Fixation and Isolation of Extracellular Nucleic Acids

Abstract

The present invention provides methods, compositions and devices for fixing extracellular nucleic acid populations in cell-containing biological samples using polyoxyethylene polymers or ethylene glycol as fixatives.

Description

[0001] This application is a divisional application of the Chinese invention patent application with application number 201580014181.6 filed on March 18, 2015 and invention name “Fixation and separation of extracellular nucleic acids”. Technical Field

[0002] The present technology relates to methods and compositions for stablizing extracellular nucleic acid populations in cell-containing samples, especially blood samples, and to methods for isolating extracellular nucleic acids from corresponding fixed biological samples. Background Art

[0003] Extracellular nucleic acids have been identified in blood, plasma, serum and other body fluids. Due to the fact that they are protected from nuclease destruction (for example, because they are secreted in the form of protein lipid complexes, bound to proteins or contained in vesicles), the extracellular nucleic acids found in each sample are difficult to degrade to a certain extent. The presence of extracellular nucleic acids such as DNA and / or RNA with elevated levels in many medical conditions, malignancies and infection processes is of particular concern for screening, diagnosis, prognosis, monitoring disease progression, for identifying potential therapeutic targets, and for monitoring therapeutic responses. In addition, fetal DNA / RNA elevated in maternal blood is used for, for example, sex identification, assessment of chromosomal abnormalities and monitoring complications associated with pregnancy. Therefore, extracellular nucleic acids are particularly useful in non-invasive diagnosis and prognosis, and can be used in many application areas, such as non-invasive prenatal genetic testing, tumors, transplant medicine or many other diseases as, for example, diagnostic markers, and therefore are diagnostically relevant (for example, nucleic acids from fetuses or tumors). However, extracellular nucleic acids have also been found in healthy humans. Conventional applications and analysis methods for extracellular nucleic acids are described in, for example, WO97 / 035589; WO97 / 34015; Swarup et al., FEBS Letters 581 (2007) 795-799; Fleischhacker Ann. NY Acad. Sci. 1075: 40-49 (2006); Fleischhacker and Schmidt, Biochmica et Biophysica Acta, 1775, (2007), 191-232; Hromadnikova et al. (2006) DNA and Cell biology, vol. 25, no. 11, pp. 635-640; Fan et al. (2010), Clinical Chemistry, 56: 8.

[0004] Extracellular nucleic acids are usually only contained in samples at low concentrations. For example, free circulating nucleic acids are present in plasma at a concentration of 1-100ng / ml plasma. In addition, extracellular nucleic acids often circulate in fragments of 500nt, 300nt (when indicating size and chain length therefrom, the term "nt" also includes "bp", in the case of DNA) or even smaller (circulating nucleosomes). For ccfDNA in plasma, the average length is usually only about 140-170bp. In addition, it is assumed that the actual target extracellular nucleic acids identified for diagnostic purposes also usually represent only a small part of the total extracellular nucleic acids. For ccfDNA, usually only a few thousand copies of amplifiable copies are present in each milliliter of blood, depending on, for example, pregnancy status or tumor grade. In particular, tumor-specific DNA fragments are very rare, and the concentration contained is often 1000 times less than the "normal" extracellular nucleic acid background. This low concentration brings challenges to the fixation of samples and the subsequent separation of extracellular nucleic acids from fixed samples.

[0005] In addition to the degradation that occurs in serum and possibly also in plasma, a major problem with analyzing circulating, cell-free nucleic acids (cfNA) from, for example, tumor or fetal origin is the possible dilution of the extracellular DNA (and RNA) by genetic material from damaged or destroyed cells after sample collection. After the sample is collected, cellular nucleic acids are released from the cells contained in the sample due to cell disruption during in vitro incubation, especially within a relatively short time after sample collection. Once cell lysis begins, the lysed cells release a large number of other nucleic acids, which mix with the extracellular nucleic acids, and it becomes increasingly difficult to recover the extracellular nucleic acids for detection. For blood samples, in particular, the lysis of white blood cells is a problem because they release a large amount of genomic DNA and RNA. Red blood cells do not contain genomic DNA. Therefore, fixation of circulating nucleic acids in whole blood must include a mechanism for fixing blood cells in order to prevent contamination of the extracellular nucleic acid population with cellular genomic DNA and RNA during fixation. In particular, the dilution of extracellular nucleic acids, especially of rare targets, is a problem and must be prevented. These issues are discussed in the prior art (see, e.g., Chiu et al. (2001), Clinical Chemistry 47:9 1607-1613; Fan et al. (2010) and US2010 / 0184069). In addition, due to degradation, the amount and recyclability of available extracellular nucleic acids can be substantially reduced over a period of time. In addition to mammalian extracellular nucleic acids from, for example, tumor cells or fetuses, cell-containing samples may also include other nucleic acids of interest that are not contained within the cells. Important, non-limiting examples are pathogen nucleic acids, such as viral nucleic acids. The protection of the integrity of viral nucleic acids in cell-containing samples, such as blood specimens in particular, during transportation and handling is also critical for subsequent analysis and viral load monitoring.

[0006] After sample collection, the release of intracellular nucleic acids is particularly problematic if the sample contains a large number of cells, such as in the case of whole blood samples. Therefore, in order to avoid and reduce the above problems, it is common practice to separate the substantially cell-free portion of the sample from the cells contained in the sample substantially immediately after obtaining the sample. For example, it is recommended to obtain plasma from whole blood substantially immediately after blood drawing and / or cool the whole blood and / or the obtained plasma or serum to maintain the integrity of the extracellular nucleic acids and avoid contamination of the extracellular nucleic acid population by intracellular nucleic acids released from the containing cells. However, obtaining the substantially cell-free portion of the sample is problematic, and separation is often a tedious and time-consuming multi-step process, because it is important to use carefully controlled conditions to prevent cell fragmentation during centrifugation (cell nucleic acids released during the fragmentation process can contaminate extracellular nucleic acids). As for sample processing, the need to separate directly from blood such as plasma is also a major drawback, because suitable equipment is not necessarily available at the site where the sample is collected. In addition, it is often difficult to remove all cells. Therefore, many processed samples that are usually and generally classified as "cell-free", such as plasma or serum, actually still contain residual amounts of cells that were not removed during the separation process. These cells may also be destroyed or may die during sample processing, thereby releasing intracellular nucleic acids, particularly genomic DNA, as described above. These residual cells also bring risks, they are destroyed during processing, so that their nucleic acid components, particularly genomic (nuclear) DNA and cytoplasmic RNA, will be partially merged with the extracellular circulating nucleic acid to contaminate and dilute them separately. In order to remove these residual cells and avoid / reduce the above-mentioned problems, it is well known to perform a second centrifugation step at a higher speed. However, again, such powerful centrifuges are often unavailable in facilities where blood is obtained. In addition, even if plasma is obtained directly after blood drawing, if nucleic acids cannot be directly separated, it is recommended to freeze at -80°C to preserve the nucleic acids contained therein. This also imposes practical limitations on the processing of samples such as plasma samples that must be transported frozen. This increases costs, and there is a risk of sample destruction when the cold chain is interrupted.

[0007] For blood fixation, the following techniques are known in the art: blood samples are usually collected in blood collection tubes containing spray-dried or liquid EDTA (such as BD Vacutainer K2EDTA). EDTA chelates magnesium, calcium and other divalent metal ions, thereby inhibiting enzyme reactions, such as, for example, blood coagulation or DNA degradation due to DNA enzymes. However, EDTA cannot effectively prevent the extracellular nucleic acid group from being diluted and contaminated by the released intracellular nucleic acids during storage. Therefore, the extracellular nucleic acid group found in the cell-free part of the EDTA-fixed sample changes during storage and is contaminated by a large amount of intracellular nucleic acids, particularly genomic DNA. Therefore, in particular, EDTA cannot fully fix the extracellular cell group because it cannot avoid the extracellular cell group from being contaminated by genomic DNA fragments such as those produced by cell degradation after blood collection and cell instability during sample transportation and storage.

[0008] It is known that blood collection tubes contain reagents for directly fixing RNA gene expression profiles and transcriptomes therefrom at the sample collection point (see, for example, US 6,617,170, US 7,270,953, Kruhoffer et al., 2007). However, these methods are based on the immediate lysis of cells contained in the sample. Therefore, these methods and other methods of inducing cell lysis are not suitable for fixing the extracellular nucleic acid groups in the cell-containing sample, so they induce the release of intracellular nucleic acids, thereby mixing with the extracellular nucleic acid groups.

[0009] Furthermore, methods for fixing cell-containing samples, such as blood or tissue samples, are known in the prior art, such as cells, transcriptomes, genomes and proteomes. Such methods are disclosed in, for example, WO 2008 / 145710. The methods are based on the use of specific fixing compounds, such as, for example, N,N-dimethylacetamide. However, N,N-dimethylacetamide is a toxic reagent. Therefore, it is necessary to provide other fixing methods that avoid the use of toxic reagents.

[0010] Methods specifically aimed at fixing extracellular nucleic acids contained in whole blood are known in the prior art. One method uses formaldehyde to fix cell membranes to reduce cell lysis, and in addition, formaldehyde inhibits nucleases. Corresponding methods are described in, for example, US7,332,277 and US7,442,506. To meet the needs of simultaneous cell fixation and nucleic acid fixation, a fixation system has been developed based on the use of a formaldehyde releaser. The corresponding fixative is commercially available from Streck, Inc., named Cell-free RNA BCT (Blood Collection Tube). At room temperature, a 10 ml blood collection tube is used to preserve and fix cell-free RNA in plasma for up to 3 days. The preservative fixes the cell-free RNA in the plasma, preventing the release of non-target background RNA from blood cells during sample processing and storage. US2011 / 0111410 describes the use of a formaldehyde-releasing component to obtain cell and DNA fixation in the same blood sample. Therefore, the technology described in this document, wherein the fixing agent fixes the blood cells in the blood sampling, thereby prevents the cellular RNA from being polluted by cell-free RNA or globulin RNA, suppresses RNA synthesis for at least 2 hours, and the cellular RNA in the blood cells is retained to keep the protein expression spectrum of the blood cells substantially unchanged with the blood sampling time. Leukocytes can be separated from the corresponding fixed samples, and then the cellular RNA is extracted from the leukocytes. However, using formaldehyde or releasing formaldehyde substances has defects, because they are through the effectiveness of the cross-linking damage of the extracellular nucleic acid separation between the inducing nucleic acid molecules or between the protein and the nucleic acid. The method for fixing the blood sample is also described in such as US 2010 / 0184069 and US 2010 / 0209930.

[0011] PCT / EP2012 / 070211 and PCT / EP2012 / 068850 describe different methods for fixing extracellular nucleic acid populations in cell-containing biological samples, such as whole blood samples. The fixation compositions described in these applications are effective for fixing extracellular cell populations, in particular by preventing the release of intracellular nucleic acids into the extracellular nucleic acid population.

[0012] There is a continuing need to develop and improve methods for obtaining fixed extracellular nucleic acid groups contained in cell-containing biological samples, including samples suspected of containing cells, particularly whole blood, plasma or serum, so as to make it easier to handle and dispose of such samples accordingly. By providing an effective and reliable sample fixation technique that preferably does not impair subsequent nucleic acid separation, the separation and detection of extracellular nucleic acids contained in such samples become more reliable, and therefore, the diagnosis and prognostic application / use of extracellular nucleic acids is improved by such fixation techniques. In particular, there is a continuing need for a scheme for preserving whole blood samples, for example, for prenatal examinations and / or for screening diseases, such as tumors, particularly pre-malignant or malignant diseases.

[0013] The object of the present invention is to provide methods and compositions for fixing extracellular populations contained in cell-containing samples. In particular, the object is to overcome at least one drawback of the prior art sample fixation methods. In addition, a particular object of the present invention is to provide a method suitable for fixing cell-containing biological samples, in particular whole blood samples, at room temperature. Further, the object of the present invention is to provide a sample collection container, in particular a blood collection tube, which can effectively fix cell-containing biological samples, and in particular can fix the extracellular nucleic acid population contained in the sample. In addition, an object of the present invention is to provide a fixation technology that subsequently allows the separation of extracellular nucleic acids from the fixed sample with a good yield. Summary of the invention

[0014] The present invention is based on the unexpected discovery that polyoxyethylene polymers, such as polyethylene glycol, can effectively fix cell-containing biological samples containing extracellular nucleic acids, particularly whole blood samples. It is found that polyoxyethylene polymers of different molecular weights and various concentrations, such as polyethylene glycol, can fix the extracellular nucleic acid population of samples containing cells, and can particularly reduce the risk of extracellular cell populations being contaminated by genomic DNA, particularly fragmented genomic DNA, after collecting the sample. The use of polyoxyethylene polymers, such as polyethylene glycol, as a fixative reduces the risk of extracellular nucleic acid populations being diluted by intracellular nucleic acids, such as particularly genomic DNA, which significantly helps to retain the atlas of extracellular nucleic acid populations during sample collection. The fixation effect is stronger than the effect of using other fixatives. In addition, it is found that polyoxyethylene polymers, such as preferably polyethylene glycol, can be conveniently used in combination with other fixatives, such as caspase inhibitors and amides, to further improve the fixation effect on cell-containing samples. Advantageously, polyoxyethylene polymers, such as preferably polyethylene glycol, are not classified as toxic, harmful or irritating agents. In a preferred embodiment, two or more polyoxyethylene alcohols (poly(oxyethylene)glycol) with different molecular weights are used. Preferably, a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 is used in conjunction with a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less for fixation. It has been found that a moderate combination of polyoxyethylene polymers of different molecular weights, such as the use of high and low molecular weight polyoxyethylene polymers, provides an effectively fixed sample from which extracellular nucleic acids can subsequently be isolated in good yield using a variety of standard nucleic acid isolation methods. In addition, the use of ethylene glycol (1,2-ethylene glycol) as a fixative for cell-containing samples is described herein. Ethylene glycol can also be used in conjunction with polyoxyethylene polymers for fixation.

[0015] According to a first aspect, a method suitable for fixing an extracellular nucleic acid population contained in a cell-containing biological sample is provided, comprising the step of contacting the cell-containing sample with at least one polyoxyethylene polymer as a fixative or ethylene glycol as a fixative.

[0016] According to a second aspect, a method for isolating extracellular nucleic acids from a cell-containing biological sample is provided, wherein the method comprises the steps of:

[0017] a) fixing a cell-containing biological sample according to the method defined in the first aspect of the present invention; and

[0018] b) Isolation of extracellular nucleic acids from fixed samples.

[0019] According to a third aspect, there is provided a composition suitable for fixing a biological sample containing cells, comprising:

[0020] i) polyoxyethylene polymers as fixatives, or

[0021] ii) ethylene glycol as a fixative,

[0022] and one or more, preferably two or more, other additives selected from the following group:

[0023] - one or more primary, secondary or tertiary amides;

[0024] - Caspase inhibitors;

[0025] - Anticoagulants and / or chelating agents.

[0026] Preferably, the composition according to the third aspect comprises a polyoxyethylene polymer as a fixative, which is preferably a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500, and further comprises one or more, preferably two or more other auxiliary agents selected from the group consisting of:

[0027] - at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first polyoxyethylene polymer, which is preferably a high molecular weight polyoxyethylene polymer, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less;

[0028] - one or more primary, secondary or tertiary amides;

[0029] - Caspase inhibitors;

[0030] - Anticoagulants and / or chelating agents.

[0031] By fixing the cells and extracellular nucleic acid groups contained in the sample, the corresponding fixed composition is particularly effective in fixing cell-containing biological samples, such as blood, blood plasma and / or serum. During fixation, the extracellular nucleic acid group contained in the cell-containing biological sample is basically maintained in the state displayed when the biological sample contacts with the fixed composition. The release of genomic DNA and other intracellular nucleic acids is significantly reduced, as shown in the embodiments. Compared with the extracellular nucleic acid separated from the unfixed sample, the extracellular nucleic acid separated from each fixed sample comprises significantly less intracellular nucleic acid, particularly fragmented genomic DNA pollution. Each fixed composition allows storage and / or processing at room temperature, such as transporting fixed biological samples, such as blood for several days, without substantial damage to the sample, including the quality of each extracellular nucleic acid group therein.

[0032] According to a fourth aspect, the composition according to the third aspect of the present invention is used for fixing an extracellular nucleic acid population in a cell-containing biological sample, preferably a blood sample.

[0033] According to a fifth aspect, a collecting device for collecting a biological sample containing cells is provided, wherein the collecting device comprises:

[0034] i) polyoxyethylene polymers as fixatives, or

[0035] ii) ethylene glycol as a fixative,

[0036] and one or more other adjuvants selected from the group consisting of:

[0037] - one or more primary, secondary or tertiary amides;

[0038] - Caspase inhibitors;

[0039] - Anticoagulants and / or chelating agents.

[0040] Preferably, the collecting device according to the fifth aspect comprises a polyoxyethylene polymer as a fixing agent, which is preferably a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500, and further comprises one or more, preferably two or more, other auxiliary agents selected from the group consisting of:

[0041] - at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first polyoxyethylene polymer, which is preferably a high molecular weight polyoxyethylene polymer, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less;

[0042] - one or more primary, secondary or tertiary amides;

[0043] - Caspase inhibitors;

[0044] - Anticoagulants and / or chelating agents.

[0045] A collection device for collecting a cell-containing biological sample, preferably a blood sample, may include the fixing composition according to the third aspect of the present invention. The advantage of providing a corresponding collection device, such as a sample collection tube, including the fixing composition is that the cell-containing biological sample is immediately fixed as soon as the sample is collected into the corresponding device.

[0046] According to a sixth aspect, a method is provided, comprising the step of collecting, preferably drawing, a biological sample, preferably blood, from a patient directly into the container of the fifth aspect of the invention.

[0047] According to a seventh aspect, a method for producing the composition of the third aspect of the present invention is provided, wherein the components of the composition are mixed, preferably mixed in solution. The term "solution" as used herein refers in particular to a liquid composition, preferably an aqueous composition. It can be a homogeneous mixture of only one phase, but it is also within the scope of the present invention that the solution includes solid components, such as a precipitate.

[0048] From the following description and the appended claims, other objects, features, advantages and aspects of the present invention will become more apparent to the present invention and the skilled person. However, it should be understood that the following description, the appended claims and the specific examples, which indicate the preferred embodiments of the present application, are given only by way of illustration. DETAILED DESCRIPTION

[0049] The present invention relates to methods, compositions and devices for fixing extracellular nucleic acid groups contained in cell-containing biological samples based on the use of polyoxyethylene polymers as fixatives and the technology thereof. Polyoxyethylene polymers of different molecular weights and various concentrations such as polyethylene glycol have been shown to be effective as fixatives. In addition, advantageous combinations with other fixatives are described. The fixation obtained by the method and composition of the present invention enables long-term storage and / or processing of fixed samples at room temperature.

[0050] The fixation technique described herein reduces the extracellular nucleic acid group included in the sample containing cells and is derived from the intracellular nucleic acid of the broken and / or dead cells contained in the sample, particularly the fragment genomic DNA pollution, and therefore the risk of being diluted. Because the composition of the extracellular nucleic acid group is fixed and therefore substantially preserved when obtaining the sample, the sample collection and nucleic acid separation interval can be changed in a suitable fixing period, and there is no obvious negative effect on the composition of the extracellular nucleic acid group. This is an important advantage, because it reduces the extracellular nucleic acid group variability caused by different treatment procedures. Compared with the extracellular nucleic acid separated from the unfixed sample, the extracellular nucleic acid separated from the corresponding fixed sample includes significantly less intracellular nucleic acid, particularly the fragment genomic DNA pollution. The fixation technique described improves the standard of diagnosis or prognosis of extracellular nucleic acid analysis, because the change of the processing / storage of the sample has less influence on the quality of the extracellular nucleic acid group contained in the cell-containing biological sample, correspondingly composition and the collection of illustrative plates thus, so that the diagnosis or prognosis application based on extracellular nucleic acid is more reliable and more independent of the storage / processing conditions used. The substantial preservation obtained is an important advantage, because it improves the accuracy of any subsequent test intended to analyze extracellular nucleic acid. Thus, the diagnosis and prognosis applicability of the extracellular nucleic acid of separation separately are improved.The specific embodiment described herein has the advantage that the ratio of some extracellular nucleic acid molecules included in the extracellular nucleic acid group can remain constant, and the ratio existing during the collection of biological specimen is more suitable thus.Therefore, advantageously, the collection of illustrative plates of the extracellular nucleic acid group can be preserved.Therefore, the corresponding cell-containing biological specimen, the analysis of the extracellular nucleic acid particularly obtained from the corresponding fixed sample becomes more comparable.

[0051] In addition, the teaching of the present invention excludes the necessity of directly separating the cells contained in the biological sample from the cell-free part of the sample after the sample is collected in order to avoid the corresponding reduction of the intracellular nucleic acid, particularly the contamination of fragmented genomic DNA, which is released by the dead or degenerated cells during storage / transportation. This advantage significantly simplifies the processing of biological samples containing cells, such as whole blood samples. However, when processing cell-eliminating biological samples, or samples commonly referred to as "cell-free", such as blood plasma or serum, the teaching of the present invention is also advantageous. The corresponding cell-eliminating or "cell-free" biological samples may also include (depending on the separation method used) residual cells, particularly leukocytes, which contain genomic DNA. The residual cells cause risks, and if (possibly) the residual cells are broken or dead during transportation or storage, the extracellular nucleic acid group becomes more and more polluted by intracellular nucleic acid, particularly fragmented genomic DNA. When the fixation method taught by the present invention is adopted, this risk is greatly reduced. Therefore, the present invention has many advantages when fixing biological samples containing a large number of cells, such as blood samples, and the present invention also has important advantages when fixing biological samples containing fewer or only a small number of cells or may only be suspected of containing cells, such as, for example, plasma, serum, urine, saliva, synovial fluid, amniotic fluid, tears, lymph fluid, alcohol, cerebrospinal fluid, etc.

[0052] Furthermore, the use of ethylene glycol for fixing an extracellular nucleic acid population in a cell-containing sample is described, wherein, optionally, the ethylene glycol is used in combination with a polyoxyethylene polymer and / or one or more other fixatives described herein.

[0053] A. Fixation Method

[0054] According to a first aspect, a method for fixing an extracellular nucleic acid population contained in a cell-containing biological sample is provided, comprising the step of contacting the cell-containing biological sample with at least one polyoxyethylene polymer as a fixative or with ethylene glycol as a fixative. The advantages of using a polyoxyethylene polymer as a fixative are described above.

[0055] Preferably, the method described in the first aspect includes the step of contacting the cell-containing biological sample with at least one polyoxyethylene polymer as a fixative. The term polyoxyethylene polymer refers in particular to an oligomer or polymer of ethylene oxide. It includes at least two ethylene oxide units. Low and high molecular weight polyoxyethylene polymers are known. Their molecular weight is usually a multiple of 44 of the molecular weight of its monomer, and can be as high as 100,000. The molecular weight is expressed in Da. The polyoxyethylene polymer may be linear or branched, or have other geometric configurations. Linear polyoxyethylene polymers are preferred. The polyoxyethylene polymer may be unsubstituted or substituted, preferably polyethylene glycol. As demonstrated in the embodiments, polyethylene glycol with various molecular weights and various concentrations has a cell fixation effect, and therefore can be used alone or in combination with other fixatives to fix the extracellular nucleic acid population in the cell-containing sample, in particular, by reducing the dilution of the extracellular nucleic acid population by intracellular nucleic acids such as, in particular, genomic DNA. However, other polyoxyethylene polymers may also be used to obtain the fixation effect shown by polyethylene glycol. As mentioned, it is also possible to use substituted polyoxyethylene polymers with a fixing effect, such as alkyl polyoxyethylene polymers, such as alkyl polyethylene glycols, and poly(oxyethylene) esters, poly(oxyethylene) amines, poly(oxyethylene) thiol compounds, poly(oxyethylene) glycerides and others. A preferred embodiment of the polyoxyethylene polymer used as a fixing agent is polyethylene glycol. It is preferably unbranched and can be unsubstituted or substituted. Known substituted forms of polyethylene glycol include alkyl polyethylene glycols, for example, which are substituted at one or both ends by C1-C5 alkyl groups. Preferably, the formula HO-(CH2CH2O) is used. n -H. Even if not explicitly stated, all disclosures describing polyoxyethylene polymers in this application generally specifically apply to and specifically refer to the preferred embodiment polyethylene glycol. Polyoxyethylene polymers of various molecular weights can be used. Preferably, the term polyethylene glycol refers to an oligomer or polymer, as indicated by the molecular weights specified herein as suitable and preferred for polyoxyethylene polymers, which also specifically apply to the preferred embodiment polyethylene glycol.

[0056] The relationship between the fixing effect of polyoxyethylene polymer and its molecular weight has been found. It has been found that higher molecular weight polyoxyethylene polymer is a more effective fixing agent compared with lower molecular weight polyoxyethylene polymer. For obtaining effective fixation with lower molecular weight polyoxyethylene polymer, it is usually recommended to have a higher concentration than that of higher molecular weight polyoxyethylene polymer. But for some applications such as blood samples, it is preferred to keep a low amount of fixing aid. Therefore, in an embodiment, higher molecular weight polyoxyethylene polymer is used as fixing agent, because they allow the use of lower concentration polyoxyethylene polymer, while producing a strong fixing effect to the extracellular nucleic acid group.

