immobile coating (preferably a coating that can be re-dissolved or otherwise dispersed upon contact with the cell-containing sample which preferably is a blood product sample).
  • liquid removal conditions may be such that they result in a material that upon contact with the sample under consideration (e.g., a whole blood sample) the protective agent will disperse in the sample, and substantially preserve components (e.g., extracellular nucleic acids) in the sample.
  • Liquid removal conditions may be such that they result in a remaining composition that is substantially free of crystallinity, has a viscosity that is sufficiently high that the remaining composition is substantially immobile at ambient temperature; or both.
  • liquid compositions may be used.
  • Liquid compositions often have the advantage that the mixture with the sample to be stabilised can be quickly achieved, thereby basically providing an immediate stabilising effect as soon as the sample comes into contact with the liquid stabilizing composition.
  • the stabilising agent(s) present in the liquid stabilizing composition remain stable in solution and require no pre-treatment—such as for example the dissolving of precipitates of limited solubility—by the user because pre-treatments of this kind pose a risk of variations in the stabilising efficiency.
  • the stabilizing composition is comprised in the container in an amount effective to provide the stabilisation of the amount of sample to be collected in said container.
  • the liquid stabilizing composition is contacted with the biological sample in a volumetric ratio selected from 10:1 to 1:20, 5:1 to 1:15, 1:1 to 1:10 and 1:2 to 1:5. It is a particular advantage of the stabilizing composition of the present invention that stabilization of a large sample volume can be achieved with a small volume of the stabilizing composition. Therefore, preferably, the ratio of stabilizing composition to sample lies in a range from 1:2 to 1:7, more preferred 1:3 to 1:5.
  • the container is evacuated.
  • the evacuation is preferably effective for drawing a specific volume of a fluid sample into the interior.
  • the container comprises a tube having an open end sealed by a septum.
  • 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.
  • the septum is constructed such that it is compatible with the standard sampling accessories (e.g. cannula, etc.).
  • a sample amount that is predetermined by the vacuum is collected in the container.
  • a respective embodiment is in particular advantageous for collecting blood.
  • a suitable container is e.g. disclosed in U.S. Pat. No. 6,776,959.
  • the container according to the present invention can be made of glass, plastic or other suitable materials.
  • Plastic materials can be oxygen impermeable materials or may contain an oxygen impermeable layer.
  • the container can be made of water- and air-permeable plastic material.
  • the container according to the present invention preferably is made of a transparent material. Examples of suitable transparent thermoplastic materials include polycarbonates, polyethylene, polypropylene and polyethyleneterephthalate.
  • the container may have a suitable dimension selected according to the required volume of the biological sample being collected. As described above, preferably, the container is evacuated to an internal pressure below atmospheric pressure. Such an embodiment is particularly suitable for collecting body fluids such as whole blood. The pressure is preferably selected to draw a predetermined volume of a biological sample into the container.
  • vacuum tubes also non-vacuum tubes
  • mechanical separator tubes or gel-barrier tubes can be used as sample containers, in particular for the collection of blood samples.
  • suitable containers and capping devices are disclosed in U.S. Pat. No. 5,860,397 and US 2004/0043505.
  • As container for collecting the cell-containing sample also further collection devices, for example a syringe, a urine collection device or other collection devices can be used.
  • the type of the container may also depend on the sample type to be collected and suitable containers are also available to the skilled person.
  • the container respectively the device is filled or is pre-filled with at least one apoptosis inhibitor, preferably a caspase inhibitor, at least one hypertonic agent, preferably at least one hydroxylated organic compound as described in detail above, e.g. dihydroxyaceton and optionally a further additive such as an anticoagulant, preferably a chelating agent, more preferred EDTA.
  • apoptosis inhibitor preferably a caspase inhibitor
  • at least one hypertonic agent preferably at least one hydroxylated organic compound as described in detail above, e.g. dihydroxyaceton and optionally a further additive such as an anticoagulant, preferably a chelating agent, more preferred EDTA.
  • at least one hypertonic agent which preferably is a hydroxylated organic compound, e.g.
  • a carbohydrate such as dihydroxyacetone and at least one caspase inhibitor, preferably Q-VD-OPH unexpectedly stabilizes extracellular nucleic acids in whole blood, plasma or serum and prevents the release of cellular nucleic acids in particular from white blood cells that are contained in such samples.
  • the extracellular nucleic acid population is preserved in the state it had shown at the time of blood draw.
  • an anticoagulant is encompassed in addition to the compound according to formula 1.
  • the anticoagulant is preferably a chelating agent such as EDTA.
  • the stabilizing composition comprised in the container may also comprise an apoptosis inhibitor, preferably a caspase inhibitor and/or at least one hypertonic agent, preferably at least one hydroxylated organic compound as described in detail above, e.g. dihydroxyaceton and optionally further additives.
  • the stabilizing composition comprised in the container comprises a caspase inhibitor and an anticoagulant.
  • the container has an open top, a bottom, and a sidewall extending therebetween defining a chamber, wherein the stabilization composition according to the present invention is comprised in the chamber. It may be comprised therein in liquid or solid form.
  • 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.
  • the closure is capable of being pierced with a needle or cannula, and the reduced pressure is selected to draw a specified volume of a liquid sample into the chamber.
  • the chamber is at a reduced pressure selected to draw a specified volume of a liquid sample into the chamber
  • 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 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.
  • the container is for drawing blood from a patient.
  • a method comprising the step of collecting a sample from a patient directly into a chamber of a container according to the fourth aspect of the present invention. Details with respect to the container and the sample were described above. It is referred to the respective disclosure. According to one embodiment, a blood sample is collected, preferably it is withdrawn from the patient.
  • the methods and compositions disclosed herein allow for the efficient preservation and isolation of extracellular nucleic acids while reducing possible mixing with nucleic acids, in particular fragmented genomic DNA, which originates from cells comprised in the biological sample and which may enter a biological sample due to cell damage, respectively cell lysis.
