KR20250020999A - Composition for the preservation of blood cells - Google Patents

Abstract

The present invention relates to a composition for fixing components in a blood sample, and according to an embodiment of the present invention, a composition for a vacuum blood collection tube can be provided, which can preserve a sample such as blood for a long time even at room temperature, while stabilizing minute components such as nucleic acids inside the sample even when exposed to external environmental shocks, and at the same time can be implemented at a realistically economical price.

Description

Composition for the preservation of blood cells

The present invention relates to a composition capable of preserving nucleic acids in a blood sample for a long period of time, and to the development of a composition that complements problems that may arise during blood delivery by preserving nucleic acids in blood while it is being transported from a hospital or clinic where blood is collected to a laboratory where testing is performed.

Liquid biopsy, which has recently emerged as a new topic for early cancer diagnosis, analyzes patients' genetic information using next-generation sequencing, and is accelerating the growth of the diagnostic field in that it enables monitoring of daily information.

'Tissue biopsy', which is currently used as a standard method for diagnosing cancer, is very invasive and causes inconvenience to patients, including pain, cost, and time.

On the other hand, Liquid Biopsy replaces liquid samples such as blood, saliva, urine, and cell washing fluid, and is a technology that separates circulating tumor cells (ctDNA) or circulating tumor cells (CTCs), exosomes, etc. present in them and analyzes various nucleic acid information inside. It is a genetic test that applies the latest technology that can detect residual disease and treatment resistance at an early stage by analyzing tumor components (circulating tumor cells or circulating tumor DNA, cell-free DNA) inside body fluids.

Since these liquid biopsies are performed only through specimen collection by medical personnel, long-term transport is inevitably performed due to the physical distance difference between the specimen (blood and urine) collection and the testing institution, the laboratory. The medium required for such long-term transport is a vacuum blood collection tube, which has a preservative inside to preserve the sample, so that the sample it receives can be stably fixed even during long-term transport, and until the test is performed, the characteristic of stabilizing the internal sample against external environments such as shock or thermal environment during transport, such as preservation of nucleic acid contained inside, is a very important factor.

However, in the case of existing vacuum blood collection tubes, many products depend on overseas imports as they are very expensive products, and they have poor preservation function that occurs during long-term transport of samples compared to the high cost, revealing limitations in terms of practicality in terms of economic feasibility.

Accordingly, there is a growing need for the popularization of vacuum blood collection tubes that can preserve samples such as blood for long periods of time even at room temperature, while stabilizing trace elements such as nucleic acids within the sample against external environmental shocks, and at a realistically economical price.

Korean Patent Publication No. 2021-0069505 Korean Patent No. 1965169

The present invention has been made to solve the above-described problems, and the purpose of the present invention is to provide a composition for a vacuum blood collection tube, which can preserve a sample such as blood for a long period of time even at room temperature, while stabilizing minute components such as nucleic acids inside the sample even when subjected to external environmental shocks, and at the same time can be implemented at a realistically economical price.

As a means for solving the above-described problem, in an embodiment of the present invention, the mixing ratio of an antibiotic and an anticoagulant is formed as 1:(1.5 to 7), and the ratio of an enzyme inhibitor is 20 to 30 wt% based on 100 parts by weight of the entire composition, and the enzyme inhibitor enables providing a composition for fixing nucleic acid in a blood sample including an RNA hydrolase inhibitor and a metabolic inhibitor.

In addition, in an embodiment of the present invention, it is possible to provide a vacuum blood collection tube including a composition that improves the preservation of blood components.

According to an embodiment of the present invention, a composition for preserving a sample such as blood for a long period of time even at room temperature is provided, and in particular, stabilization of minute components such as nucleic acids inside a blood sample can be realized even against external environmental shocks, and at the same time, a composition for a vacuum blood collection tube can be provided that can be realized at a realistically economical price.

In addition, according to an embodiment of the present invention, the composition for fixing nucleic acids in blood of the present invention can be stably implemented for a longer period of time, and a specimen treated with the composition of the present invention can minimize genomic DNA contamination due to cell lysis in blood even when the specimen is subjected to movement and shaking (e.g., external impact occurring during delivery, environmental influences).

