RU2798013C1 - Environment and device for collecting, storing and transporting biological samples, as well as their use for the stabilization of extracellular nucleic acids - Google Patents

Abstract

FIELD: biotechnology and medicine.

SUBSTANCE: invention concerns preanalytical systems for taking biological material containing a stabilizing medium for storage and transportation of extracellular nucleic acids for the purpose of their subsequent analysis and use of the results obtained in clinical practice. The invention is a medium for collecting, storing and transporting biological samples, where the biological sample containing extracellular nucleic acids to be stored is blood or plasma; where extracellular nucleic acids are extracellular DNA or RNA; containing a release gel, where a thixotropic gel is used as the release gel; and a stabilizing solution containing at least 1 cell membrane fixing agent and at least 1 anticoagulant, where the anticoagulant can function as an ionic stabilizing and/or chelating agent. The invention also relates to a device for collecting, storing and transporting biological samples containing the medium according to claim 1, which is a test tube with a medium according to claim 1, and a stabilizing solution containing a cell membrane fixing agent, which is part of the medium according to claim 1, located above the thixotropic gel and/or the specified thixotropic gel is located under the specified stabilizing solution.

EFFECT: invention can be used to stabilize extracellular nucleic acids for up to 15 days at temperatures from -20 to +37°C and up to 12 months at temperatures from -86 to -20°C.

17 cl, 5 dwg, 10 tbl, 3 ex

Description

The present invention relates to medicine and concerns preanalytical systems for taking biological material containing a stabilizing medium for storing and transporting extracellular nucleic acids for the purpose of their subsequent analysis and use of the obtained results in clinical practice. The essence of the invention lies in the development of a medium (composition) of a new composition and a device containing this medium (composition) for collecting, storing and transporting biological samples, as well as new methods for their use, which allow the preservation of extracellular nucleic acids for a long time in a wide temperature range , and at the same time are suitable for work in laboratories with different levels of equipment.

Description of the invention

Technical field

The present invention relates to the field of molecular biology, bioorganic chemistry, and genetics, and is a preanalytical system for taking biological material containing a medium for stabilizing nucleic acids in order to store and transport these biopolymers for subsequent analysis in clinical diagnostic laboratories and use of the obtained results in as diagnostic and prognostic markers in various fields of medicine.

State of the art

Analysis of free-circulating extracellular nucleic acids (DNA and RNA) is used for various tasks, including non-invasive prenatal testing - NIPT [1], for the diagnosis, prognosis and monitoring of the results of cancer treatment [2], as well as in transplantology [ 3], in sports medicine [4] and even for predicting the course of COVID-19 [5]. The following methods for ensuring the safety of extracellular DNA can be distinguished:

1 A common method to prevent the release of genomic DNA into the plasma fraction is to collect blood in standard tubes with EDTA or sodium citrate, then store the blood samples in a refrigerator, with mandatory centrifugation of the sample for 6 hours and collection of plasma after centrifugation. Once plasma has been collected, it must be frozen prior to DNA extraction. In turn, if it is necessary to analyze extracellular RNA, a whole blood sample must be centrifuged no later than 1 hour after sampling, and then either RNA isolation should be performed, followed by storage at -86 ° C, or a reverse transcription reaction should be performed to obtain complementary DNA (cDNA), which is more stable, but at the same time, storage and transportation of which must also be carried out in a frozen state. Separately, it should be noted that even during the indicated time before centrifugation (6 hours in the case of DNA and 1 hour in the case of RNA), significant degradation of extracellular nucleic acids is already observed.

The problem with using this method is that most blood sampling points do not have the necessary equipment and have to send samples to centralized laboratories for isolation or analysis of extracellular nucleic acids. At the same time, despite the fact that in most blood sampling rooms and laboratories it is possible to separate plasma, it must be stored in a frozen state until DNA extraction and subsequent diagnostic manipulations. In turn, work with extracellular RNA, both RNA isolation and cDNA production for the purpose of sending it frozen to a centralized laboratory, is practically not feasible in medical offices / biomaterial collection points due to the low level of equipment and high requirements for staff qualifications. Therefore, standard EDTA or sodium citrate tubes are not an ideal or practical solution for the widespread use of extracellular nucleic acids in diagnostic applications, especially for resource-constrained laboratories lacking the necessary equipment for immediate plasma preparation and/or RNA isolation and subsequent storage in the freezer before shipping. In addition, the shipment of such samples requires strict observance of the cold chain during transportation at negative temperatures, since when defrosting a sample of extracellular DNA/RNA, it can significantly degrade and become unsuitable for further research.

Thus, these tubes are suitable only for laboratories where either there are no tasks for long-term storage of biomaterial and the extraction of extracellular DNA/RNA is carried out within one working day, or the laboratory equipment allows you to separate the plasma / isolate RNA and freeze the sample for further transportation. 2. The second method is an improved version of the first method and consists in collecting blood in test tubes containing an anticoagulant similar to the method in paragraph 1, polyhydric alcohol as a stabilizer and a special (thixotropic) gel that creates a barrier between blood cells and plasma after centrifugation . Since the specific gravity of the thixotropic gel used in tubes is intermediate between the specific gravity of the heavier (blood cells) and lighter (plasma) phases, during centrifugation, this gel forms an intermediate layer between the blood cells that remain under the gel and the top layer. plasma. The position of the gel after centrifugation is influenced by many characteristics of the gel, such as its specific gravity, yield strength, and viscosity. The position of the gel can also be influenced by external conditions such as temperature and centrifugation speed. Additional factors associated with the in vitro whole blood sample may also be involved, such as heparin therapy, low hematocrit, elevated plasma protein, high lipids, and other factors that affect plasma specific gravity. In addition, the type of polymer that is used to create the gel can also affect its viscosity, density, and other physical properties.

This approach to stabilization, storage, and production of extracellular DNA is described in US20200196594 by delta DNA Biosciences Inc. (Canada) and implemented in a HAEM-LOK™ blood collection tube. Using a combination of a barrier matrix, a special barrier fluid, and a single centrifugation step, all blood cells (including all "buffy coat" cells) move below the physical barrier, forming cell-free plasma. Early removal of all cells prevents the subsequent release of genomic DNA from lysed cells and allows the most efficient extraction of extracellular DNA from the fraction of blood plasma that is physically separated from blood cells. After centrifugation, blood samples in HAEM-LOK™ can be stored in the original tube at ambient temperature for 2-3 weeks, simplifying transport and workflow. In addition, a whole blood sample can be stored in this tube for up to 5 days without centrifugation at a temperature from 0°C to +10°C, which allows the sample to be transported to a centralized laboratory from a medical office / biomaterial collection point with a low level of equipment, while transportation requires strict adherence to the cold chain. Separately, it should be noted that the shelf life of 5 days and the upper temperature limit of +10°C are due, in particular, to the absence of cell membrane fixatives or apoptosis inhibitors in the composition of the stabilizing solution of this tube, which does not allow cell stabilization, and, as a result, prevent release of genomic DNA from nucleated cells and lysis (hemolysis) of erythrocytes for a longer time and in a wider temperature range. 3. The third method of stabilization followed by separation of plasma from blood cells and extraction of extracellular DNA from the blood plasma fraction is given in patent application WO2017201612 and is based on the differential precipitation method in order to further concentrate extracellular DNA. It uses the principle originally described by John Lees and Robert Schleif [6]. The authors showed that exposure to DNA of a combination of polyethylene glycol and sodium chloride at various concentrations can precipitate DNA of different classes and sizes in different ways. Thus, very high molecular weight DNA (genomic DNA), which is released from cells at high sodium chloride concentrations, immediately precipitates in the presence of polyethylene glycol. Extracellular DNA, which has a much lower molecular weight, does not precipitate under these conditions, allowing the two classes of DNA to be better separated due to their size. The composition proposed by the authors of this patent for the stabilization of extracellular DNA does not contain cell membrane fixatives, but only preservatives, anticoagulants and precipitants, which make it possible to isolate extracellular DNA in a relatively high concentration. This approach to the stabilization, storage and isolation of extracellular nucleic acids is implemented in the cf-DNA & cf-RNA Collection & Preservation blood collection tube from Norgen Biotek Corp. (Canada), while the tube with a biological sample should be stored at a temperature not exceeding below +4°C to avoid cell hemolysis.

4 The fourth most widely used method to prevent the release of genomic DNA into a plasma fraction is to use chemical cell membrane stabilizing agents, such as aldehydes (eg formaldehyde or glutaraldehyde), which stabilize nucleated cells by fixing their membrane. This method has a number of disadvantages, which is due to the use of aldehydes as preservatives for the stabilization of extracellular DNA, since they can cause the formation of DNA-protein and DNA-DNA cross-links, which in turn can negatively affect subsequent DNA amplification and sequencing. In turn, in order to neutralize the potentially negative effect of aldehydes on extracellular nucleic acids and the effectiveness of molecular genetic testing, compositions using this method increasingly contain additional components that neutralize the effect of aldehydes and their derivatives. A similar approach to preserving extracellular nucleic acids by fixing the mean volume of erythrocytes, preventing lysis of leukocytes and, as a result, preventing the release of membrane vesicles into the extracellular space - plasma, is described in international patent application WO2019/079743 A1. The composition developed by the authors is intended to stabilize leukocytes and erythrocytes, preventing the release of nucleic acids and membrane vesicles from cells in a biological sample. At the same time, due to the presence of an aldehyde “quencher” in the composition of the preservative solution, the composition according to the invention proposed by the authors is suitable for stabilizing biological samples for the purpose of subsequent analysis by immunoassay, PCR, next generation sequencing (NGS), etc. A similar composition is used by the authors in the patent application US2020/0318161 A1 to stabilize free-circulating extracellular RNA, while the composition according to this invention additionally contains nuclease/protease inhibitors and transcription inhibitors that provide RNA stabilization and at the same time maintain the level of RNA expression at the time of blood sampling.

Thus, the use of test tubes with chemical stabilizers that contain precipitants (according to item 3) or membrane fixatives (according to item 4) allows solving the problem of long-term storage and transportation of a whole blood sample in the temperature range from +4 to +37°C, there is no need for pre-separation of plasma and freezing, which allows the use of these tubes in laboratories and medical offices with limited resources.

At the same time, tubes with chemical stabilizers that contain precipitants or membrane fixatives, as well as tubes with anticoagulants without and with the addition of a barrier gel, are not intended for storing a sample with extracellular DNA / RNA at temperatures below 0 ° C, and therefore, to solve logistic tasks during the cold season, regions with a temperate or subpolar climate require either 1) preliminary separation of plasma from blood cells, freezing plasma samples and transporting them frozen with strict observance of the cold chain, or 2) transporting whole blood in test tubes with chemical stabilizers, but also with strict observance of the temperature regime of storage and transportation not lower than +4°C.

At the same time, strict adherence to the temperature regime in a narrow temperature range during storage and transportation in combination with the use of thermal recorders (loggers) or thermal indicators determines the higher cost of delivering the biomaterial to the centralized laboratory, which can negatively affect the final cost of the study for the patient, and not always provides a guarantee of the safety of the biomaterial, which may lead to an increased risk of repeated blood sampling and a longer waiting time until the result of the study is received, and, therefore, before the possibility of making a medical / medical decision if necessary.

To solve the problem of storing and transporting biological samples in a wide temperature range, the closest analogue of the technical solution is the medium for storing and transporting blood samples, described in international patent application WO2004032750A1. The medium according to the invention includes a separating gel and a stabilizing solution containing, as stabilizing agents, inhibitors of caspase - proteolytic enzymes belonging to the family of cysteine proteases and cleaving proteins exclusively after aspartate (cysteinyl-aspartate-specific proteases). The application also states that the medium contains at least one stabilizing agent in an amount sufficient to prevent or eliminate the occurrence of morphological changes, cell membrane degradation, DNA fragmentation, or loss of cellular function or viability.

Caspases are known to regulate many cellular and biochemical changes in dying apoptotic cells. In particular, the DNA fragmentation observed during apoptosis is the result of cleavage by the effector caspase of a complex consisting of the cellular DNase CAD (caspase-activated DNase) and its inhibitor ICAD. When the inhibitor is cleaved, active DNase CAD begins to cleave DNA at the most accessible sites between nucleosomes, which leads to its degradation and the detection of a characteristic “ladder” of DNA fragments of different sizes, observed during apoptosis. In turn, the total inhibition of caspase can prevent the triggering of the caspase-dependent pathway of cell apoptosis and the subsequent release of intracellular nucleic acids, as well as contribute to less degradation of extracellular DNA due to the lack of release of intracellular DNases [7].

Since, in addition to the caspase-dependent pathway of apoptosis, many other mechanisms of cell death are known, in all other cases, including cell apoptosis along the caspase-independent pathway, the stabilizing solution indicated by the authors of international patent application WO2004032750A1, containing one or more caspase inhibitors as stabilizing agents, will not be able to provide preservation and stability of the initial concentration of extracellular nucleic acids, namely DNA and, especially, RNA, in a biological sample due to the absence of a universal agent in the stabilizing solution that can prevent the release of intracellular nucleic acids, as well as the degradation of extracellular nucleic acids due to the lack of release of intracellular nucleoproteases.

Perhaps for this reason, the authors of this international application for the invention do not position the medium they have developed for stabilizing extracellular nucleic acids, the precise quality of which is necessary for the subsequent conduct of such research methods that are highly sensitive to the quality of the analyzed sample, such as NIPT and liquid biopsy, but mainly for the storage of whole blood for the purpose of subsequent standard general clinical studies, while the temperature range and the period of such storage are not specified in this application.