  • the methods according to the present invention, as well as the compositions and the disclosed devices reduce the degradation of extracellular nucleic acids and also reduce cell lysis and/or release of genomic nucleic acids, in particular fragmented genomic DNA, so that the extracellular nucleic acids contained in the sample do not become contaminated with intracellular nucleic acids, respectively a respective contamination is reduced by the teachings according to the present invention.
  • an intermixing of extracellular nucleic acids and cellular nucleic acids, in particular fragmented genomic DNA may reduce the accuracy of any measurement of the amount of extracellular nucleic acids in a biological sample.
  • an important advantage of the present invention is the possibility for essentially simultaneous stabilizing of both the cells contained in the sample (in particular white blood cells in case of whole blood, plasma or serum) and the extracellular nucleic acids. This helps to prevent cellular nucleic acids such as genomic DNA from being released into the cell-free portion of the sample, and further diluting the comprised extracellular nucleic acids (and associated biomarkers) of interest, while also maintaining the structural integrity of the extracellular nucleic acids.
  • contacting the cell-containing biological sample such as whole blood or plasma with the stabilising agent(s) allows the sample to be stored for a period of time prior to isolating the extracellular nucleic acids.
  • the cell-containing biological sample e.g. blood or plasma
  • the stabilising agent(s) may be drawn at one location (e.g., a health care facility), contacted with the stabilising agent(s), and later transported to a different remote location (e.g., a laboratory) for the nucleic acid isolation and testing process.
  • the stabilization reagents provide an advantage over known state-of-the-art stabilization reagents which involve the use of cross-linking reagents, such as formaldehyde, formaldehyde releasers and the like, as the stabilization of samples according to the present invention does not involve the use to such crosslinking reagents.
  • Crosslinking reagents cause inter- or intra-molecular covalent bonds between nucleic acid molecules or between nucleic acids and proteins. This effect can lead to a reduced recovery of such stabilized and partially crosslinked nucleic acids after a purification or extraction from a complex biological sample.
  • the concentration of circulating nucleic acids in a whole blood samples is already relatively low, any measure which further reduces the yield of such nucleic acids should be avoided. This may be of particular importance when detecting and analyzing very rare nucleic acid molecules derived from malignant tumors or from a developing fetus in the first trimester of pregnancy. Therefore, according to one embodiment, no formaldehyde releaser is comprised in the stabilizing composition, respectively is not additionally used for stabilization.
  • the apoptosis inhibitor that is used in the methods and/or compositions according to the present invention is not selected from the group consisting of aurintricarboxylic acid, phenylmethylsulfonyl fluoride (PMSF), leupeptin and Na-Tosyl-Lys chloromethyl ketone hydrochloride (TLCK).
  • the apoptosis inhibitor is not selected from said group in particular if the apoptosis inhibitor is not used in combination with a hypertonic agent as additional stabilizer.
  • solution refers to a liquid composition, preferably an aqueous composition. It may be a homogenous mixture of only one phase but it is also within the scope of the present invention that a solution comprises solid additives such as e.g. precipitates.
  • nucleotides nt refer to the chain length and thus are used in order to describe the length of single-stranded as well as double-stranded molecules. In double-stranded molecules said nucleotides are paired.
  • subject matter described herein as comprising certain steps in the case of methods or as comprising certain ingredients in the case of compositions, solutions and/or buffers refers to subject matter consisting of the respective steps or ingredients. It is preferred to select and combine preferred embodiments described herein and the specific subject-matter arising from a respective combination of preferred embodiments also belongs to the present disclosure.
  • Apoptosis inhibitor Description 1 Metabolic inhibitors AICA-Riboside, Acadesine, Offers protection against cell death induced by glucose deprivation AICAr, 5-Aminoimidazole-4- carboxamide-1- ⁇ -riboside, Z- Riboside Apoptosis Inhibitor II, diarylurea prevents the active ⁇ 700-kDa apoptosome complex formation compound Bax Channel Blocker, ( ⁇ )-1- A cell-permeable dibromocarbazolo-piperazinyl derivative that (3,6-Dibromocarbazol-9-yl)-3- displays anti-apoptotic properties.
  • BH4 conserved N-terminal homology domain
  • the BH4 domain is linked to a carrier peptide, a 10-amino acid HIV-TAT48-57 sequence with a ⁇ -alanine residue as a spacer for maximum flexibility. Following its uptake, it is mainly localized to the mitochondria Bongkrekic Acid, Triammonium Acts as a ligand of the adenine nucleotide translocator.
  • a potent Salt inhibitor of mitochondrial megachannel permeability transition pore). Significantly reduces signs of apoptodsis induced by nitric oxide.
  • Pifithrin-p targets only the mitochondrial branch of the p53 pathway without affecting the important transcriptional functions of p53, it is superior to Pifithrin-a (Cat. No. 506132) in in vivo studies. Shown to selectively interact with inducible HSP70 and disrupt its functions Pifithrin- ⁇ , Cyclic- A cell-permeable and very stable analog of Pifithrin-a (Cat. No. 506132), with similar biological function, but with reduced cytotoxicity.
  • a chemical inhibitor of p53 Reversibly inhibits p53- dependent transactivation of p53-responsive genes; also reversibly blocks p53-mediated apoptosis.
  • Thr-Lys-OH Significantly lowers the DNA-binding activity of Stat3 by forming an inactive Stat3:peptide complex and reduces the levels of active Stat3:Stat3 dimers that can bind DNA. Displays greater affinity for Stat3, and to a lesser extent Stat1, over Stat5. Supplied as a trifluoroacetate salt.