FIG. 1 is a drawing illustrating a process of using a vacuum blood collection tube using a composition for fixing components in a blood sample according to an embodiment of the present invention.
Figure 2 illustrates an image of a commercialized vacuum blood collection tube example and an image of the inspection equipment used in an experimental example of the present invention.
Figures 3 and 4 illustrate the results of an experiment in which cell-free nucleic acids were extracted from blood mixed with the composition (NanoGrab) of the present invention and stored at room temperature for 0, 7, and 14 days.
Figures 5 and 6 illustrate the results of an experiment conducted in which a composition of an embodiment of the present invention was mixed with blood and a shaker (orbital shaker) was operated at a speed of 350 rpm or level 3 for 1 to 2 hours a day to apply physical shock to the blood for 0, 7, and 10 days.

The advantages and features of the present invention, and the method for achieving them, will become clear with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided so that the disclosed contents can be thorough and complete and so that the spirit of the present invention can be sufficiently conveyed to those skilled in the art.

The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprises" or "has" and the like are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms defined in commonly used dictionaries, such as those defined in common dictionaries, should be interpreted as having a meaning consistent with the meaning they have in the context of the relevant art, and shall not be interpreted in an idealized or overly formal sense unless expressly defined in this application.

A composition for fixing a component in a blood sample according to an embodiment of the present invention (hereinafter referred to as the "present invention") is formed in a mixing ratio of an antibiotic and an anticoagulant of 1:(1.5 to 7), and a ratio of an enzyme inhibitor of 20 to 30 wt% based on 100 parts by weight of the composition, wherein the enzyme inhibitor includes an RNA hydrolase inhibitor and a metabolic inhibitor, so as to provide a composition for fixing a component in a blood sample. In particular, in a preferred embodiment of the present invention, the component in the blood sample will be described using "nucleic acid" as an example.

Specifically, the composition of the present invention can be implemented with 15 to 21 wt% of an anticoagulant, 40 to 50 wt% of a formaldehyde releaser, 20 to 30 wt% of an enzyme inhibitor, and 3 to 10 wt% of an antibiotic, based on 100 wt% of the total composition.

In this case, the antibiotic may be one or a combination of two or more selected from among chloramphenicol, 5 bromo-5 nitro 1,3 dioxane, tetradecyltrimethylammonium bromide, and sodium azide. In a preferred embodiment of the present invention, chloramphenicol may be applied.

In addition, the anticoagulant in the present invention may be applied by including at least one chelating agent selected from sodium citrate, sodium borate, sodium fluoride, sodium fluoride, and EDTA. In a preferred embodiment of the present invention, it may be composed by applying a citrate-phosphate-dextrose solution with adenine (CPDA) and K ₂ EDTA.

In addition, the RNA hydrolase inhibitor in the present invention may be configured to include Dihydroxyacetone (DHA) and Dithiothreitol (DTT) mixed with 5,5'-dithiobis (2-nitrobenzoic acid). In this case, preferably, the RNA hydrolase inhibitor may be applied in an amount of 20 to 30 wt% based on 100 wt% of the total composition, or 10 to 200 g/L of the total composition.

In addition, the composition of the present invention may further comprise one or a mixture thereof of formaldehyde release control agents, namely Sodium hydroxymethylamino acetate (Sodium hydroxymethyl glycinate), Tris(hydroxymethyl) nitromethane, DMDM hydantoin (Glydant), and Benzylhemiformal.

In the case of the composition according to the present invention described above, it can be used in a manner included inside a vacuum blood collection tube.

In this case, a vacuum blood collection tube containing a composition according to the present invention can be used in the following manner.

That is, it can be used in a process of a first step of mixing a blood sample and a composition for fixing a biological component in the blood sample by contacting them in a vacuum blood collection tube containing the composition according to an embodiment of the present invention, a second step of centrifuging the vacuum blood collection tube containing the mixture of the blood sample and the composition using a centrifuge to separate plasma containing one or more target nucleic acids, and a third step of analyzing the characteristics of the target nucleic acids.

In this case, the second step may be used as a step of isolating plasma containing one or more target nucleic acids for further analysis by centrifuging the mixture at a speed of 1000 g or more and 7500 g or less for about 5 minutes or more.

