Compositions for hemolysis, coagulation regulation and stabilization of extracellular vesicles

Abstract

To provide compositions for hemolysis and coagulation regulation and stabilization of extracellular vesicles.SOLUTION: Provided are a composition and a method of use thereof, the composition comprising one or more components capable of releasing an aldehyde, one or more anticoagulants or chelating agents, and one or more polysaccharides. The composition has a pH of about 4 to about 6. The present teachings relate to a blood sample regulation composition. More particularly, the present teachings relate to a composition for regulating mechanisms in a blood sample so that components within the blood sample maintain sufficient integrity for downstream testing for a variety of diagnostic indicators.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 574,515, filed October 19, 2017, the disclosure of which is incorporated by reference in its entirety.

[0002] The present teachings relate to blood sample conditioning compositions. More specifically, the present teachings relate to compositions for conditioning the mechanisms of a blood sample such that components within the blood sample maintain sufficient integrity for downstream testing of various diagnostic indicators. [Background technology]

[0003] The advent of liquid biopsy (e.g., analysis of blood and other liquid samples from the body) has revolutionized prenatal medicine and cancer diagnosis. It is now possible to isolate and analyze circulating DNA (cfDNA or cell-free DNA) (e.g., fetal or cancer), cell-free RNA, and circulating tumor cells from a patient's blood sample. As analytical capabilities advance, the need for liquid biopsy assays is to maximize downstream results in terms of sensitivity and specificity, taking into account the associated preanalytical variables. One of the most important preanalytical variables associated with the collection of liquid biopsy samples is cellular and nucleic acid stabilization, especially given the significant lag time it takes for these samples to reach a reference laboratory. Whole blood samples are the most efficient patient sample for liquid biopsy studies, but are limited by multiple lag time factors. Most important among these is the fragility of leukocytes and erythrocytes / reticulocytes. Specifically, storage of blood results in the time-dependent release of genomic DNA from apoptotic leukocytes and cellular RNA from both deteriorating leukocytes and reticulocytes. In the case of PCR-type liquid biopsy assays, non-specific increases in cell-free RNA and DNA run the risk of diluting out target transcripts (e.g., mutated oncogene DNA, fetal DNA, etc.).

[0004] Although multiple commercial options exist for nucleic acid stabilization in whole blood, these are aimed at stabilizing either DNA or RNA, but not both simultaneously. Collection tubes specialized for stabilization of cell-free DNA include CELL-FREE DNABCT® (Streck), ccfDNA PAXgene (Qiagen), LBGard (Biomatrica), and Cell-Free DNA (Roche). These tubes are described as preventing the release of DNA from apoptotic leukocytes, and maintain plasma cell-free DNA levels at the time of aspiration up to 7–14 days after collection. Collection tubes for RNA analysis include PAXgene Blood RNA (Qiagen), RNAGard (Biomatrica), and Tempus Blood RNA (Thermo Sci.). A pitfall of available RNA tubes is the fact that they are based on complete lysis of the collected blood sample and, as a result, an overwhelming excess of erythrocyte-specific transcripts (e.g., globin α and β mRNA). Subsequent sample processing is therefore required to deplete globin and ribosomal RNA and enrich for the informative target mRNA pool. The only non-lysing RNA blood collection tube on the market is Streck's CELL-FREE RNABCT®. This tube requires a narrow temperature range and, importantly, is intended only to stabilize RNA. No blood collection tube has yet been reported that efficiently stabilizes both circulating RNA and DNA.

[0005] It would be desirable to provide a collection device and a composition disposed therein based on an anticoagulant formulation that maintains the mean cell volume (MCV) of red blood cells to prevent blood clotting and minimize hemolysis. The composition would preferably stabilize the white blood cell components, thereby preventing the release and excessive accumulation of DNA in the plasma. The composition would further act as an erythrocyte protector that maintains the structural integrity of the red blood cells and prevents the release of membrane vesicles as a function of storage time. This is important because circulating or cell-free RNA is encapsulated in membrane vesicles, including microvesicles and extracellular vesicles (including but not limited to exosomes). A desirable composition would maintain sample stability and integrity for a minimum of six days over a wide temperature range. Plasma concentrations of cfDNA and membrane vesicles, and therefore cfRNA, would ideally remain substantially at aspirated concentrations. Summary of the Invention [Means for solving the problem]

[0006] The present teachings provide compositions that maintain red blood cell MCV, substantially prevent white blood cell lysis, and substantially prevent the release of extracellular membrane vesicles (e.g., exosomes, ectosomes, etc.). The methods provided herein aim to stabilize white blood cells and red blood cells, thereby substantially preventing the release of nucleic acids and membrane vesicles from cells in biological samples. The compositions of the present disclosure are particularly suitable for use as biological sample stabilization and conditioning agents for downstream analysis by immunoassays, polymerase chain reaction, next-generation sequencing, and / or other.

[0007] The composition may include one or more components capable of releasing an aldehyde, one or more anticoagulants or chelating agents, and one or more polysaccharides. The composition may have a pH of about 4 to about 6.

[0008] The composition may include one or more amines. The composition may include a sodium citrate composition. The composition may include one or more formaldehyde donors. The composition may include ethylenediaminetetraacetic acid or a salt thereof. The composition may include a transcription inhibitor. The composition may include one or any combination of actinomycin D, alpha-amanitin, flavopiridol, DRP (5,6-dichloro-1-β-D-ribofuranosyl-1H-benzimidazole), and triptolide. The composition may include formaldehyde. The composition may be substantially free of separately added formaldehyde. The composition may be substantially free of ethylenediaminetetraacetic acid. The composition may be substantially free of sodium heparin. The polysaccharide may be selected from starch, cellulose, glycogen, or any combination thereof.

[0009] The one or more components capable of releasing aldehydes can be selected from diazolidinyl urea, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, oxazolidine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, quaternium-15, DMDM ​​hydantoin, 2-bromo-2-nitropropane-1,3-diol, 5-bromo-5-nitro-1,3-dioxane, tris(hydroxymethyl)nitromethane, hydroxymethylglycinate, polyquaternium, or combinations thereof. The composition may include imidazolidinyl urea. The composition may include imidazolidinyl urea and may be substantially free of other components capable of releasing aldehydes. The composition may include diazolidinyl urea. The composition may include both imidazolidinyl urea and diazolidinyl urea. The composition may include 2-bromo-2-nitropropane-1,3-diol.

[0010] The composition may comprise from about 0.1% to about 1% by volume of one or more polysaccharides. The amount of formaldehyde present after sample collection may be from about 10 ppm to about 10,000 ppm.

[0011] The amine may quench available free aldehydes, if present. Sample stability may be maintained for a minimum of 6 days at a temperature range of about 4°C to about 50°C. In further embodiments, sample stability is maintained for a minimum of 6 days at a temperature range of about 2°C to about 50°C, or about 2°C to about 37°C. Plasma concentrations of cfDNA and extracellular vesicles, and cfRNA are approximately the same as at the time of aspiration. Red blood cell MCV may be maintained, leukocyte lysis may be substantially inhibited, and release of extracellular vesicles (e.g., exosomes) may be substantially inhibited. Leukocytes may be substantially stabilized such that the number of exosomes recovered on days 4 and 6, and possibly beyond post-blood draw, is substantially similar to the exosome population present at the time of blood draw. Cell morphology and surface antigen expression may be maintained to allow immunophenotyping of leukocytes by flow cytometry.