The need to use additional cell stabilizing components, in addition to caspase inhibitors, is given in the patents for inventions US10724074B2 and US10144952B2 company Qiagen GmbH. In particular, we have shown that caspase inhibitors reduce the contamination of the extracellular nucleic acid population with intracellular nucleic acids, in particular, fragmented genomic DNA that is released from the cells contained in the sample, for example, from damaged or dying cells. However, only a stabilizing combination that includes an apoptosis inhibitor and an additional stabilizing agent, such as N,N-dialkylpropanamide, in particular N,N-dimethylpropanamide (DMPA), or butanamide, is effective in maintaining the population of extracellular nucleic acids contained in the sample on the moment of taking a biological sample, and to stabilize the transcriptome of the contained cells.

The mechanism of action of N,N-dialkylpropanamides, in particular DMPA, and butanamide, is not fully understood, while there is evidence that some of the butanamide derivatives demonstrate an inhibitory effect on histone deacetylases, which are key enzymes that control the proliferation or differentiation status of most cells. . In turn, N,N-dialkylpropanamides, and in particular DMPA, are able to stabilize the transcription profile of genes by inhibiting changes in transcript levels, while lymphocyte cells remain intact. Thus, these stabilizing agents, which are predominantly used in combination with caspase inhibitors, stabilize cells "from the inside", preventing their death through the apoptotic pathway, but do not exert an external stabilizing effect on nucleated cells by fixing their membrane. This explains why the compositions for stabilizing, storing and transporting nucleic acids given in the mentioned patents US10724074B2 and US10144952B2 provide a stabilizing effect for up to a maximum of seven days (mainly 2-3 days) at room temperature without the possibility of cooling / freezing or storage for more than a long period of time.

Thus, despite the progress in the stabilization of extracellular nucleic acids, there remains a need for a composition and a device for collecting, storing and transporting biological samples, the combination of which would ensure the preservation of extracellular nucleic acids for a long time in a wide range of negative and positive temperatures, and at the same time, it would be suitable for work in laboratories with any level of equipment, including for medical offices / biomaterial collection points with a minimum instrumentation base.

Such a composition (medium) and device, as well as methods of using them to stabilize extracellular nucleic acids, are provided by the present invention.

Disclosure of invention

The essence of the invention lies in the development of a Medium of a new composition and the creation of a Device containing this medium for the collection and subsequent storage and transportation of biological samples for a long time in a wide temperature range, as well as new methods for their use for stabilizing nucleic acids in laboratories with different levels of equipment.

The technical result arising from the implementation of the invention is as follows:

• Stabilization of the cell membrane due to cross-links and, as a result, fixation of the cell membrane, which prevents their hemolysis, regardless of the nature of apoptotic processes inside cells and their maturity, and therefore fixed cells not only do not release intracellular DNA or RNA during storage before centrifugation, ensuring the preservation of the concentration of nucleic acids at the time of blood sampling, but also during centrifugation, due to the fixed membrane, they become more resistant to mechanical stress, which allows not only to move all blood cells without damage under the gel barrier, obtaining cell-free plasma, but also to avoid release into plasma of intracellular nucleic acids by centrifugation.

• Increased stabilization time for extracellular nucleic acids in a wide temperature range: up to 15 days at temperatures from -20°C to +37°C and up to 12 months at temperatures from -86°C to -20°C, which allows for storage and transportation a sample that is predominantly a blood sample and contains extracellular nucleic acids that need to be preserved under virtually any environment and resource available to the laboratory.

• The invention allows centrifugation with centrifugal acceleration already from 2000 to 3600g in wider time ranges from 10 to 30 minutes without mechanical injury to the cell membrane, while in the prior art it is recommended to use a fundamentally more gentle centrifugation mode: 10 minutes at 2000g in order to avoid additional release of nucleic acids from cells due to their traumatization. In turn, a longer centrifugation with a higher acceleration makes it possible to isolate residual blood cells, extracellular structures and cellular debris, which, in turn, makes it possible, after plasma collection, not to carry out a second round of centrifugation at 13000-16000g for 10-20 minutes immediately prior to the nucleic acid extraction step. This technical result is relevant for laboratories with an extensive instrument base, a large flow of research and the need to reduce the average time for sample preparation and nucleic acid extraction.

• EFFECT: invention allows effective centrifugation of platelets without degranulation with simultaneous adsorption of platelets by the gel, which completely excludes the release of mitochondrial DNA into plasma planned for further analysis. This allows, even with the minimum recommended centrifugation time of 10 minutes and an acceleration of 2000g, to isolate all cells from the plasma, including platelets and “buffy coat” cells, and to obtain cell-free plasma without impurities of mitochondrial and apoptotic DNA, additionally introduced from cells after blood sampling. This technical result makes it possible to reduce equipment requirements to a minimum and carry out centrifugation in medical offices / biomaterial collection points with limited resources using compact laboratory centrifuges.

• Higher concentration of extracellular DNA and RNA, which is detected when extracting DNA / RNA after centrifugation from blood samples in tubes according to the invention with anticoagulant (for example, EDTA), cell membrane fixative and gel compared to tubes with EDTA without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids, in particular, with Cell-Free DNA BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel.

The studies carried out have shown that

в случае ДНК - это увеличение до 2,4 / 2,4 и 1,7 раз соответственно;

in the case of DNA, this is an increase of up to 2.4 / 2.4 and 1.7 times, respectively;

в случае РНК - это увеличение в среднем до 464 / 567 и 36 раз соответственно.

in the case of RNA, this is an increase to an average of 464 / 567 and 36 times, respectively.

• Higher concentration of extracellular DNA and RNA, which is found in the extraction of DNA / RNA after centrifugation and 1 freeze / thaw cycle of a blood sample in tubes according to the invention with an anticoagulant (for example, EDTA), cell membrane fixative and gel compared to tubes with EDTA without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids, in particular, with RNA Complete BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel without freezing cycles.

The studies carried out have shown that

в случае ДНК - это увеличение до 2,9 / 3,0 и 2,0 раз соответственно;

in the case of DNA, this is an increase of up to 2.9 / 3.0 and 2.0 times, respectively;

случае РНК - это увеличение в среднем до 1013 / 1317 и 76 раз соответственно.

in the case of RNA, this is an increase to an average of 1013 / 1317 and 76 times, respectively.

The present invention, providing the above technical result, is the following:

1. Environment for collecting, storing and transporting biological samples,

- where the biological sample containing the extracellular nucleic acids to be preserved is blood or plasma;

- where extracellular nucleic acids are extracellular DNA or RNA;

containing release gel

- where a thixotropic gel is used as a release gel;

- where thixotropic gel has a density in the range from 1.025 to 1.065 and is chemically inert with respect to biological samples;

- where the thixotropic gel has sufficient viscosity so that under centrifugal forces in the range of 2000 to 3600 g it will flow and form the desired barrier between plasma and blood cells;

stabilizing solution containing at least 1 cell membrane fixing agent

- where 2-bromo-2-nitropropane-1,3-diol, dimethylol urea, sodium hydroxymethylglycinate, diazolidinyl urea, dimethyl-dimethylol-hydantoin, imidazolidinyl urea, 1,3,5 -tris(hydroxyethyl)-s-triazine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, dimethylol-5,5-dimethylhydantoin, or combinations thereof;

- where the fixing agent releases free aldehyde and has the properties of a preservative,

and at least 1 anticoagulant

- where ethylene glycol tetraacetic acid (EGTA) and its salts, sodium citrate, ethylenediaminetetraacetic acid (EDTA) and its salts, sodium fluoride or combinations of them are used as an anticoagulant, which is part of the stabilizing solution;

- where the anticoagulant can function as an ionic stabilizing and/or chelating agent.

2. Medium according to claim. 1, where the content of the fixing agent can advantageously be from 0.2% to 63.4% by weight of the entire composition.

3. Medium according to claim 1, wherein the content of the anticoagulant can advantageously be from 0.1% to 28.3% by weight of the total composition.

4. The medium of claim 1, wherein the stabilizing solution may not necessarily include a quenching agent in an amount sufficient to react with any free aldehyde that may be present before or after sampling and which is formed from the fixing agent to form a reaction product that will not have a denaturing effect on proteins in biological samples, while the content of the quenching agent can mainly be from 0% to 7.8% by weight of the entire composition,

- where as a quenching agent can be used a compound that includes at least one functional group capable of reacting with an electron-deficient functional group of the aldehyde;

- where ethylenediamine, aminoacetic acid, lysine, arginine, adenine, guanine, cytosine, thymine, tryptophan, tyrosine, phenylalanine, ornithine, S-adenosylmethionine, alanine, arginine, cysteine, glutamic acid, aspartic acid, histidine can be used as a quenching agent , leucine, lysine, proline, serine, threonine, homocysteine, nicotinamide, or combinations thereof.

5. Medium according to claim 1, where the composition of the stabilizing solution may not necessarily include additional non-ionic stabilizing agents at a concentration of from 0% to 18.5% by weight of the entire composition, which dissolve in an aqueous solution without the formation of ions and can be selected from the group polyols (polyhydric alcohols) such as sucrose, lactose, trehalose, melibiose, mannitol, or combinations thereof.

6. Medium according to claim 1, where the composition of the stabilizing solution may not necessarily include inhibitors of enzymes - nucleases and / or proteases, including inhibitors of metabolic enzymes and inhibitors of phosphatase, which prevent the degradation of nucleic acids and ensure their stabilization, as well as inhibitors of apoptosis , including caspase inhibitors, including in combination with N,N-dialkylpropanamide, such as N,N-dimethylpropanamide (DMPA), or butanamide, which prevent cell apoptosis in a caspase-dependent pathway, and transcription inhibitors, which are designed to maintain a baseline RNA expression at the time of blood sampling by inhibiting the synthesis of new RNA transcripts, including mRNA, microRNA, IncRNA, piRNA, YRNA, circRNA and other ncRNA:

- enzyme inhibitors can be selected from the following groups of compounds: diethyl pyrocarbonate, ethanol, auryl tricarboxylic acid (ATA), glyceraldehyde, formamide, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphine hydrochloride or combinations thereof, but not limited to;

- inhibitors of metabolic enzymes can be selected from the following groups of compounds: glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, potassium oxalate or combinations of them, but not limited to this;

- protease inhibitors can be selected from the following groups of compounds: antipain, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TRCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E64, pepstatin, 1,10-phenanthroline, phosphoramodone, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, pancreatic protease inhibitor, ovostatin, cystatin, protease inhibitor, or combinations thereof;

- phosphatase inhibitors can be selected from the following groups of compounds: caliculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic (okadaic) acid, cantharidin, fostriyetsin, endothall, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, defostatin, mpV (pic) DMHV, sodium orthovanadate, or combinations thereof, but not limited to;

- the content of inhibitors of enzymes - nucleases and/or proteases, including inhibitors of metabolic enzymes and phosphatase inhibitors, can mainly range from 0% to 25.8% by weight of the entire composition.

7. Medium according to claim 1, wherein the stabilizing solution may optionally contain one or more cell permeability enhancing agents at concentrations of 0% to 5.4%, which may be selected from the following compounds or combinations thereof: DMSO (dimethyl sulfoxide ), ethylene glycol, glycerin, cellosolves, ethylene glycol dimethyl ether, phenoxyethanol, Triton X 100, Triton X 705 (non-ionic detergent), 1-methyl-2-pyrrolidinone, Tween 20, Tween 40 (non-ionic detergent), Brij 35 (non-ionic detergent) , polyoxyethylene ether (Polyox), monensin, monactin, pentachlorophenol, 2,4-dinitrophenol, saponin, SDS (sodium dodecyl sulfate).

8. Medium according to claim 1, where the stabilizing solution may optionally include the following substances or combinations thereof at a concentration of 0% to 6.2%:

- proteins such as: biotin, albumins: egg, bovine, including bovine serum albumin - BSA, gelatin and similar compounds;

additional nucleic acid stabilizers such as: guanidine hydrochloride, polycations such as polyethyleneimine and the like;

- antioxidants/reducing agents such as Trolox, α-tocopherol, nicotinamide and the like;

- nucleic acid dyes such as: DAPI (diamidino-2-phenylindole), propidium iodide, fluorescein diacetate and the like;

- antibiotics and other additional components, such as: doxycycline, sulfasalazine, curcumin, 6-aminocaproic acid, minocycline, chitosan, phytic acid, β-sitosterol, c-AMP, polylysine, biochanin A, demeclocycline, chlortetracycline, oxytetracycline, cyclohexamide, rifampicin , soy milk, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, 2-aminoethylbenzylsulfonyl fluoride (AEBSF), including but not limited to.

9. Medium according to claim 1, where the pH of the stabilizing solution can be, depending on the combination of its constituent agents and additional components, from 4 to 10, preferably from 6 to 8.

10. A device for collecting, storing and transporting biological samples containing the medium according to claim 1, which is a test tube with a medium according to claim 1, and a stabilizing solution containing a cell membrane fixing agent, which is part of the medium according to claim 1, is located above the thixotropic gel and/or said thixotropic gel is located below said stabilizing solution.