In addition, the second step may be configured as a step in which an inert gas is filled in the space inside the vacuum blood collection tube in addition to the composition, and the oxygen concentration of the mixture of the blood sample and the composition is reduced to 10% or less of the oxygen level of the initial blood sample.

Hereinafter, each component and method of use for an example of applying a composition for fixing components in a blood sample according to an embodiment of the present invention will be specifically described.

The composition for fixing a component in a sample according to an embodiment of the present invention may be included in a vacuum blood collection tube and used for the purpose of preserving nucleic acids. That is, the biological sample accommodated in a vacuum blood collection tube including the composition of the present invention may be a biological fluid.

Specifically, the sample described in the present invention may be a biological fluid to which a method for preserving nucleic acids is applied, and the biological fluid may be any one of a substrate, plasma, serum, urine, saliva, stool, milk, tear, sweat, cerebrospinal fluid, saturated fat, semen, vaginal secretion, ascites fluid, and amniotic fluid. In another preferred embodiment, the biological fluid may be whole blood. In this case, nucleic acid cells contained in the whole blood may be applied as RNA, cell-free DNA, or a combination thereof as the biological fluid. Cell-free DNA may be isolated from whole blood, and specifically, cell-free cell-free DNA is separated and applied as cell-free cell-free plasma DNA.

In an embodiment of the present invention, the combination of components according to the composition according to the present invention for preserving nucleic acids (cell free nucleic acids) from a biological sample has been found to enable preservation of cell free nucleic acids from a biological sample, such as cfDNA of plasma, at room temperature for at least 28 days, which allows for a longer period of storage of blood at room temperature compared to commercially available products such as AMPULAB™ from SOYA greentec Inc., (Republic of Korea).

According to the present invention, it was confirmed that the composition for fixing nucleic acids in blood can be stably implemented for a longer period of time, and that the specimen treated with the composition of the present invention can minimize genomic DNA contamination due to cell lysis in blood even when the specimen is subjected to movement and shaking (e.g., external impact occurring during delivery, environmental influences).

In the case of the composition according to the present invention, the composition (preservative composition) exhibits the function of reducing cell lysis and stably fixing nucleic acids, and can also be used to preserve all cells in a body fluid, such as circulating tumor cells. Compared to conventional commercial products, when a vacuum blood collection tube is implemented with the composition of the present invention, stable preservation for about 14 days is possible, so the preservation effect is excellent.

This means that the composition according to the present invention further comprises a metabolic antagonist for reducing cellular metabolism and cellular respiration within a cell, thereby preventing cellular metabolism of the sample, thereby further reducing degradation of cell-free nucleic acids and lysis of cells in the sample.

As described above, the present invention is a composition for fixing components in a blood sample (hereinafter referred to as the “present invention”), which is implemented as a material capable of preserving a sample such as blood for a long period of time even at room temperature, while stabilizing minute components such as nucleic acids inside the sample even against external environmental shocks.

That is, the present invention includes an anticoagulant, an antibiotic, and an enzyme inhibitor to implement preservation stability by contacting a sample, such as a body fluid sample (e.g., a blood component containing nucleic acids), and in this case, the enzyme inhibitor may be configured to include an RNA hydrolase inhibitor and a metabolic inhibitor. Hereinafter, examples of materials constituting the composition of the present invention will be described.

1. Antibiotics

The antibiotic of the present invention may be applied as one or a combination of two or more substances selected from chloramphenicol, 5 bromo-5 nitro 1,3 dioxane, tetradecyltrimethylammonium bromide, and sodium azide. In one embodiment of the present invention, chloramphenicol may be applied as the antibiotic. Chloramphenicol, in addition to its antibiotic function, may act as a metabolism inhibitor, and may be included in an amount of 3 to 10 wt% of the total composition.

2. Anticoagulants

The present invention comprises an anticoagulant having anticoagulant properties, which may be composed of one or more chelating agents selected from sodium citrate, sodium borate, sodium fluoride, and EDTA. In this case, the anticoagulant may be included in an amount of 15 to 21 wt% based on 100 wt% of the total composition.