[0012] Stabilization and separation of circulating tumor cells (CTCs) and tumor debris can also be facilitated. Testing of multiple components in a biological sample is also facilitated, especially when two or more blood components can be analyzed to identify indicators of the presence of a pathology or disease, the severity of the disease, or the success or failure of treating the disease.

[0013] The teachings described herein further provide methods of using the compositions described herein to stabilize white and red blood cells to substantially prevent the release of nucleic acids and extracellular vesicles from within the cells in a biological sample.

[0014] The teachings herein further provide methods of using the compositions described herein, including as stabilizing and modulating agents for biological samples for downstream analysis by immunoassays, polymerase chain reaction, next generation sequencing, and / or the like. The methods described herein may include treating a single blood sample with the composition and isolating two or more of DNA, RNA, extracellular vesicles, circulating tumor cells, circulating rare cells, and proteins from the sample.

[0015] The teachings herein further provide the use of the compositions described herein for isolating both DNA and RNA from a blood sample.The teachings herein further provide the use of the compositions described herein for isolating extracellular vesicles from a blood sample.The teachings herein further provide the use of the compositions described herein for isolating circulating tumor cells / tumor debris.

[0016] In one aspect, the present disclosure provides a composition comprising (i) a component capable of releasing aldehydes, (ii) an anticoagulant, and (iii) a cyclodextrin or its functionalized derivative.In one embodiment, the component capable of releasing aldehydes is diazolidinyl urea, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, oxazolidine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, quaternium-15, DMDM ​​hydantoin, 2-bromo-2-nitropropane-1,3-diol, 5-bromo-5-nitro-1,3-dioxane, tris(hydroxymethyl)nitromethane, hydroxymethylglycinate, polyquaternium, or combinations thereof.In one embodiment, the component capable of releasing aldehydes is imidazolidinyl urea. In further embodiments, the component capable of releasing an aldehyde is diazolidinyl urea. In yet other embodiments, the component capable of releasing an aldehyde includes imidazolidinyl urea and diazolidinyl urea. In some embodiments, the anticoagulant is a citrate-based anticoagulant including citrate and dextrose. In further embodiments, the citrate-based anticoagulant is anticoagulant citrate dextrose-A (ACD-A), anticoagulant citrate dextrose-B (ACD-B), or citrate-phosphate-dextrose-adenine (CPDA). In some embodiments, the citrate-based anticoagulant is present at a concentration of about 0.75% to about 4%. In yet other embodiments, the anticoagulant includes citric acid, trisodium citrate, and glucose. In some embodiments, the dextrose is present at a concentration of about 2% to about 20%. In a further embodiment, the citrate ion concentration is from about 200 mM to about 500 mM. In one embodiment, the citric acid is present at a concentration of from about 0.5% to about 4%. In a further embodiment, the trisodium citrate is present at a concentration of from about 3% to about 15%.

[0017] In some embodiments, the component capable of releasing an aldehyde is present at a concentration of about 10% to about 40%. In further embodiments, the cyclodextrin is α-cyclodextrin or a functionalized derivative thereof. In some embodiments, the α-cyclodextrin is present at a concentration of about 0.75% to about 4%.

[0018] In some embodiments, the composition further comprises formaldehyde. In further embodiments, the composition does not comprise separately added formaldehyde.

[0019] In further embodiments, sample stability is maintained for a minimum of 1 day at a temperature range of about 2° C. to about 37° C. In certain embodiments, sample stability is maintained for about 1 day up to 8 days at a temperature range of about 2° C. to about 37° C.

[0020] In an aspect, the present disclosure provides a method for inhibiting lysis of a cell, comprising contacting the cell with a composition of the present disclosure, and the inhibition prevents the release of nucleic acid and / or extracellular vesicles from the cell. In an embodiment, the cell is a white blood cell. In a further embodiment, the cell is an erythrocyte. In an embodiment, the nucleic acid is cell-free RNA (cfRNA), cell-free DNA (cfDNA), cellular RNA, or cellular DNA. In an embodiment, the lysis is inhibited for at least about 24 hours. In a further embodiment, the lysis is inhibited for at least 2 days, or at least 3 days, or at least 4 days. In yet a further embodiment, the method of the present disclosure further comprises maintaining the cell for at least about 24 hours, and then isolating nucleic acid from the cell. In an embodiment, the cell is maintained at room temperature.

[0021] In an aspect, the present disclosure provides a composition according to the present disclosure for use in stabilizing cells, characterized in that the composition comprises contacting cells with the composition, and the contacting prevents the release of nucleic acid and / or extracellular vesicles from the cells.In an embodiment, the cell is a white blood cell.In a further embodiment, the cell is an erythrocyte.In yet a further embodiment, the nucleic acid is cell-free RNA (cfRNA), cell-free DNA (cfDNA), cellular RNA, or cellular DNA. [Brief description of the drawings]

[0022] [Figure 1] 1 is a graph depicting hemolysis as a function of blood storage time utilizing three different formulations at three different storage temperatures. [Diagram 2] 1 is a graph showing exosome release upon use of various anticoagulants. [Diagram 3] 1 is a graph depicting plasma DNA concentration as a function of preservative percentage. [Figure 4A] FIG. 1 is a graph showing plasma RNA concentration as a function of preservative percentage. [Figure 4B] 1 is a graph showing plasma RNA concentration as a function of the percentage of α-cyclodextrin. [Figure 5A] 1 is a graph showing ubiquitin B mRNA copy number as a function of α-cyclodextrin ratio. [Figure 5B] 1 is a graph showing hemoglobin A mRNA copy number as a function of α-cyclodextrin percentage. [Figure 6] Total cell-free RNA (cfRNA) concentration as a function of whole blood storage time is shown. Plasma was isolated and cfRNA was extracted using a commercially available kit (Qiagen, QIAamp Circulating Nucleic Acid Isolation Kit). The resulting cfRNA was quantified using the Qubit RNA HS Assay (Invitrogen). Results are the mean with standard deviation from three healthy donors. [Figure 7]Figure 6 shows the fold increase in concentration of the red blood cell-specific (RBC-specific) transcript globin A1 / 2 as a function of whole blood storage time. cfRNA was isolated as shown in Figure 6 and in the description below, reverse transcribed (Bio-Rad iScript), and used in a droplet digital PCR reaction (Bio-Rad QX200 setup) using commercially available primers / probes (Thermo-Fisher). Results are the mean with standard deviation from three healthy donors. [Figure 8] Figure 1 shows the fold increase in concentration of ubiquitin B transcripts (expressed in all cells) as a function of whole blood storage time. cfRNA was isolated as shown in Figure 6 and in the description below, reverse transcribed (Bio-Rad iScript), and used in droplet digital PCR reactions (Bio-Rad QX200 setup) using commercially available primers / probes (Thermo-Fisher). Results are means with standard deviations from three healthy donors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The description and figures presented herein are intended to acquaint those skilled in the art with the teachings, their principles, and their practical applications. Those skilled in the art may adapt and apply the teachings in their numerous forms, as may best suit the requirements of a particular application. Thus, the specific embodiments of the present teachings described are not intended to be exhaustive or limiting of the teachings. Thus, the scope of the teachings should not be determined with reference to the above description, but instead with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. Other combinations are possible, as may be gleaned from the following claims, which are also incorporated by reference into this written description.