[0057] Therefore, according to one embodiment, a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 is used as a fixative and contacted with a sample containing cells. The molecular weight of the high molecular weight polyoxyethylene polymer is selected from the following ranges: 1500-50000, 1500-40000, 2000-40000, 3000-40000, 2000-30000, 2500-30000, 2500-25000, 3000-20000, 4000-20000, 3500-15000, 3000-10000, 4000-10000, 4500-10000, 4500-9000, 4500-8000, 5000-8000, 5000-7000 and 5500-7000. As shown in the examples, many different high molecular weight polyoxyethylene polymers can be used in conjunction with the present invention. In conjunction with different aspects and embodiments of the present invention, suitable high molecular weight polyoxyethylene polymers have also been described. For polyoxyethylene glycol, particularly the use of unsubstituted polyethylene glycol, these molecular weights are particularly preferred. Unsubstituted polyethylene glycol is also used in the examples. The molecular weight of the polyoxyethylene polymer with a specific molecular weight can change within a certain manufacturing condition range, which is well known to those skilled in the art.

[0058] The concentration of high molecular weight polyoxyethylene polymer used produces or supports the fixation of extracellular nucleic acid groups contained in cell-containing samples. Suitable concentrations for different sample types can be determined by those skilled in the art, for example, by testing the concentration of a specific high molecular weight polyoxyethylene polymer in a test experiment described in the examples. As shown in the examples, high molecular weight polyoxyethylene polymers are effective at various concentrations. The fixation effect obtained and the preferred concentration also depend on whether one or more other fixatives are used. The preferred combination is described herein. According to one embodiment, the mixture obtained after contacting a cell-containing biological sample with a high molecular weight polyoxyethylene polymer and optional other auxiliary agents includes a high molecular weight polyoxyethylene polymer in a concentration range selected from the following group: 0.05%-4% (w / v), 0.1%-3% (w / v), 0.2%-2.5% (w / v), 0.25%-2% (w / v), 0.3%-1.75% (w / v) and 0.35%-1.5% (w / v). According to one embodiment, the high molecular weight polyoxyethylene polymer is used in a lower concentration range, such as 0.25%-1.5% (w / v), 0.3%-1.25% (w / v), 0.35%-1% (w / v) and 0.4%-0.75% (w / v). The above concentration range is particularly suitable for fixing blood. It is advantageous in certain embodiments to use the high molecular weight polyoxyethylene polymer at a concentration of 1.5% (w / v) or less, 1.25% (w / v) or less, 1% (w / v) or less, and particularly at 0.75% (w / v) or less. In embodiments where the high molecular weight polyoxyethylene polymer is used in a higher concentration for the resulting mixture comprising a fixative and a sample containing cells, such as a blood sample, it is found that when using certain standard nucleic acid separation procedures involving, for example, the use of a silica gel column, the subsequent separation of extracellular nucleic acids may be impaired. However, this is advantageous for using a fixation technique that matches most nucleic acid separation methods, and the use of a silica gel column to separate extracellular nucleic acids is widely used and established. High molecular weight polyoxyethylene polymers are used to fix the mixture containing the sample at a concentration of 1.5% (w / v) or less, 1.25% (w / v) or less, 1% (w / v) or less, or 0.75% or less to support the use of this standard method to effectively separate extracellular nucleic acids from the fixed sample with a good yield, even if a higher volume of fixed composition is used. This is advantageous because extracellular nucleic acids, particularly specific target nucleic acids contained in the extracellular nucleic acid population, often exist only with very few copies. The fixation techniques described herein are also applicable to other nucleic acid separation methods, including those based on ion exchange. As shown in the examples, the damage observed also depends on the volume of the fixed composition containing the high molecular weight (polyoxyethylene) polymer. Using a smaller volume of fixed composition for fixation can compensate for damage, even if a high molecular weight polyoxyethylene polymer is used at a higher concentration, so that subsequent nucleic acid separation is not impaired.Therefore, to reduce or even avoid damage, reducing the total concentration of high molecular weight polyoxyethylene polymer in the mixture containing the sample and / or reducing the volume of the fixing composition containing the high molecular weight polyoxyethylene polymer is an option. The volume-dependent effect shown in the examples is significant and very surprising, because the total concentration in the mixture containing the sample is the same.

[0059] According to one embodiment, the molecular weight of the polyoxyethylene polymer used for fixation is lower than 1500, and it may be a low molecular weight polyoxyethylene polymer with a molecular weight of 1000 or lower. It is used at a concentration that can produce or support a fixation effect on the extracellular nucleic acid group of a cell-containing biological sample. The appropriate concentration for different sample types is determined by those skilled in the art, such as by testing different concentrations in the test experiments described in the embodiments. The corresponding polyoxyethylene polymer, such as a low molecular weight polyoxyethylene polymer with a molecular weight of 1000 or lower, can be present in a mixture obtained after contacting a cell-containing biological sample with the polyoxyethylene polymer and optional other auxiliary agents in a concentration range selected from the following group: 0.5%-10%, 1.5%-9%, 2%-8%, 2-7%, 2.5%-7% and 3%-6%. When the polyoxyethylene polymer is solid, the percentage value refers to (w / v), and when the polyoxyethylene polymer is liquid, the percentage value refers to (v / v). The concentration shown is particularly suitable for the case of using blood samples. To obtain (or support) fixation of the extracellular nucleic acid population, a higher concentration of at least 1%, preferably at least 1.5%, of the low molecular weight polyoxyethylene polymer is advantageous. As shown in the examples, polyoxyethylene polymers such as polyethylene glycol with a molecular weight of 1500 or less, such as 1000 or less, also exhibit a fixation effect on the extracellular nucleic acid population, particularly by reducing dilution by genomic DNA. The examples show that polyoxyethylene polymers such as polyethylene glycol with a molecular weight of 1500 or less, such as 1000 or less, are effective fixatives, particularly when used in combination with one or more other fixatives described herein, such as caspase inhibitors and at least one primary, secondary, or tertiary amide. The molecular weight range of the low molecular weight polyoxyethylene polymer is selected from: 100-1000, 150-800, 150-700, preferably 200-600 and more preferably 200-500, such as 200-400.

[0060] It is found that molecular weight is 1000 or lower, preferably 800 or 700 or lower low molecular weight polyoxyethylene polymer can be used with substantially higher concentration, because it does not hinder the subsequent separation of extracellular nucleic acid from fixed sample basically, even if adopts the fixed composition of higher volume.But, if the amount that needs to obtain fixed effect is too large, this is inconvenient for the processing and handling of sample.Usually preferably adopt quite low amount or volume of fixative fixed sample.This especially, for some sample such as blood sample, the amount of fixative that can be added to sample is limited to the standard collection tube used.For example, for the standard collection device for collecting 100 milliliters of blood, can add maximum amount of about 2 milliliters of fixative.

[0061] According to one embodiment, at least two polyoxyethylene polymers are used for fixing, and their molecular weights are different. They may be of the same type, preferably all are polyethylene glycol, such as unsubstituted polyethylene glycol. According to one embodiment, the difference between molecular weights is at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000. According to one embodiment, the difference between molecular weights is at least 2500, at least 3500, at least 5000 or at least 7500. As described in the specific embodiment of this embodiment subsequently, it is advantageous to use two polyoxyethylene polymers of different molecular weights. As described herein, the fixing effect of polyoxyethylene polymers seems to depend on their molecular weight. In the test example, it is found that the higher the molecular weight, the better the fixing effect. However, according to their molecular weight, polyoxyethylene polymers may have different effects on subsequent nucleic acid separation methods. As described above, it has been found that in certain embodiments involving the use of higher molecular weight polyoxyethylene polymers as fixatives, nucleic acid separation efficiency using certain nucleic acid separation methods is low, particularly when higher volumes of fixative compositions and higher concentrations of polymers are used in the fixative mixture containing the sample. As shown in the Examples, these problems that occur in certain situations can be overcome when mixtures of polyoxyethylene polymers of different molecular weights are used. Therefore, embodiments using at least two polyoxyethylene polymers of different molecular weights are advantageous because they can provide a balanced composition of polyoxyethylene polymers having desired characteristics associated with the fixation effect to be obtained and, for example, desired characteristics for certain downstream uses.

[0062] According to one embodiment, a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 as defined above is used in combination with a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, and a sample containing cells is contacted with the two types of polyoxyethylene polymers. Suitable embodiments are described herein. It is advantageous to use a high molecular weight polyoxyethylene polymer in combination with a low molecular weight polyoxyethylene polymer, because the low molecular weight polyoxyethylene polymer can reduce the concentration of the high molecular weight polyoxyethylene polymer required for obtaining effective fixation of the sample. Therefore, the high molecular weight polyoxyethylene polymer can be used in a mixture with the sample at a concentration that does not damage the subsequent nucleic acid separation using certain standard methods such as those involving silica gel columns. This embodiment is advantageous because it provides more freedom for the volume or amount of the fixed composition used. The low molecular weight polyoxyethylene polymer helps fixation, but compared with the high molecular weight polyoxyethylene polymer, no significant damage to subsequent nucleic acid separation is found in the test sample, when the method is used, such as those involving silica gel columns, the higher molecular weight polyoxyethylene polymer in the embodiment shows a damaging effect at certain concentrations and / or volumes. The use of a combination of high and low molecular weight polyoxyethylene polymers to fix the extracellular nucleic acid population in a cell-containing sample is a particularly preferred embodiment and has important advantages. The low molecular weight polyoxyethylene polymer can be of the same type as the high molecular weight polyoxyethylene polymer described above. And for the low molecular weight polyoxyethylene polymer, polyethylene glycol, such as unsubstituted polyethylene glycol, is preferably used. The suitable molecular weight of the high molecular weight polyoxyethylene polymer is as described above. Its molecular weight range is selected from the following group: 1500-50000, 2000-40000, 2500-30000, 2500-25000 and 3000-20000. The molecular weight range of the low molecular weight polyoxyethylene polymer is selected from the following group: 100-1000, 150-800, 150-700, preferably 200-600 and particularly preferably 200-500, such as 200-400. The suitable and preferred concentrations and concentration ranges of high and low molecular weight polyoxyethylene polymers are as described above, and can also be used in the embodiment in which the two types of polymers are used in combination.

[0063] According to one embodiment, the mixture obtained after the cell-containing biological sample is preferably blood and high and low molecular weight polyethylene oxide polymers and optional other auxiliary agents for fixation include a high molecular weight polyethylene oxide polymer with a molecular weight range of 3000-40000, preferably 4000-20000, a concentration range of 0.2%-1.5% (w / v), preferably 0.3%-1.25% (w / v), and in an embodiment, a range of 0.4 (w / v)-0.75% (w / v); and a low molecular weight polyethylene oxide polymer with a molecular weight range of 200-800, preferably 200-600, a concentration range of 1.5%-8%, preferably 2%-7%, and more preferably 2.5%-6%. The high and low molecular weight polyethylene oxide polymers are preferably polyethylene glycol, such as unsubstituted polyethylene glycol. Suitable embodiments of high and low molecular weight polyethylene oxide polymers are also described above and combined with different aspects and embodiments. In this embodiment, the cell-containing biological sample may be blood, and the blood sample is also contacted with an anticoagulant. Suitable examples of anticoagulants are described below.

[0064] As shown in the examples provided, in embodiments, the use of polyoxyethylene polymers alone, such as high molecular weight polyoxyethylene polymers, has been effective in fixing cell-containing samples and protecting extracellular nucleic acid populations from changes in their composition, particularly changes caused by contamination of fragmented genomic DNA released by damaged or dead cells during storage. However, if polyoxyethylene polymers, preferably polyethylene glycol, are used in conjunction with other fixatives and / or adjuvants, the fixation effect can be significantly improved. It was found that the combination of polyoxyethylene polymers with other fixatives significantly improved the fixation effect obtained. As shown in the examples, when combined with other fixatives such as amides and caspase inhibitors, the fixation effect is significantly supported and improved. This balanced composition significantly improves the fixation effect obtained, which is also superior to existing prior art. Suitable and preferred embodiments of other fixatives and innovative compositions are subsequently described.

[0065] According to one embodiment, the sample containing cells is also contacted with one or more primary, secondary or tertiary amides as other fixatives. Primary, secondary or tertiary amides have a favorable fixation effect on cell-containing samples and are therefore used to support the fixation of extracellular nucleic acid populations in one embodiment. Two or more primary, secondary or tertiary amides or combinations may also be used. The amide is preferably a carboxylic acid amide.

[0066] Suitable concentrations of different primary, secondary or tertiary amides and / or different sample types can be determined by those skilled in the art, such as by testing different concentrations in the test experiments described in the Examples. Typically, a mixture obtained when a cell-containing biological sample is contacted with at least one polyoxyethylene polymer and one or more primary, secondary or tertiary amides and optionally other adjuvants may include the amide (or a combination of amides) at a concentration of at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5% or at least 0.75%. Suitable concentration ranges include, but are not limited to, 0.1%-10%, 0.25%-7.5%, 0.3%-5%, 0.4%-3%, 0.5%-2%, 0.6%-1.8% and 0.75%-1.5%. As used herein, concentrations or concentration ranges indicated in percentages are, specifically, weight / volume (w / v) percentages for solid amides and volume / volume (v / v) percentages for liquid amides.

[0067] According to one embodiment, the primary, secondary or tertiary amide is a compound of formula 1:

[0068]

[0069] wherein R1 is hydrogen or alkyl, preferably C1-C5 alkyl, C1-C4 alkyl or C1-C3 alkyl, more preferably C1-C2 alkyl, R2 and R3 are the same or different, selected from hydrogen and hydrocarbon groups, preferably alkyl groups, having a carbon chain length of 1-20 atoms, in straight or branched form, and R4 is oxygen, sulfur or selenium, preferably R4 is oxygen.

[0070] In addition to the polyoxyethylene polymers used for immobilization, combinations of one or more compounds of Formula 1 can also be used.

[0071] In embodiments where R1 is alkyl, chain lengths of 1 or 2 are preferred for R1.

[0072] R2 and / or R3 of the compound shown in Formula 1 are the same or different and are selected from hydrogen and hydrocarbon groups, preferably alkyl groups. According to one embodiment, R2 and R3 are both hydrogen. According to one embodiment, one of R2 and R3 is hydrogen and the other is a hydrocarbon group. According to one embodiment, R2 and R3 are the same or different hydrocarbon groups. The hydrocarbon groups R2 and / or R3 can be independently selected from the following groups: alkyl, alkenyl, alkoxy, long-chain alkoxy, cycloalkyl, aryl, haloalkyl, alkylsilyl, alkylsiloxy, alkylene, alkenyl, arylene, carboxylate and carbonyl groups including short-chain alkyl and long-chain alkyl. In particular, the chain length n of R2 and / or R3 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. According to one embodiment, the carbon chain length of R2 and R3 is 1 to 10, preferably 1 to 5, more preferably 1 to 2. According to one embodiment, R2 and / or R3 is an alkyl group, preferably a C1-C5 alkyl group.

[0073] Preferably, the compound of formula 1 is a carboxylic acid amide, such that R4 is oxygen. It can be a primary, secondary or tertiary carboxylic acid amide.

[0074] According to one embodiment, the compound shown in formula 1 is an N,N-dialkyl-carboxylic acid amide. The preferred R1, R2, R3 and R4 groups are described above. The use of the corresponding compound shown in formula 1 has the advantage that, additionally, intracellular nucleic acids, such as in particular RNA, such as mRNA and / or miRNA transcripts can be fixed in a sample containing cells. The additional fixation of intracellular nucleic acids, in particular gene transcription levels, is advantageous because it allows subsequent analysis of target transcripts or transcription profiles contained in cells. According to one embodiment, the compound shown in formula 1 is selected from the group consisting of N,N-dimethylacetamide, N,N-diethylacetamide, N,N-dimethylformamide and N,N-diethylformamide. Also suitable are separate thio analogs, which include sulfur, replacing the oxygen of R4. For example, N,N-dimethylacetamide (DMAA) obtains good fixation results, but it is a toxic reagent. Preferably, at least one compound shown in formula 1 is combined with a polyoxyethylene polymer for fixing a cell-containing biological sample, which is not a toxic reagent according to the GHS classification.

[0075] According to one embodiment, a cell-containing biological sample is contacted with a polyoxyethylene polymer and a compound represented by Formula 1, which is N,N-dialkylpropionamide, such as N,N-dipropylpropionamide, for fixation. As shown in the examples, the corresponding combination is particularly suitable for fixing extracellular nucleic acid populations in cell-containing samples, such as blood samples.

[0076] According to one embodiment, a sample containing cells is contacted with a polyoxyethylene polymer and at least one compound shown in Formula 1, which is a primary or secondary carboxylic acid amide. From the unpublished US 61 / 803,107 and EP13160896.0 (see the disclosed WO 2014 / 146781) incorporated herein by reference, it can be seen that primary and secondary carboxylic acid amides can fix extracellular nucleic acid groups in a wide concentration range. According to one embodiment, the primary carboxylic acid amide is selected from the group consisting of formamide, acetamide, propionamide and butanamide. Preferably, the primary carboxylic acid is butanamide, because butanamide is particularly effective for fixing extracellular nucleic acid groups, as shown in the examples herein, and in addition, it is non-toxic. The fixation effect of butanamide on the extracellular nucleic acid group of a cell-containing sample is also described in detail in the unpublished applications EP 13159834.4 and EP 13180086.4 (see the disclosed WO 2014 / 146780 and WO 2014 / 146782), which are incorporated herein by reference.

[0077] According to a preferred embodiment, the cell-containing sample is contacted with a polyoxyethylene polymer, preferably polyethylene glycol, and butanamide and / or N,N-dialkylpropanamide for fixation, wherein the N,N-dialkylpropanamide is preferably N,N-dimethylpropanamide. The above-mentioned suitable and preferred concentrations and concentration ranges generally used for primary, secondary and tertiary amides are also generally particularly applicable to this embodiment. As shown by the examples, the use of butanamide and / or N,N-dialkylpropanamide in concentrations within these ranges provides a beneficial fixation effect for cell-containing samples such as blood samples.

[0078] According to one embodiment, the cell-containing biological sample is contacted with the polyoxyethylene polymer described above and at least one caspase inhibitor. It is also within the scope of the present invention to use a combination of different caspase inhibitors. As shown by the examples, the use of polyoxyethylene polymers, preferably high molecular weight polyethylene glycols, in combination with caspase inhibitors significantly improves the fixation effect obtained. In addition, it is found that in particular, contact with biological samples containing a large number of cells, and in addition, their compositions are different, such as blood samples, the fixation effect obtained is stronger and more uniform. The changes in the extracellular nucleic acid groups that occur during the in vitro treatment of blood samples such as blood samples from different hosts may be different. Strong changes (especially strong growth of genomic DNA) appear in the extracellular nucleic acid group profiles of some samples, while in other samples, the effect is not very prominent. Such samples react differently to fixation. When polyoxyethylene polymers are used in combination with caspase inhibitors and one or more primary, secondary or tertiary amides described above are preferably used for fixation or blood samples of different hosts, a more balanced fixation effect can be obtained. The caspase inhibitor present in the obtained mixture significantly supports the fixation of the extracellular nucleic acid group. In addition, the degradation of nucleic acids, especially genomic DNA, present in the sample is reduced by the combination of fixatives. Thus, the use of polyoxyethylene polymers such as polyethylene glycol in combination with at least one caspase inhibitor significantly improves the fixation effect, thereby supporting that the extracellular nucleic acid population contained in the sample is substantially maintained in the state it exhibited when the biological sample was obtained, respectively collected, or even stored for a long time.

[0079] Preferably, the caspase inhibitor is cell permeable. Members of the caspase gene family play a significant role in apoptosis. Substrate selection or specificity of each caspase has been used to develop polypeptides that successfully compete for caspase binding. It is possible to generate reversible or irreversible caspase activation inhibitors by connecting caspase-specific polypeptides to compounds such as aldehydes, nitriles or ketones. For example, fluoromethylketone (FMK)-derived polypeptides such as Z-VAD-FMK are effective irreversible inhibitors without added toxic effects. Inhibitors synthesized by reacting with benzyloxycarbonyl (BOC) at the N-terminus and O-methyl side chains show enhanced cell permeability. Other suitable caspase inhibitors are synthesized by reacting with phenoxy at the C-terminus. An example is Q-VD-OPh, which is a cell permeable, irreversible broad-spectrum caspase inhibitor, even more effective than the caspase inhibitor Z-VAD-FMK in preventing apoptosis and thus supporting fixation.

[0080] According to one embodiment, the caspase inhibitor is a pan-caspase inhibitor and is therefore a broad-spectrum caspase inhibitor. According to one embodiment, the caspase inhibitor comprises a modified caspase-specific polypeptide. Preferably, the caspase-specific polypeptide is modified by an aldehyde, nitrile or ketone compound. According to one embodiment, the caspase-specific polypeptide is modified, preferably at the carboxyl end, to react with an O-phenoxy or fluoromethylketone (FMK) group. According to one embodiment, the caspase inhibitor is selected from the group consisting of Q-VD-OPh and Z-VAD (OMe) -FMK. In a preferred embodiment, Q-VD-OPh, as a broad-spectrum inhibitor of caspases, is used for fixation. Q-VD-OPh is cell permeable and inhibits cell death caused by apoptosis. Q-VD-OPh is non-toxic to cells, even at fairly high concentrations, including carboxyl-terminal phenoxy groups coupled to the amino acids valine and aspartic acid. It is equivalent in inhibiting apoptosis mediated by three major apoptotic pathways, caspase-9 and caspase-3, caspase-8 and caspase-10, and caspase-12 (Caserta et al., 2003). Examples of caspase inhibitors are also listed in Table 1 of WO 2013 / 045457, incorporated herein by reference.

[0081] The mixture obtained after contacting the biological sample with at least one polyoxyethylene polymer and at least one caspase inhibitor and optionally other auxiliary agents may include a caspase inhibitor (or a combination of caspase inhibitors) at a concentration of at least 0.05 μM, at least 0.1 μM, at least 0.5 μM, at least 0.75 μM, at least 1 μM, at least 1.25 μM, at least 1.5 μM, at least 1.75 μM, at least 2 μM, at least 2.25 μM, at least 2.5 μM, at least 2.75 μM, at least 3 μM, at least 3.25 μM or at least 3.5 μM. Suitable concentration ranges of one or more caspase inhibitors when mixed with a biological sample containing cells and other auxiliary agents include, but are not limited to, 0.1 μM-25 μM, 0.75 μM-20 μM, 1 μM-15 μM, 1.5 μM-12.5 μM, 2 μM-10 μM and 3 μM-7.5 μM. The above mentioned concentrations apply to the use of a single caspase inhibitor as well as the use of a combination of caspase inhibitors. The above concentrations are particularly suitable when using pan-caspase inhibitors, particularly modified caspase-specific polypeptides such as Q-VD-OPh and / or Z-VAD(OMe)-FMK. The above concentrations are particularly suitable for fixed whole blood. The suitable concentration ranges of each caspase inhibitor and / or other cell-containing biological samples can be determined by a person skilled in the art, such as by testing each caspase inhibitor at different concentrations in the test assays described in the Examples.

[0082] The fixation effect observed with the combination of fixatives is stronger than that observed with any of the individual fixatives when used alone, and / or lower concentrations of the individual fixatives can be used, making the use of a combination of fixatives an attractive option. Combinations of fixatives including polyoxyethylene polymers such as polyethylene glycol described herein have a better fixation effect than individual fixatives, and are therefore an advantageous embodiment. In addition, other adjuvants can be used for fixation, such as anticoagulants and chelating agents, which are particularly useful when fixing blood samples.

[0083] As discussed in the background of the invention, extracellular nucleic acids are usually not present naked in the extracellular part of a cell-containing sample, but are fixed to a certain extent, for example by being released and protected in a complex or by being contained in a vesicle or the like. This has the effect that the extracellular nucleic acids are naturally already fixed to a certain extent and are therefore usually not rapidly degraded by nucleases in cell-containing samples such as whole blood, plasma or serum. Therefore, when aiming to fix the extracellular nucleic acids contained in a cell-containing biological sample, one of the main problems after obtaining or collecting the cell-containing biological sample is that the extracellular nucleic acid population contained in the collected cell-containing biological sample is contaminated or diluted by intracellular nucleic acids, particularly fragmented genomic DNA, originating from damaged or dead cells contained in the cell-containing biological sample. The fixation technique according to the invention is particularly advantageous in this respect, since it not only substantially preserves the extracellular nucleic acids present in the sample, and for example inhibits the degradation of the contained extracellular nucleic acids (in the case of blood, preferably inhibits at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or more preferably at least 95% during fixation compared to an unfixed sample or, for example, an EDTA-fixed sample), but furthermore, effectively reduces the release of genomic DNA from cells contained in the obtained cell-containing biological sample and / or reduces the fragmentation of each genomic DNA. According to one embodiment, the use of a polyoxyethylene polymer to fix the extracellular nucleic acid population in the cell-containing sample, optionally but preferably in combination with one or more of the above-mentioned other fixatives, has the effect of reducing the increase in DNA caused by the genomic DNA released from the cells contained in the sample during fixation compared to a non-fixed sample. According to one embodiment, compared to the corresponding sample fixed by unfixed sample or EDTA (especially blood sample or sample derived from blood such as plasma or serum), during fixation, the release of the genomic DNA is reduced by at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 10 times, at least 12 times, at least 15 times, at least 17 times or at least 20 times. According to one embodiment, compared to the corresponding sample fixed by unfixed sample or EDTA (especially blood sample or sample derived from blood such as plasma or serum), during fixation, the release of the 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%. The released DNA can be determined by quantitative ribosomal 18S DNA as described in the Examples section herein. As shown in the Examples, the fixation obtained by the teachings of the present invention significantly reduces this release of DNA during fixation.Thus, according to one embodiment, the fixation effect obtained by using a polyoxyethylene polymer, such as polyethylene glycol, optionally in combination with one or more of the above-mentioned other fixatives, such as preferably one or more primary, secondary or tertiary amides and caspase inhibitors, results in a reduction of DNA released from cells contained in the fixed sample during fixation by at least up to 8 times, at least up to 7 times, at least up to 5 times, more preferably at least up to 4 times, more preferably at least up to 3 times or preferably up to 2 times or less, as measured in the 18S DNA test described in the examples. As shown by the examples, effective fixation of the extracellular nucleic acid population for at least three days is obtained using the method of the present invention, even up to 6 days or longer when the fixative combination described herein is used. During shorter and longer storage periods of the fixed sample, the DNA release can be reduced in the examples by at least up to 2 times or less, as measured in the 18S DNA test described in the examples. Thus, according to an embodiment of the fixation technique described herein, when using the fixation method of the present invention, DNA release can be reduced to 3 times or less, or even 2 times or less, for up to 3 days or even up to 6 days of storage and longer. As shown in the examples, in an embodiment, a value of 1-1.5 is obtained. Compared with the prior art methods, this is a significant improvement in the fixation of the extracellular nucleic acid group. However, of course, the sample can also be further processed earlier, if necessary. It is not necessary to utilize a completely achievable fixation period. In addition, different standard methods can be used to effectively separate nucleic acids from the fixed samples respectively, because no cross-linked samples are produced due to fixation. This greatly simplifies and improves the standardization of molecular analysis that relies on extracellular nucleic acid analysis.