Chelating agent is a compound that chelates ions in the cell membrane, weakens the cell membrane, and makes the cell more susceptible to biocides. Therefore, the chelating agent assists the antimicrobial compound in killing bacteria and fungi. The chelating agent can also maintain the pH of the composition according to the present invention together with the buffer. That is, when the cell sample as a sample is a blood sample, the chelating agent of the present invention reduces platelet aggregation and maintains anticoagulation of the blood.

Such chelating agents may include triacetate, tetraacetate or pentaacetate substituents. The chelating agent may be EDTA, an EDTA derivative (e.g., sodium EDTA), or a combination thereof. As an EDTA derivative, an EDTA sodium salt may be included (e.g., a sodium salt and/or a potassium salt of EDTA).

The above chelating agent can be selected from EDTA disodium, EDTA4 sodium, EDTA2 potassium, EDTA4 potassium, ethylene glycol-bis-(3-amino ethyl ether) N-N-4 acetic acid, and ethylene glycol-bis-(2-amino ethyl ether) N, N, N', N'-4 acetic acid (EGTA) or their disodium salts (EGTA disodium). EDTA disodium exhibits a pH lower than pH7 when dissolved in water. EDTA4 sodium exhibits a pH higher than pH7 when dissolved in water. The pH of 5% EDTA disodium dissolved in water at 25°C can be within pH 4 to 6. EDTA disodium can be used in anhydrous form or in hydrate form (e.g., EDTA disodium dihydrate). The pH of 1% EDTA4 sodium in water at 25℃ can be within pH 10.7 to 11.7. EDTA4 sodium can be used in anhydrous form or in hydrate form (e.g., EDTA4 sodium dihydrate).

In a preferred embodiment of the present invention, K ₂ EDTA can be applied as the chelating agent, and Citrate-phosphate-dextrose solution with adenine (CPDA) can be applied as a solvent.

The chelating agent as an anticoagulant applied in the present invention can simultaneously implement an anticoagulant function as described above, and can simultaneously implement a function of preserving nucleic acids by suppressing a substance that implements nucleic acid decomposition. That is, for example, such a chelating agent includes a chelating agent such as sodium citrate, sodium borate, sodium fluoride, and EDTA. Such a chelating agent binds trace metals necessary for the protein decomposition activity of a protein hydrolyzing agent in a sample. In this case, the chelating agent and the metal necessary for the protein decomposition activity are bound, so that the protein hydrolyzing agent cannot decompose the cell wall, and thus the cell wall is maintained in its original state. As a result, the original form of the nucleic acid molecule in the cell wall is not exposed to a component that decomposes the nucleic acid molecule, and can be maintained intact.

3. Enzyme inhibitors

The present invention may include an inhibitor of RNA hydrolysis enzyme as a component of the composition, and in a preferred embodiment, 5,5'-dithiobis (2-nitrobenzoic acid) may be applied. In addition, it may include a metabolic inhibitor, and in a preferred embodiment, any one selected from Dihydroxyacetone (DHA), Dithiothreitol (DTT), and 5,5'-dithiobis (2-nitrobenzoic acid) or a combination thereof may be applied.

The composition according to the present invention further comprises an enzyme inhibitor that acts to inhibit nucleases. The enzyme inhibitors may include a chelate for inhibiting metal dependent nucleases, and the other component inhibits non-metal dependent nucleases. Examples of suitable enzyme inhibitors include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), HEDTA, citrate, oxalate, aurintricarboxylic acid (ata), dithiothreitol (DTT) and any combination thereof.

Preferably, the enzyme inhibitor may be composed of Dihydroxyacetone (DHA), Dithiothreitol (DTT) mixed with 5,5′-dithiobis (2-nitrobenzoic acid).

Alternatively, as another embodiment, the enzyme inhibitor may be a citrate salt, such as sodium citrate or potassium citrate. The enzyme inhibitor of the present invention is preferably included in an amount of 20 to 30 wt% based on 100 wt% of the total composition.