11. The device according to claim 10, which is vacuum tubes, including sterile ones, which may contain a vacuum intended for aspiration of a biological sample, while vacuum tubes can preferably have a nominal capacity of 9.0 ml, 8.5 ml, 8, 0 ml, 7.5 ml, 7.0 ml (size 16×100 mm), 6.0 ml, 5.5 ml, 5.0 ml (size 13×100 mm), 2.0 ml, 2.5 ml, 3.0 ml, 3.5 ml, 4.0 ml, 4.5 ml (13×75 mm), but not limited to.

12. The apparatus of claim. 10, wherein the content of the release gel is preferably from 0.3 g to 2.5 g per tube, but not limited to this.

13. Use of the device according to claim 10, containing the medium according to claim 1, for stabilizing extracellular nucleic acids for up to 15 days at temperatures from -20°C to +37°C and up to 12 months at temperatures from -86°C to -20 °C.

14. The use according to claim 13, where the application is carried out through the interaction of the biological sample located in the device according to claim 10, with the cell membrane fixing agent that is part of the stabilizing medium solution according to claim 1, and the separation gel, which, after centrifugation for 10-30 minutes at 2000-3600g creates a separating barrier between the supernatant and cells, mainly plasma and blood cells, making the nucleic acids in the plasma sample suitable for isolation and subsequent analysis, including, but not limited to, the use of PCR and sequencing;

15. Use according to claim 13, where the use includes taking a biological sample with the possibility of storage and transportation without centrifugation up to 24 hours after taking at a temperature from +4°C to +37°C, followed by centrifugation and further storage and transportation from 5 up to 15 days at temperatures from -20°C to +37°C with the possibility of repeated freezing / thawing of the sample after centrifugation with storage at temperatures from -86°C to -20°C for up to 12 months.

16. Use according to claim 13, where the use includes the possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking at a temperature of +4°C to +37°C and centrifugation immediately before the selection of the supernatant, mainly plasma, for removal of cells, extracellular structures and cellular debris to obtain cell-free plasma.

17. Use according to claim 13, where the use includes the possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking at a temperature of +4°C to +37°C while preserving extracellular nucleic acids, as well as circulating cells and / or extracellular structures as needed.

The main aspect of the invention is a medium for collecting, storing and transporting biological samples, containing a separating thixotropic gel, a stabilizing solution comprising at least one cell membrane fixing agent and an anticoagulant. The cell membrane fixing agent at a preferred concentration of 0.2% to 63.4% may be selected from the following groups of compounds or a combination of them: dimethyl-dimethylol-hydantoin, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, dimethylol-5,5- dimethylhydantoin, while the fixing effect of the selected compounds is mainly associated with their reaction with membrane proteins, namely with the formation of covalent bonds both within proteins and between them. This leads to the fact that the cell membrane becomes rigid and, therefore, resists degradation and mechanical stress much better, in contrast to the action of caspase inhibitors indicated in international patent application WO2004032750A1, as well as in patents for inventions US10724074B2 and US10144952B2, which are not chemically "fix" the cell membrane, making it more rigid due to the cross-linking of membrane proteins, and indirectly, by inhibiting caspase, they prevent cell apoptosis along the caspase-dependent pathway and the release of intracellular DNA, as well as the degradation of extracellular DNA.

The composition of the stabilizing solution included in the Medium according to the main aspect of the invention includes at least 1 anticoagulant

- where ethylene glycol tetraacetic acid (EGTA) and its salts, sodium citrate, ethylenediaminetetraacetic acid (EDTA) and its salts, sodium fluoride or combinations of them are used as an anticoagulant, which is part of the stabilizing solution;

- where the anticoagulant can function as an ionic stabilizing and/or chelating agent.

A medium for collecting, storing and transporting biological samples according to the main aspect of the invention, in addition to a stabilizing solution, comprising at least one cell membrane fixing agent, which is able to effectively prevent the release of genomic DNA from nucleated cells (which further distinguishes this Medium from a composition based on a thixotropic gel patent US20200196594), also contains a release thixotropic gel, which distinguishes this Medium from compositions based on a cell membrane fixing agent presented in patent applications EP1413874A1 and WO2019/079743A1. The use of a separating thixotropic gel as a barrier matrix makes it possible to separate cells from plasma and thereby prevent the subsequent release of genomic DNA from lysed cells.

Separately, it should be noted that, according to the main aspect of the invention, a thixotropic gel is used as a separating gel in the composition of the Medium for collecting, storing and transporting biological samples, mainly in an amount of 0.3 g to 2.5 g, including olefin, polyester, polyamide gel, but not limited to them. In addition, polyesters, denatured collagens, polypropylenes, polysiloxanes such as dimethylpolysiloxane and ethyltriethoxysilane, hydrocarbon gel materials such as polybutene, and combinations thereof can be used as components of the thixotropic gel. However, the thixotropic gel, also referred to herein as a release gel, has a density in the approximate range of 1.025 to 1.065 and is chemically inert with respect to biological samples, in particular, but not limited to blood components.

Thus, the proposed composition of the Medium for collecting, storing and transporting biological samples according to the main aspect of the invention provides a technical result, namely, stabilization due to cross-links and, as a result, fixation of the cell membrane, which prevents their hemolysis (including during apoptosis). In turn, during centrifugation, cells with a fixed membrane become more resistant to mechanical stress, which allows not only to move all blood cells without damage under the gel barrier, obtaining cell-free plasma, but also to avoid release into plasma during centrifugation of intracellular nucleic acids, including genomic DNA that can make a significant contribution to the total amount of extracellular nucleic acids in the analyzed sample (increase the amount of extracellular nucleic acids relative to the amount that was present in the sample at the time of blood sampling) and, as a result, significantly reduce the sensitivity of the analysis being performed, for example, violating the ratio fetal/maternal or genomic/tumor DNA. In particular, this also applies to the release of mitochondrial DNA from platelets, which occurs after the activation and subsequent degranulation of these cells. In this case, the platelet activator can be a mechanical effect, including centrifugation, in which excessive acceleration increases the percentage of activated platelets, which is accompanied by their premature degranulation. The presence of a membrane fixing agent makes the platelet membrane more resistant to mechanical stress, preventing their premature activation and release of extracellular mitochondrial DNA.

The environment for collecting, storing and transporting biological samples according to the main aspect of the invention may also contain additional components that enhance the stabilizing effect. Thus, for example, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention does not necessarily, but may include a quenching agent in an amount sufficient to react with any free formaldehyde that can be formed from the cell membrane fixing agent with the formation of a reaction product, which will not denaturate proteins in biological samples. In turn, as a quenching agent, a compound can be used that includes at least one functional group capable of reacting with an electron-deficient functional group of formaldehyde, which is selected from the group consisting of an amino compound that reacts with formaldehyde to form methylol, and/ or an imine Schiff base and/or a cis-diol compound that reacts with formaldehyde to form a cyclic acetal, or a combination of these, but not limited to.

The quenching agent at a preferred concentration of 0% to 7.8% may be selected from, but not limited to, the following compounds/groups of compounds, or a combination of them:

- alkylamines, polyamines, amino acids, primary amines, secondary amines, ammonium salts;

- ethylenediamine, urea, glycine, lysine, arginine, adenine, guanine, cytosine, thymine, spermidine, tryptophan, tyrosine, phenylalanine, ornithine, S-adenosylmethionine, aspartate, glutamine, alanine, arginine, cysteine, glutamic acid, aspartic acid, histidine , leucine, lysine, proline, serine, threonine, homocysteine, nicotinamide. Also, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention, optionally, but may include additional non-ionic stabilizing agents that dissolve in an aqueous solution without the formation of ions. Nonionic stabilizing agents at a preferred concentration of 0% to 18.5% may be selected from the group of polyols (polyhydric alcohols) such as, but not limited to, sucrose, lactose, trehalose, melibiose, mannitol, inositol, or combinations thereof.

Also, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention is optional, but may include diluting agents in a preferred concentration from 0% to 15%, including iso- or hypertonic solutions of metrizamide, an iodinated organic compound having a lipophilic substituent, or mammalian cell culture media that prevent or reverse swelling of nucleated cells, which in turn increases their buoyant density, thereby increasing plasma purity.

Additionally, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention is optional, but may include genomic DNA precipitating agents in a preferred concentration from 0% to 34.5%, such as polyethylene glycol (PEG with various molecular weights, which are known in of the art) or other polymers, including cyclodextrins, including but not limited to: polyvinylpyrrolidone (PVP with various molecular weights that are known in the art), magnesium gluconate, methylcellulose (MC), ethylcellulose (EC), hydroxyethylcellulose (HEC ), hydroxypropylcellulose (HPC), dextrins (with various molecular weights as known in the art), dextrans (with various molecular weights), polyethylene oxide, polyethyloxazoline, ficolls (with various molecular weights), α-cyclodextrin, β-cyclodextrin, Y -cyclodextrin, gelatins, sugars (without limitation), hydroxypropyl methylcellulose, hydroxyethyl methylcellulose.

Also, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention is optional, but may include in concentrations from 0% to 25.8% inhibitors of enzymes - nucleases and / or proteases, including inhibitors of metabolic enzymes and phosphatase inhibitors, which prevent the degradation of nucleic acids and ensure their stabilization, as well as inhibitors of apoptosis, including caspase inhibitors, including in combination with N,N-dialkylpropanamide, for example, with N,N-dimethylpropanamide (DMPA), or butanamide, which prevent cell apoptosis by caspase-dependent pathway, and transcription inhibitors, which are designed to maintain the basic level of RNA expression at the time of blood sampling by inhibiting the synthesis of new RNA transcripts (including mRNA, microRNA, IncRNA, piRNA, YRNA, circRNA and other ncRNA).

- Enzyme inhibitors can be selected from the following groups of compounds: diethylpyrocarbonate, ethanol, glyceraldehydes, sodium fluoride, ethylenediaminetetraacetic acid (EDTA), formamide, aurine tricarboxylic acid (ATA), vanadyl-ribonucleoside complexes, heparin, bentonite, ammonium sulfate, dithiothreitol (DTT) , beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphene hydrochloride, or combinations thereof, but not limited to.

- Inhibitors of metabolic enzymes can be selected from the following groups of compounds: glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, sodium fluoride, potassium oxalate, or a combination of them, but not limited to.

- Protease inhibitors can be selected from the following groups of compounds: antipain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TRCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E64, pepstatin, VdLPFFVdL, EDTA, 1,10-phenanthroline, phosphoramodone, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin, egg white cystatin, soy protease inhibitor, or a combination thereof, but not limited to this.

- Phosphatase inhibitors can be selected from the following groups of compounds: caliculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic (okadaic) acid, cantharidin, fostriecine, endothall, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, defostatin, mpV (pic) DMHV, sodium orthovanadate, or combinations thereof, but not limited to.

Additionally, the composition of the stabilizing solution included in the Medium according to the main aspect of the invention is optional, but may include one or more agents that increase cell permeability in concentrations from 0% to 5.4%, which can be selected from the following compounds or combinations thereof: DMSO (dimethyl sulfoxide), ethylene glycol, glycerin, cellosolve, ethylene glycol dimethyl ether, phenoxyethanol, Triton X 100, Triton X 705 (non-ionic detergent), 1-methyl-2-pyrrolidinone, Tween 20, Tween 40 (non-ionic detergent), Brij 35 ( non-ionic detergent), polyoxyethylene ether (Polyox), monensin, monactin, pentachlorophenol, 2,4-dinitrophenol, saponin, SDS (sodium dodecyl sulfate).

Also, the stabilizing solution, which is part of the Medium according to the main aspect of the invention, is optional, but may contain in a concentration from 0% to 6.2%:

- proteins such as: biotin, albumins (egg, bovine, including bovine serum albumin - BSA), gelatin and similar compounds;

additional nucleic acid stabilizers such as: guanidine hydrochloride, polycations such as polyethyleneimine and the like;

- antioxidants/reducing agents such as Trolox, α-tocopherol, nicotinamide and the like;

- nucleic acid dyes such as: DAPI (diamidino-2-phenylindole), propidium iodide, fluorescein diacetate and the like;

- antibiotics and other additional components, such as: doxycycline, sulfasalazine, curcumin, 6-aminocaproic acid, minocycline, chitosan, phytic acid, β-sitosterol, c-AMP, polylysine, biochanin A, demeclocycline, chlortetracycline, oxytetracycline, cyclohexamide, rifampicin , soy milk, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, 2-aminoethylbenzylsulfonyl fluoride (AEBSF), including but not limited to.

The pH of the stabilizing solution, which is part of the Medium according to the main aspect of the invention, can be, depending on the combination of the agents and additional components included in its composition, from 4 to 10, preferably from 6 to 8.

The second aspect of the invention is a device for collecting, storing and transporting biological samples, containing the Medium according to the main aspect of the invention. The device preferably consists of vacuum tubes with a thixotropic separation gel and a stabilizing solution containing at least one cell membrane fixing agent and located above the separation gel. The tubes can be of any nominal capacity and have any form factor that allows centrifugation and/or sedimentation of blood cells, including in combination with genomic DNA, or use other concentration methods that provide isolation of extracellular nucleic acids, including from circulating cells and /or extracellular structures.