[0084] The selection of suitable auxiliary agents that may also be beneficial to the fixation effect may also depend on the type of cell-containing sample to be fixed. For example, when processing cell-containing biological sample blood, an anticoagulant is additionally used to prevent coagulation. The anticoagulant is used in a concentration that can coagulate the amount of blood to be fixed. The anticoagulant may, for example, be selected from the following group: heparin, a chelating agent such as ethylenediaminetetraacetic acid, a salt of a carboxylic acid such as citric acid or oxalic acid, or any combination thereof. In a preferred embodiment, the anticoagulant is a chelating agent. A chelating agent is an organic compound that can form a coordination bond with a metal through two or more atoms of the organic compound. Chelating agents according to the present invention include, but are not limited to, diethyltriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycoltetraacetic acid (EGTA) and N,N-di(carboxymethyl)glycine (NTA), and in addition citrate or oxalate. According to a preferred embodiment, EDTA is used as an anticoagulant. As used herein, the term "EDTA" refers in particular to the EDTA portion of an EDTA compound such as K2EDTA, K3EDTA or Na2EDTA. The use of chelating agents such as EDTA also has a beneficial effect, such as the inhibition of nucleases such as DNA enzymes and RNA enzymes, thereby, for example, preventing extracellular nucleic acids from being degraded by nucleases. Therefore, when using non-blood cell samples, it is also advantageous to use chelating agents such as EDTA. In addition, it is found that the EDTA used / added in higher concentrations supports the fixation effect. However, EDTA alone cannot obtain enough fixation effects for the purposes described in the present invention. However, in combination with the teachings of the present invention, particularly with polyoxyethylene polymers (and one or more other fixatives described herein, such as preferred primary, secondary or tertiary amides and / or caspase inhibitors), the fixation effect can be further improved because of the reasons discussed above.

[0085] According to one embodiment, the concentration range of the chelating agent, preferably EDTA, in the mixture obtained when the cell-containing biological sample is contacted with the polyoxyethylene polymer and optionally one or more other auxiliary agents is selected from the group consisting of 0.5-40 mg / ml, 1-30 mg / ml, 1.6-25 mg / ml, 5-20 mg / ml and 7.5-17.5 mg / ml. Each concentration is particularly effective when fixing blood, plasma and / or serum samples. Suitable concentrations can also be determined by a person skilled in the art.

[0086] In order to further support the fixation of the sample containing cells, correspondingly support the preservation of the extracellular nucleic acid group, other auxiliary agents can also be used. The example of each auxiliary agent includes, but is not limited to, nuclease inhibitors, particularly RNA enzyme and DNA enzyme inhibitory compounds. When selecting corresponding other auxiliary agents for supporting fixation, it should be careful not to damage and / or eliminate the fixation effect. Therefore, auxiliary agents, such as chaotropic agents, can not be used in a concentration that causes or supports the lysis and / or degradation of nucleated cells contained in the fixed cell-containing biological sample and / or supports the degradation of nucleic acids contained in the cell-free part of the biological sample. Therefore, preferably, the fixation method described herein does not include the auxiliary agents that use (i) induce or promote the lysis of nucleated cells, (ii) induce or promote general cell lysis and / or (iii) cause the degradation of nucleic acids contained in the cell-free part of the cell-containing biological sample. Because the fixation method described herein is not based on cell lysis but preserves cells, cells can be separated from the sample containing cells after the fixation period, so that the cell-free or cell-eliminated part including the extracellular nucleic acid group can be obtained. Due to the fixation based on polyoxyethylene polymer described herein, the extracellular nucleic acid group substantially corresponds to or is at least similar to the extracellular nucleic acid group present when the sample is collected and fixed. In addition, nucleic acid can be separated from the separated cells and used for analysis. As mentioned above, the composition including the compound shown in Formula 1 also has transcriptome fixation performance. By fixing the transcriptome and the extracellular nucleic acid group, the samples fixed respectively are also suitable for gene expression profiles. In addition, if necessary, the samples fixed respectively allow the extracellular and intracellular nucleic acid groups to be separated and analyzed from the same fixed sample.

[0087] In a preferred embodiment, the cell-containing biological sample, preferably a blood sample or a sample derived from blood such as plasma or serum, is contacted with:

[0088] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of at least 1500, preferably 1500-50000, 2000-40000, 2500-30000, 2500-25000, more preferably 3000-20000 or 4500-10000;

[0089] b) one or more compounds of Formula 1, preferably at a concentration such that the concentration in the mixture with the cell-containing biological sample ranges from 0.25% to 5%, 0.3% to 4%, 0.4% to 3%, 0.5% to 2%, or 0.75% to 1.5%;

[0090] c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-Oph, preferably in a concentration such that the concentration of the caspase inhibitor in the mixture with the cell-containing biological sample is in the range of 0.1 μM-20 μM, more preferably 0.5 μM-10 μM, more preferably 1 μM-10 μM, more preferably 3 μM-7.5 μM or 3 μM-5 μM;

[0091] d) optionally at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight polyoxyethylene polymer used, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene having a molecular weight of 1000 or less, preferably a molecular weight of 200-800 or 200-600;

[0092] e) optionally a chelating agent, more preferably EDTA.

[0093] In a preferred embodiment, the cell-containing biological sample, preferably a blood sample or a sample derived from blood such as plasma or serum, is contacted with:

[0094] a) at least one high molecular weight polyethylene oxide polymer, preferably polyethylene glycol, having a molecular weight of 2000-40000, 2500-30000, 3000-25000, 3500-20000 or 4000-15000;

[0095] b) one or more compounds of formula 1, preferably butanamide and / or N,N-dialkylpropionamide;

[0096] c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-Oph;

[0097] d) at least one low molecular weight polyethylene oxide, preferably polyethylene glycol, having a molecular weight of 100-1000, 150-800 or 200-600;

[0098] e) optionally a chelating agent, more preferably EDTA,

[0099] Wherein, after the cell-containing biological sample is contacted with the auxiliary agent and optionally other auxiliary agents for fixation, the obtained mixture includes:

[0100] - high molecular weight polyoxyethylene polymers at a concentration of 0.25%-1.25% (w / v), 0.3%-1% (w / v) or 0.4%-0.75% (w / v);

[0101] - one or more compounds of formula 1, at a concentration of 0.3%-4%, 0.4-3% or 0.5-2%;

[0102] - a caspase inhibitor at a concentration of 1 μM to 10 μM, preferably 3 μM to 7.5 μM; and

[0103] - Low molecular weight polyoxyethylene polymers at concentrations of 1.5%-10%, 2%-8%, 2.5-7% and 3%-6%.

[0104] According to one embodiment, the method of the present invention is used to fix the extracellular nucleic acid group contained in the blood sample, comprising contacting the blood sample with at least one polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, preferably polyethylene glycol and an anticoagulant, wherein during fixation, the release of genomic DNA from the cells contained in the blood sample into the cell-free portion of the blood sample is reduced. In particular, the present invention provides a method for fixing the extracellular nucleic acid group contained in the blood sample, comprising contacting the blood sample with at least one polyoxyethylene polymer, one or more primary, secondary or tertiary amides (suitable and preferred embodiments and concentrations as described above), at least one caspase inhibitor and an anticoagulant, wherein the release of genomic DNA from the nucleated cells contained in the blood sample into the cell-free portion of the blood sample is reduced. In particular, white blood cell lysis is avoided / reduced during fixation. In addition, due to fixation, the degradation of nucleic acids present in the sample is reduced.

[0105] In one embodiment, the cell-containing biological sample is a blood sample, which is contacted with:

[0106] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of 2000-40000, 2500-30000, 2500-25000, 3000-20000, 3500-15000 or 3000-10000;

[0107] b) one or more compounds of formula 1;

[0108] c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-OPh;

[0109] d) at least one low molecular weight polyethylene oxide polymer having a molecular weight of 1000 or less, preferably 100-800, 200-600 or 200-500;

[0110] e) an anticoagulant, preferably a chelating agent, more preferably EDTA,

[0111] After the blood sample is contacted with the auxiliary agent and optionally other auxiliary agents for fixation, the obtained mixture includes:

[0112] - a high molecular weight polyoxyethylene polymer at a concentration of 0.2%-1.5% (w / v), 0.25%-1.25% (w / v), 0.3%-1% (w / v) or 0.4%-0.75% (w / v);

[0113] - one or more compounds of formula 1, at a concentration of 0.3%-4%, preferably 0.5-3%, more preferably 0.5%-2% or 0.75%-1.5%;

[0114] - a caspase inhibitor at a concentration of 1 μM to 10 μM, preferably 3 μM to 7.5 μM; and

[0115] - Low molecular weight polyoxyethylene polymers, in a concentration of 1.5% to 10%, preferably 2% to 6%.

[0116] According to one embodiment, the method is for fixing the extracellular nucleic acid population contained in a blood sample, comprising contacting the blood sample with at least one polyoxyethylene polymer, butanamide and / or N,N-dialkylpropionamide such as N,N-dimethylpropionamide, at least one caspase inhibitor and an anticoagulant, wherein the release of genomic DNA from cells contained in the blood sample into the cell-free portion of the blood sample is reduced. Preferably, the fixation effect is obtained for at least three days, more preferably up to six days or longer. As described above, the release of DNA from the contained cells during the entire fixation period is preferably no more than a maximum of 2 times, preferably even about 1.5 times or less.

[0117] In embodiments, an additional advantage when using the method of the present invention to fix a blood sample is that hemolysis is significantly reduced during fixation. In one embodiment, one or more other reagents and a polyoxyethylene polymer for fixation are contained in an aqueous fixation composition. The examples show that this embodiment significantly reduces red blood cell hemolysis compared to unfixed or standard EDT blood samples, and even compared to other fixed samples. Hemolysis is the rupture of red blood cells and the release of their cytoplasm into the surrounding extracellular fluid, such as plasma. The extent of hemolysis can be analyzed, for example, by visual inspection, because the released hemoglobin causes the serum or plasma to appear red. The causes of in vitro hemolysis are mostly related to specimen collection. However, if the correct fixation method is not used, in vitro hemolysis also usually occurs in blood samples during ex vivo storage. Depending on the extracellular nucleic acid of interest, hemolysis can be a considerable problem. If the extracellular nucleic acid of interest is DNA, hemolysis is a minor problem because red blood cells do not contain nuclei and therefore do not contain genomic DNA. Therefore, no intracellular DNA is released from red blood cells during hemolysis. When the extracellular nucleic acid of interest is DNA, in particular, the lysis or death of white blood cells is a problem, because in this case, in addition to the intracellular RNA, genomic DNA is also released. Therefore, when the extracellular nucleic acid of interest is extracellular DNA, in particular, the lysis of white blood cells must be avoided. White blood cells can differ from each other in their stability properties. Therefore, certain types of white blood cells are more stable than other types. However, in general, white blood cells are significantly more stable than red blood cells. Therefore, red blood cell lysis does not necessarily mean white blood cell lysis. The prior art also uses the different sensitivities of white blood cells and red blood cells to lysis to specifically lyse red blood cells while retaining white blood cells, so that, for example, white blood cells can be collected. However, if the extracellular nucleic acid of interest is RNA, hemolysis of red blood cells and thus lysis do not constitute a problem. Mature red blood cells do not contain RNA either, but their precursors (reticulocytes) do. Reticulocytes account for about 0.5%-1% of red blood cells and contain a large amount of globin RNA. Therefore, in particular, when the extracellular nucleic acid of interest is RNA, it is advantageous to reduce the dilution of the extracellular nucleic acid population, especially the extracellular RNA population, by globin mRNA, and to reduce or prevent erythrocyte and thereby reticulocyte lysis during storage. In addition, as described above, it is advantageous to maintain the composition and profile of the extracellular nucleic acid population. As shown by the examples, in an embodiment, when the fixation method of the present invention is used, hemolysis is effectively reduced. Especially when a fixation composition containing water is used. Thus, the extracellular nucleic acid population is substantially retained, and in addition, the fixed blood sample, in particular the plasma or serum obtained from the fixed blood sample, is generally also suitable for other standard laboratory analyses due to the prevention of hemolysis and cell lysis.

[0118] The polyoxyethylene polymer may be included in the fixative composition. If one or more other fixatives and adjuvants are additionally used, they may and preferably are also included in the fixative composition. Preferably, the fixative composition according to the third aspect of the present invention is used for fixing a cell-containing biological sample.

[0119] In addition, according to an alternative embodiment of the first aspect, the cell-containing biological sample to be fixed is contacted with ethylene glycol (1,2-ethylene glycol) as a fixative. Ethylene glycol may be used in the concentration described above for low molecular weight polyethylene oxide polymers. The corresponding disclosures mentioned also apply here. Specifically, the cell-containing biological sample to be fixed may be contacted with ethylene glycol and any one or more other fixatives described herein, preferably with at least one caspase inhibitor and / or at least one primary, secondary, or tertiary amide (preferably a compound shown in Formula 1 above). Ethylene glycol may be used in combination with at least one polyethylene oxide polymer. The above describes suitable embodiments and concentration ranges of each fixative, which are also applicable to embodiments in which ethylene glycol is used to fix or support the fixation of cell-containing samples and extracellular nucleic acid groups contained therein.

[0120] The components of the fixing composition can be contained in, respectively dissolved in, a solvent, such as water, a buffer, such as a biological buffer, such as MOPS, TRIS, PBS and the like. In addition, the components are dissolved in or the fixing composition may contain a polar aprotic solvent, such as dimethyl sulfoxide (DMSO). As shown in the examples, when fixing blood samples, it is particularly preferred to use a fixing composition comprising water, because this embodiment significantly reduces hemolysis.

[0121] To collect cell-containing biological samples, polyoxyethylene polymers and other optional fixatives and / or adjuvants can be present in a device, preferably a container. Polyoxyethylene polymers and one or more compounds optionally used for fixation can be present in a fixative composition in a corresponding device, or exist as independent entities. In addition, they can be added to a corresponding collection device before collecting cell-containing biological samples, or they can be added to a collection device after collecting cell-containing biological samples. It is also within the scope of the present invention to add fixatives and other optional adjuvants used separately to cell-containing biological samples. However, for ease of handling, it is preferred to provide polyoxyethylene polymers and any other fixatives and / or adjuvants used in a corresponding collection device, such as in a single composition form. However, they may be present in a collection device as a separate component or composition. In a preferred embodiment, before adding a cell-containing biological sample, at least one polyoxyethylene polymer, one or more primary, secondary or tertiary amides, at least one caspase inhibitor and optionally one or more other adjuvants, such as anticoagulants, such as EDTA are present in a collection device. This ensures that the cell-containing biological sample is quickly fixed once it contacts the fixative used according to the teaching of the present invention. The fixing agent is present in the container in an amount that can effectively fix the cell-containing biological sample to be collected and respectively contained in the collection device. Subsequently, suitable and preferred embodiments of each collection device are also described in conjunction with the fifth aspect, with reference to the disclosure. When ethylene glycol is used as the fixing agent, the same applies.

[0122] Preferably, the cell-containing biological sample is contacted with the polyoxyethylene polymer and optionally other auxiliary agents directly after collection and / or during collection. Therefore, as described above, preferably, the reagent for fixation is provided in the form of a composition. Preferably, the fixing composition is provided in liquid form. For example, it can be pre-filled in a sample collection device to quickly fix the cell-containing biological sample during collection. According to one embodiment, the fixing composition is contacted with the sample containing cells in a volume ratio selected from the following group: 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5 and 1:7-1:5, especially about 1:6. These ratios are particularly useful for fixing blood samples. For fixing blood samples, the fixing composition preferably contains water. The above describes the suitable and preferred concentrations of auxiliary agents in the mixture containing cell-containing samples, especially blood samples, with reference to the corresponding disclosure. The special advantage of the teaching of the present invention is that a small volume of the fixing composition of the present invention can be used to obtain a large sample volume fixation. This is particularly, if the above-mentioned high molecular weight polyoxyethylene polymer is used, it allows the use of lower concentrations compared to low molecular weight polyoxyethylene polymers. Therefore, preferably, the ratio of the fixing composition to the sample is 1:10-1:5, in particular 1:7-1:5, such as about 1.6.

[0123] As used herein, the terms "cell-containing biological sample", "cell-containing sample" and similar terms refer in particular to a sample comprising at least 50, 250, at least 500, at least 1000, at least 1500, at least 2000 or at least 5000 cells. According to one embodiment, the cell fraction accounts for at least 1%, at least 2%, at least 2.5%, at least 5%, preferably at least 10%, at least 15%, at least 20%, more preferably at least 25%, at least 30%, at least 35% or at least 40% of the cell-containing biological sample. Cell-containing samples comprising a considerable number of cells, wherein the cell fraction accounts for more than 40%, can also be fixed using the teachings described herein. However, the term "cell-containing biological sample" also refers to and thus includes cell-depleted samples, including cell-depleted samples that are usually referred to as "cell-free", such as plasma, since the corresponding samples often include residual cells. At least, it often cannot be completely excluded that even if a so-called "cell-free" sample such as plasma contains residual amounts of cells, it accordingly constitutes a risk that the extracellular nucleic acid population is contaminated by intracellular nucleic acids released from said residual cells. Therefore, according to one embodiment, each cell-eliminated and "cell-free" sample is also covered by the term "cell-containing biological sample". Therefore, a "cell-containing sample" may include a large number of cells, such as in the case of whole blood, or may only include a small number of cells. Therefore, the term "cell-containing biological sample" also includes samples that are only suspected of containing cells or are at risk of containing cells. As discussed above, for biological samples that only include a small amount, a corresponding residual amount of cells, such as plasma (plasma contains - depending on the preparation method - usually a small residual amount of cells, even if it is usually referred to as cell-free), the method according to the present invention has considerable advantages, because these residual cells may also lead to undesirable contamination of the contained extracellular nucleic acids. The use of the fixation method according to the present invention has the advantage that, essentially regardless of the composition of the cell-containing biological sample and the number of cells contained therein, the extracellular nucleic acid group contained in the sample can be essentially preserved, correspondingly fixed, thereby allowing standardized subsequent separation and / or analysis of the contained extracellular nucleic acids.

[0124] According to one embodiment, the cell-containing biological sample is selected from the group consisting of body fluids and cell-containing samples derived from body fluids, in particular whole blood, samples derived from blood such as plasma or serum, buffy coat, urine, sputum, tears, lymph, sweat, wine, cerebrospinal fluid, ascites, milk, feces, bronchial lavage fluid, saliva, amniotic fluid, nasal secretions, vaginal secretions, sperm / semen, wound secretions, cell cultures and swab samples. According to one embodiment, the cell-containing biological sample is a body fluid, a body secretion or a body excretion, preferably a body fluid, more preferably urine, lymph, blood, buffy coat, plasma or serum. In particular, the cell-containing biological sample can be a circulating body fluid such as blood or lymph. Preferably, the cell-containing biological sample fixed using the teachings described herein is a blood sample, sometimes also referred to as whole blood. The blood sample is preferably not diluted or fractionated before fixation. According to one embodiment, the blood sample is peripheral blood. According to one embodiment, the cell-containing biological sample is obtained from a human. The cell-containing biological sample includes extracellular nucleic acids in the extracellular portion.

[0125] The term "extracellular nucleic acid" or "extracellular nucleic acid" is used herein to refer in particular to nucleic acids that are not contained in cells. Each extracellular nucleic acid also often refers to a cell-free nucleic acid. These terms are used herein as synonyms. Therefore, extracellular nucleic acids are usually present outside a cell or multiple cells in a sample. The term "extracellular nucleic acid" refers to, for example, extracellular RNA and extracellular DNA. Examples of typical extracellular nucleic acids found in a cell-free portion (corresponding portion) of a biological sample such as a body fluid include, but are not limited to, mammalian extracellular nucleic acids, such as extracellular tumor-related or tumor-derived DNA and / or RNA, other extracellular disease-related DNA and / or RNA, epigenetic modified DNA, fetal DNA and / or RNA, small interfering RNA, such as miRNA and siRNA, and non-mammalian extracellular nucleic acids, such as viral nucleic acids, pathogen nucleic acids, such as those released from prokaryotes (such as bacteria), viruses, eukaryotic parasites or fungi into extracellular nucleic acid groups. Extracellular nucleic acid groups typically include a certain amount of intracellular nucleic acids released from damaged or dead cells. For example, the extracellular nucleic acid group present in blood typically includes intracellular globulin mRNA released from damaged or dead cells. This is a natural process that occurs in vivo. Such intracellular nucleic acids present in the extracellular nucleic acid group can even be used as a control in subsequent nucleic acid detection methods. The fixation method described herein can especially reduce the risk of significantly increasing the amount of intracellular nucleic acids, such as genomic DNA, contained in the extracellular nucleic acid group after collecting a sample containing cells due to in vitro treatment of the sample. Therefore, the change of the extracellular nucleic acid group caused by in vitro treatment is reduced or even prevented. According to one embodiment, the cell-containing biological sample is or is derived from a body fluid, such as blood, plasma, serum, saliva, urine, wine, cerebrospinal fluid, sputum, lacrimal fluid, sweat, amniotic fluid or lymph. Therefore, we talk about the extracellular nucleic acids obtained from circulating body fluids such as blood, or lymph as circulating extracellular nucleic acids or circulating cell-free nucleic acids. According to one embodiment, the term extracellular nucleic acids refers in particular to mammalian extracellular nucleic acids. Examples include, but are not limited to, disease-related or disease-derived extracellular nucleic acids, such as tumor-related or tumor-derived extracellular nucleic acids, due to inflammation or injury, especially trauma-released extracellular nucleic acids, other diseases-related and / or due to other diseases-released extracellular nucleic acids, or fetal extracellular nucleic acids. The term "extracellular nucleic acid" or "extracellular nucleic acid" as used herein also refers to extracellular nucleic acids obtained from other cell-containing biological samples, particularly non-body fluid biological samples. Typically, the sample contains more than one type or class of extracellular nucleic acids.

[0126] As used herein, the term "extracellular nucleic acid group" refers in particular to a collection of different extracellular nucleic acids contained in a cell-containing sample. A cell-containing sample generally includes a unique and therefore unique extracellular nucleic acid group. Therefore, the type, kind, ratio and / or amount of one or more extracellular nucleic acids contained in a specific sample of an extracellular nucleic acid group may be an important sample characteristic. As discussed above, it is therefore important for fixing and thus maintaining the extracellular cell group in a state when the sample is collected, because its composition and / or the amount of one or more extracellular nucleic acids contained in the extracellular nucleic acid group sample can provide valuable medical treatment, prognosis or diagnostic information. Therefore, it is advantageous if the atlas of the extracellular nucleic acid group is effectively fixed in the expected stable period. The fixation technique described herein reduces the contamination and dilution of the extracellular nucleic acid group by intracellular nucleic acids, particularly genomic DNA, after sample collection and fixation. Therefore, it is achieved that the extracellular nucleic acid group is substantially retained. As shown by the examples, changes in the amount, quality and / or composition of the extracellular nucleic acid population, especially changes caused by the increase in released genomic DNA, are significantly reduced during fixation, compared to blood samples or samples derived from blood, such as corresponding samples fixed with EDTA or unfixed samples. According to one embodiment, the increase in genomic DNA from T0 (fixation point) to the end of the fixation period (preferably 48h, 72h or 96h after T0) is reduced to at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95%, compared to blood samples (such as 1.5mg EDTA / ml fixed blood samples) or samples derived from blood, such as corresponding samples fixed with EDTA or unfixed samples. As shown by the examples, in particular in the embodiment of the combined use of a caspase inhibitor and at least one primary, secondary or tertiary amide, a value of more than 80% or more is obtained.