Enzyme inhibitors that act to inhibit nucleases preferably further comprise at least one stabilizing agent at a concentration capable of stabilizing nucleic acids in a biological sample, particularly a whole blood sample. Typically, the gene induction inhibitor is an aqueous solution of one stabilizing agent or a mixture of multiple stabilizing agents. It is preferred that the stabilizing agent effectively stabilizes DNA and RNA, including mRNA, tRNA, snRNA, low molecular weight (LMW) RNA, rRNA, and cRNA, so as to inhibit or suppress ex vivo gene induction in a biological sample during storage at room temperature. Examples of suitable stabilizing agents for stabilizing and protecting nucleic acids include cationic compounds, detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, quaternary amines, and mixtures thereof. A suitable ribonuclease inhibitor is placental ribonuclease inhibitor protein. Examples of chaotropic salts include urea, formaldehyde, guanidinium isothiocyanate, guanidinium hydrochloride, formamide, dimethyl sulfoxide, ethylene glycol, and tetrafluoroacetate. In another embodiment, the gene induction inhibitor is an organic solvent or an organic reducing agent. Examples of suitable organic solvents are selected from the group consisting of phenol, chloroform, acetone, and alcohols. The alcohols are typically lower alcohols. Examples of organic reducing agents can be selected from the group consisting of mercapto alcohol, dithiothreitol (DTT), and mixtures thereof.

The stabilizer may also optionally include other components that process the biological sample. For example, it is desirable for the stabilizer to lyse reticulated red blood cells, bacteria, red blood cells, and white blood cells, since it renders the cells in the biological sample membrane-permeable. Such other components include proteases, phenols, phenol/chloroform mixtures, alcohols, aldehydes, ketones, and organic acids.

In another preferred embodiment of the present invention, the stabilizer is a cationic compound and comprises a proton donor in an amount effective to stabilize nucleic acids. The addition of a proton donor to the cationic compound enhances the ability of the cationic compound to stabilize nucleic acids in a biological sample. Examples of such suitable proton donors include monocarboxylic acids, dicarboxylic acids, tricarboxylic acids, aliphatic keto dicarboxylic acids, amino acids, mineral acids, and mixtures thereof. In one embodiment, the proton donor is selected from the group consisting of an alkenyl carboxylic acid, a C ₁ -C ₆ aliphatic monocarboxylic acid, an aliphatic C ₂ -C ₆ dicarboxylic acid, a tricarboxylic acid, a hydroxy monocarboxylic acid, a hydroxy dicarboxylic acid, a hydroxy tricarboxylic acid, an aliphatic keto monocarboxylic acid, an aliphatic keto dicarboxylic acid, an amino acid, and mixtures thereof. Examples of suitable aliphatic carboxylic acids include C 1 to C 6 alkyl carboxylic acids, such as acetic acid, propionic acid, n-butanoic acid, n-pentanoic acid, isopentanoic acid, 2-methylbutanoic acid, 2,2-dimethylpropionic acid, _n -hexanoic acid, n-octanoic acid, n-decanoic acid, _and dodecanoic acid. Examples of alkenyl carboxylic acids include acrylic acid, methacrylic acid, butyric acid, isobutenoic acid, and mixtures thereof.

In a composition comprising an “enzyme inhibitor” according to the present invention, the enzyme inhibitor can be applied to form a complex with a metal ion.

For example, to reduce the clotting properties of blood, calcium, magnesium, manganese or zinc inhibit nucleases and/or reduce enzymatic cell lysis.

Accordingly, as described above, enzyme inhibitors in the present invention include, but are not limited to, examples of ethylenediaminetetraacetic acid (eta), hydroxyethylethylenediaminetriacetic acid (HEDTA), dithiothreitol (DTT), ethylene glycol bis (β-aminoethyl ether) -n, N, N′, N′-tetraacetic acid (EGTA), citric acid, oxalate, aurintricarboxylic acid (ata), tartaric acid, glucaric acid, or any salt thereof.

Additionally, inhibition of nucleases can prevent or reduce degradation of cell-free nucleic acids within a biological sample. Examples of enzymes inhibited by the enzyme inhibitors herein include lysostaphin, zymolase, protease, glycanase, and the like, which act to preserve cells in a blood or other biological sample.

4. Optional Inclusion Substance_Platelet Inhibitor

The present invention relates to an inhibitor system used to prolong the preservation of human platelets, wherein the inhibitor system can inhibit platelet activity during storage, but allows platelet activity to resume after removal from the platelets, and may further comprise amiloride, sodium nitroprusside, adenosine, dipyridamole, quinacrine, ticlopidine and heparin.