[0024] The teachings herein relate to blood sample conditioning and cell stabilizing compositions that can maintain a baseline level of extracellular vesicles, including exosomes, microvesicles, and apoptotic bodies (collectively referred to hereinafter as exosomes or extracellular vesicles). The baseline level of exosomes refers to the amount or concentration of exosomes in a blood sample immediately after blood collection. Extracellular vesicles carry nucleic acids, and therefore act as a protective membrane for cell-free nucleic acids. The amount of extracellular vesicles and nucleic acids in a blood sample increases with time after blood collection due to the release of vesicles from both white and red blood cells. The compositions featured herein aim to preserve the individual components, including extracellular vesicles and nucleic acids, and prevent cell lysis to allow intracellular material to leave the cell and contaminate the extracellular material present at the time of blood collection. The compositions disclosed herein can substantially stabilize blood cells, such that the number of extracellular vesicles recovered on days 4 and 6, and possibly beyond post-blood collection, is substantially similar to the extracellular vesicle population present at the time of blood collection. Thus, the concentration of cell-free RNA (cfRNA), cell-free DNA (cfDNA), proteins, and other cellular material inside the extracellular vesicles (in addition to the acellular material outside the extracellular vesicles) is substantially similar to that at the time of blood collection on days 4 and 6, and possibly beyond the time of blood collection. Thus, the compositions of the present disclosure facilitate the stabilization of cfRNA, cfDNA, proteins, and other cellular material within the extracellular vesicles, genomic DNA and RNA, as well as other RNA species (e.g., mRNA, miRNA, piRNA, siRNA, shRNA, and others), allowing downstream separation and testing of both acellular material and cellular material (e.g., DNA and RNA within white blood cells, circulating tumor cells (CTCs), circulating fetal cells, or other rare circulating cells). In further embodiments, the compositions of the present disclosure are useful for direct and indirect extracellular vesicle analysis. Extracellular vesicle analysis is performed, for example, but not limited to, by flow cytometry analysis.

[0025] Many chemical components are commonly used in the steps of biological sample collection, biological sample processing, and biological sample analysis. However, many of these known components are harmful to white blood cells, red blood cells, rare circulating cells, nucleic acids, proteins, or extracellular vesicles. In addition, many of these components can combine with other components to have harmful effects on white blood cells, red blood cells, nucleic acids, proteins, extracellular vesicles, circulating tumor cells, circulating endothelial cells, circulating progenitor cells, or circulating fetal cells. Furthermore, certain components are beneficial at certain concentrations but have other problems for various reasons, such as increased MCV, induction of apoptosis in white blood cells, and the volume required for efficacy. As a result, it is very difficult to identify combinations of chemical components that effectively stabilize the above sample components without harmful effects.

[0026] Because chemical components that function and / or act as stabilizing compounds are solids in solution, their potential use is limited by solubility and the amount of dilution that can be tolerated for downstream analysis. To be utilized as an effective stabilizer, many chemical components would need to be present in sufficient amounts to stabilize the sample. Furthermore, to produce a direct draw blood tube, the stabilizing composition must be present in very small amounts, so that most of the tube is filled with the sample. Thus, the amount of composition in the tube is ideally about 0.02 to about 2 mL. The amount of composition in the tube can be at least about 0.5 mL, or even at least about 1 mL. The amount of composition in the tube can be less than about 3.0 mL, or even less than about 2.0 mL.

[0027] Often, for a particular chemical component to be effective, it must be present in a high enough concentration for efficacy. Unfortunately, high concentrations of many chemical components can cause (as one example) red blood cell clumping and rupture, increasing MCV, and increasing hemolysis, among other problems.

[0028] Balancing these issues requires identifying chemical components that (1) can be effective at low concentrations that meet the demands of relatively small amounts of the composition, and (2) do not react with other components in a way that damages white blood cells, red blood cells, other rare circulating cells such as tumor and fetal cells, nucleic acids, exosomes, or proteins.

[0029] Additionally, because the compositions described herein maintain cell morphology and surface antigen expression, the compositions according to the teachings herein can facilitate immunophenotyping of white blood cells by flow cytometry. Stabilization and isolation of circulating tumor cells and other rare circulating cells / materials (e.g., circulating endothelial cells, circulating progenitor cells, circulating fetal nucleic acids in maternal blood) can also be facilitated utilizing compositions according to the present teachings. The compositions described herein can also facilitate testing of multiple components in a biological sample, particularly where two or more blood components can be analyzed to identify indicators of the presence of a pathology or disease, the severity of the disease, and the success or failure of treatment of the disease. As a non-limiting example, mechanical separation may be used to separate the CTCs stabilized with the compositions disclosed herein. Following this process, the remaining sample can be spun down or centrifuged to collect the plasma, after which exosomes (which may contain RNA) and / or cell-free DNA can be isolated from the sample. Alternatively, the plasma may be isolated first, followed by isolation of the CTCs and WBCs from the buffy coat. Proteins, DNA and RNA from remaining white blood cell populations or other rare cell populations, including CTCs, can be separated and analyzed from the remaining cell sample. Proteomic analysis can also be facilitated in samples treated with the compositions described herein. Thus, the compositions described herein can facilitate the ability to use a single sample for analysis that far exceeds what is understood to be possible today. The ability of the compositions herein to substantially prevent hemolysis, stabilize exosomes and associated cell-free RNA, stabilize cell-free DNA and stabilize protein allows such multi-step analysis.

[0030] Hemolysis is a common problem, especially as it relates to blood samples. Hemolysis can occur during one or more of the following: sample collection, sample transport, sample storage tubes, and downstream processing of the sample. Red blood cell lysis (e.g., hemolysis) can cause many challenges to cellular component analysis, and therefore the quality of the analysis improves as hemolysis is reduced. Many blood collection tubes contain ethylenediaminetetraacetic acid (EDTA). While EDTA has been found to reduce hemolysis somewhat, the test results identified herein (see, e.g., FIG. 1) indicate that the amount of hemolysis can actually be reduced by carefully selecting the correct anticoagulant. There are many anticoagulants, including but not limited to those based on EDTA, heparin-based, oxalate-based, and citrate-based. The compositions herein, in various embodiments, are based on a particular type of citrate-based anticoagulant (e.g., anticoagulant citrate dextrose-A or ACD-A) due to its dual ability to exhibit reduced hemolysis and stabilize red blood cell membranes. The mean cell volume (MCV) of red blood cells may be decreased or stabilized with certain citrate-based anticoagulants, specifically ACD-A and ACD-B. ACD-A and ACD-B each contain citric acid, trisodium citrate, and glucose. The ACD in solution A is concentrated for 8.5 milliliters of blood, while the ACD in solution B is concentrated for 6 milliliters of blood. The molar concentration of citrate utilized in the compositions herein is preferably about 0.05M to about 0.2M. The molar concentration of citrate utilized in the compositions herein may be about 0.11M. The remaining composition to form the compositions described herein may be diluted to have a final molar concentration of about 0.0025M to about 0.1M.

[0031] The desire to reduce hemolysis may also necessitate the identification of preservatives that do not cause (or minimize) lysis of red blood cells. Certain preservatives may increase the occurrence of red blood cell lysis and therefore may not be preferred preservatives for inclusion in the compositions described herein.

[0032] The concentration of the preservative can be selected such that leukocyte lysis is minimized. Leukocyte lysis is time dependent, with most samples eventually undergoing leukocyte lysis, and the amount of preservative should be sufficient to minimize, if not eliminate, leukocyte lysis during the period from blood collection until the sample is processed to separate sample components (e.g., 24 hours to a week, or possibly longer). Thus, the concentration of the preservative in the composition (before blood collection) can be from about 0.25% to about 2%. The concentration can be from about 2.5% to about 10%. The concentration can be from about 4% to about 7%. The concentration can be from about 2.5% to about 50%. The concentration can be from about 2% to about 100%.