Separately, it should be noted that the biological sample containing the nucleic acids to be preserved is a biological fluid. In turn, the biological fluid is predominantly blood, plasma, serum, urine, saliva, stool, breast milk, tears, sweat, cerebrospinal fluid, synovial fluid, semen, vaginal fluid, ascitic fluid, amniotic fluid or cell culture media, but not limited to this. The collection of biological fluid in the Device for collecting, storing and transporting biological samples according to the second aspect of the invention can be carried out both by venipuncture using standard phlebotomy equipment for taking whole blood from a vein, and by adding (adding) an appropriate volume of biological fluid to the Device containing Environment according to the main aspect of the invention. In this case, the nucleic acids that need to be preserved are mainly located in the blood plasma, but not limited to this.

In turn, the presence in the Device for collecting, storing and transporting biological samples proposed for patenting according to the second aspect of the invention, containing the Medium according to the main aspect of the invention, of a separating thixotropic gel in combination with a cell membrane fixing agent causes not only effective centrifugation of platelets without degranulation, but as well as the adsorption of platelets by the gel, which completely excludes the release of mitochondrial DNA into the plasma planned for further analysis. This allows, even with the minimum recommended centrifugation time of 10 minutes and an acceleration of 2000g, to isolate all cells from the plasma, including platelets and “buffy coat” cells, and to obtain cell-free plasma without impurities of mitochondrial and apoptotic DNA, additionally introduced from cells after blood sampling. This technical result makes it possible to reduce equipment requirements to a minimum and carry out centrifugation in medical offices / biomaterial collection points with limited resources using compact laboratory centrifuges.

At the same time, the presence in the stabilizing solution of the Device for collecting, storing and transporting biological samples according to the second aspect of the invention, containing the Medium according to the main aspect of the invention, the cell membrane fixative allows centrifugation with centrifugal acceleration already from 2000 to 3600g in wider time ranges from 10 up to 30 minutes without mechanical injury to the cell membrane, while in the case of taking whole blood into a stabilizing solution without a component fixing the cell membrane, the authors of patent US20200196594 recommend using a fundamentally more gentle centrifugation mode: 10 minutes at 2000g to avoid additional release of nucleic acids from cells due to their traumatization . In turn, longer centrifugation with higher acceleration makes it possible to isolate residual blood cells, extracellular structures and cellular debris, which, in turn, makes it possible, after plasma collection, not to carry out a second round of centrifugation at 13000-16000g for 10-20 minutes immediately prior to the nucleic acid extraction step. This technical result is relevant for laboratories with an extensive instrument base, a large flow of research and the need to reduce the average time for sample preparation and nucleic acid extraction.

The third aspect of the invention is the use of the device according to the invention for stabilizing nucleic acids for a long time in a wide temperature range (up to 15 days at temperatures from -20°C to +37°C and up to 12 months at temperatures from -86°C to -20 °C) using the Medium according to the main aspect of the invention contained in the Device according to the second aspect of the invention for collecting, storing and transporting biological samples in laboratories with different levels of equipment.

The technical result according to the third aspect of the invention is achieved due to the balanced composition of the Medium according to the main aspect of the invention, in which a combination of a separating gel and a stabilizing solution is implemented, which is located above the separating gel and contains a universal cell membrane fixing agent that has a stabilizing effect on the cell membrane, which allows using Devices according to the second aspect of the invention for collecting, storing and transporting biological samples, which is mainly a vacuum tube, to provide storage and transport of a sample, which is mainly a blood sample and contains extracellular nucleic acids that you want to save, under any conditions and resources that the laboratory has:

- the possibility of storing and transporting a biological sample without centrifugation up to 24 hours after taking at a temperature of +4°C to +37°C, followed by centrifugation and then storage and transportation from 5 to 15 days, depending on the combination of components of the stabilizing solution at a temperature of - 20°С to +37°С;

- the possibility after centrifugation at any period of storage of a biological sample ranging from 5 to 15 days, depending on the combination of components of the stabilizing solution, repeated freezing / thawing of the sample with storage at a temperature of -86°C to -20°C for up to 12 months.

- additional possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking, depending on the combination of components of the stabilizing solution at a temperature of +4°C to +37°C, while preserving extracellular nucleic acids, as well as circulating cells and/or extracellular structures, if necessary;

- additional possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking, depending on the combination of components of the stabilizing solution at a temperature of +4°С to +37°С and centrifugation immediately before plasma sampling to remove cells, extracellular structures and cellular debris to obtain cell-free plasma.

As a result, the main objective of the present invention is solved, which is to create a composition (medium) and a device for collecting, storing and transporting biological samples containing this medium, which would ensure the safety of extracellular nucleic acids for a long time in a wide temperature range, including negative , and at the same time would be suitable for work in laboratories with any level of equipment, including for medical offices / biomaterial collection points with a minimum set of equipment:

- storage and transportation of biological samples after centrifugation up to 15 days in the temperature range from -86°C to +37°C with the possibility of multiple freezing / thawing of the sample, which is important for any laboratory in the cold season, allows you not to depend on strict adherence to a certain temperature storage regime, as in the case of transporting a frozen plasma sample at negative temperatures or a whole blood sample at temperatures of + (4-37) ° C, and ensure the necessary quality of extracellular nucleic acids in the sample, thereby reducing the likelihood of repeated blood sampling from patients, including including due to cell hemolysis;

- additional possibility of storage and transportation of biological samples without centrifugation for up to 15 days at temperatures from +4°C to +37°C, which is important for any laboratories in the warm season, but especially for blood sampling rooms / medical rooms with limited resources and low equipment level.

Implementation of the invention

The following paragraphs contain definitions of terms in accordance with this invention and are intended to be applied uniformly throughout the scope of the description and claims, except where broader definitions are given.

As used herein, the term "nucleic acid" includes both ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), and also includes RNA and/or DNA that is linear or branched, single or double stranded, or fragments thereof. In particular, the nucleic acids may be extracellular DNA, extracellular RNA, or any combination thereof. The biological sample containing the nucleic acids to be preserved may be any biological fluid, and in particular may be blood. More specifically, extracellular nucleic acids may be present in plasma.

In more detail, nucleic acids can be extracellular DNA or RNA from a subject containing its own nucleic acid, and/or a subject to be detected and/or monitored for a cancer, and/or a subject to be detected and/or monitored for a neurodegenerative disease, or a subject subject to detection and/or monitoring for mental illness, monitoring for organ transplant rejection, or a subject who is pregnant and requires self- and/or fetal DNA testing, including to identify markers of genetic disease, disease susceptibility, gender/rhesus -phenotype / genetic profile / determination of paternity during pregnancy, but not limited to this.

Biological samples may also contain circulating cells and/or any extracellular structures (including ectosomes and exosomes) of the subject to be detected and/or monitored for oncological disease, but not limited to this.

The term "release thixotropic gel" means a gel that becomes thinner as a result of agitation or pressure, that is, the viscosity of which decreases as a result of agitation or pressure. Thus, the term "release thixotropic gel" as used herein refers to a gel-like substance that is thick or viscous under static conditions, but will flow and become less viscous when shaken, agitated, sheared or otherwise stressed (RU2667964C1). The gel creates a reliable barrier between blood cells and plasma, which prevents the release of genomic DNA into plasma during cell hemolysis during sample storage and transportation). In accordance with this invention, the thixotropic gel may be an olefin, polyester, polyamide gel (including, but not limited to). Polyesters, denatured collagens, polypropylenes, polysiloxanes such as dimethylpolysiloxane and ethyltriethoxysilane, hydrocarbon gel materials such as polybutene, and combinations thereof (including but not limited to) may also be used as thixotropic gel components. The thixotropic gel, or mixtures thereof based on it, must be insoluble in water and chemically inert with respect to blood components that can be used in accordance with this invention.

The objective of the present invention is to provide an environment and a device for collecting, storing and transporting biological samples containing this environment, which would ensure the safety of extracellular nucleic acids for a long time in a wide range of temperatures and at the same time would be suitable for work in laboratories with any level of equipment, including for medical offices / biomaterial collection points with a minimum set of equipment.

In turn, the use of the main aspects of the invention proposed in the framework of the present application, namely, the Medium of a new composition containing a separating thixotropic gel and a stabilizing solution, including at least one agent fixing the cell membrane, as well as Devices for collecting, storing and transporting biological samples, containing this medium, and methods of their use for the stabilization of nucleic acids can reduce the likelihood that the population of extracellular nucleic acids in a biological sample will be diluted with intracellular nucleic acids, in particular, fragmented genomic DNA originating from damaged and/or dying (apoptotic) cells, contained in the test sample.

The invention is illustrated by the following particular examples, which confirm the practical possibility of using one of the embodiments of the Medium according to the main aspect of the invention in a Device for collecting, storing and transporting biological samples, to stabilize biological samples, in this case, whole blood samples containing extracellular nucleic acids and cells, during centrifugation at different periods of storage, as well as during transportation and storage in different temperature ranges.

The exemplary embodiment of the Medium contains a stabilizing solution (0.1 ml per 1 ml of blood) and a separating thixotropic gel in the Device for collecting, storing and transporting biological samples in the concentration ranges indicated in Table 1.

Separating thixotropic gel is a special inert separating gel, which is heavier than serum, but lighter than a blood clot (blood cells), therefore, after centrifugation, the gel in the form of a thin strip occupies an intermediate position and serves as a separating barrier. Allows cells to be isolated from plasma / serum and other biological fluids, which prevents active enzymes from degrading cells from escaping into solution along with additional intracellular DNA and / or RNA, which significantly degrade the quality and reduce the concentration of the primary free-circulating extracellular DNA / RNA present on the moment of taking a biological sample, and also increase the concentration of intracellular DNA and/or RNA.

Ethylenediaminetetraacetic acid (EDTA) - chelates (binds) divalent metals in solution (magnesium, calcium, etc.) and thereby inactivates the action of metal-dependent enzymes. It prevents blood coagulation (one of the most common anticoagulants), and also inhibits the work of nucleases, which are metal-dependent, thereby preventing the breakdown / degradation of extracellular nucleic acids.

Dimethylol-5,5-dimethylhydantoin, diazolidinyl urea - broad-spectrum preservatives, stabilize the cell membrane through chemical fixation, and also prevent the growth of solutions, as they have activity against a wide range of gram-negative and gram-positive bacteria, molds and fungi.

Additionally, Table 2 shows the composition of the Environment for the collection, storage and transportation of biological samples in the other three possible embodiments.

However, the examples provided are for illustrative purposes only and should not be construed as limiting the invention in any way.

According to the present invention, all possible combinations of the composition of the stabilizing solution were analyzed, as described in paragraph 1:

cell membrane fixer

1. 2-bromo-2-nitropropane-1,3-diol

2. dimethylol urea

3. sodium hydroxymethylglycinate

4. diazolidinyl urea

5. dimethyl-dimethylol-hydantoin

6. imidazolidinyl urea

7. 1,3,5-tris(hydroxyethyl)-s-triazine

8. 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione

9. dimethylol-5,5-dimethylhydantoin

anticoagulant/ionic stabilizing agent

10. ethylene glycol tetraacetic acid (EGTA) and its salts

11. sodium citrate

12. ethylenediaminetetraacetic acid (EDTA) and its salts

13. sodium fluoride

In total, variations in the composition of the stabilizing solution were obtained and analyzed according to Table 3.

As illustrating the invention a detailed implementation example, the composition according to table 1 is selected.

For each of the presented variations, studies were conducted similar to examples 1 and 2.

In all variants, the compliance of all combinations with the required parameters was revealed, each of the possible combinations ensures the achievement of the claimed technical result.

For all variants, cell-free DNA was demonstrated to be resistant to all tested climatic factors under the specified application conditions and the ability to preserve the sample until analysis.

The data obtained for all variants according to the invention also indicate the resistance of extracellular RNA to all tested climatic factors under the indicated conditions of use and the ability to save the sample until analysis.

At the same time, the design of the Device for collecting, storing and transporting biological samples according to the second aspect of the invention, in this embodiment of the invention, is a transparent vacuum tube (PET) containing the Medium according to the main aspect of the invention, equipped with a stopper (butyl rubber) and a protective cap (polypropylene). The stabilizing solution in this embodiment is located above the release gel. The construction in this embodiment corresponds to the drawing in FIG. 1.

Example 1 Stabilization of extracellular DNA

Blood collection

Blood sampling from healthy donors was carried out on the basis of informed voluntary consent in the Device for collecting, storing and transporting biological samples according to the second aspect of the invention, which in this embodiment is a vacuum tube containing the Medium according to the main aspect of the invention, having the composition as indicated in table 1 .

Within the framework of this experiment, the samples were intended for: - checking the possibility of storing and transporting the sample without centrifugation for 8 hours after blood sampling at a temperature from +4°C to +37°C, followed by centrifugation and further storage and transportation for 15 days at temperature from -20°С to +37°С;

- checking the possibility after centrifugation at any period of storage of the sample within 15 days of triple freezing / thawing of the sample with storage at a temperature of -86°C to -20°C for 6 months.

To solve these problems, the following blood samples were taken:

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре +(15-25)°С после центрифугирования - сроки проверки 0, 5, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S1, S2;

2 test tubes of size 16×100 from each donor for subsequent storage at room temperature +(15-25)°C after centrifugation - check periods 0, 5, 10, 15 days with triple freezing / thawing of the sample tube at a temperature of -86° C - samples S1, S2;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в термостате суховоздушном при температуре +37°С после центрифугирования - сроки проверки 0, 5, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S3, S4;

2 test tubes of size 16×100 from each donor for subsequent storage in a dry-air thermostat at a temperature of +37°C after centrifugation - test periods 0, 5, 10, 15 days with triple freezing / thawing of the sample tube at a temperature of -86°C - samples S3, S4;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в холодильнике при +(4-8)°С после центрифугирования - сроки проверки 0, 5, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S5, S6;

2 test tubes of size 16×100 from each donor for subsequent storage in a refrigerator at +(4-8)°C after centrifugation - check periods 0, 5, 10, 15 days with triple freezing / thawing of the sample tube at a temperature of -86° C - samples S5, S6;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в морозильной камере при температуре -20°С после центрифугирования - сроки проверки 0, 5, 10, 15 сутки - образцы S7, S8.