5. Optional Inclusions_Blood Protein Preservatives

The present invention may further comprise at least one protective agent selected from the group consisting of albumin, triglyceride, glycerol, dextran, propylene glycol, galactose, alginate and trehalose as a preservative for protecting serum proteins during storage.

6. Preservative of the base sequence of nucleic acids in the sample

The present invention further includes an acid-removed formaldehyde solution, which is prepared by treating a concentrated formaldehyde solution with a basic ion-exchange resin to preserve the base sequence of nucleic acids in a blood sample accommodated in a vacuum blood collection tube, and diluting the acid-removed formaldehyde solution thus produced with a phosphate buffer having a pH of 7.2 to 7.4 to a concentration in the range of 2 to 4%. The acid-removed formaldehyde diluted solution used in the present invention can be applied as a buffered solution containing a detergent that dissolves cell membranes. In this case, the detergent can be composed of an anionic detergent such as sodium dodecyl sulfate (SDS) and lithium lauryl sulfate (LLS). More specifically, such detergent can selectively adopt an anionic detergent, a cationic detergent, or a nonionic detergent. The anionic detergent can be applied, for example, to a dodecanedioic acid plant. Nonionic detergents can be applied as ethylene oxide condensation products, such as ethoxylated fatty acid esters of polyhydric alcohols, for example. Preferred nonionic detergents are polyoxyethylene sorbitan monolaurate or dodecanedioic acid plant. The detergent is included in an amount effective to lyse cells. The detergent can also form micelles and other complexes with nucleic acids and protect RNA and/or DNA by other mechanisms.

7. Composition and use of vacuum blood collection tube

The composition according to the present invention is accommodated in a vacuum-treated blood collection tube, and after contacting a biological sample (e.g., whole blood), is transferred, and plasma containing a target nucleic acid is separated through centrifugation and tested. The target nucleic acid may be DNA or RNA or a combination thereof.

The method of using the vacuum blood collection tube described above can be carried out in the following order.

Specifically, the method comprises the steps of: 1) contacting and mixing a blood sample and a composition for fixing a biological component in the blood sample into a vacuum blood collection tube; 2) centrifuging the vacuum blood collection tube containing the mixture of the blood sample and the composition through a centrifuge and separating plasma containing one or more target nucleic acids; and 3) analyzing the characteristics of the target nucleic acids. By using the vacuum blood collection tube equipped with the composition for fixing a component in a blood sample of the present invention as described above, information on target nucleic acids can be derived.

In this case, the second step may consist of a step of centrifuging the mixture at a speed of 1000 g or more and 7500 g or less for about 5 minutes or more to separate plasma containing one or more target nucleic acids for further analysis.

Additionally, an inert gas may be filled in the space inside the vacuum blood collection tube in addition to the composition, in which case the oxygen concentration of the mixture of the blood sample and the composition may be reduced to 10% or less of the oxygen level of the initial blood sample.

8. Optional Buffer

The composition of the present invention may further comprise one or more buffers. Examples of such additional buffers may optionally include tris(hydroxymethyl)aminomethane ( Tris; tris base, 2-amino-2-(hydroxymethyl)-1,3-propanediol, THAM, tromethamol) or (e. g. tris(hydroxymethyl)aminomethane hydrochloride or Tris-HCl ), Trizma® base (e. g. 40% (w/w) stock solution of Tris in water ), HEPES, BES, MOPS, HEPES, TAE, TBE, phosphate buffer, sodium borate buffer, sodium cacodylate buffer.

9. Red Blood Cell Preservation Solution

The composition of the present invention may further comprise one or more components for preserving red blood cells. The components for preserving red blood cells may comprise at least one component selected from the group consisting of glutathione, glutamine, cacodylates, barbituric acid, barbiturates, and additional buffers.

[Experimental example]

Below, the results of comparing the preservation performance of a vacuum blood collection tube using a composition according to an embodiment of the present invention and a commercially available blood collection tube will be described.

In the case of a vacuum blood collection tube using a composition according to an embodiment of the present invention, it is used in the same process as illustrated in Fig. 1. First, a blood sample is collected, placed into a blood collection tube, and the blood and composition are mixed (step 1). Next, after the transport process, the blood is centrifuged and the process of removing plasma and impurities is performed (step 2).