[0033] If present, the concentration of the amine may be directly dependent on the concentration of the preservative. In essence, the presence of the amine may act to chemically bind to molecular components within the preservative. Thus, it may be preferable for sufficient amine to be present in the composition so that all of the preservative components that are free and react with the amine are sufficiently reacted and therefore non-reactive within the sample. Such molecular binding activity may reduce the presence of the component (which may be formaldehyde), thereby reducing the detrimental effect that reactive free formaldehyde may have on the sample components that need to be isolated for analysis. Thus, the concentration of the amine in the composition (before blood collection) may be about 0.01% to about 0.2%. The concentration may be about 0.03% to about 0.1%. The concentration may be about 0.05% to about 0.09%. The concentration may be about 0.75% to about 3.75%.

[0034] In an effort to stabilize and / or protect exosome concentration and simultaneously minimize leukocyte and erythrocyte lysis, many chemicals traditionally used in biological sample collection and testing will damage exosomes, increase leukocyte lysis, or interact with the sample in a way that increases erythrocyte lysis. Many traditional chemicals interfere with the actual separation of exosomes and nucleic acids, while others increase hemolysis. Furthermore, the concentrations of some chemicals have different effects at different levels. For example, certain polysaccharides utilized in the compositions described herein are known to increase hemolysis, but at certain lower concentrations, they have been discovered to act to reduce erythrocyte lysis.

[0035] The compositions herein may also include one or more transcription inhibitors. Typically, compositions for stabilizing biological samples include nuclease, metabolic, and / or protease inhibitors that block the degradation of nucleic acids and / or changes in their relative concentrations (e.g., gene expression). However, the teachings herein relate, at least in part, to incorporating the use of transcription inhibitors instead, since the purpose of the formulation is to maintain baseline RNA levels. White blood cells (and other rare circulating cells) undergo cell signaling after blood collection, altering their RNA gene expression profile. As little as two hours after blood collection, the RNA profile is altered. To obtain meaningful results, a baseline RNA profile (at or close to the time of aspiration) must be maintained to identify actionable targets. To do this, not only is inhibition of RNA degradation necessary, but inhibition of new RNA transcripts (including mRNA, microRNA, lncRNA, piRNA, YRNA, circRNA and other ncRNAs) is critical.

[0036] In an effort to prevent red blood cell lysis, the composition according to the teachings herein may be able to help to substantially prevent the size change of red blood cells. This may also be true for white blood cells. The ability of the composition disclosed herein to maintain the size of red blood cells and white blood cells may contribute to the ability to substantially prevent the lysis of red blood cells and white blood cells, thereby avoiding hemolysis and avoiding the release of background genomic nucleic acid into blood samples after blood collection.

[0037] The compositions described herein can further act to preserve nucleic acid, regardless of origin.As an example, the compositions described herein can stabilize RNA of platelet origin.Platelets contain a large amount of important RNA species, such as mRNA, miRNA, circRNA, and there is increasing evidence that platelet mRNA expression patterns change in human disease.Therefore, the advantage of the compositions disclosed herein is that mRNA of platelet origin can be analyzed and isolated with RNA of other origins.

[0038] One or more components of the compositions described herein can provide effectiveness in two or more aspects of sample stabilization. As an example, the polysaccharides described herein can act to maintain the integrity of extracellular vesicles (and thus the RNA located therein) and act to reduce red blood cell lysis. Certain components (including various disaccharides) have been considered, but have not been able to reduce or reduce hemolysis in an effective manner.

[0039] The present teachings generally relate to methods for preserving biological samples, including blood or other biological materials. The compositions described herein may include one or more preservatives, one or more anticoagulants or chelating agents, one or more polysaccharides, one or more amines, and one or more transcription inhibitors, and one or more ribonuclease inhibitors. The compositions may include two or more, three or more, four or more, or even all of these components.

[0040] The preservative can be selected from diazolidinyl urea, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, oxazolidine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione, quaternium-15, DMDM ​​hydantoin, 2-bromo-2-nitropropane-1,3-diol, 5-bromo-5-nitro-1,3-dioxane, tris(hydroxymethyl)nitromethane, hydroxymethylglycinate, polyquaternium, or combinations thereof. The preservative may include a mixture of imidazolidinyl urea and diazolidinyl urea. The preservative may include imidazolidinyl urea or may be free of other preservatives. The preservative may include diazolidinyl urea or may be free of other preservatives. The preservative may include oxazolidine. The preservative may comprise a mixture of oxazolidine compounds.The preservative may comprise a bicyclic oxazolidine composition.The preservative may comprise a triazine.

[0041] The preservative may be a derivative of uric acid. The preservative may be a reaction product of an aldehyde and a derivative of uric acid. The preservative may be selected such that at least a portion of the aldehyde (which may be formaldehyde) is reactive when contacted with a biological sample. However, the amount of formaldehyde present (before or after sample collection) must be low enough to avoid damage to nucleic acids, extracellular vesicles, or proteins present in the biological sample. The release of formaldehyde may increase the pH of the composition and / or the composition-sample combination. In an effort to control the pH of the composition and / or the composition-sample combination, the formaldehyde may be released in a controlled manner. The composition and / or sample may be contacted with ingredients to cause the release of formaldehyde over time in a predetermined manner. The formaldehyde may be encapsulated as discussed herein to allow for the release and / or controlled release of formaldehyde. The composition and / or the combination of the composition and the sample can also be exposed to elevated temperatures (e.g., temperatures above ambient or room temperature) such that the increase in temperature causes the release or increased release of formaldehyde (and thus an increase in pH). Alternatively, the formaldehyde may be released immediately upon contact of the formaldehyde releasing agent with the composition. The formaldehyde may be released upon contact of the composition with the sample. The formaldehyde releasing agent can be selected such that the formaldehyde is essentially released over time, without the need to encapsulate the formaldehyde or treat the composition in any way to force a controlled release.

[0042] The composition and / or sample and composition may further include one or more by-products of the components contained in the stabilization tube according to the teachings herein. One or more of these by-products may include aldehydes. The one or more by-products may include urea. The one or more by-products may be selected from allantoin, (4-hydroxymethyl-2,5-dioxo-imidazolidin-4-yl)-urea, (3,4-bis-hydroxymethyl-2,5-dioxo-imidazolidin-4-yl)-urea, and (3-hydroxymethyl-2,5-dioxo-imidazolidin-4-yl)-urea.

[0043] Aldehydes present either before or after sample collection may be in amounts of less than 40,000 ppm, less than 30,000 ppm, less than 20,000 ppm, or even less than 10,000 ppm. That being said, some formaldehyde may be present to effectively crosslink and stabilize one or more of the white blood cells and / or red blood cells. Thus, formaldehyde may be present in an amount of at least 2,000 ppm, at least 5,000 ppm, or even at least 10,000 ppm. The amount of free formaldehyde in the composition before contact with the sample may be up to about 0.5 weight percent, up to about 1.0 weight percent, up to about 2 weight percent, or even up to about 3 weight percent of the composition. The composition may include less than about 20 ppm of aldehyde. The composition may include less than about 15 ppm of aldehyde. The composition may include less than about 10 ppm of aldehyde. The composition may comprise less than about 5 ppm of aldehyde. The composition may comprise at least about 0.1 ppm to about 20 ppm of aldehyde. The composition may comprise at least about 0.5 ppm to about 15 ppm of aldehyde. The composition may comprise at least about 1 ppm to about 10 ppm of aldehyde.