2 test tubes of size 16×100 from each donor for subsequent storage in a freezer at a temperature of -20°C after centrifugation - check times 0, 5, 10, 15 days - samples S7, S8.

- checking the additional possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after blood sampling, depending on the combination of components of the stabilizing solution at a temperature of +4°C to +37°C, while preserving extracellular nucleic acids, as well as circulating cells and/ or extracellular structures as needed.

To solve this problem, the following samples were taken:

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре без центрифугирования - сроки проверки 0, 5, 10, 15 сутки - S9, S10 (сохранность клеток и внеклеточных структур оценивали визуально по наличию гемолиза на разных сроках хранения).

2 test tubes of size 16×100 from each donor for subsequent storage at room temperature without centrifugation - check dates 0, 5, 10, 15 days - S9, S10 (the safety of cells and extracellular structures was assessed visually by the presence of hemolysis at different storage periods).

- checking the additional possibility of storing and transporting the sample without centrifugation from 5 to 15 days after blood sampling, depending on the combination of components of the stabilizing solution at a temperature of +4°C to +37°C and centrifugation immediately before plasma sampling to remove cells, extracellular structures and cell debris to obtain cell-free plasma. To solve this problem, the following samples were taken:

по 4 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре с центрифугированием непосредственно перед отбором плазмы - сроки проверки 0, 5, 10, 15 сутки - S11-S14. Пробы крови отбирали в вакуумные пробирки со Средой по основному аспекту изобретения процедурой венепункции стандартными двухсторонними иглами. Заполняли вакуумную пробирку до метки (квадрат черного цвета на этикетке вакуумной пробирки). Далее снимали пробирку с держателя / адаптера иглы и сразу плавно перемешивали, осторожно перевернув пробирку 10 раз (не встряхивая). Одно переворачивание пробирки - это полный поворот запястья на 180 градусов и обратно.

4 test tubes of size 16×100 from each donor for subsequent storage at room temperature with centrifugation immediately before plasma collection - test dates 0, 5, 10, 15 days - S11-S14. Blood samples were taken into vacuum tubes with Medium according to the main aspect of the invention by a venipuncture procedure with standard double-ended needles. The vacuum tube was filled to the mark (black square on the label of the vacuum tube). The tube was then removed from the needle holder/adapter and immediately mixed gently by gently inverting the tube 10 times (without shaking). One inversion of the test tube is a complete turn of the wrist 180 degrees and back.

Plasma preparation

Samples S1-S8:

8 hours after taking venous blood, the tubes S1-S8 were centrifuged at room temperature +(15-25)°C for 15 minutes at 3000g, followed by placing the tubes for storage under different temperature conditions:

- at room temperature: 0 and 5 days - S1, 10 and 15 days - S2;

- in a dry-air thermostat at a temperature of +37°С: 0 and 5 days - S3, 10 and 15 days - S4;

- in the refrigerator at +(4-8)°C: 0 and 5 days - S5, 10 and 15 days - S6;

- in a freezer at a temperature of -20°C: 0 and 5 days - S7, 10 and 15 days - S8.

The selection and preparation of plasma for the study was carried out on days 0, 5, 10 and 15 of storage as follows:

- ½ of the plasma volume was taken into a clean Eppendorf-type test tube, if the total volume of ≈3-3.5 ml of plasma was in the test tubes with a separating thixotropic gel before testing at the analyzed storage period (if there was already ½ of the plasma volume in the test tube, then the entire remaining volume)

- the analyzed sample was centrifuged at a temperature of +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular DNA was isolated from 1.5 ml of plasma according to the manufacturer's instructions.

In the case of tubes S7 and S8 stored at -20°C, as well as in the analysis of samples from tubes frozen at -86°C, the following was additionally given:

- defrosting the test tube at room temperature;

- smooth mixing 4-6 times or soft pipetting;

далее аналогично пробиркам S1-S6 проводили отбор и подготовку плазмы с последующим выделением внеклеточной ДНК согласно инструкции производителя;

then, similarly to test tubes S1-S6, plasma was collected and prepared, followed by isolation of extracellular DNA according to the manufacturer's instructions;

- re-freezing of the tube with the remaining ½ volume of plasma, gel and blood cells, if necessary.

Samples S9, S10: Stabilized blood samples were stored at room temperature in an upright position. The selection and preparation of plasma for the study was carried out on days 0.5 (tube S9) and 10.15 days of storage (tube S10) as follows:

- smooth mixing was carried out 6-8 times and the subsequent sampling of ½ of the blood volume into an Acti-Fine® vacuum tube without filler produced by Granat Bio Tech (SEZ Dubna, Russia), if there was a full volume of blood in the tube before testing at the planned storage period ≈7.5 ml (if there was already ½ volume of whole blood in the tube, then smooth mixing was carried out 6-8 times and the entire remaining volume was transferred to the Acti-Fine® tube without filler);

- carried out centrifugation of the selected blood sample in test tubes at room temperature for 15 minutes at 1600g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube and re-centrifuged to separate the cell debris at +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular DNA isolation was performed according to the manufacturer's instructions.

Samples S11-S14:

Stabilized blood samples were stored at room temperature in an upright position. The selection and preparation of plasma for the study was carried out on days 0, 5, 10 and 15 of storage (tubes S11, S12, S13 and S14, respectively) as follows:

- immediately before plasma sampling, the tubes were centrifuged at room temperature +(15-25)°C for 15 minutes at 3000g;

- carried out the selection of 1.5-2 ml of plasma in a clean tube type Eppendorf;

- the analyzed sample was centrifuged in test tubes at a temperature of +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular DNA isolation was performed according to the manufacturer's instructions.

Examples of photographs of test tubes with the medium according to the main aspect of the invention before and after centrifugation are shown in Figs. 2.

Isolation of extracellular DNA

Isolation of extracellular DNA from plasma was performed using the MagMAX™ Cell-Free DNA Isolation Kit (ThermoFisher Scientific, USA). Isolation was carried out according to Protocol No. 2 without the addition of protein kinase. The kit includes buffer solutions and magnetic particles with which extracellular DNA molecules bind. At the first stage, a 1.5 ml plasma sample was placed into 15 ml Falcon type tubes. 2.53 ml of a pre-prepared solution containing the Lysing/Binding buffer and magnetic particles, which were preliminarily mixed in a Microspin FV-2400 mini-vortex centrifuge (Biosan, Latvia), was added to each tube. The tubes were carefully inverted 10 times, and then all the tubes were vigorously shaken on a vortex for 10 minutes (this step is necessary for the binding of extracellular DNA to magnetic particles). Next, the tubes were placed and incubated in a magnetic rack for 5 minutes and then the clear supernatant was removed, while the magnetic particles remained on the walls of the tubes.

The next steps involved washing the magnetic particles with a special wash buffer (MagMAX™ Cell Free DNA Wash Solution). To do this, 1 ml of buffer was added to the magnetic particles and the contents of the tubes were mixed on a vortex for 30 seconds. The resulting solution containing wash buffer and magnetic particles was transferred into 2 ml Eppendorf tubes.

These tubes were placed on a magnetic rack for 2 ml tubes and then left for 3 minutes until the buffer became transparent and the magnetic particles were completely collected on the walls of the tube. Thereafter, the buffer was aspirated.

перемешивали содержимое на вортексе в течение 30 секунд

возвращали пробирки на магнитный штатив

проводили аспирацию буфера.The second washing step included a repeat of the first: 1 ml of wash buffer was added to the magnetic particles

vortex the contents for 30 seconds

returned the tubes to the magnetic rack

buffer was aspirated.

перемешивали содержимое на вортексе в течение 30 секунд

возвращали пробирки на магнитный штатив

проводили аспирацию этанола

данный этап проводили 2 раза.The next 2 wash steps involved using freshly made 70% ethanol. To do this, 1 ml of 70% ethanol was added to 2 ml of Eppendorf type tubes.

vortex the contents for 30 seconds

returned the tubes to the magnetic rack

ethanol was aspirated

this step was carried out 2 times.

Next, the magnetic particles were dried from residual ethanol for 2-3 minutes and 15 µl of extracellular DNA elution buffer was added. At the same time, for efficient transfer of DNA into the buffer solution, after adding the elution buffer to the magnetic particles, the contents of the tubes were pipetted and left in a thermal shaker for microtubes and PCR plates TS-100 (Biosan, Latvia) at maximum speed for five minutes. Next, the tubes were again placed in a magnetic rack until the liquid became transparent and all magnetic particles were collected on the side walls of the tubes. The liquid containing the eluted DNA was carefully aspirated and transferred to a new 0.5 ml Eppendorf tube (DNA LoBind).

Evaluation of the stabilizing effect of one of the embodiments of the Environment for the collection, storage and transport of biological samples using Tare Station 2200

To evaluate the effectiveness of the stabilizing effect of one of the embodiments of the Medium (according to Table 1) on the main aspect of the invention in the Device for collecting, storing and transporting biological samples, namely the ability to stabilize extracellular nucleic acids and cells, a biological sample, in this case a whole blood sample, was a qualitative determination of the presence of extracellular DNA in tubes S1-S14 from different donors without/with centrifugation at different periods of storage, as well as during transportation and storage in different temperature ranges, was carried out. Qualitative determination of the presence of extracellular DNA was performed by capillary electrophoresis on TapeStation 2200 equipment (Agilent, USA) using ready-made reagent kits in accordance with the manufacturer's instructions for use. The determination of extracellular DNA was carried out by the presence of characteristic fluorescence peaks on the electrophoregram, while the fluorescent peaks had to meet the requirements of Table 4.

In turn, the assessment of the presence and determination of the length of fragments of extracellular DNA isolated from plasma by capillary electrophoresis was performed using the D1000 ScreenTape System kit and preliminary sample preparation according to the following procedure. In 0.2 ml tubes in strip format, consisting of 8 tubes with individually attached striped flat caps, 2 μl of buffer (D1000 Sample Buffer) was added to each tube. Next, 2 µl of a DNA fragment length marker (D1000 Sample Ladder) was added to the first tube, and extracellular DNA samples were added to the remaining tubes in an amount of approximately 0.5-1 ng per well, while the sample volume was 2 µl. The concentration of extracellular DNA was preliminarily measured using a Qubit 4.0 fluorimeter (ThermoFisher Scientific, USA) using a High Sensitivity dsDNA kit according to the protocol recommended by the manufacturer. The total volume of the contents of each tube was 4 μl. Test tubes in strips of 2 pieces were loaded into the automatic TapeStation 2200 system together with dosing tips and a special chip for capillary electrophoresis, and then the program was launched to obtain and analyze the results. Examples of results obtained using the TapeStation 2200 are shown in FIG. 3-5.

Additional examples of the results of testing one of the embodiments of the Medium according to the main aspect of the invention in the Device for collecting, storing and transporting biological samples, for its ability to stabilize extracellular DNA in whole blood samples without / with centrifugation at different storage periods in different temperature ranges are shown in tables 5 and 6.

As a result of the testing of one of the embodiments of the Medium according to the main aspect of the invention, containing a stabilizing solution and a separating thixotropic gel in the Device for collecting, storing and transporting biological samples in the concentration ranges indicated in Table 1, the resistance of extracellular DNA to the effects of all tested climatic conditions was demonstrated. factors under specified conditions of use and the ability to preserve the sample prior to analysis.

For all variants of the medium, similar tests were carried out. In all versions of the compositions of the medium, the resistance of extracellular DNA to the effects of all tested climatic factors under the indicated conditions of use and the ability to save the sample until analysis was demonstrated.

In the case of extracellular DNA, all given combinations were checked for compliance with the parameters indicated in table 4.

Example 2 Stabilization of extracellular RNA

Blood collection

Blood sampling from healthy donors was carried out on the basis of informed voluntary consent in the Device for collecting, storing and transporting biological samples according to the second aspect of the invention, which in this embodiment of the invention is a vacuum tube containing the Medium according to the main aspect of the invention, having a composition according to table 1.

As part of this experiment, the samples were intended for:

- checking the possibility of storing and transporting the sample without centrifugation for 8 hours after blood sampling at a temperature of +4°C to +37°C, followed by centrifugation and then storage and transportation for 15 days at a temperature of -20°C to +37 °C;

- checking the possibility after centrifugation at any period of storage of the sample within 15 days of triple freezing / thawing of the sample with storage at a temperature of -86°C to -20°C for 6 months.