Thereafter, cell-free nucleic acid extraction and analysis are performed on the centrifuged sample, and in this case, the analysis indices are cell-free nucleic acid expression score, cell-free nucleic acid concentration, and cell-free nucleic acid peak results.

In the experiments for the following examples of the present invention, the preservation power is read according to the time of introduction into the sample and the preservation process.

To this end, as shown in Fig. 2, a composition made as an example of the present invention was mixed with blood collected from a hospital or clinic, and then left at room temperature for 0 to 14 days, after which cell-free nucleic acids were extracted using commercially available kits.

Thereafter, in order to verify whether the cell-free nucleic acids were well preserved, a quality test was performed based on the concentration of cell-free nucleic acids, expression pattern, and cell-free nucleic acid score (%) calculated by the software in the device using the 4150 or 4200 TapeStation System device of Agilent (California, USA) as shown in Fig. 2. When the cell-free nucleic acid extraction results are confirmed using the 4150 or 4200 TapeStation System device of Agilent and the dedicated software, the concentration of nucleic acids between the sizes of cell-free nucleic acids (50 to 700 bp) and the percentage (%) of cell-free nucleic acids among the total nucleic acids are expressed as the cell-free nucleic acid score (%Cf-DNA), which is designed to allow the quality of cell-free nucleic acids to be checked at a glance.

According to Agilent, more than 80% of cell-free nucleic acids must be used based on 100% to proceed to the next step, Quantitative PCR (qPCR) or Next generation sequencing (NGS). Therefore, since the equipment enables qualitative and quantitative analysis of cell-free nucleic acids, it was used to verify the examples of the present invention.

In the case of the composition used in this experiment, when implementing the entire composition, 15 to 21 wt% of an anticoagulant, 40 to 50 wt% of a formaldehyde releaser, 20 to 30 wt% of an enzyme inhibitor, and 3 to 10 wt% of antibiotics were mixed based on 100 wt% of the entire composition.

In this case, the anticoagulant was K2EDTA, the antibiotic was chloramphenicol mixed in a ratio of 1:10, and the enzyme inhibitor was Dihydroxyacetone (DHA) and Dithiothreitol (DTT) mixed with 5,5′-dithiobis (2-nitrobenzoic acid), an inhibitor of RNA hydrolase. The formaldehyde release controlling agent was Sodium hydroxymethylamino acetate (Sodium hydroxymethyl glycinate).

The tube body for the vacuum blood collection tube used may be made of one or more of polypropylene, polystyrene, polycarbonate, and acrylonitrile resin. However, in the present invention, a ready-made product made of polypropylene was used, and an inert gas was filled inside.

As a negative control, K2EDTA, which is currently widely used as an anticoagulant vacuum blood collection tube, was used, and as a positive control, an experiment was conducted using a competing product (AMPULAB™ of SOYA greentec Inc., (Korea)), a vacuum blood collection tube dedicated for cell-free nucleic acids. The amount of composition in the vacuum blood collection tube was applied to all the tubes equally, containing 10 g/L.

Figures 3 and 4 show the results of an experiment in which cell-free nucleic acids were extracted from blood mixed with the composition (NanoGrab) of the present invention and then stored at room temperature for 0, 7, and 14 days. As a negative control group, K2EDTA, which is currently widely used as an anticoagulant vacuum blood collection tube, was used, and as a positive control group, a competitor's product, a dedicated vacuum blood collection tube for cell-free nucleic acids, was used for the experiment.

The blood used in the experiment was obtained from one person, and the control and experimental groups were tested under the same conditions. Cell-free nucleic acids were extracted using a commercial cell-free nucleic acid extraction kit, and the extracted cell-free nucleic acids were subjected to qualitative and quantitative analysis using Agilent's 4150 or 4200 TapeStation System equipment and software.

In the negative control group, the nucleic acids in the cell-free nucleic acid region (50–700 bp) gradually became faint and disappeared after 7 days of storage, whereas in the experimental group (NanoGrab), the cell-free nucleic acids were stably preserved for up to 14 days at room temperature. In addition, compared to the competitive product, which is the positive control group, the cell-free nucleic acid score (%Cf-DNA) was maintained high at over 80%, and it was also confirmed that the HMW DNA region, which is the genomic DNA region, did not increase and only the cell-free nucleic acid region was maintained for 14 days.