[0127] As described above and as demonstrated in the examples, the use of the method of the present invention allows the fixation of samples containing cells without refrigeration or freezing for a long time. Therefore, the sample can be kept at room temperature, even at elevated temperatures such as up to 30°C or even up to 40°C. According to one embodiment, the fixation effect is obtained for at least three days, preferably at least four days, more preferably at least 6 days. Preferably, during the fixation period, the fixation method of the present invention has the effect that the cells contained in the sample are fixed, the release of genomic DNA from the cells contained in the sample into the cell-free part of the sample is reduced and / or the degradation of nucleic acids present in the sample due to fixation is reduced. In particular, during the described fixation period, fixation reduces the dilution of the extracellular DNA group contained in the biological sample during fixation by the genomic DNA derived from the cells contained in the fixed sample. The fixation effect achieved according to the method of the present invention is described in detail above, with reference to the above disclosure, preferably, during the fixation period, fixation reduces the contamination of the extracellular cell group contained in the biological sample during fixation by the intracellular nucleic acid, especially genomic DNA, derived from the cells contained in the fixed sample. As shown in the examples, the blood sample can be fixed at room temperature for up to 3 days or more. Even at room temperature for up to 6 days, or even longer storage periods, when using polyoxyethylene polymers, preferably in combination with caspase inhibitors and one or more primary, secondary or tertiary amides, the extracellular nucleic acid population is substantially fixed (especially compared to non-fixed samples or, for example, compared to samples fixed by standard methods, such as EDTA treatment). As shown in the examples, even after a very long fixation time of 10 days, the fixation effect is maintained. Although it is generally possible that the fixation effect decreases over time, this also depends on the source, such as the host from which the cell-containing biological sample is obtained, and it is generally sufficient to maintain the composition of the extracellular nucleic acid population, so that the extracellular nucleic acid can be analyzed and / or further processed. Therefore, the cell-containing biological sample fixed according to the method of the present invention, in particular, the sample fixed with polyoxyethylene polymers, one or more primary, secondary or tertiary amides and caspase inhibitors is still suitable for separation and analysis of the extracellular nucleic acids contained therein, even after long-term storage at room temperature. Therefore, even longer storage / transportation times are possible. However, in general, longer periods are unnecessary because conventional storage and, for example, the time for transporting to a laboratory for implementing nucleic acid separation and optional analysis is usually no more than 6 or 7 days, but usually can be completed after two or three days. As shown in the examples, the fixation efficiency is particularly good within this period. However, the fixation efficiency and the long fixation time achieved with the process of the invention provide an important safety factor.

[0128] The method according to the present invention and the fixing composition described subsequently can fix a large volume of biological samples containing cells using a small volume / amount of fixing agent added, because the combination of at least one polyoxyethylene polymer, preferably polyethylene glycol and the fixing agent used according to the teachings of the present invention is highly active for fixing, especially the combination. This is an important advantage, because the size / volume of the sample brings considerable restrictions to the subsequent nucleic acid separation procedure, especially when it is intended to use an automatic procedure to separate the extracellular nucleic acids contained in the sample. In addition, one must consider that the extracellular nucleic acids are usually only contained in a small amount in the biological sample containing cells. Therefore, it is advantageous to process a larger volume of biological samples containing cells, such as blood samples, that is, more extracellular nucleic acids can be separated from the sample, so that they can be used for subsequent analysis. According to one embodiment, the sample volume fixed by the method of the first aspect is selected from the group consisting of 1 to 50 ml, 2 to 35 ml, 3 to 25 ml, 4 to 20 ml and 5 to 15 ml.

[0129] The fixation method described herein provides significant advantages over the most advanced fixation methods for fixing extracellular nucleic acid populations in cell-containing samples, which are based on the use of crosslinking agents, such as formaldehyde, formalin, formaldehyde releasers and the like. Crosslinking agents cause the formation of intermolecular or intramolecular covalent bonds between nucleic acid molecules or between nucleic acids and proteins. This crosslinking can significantly impair the subsequent separation of nucleic acids from these fixed samples, and a specially adjusted nucleic acid separation procedure is usually required to allow separation from these samples. Since, for example, the concentration of circulating nucleic acids in whole blood samples is already relatively low, any detection that further reduces the yield of such nucleic acids should be avoided. This may be particularly important when detecting or analyzing very rare nucleic acid molecules derived from, for example, malignant tumors or a developing fetus in the first three months of pregnancy. As shown by the examples, the method of the present invention does not require a crosslinking agent for fixation. Therefore, according to one embodiment, the fixation method according to the present invention does not involve the use of a crosslinking agent that induces protein-nucleic acid and / or protein-protein crosslinking. In particular, fixation does not involve the use of formaldehyde, formalin, paraformaldehyde or a formaldehyde releaser. Furthermore, as described above, according to one embodiment, the immobilization method according to the present invention does not involve the use of auxiliary agents that are classified as toxic agents.

[0130] After the fixation period, the method may include one or more of the following steps:

[0131] a) fixing the sample and subjecting it to nucleic acid analysis and / or detection methods;

[0132] b) isolating extracellular nucleic acids from fixed samples;

[0133] c) isolating extracellular nucleic acids from the fixed sample and analyzing and / or detecting the isolated nucleic acids;

[0134] d) removing cells contained in the fixed sample;

[0135] e) removing cells contained in the fixed sample before performing the nucleic acid isolation, analysis and / or detection steps;

[0136] f) removing cells from the fixed sample and isolating extracellular nucleic acids from the cell-free or cell-depleted portion of the fixed sample;

[0137] g) storing (i) a fixed sample, (ii) a fixed sample from which cells have been removed, and / or (iii) cells removed from a sample;

[0138] h) removing cells from the fixed sample and discarding them; and / or

[0139] i) removing cells from the fixed sample, and isolating nucleic acid from the cells removed from the fixed sample;

[0140] j) Removing cells from the fixed sample and isolating extracellular nucleic acids from the cell-free or cell-depleted fraction of the fixed sample using a size-selective nucleic acid isolation method.

[0141] Thus, a cell-containing biological sample fixed by the method of the present invention can be analyzed in a nucleic acid analysis and / or detection method; and / or may be further processed. The nucleic acids may be analyzed directly after fixation of the biological sample, or the nucleic acids may be first separated from the fixed sample. Details on nucleic acid separation and analysis are also described below in conjunction with the second aspect of the present invention, with reference to the disclosure.

[0142] B. Nucleic Acid Isolation Methods

[0143] According to a second aspect, a method for isolating extracellular nucleic acids from a cell-containing biological sample is provided, comprising the steps of:

[0144] a) fixing a cell-containing biological sample according to the method defined in the first aspect of the present invention; and

[0145] b) Isolation of extracellular nucleic acids from fixed samples.

[0146] In step a), the extracellular nucleic acid group contained in the sample containing cells is fixed according to the method described in the first aspect of the present invention. As discussed above, the fixation according to the present invention has the effect that the extracellular nucleic acid group contained in the sample can be basically maintained during the fixation period in the state when the biological sample is collected separately. In particular, during storage / processing, the high growth of nucleic acids usually observed caused by intracellular nucleic acids, particularly genomic DNA, and more particularly fragmented genomic DNA is reduced or even avoided, as shown in the embodiments. Without being bound by theory, it is believed that the fixation based on polyoxyethylene polymers described herein fixes cells and / or reduces cell damage during fixation, thereby reducing the release of intracellular nucleic acids. The method allows the separation of cell parts from the fixed sample after the desired fixation period. Therefore, compared with non-fixed samples, the extracellular nucleic acids obtained by the corresponding fixed samples contain very little contamination of intracellular nucleic acids derived from degenerated or dead cells, and in particular, contain a small amount of fragmented genomic DNA. In addition, the fixation according to the present invention does not require and preferably does not involve the use of cross-linking agents. This is an important advantage over the prior art involving the use of cross-linking agents such as formaldehyde, formalin or formaldehyde releasers, because when using standard nucleic acid isolation techniques, these reagents generally reduce the recoverable amount of extracellular nucleic acids due to cross-linking. In addition, as described above, the fixation described herein allows the sample to be stored and / or processed, such as transported for a long time, even at room temperature, before separating the cells contained in the sample and / or before separating the nucleic acids contained therein in step b). For details on the fixation performed in step a), reference is made to the above disclosure which is also applicable here. The following briefly describes non-limiting embodiments.

[0147] According to one embodiment, in step a), the cell-containing biological sample, such as a whole blood sample, is fixed using:

[0148] - a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 as described above, optionally in combination with other polyoxyethylene polymers having a molecular weight at least 100 lower than the high molecular weight polymer, such as a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less;

[0149] - At least one caspase inhibitor;

[0150] - one or more primary, secondary and tertiary amides, and

[0151] - Optionally, further fixing agents and / or auxiliaries.

[0152] Suitable and preferred embodiments of the fixation method according to the invention carried out in step a) and the compounds used for fixation are described above, with reference to the above disclosure also applicable here. Particularly preferred is the additional use of low molecular weight polyoxyethylene polymers, at least one caspase inhibitor and butanamide and / or N,N-dialkylpropionamide, such as preferably N,N-dimethylpropionamide for fixation. When fixing whole blood samples, a combination with an anticoagulant, preferably a chelating agent, such as EDTA is preferred. In addition, as described above, according to an alternative to the first aspect, the cell-containing biological sample to be fixed is contacted with ethylene glycol (1,2-ethylene glycol) as a fixative. Reference is made to the above disclosure.

[0153] If the cell-containing biological sample includes a large number of cells, such as in the case of whole blood, the cells are separated from the remaining sample to obtain a cell-free, correspondingly cell-reduced or cell-eliminated portion of the fixed sample, and then extracellular nucleic acids are separated therefrom in step b). Therefore, according to one embodiment, cells are removed from the cell-containing sample between step a) and step b). If the sample is processed to include only a small amount of residual cells, such as plasma or serum, and / or the extracellular nucleic acid of interest is DNA, the intermediate step can be omitted. Due to the fixation of the present invention, the genomic DNA released from the included cells during the fixation period is reduced or even prevented, and in addition, the fragmentation of genomic DNA is reduced, especially when caspase inhibitors are used in addition. As described herein, due to its relatively large size, unfragmented genomic DNA can be distinguished from smaller extracellular DNA. This allows the extracellular DNA to be selectively separated by using a size selection separation scheme, even in the presence of unfragmented genomic DNA. However, in order to improve the effect, it is preferred to remove cells (or potential residual cells) from the fixed sample before separating extracellular nucleic acids in step b), thereby reducing the contamination of the extracellular nucleic acid population by intracellular nucleic acids released from cells during nucleic acid separation. If the extracellular nucleic acid of interest is RNA, it is also advantageous to remove the contained cells, because it is difficult to distinguish between extracellular RNA and intracellular RNA, and in addition, dilution of the extracellular RNA can be prevented. In addition, if the extracellular nucleic acid of interest is DNA, it is usually advantageous to remove the cells before step b), and therefore preferred, because this allows the use of standard nucleic acid isolation procedures in step b).

[0154] Depending on the type of cell-containing biological sample, cells, including residual cells, can be separated and removed by centrifugation, preferably high-speed centrifugation, or if the centrifugation step is to be avoided, by using non-centrifugal means, such as filtration, precipitation or binding to a surface, such as (optionally magnetic) particles. Each cell separation method is known in the prior art, and therefore, no detailed description is required. Each cell removal step can also be easily included in the automated sample preparation scheme. If necessary, it is also possible to further process each removed cell. Cells can be, for example, stored, analyzed and / or biomolecules, such as nucleic acids or proteins, can be separated from the removed cells. In addition, it is found that, in particular, when additional compounds such as DMPA shown in Formula 1 are used to fix samples containing cells, intracellular nucleic acids such as intracellular RNA can be fixed. The additional fixation of the transcriptome is advantageous because it allows the analysis of the spectrum of transcripts in the separated intracellular nucleic acids, which are also important biomarkers for in vitro diagnosis.

[0155] Furthermore, it is within the scope of the present invention to include further intermediate steps to process the fixed sample.

[0156] In step b), the extracellular nucleic acids are separated, preferably from a cell-free, respectively cell-depleted portion of the fixed sample, for example from the supernatant, or from plasma and / or serum, in case the fixed cell-containing sample is a blood sample. For the separation of extracellular nucleic acids, any known nucleic acid separation method suitable for isolating nucleic acids from a fixed sample, respectively from an obtained cell-depleted sample, can be used. Examples of various purification methods include, but are not limited to, extraction, solid phase extraction, silica-based purification methods, purification based on magnetic particles, phenol-chloroform extraction, chromatography, anion exchange chromatography (using anion exchange surfaces), electrophoresis, filtration, precipitation and combinations thereof. It is also within the scope of the present invention to specifically separate specific extracellular nucleic acids, for example by using suitable probes attached to a solid support capable of sequence-specific binding.

[0157] According to one embodiment, in step b), the nucleic acids are separated using a chaotropic agent and / or ethanol. Preferably, the nucleic acids are separated by binding them to a solid phase, preferably a solid phase comprising silica or carrying an anion exchange functional group. Each method is known in the prior art and, therefore, does not need to be described in detail. Methods and kits for separating extracellular nucleic acids are commercially available, such as Circulating Nucleic Acid Kit (QIAGEN), Chemagic Circulating NA Kit (Chemagen), NucleoSpin Plasma XS Kit (Macherey-Nagel, MN), Plasma / Serum Circulating DNA Purification Kit (NorgenBiotek, NB), Plasma / Serum Circulating RNA Purification Kit (NB), High-purity Viral Nucleic Acid Large-capacity Kit (Roche), and other commercially available kits for extracting and purifying extracellular nucleic acids. As shown above, in particular, the embodiment of using a high molecular weight polyoxyethylene polymer in combination with a low molecular weight polyoxyethylene polymer is advantageous because it allows the use of a wide range of nucleic acid separation procedures to separate extracellular nucleic acids in high yield. As described above, for example, in conjunction with a subsequent nucleic acid separation method involving the use of a silica gel column, it is preferred to use a high molecular weight polyoxyethylene polymer having a molecular weight of at least 2000, preferably at least 3000, and more preferably 4500-10000 in conjunction with a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, preferably 150-700, and more preferably 200-600. As described above, this combination of polymers can reduce the amount of high molecular weight polyoxyethylene polymer required for effective fixation in a fixed mixture including a sample containing cells to be fixed to, for example, 1.5% (w / v) or less, 1.25% (w / v) or less, 1% (w / v) or less, or 0.75% (w / v) or less. In the test examples, these lower concentrations of high molecular weight polymers in combination with low molecular weight polymers did not substantially impair subsequent nucleic acid separation, even when using a silica gel column, while achieving a stronger fixation effect. In addition, due to the use of a combination of polymers, the amount of low molecular weight polymers can also be reduced, so that the required volume of the fixed composition remains within an acceptable range. However, as shown in the Examples, lower concentrations of the high molecular weight polyoxyethylene polymer of 1.5% (w / v) or less, 1.25% (w / v) or less, 1% (w / v) or less, or 0.75% (w / v) or less, respectively, may also be used in the absence of the low molecular weight polyoxyethylene polymer, if other fixatives are used, preferred embodiments of such fixatives are described herein.

[0158] According to one embodiment, in step b), the nucleic acid is separated by binding the nucleic acid to a solid phase comprising an anion exchange group. For example, a suitable anion exchange group is an amino group. Preferably, the binding occurs at a pH below 7. Such anion exchange-based nucleic acid separation method is known to those skilled in the art. According to one embodiment, the nucleic acid is an extracellular nucleic acid. Suitable anion exchange-based methods, such as the method described in WO2013045432, incorporated herein by reference, are particularly suitable for separating extracellular nucleic acids, such as extracellular DNA, from plasma obtained from a blood sample fixed using the fixation method described herein.

[0159] According to one embodiment, the total nucleic acid is separated from a fixed sample containing cells obtained after step a) or optionally, in an intermediate step, from a fixed sample containing cells after removing cells. Preferably, nucleic acid is separated from a fixed sample, or a cell-free, correspondingly cell-eliminated portion of a fixed sample. As, total nucleic acid can be separated from blood plasma or serum, and extracellular nucleic acid will be included in these extracted nucleic acids as a part. If the cells contained in the fixed sample are effectively removed before nucleic acid separation, the total nucleic acid separated will mainly include or even consist of extracellular nucleic acid.

[0160] It is also within the scope of the present invention to separate at least a major specific target nucleic acid. The target nucleic acid can be, for example, a certain type of extracellular nucleic acid, such as DNA or RNA, including mRNA and microRNA, other non-coding nucleic acids, epigenetically modified nucleic acids, and other nucleic acids. For example, the target extracellular nucleic acid can be DNA, and the non-target extracellular nucleic acid can be RNA or vice versa. In particular, target-specific nucleic acid separation methods intended to separate DNA or RNA are known in the prior art and therefore do not need to be described in detail herein. According to one embodiment, the non-target nucleic acid is destroyed by adding a suitable enzyme that specifically destroys the non-target nucleic acid, such as, if the target nucleic acid is DNA, adding RNA enzyme, if the target nucleic acid is RNA, adding DNA enzyme. The enzyme can be added to the cleavage or binding mixture, or added after the extracellular nucleic acid is bound to the solid phase. Suitable embodiments for implementing each non-target nucleic acid decomposition step are known in the prior art and therefore do not need to be described in any detail herein. According to a feasible embodiment, if DNA and RNA are bound to a solid support, the selective elution conditions of the target nucleic acid can be applied to mainly and selectively recover the target nucleic acid from the solid phase. According to one embodiment, a separation method is used, wherein under non-target nucleic acid, such as RNA, the target nucleic acid, such as DNA is selectively bound to a solid phase. Suitable binding conditions are known in the prior art, as described in WO 95 / 21849. According to one embodiment, by binding at least a portion of the non-target nucleic acid to a solid phase under suitable conditions, the remaining sample including the target extracellular nucleic acid and the non-target nucleic acid bound to the solid phase are then separated to remove the non-target nucleic acid. This can be achieved, for example, by adding a suitable solid phase under the following conditions: wherein the main non-target nucleic acid, such as DNA, is bound to the solid phase but not the target nucleic acid, such as RNA is retained in the sample and recovered therefrom in the separation step. Suitable methods for selectively removing non-target nucleic acids from target nucleic acids, such as described in EP 0 880 537 and WO 95 / 21849, are incorporated herein by reference. If necessary, the non-target nucleic acid may also be used further, such as further processed, such as eluted from the solid phase. However, it may also be discarded. It is also within the scope of the present invention to adopt nuclease digestion of non-target nucleic acids or their residues after separating the target nucleic acid.

[0161] The term target nucleic acid may also refer to a specific type of nucleic acid, such as a specific extracellular nucleic acid, known to be a disease marker. As discussed above, separation of extracellular nucleic acids may also include specific separation of each target nucleic acid, such as by using suitable capture probes that support selective separation of target nucleic acids.

[0162] The term target nucleic acid may also refer to a nucleic acid with a certain length, such as a nucleic acid of 5000nt or shorter, 2000nt or shorter, 1000nt or shorter, 900nt or shorter, 800nt or shorter, 700nt or shorter, 600nt or shorter, 500nt or shorter, 400nt or shorter or 350nt or shorter in length. It is advantageous to separate a target nucleic acid of a certain maximum size in the context of the present invention. It is known that the size of extracellular nucleic acid is usually 1000nt or shorter, and usually even 500nt or shorter. The size, respectively, size range referred to herein refers to chain length. That is, in the case of double-stranded nucleic acids such as double-stranded DNA, it refers to base pairs. In step b), selective separation of smaller nucleic acids can enhance the extracellular nucleic acid portion obtained in the separated nucleic acid. According to the fixation method of the present invention, it is possible to more effectively separate such high molecular weight genomic DNA from smaller extracellular nucleic acid groups due to the suppression of genomic DNA release and / or the suppression of released genomic DNA fragmentation. Due to the use of the immobilization technology of the present invention, the significant size difference between genomic DNA and extracellular nucleic acid is basically preserved, and the use of size-selective nucleic acid separation scheme can more effectively remove genomic DNA. Due to effective fixation, the size difference between genomic DNA (usually greater than 10000bp) and extracellular nucleic acid (usually less than 1000nt / bp) in the fixed sample as described herein is usually relatively large, so the known method of selectively separating a certain target length nucleic acid can be used. Therefore, according to one embodiment, step b) includes selectively separating target nucleic acids with a length of 2000nt or shorter, 1500nt or shorter, 1000nt or shorter, 900nt or shorter, 800nt or shorter, 700nt or shorter, 600nt or lower, or 500nt or lower. For example, suitable methods for selectively separating nucleic acids of various sizes by eliminating high molecular weight genomic DNA are known in the prior art, so it is not necessary to describe in detail herein. The classical method for separating DNA of target size involves separating DNA in a gel, cutting out one or more required gel bands, and then separating DNA of target size from the gel fragment. Another widely used technique is size selective precipitation with polyethylene glycol-based buffers (Lis and Schleif, Nucleic Acids Res. 1975 03; 2(3): 383-9) or binding / precipitation on carboxyl-functionalized beads (DeAngelis et al., Nuc. Acid. Res. 1995, Vol. 23(22), 4742-3; US 5,898,071 and US 5,705,628, commercialized by Beckman-Coulter (AmPure XP; SPRIselect) and US 6,534,262).In addition, size selective separation methods based on the use of solid supports including anion exchange groups and varying pH values ​​are known. Size selective separation provides more opportunities to reduce the amount of intracellular nucleic acids in the separated extracellular nucleic acids. For example, when the target extracellular nucleic acid of interest is DNA, during the nucleic acid separation step b), the removal of genomic DNA can also supplement or even replace the separate high G force centrifugation of the plasma sample before starting nucleic acid extraction to remove residual cells. Due to the fixation of the present invention, the genomic DNA released from the residual cells is prevented from large-scale degradation, especially if caspase inhibitors are used additionally, and therefore, the unfragmented or less fragmented genomic DNA can be removed by using a size selective nucleic acid separation scheme in step b). This is particularly advantageous because many clinical trial laboratories do not have a centrifuge that can implement such high G force centrifugation or other devices for removing, in particular, trace residual cells.

[0163] Then in step c), use suitable test and / or analytical method to analyze and / or further process the extracellular nucleic acid of separation.For example, they can be identified, modified, contacted with at least one enzyme, amplified, reverse transcribed, cloned, sequenced, contacted with probe, detected (whether they exist or not) and / or can be quantified.Each method is known in the prior art and is generally used in medical science, diagnosis and / or prognosis field to analyze extracellular nucleic acid (also referring to the detailed description in the background of the present invention).Therefore, after step b), separation of extracellular nucleic acid, optionally as part of total nucleic acid, total RNA and / or longitudinal DNA (see above), they can be analyzed, for example, thereby identifying disease state including but not limited to numerous tumor diseases, particularly precancer and malignant tumor as different forms of tumor or cancer presence or absence or severity. For example, the isolated extracellular nucleic acids can be analyzed to detect diagnostic and / or prognostic markers (such as fetal- or tumor-derived extracellular nucleic acids) in many application areas, including but not limited to non-invasive prenatal genetic testing, screening, disease screening, pathogen screening, oncology, cancer screening, early cancer screening, tumor treatment monitoring, genetic testing (typing), infectious disease detection, injury diagnosis, trauma diagnosis, transplant medicine or many other diseases, therefore, are diagnostic and / or prognostic related. According to one embodiment, the isolated extracellular nucleic acids are analyzed to identify and / or characterize disease or fetal characteristics. Therefore, as discussed above, the separation method described herein can further include a step c) of nucleic acid analysis and / or processing.

[0164] Thus, according to one embodiment, the isolated extracellular nucleic acids are analyzed in step c) to identify, detect, screen, monitor or rule out a disease and / or at least one fetal characteristic. Analysis / further processing of the isolated extracellular nucleic acids may be performed using any nucleic acid analysis / processing method, including but not limited to amplification techniques, 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 spectroscopy, hybridization assays, DNA or RNA sequencing, next generation sequencing, restriction analysis, reverse transcription, nucleic acid sequence-based amplification (NASBA), allele-specific polymerase chain reaction, polymerase cycle assembly (PCA), asymmetric polymerase chain reaction, linear exponential polymerase chain reaction (LATE-PCR), helicase-dependent amplification (HDA), hot-start polymerase chain reaction, inter-sequence-specific polymerase chain reaction (ISSR), reverse polymerase chain reaction, ligation-mediated polymerase chain reaction, methylation-specific polymerase chain reaction (MSP), multiplex polymerase chain reaction, nested polymerase chain reaction, solid phase polymerase chain reaction, or any combination thereof. Each technique is known to those skilled in the art and thus requires no further description herein.

[0165] According to one embodiment, after collecting and fixing the cell-containing biological sample according to the teachings of the present invention, either or both of step b) separation and step c) analysis occurs in at least one day up to three days or two days up to ten days. The suitable time period for fixing the cell-containing biological sample, especially the blood sample, respectively, and the fixation method of the extracellular nucleic acid group contained therein using the method of the present invention is also described above, and each disclosure also applies here. According to one embodiment, after collecting and fixing the cell-containing sample according to the method of the present invention, the nucleic acid separation step b) is performed for at least one day, at least two days or at least three days.

[0166] C. Fixed composition

[0167] According to a third aspect, there is provided a composition suitable for fixing a biological sample containing cells, comprising:

[0168] i) polyoxyethylene polymers as fixatives, or

[0169] ii) ethylene glycol as a fixative,

[0170] and one or more, preferably two or more other adjuvants selected from the following group:

[0171] - one or more primary, secondary or tertiary amides;

[0172] - Caspase inhibitors;

[0173] - Anticoagulants and / or chelating agents.

[0174] Preferably, the composition according to the third aspect comprises a polyoxyethylene polymer as a fixative, which is preferably a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500, and further comprises one or more, preferably two or more other auxiliary agents selected from the group consisting of:

[0175] - at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first polyoxyethylene polymer, which is preferably a high molecular weight polyoxyethylene polymer, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less;

[0176] - one or more primary, secondary or tertiary amides;

[0177] - Caspase inhibitors;

[0178] - Anticoagulants and / or chelating agents.