[0044] The preservative may have a pH of at least about 4, at least about 5, or even at least about 6. The preservative may have a pH of less than 8, less than 7, or less than 6. The preservative may have a pH of from about 4 to about 6. The preservative may have a molecular weight of at least about 20 g / mole, at least about 50 g / mole, or an average molecular weight of at least about 80 g / mole. The preservative may have a molecular weight of less than about 500 g / mole, less than about 400 g / mole, or less than about 300 g / mole.

[0045] The one or more preservatives may be present in the composition at a concentration of about 0.5% to about 5.0% by volume. The one or more preservatives may be present in the composition at a concentration of about 0.5% to about 1.0% by volume. The one or more preservatives may be present in the composition at a concentration of about 5% by volume. The one or more preservatives may be present in the composition at a concentration of about 2.5% by volume.

[0046] The pH of the composition may change before and after contact with the sample. The composition may include one or more components to bring the pH of the composition (before contact with the sample) to a range of at least about 3, at least about 4, at least about 5 to less than about 11, less than about 10, or even less than about 9. For example, the pH of the composition before contact with the sample may be 3 to about 6. The composition may include one or more agents responsible for causing the release of acidic compounds upon contact with the sample. The pH of the combined sample and composition may be at least about 5, at least about 6, or even at least about 7. The pH of the combined sample and composition may be less than about 10, less than about 9, or even less than about 8.

[0047] The one or more amines can be selected from one or more of tryptophan, tyrosine, phenylalanine, glycine, ornithine, and S-adenosylmethionine, aspartic acid, glutamine, alanine, arginine, cysteine, glutamic acid, glutamine, histidine, leucine, lysine, proline, serine, threonine, or combinations thereof. The one or more amines can be selected based on their reactive capabilities. As a non-limiting example, the one or more amines can be aldehyde reactive agents. For example, the aldehyde reactive agent can be selected from one or any combination of tris, lysine, glycine, urea, or derivatives (e.g., salts and / or esters) of either or both. The aldehyde reactive agent can be selected to react with any free formaldehyde that may be present either before or after sample collection. The concentration of the amine in the composition (before blood collection) can be about 0.25% to about 1.5%. The concentration may be from about 0.3% to about 0.8%. The concentration may be from about 0.4% to about 0.7%.

[0048] The one or more anticoagulants or chelating agents may be selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) and its salts, ethylene glycol tetraacetic acid (EGTA) and its salts, hirudin, heparin, citric acid, and salts of citric acid. The anticoagulant may be selected from the group consisting of acid, oxalic acid, salts of oxalic acid, acid citrate dextrose (ACD), citric acid, citrate-theophylline-adenosine-dipyridamole (CTAD), citrate-pyridoxal phosphate-tris, heparin-β-hydroxy-ethyl-theophylline, polyanethol sulfonic acid, sodium fluoride, heparin sodium, thrombin, and PPACK (D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone, and any combination thereof. Thus, the concentration of the anticoagulant in the composition (before blood collection) may be from about 2.5% to about 10%. The concentration may be from about 3% to about 8%. The concentration may be from about 4% to about 7%.

[0049] The one or more polysaccharides can be selected from starch, cellulose, glycogen, or a combination thereof. The one or more polysaccharides can act as a red blood cell protective agent. The one or more polysaccharides can help stabilize the red blood cell membrane such that cell lysis is slowed, minimized, substantially prevented, or some combination thereof. The concentration of the polysaccharide in the composition (before blood collection) can be from about 0.001% to about 5.0%. The concentration can be from about 0.02% to about 3.0%. The concentration can be from about 0.5% to about 2.0%.

[0050] The one or more transcription inhibitors can be selected to promote the stability of one or more components within the sample. The one or more transcription inhibitors can be selected from actinomycin D, α-amanitin, triptolide, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB), flavopiridol, or any combination thereof. The concentration can be from about 0.5 μM to about 500 μM.

[0051] The sample collection tube may include one or more enzyme inhibitors. The one or more enzyme inhibitors may be selected from the group consisting of diethyl pyrophosphate, ethanol, aurintricarboxylic acid (ATA), glyceraldehyde, sodium fluoride, ethylenediaminetetraacetic acid (EDTA), formamide, vanadyl-ribonucleoside complex, macaloid, heparin, hydroxylamine-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), β-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphene hydrochloride, divalent cations such as Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, Cu+2, and any combination thereof.

[0052] The sample collection tube may include one or more metabolic inhibitors, which may be selected from the group consisting of glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvic acid and glyceryl dihydroxyacetate, sodium fluoride, K2C2O4, and any combination thereof.

[0053] The sample collection tube can include one or more protease inhibitors. The one or more protease inhibitors can include anti-pain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indole acetic acid (IAA), E-64, pepstatin, VdLPFFVdL, EDTA, 1,10-phenanthroline, phosphoramormodon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin, egg white cystatin, doxorubicin, cytostatin, cytosine ... The inhibitor may be selected from the group consisting of cycycline, sulfasalazine, curcumin, homocysteine, 6-aminocaproic acid, doxycycline, minacycline HCl, nicotinamide, chitosan, lysine, glyceraldehyde, phytic acid, b-cytocerol, C-AMP, polylysine low molecular weight, biochanin A, sulfasalazine, tetracycline, tetracycline, cyclohexamide, rifampicin, soy milk, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, AEBSF, and any combination thereof. The protectant may comprise a phosphatase inhibitor selected from the group consisting of calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic acid, cantharidin, microcystin LR, okadaic acid, hostolysin, tautomycin, cantharidin, endosole, nodularin, cyclosporin A, FK506 / immunophilin complex, cypermethrin, deltamethrin, fenvalerate, bpV(phen), dephostatin, mpV(pic)DMHV, sodium orthovanadate, and any combination thereof.

[0054] The sample collection tube may include one or more nuclease inhibitors. The one or more nuclease inhibitors may be selected from the group consisting of diethyl pyrocarbonate, ethanol, aurintricarboxylic acid (ATA), formamide, vanadyl-ribonucleoside complex, macaloid, ethylenediaminetetraacetic acid (EDTA), proteinase K, heparin, hydroxylamine-cupric ion, bentonite, ammonium sulfate, dithiothreitol (DTT), beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphene hydrochloride, or divalent cations such as Mg+2, Mn+2, Zn+2, Fe+2, Ca+2, Cu+2, and any combination thereof.

[0055] The sample collection tube may include one or more phosphatase inhibitors. The one or more phosphatase inhibitors may be selected from the group consisting of calyculin A, nodularin, NIPP-1, microcystin LR, tautomycin, okadaic acid, cantharidin, imidazole, microcystin LR, okadaic acid, hostolysin, tautomycin, cantharidin, endosole, nodularin, cyclosporin A, FK506 / immunophilin complex, cypermethrin, deltamethrin, fenvalerate, bpV(fen), dephostatin, mpV(pic)DMHV, sodium orthovanadate, and any combination thereof.

[0056] The sample collection tube may include bicyclic oxazolidine, DMDM ​​hydantoin, sodium hydroxymethylglycinate, hexamethylenetetramine allyl chloride, biocides, water-soluble zinc salts, or any combination thereof. The sample collection tube may include polyacrylic acid or a suitable acid having a pH ranging from about 1 to about 7. The sample collection tube may include amines, amino acids, alkylamines, polyamines, primary amines, secondary amines, ammonium salts, or any combination thereof. The sample collection tube may include one or more primary amines. The sample collection tube may include one or more amides (e.g., butanamides). The sample collection tube may include one or more apoptosis inhibitors. The sample collection tube may include one or more caspase inhibitors.