  • STAT3 Inhibitor Peptide Cell- A cell-permeable analog of the Stat3-SH2 domain-binding Permeable phosphopeptide (Cat. No. 573095) that contains a C-terminal mts (membrane translocating sequence) and acts as a highly selective, Peptide sequence: potent blocker of Stat3 activation.
  • TNF ⁇ Tumor necrosis factor a
  • TNFR1 TNF Receptor 1
  • Maslinic Acid A pentacyclic triterpene with antioxidant and anti-inflammatory properties. Shown to block the generation of nitric oxide, and inhibits the secretion of IL-6 and TNF-a induced by lipopolysaccharides Naringin hydrate A citrus bioflavonoid found to inhibit cytochrome P450 monooxygenase activity in mouse liver. It prevents toxin-induced cytoskeletal disruption and apoptotic liver cell death. Necrostatin-1 An inhibitor of necroptosis, a non-apoptotic cell death pathway. Does not affect Fas/TNFR-triggered apoptosis.
  • said compound is not used as stabilizer according to the present invention.
  • NSC348884 hydrate, N1,N2- This product is a nucleolar phosphoprotein that displays several bis((3-imino-6-methyl-3H-indol- biological activities in ribosome biogenesis, cell proliferation, 2-yl)methyl)-N1,N2-bis((6- cytoplasmic/nuclear shuttle transportation, nucleic acid binding, methyl-1H-benzo[d]imidazol-2- ribonucleic cleavage, centrosome duplication and molecular yl)methyl)ethane-1,2-diamine chaperoning, and is found in higher levels in tumor cells.
  • NSC34884 upregulates p53.
  • Orsellinic acid Benzoic acid Blocks PAF-mediated neuronal apoptosis. Shows free radical scavenging activity.
  • tetramethyl A synthetic derivative of NDGA and a non-selective lipoxygenase Nordihydroguaiaretic Acid inhibitor It inhibits Sp1 transcription factor binding at the HIV long terminal repeat promoter and at the ⁇ -ICP4 promoter (a gene essential for HSV replication).
  • This agent exhibits greater than 300-fold Anthrapyrazolone selectivity for JNK against related MAP kinases ERK1 and p38-2, and the serine threonine kinase PKA.
  • SP600125 is a reversible ATP-competitive inhibitor.
  • Mdivi-1, 3-(2,4-Dichloro-5- Mdivi-1 is a selective inhibitor of mitochondrial division in yeast and methoxyphenyl)-2,3-dihydro-2- mammalian cells which acts via inhibiting the mitochondrial division thioxo-4(1H)-quinazolinone, 3- dynamin.
  • Mdivi-1 inhibits apoptosis by inhibiting (2,4-Dichloro-5-methoxyphenyl)- mitochondrial outer membrane permeabilization.
  • time-lapse fluorescence microscopy revealed no detectable mitochondrial division after treatment with Mdivi-1 Minocycline .
  • hydrochloride Tetracycline derivative with antimicrobial activity.
  • Anti-inflammatory and neuroprotective Ro 08-2750 (C13H10N4O3) Inhibitor of NGF-induced apoptosis.
  • RKTS-33 (C7H8O4) selective inhibition of Fas ligand-dependent pathway alone 2.
  • actinomycin promotes induction of apoptosis by some specific stimuli, for example, TRAIL and Fas (CD95).
  • Actinomycin D can also alleviate or block the apoptotic process and decrease the cytotoxicity induced by several stimuli such as the dihydrofolate reductase inhibitor aminopterin and the prostaglandin derivative 15-deoxy-D12,14-prostaglandin J2, thus it can have both pro and anti-apoptotic activities in some systems.
  • said compound is not used as stabilizer according to the present invention.
  • PMSF causes sulfonylation of the active-site serine residues. Also reported to inhibit internucleosomal DNA fragmentation in immature thymocytes. For a related, more stable inhibitor, see AEBSF ( ⁇ )-Huperzine A An inhibitor of AChE. Antagonist of NMDA receptors. Protects against glutamate-mediated excitotoxicity. Razoxane Inhibits topoisomerase II without inducing DNA strand breaks (topo II catalytic inhibitor). Suptopin-2 Suppressor of topoisomerase II inhibition. Reverses cell cycle arrest; bypass of checkpoint function. Has inherent fluorescence and a distinct advantage in identification of molecule targets; effective concentraion in the pM range. 3.
  • Enzymes 3.1. Caspases Apoptosis Inhibitor; 2-(p- Effects attributable to the inhibition of caspase-3 activation Methoxybenzyl)-3,4- pyrrolid inedio1-3-acetate cIAP-1, Human, Recombinant, Recombinant, human cIAP-1 (amino acids 1-618) fused to the E. coli peptide sequence MATVIDH10SSNG at the N-terminus and expressed in E. coli.
  • clAP is a member of the inhibitor of apoptosis family of proteins that inhibits proteolytic activity of mature caspases by interaction of the BIR domain with the active caspase CrmA
  • Recombinant CrmA cowpox viral serpin cytokine response modifier A
  • CrmA is a natural inhibitor of human caspase-1 and granzyme B, enzymes that are involved in apoptosis Group III Caspase Inhibitor I
  • a potent, cell-permeable, and irreversible inhibitor of Group III caspases (caspase-6, -8, -9, and -10), although more effective Peptide sequence: towards caspases-6 and -8.
  • IEPD-CHO Caspase-8 inhibitor pretreatment with an esterase is required.
  • Kaempferol A cell-permeable phytoestrogen that inhibits topoisomerase I- catalyzed DNA religation in HL-60 cells. Offers protection against A ⁇ 5-35-induced cell death in neonatal cortical neurons. Its protective effects are comparable to that of estradiol. Blocks the A ⁇ -induced activation of caspase-2, -3, -8, and -9, and reduces NMDA-induced neuronal apoptosis. Reported to be a potent inhibitor of monoamine oxidases.