[0179] Advantages and suitable and preferred embodiments are discussed above in conjunction with the fixation method of the first aspect, and reference is also applied to the above disclosure herein. As discussed above, by the present invention, a fixation composition is provided, particularly comprising a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and at least one caspase inhibitor, optionally but preferably in combination with one or more primary, secondary or tertiary amides, in fixing a cell-containing biological sample, particularly blood, plasma and / or serum, which is particularly effective, by fixing the included cells and the included extracellular nucleic acids so as to substantially retain, during fixation, the corresponding fixed extracellular nucleic acid population. Each fixation composition enables the cell-containing biological sample, preferably blood, to be stored and / or processed at room temperature, such as transported for at least two days, preferably at least three days or even longer, without substantially affecting the quality of the blood sample, the corresponding extracellular nucleic acid population contained therein. In particular, during fixation, the extracellular nucleic acid composition is diluted by intracellular nucleic acids, particularly fragmented genomic DNA, and the corresponding contamination is reduced or even avoided. Preferably, the fixation composition is immediately contacted with the sample containing cells during or after the collection of the cell-containing biological sample. In addition, as described above, ethylene glycol may be used as a fixative or together with a polyoxyethylene polymer as a fixative. The preferred combinations containing other fixatives are substantially the same.

[0180] According to one embodiment, the fixing composition comprises a high molecular weight polyoxyethylene polymer. Details are described above in conjunction with the fixing method of the first aspect, with reference to each disclosure. The high molecular weight polyoxyethylene polymer is preferably polyethylene glycol, such as unsubstituted polyethylene glycol. In an embodiment, the molecular weight may be selected from the group consisting of 1500-40000, 2000-30000, 2500-25000, 3000-20000, 3500-15000, 4000-10000, 4500-9000 and 5000-8000. As mentioned above, higher molecular weights of more than 40000 can be used.

[0181] According to one embodiment, the fixing composition comprises a polyoxyethylene polymer having a molecular weight of 1500 or less, in an embodiment, a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less. The molecular weight of the low molecular weight polyoxyethylene polymer is selected from the group consisting of 100-1000, 150-800, 150-700, preferably 200-600, more preferably 200-500, such as 200-400. As shown in the examples, the fixing composition comprising the polyoxyethylene polymer, preferably polyethylene glycol, is an effective fixing agent when used in combination with one or more other fixing agents, such as preferably a caspase inhibitor described herein and one or more primary, secondary or tertiary amides. If the fixing composition comprises each polyoxyethylene polymer having a molecular weight of 1500 or less, such as a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, the fixing effect of these fixing agents is improved.

[0182] According to one embodiment, the fixing composition includes a high molecular weight polyoxyethylene polymer and further includes at least one other polyoxyethylene polymer having a molecular weight lower than the molecular weight of the high molecular weight polyoxyethylene polymer by at least 100, preferably at least 200, at least 300 or at least 400. According to one embodiment, the difference in molecular weight is at least 2500, at least 3500, at least 5000 or at least 7500. As described above in conjunction with the fixing method of the first aspect, it is advantageous to use a composition of polyoxyethylene polymers with different molecular weights. Preferably, the two polyoxyethylene polymers are polyethylene glycols, such as unsubstituted polyethylene glycol. According to an advantageous embodiment, the fixing composition including a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 also includes a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less. The details are described above in conjunction with the fixing method of the first aspect, and reference is also applied to the various disclosures herein. The low molecular weight polyoxyethylene polymer is preferably polyethylene glycol, such as unsubstituted polyethylene glycol. The molecular weight of the low molecular weight polyoxyethylene polymer may be selected from the group consisting of 100-1000, 150-800, preferably 200-600.

[0183] According to one embodiment, the fixing composition of the polyoxyethylene polymer, which is preferably a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500, further comprises one or more primary, secondary or tertiary amides. Advantages of using one or more of the amides in combination with the polyoxyethylene polymer and suitable and preferred embodiments are described in detail above together with the fixing method of the first aspect, with reference to the above disclosure also applied hereto. According to one embodiment, at least one primary, secondary or tertiary amide contained in the fixing composition is a compound of formula 1:

[0184]

[0185] Wherein, R1 is hydrogen or alkyl, preferably C1-C5 alkyl, C1-C4 alkyl or C1-C3 alkyl, more preferably C1-C2 alkyl, R2 and R3 are the same or different, selected from hydrogen and hydrocarbon groups, preferably alkyl groups, having a carbon chain length of 1-20 atoms, in straight or branched form, and R4 is oxygen, sulfur or selenium. Preferably, the amide is a carboxylic acid amide, so that R4 is oxygen. Preferred embodiments are described above together with the fixation method, and reference is also applied to the above disclosures herein. Preferably, a compound of formula 1 that is not classified as a toxic agent is used. Preferably, the fixation composition comprising a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and comprising a compound of formula 1 also includes a caspase inhibitor.

[0186] The compound of formula 1 may be a carboxylic acid amide selected from primary carboxylic acid amides and secondary carboxylic acid amides. According to one embodiment, the composition includes a primary carboxylic acid amide selected from formamide, acetamide, propionamide and butyramide. Preferably, the carboxylic acid amide is selected from butyramide and formamide. More preferably, it is butyramide, because this agent is particularly effective in fixing the extracellular nucleic acid population.

[0187] According to one embodiment, at least one compound according to formula 1 is an N,N-dialkyl-carboxylic acid amide. According to one embodiment, the compound represented by formula 1 is an N,N-dialkyl propionamide, preferably N,N-dimethyl propionamide. N,N-dimethyl propionamide is not classified as a toxic agent. In addition, N,N-dimethyl propionamide has the beneficial effect that it can also fix intracellular nucleic acids, especially if used at appropriate concentrations, it is possible to fix the transcriptional profile.

[0188] According to one embodiment, the fixing composition comprises butanamide and / or N,N-dialkylpropionamide, wherein the N,N-dialkylpropionamide is preferably N,N-dimethylpropionamide. As shown in the examples, the two amides alone and in combination significantly improve the observed fixing effect.

[0189] According to one embodiment, the fixation composition comprising the polyoxyethylene polymer further comprises a caspase inhibitor. The benefits of using a caspase inhibitor and suitable and preferred embodiment combinations of caspase inhibitors are described in detail above together with the fixation method of the first aspect, with reference to the above disclosure also being used therefor. Preferably, the caspase inhibitor is a pan-caspase inhibitor. Preferably, the caspase inhibitor is a modified caspase-specific polypeptide, preferably modified by an O-phenoxy group at the C-terminus, such as Q-VD-OPh. According to a preferred embodiment, a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 is used as the polyoxyethylene polymer. Preferably, polyethylene glycol is used. Preferably, unsubstituted polyethylene glycol is used.

[0190] According to one embodiment, the fixation composition comprises a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and at least one anticoagulant. This embodiment is particularly suitable for fixing blood samples or cell-containing samples derived from blood. According to one embodiment, the fixation composition comprises a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and a chelating agent. Suitable chelating agents, also acting as anticoagulants and suitable concentrations for fixation are described above in conjunction with the method of the invention, reference being made to the above disclosure also for this purpose. Preferably, EDTA is used as chelating agent.

[0191] The fixing composition may also include other adjuvants, as described above in conjunction with the fixing method.

[0192] For fixing blood, the fixing composition preferably includes a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, at least one caspase inhibitor and an anticoagulant, and optionally at least one primary, secondary, or tertiary amide described above. The polyoxyethylene polymer is preferably polyethylene glycol. As described, the amide is preferably a compound shown in Formula 1. It is preferred to use butyramide and / or N,N-dialkylpropionamide, preferably N,N-dimethylpropionamide. According to one embodiment, the composition includes a high molecular weight polyoxyethylene polymer, preferably polyethylene glycol, and also includes a low molecular weight polyoxyethylene polymer, preferably polyethylene glycol. The preferred molecular weight is described above, with reference to each disclosure.

[0193] According to one embodiment, the fixing composition comprises:

[0194] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of at least 1500, preferably 1500-50000, 2000-40000, 2500-30000, 2500-25000, more preferably 3000-20000, 3500-15000 or 4500-10000;

[0195] b) one or more compounds of formula 1;

[0196] c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-OPh;

[0197] d) at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the high molecular weight polyoxyethylene polymer used, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene having a molecular weight of 1000 or less, preferably 200-800 or 200-600;

[0198] e) optionally an anticoagulant and / or a chelating agent, more preferably EDTA.

[0199] According to one embodiment, the fixing composition comprises:

[0200] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of 2,000-40,000, 2,500-30,000, 2,500-25,000, 3,000-20,000 or 3,500-15,000;

[0201] b) one or more compounds of formula 1, preferably butanamide and / or N,N-dimethylpropionamide;

[0202] c) at least one caspase inhibitor;

[0203] d) at least one low molecular weight polyethylene oxide polymer having a molecular weight of 100-1000, 150-800 or 200-600;

[0204] e) Anticoagulants and / or chelating agents, preferably EDTA.

[0205] According to one embodiment, the fixing composition comprises:

[0206] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of 4500-10000;

[0207] b) one or more compounds of formula 1, preferably butanamide and / or N,N-dimethylpropionamide;

[0208] c) at least one caspase inhibitor;

[0209] d) at least one low molecular weight polyethylene oxide polymer having a molecular weight of 100-800, preferably 200-600;

[0210] e) Anticoagulants and / or chelating agents, preferably EDTA.

[0211] Suitable and preferred embodiments of high and low molecular weight polyethylene oxide polymers, caspase inhibitors, compounds of formula 1 and anticoagulants and chelating agents are described in detail above together with the immobilization method, reference is also made to the above disclosures for this purpose. Preferably, the anticoagulant is a chelating agent, more preferably EDTA.

[0212] Suitable and preferred concentrations of the various agents that can be used for fixation in a fixation mixture comprising a fixation composition and a cell-containing biological sample are described above and also apply here. When the expected amount of the fixation composition is mixed with the expected amount of the sample containing cells to be fixed, the skilled person can select the appropriate concentration of the agent in the fixation composition so as to obtain the concentration in the mixture comprising the cell-containing sample. Reference is also made to the above disclosure about the fixation composition for this purpose.

[0213] According to one embodiment, the fixing composition is a liquid. Thereafter, the indicated concentrations of the respective reagents, if present in the fixing composition, are particularly preferred for fixation of blood samples. For example, 0.5 ml-2.5 ml, 0.5 ml-2 ml, preferably 1 ml-2 ml or 1 ml-1.5 ml of a liquid fixing composition can be used. The fixing composition comprising the fixing agents in the following indicated concentrations can be used to fix, for example, 10 ml of blood.

[0214] According to one embodiment, the liquid fixative composition comprises a high molecular weight polyoxyethylene polymer, preferably polyethylene glycol, in a concentration selected from the group consisting of 0.4%-35% (w / v), 0.8%-25% (w / v), 1.5%-20% (w / v), 2.5%-17.5% (w / v), 3%-15% (w / v), 4%-10% (w / v) and 3%-5% (w / v). Suitable concentrations can be determined by a person skilled in the art and may depend in particular on whether the high molecular weight polyoxyethylene alcohol is used alone or in combination with another polyoxyethylene polymer, such as an oligooxyethylene polymer, and the amount of the fixative composition used to fix a certain amount of a sample containing cells, such as a volume. When a high molecular weight polyoxyethylene polymer is used alone, examples of suitable concentration ranges include, but are not limited to, concentrations selected from the group consisting of 2.2%-33.0% (w / v), 4.4%-22.0 (w / v)%, 6.6%-16.5% (w / v), and 8.8%-13.2% (w / v). When a high molecular weight polyoxyethylene polymer is used in combination with a low molecular weight polyoxyethylene polymer, examples of suitable concentration ranges include, but are not limited to, concentrations selected from the group consisting of 0.4%-30.7%, 0.8%-15.3%, 1%-10%, 1.5%-7.7%, 2.5%-6%, 3.1%-5.4%, and 3%-4%.

[0215] In a specific embodiment, the liquid fixing composition comprises 5 mg-500 mg, in particular 10 mg-250 mg, 25 mg-150 mg or 40 mg-100 mg of a high molecular weight polyethylene oxide polymer, preferably polyethylene glycol. In particular, the liquid fixing composition may comprise 0.5 μmol-50 μmol, in particular 1 μmol-25 μmol, 2 μmol-20 μmol or 3 μmol-10 μmol of a high molecular weight polyethylene oxide polymer. The liquid fixing composition may be filled in a collection device and adapted to fix the sample unit.

[0216] According to one embodiment, the liquid fixative composition comprises a low molecular weight polyoxyethylene polymer, preferably polyethylene glycol, in a concentration selected from the group consisting of 0.8%-92.0%, 3.8%-76.7%, 11.5%-53.7%, 19.2%-38.3%, 20%-30% and 20%-27.5%. Depending on whether the low molecular weight polyoxyethylene polymer is liquid or not, the aforementioned concentration refers to (w / v) or (v / v). As shown in the examples, the low molecular weight polyoxyethylene polymer can effectively support the fixation of cell-containing samples, particularly when used in combination with one or more other fixatives described herein.

[0217] In a specific embodiment, the liquid fixative composition comprises 40 μl-4000 μl, in particular 100 μl-2000 μl, 150 μl-1500 μl, 200 μl-1000 μl or 250 μl-750 μl of a low molecular weight polyethylene oxide polymer. In particular, the liquid fixative composition may comprise 0.2 mmol-15 mmol, in particular 0.5 mmol-10 mmol, 0.75 mmol-5 mmol, 1 mmol-3 mmol or 1.2 mmol-2 mmol of a low molecular weight polyethylene oxide polymer. The liquid fixative composition may be filled in a collection device and adapted to fix the sample unit.

[0218] According to one embodiment, the liquid fixing composition comprises one or more primary, secondary or tertiary amides in a concentration selected from the group consisting of 0.4%-38.3%, 0.8%-23.0%, 2.3%-11.5%, 3.8%-9.2%, 5%-15% and 7.5%-12.5%. Depending on whether the primary, secondary or tertiary amide is liquid or not, the aforementioned concentrations refer to (w / v) or (v / v). As mentioned above, preferably the fixing composition also includes one or more primary, secondary or tertiary amides, suitable and preferred embodiments are as described above.

[0219] In a particular embodiment, the liquid fixing composition comprises 10 μl-2000 μl, in particular 50 μl-1000 μl, 100 μl-750 μl or 125 μl-500 or 150-250 μl of primary, secondary or tertiary amide. In particular, the liquid fixing composition may comprise 0.2 mmol-15 mmol, in particular 0.5 mmol-10 mmol, 0.75 mmol-5 mmol, 1 mmol-3 mmol or 1.2 mmol-2 mmol of primary, secondary or tertiary amide. The liquid fixing composition may be filled in a collection device and adapted to fix the sample unit.

[0220] According to one embodiment, the liquid fixative composition comprises a caspase inhibitor at a concentration selected from the group consisting of 0.1 μM-220 μM, 0.8 μM-115.0 μM, 7.7 μM-76.7 μM and 23.0 μM-50 μM. In a specific embodiment, the liquid fixative composition comprises 1 nmol-1000 nmol, particularly 5 nmol-500 nmol, 10 nmol-200 nmol or 25 nmol-100 nmol of a caspase inhibitor. The liquid fixative composition can be filled as in a collection device and as suitable for fixing a sample unit.

[0221] According to one embodiment, the liquid fixative composition comprises a chelating agent, preferably EDTA, such as K2EDTA, at a concentration selected from the group consisting of 9.5mM-1100mM, 20mM-750mM, 50mM-600mM, 75mM-550mM, 100mM-500mM, 125mM-450mM, 130mM-300mM and 140mM-250mM. In a specific embodiment, the liquid fixative composition comprises 10nmol-3000nmol, in particular 50nmol-1500nmol, 100nmol-1000nmol or 150nmol-500nmol of the chelating agent. The liquid fixative composition can be filled in a collection device and adapted to fix the sample unit.

[0222] As described above, the liquid fixing composition comprises a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and preferably one or more primary, secondary or tertiary amides, a caspase inhibitor and a chelating agent. According to one embodiment, the liquid composition comprises a high molecular weight polyoxyethylene polymer and further comprises a low molecular weight polyoxyethylene alcohol.

[0223] According to an alternative, the composition includes ethylene glycol (1,2-ethylene glycol) as a fixative. Ethylene glycol can be used at the concentrations described above for low molecular weight polyoxyethylene polymers. Reference is also made to the various disclosures herein. In particular, the composition may include ethylene glycol and any one or more of the above-mentioned other fixatives together with the composition. According to one embodiment, the composition also includes at least one caspase inhibitor and / or at least one primary, secondary, or tertiary amide, in particular a compound as shown in Formula 1 above. Preferably, the composition includes both. Compositions including ethylene glycol may also include anticoagulants, preferably chelating agents, and / or polyoxyethylene polymers, such as high and / or low molecular weight polyoxyethylene polymers, as described above. Suitable embodiments and concentration ranges of each fixative are as described above and are also used in this embodiment, wherein the composition includes ethylene glycol as a fixative.

[0224] The fixing composition provided by the present invention fixes the cell-containing biological sample, and therefore does not induce the lysis and / or destruction of the nucleated cells and preferably the anucleated cells contained in the sample. Therefore, the fixing composition does not include such a concentration of auxiliary agents: wherein the auxiliary agent will induce or promote the cell lysis of each cell and preferably the cell. The fixing composition induces the damage of the cells contained in the sample, as determined by the test method described in the embodiment section. In particular, the fixing composition described herein can reduce the release of genomic DNA from the cells contained in the cell-containing biological sample into the cell-free part of the sample. In addition, especially when it also includes a preferred caspase inhibitor, the fixing composition may be able to reduce the degradation of nucleic acids present in the fixed sample, especially genomic DNA. As described, the fixing composition can reduce or prevent the extracellular nucleic acid group contained in the biological sample from being contaminated by intracellular nucleic acids, especially DNA and RNA, derived from the cells contained in the fixed sample. Preferably, the fixing composition does not include a crosslinking agent that induces protein-DNA and / or protein-protein crosslinking. In particular, the fixing composition does not include formaldehyde, formalin, paraformaldehyde or formaldehyde releaser or similar crosslinking agents. Preferably, the fixing composition does not include reagents classified as toxic reagents according to GHS. Preferably, the fixation composition of the present invention can fix the extracellular nucleic acid population contained in the cell-containing biological sample for a period of time selected from the group consisting of at least two days, at least three days, at least four days, at least five days and / or at least six days, preferably at room temperature without centrifugation. In particular, one or more, preferably all of the above-mentioned fixation effects are obtained within the defined fixation time.

[0225] According to one embodiment, the fixative composition is a fixed blood, essentially comprising one or more polyoxyethylene polymers, one or more primary, secondary or tertiary amides, at least one caspase inhibitor and an anticoagulant, preferably a chelating agent, such as EDTA, and optionally, a solvent and / or a buffer. As mentioned, water is preferably used as solvent, because it reduces hemolysis during storage. The same applies to modified forms of the embodiment in which ethylene glycol is used as the fixative.

[0226] The fixing composition can be provided in solid form, semi-liquid form or in liquid. If the cell-containing biological sample to be fixed contains a liquid to dissolve the solid (such as, for example, cell-containing body fluid, cells in culture medium, urine) or if a liquid, such as water, is added thereto to dissolve the solid composition, the solid composition is, for example, a suitable choice. As shown in the examples, the solid composition can be used in solid form. However, liquid compositions may also be used. Liquid compositions have the advantage that a mixture containing the sample to be fixed can be quickly obtained, so that an immediate fixing effect can be provided as soon as the sample contacts the liquid fixing composition. Preferably, the one or more fixing agents present in the liquid fixing composition remain stable in solution and do not require user pretreatment, such as, for example, dissolving precipitates of limited solubility, because such pretreatment poses a risk of variation in the fixing efficiency. As shown in the examples, when fixing blood samples, a fixing composition comprising water is particularly advantageous because hemolysis is reduced during storage.

[0227] The present invention also provides a mixture comprising the fixing composition of the third aspect of the present invention mixed with a cell-containing biological sample. Suitable and preferred embodiments of cell-containing biological samples and suitable concentrations of one or more fixing agents when mixed with cell-containing biological samples are described above, especially in conjunction with the fixing method of the present invention. Reference is also made to the above disclosure for this purpose. As described, preferably the sample containing cells is a blood sample.

[0228] According to one embodiment, the fixing composition of the present invention is pre-installed in a sample collection device to fix the sample immediately upon collection or during collection. According to one embodiment, the fixing composition is contacted with the cell-containing biological sample in a volume ratio selected from the group consisting of: 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5 and 1:7-1:5, especially about 1:6. A particular advantage of the fixing composition of the present invention is that a large sample volume can be fixed using a small volume of the fixing composition. Therefore, preferably, the ratio of the fixing composition to the sample is in the range of 1:10-1:5, especially 1:7-1:5, such as about 1:6.

[0229] The fixing composition of the third aspect of the present invention can be used to fix the extracellular nucleic acid group contained in the cell-containing sample, such as preferably a blood sample. In addition, as mentioned above, the fixing composition fixes the cells contained, thereby particularly reducing the release of genomic DNA and other intracellular nucleic acids from the cells included in the cell-containing biological sample. Therefore, the extracellular nucleic acid group is reduced by genomic DNA and other intracellular nucleic acid contamination.

[0230] The fixing composition of the present invention may also be incorporated into a sample collection device, particularly a blood collection device, such as a blood collection container, thereby providing a new and useful version of the device. The container is preferably a blood collection tube. The container type also depends on the sample to be collected, and other suitable forms are described below.

[0231] D. Application

[0232] According to a fourth aspect, the present invention relates to the use of a polyoxyethylene polymer, preferably polyethylene glycol, and / or the use of ethylene glycol, for fixing a cell-containing biological sample, in particular a population of extracellular nucleic acids contained in a cell-containing biological sample. In particular, the fixation composition of the third aspect can be used for said purpose, for example in the method of the fourth aspect of the present invention. The details of the method are described above, with reference to the above disclosure also being used for this. Preferably, as described above, if the cell-containing biological sample is a preferred embodiment, blood, the composition comprises an anticoagulant.

[0233] E. Collection device

[0234] According to a fifth aspect, the present invention provides a collection device for collecting a biological sample containing cells, wherein the collection device comprises:

[0235] i) polyoxyethylene polymers as fixatives, or

[0236] ii) ethylene glycol as a fixative,

[0237] and one or more, preferably two or more other adjuvants selected from the following group:

[0238] - one or more primary, secondary or tertiary amides;

[0239] - Caspase inhibitors;

[0240] - Anticoagulants and / or chelating agents.

[0241] Preferably, the collecting device of the fifth aspect comprises as a fixing agent a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500, and further comprises one or more, preferably two or more, other auxiliary agents selected from the group consisting of:

[0242] - at least one other polyoxyethylene polymer having a molecular weight at least 100, preferably at least 200, at least 300 or at least 400 lower than the molecular weight of the first polyoxyethylene polymer, which is preferably a high molecular weight polyoxyethylene polymer, wherein the other polyoxyethylene polymer is preferably a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less;

[0243] - one or more primary, secondary or tertiary amides;

[0244] - Caspase inhibitors;

[0245] - Anticoagulants and / or chelating agents.

[0246] Providing a corresponding collecting device, such as a sample collecting tube, has the advantage that the sample is immediately fixed when it is collected in the collecting device. The collecting device for collecting a cell-containing biological sample, preferably a blood sample, may include a fixing composition according to the third aspect of the invention. Details about the use of a polyoxyethylene polymer and optionally one or more other adjuvants for fixing and fixing compositions are described above together with other aspects, and reference is also made to the above disclosure for this purpose. Similarly, for alternatives for using ethylene glycol as a fixing agent, details of the embodiments have been described above. Ethylene glycol can also be used in combination with at least one polyoxyethylene polymer. Subsequently, the collecting device is also referred to as a container.

[0247] According to one embodiment, the collection device comprises a polyoxyethylene polymer, preferably polyethylene glycol, one or more primary, secondary or tertiary amides, preferably one or more compounds of formula 1 and a caspase inhibitor. In addition, if the collection container is used to collect blood, it preferably also comprises an anticoagulant, preferably a chelating agent.

[0248] According to one embodiment, a collection device for receiving and collecting a biological sample containing cells is provided, comprising:

[0249] a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of at least 1500, at least 2000, preferably at least 3000, wherein the molecular weight is preferably selected from the group consisting of 2000-40000, 2500-30000, 2500-25000, 3000-20000, 3500-15000, 4000-10000, 4500-8000 and 5000-7000;

[0250] b) one or more primary, secondary or tertiary amides;

[0251] c) at least one caspase inhibitor, preferably a pan-caspase inhibitor, more preferably Q-VD-OPh;

[0252] d) optionally at least one low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, wherein the molecular weight is preferably selected from the group consisting of 100-800, 150-700, 150-600, 200-500 and 200-400;

[0253] e) optionally an anticoagulant and / or a chelating agent, more preferably EDTA.

[0254] In this embodiment, the high molecular weight polyoxyethylene polymer (component a)) which is preferably polyethylene glycol may be contained in the collection device at a concentration such that when the biological sample containing cells is collected into the device, the concentration of the high molecular weight polyoxyethylene polymer in the resulting mixture is selected from the group consisting of 0.05%-4% (w / v), 0.1%-3% (w / v), 0.2%-2.5% (w / v), 0.25%-2% (w / v), 0.3%-1.75% (w / v) and 0.35%-1.5% (w / v). According to one embodiment, the high molecular weight polyoxyethylene polymer is contained in the collection device at a concentration such that when the biological sample containing cells is collected into the device, the concentration of the high molecular weight polyoxyethylene polymer in the resulting mixture is selected from the group consisting of 0.25%-1.5% (w / v), 0.3%-1.25% (w / v), 0.35%-1% (w / v) and 0.4%-0.75% (w / v). These concentration ranges are particularly suitable for fixing blood, the advantages of which are discussed above along with the fixation methods.