To solve these problems, the following blood samples were taken:

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре +(15-25)°С после центрифугирования - сроки проверки 0, 4, 7, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S15, S16;

2 test tubes of size 16×100 from each donor for subsequent storage at room temperature +(15-25)°C after centrifugation - check times 0, 4, 7, 10, 15 days 86°C - samples S15, S16;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в термостате суховоздушном при температуре +37°С после центрифугирования - сроки проверки 0, 4, 7, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S17, S18;

2 test tubes of size 16×100 from each donor for subsequent storage in a dry-air thermostat at a temperature of +37°C after centrifugation - check periods 0, 4, 7, 10, 15 days with triple freezing / thawing of the sample tube at a temperature of -86° C - samples S17, S18;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в холодильнике при +(4-8)°С после центрифугирования - сроки проверки 0, 4, 7, 10, 15 сутки с трехкратным замораживанием / размораживанием пробирки с образцом при температуре -86°С - образцы S19, 20;

2 test tubes of size 16×100 from each donor for subsequent storage in a refrigerator at +(4-8)°C after centrifugation - check periods 0, 4, 7, 10, 15 days with triple freezing / thawing of the sample tube at a temperature - 86°C - samples S19, 20;

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения в морозильной камере при температуре -20°С после центрифугирования - сроки проверки 0, 4, 7, 10, 15 сутки - образцы S21, S22;

2 test tubes of size 16×100 from each donor for subsequent storage in a freezer at a temperature of -20°C after centrifugation - test dates 0, 4, 7, 10, 15 days - samples S21, S22;

- checking the additional possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after blood sampling, depending on the combination of components of the stabilizing solution at a temperature of +4°C to +37°C, while preserving extracellular nucleic acids, as well as circulating cells and/ or extracellular structures if necessary;

To solve this problem, the following samples were taken:

по 2 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре без центрифугирования - сроки проверки 0, 4, 7, 10, 15 сутки - S23, S24 (сохранность клеток и внеклеточных структур оценивали визуально по наличию гемолиза на разных сроках хранения).

2 test tubes of size 16×100 from each donor for subsequent storage at room temperature without centrifugation - check dates 0, 4, 7, 10, 15 days - S23, S24 (the safety of cells and extracellular structures was assessed visually by the presence of hemolysis at different storage periods ).

- checking the additional possibility of storing and transporting the sample without centrifugation from 5 to 15 days after blood sampling, depending on the combination of components of the stabilizing solution at a temperature of +4°C to +37°C and centrifugation immediately before plasma sampling to remove cells, extracellular structures and cell debris to obtain cell-free plasma. To solve this problem, the following samples were taken:

по 4 пробирки типоразмера 16×100 от каждого донора для последующего хранения при комнатной температуре с центрифугированием непосредственно перед отбором плазмы - сроки проверки 0, 4,7, 10, 15 сутки - S25-S29.

4 test tubes of size 16×100 from each donor for subsequent storage at room temperature with centrifugation immediately before plasma collection - check times 0, 4.7, 10, 15 days - S25-S29.

Blood samples were taken into vacuum tubes with Medium according to the main aspect of the invention of the composition according to table 1 by the procedure of venipuncture with standard double-ended needles. The vacuum tube was filled to the mark (black square on the label of the vacuum tube). The tube was then removed from the needle holder/adapter and immediately mixed gently by gently inverting the tube 10 times (without shaking). One inversion of the test tube is a complete turn of the wrist 180 degrees and back.

Additionally, a blood sample was taken from each donor in BD Vacutainer® Plus vacuum tubes with EDTA as an additional control on day 0 of storage. Tubes with EDTA and biological samples were kept on ice and all manipulations were carried out within 1 hour after blood sampling. Fractionation of whole blood in tubes with EDTA, namely, obtaining plasma free of cellular elements, was carried out by centrifugation at room temperature for 20 minutes at low speed (900g), since centrifugation of blood at high speed inevitably leads to the destruction of some of the cells and the entry of cells contained in of them nucleic acids into plasma. Plasma free of formed elements was carefully collected and immediately used for RNA isolation. In turn, vacuum tubes with the Medium according to the main aspect of the invention and the biological sample were subjected to centrifugation at higher speeds (3000g) due to the presence of a cell membrane fixing agent in the stabilizing solution, which prevents the destruction of cells and the release of intracellular RNA from them.

Plasma preparation

Samples S15-S22:

8 hours after taking venous blood, the tubes S15-S22 were centrifuged at room temperature +(15-25)°C for 15 minutes at 3000g, followed by placing the tubes for storage under different temperature conditions:

- at room temperature: 0, 4, 7 days - S15; 10, 15 days - S16;

- in a dry-air thermostat at a temperature of +37°С: 0, 4, 7 days - S17; 10, 15 days - S18;

- in the refrigerator at +(4-8)°С: 0, 4, 7 days - S19; 10, 15 days - S20;

- in a freezer at a temperature of -20°C: 0, 4, 7 days - S21; 10, 15 days - S22.

The selection and preparation of plasma for the study was carried out on days 0, 4, 7, 10 and 15 of storage as follows:

- carried out the selection of 1 ml of plasma in a clean tube type Eppendorf;

- the analyzed sample was centrifuged at a temperature of +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular RNA isolation was performed according to the manufacturer's instructions. In the case of tubes S21 and S22 stored at -20°C, as well as in the analysis of samples

from tubes frozen at -86°C, the following was additionally given:

- defrosting the test tube at room temperature;

- smooth mixing 4-6 times or soft pipetting;

далее аналогично пробиркам S15-S20 проводили отбор и подготовку плазмы с последующим выделением внеклеточной РНК согласно инструкции производителя;

then, similarly to test tubes S15-S20, plasma was collected and prepared, followed by isolation of extracellular RNA according to the manufacturer's instructions;

- re-freezing of the tube with the remaining ½ volume of plasma, gel and blood cells, if necessary.

Samples S23, S24:

Stabilized blood samples were stored at room temperature in an upright position. The selection and preparation of plasma for the study was carried out on days 0.4, 7 (tube S23) and 10, 15 days of storage (tube S24) as follows:

- carried out smooth mixing 6-8 times and subsequent sampling of 2.5 ml of blood into a vacuum test tube Acti-Fine® without filler manufactured by Granat Bio Tech (SEZ Dubna, Russia);

- carried out centrifugation of the selected blood sample in test tubes at room temperature for 15 minutes at 1800g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube and re-centrifuged to separate the cell debris at +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular RNA isolation was performed according to the manufacturer's instructions. Samples S25-S29:

Stabilized blood samples were stored at room temperature in an upright position. The selection and preparation of plasma for the study was carried out on days 0, 4, 7, 10 and 15 of storage (tubes S25, S26, S27, S28 and S29, respectively) as follows:

- immediately before plasma sampling, the tubes were centrifuged at room temperature +(15-25)°C for 15 minutes at 3000g;

- carried out the selection of 1 ml of plasma in a clean tube type Eppendorf;

- the analyzed sample was centrifuged in test tubes at a temperature of +4°C for 10 minutes at 16000g;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular RNA isolation was performed according to the manufacturer's instructions. Isolation of RNA

Isolation of RNA from plasma for further staging of the reverse transcription reaction was carried out using the Proba-NK kit (OOO DNA-Technology, Russia) in triplicate. In pre-marked 1.5 ml Eppendorf tubes, 300 μl of lysing solution were added without touching the edge of the tube. Next, 100 µl of the studied plasma samples were added to the test tubes, the caps of the tubes were tightly closed and shaken on a vortex for 3-5 sec. Next, the tubes were thermostated at a temperature of 65°C for 15 minutes and the condensate was precipitated by centrifugation at 13,000 rpm for 30 seconds at room temperature.

After that, 400 µl of the precipitation reagent was added to each tube, vortexed for 3-5 seconds, and the tubes were centrifuged at 13,000 rpm for 15 minutes at room temperature. Without touching the sediment, the supernatant was completely removed from each tube with a separate tip, 500 µl of washing solution No. 1 was added to the sediment, the caps of the tubes were closed and mixed, gently inverting the tubes 3-5 times. Next, the tubes were centrifuged at 13,000 rpm for 5 min at room temperature. Without touching the sediment, the supernatant was completely removed from each tube with a separate tip, 300 µl of washing solution No. 2 was added to the sediment, the caps of the tubes were closed and mixed, gently inverting the tubes 3-5 times. Next, the tubes were centrifuged at 13,000 rpm for 5 min at room temperature. Without touching the precipitate, the supernatant was completely removed from each tube with a separate tip, and the precipitate was dried at a temperature of 65°C for 5 min in tubes with open lids.

After that, 16.5 μl of dissolution buffer was added to the sediment, the tubes were shaken on a vortex for 3-5 sec, and drops were precipitated by centrifuging the tubes for 3-5 sec. The resulting RNA preparations were immediately used to set up the reverse transcription reaction.

Setting up a reverse transcription reaction (obtaining cDNA)

In the course of reverse transcription of RNA, cDNA was obtained with significantly greater stability, which made it possible to store samples in the future or to analyze them without significant degradation of nucleic acids for a long time.

To carry out the reverse transcription reaction, 0.5 ml Eppendorf tubes were labeled, and the contents of the tubes "OT-buffer" and "Primers OT-RANDOM + dNTP" from the set of reagents for reverse transcription (OOO "DNA-Technology", Russia) were thawed. ) at room temperature, then the tubes were shaken for 3-5 sec and centrifuged at 1000 rpm for 3-5 sec on a Microspin FV-2400 microcentrifuge-vortex (Biosan, Latvia). In a separate tube, an OT mixture was prepared for setting up the reverse transcription reaction by mixing 2.0 × (N + 1) μl of RT buffer, 1.0 × (N + 1) μl of primers OT-RANDOM + dNTP, 0.5 × ( N+1) µl of reverse transcriptase, where N is the number of analyzed samples (N) with a margin for 1 sample. Then, 3.5 µl of the RT mixture was added to the pre-labeled tubes, 16.5 µl of isolated RNA samples were added, vortexed for 3–5 sec, and drops were precipitated by centrifugation at 1000 rpm for 3–5 sec.

After that, the tubes were placed in a thermostat and incubated at a temperature of +40°C for 30 minutes, then at a temperature of +95°C for 5 minutes. Next, to precipitate the condensate, centrifugation was performed at 13,000 rpm for 30 seconds, and 35 μl of the dissolution buffer from the nucleic acid isolation kit were added to the resulting cDNA. The tubes were shaken on a vortex for 3-5 sec and drops were precipitated by centrifugation at 1000 rpm for 3-5 sec. The resulting cDNA samples were used as a template for real-time polymerase chain reaction (PCR).

Setting up real-time PCR

4). Детекцию флюоресценции проводили при 72°С в конце каждого цикла. Кривые накопления флуоресцентного сигнала анализировали по каналу FAM. Для каждого образца были выполнены три технических повтора ПЦР, при этом полученные результаты представляли собой средний пороговый цикл (Ct) прохождения реакции с определенными стандартными отклонениями (SD). Анализ полученных результатов проводили с помощью программного обеспечения используемого амплификатора. Дополнительно определяли значение ΔCt=Ct^ЭДТА (средний пороговый цикл образца кДНК, полученного с использованием вакуумных пробирок BD Vacutainer® Plus с ЭДТА на 0 сутки хранения) - Ct^{образца} (средний пороговый цикл исследуемого образца кДНК, полученного с использованием одного из вариантов воплощения Среды по основному аспекту изобретения в Устройстве для сбора, хранения и транспортировки биологических образцов, на разных сроках хранения в разных температурных условиях). По результатам оценки средних значений экспрессии гена b2M делали заключение о сохранности образцов внеклеточной РНК.The cDNA samples obtained as a result of reverse transcription were used to assess the level of expression of the housekeeping gene - beta-2-microglobulin (b2M) by real-time PCR. Real-time quantitative PCR was performed using a ready-made iQ SYBR Green Supermix PCR system (Bio-Rad, USA). The reaction mixture for PCR with a total volume of 25 µl contained 12.5 µl of twofold iQ SYBR Green Supermix (Bio-Rad, USA), 5 pmol of forward (5'-CGTACTCCAAAGATTCAGGTT-3') and reverse (5'-TGCTGCTTTACATGTCTCGAT-3 ') primers to the regions present in the b2M gene transcript, the sequences and optimal amplification conditions of which were obtained from the literature data [8], 5 µl of water without nucleases (Thermo Scientific, USA) and 5 µl of cDNA per reaction. PCR was performed using a DT-Prime amplifier (OOO DNA Technology, Russia) under the following conditions: 1) 95°С, 5 min; 2) 90°С, 10 s; 3) 64°С, 15 s; 4) 72°C, 20 s (45 cycles 2

4). Fluorescence detection was performed at 72°C at the end of each cycle. Fluorescent signal accumulation curves were analyzed using the FAM channel. For each sample, three technical replicates of the PCR were performed, with the results obtained representing the average threshold cycle (Ct) of the passage of the reaction with certain standard deviations (SD). The analysis of the obtained results was carried out using the software of the cycler used. Additionally, the value of ΔCt=Ct ^EDTA (average threshold cycle of the cDNA sample obtained using vacuum tubes BD Vacutainer® Plus with EDTA on day 0 storage) - Ct ^{of the sample} (average threshold cycle of the studied cDNA sample obtained using one of the embodiments of the Medium according to the main aspect of the invention in the Device for collecting, storing and transporting biological samples, at different periods of storage at different temperature conditions). Based on the results of evaluating the average values of the b2M gene expression, a conclusion was made about the safety of extracellular RNA samples.

Examples of test results of one of the embodiments of the Medium according to the main aspect of the invention according to table 1 in the Device for collecting, storing and transporting biological samples, for its ability to stabilize extracellular RNA in whole blood samples without/with centrifugation at different storage periods in different temperature ranges are given in tables 7 and 8.