[0057] The sample collection tube may include one or more polymeric components. The polymers may include, but are not limited to, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), and cyclodextrin.

[0058] The compositions described herein may be any of the following: doxycycline, polyethylene glycol, sulfasalazine, polyvinylpyrrolidone, curcumin, magnesium gluconate, homocysteine, methylcellulose (MC), 6-aminocaproic acid, ethylcellulose, aprotinin, hydroxyethylcellulose, doxycycline, hydroxypropylcellulose, minocycline HCl, dextrin, nicotinamide, dextran, chitosan, polyethylene oxide, lysine, polyethyloxazoline, glyceraldehyde, ficoll, phytic acid, α-cyclodextrin, b-cytocerol, β-cyclodextrin, C-AMP, Y-cyclodextrin, polylysine, gelatin, biochanin A, The additives may include one or more of the following: sugars (e.g., sucrose, mannitol, lactose, trehalose), sulfasalazine, hydroxypropylmethylcellulose, demeclocycline, hydroxyethylmethylcellulose, chlortetracycline, oxytetracycline, cyclohexamide, rifampicin, soy milk, soy protease inhibitors, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, AEBSF, alpha-2-macroglobulin, or combinations thereof. It is contemplated that one or more of the aforementioned compounds may be substituted for cyclodextrin in the compositions of the present disclosure.

[0059] The sample collection tube may contain one or more detergents. The one or more detergents are DMSO (dimethyl sulfoxide), ethylene glycol, polyethylene glycol, glycerin, Cellosolves (ethylene glycol dimethyl ether) (phenoxyethanol), TritonX100, TritonX705 (non-ionic detergent), 1-methyl-2-pyrrolidinone, Tween20, Tween40 (non-ionic), Brij35 (detergent), polyoxyethylene ether (Polyox), sodium cholate, ethylene oxide polymers, monensin, monactin, pentachlorophenol, 2,4 dinitrophenol, saponin, SDS (sodium dodecyl sulfate), or combinations thereof. The effect of the aforementioned detergents is believed to be concentration-dependent herein. For example, and without limitation, in certain embodiments, lower concentrations of the aforementioned detergents are believed to be useful for inhibiting the lysis of cells by contacting the cells with the composition of the present disclosure, which inhibits the release of nucleic acids and / or extracellular vesicles from the cells.

[0060] The sample collection tube may contain proteins such as biotin, albumin (egg, bovine), gelatin, and similar such compounds. The sample collection tube may contain RNAse inhibitors such as RNAse inhibitor from human placenta, and similar such compounds. The sample collection tube may contain nucleic acid stabilizers such as polycations such as guanidinium hydrochloride, polyethyleneimine, and similar such compounds. The sample collection tube may contain amino acids / polypeptides such as glutamic acid, glycine, aspartic acid, and similar such compounds. The sample collection tube may contain fixatives such as aldehydes (formaldehyde and glutaraldehyde), alcohols (ethanol, methanol), and similar such compounds. The sample collection tube may contain anticoagulants such as EDTA (ethylenediaminetetraacetic acid), and similar compounds. The sample collection tube may contain ACD (Acid Citrate Dextrose), heparin, and similar such compounds. The sample collection tube may contain protease inhibitors such as EDTA, PMSF (phenylmethylsulfonyl fluoride), AEBSF (2-aminoethylbenzenesulfonyl fluoride), and similar such compounds. Antioxidants / reducing agents such as ethanol, B-mercaptoethanol, and similar such compounds The sample collection tube may contain a nucleic acid dye, such as DAPI (diamidino-2-phenylindole), propidium iodide, fluorescein diacetate, and similar such compounds. The sample collection tube may contain a carbohydrate, such as sugar (sucrose), cellulose, and similar such compounds. It should be understood that the above specific list of compounds may include a measure of overlap that recognizes the sometimes overlapping functions of certain compounds. Those skilled in the art should understand and appreciate this aspect of the present disclosure.

[0061] The aldehyde reactive agent may be present in a ratio (by weight) of about 1:20 to about 1:1 relative to the preservative. The aldehyde reactive agent may be present in a ratio (by weight) of about 1:25 to about 5:1 relative to the anticoagulant. The preservative may be present in a ratio (by weight) of about 1:10 to about 15:1 relative to the anticoagulant.

[0062] One or more components in the composition may be in gel form, liquid form, solid form, or some combination thereof. One or more components of the composition may be particulate and / or may form a film on the wall of the sample collection container and / or sample test container. One or more components may be freeze-dried and then sprayed onto one or more portions of the sample collection container. One or more components in the composition may be encapsulated in a mounting medium. The mounting medium may be selected to degrade or otherwise expose the encapsulating material upon contact with the sample, thereby delivering the material within the mounting medium to the sample. The composition may include an inactive component such as a buffer or water.

[0063] The one or more preservatives may be present in the composition at a concentration of about 0.5% to about 5.0% by volume. The one or more preservatives may be present in the composition at a concentration of about 0.5% to about 1.0% by volume. The one or more preservatives may be present in the composition at a concentration of about 0.5% by volume. The one or more preservatives may be present in the composition at a concentration of about 0.75% by volume. The one or more preservatives may be present in the composition at a concentration of about 1% by volume. The one or more preservatives may be present in the composition at a concentration of about 2.5% by volume. The one or more preservatives may be present in the composition at a concentration of about 5% by volume.

[0064] The composition may be substantially free of any or more of detergents and chaotropic agents, and substances that result in lysis of RBCs, WBCs, platelets, and other rare circulating cells. The composition may be substantially free of any or more of substances that result in activation of cells and subsequent release of membrane vesicles containing calcium ionophores and chemotherapeutic drugs. The composition may be substantially free of any or more of substances that result in RBC hemolysis and increase in RBC MCV, including antimalarials, nitrites, and heavy metals. The composition may be substantially free of any one or more substances that result in downstream PCR inhibition, including anticoagulants heparin, calcium ions, heme, and hemoglobin. The composition may be substantially free of any or more of substances that result in coagulation, including thrombin, calcium ions, silica, and celite. The composition may further be substantially free of one or more of Tris buffer, bovine serum albumin, polysorbate 20, sodium azide, sodium chloride, serum separator, clot activator, and gentamicin.

[0065] The biological sample may be stabilized for at least 3 days. The biological sample may be stabilized for at least 5 days. The biological sample may be stabilized for at least 7 days. Preferably, hemoglobin degradation of the biological sample comprising blood material is stabilized for at least 5 days.

[0066] The compositions described herein may be utilized in a method that may include handling a biological sample that remains suspended in the composition for delivery to a remote site where analysis of the blood material will be performed. The method may include transporting (e.g., shipping) the biological sample, which remains suspended in the composition for delivery to a site where analysis of the blood material will be performed. The method may include exposing the sample to temperature fluctuations during handling and / or transport. For example, such exposure may be to a temperature of about 4°C to about 37°C, or about 2°C to about 37°C. The method may include exposing the sample to temperatures up to 37°C during handling and / or transport. The remote site may be a site where downstream analysis of the blood material is to be performed. The site may be a site where immunoassays or nucleic acid analysis of the blood material is to be performed.