[0255] In this embodiment, one or more primary, secondary or tertiary amides (component b)) are contained in the collection device at a concentration such that when the cell-containing biological sample is collected into the device, the concentration of the amide (or combination of amides) in the resulting mixture is selected from the group consisting of: 0.25%-5%, 0.3%-4%, 0.4%-3%, 0.5%-2% or 0.75%-1.5%. At least one amide is preferably a compound of Formula 1:

[0256]

[0257] Wherein R1 is hydrogen or alkyl, preferably C1-C5 alkyl, C1-C4 alkyl or C1-C3 alkyl, more preferably C1-C2 alkyl, R2 and R3 are the same or different, selected from hydrogen and hydrocarbon, preferably alkyl, having a carbon chain length of 1-20 atoms, in straight or branched form, and R4 is oxygen, sulfur or selenium. Preferably, the amide is a carboxylic acid amide so that R4 is oxygen. Preferred embodiments of the compound shown in formula 1 are described above together with the fixation method, and reference is also made to the above disclosure for this purpose. Preferably, the collecting device includes butanamide and / or N,N-dialkylpropionamide, preferably N,N-dimethylpropionamide, as the compound shown in formula 1.

[0258] In this embodiment, at least one caspase inhibitor (component c)) is advantageously contained in the collection device in a concentration such that when the cell-containing biological sample is collected into the device, the concentration of the caspase inhibitor in the resulting mixture is in the range of 0.1 μM-20 μM, more preferably 0.5 μM-10 μM, more preferably 1 μM-10 μM, more preferably 3 μM-7.5 μM or 3 μM-5 μM. As shown by the examples, the fixation composition comprising a polyoxyethylene polymer and a caspase inhibitor is particularly effective in fixing cell-containing biological samples, in particular whole blood samples. Preferably, one or more primary, secondary or tertiary amides as described above are additionally used.

[0259] If in this embodiment, the collecting device further comprises a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less as component b), when the cell-containing biological sample is collected into the device, it is preferably contained in the collecting device in a concentration of 0.5%-10%, 1.5%-9%, 2%-8%, 2.5%-7% and 3%-6% of the low molecular weight polyoxyethylene polymer in the resulting mixture. When the polyoxyethylene polymer is solid, the percentage refers to (w / v), and when the polyoxyethylene polymer is liquid, the percentage refers to (v / v). Preferably, the polyoxyethylene polymer is polyethylene glycol. The advantages of the embodiment in which a high molecular weight polyoxyethylene polymer is used in combination with a low molecular weight polyoxyethylene polymer are described in detail above. In a preferred embodiment, the collection container comprises a high molecular weight polyoxyethylene polymer (component a)) and a low molecular weight polyoxyethylene polymer (component d)) in concentrations such that when a cell-containing biological sample is collected into the device, the concentration of the high molecular weight polyoxyethylene polymer in the resulting mixture is in a range selected from the group consisting of 0.2%-1.5% (w / v), 0.3%-1.25% (w / v) and 0.4%-0.75% (w / v) for high molecular weight polyoxyethylene alcohol, and in a range selected from the group consisting of 1.5%-8%, 2%-7% and 2.5%-6% for low molecular weight polyoxyethylene alcohol. Preferably, the high and low molecular weight polyoxyethylene polymers are polyethylene glycols, as described above.

[0260] According to one embodiment, the collection device also includes an anticoagulant and / or a chelating agent. This embodiment is particularly suitable if the container is used to collect blood or a sample derived from blood, such as plasma or serum. The anticoagulant is contained in a concentration that can prevent blood from clotting. Suitable anticoagulants are described above together with the method of the first aspect, and reference is also made to the above disclosures for this purpose. As described, the anticoagulant is preferably a chelating agent, and suitable embodiments are described above, with reference to each disclosure. According to one embodiment, the container includes a chelating agent, preferably EDTA, in a concentration such that when a cell-containing biological sample is collected into the container, the concentration of the chelating agent in the resulting mixture is within a range selected from the group consisting of 0.5-40 mg / ml, 1-30 mg / ml, 1.6-25 mg / ml, 5-20 mg / ml, and 7.5-17.5 mg / ml.

[0261] According to one embodiment, the collecting device includes ethylene glycol as a fixing agent. Ethylene glycol can be used in the concentrations described above for low molecular weight polyoxyethylene polymers. Reference is also made to the various disclosures herein. In particular, the collecting device may include ethylene glycol and one or more other fixing agents described herein, preferably at least one caspase inhibitor and / or at least one primary, secondary, or tertiary amide, which is preferably a compound shown in Formula 1 defined above. The collecting device may also include ethylene glycol and at least one polyoxyethylene polymer. Suitable embodiments and concentration ranges for each fixing agent are described above, and are also used for embodiments in which ethylene glycol is contained in a collecting container to fix or support the fixation of a cell sample and an extracellular nucleic acid group contained therein.

[0262] The fixing composition and / or each compound for fixing contained in the collection container can be provided in liquid, semi-liquid or dry form. As discussed above, polyoxyethylene polymers and other auxiliary agents for fixing can be provided in the form of fixing composition. The compound for fixing may also be provided in the container as a separate part, and may also be provided in the container in different forms. For example, a component may be provided in dry form, and another compound is provided in liquid. Other combinations are also possible. Suitable formulations and manufacturing selections are known to those skilled in the art.

[0263] For fixing whole blood, it is preferred to include an anticoagulant, such as EDTA, in the container, for example, in the fixing composition. The dried form is, for example, a suitable choice, if the biological sample to be fixed contains a liquid to dissolve the solid (such as, for example, a cell-containing body fluid, cells in a culture medium, urine), or if a liquid, such as water or other solvent is added thereto to dissolve the solid. The advantage of using a solid fixing composition is that solids are generally more stable than liquid chemistry. According to one embodiment, the inner wall of the container is treated / coated with a fixing composition of the present invention or its individual components, such as an anticoagulant. The composition or component can be applied to the inner wall using, for example, a spray drying method. Liquid removal techniques can be implemented on the fixing composition to obtain a protective composition that is substantially in a solid state. Liquid removal conditions can be conditions that obtain at least about 50% by weight, at least about 75% by weight, or at least about 80% by weight of the original amount of the dispersed liquid composition. Liquid removal conditions can be conditions that obtain sufficient liquid removal so that the resulting composition is a film, gel, or other substantially solid or high viscosity layer form. For example, it is possible to obtain a substantially fixed coating (preferably a coating that can be redissolved or not dispersed once in contact with a sample containing cells, preferably a blood product sample). Lyophilization or other techniques may be used to achieve a substantially solid form of the protective agent (e.g., in the form of one or more pellets). Thus, the liquid removal conditions may be such that once the resulting material is contacted with a sample (e.g., a whole blood sample) under the conditions of interest, the protective agent will be dispersed in the sample and fully protect the components of the sample (e.g., extracellular nucleic acids). The liquid removal conditions may be such that a substantially amorphous residual composition is obtained with a sufficiently high viscosity that the residual composition is substantially fixed at ambient temperature; or both.

[0264] According to one embodiment, a liquid composition is used. There are benefits for specific samples such as blood samples. Liquid compositions have the advantage of being able to quickly obtain a mixture with a cell-containing biological sample to be fixed, so that once the sample is in contact with the liquid fixative composition, a substantially immediate fixation effect is provided. In addition, if a larger amount of a fixative composition is used, it is therefore not possible or difficult to spray dry or if the composition interferes with providing a dried composition, a liquid composition is advantageous. Preferably, the fixative present in the liquid fixative composition remains stable in solution and does not require user pretreatment, such as, for example, dissolving a precipitate of limited solubility, because such pretreatment poses a risk of changes in fixation efficiency. To fix blood, according to one embodiment, all compounds are present in the fixative composition. As discussed above, in the case of blood, it is advantageous to use a fixative composition comprising sufficient water to reduce hemolysis during storage of the fixed sample.

[0265] The fixing composition is contained in the container, and its content is effective to provide a fixation effect for the sample to be collected in the container. According to one embodiment, the liquid fixing composition is contacted with the biological sample, and the volume ratio is selected from 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5 and 1:7-1:5, especially about 1:6. A particular advantage of the fixing composition of the present invention is that a small volume of the fixing composition can be used to achieve fixation of a large sample volume. Therefore, preferably, the ratio of the fixing composition to the sample is 1:10-1:5, especially 1:7-1:5, such as about 1:6.

[0266] According to one embodiment, the collecting device is evacuated. Preferably, evacuation is effective for placing a specific volume of liquid cell-containing biological sample inside. Therefore, it ensures that the correct amount of sample contacts the pre-filled amount of fixative composition contained in the container, and therefore, fixation is effective. According to one embodiment, the container includes a tube, and its open end is sealed by a diaphragm. For example, the container is pre-filled with a limited amount of fixative composition, either in solid form or in liquid form, providing a limited vacuum and being sealed by a diaphragm. The diaphragm is constructed so that it is compatible with standard sampling accessories (such as cannula, etc.). When in contact with a cannula, the sample volume predetermined by the vacuum is collected in the container. Each embodiment is particularly advantageous for collecting blood. Suitable containers are disclosed in US6,776,959.

[0267] The container of the present invention can be made of glass, plastic or other suitable materials. The plastic material can be an oxygen permeable material or may contain an oxygen permeable layer. Alternatively, the container can be made of an air permeable plastic material. The container of the present invention is preferably made of a transparent material. Examples of suitable transparent thermoplastic materials include polycarbonate, polyethylene, polypropylene and polyethylene terephthalate. The container can have a suitable size selected according to the required volume of the collected biological sample. As described above, preferably, the container is evacuated to an internal pressure below atmospheric pressure. This embodiment is particularly suitable for collecting body fluids, such as whole blood. The pressure is preferably selected to collect a predetermined volume of biological samples into the container. In addition to this vacuum tube, a non-vacuum tube, a mechanical separator tube or a gel barrier tube can be used as a sample container, particularly for collecting blood samples. Examples of suitable containers and capping devices are disclosed in US 5,860,397 and US 2004 / 0043505. As with containers for collecting cell-containing samples, other collection devices can be used, such as syringes, urine collection devices or other collection devices. The type of container may also depend on the type of sample to be collected, and appropriate containers are available to those skilled in the art.

[0268] According to one embodiment, the container has an open top, a bottom and a sidewall extending therebetween, defining a chamber, wherein a polyoxyethylene polymer, preferably a high molecular weight polyoxyethylene polymer, and one or more other fixatives mentioned above or a fixative composition of the third aspect are contained in the chamber. It may be contained in liquid or solid form. According to one embodiment, it is a liquid. According to one embodiment, the container is a tube, the bottom is a closed bottom, the container further comprises a cover at the open top, and the chamber is under reduced pressure. The benefits of reduced pressure in the chamber are described above. Preferably, the cover can be pierced by a needle or cannula, and the reduced pressure is selected to draw a specific volume of liquid sample into the chamber. According to one embodiment, the chamber is selected to be under reduced pressure to draw a specific volume of liquid sample into the chamber, and the fixative composition is a liquid, placed in the chamber so that the volume ratio of the fixative composition to the specific volume of the sample containing cells is selected from: 10:1-1:20, 5:1-1:15 and 1:1-1:10, and preferably 1:10-1:5, more preferably 1:7-1:5. The relevant advantages are described above.

[0269] Preferably, the container is used to draw blood from a patient. According to one embodiment, it draws 10 ml of blood from a patient. According to one embodiment, the fixing composition is a liquid, and the volume is 2 ml or less, which can be between 0.5 ml and 2 ml, 0.75 ml and 1 ml and 1.5 ml.

[0270] F. Methods for Collecting Cell-Containing Samples

[0271] According to a sixth aspect, a method is provided, comprising the step of collecting a biological sample containing cells from a patient and placing it directly into a chamber of a container of the fifth aspect of the invention. Details about the container and the biological sample containing cells are described above. Reference is made to each disclosure. According to one embodiment, a blood sample is collected, preferably drawn from a patient and placed into a container.

[0272] G. Preparation method

[0273] According to a seventh aspect, a method for preparing the fixing composition of the third aspect of the invention is provided, wherein the components of the fixing composition are mixed. Preferably, they are mixed to provide a liquid solution. As described, a fixing composition comprising water is particularly preferred for fixing blood samples because it can effectively reduce hemolysis.

[0274] The present invention is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used for the practice or test of embodiments of the present invention. Numerical ranges include the numbers defining the range. The titles provided herein are not limitations on the various aspects of the present invention or embodiments that can be read as a whole by reference to the specification.

[0275] Unless the context indicates otherwise, when a solid compound is contained in a liquid mixture or composition, the percentage values ​​described herein refer to (w / v); when a liquid compound is contained in a liquid mixture or composition, such as a mixture obtained by contacting a fixative or a fixative composition comprising the fixative with a sample comprising cells, the percentage values ​​refer to (v / v).

[0276] As used in this specification, the singular forms "a", "an", and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a polyoxyethylene polymer" includes a single type of polyoxyethylene polymer, as well as two or more polyoxyethylene polymers. Likewise, reference to an "agent", "adjuvant", "compound", etc. includes both a single entity and a combination of two or more such entities. References to "the disclosure", "the present invention", etc. include single or multiple aspects of the teachings herein; and so forth. Aspects taught herein are encompassed by the term "invention".

[0277] The term "solution" as used herein refers in particular to a liquid composition, preferably an aqueous composition. It may be a homogeneous mixture of only one phase, but it is also within the scope of the present invention that the solution includes solid adjuvants, such as precipitates, and in particular contains chemical substances, such as fixatives.

[0278] The sizes (sizes) referred to herein with respect to nucleotides, respectively size (size) ranges (nt) refer to the chain length, and are thus used to describe the length of single-stranded as well as double-stranded molecules. In double-stranded molecules, the nucleotides are paired.

[0279] According to one embodiment, in the case of a method, the subject matter described herein comprises certain steps, or in the case of a composition, comprises certain ingredients, protocols and / or buffers refer to the subject matter consisting of the individual steps or ingredients. The preferred selection and combination of the preferred embodiments described herein and the specific subject matter resulting from each combination of the preferred embodiments also belong to the present invention.

[0280] This application claims the priority of EP 14 000 990.3 and US 61 / 955,200 (filing date: March 18, 2014), the disclosures of which are incorporated herein by reference.

[0281] Example

[0282] It should be understood that the following examples are for illustrative purposes only and are not intended to limit the present invention in any way.

[0283] Abbreviations used:

[0284] BA: Butyramide

[0285] ccfDNA: circulating, cell-free DNA

[0286] DMPA: dimethylpropionamide

[0287] EDTA: Ethylenediaminetetraacetic acid

[0288] PEG: polyethylene glycol

[0289] The ability of polyethylene glycol (PEG) to fix cell-containing biological samples, here blood samples, was examined either alone or in combination with various fixatives, including caspase inhibitors and / or various primary and / or tertiary amides. PEG was found to effectively fix leukocytes in whole blood samples in a manner that prevented the release of genomic DNA into the extracellular nucleic acid population compared to a control sample (EDTA blood). This fixation effect was demonstrated for PEG of different molecular weights and when used at various concentrations. It was demonstrated that PEG could be added as a non-aqueous powder or liquid, such as a pure reagent or dissolved in an aqueous solution. PEG alone had a strong fixation effect by itself, but combination with other fixatives, including caspase inhibitors and various amides significantly improved the fixation, reproducibly reducing the increase in DNA released from leukocytes into plasma to 2-fold or even lower levels from day 0 (just after blood draw) to day 6 (storage for 6 days at room temperature). The extended effective fixation achieved is an important advantage because it provides a uniform, reliable fixation method for cell-containing samples, such as blood samples. Furthermore, the examples show that the use of a combination of high molecular weight PEG and low molecular weight PEG is advantageous because a strong immobilization effect is achieved and extracellular nucleic acids can be effectively isolated from the fixed sample using, for example, a silica column-based nucleic acid isolation method.

[0290] I. Materials and Methods

[0291] If not otherwise stated, the following procedures were employed in the Examples.

[0292] 1. Blood Collection and Fixation

[0293] Draw blood into 10 ml spray-dried EDTA tubes (BD) containing 1.8 mg K2EDTA per ml whole blood. 30 minutes after blood draw, either add fixative directly or pour blood gently into a new tube containing fixative. Mix blood and reagent by inverting the tube ten times. Store fixed blood specimens in an upright position at room temperature.

[0294] 2. Preparation of Plasma

[0295] Centrifuge the whole blood sample at 3.000 rpm (revolutions per minute) at ambient temperature. Remove the clear plasma fraction by pipetting and transfer to a new centrifuge tube. In a second round, centrifuge the plasma sample at 16.000 x g for 10 minutes at 4°C. Transfer the supernatant to a new tube and either use it directly for ccfDNA purification or store it at -20°C until use.

[0296] 3. ccfDNA Purification

[0297] Plasma-derived DNA was isolated using the QIAamp circulating nucleic acid kit (QIAGEN GmbH) using the protocol for purification of circulating nucleic acids from 1 ml, 2 ml or 3 ml serum or plasma. If not otherwise stated, 2 ml of plasma was mixed with proteinase K and lysis buffer ACL, incubated at 60°C for 30 min, mixed with buffer ACB, bound to a QIAamp microcolumn (which contains a silica solid phase for binding nucleic acids), washed and eluted using a QIAvac24 plus vacuum manifold with 60 μl elution buffer AVE according to the manufacturer's recommendations.

[0298] 4. Quantitative Real-time PCR Analysis of Isolated Extracellular DNA

[0299] The isolated extracellular DNA was analyzed by real-time PCR experiment on ABI PRISM HT7900 (Life technologies) with 3 μl of eluate. Two fragments of the human 8S rDNA gene, 66 bp and 500 bp, were amplified by multiple PCR using the QuantiTect Multiplex PCR Kit (Qiagen) in a 20 μl experimental volume. The cycle threshold (Ct value) of each sample was translated into the amount of gDNA in the eluate according to the gDNA standard curve: the total amount was obtained by comparing with the standard curve generated by human genomic DNA diluted from 3000 to 0.3 genome equivalents (1 genome equivalent is equivalent to about 6.6 pg human genomic DNA). The amount of gDNA at the storage time point (generally 6 days after blood collection) was compared with the gDNA level at time zero from the same donor.

[0300] Table 1: Summary of DNA target sequence information used in quantitative real-time PCR assays

[0301]

[0302] Quantification of 66bp fragments is used to reflect the total amount of 18S rDNA copies in plasma. Quantification of 500bp is used to determine the copy amount of 18S rDNA derived from apoptotic or mechanically lysed leukocytes of whole blood. The typical length of cell-free DNA is 140-170bp. Therefore, 500bp fragments are considered to be derived from apoptotic, lysed or otherwise damaged blood cells. The increase in the number of 500bp copies during storage from T0 to 6 days is used to measure the fixation efficiency. Therefore, the lower the amount of 500bp DNA released, the better the performance of the fixation method. A higher amount of released 500bpDNA indicates that lysis of white blood cells has occurred and the extracellular nucleic acid population is contaminated by intracellular genomic DNA.

[0303] Blood samples obtained from multiple different individual donors are used for subsequent experiments. For each individual donor sample, the average multiple of the copy number of 66bp and 500bp fragments of 18S rDNA gene in the fixed or unfixed blood stored at different time points (days) at room temperature is calculated relative to time point 0 (0 day) after blood drawing changes. The corresponding single calculated mean value (fold change) is used to detect the fixation efficiency of the different fixed compositions used. Because there is a natural individual difference in the composition and the amount of extracellular nucleic acid included depending on the donor behind the blood sample, this may cause the standard deviation of the increase.

[0304] 5. Hemoglobin test

[0305] The absorbance at 414 nm was measured on a SpectraMax photometer and found to be linearly correlated with the hemolytic color change of plasma.

[0306] II. Embodiment

[0307] 1. Example 1

[0308] In Example 1, the fixation effect of different molecular weight polyethylene glycols (PEG) in combination with BA, EDTA and a caspase inhibitor (Q-VD-OPh) in the absence of water was tested and compared to a combined BA, EDTA and a caspase inhibitor (Q-VD-OPh) regimen. In addition, the effect of the fixation mixture on hemolysis in plasma samples was tested in parallel. EDTA blood samples were used as unfixed reference controls.

[0309] Blood collection and fixation

[0310] 10 ml whole blood samples from eight donors, collected in 10 ml spray-dried K2EDTA tubes, were fixed with a mixture of butanamide (BA) and EDTA with or without PEG of different molecular weights without the addition of water. Caspase inhibitor (Q-VD-OPh) dissolved in DMSO was added by pipetting. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0311] All fixed blood samples were set up in triplicate for each condition and time point tested. At time point 0 (reference time point), plasma was immediately generated after mixing the fixative solution and blood, and circulating extracellular DNA was extracted. As a reference control, EDTA-fixed blood samples (collected in K2 EDTA tubes without other additives) were also stored for 0 or 6 days and analyzed in triplicate.

[0312] Composition of fixative mixture (for each 10 ml K2EDTA whole blood)

[0313] - Unfixed: 1.8mg / ml K2EDTA

[0314] -BA, EDTA, Q-VD-OPh: 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0315] -PEG (600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 250 mg PEG (600, 1000 or 3000), 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO) (anhydrous)

[0316] Thereby, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0317] - Unfixed: 1.8mg / ml K2EDTA

[0318] -BA, EDTA, Q-VD-OPh: 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0319] -PEG (600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 2.5% (w / v) PEG (600, 1000 or 3000), 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0320] result

[0321] The average change in copy number of the 66 bp and 500 bp fragments of 18S rDNA in fixed or unfixed blood from eight donors stored at room temperature for 6 days relative to time point 0 (0 day) after blood draw (x-fold change) was calculated for each of the eight blood donors. Figure 1 The corresponding average fold change of copy number from 8 donors for each condition is shown. All fixation compositions showed significantly lower amounts of genomic DNA release after 6 days of storage at room temperature compared to the unfixed control (EDTA blood), as the average fold change increase was significantly reduced. Thus, a fixation effect was obtained even over a long fixation period of 6 days. Figure 1It is shown that additionally contacting the blood samples with polyethylene glycol significantly improves the fixation effect obtained. Thus, PEG of different molecular weights is highly effective in improving the fixation effect, since the average fold change increase is always reduced below 2-fold, and in embodiments even below 1-fold. That is, the DNA levels at day 6 are comparable to the DNA levels at the basal time point (day 0).

[0322] In summary, the fixation effect of the fixation composition including a caspase inhibitor and an amide, here a primary carboxylic acid amide, can be significantly increased when used in combination with polyethylene glycol. Polyethylene glycols of different molecular weights are effective. In addition, the results show that the PEG fixation performance increases with increasing PEG molecular weight, indicating that there is a positive correlation between the molecular weight of the PEG used and the sample fixation effect obtained. Higher molecular weights improve the fixation effect obtained.

[0323] 2. Example 2

[0324] In Example 2, the fixation effect of a combination of BA, EDTA and a caspase inhibitor (Q-VD-OPh) in the absence of water was examined and compared to compositions further comprising different amounts (0.2 g, 0.3 g or 0.4 g) of PEG of molecular weight 600 (PEG600). EDTA blood was used as an unfixed reference.

[0325] Blood collection and fixation

[0326] 10 ml whole blood samples from six donors, collected in 10 ml spray-dried K2EDTA tubes, were fixed with a mixture of butanamide (BA) and EDTA, a mixture of butanamide (BA) and EDTA and different amounts of PEG 600 (PEG600) without the addition of water. In addition, a caspase inhibitor (Q-VD-OPh) dissolved in DMSO was added by pipetting. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma, and the copy number of the 18SrDNA gene was determined by real-time PCR for each condition and time point tested in triplicate. As a reference control, EDTA-fixed blood samples (collected in K2 EDTA tubes without other additives) were also stored for 6 days.

[0327] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0328] -BA, EDTA, Q-VD-OPh: 100 mg BA, 182 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0329] -PEG600 (0.2-0.4 g), BA, EDTA, Q-VD-OPh: 200, 300 and 400 mg PEG600, 100 mg BA, 188 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO)

[0330] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0331] - Unfixed: 1.8mg / ml K2EDTA

[0332] -BA, EDTA, Q-VD-OPh: 1% (w / v) BA, 20mg / ml K2EDTA, 5μM Q-VD-OPh

[0333] -PEG600 (0.2-0.4g), BA, EDTA, Q-VD-OPh: 2, 3 and 4% (w / v) PEG600, 1% (w / v) BA, 20mg / ml K2EDTA, 5μM Q-VD-OPh

[0334] result

[0335] Quantitative real-time PCR results for six individual donor samples, shown as average fold changes, are shown in Figure 2 . Show relative to time 0, adopt 0.2g, 0.3g or 0.4g PEG600 DNA (66bp and 500bp fragment) increase (average fold change).Compared with reference to EDTA blood, all tested fixed compositions show significantly lower amount of genomic DNA release after 6 days of storage at room temperature.If the sample containing cells is also contacted with polyethylene glycol, the fixation effect is significantly improved.In all three groups of PEG-based fixation schemes, compared with the composition (BA, EDTA, Q-VD-OPh) without PEG, the average fold change of two 18S rDNA amplicon copy numbers is significantly smaller.In all cases, the X fold change is less than 2 times.This embodiment shows that the additional use of different amounts of polyethylene glycol for fixing extracellular nucleic acid groups significantly improves the fixation results obtained using caspase inhibitors and primary carboxylic acid amide butyramide.