  • Peptide sequence Ac-Leu-Glu-Glu-Asp-CHO Caspase-13 Inhibitor II A cell-permeable, irreversible inhibitor of caspase-13.
  • esterase is required.
  • Caspase-1 Inhibitor I Cell- A cell-permeable inhibitor of caspase-1 (ICE; Interleukin-1 ⁇ Permeable Converting Enzyme), caspase-4, and caspase-5.
  • ICE Cell- A cell-permeable inhibitor of caspase-1
  • caspase-4 Cell- A cell-permeable Converting Enzyme
  • caspase-5 Cell- A cell-permeable inhibitor of caspase-1 (ICE; Interleukin-1 ⁇ Permeable Converting Enzyme)
  • caspase-4 caspase-5.
  • Fas-mediated apoptosis Peptide sequence and acidic sphingomyelinase activation Ac-Tyr-Val-Ala-Asp-CM K
  • Caspase-1 Inhibitor IV A highly selective, competitive, cell-permeable, and irreversible inhibitor of caspase-1, caspase-4, and caspase-5.
  • Peptide sequence Ac-Asp-Glu-Val-Asp-CHO Caspase-3 Inhibitor I, Cell- A cell-permeable inhibitor of caspase-3, as well as caspase-6, Permeable caspase-7, caspase-8, and caspase-10.
  • the N-terminal Leu-Ala-Pro-Asp-Glu-Val-Asp- sequence corresponds to the CHO hydrophobic region (h-region) of the signal peptide of Kaposi fibroblast growth factor (K-FGF) and confers cell-permeability to the peptide.
  • K-FGF Kaposi fibroblast growth factor
  • a 5 mM (1 mg/100 ⁇ l) solution of Caspase-3 Inhibitor I, Cell-permeable (Cat. No. 235427) in DMSO is also available.
  • Caspase-3 Inhibitor II A potent, cell-permeable, and irreversible inhibitor of caspase-3 as well as caspase-6, caspase-7, caspase-8, and caspase-10.
  • This tetrapeptide inhibitor has been used with the caspase-6 inhibitor Ac-VEID-CHO to dissect Peptide sequence.
  • the pathway of caspase activation in Fas-stimulated Jurkat cells Ac-Asp-Met-Gln-Asp-CHO Caspase-3 Inhibitor V
  • a potent, cell-permeable, and irreversible inhibitor of caspase-3 also recognizes caspase-1.
  • Caspase-4 Inhibitor I A reversible caspase-4 inhibitor Peptide sequence: Ac-Leu-Glu-Val-Asp-CHO Caspase-4 Inhibitor I, Cell- A potent, cell-permeable, and reversible inhibitor of caspase-4. Permeable The N-terminal sequence (amino acid residues 1-16) corresponds to the hydrophobic region of the signal peptide of Kaposi fibroblast Peptide sequence: growth factor and confers cell permeability to the peptide.
  • Caspase-5 Inhibitor I A potent, cell-permeable, and irreversible inhibitor of caspase-5. Strongly inhibits caspase-1. Also inhibits caspase-4 and caspase-8 Peptide sequence: Z-Trp-Glu(OMe)-His-Asp(OMe)- CH2F* Caspase-6 Inhibitor I A cell-permeable, irreversible inhibitor of caspase-6.
  • the N-terminal sequence corresponds to the hydrophobic region of the signal peptide of Peptide sequence: Kaposi fibroblast growth factor and confers cell permeability to the Ac-Ala-Ala-Val-Ala-Leu-Leu- peptide Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-lle-Glu-Thr-Asp- CHO Caspase-8 Inhibitor II A potent, cell-permeable, and irreversible inhibitor of caspase-8 and granzyme B. Effectively inhibits influenza virus-induced Peptide sequence: apoptosis in HeLa cells. Also inhibits granzyme B.
  • a 5 mM (250 ⁇ g/72 ⁇ l) solution of Z-LEHD- CH2F* FMK (Cat. No. 218841) in DMSO is also available Caspase-9 Inhibitor II, Cell- A potent, cell-permeable, and reversible inhibitor of caspase-9. Permeable May also inhibit caspase-4 and caspase-5.
  • the N-terminal sequence corresponds to the hydrophobic Peptide sequence: region of the signal peptide of Kaposi fibroblast growth factor and Ac-Ala-Ala-Val-Ala-Leu-Leu- confers cell permeability to the peptide Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-Leu-Glu-His-Asp- CHO Caspase-9 Inhibitor III A potent, irreversible inhibitor of caspase-9. Reported to reduce myocardial infarct size during reperfusion ( ⁇ 70 nM).
  • Caspase Inhibitor I A cell-permeable, irreversible, pan-caspase inhibitor. Inhibits Fas- mediated apoptosis in Jurkat cells and staurosporine-induced cell Peptide sequence: death in corneal epithelial cells. When using with purified native or Z-Val-Ala-Asp(OMe)-CH2F* recombinant enzyme, pre-treatment with an esterase is required.
  • Caspase Inhibitor II A potent and reversible pan-caspase inhibitor.
  • Peptide sequence Ac-Val-Ala-Asp-CHO Caspase Inhibitor II, Cell- A cell-permeable, reversible pan-caspase inhibitor produced by Permeable attaching the N-terminal sequence (amino acids 1-16) of the Kaposi fibroblast growth factor signaling peptide, which imparts Peptide sequence: cell-permeability to VAD peptide.
  • Ac-Ala-Ala-Val-Ala-Leu-Leu- Pro-Ala-Val-Leu-Leu-Ala-Leu- Leu-Ala-Pro-Val-Ala-Asp-CHO Caspase Inhibitor III A cell-permeable, irreversible, broad-spectrum caspase inhibitor.
  • Peptide sequence Boc-Asp(OMe)-CH2F* Caspase Inhibitor IV A general, irreversible caspase inhibitor.