[0067] The composition and / or sample may be placed in a sample container. The sample container may contain the composition or the composition combined with the sample in an amount of at least 0.5 mL, at least 1 mL, at least 3 mL, at least 5 mL, at least 10 mL, or even at least 20 mL. The sample container may contain the composition or the composition combined with the sample in an amount of less than 150 mL, less than 100 mL, less than 50 mL, or even less than 20 mL. The compositions described herein may be present in a direct draw blood tube. The compositions described herein may be placed in any device for collecting biological samples. The compositions described herein may be placed in a sample receiving container before the addition of the sample or may be added to the sample after collection.

[0068] The sample vessel may be a tube. The sample vessel may be cylindrical. The sample vessel may be glass or polymeric. The sample vessel may have a barrier coating, which may be a chemically enhanced plasma deposited coating, as taught in U.S. Provisional Application Nos. 62 / 454,451 and 62 / 454,460, filed February 13, 2017, and PCT Patent Publication No. 2017 / 031354, the entire contents of which are incorporated herein by reference for all purposes.

[0069] The sample container may be part of a kit, which may include a cap or cover for the sample container such that the sample remains in the sample container when the sample is shipped to a testing facility in the sample container.

[0070] After blood collection, the sample container may contain a combination of a blood sample and formaldehyde. The sample container may contain a combination of a blood sample, an anticoagulant, and formaldehyde. The sample container may contain a combination of a blood sample, formaldehyde, an anticoagulant, and a polysaccharide. The sample container may contain a combination of a blood sample, formaldehyde, an anticoagulant, a polysaccharide, and a transcription inhibitor. The sample container may contain a combination of a blood sample, formaldehyde, an anticoagulant, a polysaccharide, a transcription inhibitor, and an amino acid.

[0071] The method may include analyzing the biological sample for one or more indicators of a disease state. The biological sample may be an abnormal biological sample. For example, the abnormal result may include rare circulating cells, including altered DNA, altered RNA, and / or altered protein and / or CTCs, circulating endothelial cells, circulating progenitor cells, and circulating fetal cells. Circulating cells may also be understood to include circulating cellular components that include nucleic acids.

[0072] The method may include analysis of a stabilized biological sample including a blood material. The analysis includes one or more test methods. The method may include immunoassay of the blood material and / or one or more components in the blood material (e.g., blood cells, rare cells, nucleic acids, circulating tumor cells / fragments, circulating endothelial cells, circulating progenitor cells, fetal cells in maternal blood, extracellular vesicles). The immunoassay may be selected from the group consisting of enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), counting immunoassay (CIA), fluoroimmunoassay (FIA), chemiluminescence (CLIA) and flow cytometry. The method may include nucleic acid analysis of the blood material. The nucleic acid analysis may be selected from the group consisting of gene sequencing, polymerase chain reaction, and next-generation sequencing.

[0073] The sample collection tubes of the present teachings can resist adhesion of the contents of the collected biomedical sample to the walls of the sample collection tube. For example, the inner surface of a blood collection tube can resist adhesion of blood cells of the collected blood sample. The blood collection tube can resist adhesion of nucleic acids (e.g., DNA) of the collected blood sample to the tube walls.

[0074] A sample collection tube of the present teachings may include a tube having a closed base, an identical elongated sidewall extending from the base and terminating at an open end, defining a hollow chamber having an inner wall, the hollow chamber configured to collect a blood sample, at least the elongated sidewall of the tube being made of a material including a thermoplastic polymeric material having a moisture barrier and low moisture absorption rate, optical transparency and chemical resistance allowing viewing of the sample within the tube, and optionally a transparent silicone-containing coating and an elastomeric stopper on the majority of the sidewall of the tube. Prior to collecting the blood sample, the hollow chamber of the tube is under vacuum to ambient pressure, and the hollow chamber is partially filled with an initial state composition selected from a solid, liquid or gel, a composition including an optional anticoagulant, and a preservation composition adapted to stabilize blood cells of the blood sample to allow isolation of nucleic acids or rare cells circulating in the blood sample and / or flow cytometry testing of the blood sample. The composition can retain its initial state for a period of at least one month over a temperature range of about 2°C to about 30°C. The composition may be included, or the tube may be adapted, to contain one or more materials to allow for isolation of cellular or acellular components of the blood sample. Such materials may include proteases, including but not limited to proteinase K, or amino acids.

[0075] The blood sample collection tube may be a disposable tube. The blood sample collection tube may be non-pyrogenic or endotoxin-free. The blood sample collection tube may be used to collect a blood sample for analytical purposes.

[0076] The blood sample collection tube is a vacuum direct suction blood collection tube used for stabilization and preservation. The blood sample collection tube can be used for stabilization and preservation of nucleic acids. For example, the blood collection tube of the present teachings may be used for non-invasive prenatal testing (NIPT) to analyze cell-free fetal DNA circulating in maternal blood. Prenatal testing includes fetal sex determination, genetic screening for one or more chromosomal conditions (e.g., trisomy), prenatal DNA paternity testing, etc. The tube can be part of a kit adapted for non-invasive testing of maternal blood samples of pregnant women. The blood sample collection tube can be used for stabilization and preservation of circulating tumor cells and / or tumor DNA. The blood sample collection tube can be used for flow cytometry testing and analysis. The tube can be part of a kit for detecting circulating tumor cells. The tube can be part of a kit for detecting cancer exhibiting RNA and / or DNA nucleic acid activity. The tube can be part of a kit for determining the effectiveness of a treatment for a medical condition. The tube can be part of a kit for testing for the presence of a biomarker or the presence of a cardiac disease. The tube may be part of a kit for testing the suitability of the implant.

[0077] Evacuated blood collection tubes usually have an expiration date. Expired tubes may have reduced vacuum, resulting in shorter blood collection times and improper blood-to-composition ratios. Therefore, it is important that blood collection tubes can maintain vacuum for extended periods of time. The blood collection tubes of the present teachings may provide evacuated blood collection tubes that maintain vacuum for extended periods of time (e.g., at least 24 months). Additionally, the suction tolerance of the blood collection tube must be accurate (e.g., ±10% of the listed mL of collection volume). It is also important that the structure of the tube, including its closing mechanism, facilitates the maintenance of the required pressure within the tube despite environmental variations during tube transportation. Such variations include temperature changes, air pressure changes, humidity changes, and other environmental factors. More specifically, the tube must remain tightly closed despite the reduced humidity and air pressure that may occur at high altitudes. As an example, the tube must remain sealed and maintain the required internal pressure when the environmental air pressure is in the range of 400mmHg to 800mmHg.

[0078] The blood collection tube may be adapted for use with a stopper. The stopper may comprise an elastomeric material. The stopper may comprise a butyl rubber derivative. The stopper may comprise a halogenated butyl. The stopper may comprise a bromobutyl rubber. The stopper may comprise a pharmaceutical bromobutyl rubber. The stopper may be used with a stopper lubricant. The stopper may be coated. The stopper may be partially coated. For example, the coating may comprise silicone. The tube stopper may comprise a silicone oil coating covering at least a portion of its exterior surface that contacts the inner wall of the tube.

[0079] The present teachings contemplate a blood collection tube assembly including a blood collection tube and a stopper of the present teachings. It is believed that the seal or interface between the outer wall of the cap and the inner wall of the tube is such that moisture transmission rate is substantially reduced. It is believed that the seal or interface between the outer wall of the stopper and the inner wall of the tube is such that oxygen transmission rate is substantially reduced. An effective seal should provide both a moisture barrier and a gas barrier. The present teachings provide a blood collection tube assembly in which the stopper resists being pulled out of the opening of the tube. The present teachings provide a blood collection assembly that provides an effective moisture transmission barrier, thereby preventing moisture from escaping from the inside of the tube filled with the composition, and an effective oxygen transmission barrier, thereby preventing oxygen from penetrating the tube.