Based on the results of amplification of the b2M gene transcript site in cDNA samples obtained using one of the embodiments of the Medium according to the main aspect of the invention in the concentration ranges indicated in Table 1, in the Device for collecting, storing and transporting biological samples, at different storage periods at different temperature conditions, as well as on the basis of data from a comparative analysis of the average values of the expression of this gene in cDNA samples obtained using vacuum tubes BD Vacutainer® Plus with EDTA on the 0th day of storage at room temperature, it was concluded that extracellular RNA is resistant to all tested climatic conditions. factors under specified conditions of use and the ability to preserve the sample prior to analysis. Separately, it should be noted that on the 15th day of storage of whole blood samples, the level of expression of the b2M gene in the Device proposed in the framework of this application according to the second aspect of the invention is higher or comparable to the expression of this gene on the 0th day of storage of blood in tubes with EDTA, which completely solves the problem of transporting extracellular RNA, the analysis of which is mainly carried out as part of a liquid biopsy in oncology, up to 15 days in a wide range of negative and positive temperatures to the place of high-tech research - to world-class genomic research centers.

The analysis of possible variants of environments, as they are described in paragraph 1 of the claims, was also carried out for:

- confirmation of a higher concentration of extracellular DNA and RNA, which is detected when extracting DNA / RNA after centrifugation from blood samples in tubes according to the invention with an anticoagulant (for example, EDTA), cell membrane fixative and gel compared to tubes with EDTA without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids, namely, Cell-Free DNA BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel;

- confirmation of a higher concentration of extracellular DNA and RNA, which is found in DNA / RNA isolation after centrifugation and 1 freeze / thaw cycle of a blood sample in tubes according to the invention with anticoagulant (for example, EDTA), cell membrane fixative and gel compared to tubes with EDTA without / with separating gel and specialized tubes for stabilization of extracellular nucleic acids, in particular, with RNA Complete BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel without freezing cycles.

Example 3. Stabilization of extracellular DNA and RNA using the Medium and Device for collecting, storing and transporting biological samples according to the present invention and commercially available analogues (comparative analysis)

Blood collection

Blood sampling from healthy donors was carried out on the basis of informed voluntary consent in the Device for collecting, storing and transporting biological samples according to the second aspect of the invention, which in this embodiment is a vacuum tube containing the Medium according to the main aspect of the invention, having the composition as indicated in table 1 .

As part of this experiment, the samples were intended for:

- checks for a higher concentration of extracellular DNA and RNA, which should be detected at shelf life 0 in the isolation of DNA / RNA after centrifugation from blood samples in tubes according to the invention with anticoagulant (for example, EDTA), cell membrane fixative and gel compared to tubes with EDTA without / with separating gel and specialized tubes for stabilization of extracellular nucleic acids, namely with Cell-Free DNA BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel;

- checks for a higher concentration of extracellular DNA and RNA, which should be detected at 0 shelf life during DNA / RNA isolation after centrifugation and 1 freeze / thaw cycle of a blood sample in tubes according to the invention with an anticoagulant (for example, EDTA), cell membrane fixative and gel according to the invention compared with EDTA tubes without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids, in particular, with RNA Complete BCT® tubes manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel without freezing cycles.

To solve these problems, the following blood samples were taken, followed by centrifugation of the tubes for 1 hour after blood sampling:

по 2 пробирки с антикоагулянтом, фиксатором клеточной мембраны и гелем согласно настоящему изобретению типоразмера 16×100 от каждого донора с последующим хранением при комнатной температуре +(15-25)°С до проведения центрифугирования и далее выделением нуклеиновых кислот - образцы S30, S31;

2 tubes with an anticoagulant, a cell membrane fixative and a gel according to the present invention, size 16×100 from each donor, followed by storage at room temperature + (15-25) ° C until centrifugation and further extraction of nucleic acids - samples S30, S31;

по 2 пробирки с антикоагулянтом, фиксатором клеточной мембраны и гелем согласно настоящему изобретению типоразмера 16×100 от каждого донора с последующим хранением при комнатной температуре +(15-25)°С до проведения центрифугирования и далее выделением нуклеиновых кислот после 1 цикла замораживания /размораживания - образцы S32, S33;

2 tubes with anticoagulant, cell membrane fixative and gel according to the present invention, size 16×100 from each donor, followed by storage at room temperature +(15-25)°C until centrifugation and further nucleic acid isolation after 1 freeze/thaw cycle - samples S32, S33;

по 2 пробирки BD Vacutainer® Plus с ЭДТА типоразмера 16×100 от каждого донора с последующим хранением на льду до проведения центрифугирования и далее выделением нуклеиновых кислот - образцы S34, S35;

2 BD Vacutainer® Plus tubes with 16×100 EDTA from each donor, followed by storage on ice until centrifugation and further nucleic acid isolation - samples S34, S35;

по 2 пробирки BD Vacutainer® PPT™ с ЭДТА и разделительным гелем типоразмера 16×100 от каждого донора с последующим хранением на льду до проведения центрифугирования и далее выделением нуклеиновых кислот - образцы S36, S37;

2 BD Vacutainer® PPT™ tubes with EDTA and separation gel, size 16×100 from each donor, followed by storage on ice until centrifugation and subsequent nucleic acid isolation - samples S36, S37;

по 1 пробирке Cell-Free DNA ВСТ® производства компании Streck (США) с антикоагулянтом и фиксатором клеточной мембраны без геля объемом 10 мл от каждого донора с последующим хранением при комнатной температуре +(15-25)°С до проведения центрифугирования и далее выделением внеклеточной ДНК - образец S38;

1 tube of Cell-Free DNA BCT® manufactured by Streck (USA) with an anticoagulant and a cell membrane fixative without gel, 10 ml from each donor, followed by storage at room temperature + (15-25) ° C until centrifugation and then isolation of extracellular DNA - sample S38;

по 1 пробирке RNA Complete ВСТ® производства компании Streck (США) с антикоагулянтом и фиксатором клеточной мембраны без геля объемом 10 мл от каждого донора с последующим хранением при комнатной температуре +(15-25)°С до проведения центрифугирования и далее выделением внеклеточной РНК - образец S39.

1 tube of RNA Complete BCT® manufactured by Streck (USA) with an anticoagulant and a cell membrane fixative without gel, 10 ml from each donor, followed by storage at room temperature + (15-25) ° C until centrifugation and then isolation of extracellular RNA - sample S39.

Blood samples were taken into vacuum tubes by venipuncture with standard double-ended needles. Vacuum tubes were filled to the mark, then the tube was removed from the needle holder/adapter and immediately mixed gently by gently inverting the tube 8-10 times (without shaking).

Plasma preparation

Samples S30-S33:

Within 1 hour after taking venous blood, the tubes S30-S33 were centrifuged at room temperature +(15-25)°C for 15 minutes at 2800g, followed by placing the tubes for storage under different temperature conditions:

- at room temperature: S30, S31;

- in a freezer at a temperature of -20°C until the contents of the tube are completely frozen, then thawed at room temperature and then gently stirred 4-6 times or soft pipetting: S32, S33. Next, the plasma was prepared for the study as follows:

- carried out the selection of ½ volume of plasma in a clean tube type Eppendorf;

- performed centrifugation of the analyzed sample

при температуре +4°С в течение 10 минут при 16000g в случае проб S30 и S32, которые были предназначены для выделения внеклеточной ДНК;

at a temperature of +4°C for 10 minutes at 16000g in the case of samples S30 and S32, which were intended for isolation of extracellular DNA;

при комнатной температуре в течение 15 минут при 2800g в случае проб S31 и S33, которые были предназначены для выделения внеклеточной РНК;

at room temperature for 15 minutes at 2800g for samples S31 and S33, which were designed to isolate extracellular RNA;

- the supernatant was carefully separated, transferred to a clean Eppendorf tube;

- extracellular DNA (tubes S30, S32) and RNA (tubes S31, S33) were isolated from plasma according to the respective manufacturer's instructions.

Samples S34, S35:

Within 1 hour after taking venous blood, centrifugation of tubes S34, S35 was performed at room temperature +(15-25)°C for 20 minutes at low speed (900g), since blood centrifugation at high speed inevitably leads to the destruction of some cells and the nucleic acids they contain into plasma.

Next, plasma was collected and prepared, followed by isolation of extracellular DNA (tube S34) similarly to tubes S30, S32 and extracellular RNA (tube S35) similarly to tubes S31, S33 from plasma according to the relevant manufacturer's instructions.

Samples S36, S37:

Within 1 hour after taking venous blood, tubes S36, S37 were centrifuged at room temperature +(15-25)°C for 20 minutes at 1500g, since this value of relative centrifugal acceleration is declared by the manufacturer as the maximum allowable (recommended range from 1100 to 1500g), in which there is no destruction of cells and no entry of the nucleic acids contained in them into the plasma.

Next, plasma was collected and prepared, followed by isolation of extracellular DNA (tube S36) similarly to tubes S30, S32, S34 and extracellular RNA (tube S37) similarly to tubes S31, S33, S35 from plasma according to the relevant manufacturer's instructions.

Samples S38, S39:

Within 1 hour after taking venous blood, centrifugation was performed

пробирок S38, предназначенных для последующего выделения внеклеточной ДНК, при комнатной температуре +(15-25)°С в течение 10 минут при 1600g;

tubes S38 intended for subsequent extraction of extracellular DNA, at room temperature +(15-25)°C for 10 minutes at 1600g;

пробирок S39, предназначенных для последующего выделения внеклеточной РНК, при комнатной температуре +(15-25)°С в течение 15 минут при 1800g. Далее проводили отбор и подготовку плазмы с последующим выделением внеклеточной ДНК (пробирка S38) аналогично пробиркам S30, S32, S34, S36 и РНК (пробирка S39) аналогично пробиркам S31, S33, S35, S37 из плазмы согласно соответствующим инструкциям производителей. Выделение внеклеточной ДНК

tubes S39, intended for the subsequent isolation of extracellular RNA, at room temperature +(15-25)°C for 15 minutes at 1800g. Next, plasma was collected and prepared, followed by isolation of extracellular DNA (tube S38) similarly to tubes S30, S32, S34, S36 and RNA (tube S39) similarly to tubes S31, S33, S35, S37 from plasma according to the relevant manufacturer's instructions. Isolation of extracellular DNA

Isolation of extracellular DNA from plasma was performed using the MagMAX™ Cell-Free DNA Isolation Kit (ThermoFisher Scientific, USA) according to the methodology described in Example 1.

The presence of a fluorescence peak in the length range of fragments of 150-250 bp, characteristic of extracellular DNA, was assessed by capillary electrophoresis using TapeStation 2200 equipment (Agilent, USA) using ready-made reagent kits in accordance with the manufacturer's instructions and according to the methodology specified in Example 1. The concentration of extracellular DNA was measured using a Qubit 4.0 fluorimeter (ThermoFisher Scientific, USA) using the High Sensitivity dsDNA kit according to the protocol recommended by the manufacturer. For all samples, the concentration of extracellular DNA measured using a Qubit 4.0 fluorimeter corresponded to the concentration of extracellular DNA calculated from the area of the main fluorescence peak on the electropherogram obtained by capillary electrophoresis on TapeStation 2200 equipment, in the range of lengths of fragments of 150-250 bp, characteristic for extracellular DNA.

The results of determining the concentration of extracellular DNA, the isolation of which was carried out after centrifugation at 0 shelf life without freezing and with 1 freeze / thaw cycle from blood samples in tubes with anticoagulant, cell membrane fixative and gel according to the present invention in comparison with tubes with EDTA without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids Cell-Free DNA BCT® manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel without freezing cycles are shown in Table 9.

As a result of the testing of one of the embodiments of the Medium according to the main aspect of the invention, containing a stabilizing solution and a separating thixotropic gel in the Device for collecting, storing and transporting biological samples in the concentration ranges indicated in Table 1, a higher concentration of extracellular DNA was confirmed, which is detected in DNA extraction after centrifugation without freezing and with 1 freeze/thaw cycle of a blood sample in test tubes according to the invention compared to:

- tubes with EDTA without gel - an increase of up to 2.43 and 2.91 times, respectively;

- tubes with EDTA with separating gel - an increase of up to 2.37 and 3.00 times, respectively;

- Cell-Free DNA BCT® tubes manufactured by Streck (USA) with an anticoagulant and a cell membrane fixative without gel - an increase of up to 1.67 and 2.00 times, respectively.

RNA isolation, cDNA production and real-time PCR. Isolation of RNA from plasma for further staging of the reverse transcription reaction was carried out using the Proba-NK kit (OOO DNA-Technology, Russia) in three repetitions according to the methodology indicated in Example 2.

A qualitative assessment of the purity of circulating extracellular RNA preparations was performed by measuring the optical density of aliquots of the obtained samples at wavelengths of 260 and 280 nm on a NanoDrop 2000 spectrophotometer in accordance with the manufacturer's instructions for use. The absorbance ratio 260/280 for all analyzed samples was 1.8-2.0.

The reverse transcription reaction was carried out using a reverse transcription reagent kit (OOO DNA-Technology, Russia) according to the methodology indicated in Example 2. The cDNA samples obtained as a result of reverse transcription were used to assess the level of expression of the housekeeping gene - beta-2-microglobulin (b2M) by real-time PCR.