  • Peptide sequence Boc-Asp(OBzl)-CMK Caspase Inhibitor VI An irreversible general caspase inhibitor. Useful for studies involving recombinant, isolated, and purified caspase enzymes.
  • Peptide sequence Unlike Caspase Inhibitor I (Cat. No. 627610), this inhibitor does not Z-Val-Ala-Asp-CH2F* require pretreatment with esterase for in vitro studies.
  • a 10 mM (1 mg/221 ⁇ l) solution of Caspase Inhibitor VI (Cat. No.
  • the benzodioxane moiety is shown to fit in the ‘aspartate hole’ of the caspases and possibly disrupt caspase-8 assisted cleavage of BID, a proapoptotic protein.
  • Caspase-1 Inhibitors Including, but not limited to Ac-N-Me-Tyr-Val-Ala-Asp-aldehyde (pseudo acid) Ac-Trp-Glu-His-Asp-aldehyde (pseudo acid) Ac-Tyr-Val-Ala-Asp-aldehyde (pseudo acid) Ac-Tyr-Val-Ala-Asp-chloromethylketone Ac-Tyr-Val-Ala-Asp-2,6-dimethylbenzoyloxymethylketone Ac-Tyr-Val-Ala-Asp(OtBu)-aldehyde-dimethyl acetal Ac-Tyr-Val-Lys-Asp-aldehyde (pseudo acid) Ac-Tyr-Val-Lys-Asp-aldehyde (pseudo acid) Ac-Tyr
  • iNOS inducible nitric oxide synthase
  • BAPTA/AM Membrane-permeable form of BAPTA Can be loaded into a wide variety of cells, where it is hydrolyzed by cytosolic esterases and is trapped intracellularly as the active chelator BAPTA. Prevents cocaine-induced ventricular fibrillations. Abolishes vitamin D3- induced increase in intracellular Ca2+. Induces inactivation of protein kinase C.
  • caspase-1 ⁇ 6 nM
  • caspase-6 5.6 nM
  • Peptide sequence caspase-10 (27 nM).
  • Ac-Ile-Glu-Thr-Asp-CHO Granzyme B Inhibitor IV A reversible inhibitor of granzyme B and caspase-8 Peptide sequence: Ac-Ile-Glu-Pro-Asp-CHO Leupeptin, Hemisulfate, A reversible inhibitor of trypsin-like proteases and cysteine Microbial proteases.
  • TLCK Ketone, Hydrochloride
  • a cell-permeable furfurylidine-thiobarbituric acid compound that Ucf-101 acts as a potent, specific, competitive, and reversible inhibitor of the pro-apoptotic, heat-inducible, mitochondrial serine protease Omi/HtrA2 (IC50 9.5 ⁇ M for His-Omi134-458). Shows very little activity against various other serine proteases tested (IC50 ⁇ 200 ⁇ M).
  • Phorbol-12,13-dibutyrate Strong irritant for mouse skin, but only moderately active as a tumor promoter. Activates protein kinase C. Stimulates the phosphorylation of Na+,K+-ATPase, thereby inhibiting its activity. Promotes the expression of inducible NOS in cultured hepatocytes. Commonly used in binding studies or in applications requiring high concentrations of phorbol compounds. Hypericin Inhibits PKC, CKII, MAP Kinase, Insulin R, EGFR, PI-3 Kinase and also noted to possess antiviral activity.
  • Cdks cyclin-dependent protein kinases
  • Nilotinib Spezifischer BCR-ABL-Tyrosinkinase-lnhibitor Quercetin(Sophoretin) Quercetin is a PI3K and PKC inhibitor with IC50 of 3.8 ⁇ M and 15 ⁇ g/ml. It strongly abrogated PI3K and Src kinases, mildly inhibited Akt1/2, and slightly affected PKC, p38 and ERK1/2.
  • Quercetin is a naturally-occurring polar auxin transport inhibitor with IC50 of 0.8, 16.7, 6.1, 11.36 ⁇ M for the inhibition of LDH% release, the inhibition of TNF-induced PMN-EC adhesion, TNF- induced inhibition of DNA synthesis and proliferation. It is a type of plant-based chemical, or phytochemical, known as a flavonol and a plant-derived flavonoid found in fruits, vegetables, leaves and grains. It also may be used as an ingredient in supplements, beverages or foods. In several studies, it may have anti- inflammatory and antioxidant properties, and it is being investigated for a wide range of potential health benefits
  • test system was designed, wherein cell-containing biological samples, here whole blood samples, were incubated at room temperature (RT) for up to 6 or 7 days. Therein, the sample stabilizing properties of the additives of the present invention were tested on day 0, day 3 and day 6/7 the samples.
  • the samples were processed according to the following protocols, where applicable (for details, see also the specific examples in the results section):
  • Red blood cells are lysed because otherwise, the decisive cell populations (which can release e.g. genomic DNA) are not distinguishable in the FACS analysis due to the high amount of red blood cells.
  • the blood samples were centrifuged for 15 min at 5000 rpm, and the obtained plasma samples were again centrifuged for 10 min at 16.000 ⁇ g at 4° C.
  • the resulting blood plasma was used for isolating the nucleic acids contained therein.
  • the circulating, extracellular nucleic acids were purified from the obtained plasma samples using the QIAamp® Circulating NA Kit (according to the handbook).
  • QIAamp® Circulating NA Kit according to the handbook.
  • the DNA duplex assay was carried out according to the QuantiTect® Multiplex PCR handbook (Qiagen) with the following adaptions:
  • TABLE 2 shows compositions of PCR reagents and cycling conditions of the p53 mRNA one step real time PCR.
  • TaqMan MasterMix MM single master-mix component reaction (x-fold) c p53 FAM-BHQ + HEX-BHQ x-fach 1 x 182 1 x 20.00 mastermix/reaction RNA 5.000 / var. 5 ⁇ l RNA A. dest (PCR grade) 3.813 693.9 / 25.00 ⁇ l reaction volume 2x QuantiTect Probe RT-PCR MasterMix (Puffer) 12.500 2275.0 1 x forw. primer (20 ⁇ M) 0.500 91.0 400 nM rev.