Figures 5 and 6 show the results of an experiment conducted by mixing a composition of an embodiment of the present invention with blood and then applying physical shock to the blood by operating an orbital shaker at a speed of 350 rpm or level 3 for 1 to 2 hours a day for 0, 7, and 10 days.

This experiment was conducted to confirm whether cell-free nucleic acids are well maintained despite the continuous physical shock applied to blood as it is continuously shaken while being transported in a vacuum blood collection tube from a hospital or clinic to a research institute.

Assuming that the longest transport time for vacuum blood collection tubes containing blood domestically or internationally was 10 days, the storage conditions were planned to be 0, 7, and 10 days. In preparation for transport by vehicle or airplane, the blood was stored in a shaker set to 350 rpm or level 3, similar to vehicle vibration, for 1 to 2 hours per day for 0 to 10 days.

The experimental group maintained cell-free nucleic acids better than the competitive product, which was the positive control group, despite continuous shaking of the blood in the vacuum blood collection tube for 10 days, and it was found that the cell-free nucleic acid score (%Cf-DNA), which is Agilent's standard, was maintained at 85% for 10 days.

As shown in the above results, it can be confirmed that the composition according to the embodiment of the present invention has excellent stabilization through fixation of nucleic acids in blood samples even in an environment where they are stored at room temperature and continuously subjected to physical impact.

The composition for fixing nucleic acids in blood of the present invention can be stably implemented for a longer period of time, and a sample treated with the composition of the present invention can minimize genomic DNA contamination due to cell lysis in blood even when the sample is subjected to movement and shaking (e.g., external impact occurring during delivery, environmental influences).

As described above, the technical idea of the present invention has been specifically described in preferred embodiments, but the above preferred embodiments are for the purpose of explanation and not for the purpose of limitation. In this way, a person skilled in the art will be able to understand that various embodiments are possible by combining the embodiments of the present invention within the scope of the technical idea of the present invention.

Claims (8)

In a composition for fixing nucleic acid in a blood sample,
Anticoagulants; including antibiotics and enzyme inhibitors;
The mixing ratio of the above antibiotic and anticoagulant is formed as 1: (1.5~7).
The above enzyme inhibitor includes an RNA hydrolase inhibitor and a metabolic inhibitor.
A composition for fixing components in a blood sample.
In claim 1,
The above composition,
Based on 100 parts by weight of the total composition, 15 to 21% by weight of anticoagulant, 40 to 50% by weight of formaldehyde releaser, 20 to 30% by weight of enzyme inhibitor, and 3 to 10% by weight of antibiotics.
A composition for fixing components in a blood sample.
The above antibiotics are,
Applying one or more substances selected from chloramphenicol, 5 bromo-5 nitro 1,3 dioxane, tetradecyltrimethylammonium bromide, and sodium azide.
A composition for fixing components in a blood sample.
In claim 2,
The above anticoagulants are,
Comprising at least one chelating agent selected from the group consisting of sodium citrate, sodium borate, sodium fluoride, sodium fluoride and EDTA.
A composition for fixing components in a blood sample.
In claim 4,
The above enzyme inhibitor is,
A composition comprising one or a mixture of two or more selected from Dihydroxyacetone (DHA) and Dithiothreitol (DTT) mixed with 5,5′-dithiobis (2-nitrobenzoic acid) as an inhibitor of the above RNA hydrolase.
A composition for fixing components in a blood sample.
In claim 4,
The above RNA hydrolytic enzyme inhibitor is applied in an amount of 10 to 200 g/L of the total composition.
A composition for fixing components in a blood sample.
In claim 6,
The above composition,
Further comprising one or a mixture of Sodium hydroxymethylamino acetate (Sodium hydroxymethyl glycinate), Tris(hydroxymethyl) nitromethane, DMDM hydantoin (Glydant), and Benzylhemiformal, which are formaldehyde emission control agents.
A composition for fixing components in a blood sample.
A vacuum blood collection tube comprising a composition according to any one of claims 1 to 7.