[0336] 3. Example 3

[0337] In Example 3, the fixation effect of a reagent mixture comprising high molecular weight PEG (PEG3000), EDTA, BA and a caspase inhibitor (Q-VD-OPh) lyophilized directly into a blood collection tube in the absence of water was tested and compared with samples simultaneously treated with a solution comprising BA, EDTA and a caspase inhibitor (Q-VD-OPh).

[0338] Blood collection and fixation

[0339] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of butanamide (BA) and EDTA with or without PEG3000 and a mixture of a caspase inhibitor (Q-VD-OPh). For freeze drying, all components including caspase inhibitors, EDTA, BA and PEG were dissolved in water. 1 ml volume (total concentration see below) was freeze dried in an Epsilon 2-25D freeze dryer (Christ GmbH) in a 5 ml tube. The blood was transferred from the K2EDTA tube into a 5 ml tube containing freeze-dried fixative and fixed by inverting the tube 10 times. As a reference, the reagent was freshly prepared and the caspase inhibitor (Q-VD-OPh) dissolved in DMSO was added by pipetting.

[0340] Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of 18S rDNA gene was determined by real-time PCR in triplicate.

[0341] Composition of fixative mixture (for each 10 ml K2EDTA whole blood)

[0342] - Freshly prepared BA, EDTA, Q-VD-OPh: 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0343] - Freshly prepared PEG3000, BA, EDTA, Q-VD-OPh: 250 mg PEG3000, 100 mg BA, 132 mg K2EDTA, 10 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO) (anhydrous)

[0344] Composition of 0.5 ml of fixative mixture lyophilized into 5 ml tube:

[0345] - Lyophilized: 0.5 ml fixative containing 125 mg PEG3000, 50 mg BA, 67.5 mg K2EDTA, 5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO)

[0346] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0347] - Unfixed: 1.8mg / ml K2EDTA

[0348] -BA, EDTA, Q-VD-OPh: 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0349] -PEG3000, BA, EDTA, Q-VD-OPh: 1% (w / v) PEG 3000, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0350] result

[0351] The results are shown in Figure 3 . The DNA increase (average fold change) at 6 days after blood draw relative to time 0 based on different amplicon lengths of the 18S rDNA gene is shown. The results again show that the fixation effect is significantly improved if polyethylene glycol is additionally used for fixation, and the fixation effect obtained by BA, EDTA and caspase inhibitor (Q-VD-OPh) is enhanced. In addition, in the extended fixation period tested (6 days), the x-fold change is less than 2 times. In addition, the examples show that these fixation compositions can be freshly prepared or used in lyophilized form.

[0352] 4. Example 4

[0353] In Example 4, the fixation effect of PEG6000 (high molecular weight PEG) alone or in combination with a caspase inhibitor (Q-VD-OPh) on EDTA-fixed samples was tested in an aqueous fixative solution and compared with EDTA-fixed blood alone or BA, EDTA-fixed blood.

[0354] Blood collection and fixation

[0355] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with an aqueous solution containing butanamide or PEG in combination with EDTA and an optional caspase inhibitor (Q-VD-OPh). Plasma was obtained directly from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another 3 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was detected by real-time PCR in triplicate.

[0356] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0357] -BA, EDTA, Q-VD-OPh: 360 mg BA, 68.4 mg K2EDTA, with or without 2.4 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0358] -PEG6000, EDTA, Q-VD-OPh: 137.5 mg PEG6000, 132 mg K2EDTA, with or without 2.2 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1 ml

[0359] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0360] - Unfixed: 1.8mg / ml K2EDTA

[0361] -BA, EDTA: 3% (w / v) BA, 7.2mg / ml K2EDTA

[0362] -BA, EDTA, Q-VD-OPh: 3% (w / v) BA, 7.2mg / ml K2EDTA, 1μM Q-VD-OPh

[0363] -PEG6000, EDTA: 1.25% (w / v) PEG6000, 7.2mg / ml K2EDTA

[0364] -PEG6000, EDTA, Q-VD-OPh: 1.25% (w / v) PEG6000, 7.2mg / ml K2EDTA, 1μM Q-VD-OPh

[0365] result

[0366] The results of qPCR analysis of eight different donors are shown in Figure 4 . The average change (fold change) in the number of copies of DNA copies of different 18S rDNA gene amplicons (66bp or 500bp) at time point 0 (0 day) after blood draw for fixed or unfixed blood from eight donors stored at room temperature for three days is shown. The fixed composition containing high molecular weight polyethylene glycol shows a significantly lower amount of DNA release when stored at room temperature for three days compared with unfixed EDTA blood. The fixation effect obtained by the fixed composition containing only high molecular weight PEG as a fixative during the three-day storage period is better than the effect obtained by using the fixative butanamide. This shows that if fixation is to be obtained in a shorter fixation period, polyethylene glycol is also effective as a fixative alone. When polyethylene glycol is used in conjunction with a caspase inhibitor (Q-VD-OPh), the fixation effect is improved. The fixation effect obtained by using a combination of high molecular weight PEG and a caspase inhibitor is even better than the fixation scheme using a combination of butanamide and a caspase inhibitor. The results show that PEG soluble in an aqueous solution only combines an anticoagulant (EDTA) to fix both ccfDNA and white blood cells (thereby preventing cellular DNA from being released into plasma). Furthermore, it was found that the fixation effect was even more pronounced compared to butanamide.

[0367] 5. Example 5

[0368] In Example 5, the fixation effect of PEG of different molecular weights (PEG300, PEG600, PEG1000) in an aqueous fixative solution also including BA, EDTA and a caspase inhibitor (Q-VD-OPh) was tested and compared to samples co-treated with BA, dimethylpropionamide (DMPA), EDTA and a caspase inhibitor (Q-VD-OPh). Unfixed EDTA blood was used as a reference comparison.

[0369] Blood collection and fixation

[0370] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of butanamide, EDTA and a caspase inhibitor (Q-VD-OPh) in an aqueous solution with or without PEG of different molecular weights. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0371] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0372] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0373] -PEG (300, 600 or 1000), BA, EDTA, Q-VD-OPh: 287.5 mg PEG (300, 600 or 1000), 115 mg BA, 154.5 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0374] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0375] - Unfixed: 1.8mg / ml K2EDTA

[0376] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0377] -PEG (300, 600 or 1000), BA, EDTA, Q-VD-OPh: 2.5% (w / v) PEG, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0378] result

[0379] Figure 5 The fixation results obtained are shown. It can be seen that the aqueous fixation composition containing PEG of different molecular weights fixes the blood sample and further improves the fixation effect obtained using butanamide and caspase inhibitors. From the reduction in the amount of contaminating genomic DNA, it can be seen that the fixation of white blood cells is significantly improved. The results also show that as the molecular weight of the PEG used increases, the fixation effect is enhanced. When a polyethylene glycol with a molecular weight of 1000 is used, the increase in the 500bp fragment is reduced by less than 2 times.

[0380] 6. Example 6

[0381] Here, the fixation effect of decreasing PFG concentrations was tested in aqueous fixative solutions in combination with butanamide, EDTA and a caspase inhibitor (Q-VD-OPh). Unfixed EDTA blood was used as a reference control. Compositions containing BA, EDTA and Q-VD-OPh were tested in parallel.

[0382] Blood collection and fixation

[0383] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of butanamide, EDTA and a caspase inhibitor (Q-VD-OPh) in an aqueous solution with or without different concentrations of PEG6000. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0384] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0385] -BA, EDTA, Q-VD-OPh: 110 mg BA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1 ml

[0386] -PEG6000 (2–0.7%), BA, EDTA, Q-VD-OPh: 220, 165, 110, 77 mg PEG6000, 110 mg BA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1 ml

[0387] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0388] - Unfixed: 1.8mg / ml K2EDTA

[0389] -BA, EDTA, Q-VD-OPh: 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0390] -PEG6000 (2–0.7%), BA, EDTA, Q-VD-OPh: 2, 1.5, 1, 0.7% (w / v) PEG6000, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0391] result

[0392] In Example 6, the effects of decreasing concentrations of higher molecular weight PEG6000 on white blood cell fixation were examined when used in combination with butanamide, EDTA, and a caspase inhibitor. Figure 6 Describe the fixation results of the extracellular nucleic acid population obtained by increasing detection of 18S rDNA by quantitative real-time PCR analysis. Compared with the fixation scheme involving butanamide, EDTA and caspase inhibitors, the entire fixation composition of the present invention including PEG showed significantly lower DNA release after 6 days of storage at room temperature. In addition, it can be seen that high molecular weight polyethylene glycol can be used in different concentrations in aqueous solution to fix white blood cells and thus reduce genomic DNA contamination of the extracellular nucleic acid population. In addition, it is shown again that PEG enhances the fixation effect of butanamide and caspase inhibitors to provide a very effective fixation scheme.

[0393] 7. Example 7

[0394] In Example 7, the fixation effect of PEG in different volumes of aqueous fixative solution and in combination with EDTA, a caspase inhibitor (Q-VD-OPh) and different amides (BA or DMPA) was tested. Unfixed EDTA blood was used as a reference control. Compositions containing BA, DMPA, EDTA and Q-VD-OPh were tested in parallel.

[0395] Blood collection and fixation

[0396] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of PEG6000, butanamide or DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) in different volumes of aqueous solution (0.8 ml and 1.2 ml). Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another 6 days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was detected by real-time PCR in triplicate.

[0397] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0398] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0399] -PEG6000, BA, EDTA, Q-VD-OPh: 112 mg PEG6000, 56 mg BA, 150 mg K2EDTA, 2.23 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.2 or 0.8 ml

[0400] -PEG6000, DMPA, EDTA, Q-VD-OPh: 112 mg PEG6000, 112 μl DMPA, 150 mg K2EDTA, 2.23 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.2 or 0.8 ml

[0401] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0402] - Unfixed: 1.8mg / ml K2EDTA

[0403] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0404] -PEG6000, BA or DMPA, EDTA, Q-VD-OPh to 1.2 ml: 1% (w / v) PEG6000, 0.5% (w / v) BA or 1% (v / v) DMPA, 15 mg / ml K2EDTA, 1 μM Q-VD-OPh

[0405] -PEG6000, BA or DMPA, EDTA, Q-VD-OPh to 0.8 ml: 1.04% (w / v) PEG6000, 0.52% (w / v) BA or 1.04% (v / v) DMPA, 15.6 mg / ml K2EDTA, 1.03 μM Q-VD-OPh

[0406] result

[0407] Figure 7 Results of these fixation experiments are shown. The average change (fold change) in the copy number of DNA copies of different 18S rDNA gene amplicons (66 bp or 500 bp) at time point 0 (0 day) after blood draw for fixed or unfixed blood stored at room temperature for six days is shown. The results show that polyethylene glycol can be combined with different amides to fix white blood cells in different volumes of aqueous solutions, thereby providing fixed blood samples in which the extracellular nucleic acid population is preserved by preventing dilution by intracellular nucleic acids.

[0408] 8. Example 8

[0409] In Example 8, the effect of the fixative in the aqueous fixative solution was tested by a hemolysis test.

[0410] Blood collection and fixation

[0411] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with butanamide or a mixture of DMPA and EDTA with or without PEG added to water. Caspase inhibitor (Q-VD-OPh) dissolved in DMSO was added by pipetting. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another 3, 6 and 10 days before plasma production. Hemoglobin content was determined by measuring the absorbance at 414 nm on a spectrophotometer.

[0412] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0413] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0414] -PEG6000, BA, EDTA, Q-VD-OPh: 137.5 mg PEG6000, 55 mg BA, 165 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.0 ml

[0415] -PEG6000, DMPA, EDTA, Q-VD-OPh: 110 mg PEG6000, 110 μl DMPA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.0 ml

[0416] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0417] - Unfixed: 1.8mg / ml K2EDTA

[0418] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0419] -PEG6000, BA, EDTA, Q-VD-OPh: 1.25% (w / v) PEG6000, 0.5% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0420] -PEG6000, DMPA, EDTA, Q-VD-OPh: 1.0% (w / v) PEG6000, 1.0% (v / v) DMPA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0421] result

[0422] Figure 8 Shown are the hemolytic effects of plasma samples from 8 different donors after storage for 0, 3, 6 and 10 days. Figure 8 Depicted are the average increase in hemolysis from eight donors as the absorbance at 414 nm increases in plasma fractions.

[0423] However, in the EDTA control test, hemolysis increased during storage, and the use of the aqueous fixative solution of the present invention comprising PEG in combination with EDTA and BA or DMPA reduced hemolysis at the time point analyzed compared to the EDTA reference, as shown in FIG. Figure 8 As shown. Notably, the increase in hemolysis seen in the EDTA control from 6 days to 10 days of storage was substantially reduced in all tested solutions comprising the fixative composition of the present invention and water. The extent of reduction in hemolysis in the PEG-containing aqueous solution was comparable to the combined BA, DMPA, EDTA, caspase inhibitor (Q-VD-OPh) fixed samples. Therefore, dissolving PEG in the aqueous fixative composition is advantageous because it effectively reduces hemolysis.

[0424] 9. Example 9

[0425] In Example 9, the effects of different molecular weight PEGs (PEG300, PEG600, PEG1000, PEG3000) in combination with BA, EDTA and a caspase inhibitor (Q-VD-OPh) on ccfDNA copy number were tested and compared with unfixed EDTA control blood. Compositions containing BA, DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) were tested in parallel.

[0426] Blood collection and fixation

[0427] 10 ml whole blood samples from eight donors, collected in 10 ml spray-dried K2EDTA tubes, were fixed using a combination of butanamide, EDTA, and a caspase inhibitor (Q-VD-OPh) in an aqueous solution containing PEG of increasing molecular weight. Plasma was obtained directly from 5 ml fixed or unfixed blood samples one hour after blood collection. ccfDNA was purified from 2 ml plasma, and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0428] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0429] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0430] -PEG (300, 600, 1000 or 3000), BA, EDTA, Q-VD-OPh: 287.5 ml PEG 300 or 287.5 mg PEG (600, 1000 or 3000), 115 mg BA, 154.5 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0431] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0432] - Unfixed: 1.8mg / ml K2EDTA

[0433] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0434] -PEG (300, 600, 1000or 3000), BA, EDTA, Q-VD-OPh: 2.5% (v / v or w / v) PEG, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0435] result

[0436] The results of the fully quantitative real-time PCR analysis of eight different donors are shown as averages. Fig. 9 In particular, the mean values ​​of the absolute copy numbers of different 18S rDNA gene amplicons (66 bp or 500 bp) in fixed or unfixed blood samples of donors on day 0 after blood draw are shown. The aim was to test the effect of the fixation method used on the subsequent nucleic acid yield when using a nucleic acid isolation method based on a silica column. Fig. 9 It is shown that the addition of higher molecular weight PEG results in a decrease in the number of detectable amplicon gene copies in plasma compared to EDTA reference samples (unfixed protocols) or fixed blood solutions containing BA, DMPA, EDTA and caspase inhibitors (Q-VD-OPh). The results indicate that for the use of increased molecular weight or chain length PEG for fixation, when used at higher concentrations, it may result in a decrease in the number of detectable ccfDNA gene copies in plasma when using a nucleic acid separation method based on a silica column to separate extracellular nucleic acids from fixed samples.

[0437] 10. Example 10

[0438] In Example 10, PEG6000 was tested at different concentrations (1.0%, 1.25%, or 1.5%) in combination with BA, EDTA, and a caspase inhibitor (Q-VD-OPh). EDTA-fixed blood samples were used as reference controls. Blood mixtures containing BA, DMPA, EDTA, and a caspase inhibitor (Q-VD-OPh) were analyzed in parallel.

[0439] Blood collection and fixation

[0440] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with combinations of amides (DMPA and / or BA), EDTA, caspase inhibitors (Q-VD-OPh), with or without PEG6000 in 1.5 ml-10 ml blood volumes with increasing PEG concentrations. Plasma was obtained directly from 5 ml fixed or unfixed blood samples 1 hour after blood collection. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was detected by real-time PCR in triplicate.

[0441] Composition of the K2EDTA whole blood fixative mixture for each 10 ml:

[0442] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0443] -PEG6000 (1–1.5%), BA, EDTA, Q-VD-OPh – to 1.5 ml: 115, 144 and 172 mg PEG6000, 115 mg BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0444] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0445] - Unfixed: 1.8mg / ml K2EDTA

[0446] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0447] -PEG6000, BA, EDTA, Q-VD-OPh: 1, 1.25 and 1.5% (w / v) PEG6000, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0448] result

[0449] The results of Example 10 are shown in Fig.10 Shown is the average reduction in absolute copy number of ccfDNA at day 0 after blood draw from 8 donors based on different amplicon lengths of the 18S rDNA gene, using a fixation composition of the invention comprising different concentrations of PEG6000 (1.0%, 1.25% or 1.5%) and BA, EDTA and a caspase inhibitor (Q-VD-OPh). Fig.10 The reduction of the absolute copy number of the 66bp and 500bp fragments of the 18S rDNA gene in the fixed blood-plasma containing PEG occurs in a PEG concentration-dependent manner. This indicates that when using a nucleic acid separation scheme based on a silica gel column, an increased concentration of higher molecular weight PEG (PEG6000) in the fixed solution can lead to a reduction in the number of ccfDNA gene copies detectable in plasma.

[0450] 11. Example 11

[0451] In Example 11, the fixation effect of different volumes of an aqueous fixative composition comprising a high molecular weight PEG (PEG6000) in combination with BA, EDTA, and a caspase inhibitor (Q-VD-OPh) was analyzed. Blood incubated with a fixative solution comprising a combination of two amides (DMPA, BA), EDTA, and a caspase inhibitor (Q-VD-OPh) was analyzed in parallel. EDTA blood was used as an unfixed reference.

[0452] Blood collection and fixation

[0453] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with combinations of amides (DMPA and / or BA), EDTA, caspase inhibitors (Q-VD-OPh), with or without PEG6000 in different volumes of aqueous solutions. Plasma was obtained directly from 5 ml fixed or unfixed blood samples 1 hour after blood collection. ccfDNA was purified from 2 ml plasma and the copy number of 18S rDNA gene was determined by real-time PCR in triplicate.

[0454] Composition of the K2EDTA whole blood fixative mixture for each 10 ml:

[0455] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0456] -PEG6000, BA, EDTA, Q-VD-OPh – to 1 ml: 110 mg PEG6000, 110 mg BA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1 ml

[0457] -PEG6000, BA, EDTA, Q-VD-OPh – to 1.5 ml: 115 mg PEG6000, 115 mg BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0458] -PEG6000, BA, EDTA, Q-VD-OPh – to 2 ml: 120 mg PEG6000, 120 mg BA, 162 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0459] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0460] - Unfixed: 1.8mg / ml K2EDTA

[0461] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0462] -PEG6000, BA, EDTA, Q-VD-OPh to 1, 1.5 or 2 ml: 1% (w / v) PEG6000, 1% (w / v) BA, 15 mg / ml K2EDTA, 5 μM Q-VD-OPh

[0463] result

[0464] In Example 11, different volumes (1 ml, 1.5 ml or 2 ml) of the fixative solution of the present invention comprising PEG6000 in combination with BA, EDTA and a caspase inhibitor (Q-VD-OPh) were tested. Fig.11 The results show that the reduction in the absolute copy number of the two test fragments of the 18SrDNA gene in the fixed sample depends on the fixative volume. Therefore, increasing the volume of the fixed composition containing high molecular weight PEG (therefore increasing the ratio of the fixed composition to blood) leads to a reduction in the number of ccfDNA gene copies detectable in plasma. From the results of the 1ml fixed solution shown, it can be seen that the copy number is not significantly reduced when a smaller volume is used. This result is surprising because the total concentration is the same in the mixture containing the sample.

[0465] 12. Example 12

[0466] Example 12 shows the fixation effect of a fixation composition comprising, in addition to DMPA, EDTA and a caspase inhibitor (Q-VD-OPh), a combination of high molecular weight polyethylene glycol (PEG6000) and low molecular weight polyethylene glycol (PEG300) in two different volumes (1.5 ml and 2.0 ml) of aqueous solution. EDTA blood was used as an unfixed reference control.

[0467] Blood collection and fixation

[0468] 10 ml whole blood samples from eight donors, collected in 10 ml spray-dried K2EDTA tubes, were fixed with a mixture of PEG300 and PEG6000, DMPA, EDTA, and a caspase inhibitor (Q-VD-OPh) in different volumes of 1.5 and 2.0 ml of aqueous solution. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another six days before plasma production. ccfDNA was purified from 2 ml plasma, and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0469] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0470] -0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh – 2ml: 60mg PEG6000, 300 or 600μl PEG300, 120μl DMPA, 162mg K2EDTA, 12μl Q-VD-OPh (1mg dissolved in 388μl DMSO), add water to 2ml

[0471] -0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh – 1.5 ml: 57.5 mg PEG6000, 287.5 μl or 575 μl PEG300, 115 μl DMPA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0472] Thus, the following final concentrations of the different components were obtained in the mixture after contact with blood:

[0473] - Unfixed: 1.8mg / ml K2EDTA

[0474] -PEG6000, PEG300, DMPA, EDTA, Q-VD-OPh – 2ml: 0.5% (w / v) PEG6000, 2.5 or 5% (v / v) PEG300, 1% (v / v) DMPA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0475] result

[0476] Fig.12The quantitative real-time PCR analysis results of 8 different donors are described as the copy number changes (average fold changes) of two kinds of test 18S rDNA gene amplicons (66bp or 500bp) in the fixed or unfixed blood derived from 8 donors stored at room temperature for 6 days relative to time point 0 (day 0) after blood sampling. All fixed samples show a comparable low increase in the copy number fold change of 18S rDNA gene amplicons. The fixation effect obtained in all cases is very high. The increase of 500bp fragments is much lower than 2 times, even lower than 1.5 times, although the sample has been stored for a long period of 6 days of fixation.

[0477] Each immobilization composition was also tested for changes in the absolute copy number of 18S rDNA amplicons at day 0 after blood draw to analyze whether the detectable ccf DNA gene copy number was reduced. Fig.13 Results are shown. It can be seen that all test fixing compositions show an absolute copy number comparable to or even better than that of unfixed samples. Therefore, when using a nucleic acid separation method based on a silica gel column from a fixed sample to separate nucleic acids, the nucleic acid yield will not decrease. When using a higher concentration of high molecular weight polyethylene glycol, a nucleic acid yield decrease is observed, which is not seen when a high molecular weight polyethylene glycol and a low molecular weight polyethylene glycol combination are used. Using each combination can reduce the concentration of high molecular weight polyethylene glycol without damaging the fixation effect supported by low molecular weight polyethylene glycol. Low molecular weight polyethylene glycol can also be used in higher concentrations without damaging the subsequent nucleic acid separation procedures involving silica gel columns. The standard deviation observed for the increase is due to the fact that there is variation in the amount of ccfDNA between donor donors.

[0478] This indicates that the combination of high molecular weight polyethylene glycol and low molecular weight polyethylene glycol is very beneficial for the obtained fixation effect and the yield of nucleic acid that can be isolated from the fixed sample. Therefore, a mixture of different polyethylene glycols can be used in combination to effectively fix blood samples without reducing the absolute ccfDNA copy number.

[0479] 13. Example 13

[0480] In Example 13, the fixation effect of a combination of high molecular weight PEG (0.5% PEG6000) and low molecular weight PEG (2.5% or 5% PEG300) bound to an aqueous fixative solution containing BA, EDTA, and a caspase inhibitor (Q-VD-OPh) was analyzed. Blood incubated with a fixative solution comprising a mixture of two amides (DMPA, BA), EDTA, and a caspase inhibitor (Q-VD-OPh) was co-analyzed. EDTA blood was used as an unfixed reference control.

[0481] Blood collection and fixation

[0482] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of PEG300 and PEG6000, BA, EDTA and a caspase inhibitor (Q-VD-OPh) in an aqueous solution with a volume of 1.5 ml. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. Residual blood was stored at room temperature for another six days before plasma production. ccfDNA was purified from 2 ml plasma and the copy number of the 18S rDNA gene was determined by real-time PCR in triplicate.

[0483] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0484] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0485] -0.5% PEG6000, 2.5 or 5% PEG300, BA, EDTA, Q-VD-OPh – 1.5 ml: 57.5 mg PEG6000, 287.5 μl or 575 μl PEG300, 115 mg BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0486] Thus, the following final concentrations of the different components were obtained in the whole blood / fixative mixture:

[0487] - Unfixed: 1.8mg / ml K2EDTA

[0488] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0489] -PEG6000, PEG300, BA, EDTA, Q-VD-OPh – 1.5ml: 0.5% (w / v) PEG6000, 2.5 or 5% (v / v) PEG300, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0490] result

[0491] Fig.14The results of qPCR analysis of 8 donors are shown as the average change (fold change) in the copy number of 66bp or 500bp long 18S rDNA gene amplicons in fixed or unfixed blood from donors stored at room temperature for 6 days relative to time point 0 (day 0) after blood collection. However, unfixed EDTA blood showed an increase in the average fold change in copy number, and all fixed compositions containing PEG only showed a low increase in the average fold change in the copy number of 18S rDNA gene amplicons. The results show similar fixation capabilities of the fixed compositions containing PEG tested. The obtained fixation effect is better than the fixed compositions containing BA, DMPA, EDTA and caspase inhibitors, thus proving again the important advantages achieved by the present invention.