  • Each tested caspase inhibitor was added to whole blood samples (20 ⁇ M end concentration in 10 ml blood; blood was collected into Vacutainer K2E Tubes; BD). The whole blood sample was processed as described in section I, see 2. (plasma preparation) and 3. (nucleic acid isolation).
  • FIG. 1 a shows the obtained results.
  • the DMSO control and the K2E blood (not treated according to the teachings of the present invention) show the same ladder-like pattern of bands. This pattern occurs in samples where apoptosis takes place. During apoptosis, endonucleases degrade genomic DNA at inter-nucleosomal linker regions and produce DNA fragments of circa 180 bp or multiples of 180 bp. Thus, apoptosis occurs in samples which show a clear ladder-like pattern. Furthermore, the strength (darkness) of the pattern is decisive. The darker the bands, the more genomic DNA was released from the cells and thus contaminates the extracellular nucleic acid population.
  • FIG. 1 a shows that the DMSO control and the K2E blood samples show a strong ladder-like pattern already on day 3, which becomes even stronger on day 7.
  • genomic DNA was released from the cells contained in the sample and was also degraded. This released and degraded DNA contaminates the cell-free nucleic acids contained in the sample. Hence, no acceptable stabilisation is achieved with these samples.
  • FIG. 1 b shows the effect of the tested caspase-inhibitors on the stabilisation of the extracellular nucleic acid population (18S DNA duplex assay) within 7 days of storage at RT, here the increase in DNA.
  • FIG. 2 a shows that compared to the control samples, wherein no caspase inhibitor was added, already 1 ⁇ M caspase inhibitor significantly reduced the genomic DNA release/fragmentation on day 7. The effect is improved if 4 ⁇ M caspase inhibitor is used. Thus, already very low concentrations of the caspase inhibitor are effective in stabilising the blood sample, in particular when combined with a carbohydrate.
  • FIG. 2 b shows the effects of the tested concentrations of the caspase-inhibitor Q-VD-OPh in combination with 21 mM glucose on the increase of genomic DNA in the plasma (18S DNA duplex assay) within 7 days of storage at RT.
  • the addition of Q-VD-OPh in combination with glucose significantly reduces the release of genomic DNA into plasma.
  • FIG. 2 b shows only a minor increase of genomic DNA within 7 days of storage even if only 1 ⁇ M Q-VD-OPH was added to the whole blood sample for stabilisation.
  • the addition of 4 ⁇ M Q-VD-OPh inhibits the release of genomic DNA to plasma to a maximum of a 4-fold increase.
  • drawing whole blood in K2E Tubes without stabilisation according to the present invention leads to approximately 40-fold increase of DNA in plasma.
  • FIG. 2 b confirms that the caspase inhibitor has a stabilisation effect on whole blood even at low concentrations.
  • blood cells can be stabilized by adding a reagent that acts as a hypertonic medium in whole blood.
  • Generating a hypertonic medium by the addition of, for example, hydroxylated organic compound(s) to whole blood results in a slight release of water from the contained blood cells and results in increased stability by cell shrinking. It is assumed that said cell shrinking stabilises the cells against mechanical forces.
  • DHA Dihydroxyacetone
  • DHAP dihydroxyacetone phosphate
  • PBS purchased from SIGMA-Aldrich Kat. No: D8537
  • 3 ⁇ MOPS diluted from 1 litre of 10 ⁇ MOPS: 200 mM MOPS; 50 mM NaAc, 10 mM EDTA; pH 5; assuming that an acid medium also stabilizes ccf RNA
  • FIG. 3 shows the blood cell integrity measured by flow cytometry.
  • the Dot-Plots visualize three different cell populations: granulocytes (1), monocytes (2) and lymphocytes (3).
  • the cloud (4) in the lower left field of the plot represents the debris, mainly generated by the lysis of erythrocytes.
  • FIG. 3 show that blood cells collected and stored in PAXgene® Blood DNA tubes are not distinguishable from each other and the debris on day 6 of storage.
  • the addition of DHA enables a differentiation of the subpopulations of blood cells on day 6 of storage even though these cells become smaller as a result of the cell shrinking. This indicates that the cells contained in the sample were stabilised by the addition of DHA.
  • FIG. 4 a shows a stabilisation of the blood samples by the addition of DHA, because the release of genomic DNA is significantly lower with the DHA treated samples than in samples stored in PAXgene® Blood DNA tubes. Furthermore, as is evident from FIG. 4 a , DHA-stabilized samples do not show ladder-like degradation pattern suggesting that apoptosis, respectively a degradation of DNA is efficiently prevented.
  • FIG. 4 b shows the effect of DHA on the increase of DNA (18S DNA duplex assay) within 6 days of storage at RT.
  • DHA dissolved in 3 ⁇ MOPS provided the best results, because the level of ribosomal 18S DNA seems to remain constant till day 3 of storage.
  • the division of short amplicon copy number by long amplicon copy number indicates whether the amount of detected short or long amplicons changes over time in a similar way. A decrease of this ratio implies a stronger release of longer rather than of shorter DNA molecules and can be interpreted as release of high molecular weight genomic DNA from blood cells.
  • the diagram shown in FIG. 4 b indicates the release of genomic DNA for all three conditions. The results show that the presence of DHA slows this process down. Thus, also this experiment shows that the addition of DHA to whole EDTA blood stabilizes blood cells and hence preserves the ccfDNA population in the cell-free plasma fraction and avoids contaminations with DNA released from the cells contained in the sample e.g. due to mechanical breakup.
  • FIG. 5 shows the blood cell integrity measured by flow cytometry.