The analysis of the obtained results was carried out using the software of the cycler used. Additionally determined:

- value of ΔCt ¹ =Ct ^{of the reference sample} (average threshold cycle of a cDNA sample obtained using the BD Vacutainer® Plus with EDTA reference tube, BD Vacutainer® PPT™ with EDTA and separation gel or RNA Complete BCT® with anticoagulant and cell membrane fixative without gel on day 0 of storage) - ^sample Ct (average threshold cycle of the test cDNA sample obtained using one of the embodiments of the Medium according to the main aspect of the invention in the Device for collecting, storing and transporting biological samples after centrifugation at room temperature + (15-25) ° C at 0 shelf life without freezing);

- value of ΔCt ² =Ct ^{of the reference sample} (average threshold cycle of the cDNA sample obtained using the BD Vacutainer® Plus with EDTA reference tube, BD Vacutainer® PPT™ with EDTA and separation gel or RNA Complete BCT® with anticoagulant and cell membrane fixative without gel on day 0 of storage) - ^sample Ct (average threshold cycle of the test cDNA sample obtained using one of the embodiments of the Medium according to the main aspect of the invention in the Device for collecting, storing and transporting biological samples after centrifugation at room temperature + (15-25) ° C at 0 shelf life with 1 freeze/thaw cycle);

- the ratio of extracellular RNA concentration in the device according to the main aspect of the invention in relation to the concentration of extracellular RNA in the corresponding reference tube at 0 shelf life without freezing or with 1 freeze / thaw cycle = 1.933 ^ΔCt1 or 1.933 ^ΔCt2 , respectively, where 1.933 is the coefficient of efficiency of PCR by titration .

Examples of the results of determining the concentration of extracellular RNA, the isolation of which was carried out after centrifugation at room temperature + (15-25) ° C at 0 shelf life without freezing and with 1 freeze / thaw cycle from blood samples in test tubes with anticoagulant, cell membrane fixative and gel according to the present invention, in comparison with tubes with EDTA without / with separation gel and specialized tubes for stabilization of extracellular nucleic acids RNA Complete BCT® manufactured by Streck (USA) with anticoagulant and cell membrane fixative without gel without freezing cycles are shown in Table 10.

As a result of the testing of one of the embodiments of the Medium according to the main aspect of the invention, containing a stabilizing solution and a separating thixotropic gel in the Device for collecting, storing and transporting biological samples in the concentration ranges indicated in table 1, geometric averages were calculated for the values of 1.933^ΔCt ¹ and 1.933^ΔCt ² obtained from the analysis of 5 blood samples from healthy donors (Table 10), thus confirming the higher concentrations of extracellular RNA found in RNA isolation after centrifugation without freezing and with 1 freeze/thaw cycle of the blood sample in test tubes according to the invention compared to:

- tubes with EDTA without gel - an increase of an average of 464 and 1013 times, respectively;

- test tubes with EDTA with separating gel - an average increase of 567 and 1317 times, respectively;

- RNA Complete BCT® tubes manufactured by Streck (USA) with an anticoagulant and a cell membrane fixative without gel - an average increase of 36 and 76 times, respectively.

Brief description of the figures

Fig. 1. General view of the Device containing the Medium for collecting, storing and transporting biological samples containing a separating gel and a stabilizing solution containing at least 1 cell membrane fixing agent.

Fig. 2. Photographs of test tubes with the Medium according to the main aspect of the invention and whole blood samples on the 0th day of storage: tube S11 - after centrifugation at room temperature +(15-25)°C for 15 minutes at 3000g; tubes S12 and S13 - without centrifugation.

Fig. Fig. 3. Examples of electrophoregrams obtained using the TapeStation 2200 automatic capillary electrophoresis system for extracellular DNA samples on days 0 (A) and 5 (B) of storage at room temperature +(15-25)°C after centrifugation immediately before plasma sampling (tubes S11 and S12, respectively).

Fig. Fig. 4. Examples of electrophoregrams obtained using the TapeStation 2200 automatic capillary electrophoresis system for extracellular DNA samples on days 10 (A) and 15 (B) of storage at room temperature +(15-25)°C after centrifugation immediately before plasma sampling (tubes S13 and S14, respectively).

Fig. Fig. 5. Results of a comparative analysis of the safety of extracellular DNA for samples that were stored without prior centrifugation for 0, 5, 10, and 15 days at room temperature +(15-25)°C and that were subjected to centrifugation immediately before plasma sampling (tubes S11 , S12, S13 and S14, respectively).

List of sources used

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Claims (41)

1. Environment for collecting, storing and transporting biological samples, - where the biological sample containing the extracellular nucleic acids to be preserved is blood or plasma; - where extracellular nucleic acids are extracellular DNA or RNA; containing separating gel, - where a thixotropic gel is used as a release gel; - where the thixotropic gel has a density in the range from 1.025 to 1.065 and is chemically inert with respect to biological samples; - where the thixotropic gel has sufficient viscosity so that under centrifugal forces in the range of 2000 to 3600 g it will flow and form a barrier between plasma and blood cells; and a stabilizing solution containing at least 1 cell membrane fixing agent, - where 2-bromo-2-nitropropane-1,3-diol, dimethylol urea, sodium hydroxymethylglycinate, diazolidinyl urea, dimethyl-dimethylol-hydantoin, imidazolidinyl urea, 1,3,5 -tris(hydroxyethyl)-s-triazine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, dimethylol-5,5-dimethylhydantoin or combinations thereof; - where the fixing agent releases free aldehyde and has the properties of a preservative, and at least 1 anticoagulant, - where ethylene glycol tetraacetic acid (EGTA) and its salts, sodium citrate, ethylenediaminetetraacetic acid (EDTA) and its salts, sodium fluoride or combinations of them are used as an anticoagulant, which is part of the stabilizing solution; - where the anticoagulant can function as an ionic stabilizing and/or chelating agent. 2. Medium according to claim 1, wherein the content of the fixing agent can advantageously be from 0.2 to 63.4% by weight of the entire composition. 3. Medium according to claim 1, wherein the content of the anticoagulant can advantageously be from 0.1 to 28.3% by weight of the total composition. 4. The medium of claim 1, wherein the stabilizing solution may optionally include a quenching agent in an amount sufficient to react with any free aldehyde that may be present before or after sampling and which is formed from the fixing agent to form a reaction product that does not will have a denaturing effect on proteins in biological samples, while the content of the quenching agent can mainly be from 0 to 7.8% by weight of the entire composition, - where as a quenching agent can be used a compound that includes at least one functional group capable of reacting with an electron-deficient functional group of the aldehyde; - where ethylenediamine, aminoacetic acid, lysine, arginine, adenine, guanine, cytosine, thymine, tryptophan, tyrosine, phenylalanine, ornithine, S-adenosylmethionine, alanine, arginine, cysteine, glutamic acid, aspartic acid, histidine can be used as a quenching agent , leucine, lysine, proline, serine, threonine, homocysteine, nicotinamide, or combinations thereof. 5. Medium according to claim 1, where the composition of the stabilizing solution may optionally include additional non-ionic stabilizing agents at a concentration of 0 to 18.5% by weight of the entire composition, which dissolve in an aqueous solution without the formation of ions and can be selected from the group of polyols ( polyols), such as sucrose, lactose, trehalose, melibiose, mannitol, or combinations thereof. 6. The medium according to claim 1, where the composition of the stabilizing solution may optionally include inhibitors of enzymes - nucleases and / or proteases, including inhibitors of metabolic enzymes and inhibitors of phosphatase, which prevent the degradation of nucleic acids and ensure their stabilization, as well as inhibitors of apoptosis, including caspase inhibitors, including in combination with N,N-dialkylpropanamide, such as N,N-dimethylpropanamide (DMPA), or butanamide, which prevent cell apoptosis in a caspase-dependent pathway, and transcription inhibitors, which are designed to maintain a basic level of RNA expression at the time of blood sampling by inhibiting the synthesis of new RNA transcripts, including mRNA, miRNA, IncRNA, piRNA, YRNA, circRNA and other ncRNA: - enzyme inhibitors can be selected from the following groups of compounds: diethyl pyrocarbonate, ethanol, auryl tricarboxylic acid (ATA), glyceraldehyde, formamide, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphine hydrochloride or combinations thereof, but not limited to; - inhibitors of metabolic enzymes can be selected from the following groups of compounds: glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde-3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate and glycerate dihydroxyacetate, potassium oxalate or combinations of them, but not limited to this; - protease inhibitors can be selected from the following groups of compounds: antipain, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TRCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid (IAA), E64, pepstatin, 1,10-phenanthroline, phosphoramodone, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, pancreatic protease inhibitor, ovostatin, cystatin, protease inhibitor, or combinations thereof; - phosphatase inhibitors can be selected from the following groups of compounds: caliculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic (okadaic) acid, cantharidin, fostriyetsin, endotall, cyclosporin A, FK 506/immunophilin complexes, cypermethrin, deltamethrin, fenvalerate, defostatin, mpV (pic) DMHV, sodium orthovanadate, or combinations thereof, but not limited to; - the content of inhibitors of enzymes - nucleases and/or proteases, including inhibitors of metabolic enzymes and phosphatase inhibitors, can mainly range from 0 to 25.8% by weight of the entire composition. 7. Medium according to claim 1, wherein the stabilizing solution may optionally contain, at a concentration of 0 to 5.4%, one or more cell permeability enhancing agents, which may be selected from the following compounds or combinations thereof: DMSO (dimethyl sulfoxide), ethylene glycol, glycerin, cellosolves, ethylene glycol dimethyl ether, phenoxyethanol, Triton X 100, Triton X 705 (non-ionic detergent), 1-methyl-2-pyrrolidinone, Tween 20, Tween 40 (non-ionic detergent), Brij 35 (non-ionic detergent), polyoxyethylene ether (Polyox), monensin, monactin, pentachlorophenol, 2,4-dinitrophenol, saponin, SDS (sodium dodecyl sulfate). 8. Medium according to claim 1, where the stabilizing solution may optionally include the following substances or combinations thereof at a concentration of 0 to 6.2%: - proteins such as: biotin, albumins: egg, bovine, including bovine serum albumin - BSA, gelatin and similar compounds; additional nucleic acid stabilizers such as: guanidine hydrochloride, polycations such as polyethyleneimine and the like; - antioxidants/reducing agents such as Trolox, α-tocopherol, nicotinamide and the like; - nucleic acid dyes such as: DAPI (diamidino-2-phenylindole), propidium iodide, fluorescein diacetate and the like; - antibiotics and other additional components, such as: doxycycline, sulfasalazine, curcumin, 6-aminocaproic acid, minocycline, chitosan, phytic acid, β-sitosterol, c-AMP, polylysine, biochanin A, demeclocycline, chlortetracycline, oxytetracycline, cyclohexamide, rifampicin , soy milk, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, 2-aminoethylbenzylsulfonyl fluoride (AEBSF), including but not limited to. 9. Medium according to claim 1, where the pH of the stabilizing solution can be, depending on the combination of its constituent agents and additional components, from 4 to 10, preferably from 6 to 8. 10. A device for collecting, storing and transporting biological samples containing the medium according to claim 1, which is a test tube with a medium according to claim 1, and a stabilizing solution containing a cell membrane fixing agent, which is part of the medium according to claim 1, is located above the thixotropic gel and/or said thixotropic gel is located below said stabilizing solution. 11. The device according to claim 10, which is vacuum tubes, including sterile ones, which may contain a vacuum intended for aspiration of a biological sample, while vacuum tubes can preferably have a nominal capacity of 9.0 ml, 8.5 ml, 8, 0 ml, 7.5 ml, 7.0 ml (size 16×100 mm), 6.0 ml, 5.5 ml, 5.0 ml (size 13×100 mm), 2.0 ml, 2.5 ml, 3.0 ml, 3.5 ml, 4.0 ml, 4.5 ml (13×75 mm), but not limited to. 12. The device according to claim 10, wherein the content of the release gel is preferably from 0.3 to 2.5 g per tube, but not limited to this. 13. Use of the device according to claim 10, containing the medium according to claim 1, for stabilizing extracellular nucleic acids for up to 15 days at a temperature of -20 to +37°C and up to 12 months at a temperature of -86 to -20°C. 14. The use according to claim 13, where the application is carried out through the interaction of the biological sample located in the device according to claim 10, with the cell membrane fixing agent that is part of the stabilizing medium solution according to claim 1, and the separation gel, which, after centrifugation for 10-30 min at 2000-3600g creates a separating barrier between the supernatant and cells, mainly plasma and blood cells, making the nucleic acids in the plasma sample suitable for isolation and subsequent analysis, including, but not limited to, the use of PCR and sequencing. 15. Use according to claim 13, where the use includes taking a biological sample with the possibility of storage and transportation without centrifugation up to 24 hours after taking at a temperature of +4 to +37 ° C, followed by centrifugation and then storage and transportation from 5 to 15 days at a temperature of -20 to +37°C with the possibility of repeated freezing / thawing of the sample after centrifugation with storage at a temperature of -86 to -20°C for up to 12 months. 16. Use according to claim 13, where the use includes the possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking at a temperature of +4 to +37 ° C and centrifugation immediately before the selection of the supernatant, mainly plasma, to remove cells , extracellular structures and cellular debris to obtain cell-free plasma. 17. Use according to claim 13, where the use includes the possibility of storing and transporting a biological sample without centrifugation from 5 to 15 days after taking at a temperature of +4 to +37 ° C while preserving extracellular nucleic acids, as well as circulating cells and / or extracellular structures if necessary.

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