[0492] In addition, it was confirmed that the described favorable fixation capacity of the used fixation solutions containing a combination of high and low molecular weight PEG was not accompanied by a decrease in the ccfDNA copy number. This was analyzed by testing the same solutions for changes in the absolute copy number of 18SrDNA amplicons in plasma at day 0 after blood draw. Fig.15 The results obtained are shown. The absolute copy number obtained is similar to the fixed composition including BA, DMPA, EDTA and caspase inhibitors. Therefore, no obvious reduction in absolute ccfDNA copy number was detected in this assay. Therefore, when using the standard nucleic acid separation procedure involving silica gel membrane, the combination of different molecular weight PEGs, particularly high and low molecular weight PEGs can be combined in aqueous solutions of different volumes containing BA to effectively fix the extracellular nucleic acid population of blood samples, particularly by fixing white blood cells, without significantly reducing absolute ccfDNA copy number.

[0493] 14. Example 14

[0494] In Example 14, the effect of aqueous fixative solutions was tested by hemolysis experiments. Here, combinations of high molecular weight PEG (0.5% PEG6000) and low molecular weight PEG (2.5% or 5% PEG300) were analyzed with BA or DMPA and EDTA and a caspase inhibitor (Q-VD-OPh). Blood incubated with a fixative solution comprising a mixture of two amides (DMPA, BA), EDTA, and a caspase inhibitor (Q-VD-OPh) was analyzed together. EDTA blood was used as an unfixed reference control.

[0495] Blood collection and fixation

[0496] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and fixed with a mixture of PEG300 and PEG6000, BA or DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) in a volume of 1.5 ml aqueous solution. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another six days before plasma production. The hemoglobin content was determined by measuring the absorbance at 414 nm on a spectrophotometer.

[0497] Composition of fixative mixture (for each 10 ml K2EDTA whole blood):

[0498] -BA, DMPA, EDTA, Q-VD-OPh: 180 mg BA, 180 μl DMPA, 68.4 mg K2EDTA, 12 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 2 ml

[0499] -0.5% PEG6000, 2.5 or 5% PEG300, DMPA, EDTA, Q-VD-OPh: 57.5 mg PEG6000, 287.5 μl or 575 μl PEG300, 115 μl DMPA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0500] -0.5% PEG6000, 2.5 or 5% PEG300, BA, EDTA, Q-VD-OPh: 57.5 mg PEG6000, 287.5 μl or 575 μl PEG300, 115 mg BA, 155 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0501] Thus, the following final concentrations of the different components were obtained in the whole blood / fixative mixture:

[0502] - Unfixed: 1.8mg / ml K2EDTA

[0503] -BA, DMPA, EDTA, Q-VD-OPh: 1.5% (w / v) BA, 1.5% (v / v) DMPA, 7.2mg / ml K2EDTA, 5μM Q-VD-OPh

[0504] -PEG6000, PEG300, DMPA, EDTA, Q-VD-OPh: 0.5% (w / v) PEG6000, 2.5 or 5% (v / v) PEG300, 1% (v / v) DMPA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0505] -PEG6000, PEG300, BA, EDTA, Q-VD-OPh: 0.5% (w / v) PEG6000, 2.5 or 5% (v / v) PEG300, 1% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0506] result

[0507] Fig.16 Shown is the effect of the analyzed aqueous solutions including different molecular weight PEG and either DMPA or BA on hemolysis of plasma samples from 8 different donors at 0 day after blood draw and after 6 days of storage. Fig.16 It is shown that hemolysis increases with the increase in absorbance at 414 nm of the plasma portion after 0 and 6 days of blood storage.

[0508] As in Fig.16 As can be seen in the EDTA control experiment, the blood samples were stored for 6 days and the hemolysis was increased (increase in absorption at 414 nm) compared to the initial time point of storage day 0. With a fixative composition comprising a mixture of 0.5% PEG6000 (a higher molecular weight PEG) and 5% concentration of PEG300 (a lower molecular weight PEG) in combination with EDTA, a caspase inhibitor (Q-VD-OPh) and BA or DMPA, the hemolysis was similar to the EDTA reference sample. In contrast, with a fixative composition of the invention comprising a lower concentration of PEG300 (here: 2.5%) in combination with 0.5% PEG6000, EDTA, a caspase inhibitor (Q-VD-OPh) and BA or DMPA, the hemolysis was reduced after 6 days of blood storage. This demonstrates that hemolysis can be prevented in a fixative composition comprising a balanced composition of high and low molecular weight PEGs when dissolved in an aqueous solution.

[0509] 15. Example 15

[0510] A fixative composition including PEG6000, BA, EDTA and a caspase inhibitor (Q-VD-OPh) prefilled in a vacutainer blood collection tube (α tube) was compared to a commercially available Streck cell-free DNA BCT tube containing a fixative composition based on the use of a formaldehyde releaser as a fixative.

[0511] Blood collection and fixation

[0512] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA, pre-filled with α tubes and Streck cell-free DNA BCT tubes containing the amount of the fixation composition of the present invention including PEG6000, EDTA, caspase inhibitor (Q-VD-OPh) and BA or DMPA. Plasma was obtained directly from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another 3, 6 and 10 days before plasma production. CcfDNA was purified from 2 ml plasma, and the copy number of the 18SrDNA gene was detected by real-time PCR in triplicate.

[0513] Composition of the fixative mixture in different tubes (total drawn volume 10 ml blood):

[0514] -EDTA – 10 ml spray dried EDTA

[0515] -α1 tube (PEG6000, BA, EDTA, Q-VD-OPh): 137.5 mg PEG6000, 55 mg BA, 165 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.0 ml

[0516] -α2 tube (PEG6000, DMPA, EDTA, Q-VD-OPh): 110 mg PEG6000, 110 μl DMPA, 147 mg K2EDTA, 11 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.0 ml

[0517] -Streck Cell-Free DNA BCT Tubes: Contains a formaldehyde releaser as a fixative

[0518] Thus, the following final concentrations of the different components were obtained in the whole blood / fixative mixture:

[0519] -EDTA tube: 1.8mg / ml K2EDTA

[0520] -α1 tube (PEG6000, BA, EDTA, Q-VD-OPh): 1.25% (w / v) PEG6000, 0.5% (w / v) BA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0521] -α2 tube (PEG6000, DMPA, EDTA, Q-VD-OPh): 1.0% (w / v) PEG6000, 1.0% (v / v) DMPA, 15mg / ml K2EDTA, 5μM Q-VD-OPh

[0522] -Streck Cell-Free DNA BCT Tube: Concentration not applicable

[0523] result

[0524] The results are shown in Fig.17 and 18 Relative to time point 0 (day 0) after blood was drawn from eight blood donors, a 66 bp fragment of the 18S rDNA gene was analyzed in fixed or unfixed blood from eight donors stored at room temperature for 3, 6 or 10 days ( Fig.17 ) and 500 bp fragment ( Fig.18 ) copy number changes (mean fold change). The bars represent the corresponding standard deviations of the mean fold change of the copy number of eight donors per condition. Fig.17 and 18 As shown, the two tested fixation compositions, including PEG6000, EDTA, a caspase inhibitor (Q-VD-OPh) and BA or DMPA, are very effective in fixing the extracellular nucleic acid population in blood. For all tested time points, the average fold change in the copy number of the 66bp fragment of 18S rDNA stays at the basal level of time point zero (fold change of about 1.0). Fig.18 The results of the 500bp fragment level of the 18S rDNA gene are shown to be equivalent, that is, the test fragment of the cellular nucleic acid released during cell disruption stays in the state of time point zero during the test period. This fixation effect is equivalent to the results of using Streck cell-free DNA BCT tubes. Therefore, the fixation composition of the present invention effectively fixes the extracellular nucleic acid population in the blood sample by reducing the release of intracellular nucleic acids such as genomic DNA in particular from white blood cells, similar to the Streck cell-free DNA BCT tubes including formaldehyde releasers. However, as explained above, the use of substances that release formaldehyde has disadvantages because they damage the efficiency of extracellular nucleic acid separation by inducing crosslinking between nucleic acid molecules or between proteins and nucleic acids. Therefore, a specific nucleic acid separation method must be used. The fixation composition of the present invention based on the use of polyoxyethylene polymers does not involve the use of such crosslinking substances and has important advantages over crosslinking-based fixation techniques.

[0525] 16. Example 16

[0526] The fixation effect of different high molecular weight PEGs (0.5% PEG6000, PEG10000 or PEG20000) in combination with low molecular weight PEG (3.5% PEG300) in an aqueous fixative solution containing BA or DMPA, EDTA and a caspase inhibitor (Q-VD-OPh) was analyzed. The fixative aid was prefilled into vacuum blood collection tubes (α tubes). EDTA blood was used as an unfixed reference control.

[0527] Blood collection and fixation

[0528] 10 ml whole blood samples from eight donors were collected in 10 ml spray-dried K2EDTA tubes and pre-filled with α tubes containing the amount of the fixed composition of the present invention including or PEG6000, PEG10000 or PEG20000, as well as PEG300, EDTA, caspase inhibitor (Q-VD-OPh) and BA or DMPA. Plasma was quickly generated from 5 ml fixed or unfixed blood samples. The residual blood was stored at room temperature for another six days before plasma production. CcfDNA was purified from 2 ml plasma, and the copy number of the 18SrDNA gene was detected by real-time PCR in triplicate.

[0529] Composition of the fixative mixture in different tubes (total drawn volume 10 ml blood):

[0530] -EDTA (unfixed) – 18 mg spray dried EDTA

[0531] -α3 tube (0.5% PEG6000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh): 57.5 mg PEG6000, 402.5 μl PEG300, 115 μl DMPA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0532] -α4 tube (0.5% PEG6000, 3.5% PEG300, BA, EDTA, Q-VD-OPh): 57.5 mg PEG6000, 402.5 μl PEG300, 115 μg BA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0533] -α5 tube (0.5% PEG10000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh): 57.5 mg PEG10000, 402.5 μl PEG300, 115 μl DMPA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0534] -α6 tube (0.5% PEG10000, 3.5% PEG300, BA, EDTA, Q-VD-OPh): 57.5 mg PEG10000, 402.5 μl PEG300, 115 μg BA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0535] -α7 tube (0.5% PEG20000, 3.5% PEG300, DMPA, EDTA, Q-VD-OPh): 57.5 mg PEG20000, 402.5 μl PEG300, 115 μl DMPA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0536] -α8 tube (0.5% PEG20000, 3.5% PEG300, BA, EDTA, Q-VD-OPh): 57.5 mg PEG20000, 402.5 μl PEG300, 115 μg BA, 152 mg K2EDTA, 11.5 μl Q-VD-OPh (1 mg dissolved in 388 μl DMSO), add water to 1.5 ml

[0537] Thus, the following final concentrations of the different components were obtained in the whole blood / fixative mixture:

[0538] - Unfixed: 1.8mg / ml K2EDTA

[0539] -PEG6000, PEG10000 or PEG20000, PEG300, DMPA or BA, EDTA, Q-VD-OPh – 1.5 ml: 0.5% (w / v) PEG6000 (or PEG10000, or PEG20000), 5.5% (v / v) PEG300, 1% (v / v) DMPA or (w / v) BA, 13,2 mg / ml K2EDTA, 5 μM Q-VD-OPh

[0540] result

[0541] The results are shown in Fig.19 and 20 . Fig.19 Shown are the copy number changes (average fold change) of a 66 bp fragment or a 500 bp fragment of the 18S rDNA gene in fixed or unfixed blood from 8 donors stored at room temperature for 6 days relative to time point 0 (day 0). Fig. 20 The absolute copy number in plasma on day 0, just after blood was drawn, is shown. Fig.19As shown, PEG with a molecular weight of up to 20.000 in a composition also including PEG300, EDTA, a caspase inhibitor (Q-VD-OPh) and BA or DMPA is very effective in fixing the extracellular nucleic acid population in blood. In contrast to unfixed EDTA blood, for the fixed blood of α tubes 3-8, after 6 days of storage, the average fold change in the copy number of the 66bp fragment and the 500bp fragment of 18S rDNA remained at the basal level of day storage at time point zero (fold change of approximately 1.0). In addition, the balanced composition of high and low molecular weight PEG did not reduce the absolute copy number of ccfDNA compared to plasma from unfixed EDTA just after blood was drawn (see Fig. 20 ), thus indicating that extracellular nucleic acids can be effectively isolated from fixed samples. The lower absolute copy number in the fixed samples compared to the unfixed reference samples is due to the fact that the obtained plasma is diluted by the liquid fixation composition. Due to this dilution, the initial amount of nucleic acids in the fixed samples is lower.

[0542] 17. Example 17

[0543] The fixation technology of the present invention is also compatible with nucleic acid separation schemes based on anion exchange. Extracellular nucleic acids are separated from plasma obtained from fixed blood samples. A fixation composition comprising K2EDTA, Q-VD-OPH, DMPA, PEG 10000, and PEG300 in water is used for fixation. The QIAamp circulating nucleic acid kit (Qiagen) is used to separate extracellular nucleic acids from fixed plasma samples according to the manufacturer's instructions (2 ml plasma volume, 60 microliter elution volume). Alternatively, magnetic particles containing anion exchange groups (tertiary amine groups) are used as nucleic acid binding solid phases to separate extracellular nucleic acids from fixed plasma samples (2 ml). The sample is broken and combined at an acidic pH (4.5), and the combined nucleic acids are washed three times and eluted with 75 microliters of alkaline elution buffer (pH 12.5). The scheme is performed using an automatic system (QIAsymphony).

[0544] The yields of nucleic acids (18s rDNA (66 bp) and 18s rDNA (500 bp)) obtained using the anion exchange-based nucleic acid protocol were comparable to those obtained by PCR analysis (against a genomic DNA dilution series to determine copy number) using the QIAamp Circulating Nucleic Acid Kit (set to 100%). The results are shown in the table below.

[0545] Anion exchange-based nucleic acid separation protocol QIAamp Circulating Nucleic Acid Kit 18s rDNA (66 bp) 98.64 100.00 18s rDNA (500bp) 88.04 100.00

[0546] It can be seen that the anion exchange-based nucleic acid separation scheme can be used to obtain extracellular nucleic acids from fixed samples with high yield. Sequence Listing <110> Qiagen Co., Ltd. <120> Fixation and Isolation of Extracellular Nucleic Acids <130> 55 897 K <150> US 61 / 955,200 <151> 2014-03-18 <150> EP 14 000 990.3 <151> 2014-03-18 <160> 6 <170> PatentIn Version 3.5 <210> 1 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Human RNA forward primer <400> 1 gccgctagag gtgaaattct tg 22 <210> 2 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Human RNA reverse primer <400> 2 cattcttggc aaatgctttc g 21 <210> 3 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Human ribosomal DNA probe <400> 3 accggcgcaa gacggaccag a 21 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Human RNA forward primer <400> 4 gtcgctcgct cctctcctac tt 22 <210> 5 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Human RNA reverse primer <400> 5 ggctgctggc accagactt 19 <210> 6 <211> 25 <212> DNA <213> Artificial sequence <220> <223> Human ribosomal DNA probe <400> 6 ctaatacatg ccgacgggcg ctgac 25

Claims

1. A collection device for collecting a cell-containing biological sample, comprising a composition suitable for fixing a cell-containing biological sample, the composition comprising: a polyoxyethylene polymer which is a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 as a fixing agent and a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, and one or more other auxiliary agents selected from the group consisting of - one or more primary, secondary or tertiary amides; - Caspase inhibitors; - Anticoagulants and / or chelating agents.

2. A collection device for collecting cell-containing biological samples, characterized in that: A composition suitable for fixing a biological sample containing cells is provided, the composition comprising: Polyoxyethylene polymers, which are high molecular weight polyoxyethylene polymers having a molecular weight of at least 1500 as a fixing agent, and low molecular weight polyoxyethylene polymers having a molecular weight of 1000 or less; and one or more other adjuvants selected from the group consisting of: - one or more secondary or tertiary amides; - Caspase inhibitors.

3. The collecting device according to claim 2, characterized in that The collecting device additionally comprises: - anticoagulants and / or chelating agents; and / or -Primary amine.

4. The collecting device according to claim 1 or 2, characterized in that: The polyoxyethylene polymer is polyethylene glycol.

5. The collecting device according to claim 4, characterized in that The polyoxyethylene polymer is unsubstituted polyethylene glycol.

6. The collecting device according to claim 1 or 2, characterized in that: The collection device also includes a caspase inhibitor and one or more primary, secondary or tertiary amides, as shown in Formula 1: wherein R1 is hydrogen or alkyl; R2 and R3 are the same or different and are selected from hydrogen and hydrocarbon groups, having a carbon chain length of 1 to 20 atoms, in straight or branched form; and R4 is oxygen, sulfur or selenium.

7. The collecting device according to claim 6, characterized in that R1 is a C1-C5 alkyl group, a C1-C4 alkyl group or a C1-C3 alkyl group, or a C1-C2 alkyl group.

8. The collecting device according to claim 6, characterized in that The hydrocarbon group is an alkyl group.

9. The collecting device according to claim 6, characterized in that The R4 is oxygen.

10. The collecting device according to claim 6, characterized in that The collecting device comprises butanamide and / or N,N-dialkylpropionamide.

11. The collecting device according to claim 10, characterized in that The N,N-dialkylpropionamide is N,N-dimethylpropionamide.

12. The collecting device according to claim 1 or 2, characterized in that: The molecular weight of the high molecular weight polyoxyethylene polymer is selected from the group consisting of 1500-50000, 1500-40000, 2000-30000, 2500-25000, 3000-20000, 3500-15000 and 4000-10000; and / or The molecular weight of the low molecular weight polyoxyethylene polymer is selected from the group consisting of 100-1000, 150-800, 150-700, 200-600, 200-500 and 200-400.

13. The collecting device according to claim 1 or 2, characterized in that: The composition includes a polyoxyethylene polymer which is a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 as a fixative and a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, at least one caspase inhibitor and an anticoagulant.

14. The collecting device according to claim 13, characterized in that The collecting device comprises at least one primary, secondary or tertiary amide.

15. The collecting device according to claim 14, characterized in that The amide is a compound represented by Formula 1.

16. The collecting device according to claim 15, characterized in that The amide is butanamide and / or N,N-dialkylpropionamide.

17. The collecting device according to claim 16, characterized in that The N,N-dialkylpropionamide is N,N-dimethylpropionamide.

18. The collecting device according to claim 1 or 2, characterized in that: The collecting device comprises: a) at least one high molecular weight polyethylene oxide polymer having a molecular weight of at least 1500; b) one or more primary, secondary or tertiary amides; c) at least one caspase inhibitor; d) at least one other polyoxyethylene polymer having a molecular weight of 1000 or less.

19. The collecting device according to claim 18, characterized in that The molecular weight of the at least one high molecular weight polyoxyethylene polymer is in the range of 2000-40000, 2500-30000, 2500-25000 or 3000-20000.

20. The collecting device according to claim 18, characterized in that The one or more primary, secondary, or tertiary amides are compounds of formula I.

21. The collecting device according to claim 18, characterized in that The at least one caspase inhibitor is a pan-caspase inhibitor.

22. The collecting device according to claim 18, characterized in that The at least one caspase inhibitor is Q-VD-OPh.

23. The collecting device according to claim 18, characterized in that The molecular weight of the at least one other low molecular weight polyethylene oxide polymer is 200-800 or 200-600.

24. The collecting device according to claim 18, characterized in that The collection device also comprises an anticoagulant and / or a chelating agent.

25. The collecting device according to claim 24, characterized in that The anticoagulant and / or chelating agent is EDTA.

26. The collecting device according to claim 18, characterized in that a) a high molecular weight polyoxyethylene polymer is contained in the collection device at a concentration such that when the cell-containing biological sample is collected into the device, the concentration of the high molecular weight polyoxyethylene polymer in the resulting mixture is in a range selected from the group consisting of 0.05%-4% (w / v), 0.1%-3% (w / v), 0.2%-2.5% (w / v), 0.25%-2% (w / v), 0.3%-1.75% (w / v) and 0.35%-1.5% (w / v); b) one or more primary, secondary or tertiary amides are contained in the collection device at a concentration such that when the cell-containing biological sample is collected into the device, the concentration range of the amide (or combination of amides) in the resulting mixture is selected from the following group: 0.25%-5%, 0.3%-4%, 0.4%-3%, 0.5%-2% or 0.75%-1.5%.

27. The collecting device according to claim 26, characterized in that At least one caspase inhibitor is contained in the collection device at a concentration such that when the cell-containing biological sample is collected into the device, the concentration of the caspase inhibitor in the resulting mixture ranges from 0.1 μM to 20 μM, more preferably from 0.5 μM to 10 μM, more preferably from 1 μM to 10 μM, more preferably from 3 μM to 7.5 μM or from 3 μM to 5 μM.

28. The collecting device according to claim 1 or 2, characterized in that The collection device comprises a caspase inhibitor, wherein the caspase inhibitor has one or more characteristics selected from the group consisting of: (i) it is a caspase-specific polypeptide modified with an aldehyde, nitrile or ketone compound; (ii) it is a caspase-specific peptide modified with an O-phenoxy (OPh) or fluoromethylketone (FMK) group; (iii) It is selected from the group consisting of Q-VD-OPh and Z-VAD(OMe)-FMK.

29. The collecting device according to claim 1 or 2, characterized in that The composition has one or more of the following characteristics: a) the fixation composition does not contain an auxiliary agent at a concentration that will induce or promote cell lysis; b) the fixation composition does not contain a cross-linking agent that induces protein-DNA and / or protein-protein cross-linking; c) the fixing composition does not contain toxic agents; and / or d) The composition further comprises a sample comprising cells to be fixed.

30. The collecting device according to claim 29, characterized in that The cross-linking agent is formaldehyde, formalin, paraformaldehyde or a formaldehyde releaser.

31. The collecting device according to claim 29, characterized in that The composition further comprises a cell-containing sample to be fixed, wherein the cell-containing sample is blood.

32. The collecting device according to claim 1 or 2, characterized in that The fixative composition is a liquid and is contained within the collection device in an amount effective to provide fixation for the sample to be collected within the device.

33. The collecting device according to claim 32, characterized in that The fixation composition is contacted with the biological sample in a volume ratio selected from 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5 and 1:7-1:5, or about 1:

6.

34. The collecting device according to claim 1 or 2, characterized in that a) the collecting device is a collecting device for collecting blood, plasma or serum samples; b) the collection device is used to draw blood from a patient; and / or c) The biological cell-containing sample is a blood sample.

35. The collecting device according to claim 34 a), characterized in that The collection device comprises an anticoagulant.

36. The collecting device according to claim 35, characterized in that The anticoagulant is a chelating agent.

37. The collecting device according to claim 36, characterized in that The anticoagulant is EDTA.

38. The collecting device according to claim 1 or 2, characterized in that The collection device is evacuated.

39. The collecting device according to claim 1 or 2, characterized in that The collecting device is prefilled with a limited amount of a fixing composition, either in solid or liquid form, provided with a defined vacuum and sealed by a septum.

40. The collecting device according to claim 1 or 2, characterized in that The collection device has an open top, a bottom, and a sidewall extending therebetween, defining a chamber, wherein the polyoxyethylene polymer and one or more other adjuvants are contained within the chamber.

41. The collecting device according to claim 40, characterized in that The collecting device is a tube with a closed bottom, the collecting device further comprising a cover at an open top, and the chamber is at a reduced pressure.

42. The collecting device according to claim 41, characterized in that The shield can be pierced by a needle or cannula, and the reduced pressure selected to draw a specific volume of liquid sample into the chamber.

43. The collecting device according to claim 42, characterized in that The device comprises a fixation composition which is a liquid and is placed in the chamber so that the volume ratio of the fixation composition to a specific volume of a sample comprising cells is selected from the group consisting of: 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5 and 1:7-1:

5.

44. The collecting device according to claim 1 or 2, characterized in that A fixative composition is included that is mixed with a biological sample containing cells.

45. The collecting device according to claim 44, characterized in that The volume ratio of the fixation composition to the cell-containing biological sample is selected from 10:1-1:20, 5:1-1:15, 1:1-1:10, 1:10-1:5, 1:7-1:5, and about 1:6; and / or the cell-containing biological sample is whole blood.

46. ​​A method comprising the step of collecting a biological sample containing cells from a patient and placing it directly into a collection device as claimed in claim 1 or 2.

47. Use of a collection device for collecting biological samples containing cells, in, The collecting device comprises a composition comprising a polyoxyethylene polymer, which is a high molecular weight polyoxyethylene polymer having a molecular weight of at least 1500 as a fixing agent and a low molecular weight polyoxyethylene polymer having a molecular weight of 1000 or less, Characterized in that the collection device is used to fix the extracellular nucleic acid group contained in the cell biological sample.

48. The use according to claim 47, characterized in that The collecting device comprises one or more other auxiliary agents selected from the group consisting of: - one or more primary, secondary or tertiary amides; - Caspase inhibitors; - Anticoagulants and / or chelating agents.

49. The use according to claim 48, characterized in that - the amide is an amide as defined in claim 6; - the caspase inhibitor is a caspase inhibitor as defined in claim 28; - The anticoagulant and / or chelating agent is EDTA.

50. The use according to any one of claims 47, 48 or 49, characterized in that i) the polyoxyethylene polymer is polyethylene glycol; ii) the molecular weight of the high molecular weight polyoxyethylene polymer is selected from the group consisting of 1500-50000, 1500-40000, 2000-30000, 2500-25000, 3000-20000, 3500-15000 and 4000-10000; and / or iii) the molecular weight of the low molecular weight polyoxyethylene polymer is selected from the group consisting of 100-1000, 150-800, 150-700, 200-600, 200-500 and 200-400.

51. The use according to claim 50, characterized in that The polyoxyethylene polymer is unsubstituted polyethylene glycol.

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