  • the Dot-Plots visualize three different cell populations: granulocytes (1), monocytes (2) and lymphocytes (3).
  • the cloud in the lower left field of the plot represents the debris, mainly caused by the lysis of erythrocytes.
  • results presented in FIG. 6 a also show a stabilisation of the blood samples by the addition of the different concentrations of DHA, because the release of genomic DNA and the degradation of the DNA is efficiently prevented.
  • FIG. 6 b shows the effect of different DHA concentrations on the increase of DNA (18S DNA duplex assay) within 6 days of storage at RT.
  • 0.5M DHA in whole blood prevents most efficiently the release of genomic DNA.
  • the ratio of short to long amplicon copy numbers stays constant for up to 3 days and only decreases slightly till day 6.
  • the combination of these reagents results in an improved stabilization of extracellular nucleic acids, in particular extracellular DNA, in whole blood that lasts at least for 6 days, and furthermore, results in an efficient stabilization of blood cells, thereby preventing the release of genomic DNA, what otherwise would result in a dilution of the natural extracellular nucleic acid level in plasma.
  • DHA was dissolved in 2 ml 3 ⁇ MOPS (3M DHA in 2 ml 3 ⁇ MOPS), 50 mg K 2 EDTA and 2.4 ⁇ l of 5 nM Q-VD-OPh were added and then transferred into 10 ml whole blood, that was collected in K2E Tubes. Plasma samples were centrifuged for 10 min at 16.000 ⁇ g, 4° C. and then purified using the QIAamp® Circulating NA Kit (Qiagen) (details are described above in section I).
  • FIG. 7 a shows the blood cell integrity measured by flow cytometry.
  • the Dot-Plots visualize three different cell populations: granulocytes (1), monocytes (2) and lymphocytes (3).
  • the cloud in the lower left field of the plot represents the debris, mainly caused by the lysis of remaining erythrocytes.
  • FIG. 7 b shows the effect of the combination of EDTA, DHA and the caspase-inhibitor Q-VD-OPH on the increase of DNA (18S DNA duplex assay) within 6 days of storage at RT.
  • the results indicate that the combination of EDTA, DHA and Q-VD-OPH leads to a remarkably strong stabilization of extracellular DNA in plasma (level of measured 18S rDNA remains constant till day 6) and to a strong prevention of the release of genomic DNA from blood cells (ratio of short to long amplicon copy numbers remains constant) till day 3 of storage. Only a slight increase of genomic DNA into plasma becomes visible between day 3 and day 6 of storage.
  • the tested combination of stabilising agents is particularly efficient in stabilising whole blood samples.
  • the stabilizing reagent(s) should not only protect RNAs from degradation and prevent the release of RNAs from decaying blood cells, but should also inhibit the metabolic pathways, respectively have the effect that changes in the metabolic pathway do not affect the extracellular RNA plasma level, respectively should reduce respective effects.
  • experiment 5 was repeated and the level of mRNA was measured by real time RT-PCR.
  • FIG. 8 shows the effect of the combination of EDTA, DHA and the tested caspase-inhibitor on the transcript level in plasma within 6 days of storage.
  • target mRNAs were referred to as reference target (18S rRNA) by calculating a ⁇ Ct between p53, IL8 or c-fos and the internal standard (18S rRNA). Subtracting the ⁇ Ct of day 3 or 6 samples with the ⁇ Ct of day 0 samples defines the ⁇ Ct visualizing a relative decrease ( ⁇ values) or increase (+ values) of mRNA transcript levels.
  • IL8 and c-fos are genes whose transcription is induced after blood draw.
  • transcript levels of these targets would rise dramatically when cells release their contents; the addition of the stabilizing solution according to the preferred embodiment of present invention (combination of elevated EDTA, dihydroxyacetone, caspase inhibitor Q-VD-OPh) strongly prevents nucleic acid release from blood cells till day 3 of storage. But the data in the diagram above show—surprisingly—no significant increase of c-fos and IL8 mRNA till day 6 of storage. Thus, apparently the stabilization prevents the degradation of RNA (p53) and the release of mRNA (IL8/c-fos)
  • K2E BD 18 mg K 2 EDTA
  • DMAA was added to replicates of whole blood samples (0.75% and 1.5% end concentration in 10 ml blood; blood was collected into Vacutainer K2E Tubes; BD).
  • FIG. 11 shows the effects of the tested concentrations of DMAA on the increase of genomic DNA in the plasma.
  • Addition of DMAA significantly reduces the release of genomic DNA into plasma. The more DMAA is added to whole blood, the less DNA is released. Only a minor increase of cell-free DNA within 6 days of storage was observed if 1.5% DMAA was added to the whole blood sample. Furthermore, as the addition of 1.5% DMAA stabilizes cell-free DNA levels in whole blood samples more efficiently than 0.75% and the ratio of short to long measured 18S DNA copies decreases from day 0 to day 6, higher DMAA concentrations of than 1.5% can result in more efficient stabilization effects.
  • DMAA reduces the release of genomic DNA into blood plasma.
  • adding DMAA to a blood sample is effective in stabilising the sample even at room temperature.
  • the respectively stabilized samples were incubated at room temperature for up to six days. On day 0, day 3 and day 6, replicates were processed as follows. The samples were centrifuged at 3.000 rpm for 10 minutes at room temperature in order to collect plasma. The collected plasma was centrifuged at 16,000 ⁇ g for 10 minutes at 4° C. The cleared plasma fraction was collected and the extracellular nucleic acids were isolated using the QIAamp Circulating nucleic acid kit (1 ml input material, 60 ⁇ l elution volume). The results are shown in relative change compared to the test time point 0 days (day X copies/day 0 copies) in FIG. 10 . Values that are close to 1 imply preserved levels of ccfDNA. The higher the value, the less stabilization is achieved.
  • FIG. 13 shows the influence of combinations of DMAA and OPH concentrations on ccfDNA levels (different scaling due to exclosure of reference data).
  • EDTA reference (BD Vacutainer K2E); 2: QGN mixture (0.01 M DHA, 14 mM EDTA, 1 ⁇ M OPH);
  • Extracellular nucleic acids are often comprised in very small amounts in the sample. Therefore, it is important to have a stabilization procedure which not only efficiently preserves the extracellular nucleic acids within the stabilized sample, but additionally allows to subsequently isolate the extracellular nucleic acids with high yield from the stabilized sample.
  • Example 14 demonstrates that the stabilization method according to the present invention is superior to prior art stabilization methods in that the extracellular nucleic acids can be isolated with higher yield from the stabilized samples. This advantageously reduces the limit of detection and thus, allows to reliably determine also rare target nucleic acids within the population of extracellular nucleic acids.
  • RNA BCT and BD Vacutainer K2E tubes Whole blood samples were collected in cell-free RNA BCT and BD Vacutainer K2E tubes. To one half of blood collected in BD tubes, the QGN stabilization solution was added. Thus, the sample stabilized according to the invention comprise an additional amount of EDTA that is contributed by the BD Vacutainer stabilization. The samples were centrifuged at 3.000 ⁇ rpm for 10 minutes, and the obtained plasma was aliquoted to 1.5 ml replicates. Afterwards, the following amounts of DNA spike-in control (1.000 bp) were added per sample: 1.000 copies, 5000 copies, 100 copies, 50 copies and 10 copies.
  • FIG. 18 The results are shown in FIG. 18 .
  • 100% hit ⁇ 1.000 copies per sample was obtained when using either the BD EDTA tubes or the stabilization solution according to the present invention.
  • the stabilization that is based on the use of a formaldehyde releaser (Streck) shows a strong inhibition of the nucleic acid isolation.
  • significantly less nucleic acids could be isolated from the respective samples, even with those samples wherein 500 or even 1.000 copies were spiked in.
  • FIG. 18 shows that the best sensitivity was obtained with a sample stabilized according to the present invention.
  • the method according to the present invention not only efficiently stabilizes the samples such as blood samples but furthermore allows the subsequent recovery of even very low-abundant extracellular nucleic acids.
  • This is an important advantage because it makes this method particularly suitable for diagnostic applications and e.g. the detection of rare target extracellular nucleic acids such as e.g. tumor derived extracellular nucleic acids or fetal nucleic acids.
  • the stabilization solution that is based on the use of formaldehyde releasers had a very low performance and showed the highest limit of detection.
  • the stabilization according to the invention does not impair the subsequent isolation of nucleic acids.
  • Stabilization using a formaldehyde releaser showed the highest limit of detection and thus demonstrates that the subsequent isolation of the nucleic acid was strongly impaired. Therefore, the stabilization according to the present invention is suitable for sensitive detection of rare ccfDNA targets, which is not achieved by using state of the art methods.
  • the samples were incubated at room temperature for up to six days at 37° C. On day 0 and day 3, replicates were processed as follows: the samples were centrifuged at 3.000 rpm, for 15 minutes at room temperature to collect the plasma. The obtained plasma was then again centrifuged at 16.000 ⁇ g for 10 minutes, at 4° C. Extracellular nucleic acids obtained from the cleared plasma supernatant was purified using the QIAsymphony virus/bacteria Cell-free 1000 protocol. 1 ml plasma was used as input material, 60 ⁇ l volume was used for elution. The results are shown in FIG. 21 .
  • the method according to the present invention is particularly suitable for diagnostic applications and is also suitable for stabilizing the samples in environments wherein potentially no refrigerating facilities are available.
  • ⁇ Ct between day 0 and day 3 is reduced ( ⁇ Ct of approximately 2.5 to ⁇ Ct of approximately 1) compared to the EDTA blood reference.
  • stabilization effects were seen with a combination of Sorbitol in combination with Inositol ( ⁇ Ct of approximately 1 to 1.4).
  • FIG. 22 shows the decrease of HCV in whole blood that was incubated at 37° C. Again, it is shown that when combining DMAA, EDTA and OPH with sugar alcohols, the HCV nucleic acid level is stabilized, indicated by a slowed decline in viral RNA levels, for three days at 37° C. ⁇ Ct between day 0 and day 3 is reduced ( ⁇ Ct of approximately 1) compared to the EDTA blood reference ( ⁇ Ct of approximately 2-3). Furthermore, good stabilizing effects were achieved for Sorbitol in combination with Inositol.
  • All stabilized blood samples were set up in triplicates per condition and test time point. At time point 0 (reference), immediately after mixing the stabilization solution and blood, plasma was generated and the ccfDNA was extracted. The residual blood sample was stored for three days and six days at room temperature. As a control, the EDTA stabilized blood sample was also stored for 3 and 6 days. The plasma was generated from the stabilized and unstabilized (EDTA) blood samples by inverting the blood containing tubes for four times. Then, the tubes were centrifuged for 15 minutes at 3.000 rpm/1912 ⁇ g. 2.5 ml of the plasma fraction was transferred into a fresh 15 ml falcon tube and centrifuged for 10 minutes at 16.000 ⁇ g. 2 ml of the respectively cleared plasma was used for isolating the extracellular nucleic acid using the QIAamp circulating nucleic acid kit.
  • EDTA stabilized and unstabilized
  • FIGS. 23 and 24 Shown is the increase of DNA relative to time point 0 with 2.5%, 5% and 7.5% N,N dimethylpropanamide or 5% DMAA (fold change) using different amplicon lengths of 18SrRNA gene. Bars indicate the mean of the triplicate samples per condition and test time point. All solutions according to the present inventions show significantly lower amounts of released DNA after storage for 3 and 6 days at room temperature compared to the unstabilized EDTA blood.
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