[0080] The compositions described herein may be capable of retaining their initial state for a period of at least one month over a temperature range of about 2° C. to about 30° C. when the tube is exposed to a relative humidity of up to about at least 50%. The compositions may be capable of retaining their initial state for a period of at least one month over a temperature range of about 2° C. to about 30° C. when the tube is exposed to a relative humidity of up to about 75%.

[0081] The dimensions of the sample collection tube are approximately 13 mm by 75 mm. The tube can have an outer diameter up to a length (D×L) dimension of approximately 13 mm by 75 mm, measured at the same elongated sidewall adjacent the open end. The dimensions of the sample collection tube are approximately 16 mm by 100 mm. The tube can have an outer diameter up to a length (D×L) dimension of approximately 16 mm by 100 mm, measured at the same elongated sidewall adjacent the open end. The blood sample collection tube may be suitable for blood collection of approximately 2 mL to approximately 10 mL. For example, a small blood collection tube may be suitable for blood sample collection of approximately 2 mL. For example, a large blood collection tube may be suitable for blood sample collection of approximately 10 mL.

[0082] The blood collection tube may comprise a composition filling capacity of about 50 μL to about 70 μL. The blood collection tube may comprise a composition filling capacity of about 54 μL to about 66 μL. The blood collection tube may comprise a composition filling capacity of about 60 μL. The tube may comprise a composition filling capacity of about 160 μL to about 220 μL. The tube may comprise a composition filling capacity of about 180 μL. The tube may comprise a composition filling capacity of about 200 μL. The tube may comprise a composition filling capacity of about 200 μL to about 1,000 μL. The tube may comprise a composition filling capacity of about 750 μL. The tube may comprise a composition filling capacity of about 0.0600 g to about 0.0800 g by weight. The tube may comprise a composition filled by about 0.0700 g by weight. The tube may contain a composition filled at a weight of 0.0650g plus or minus 10%. The tube may contain a composition filled at a weight of 0.0700g plus or minus 10%. The tube may contain a composition fill of 0.0750g plus or minus 10% by weight. The tube may contain about 0.200g to about 0.300g of the composition by weight. The tube may contain a composition filled at a weight of about 0.250g. The tube may contain a composition filled at a weight of 0.225g plus or minus 10%. The tube may contain a composition fill of 0.250g plus or minus 10% by weight. The tube may contain a composition fill of 0.275g plus or minus 10% by weight.

[0083] The blood collection tube may comprise a suction tolerance of about 3 ml to about 5 ml. The blood collection tube may comprise a suction tolerance of about 4 ml. The blood collection tube may comprise a suction tolerance of about 7 ml to about 13 ml. The blood collection tube may comprise a suction tolerance of about 10 ml. The blood collection tube may comprise a composition fill volume of about 200 μL. The blood collection tube may comprise a composition fill volume of about 750 μL. The blood collection tube may comprise a composition fill volume of about 200 μL to about 750 μL. The blood collection tube may comprise a composition fill volume of about 500 μL. The blood collection tube may comprise a composition fill volume of about 500 μL to about 750 μL. The blood collection tube may comprise a composition fill volume of about 1 ml. The blood collection tube may comprise a composition fill volume of about 500 μL to about 1 ml. The blood collection tube may comprise a composition fill volume of about 200 μL to about 1 ml. The blood collection tube may have a composition filling volume of about 2 mL. The blood collection tube may have a composition filling volume of about 1 mL to about 2 mL. For example, a 10 mL blood collection tube may have a composition filling volume of about 1 mL to about 2 mL. For example, a 4 mL blood collection tube may have a composition filling volume of about 1 mL to about 2 mL.

[0084] The polymeric material of the tube containing the composition described herein may be characterized as having one or more of the following attributes: moisture barrier and low moisture absorption, purity, transparency, chemical resistance, heat resistance, and strength. The polymeric material may have one or more of the following attributes: low density, high transparency, low birefringence, very low water absorption, excellent water vapor barrier properties, variable heat deformation properties, high stiffness, strength and hardness, very good blood compatibility, excellent biocompatibility, very good resistance to acids and alkalis, very good electrical insulation properties, and very good processability / flowability.

[0085] The polymeric material may include a cyclic olefin. The polymeric material may include a cyclic olefin copolymer (COC). The polymeric material may include a homopolymer or copolymer including polyethylene, polypropylene, or both. The polymeric material may include a cyclic moiety. The polymeric material may include a polyester. The polymeric material may include a polyester terephthalate or a polyethylene terephthalate. The polymeric material may include a polycarbonate. The polymeric material may include a poly(methyl methacrylate).

[0086] The polymeric material may have excellent water vapor barrier properties. The polymeric material may have a low water vapor transmission rate (MVTR) or water vapor transmission rate (WVTR). The water vapor transmission rate may be determined according to conventional test methods, such as DIN 53 122. The polymeric material may have a water vapor transmission rate of about 0.023 g mm / m at 23°C and 85% relative humidity, as measured by DIN 53 122. 2 ·d~approx.0.045g·mm / m 2 The polymeric material may have a water vapor transmission rate of about 0.023 g mm / m at 23° C. and 85% relative humidity. 2 The polymeric material may have a water vapor transmission rate of about 0.023 g mm / m at 23° C. and 85% relative humidity. 2 ·d may have a water vapor transmission rate of less than d.

[0087] The sample collection tube may include a coating. The sample collection tube may be uncoated. The sample collection tube may be a blood collection tube with a coating on the inner wall of the tube. The coating may include one or more layers. The coating may be generally transparent. The coating may include a silicone-containing material.

[0088] The coating may serve one or more functions. The coating of the blood collection tube may be such that it changes one or more properties of the inner surface of the blood collection tube. For example, the coating may change the surface energy of the inner wall of the tube. The coating serves to prevent the adhesion of nucleic acids to the wall of the tube.

[0089] The coating, including one or more layers, can be deposited on the inner wall of the tube via any suitable mechanism. For example, the coating can be deposited on the polymer substrate by plasma, spray, and / or sputter deposition; can be applied by vapor deposition; can be applied by chemical vapor deposition; or can be applied by physical vapor deposition.

[0090] The present teachings provide a blood collection tube that does not allow the composition to lose moisture over time, which can cause the stabilized composition to thicken and compromise the integrity of all cells, including red blood cell lysis, resulting in excessive hemolysis.

[0091] The blood collection tube of the present teachings may include a composition. The tube may include a composition including one or more components. The ratio of one or more components of the composition may be about 5:1 to about 15:1 (e.g., 10:1). The composition may be a solid. The composition may be a substantially solid. The detergent composition may be a liquid. The composition may be a gel. The composition may be a film. The composition may include an aqueous material. The composition may include one or more drugs in the composition. Suitable solvents may include water, saline, dimethyl sulfoxide, alcohol, and any mixture thereof.

[0092] The blood collection tube may contain a blood collection volume of about 4 mL to about 10 mL. The blood collection tube may contain a composition volume of about 0.05 mL to about 1 mL. The blood collection tube may contain a composition volume of about 0.20 mL to about 0.90 mL. The blood collection tube may contain about 0.20 mL to about 0.30 mL of the composition for a blood collection volume of about 8 mL to about 10 mL. The blood collection tube may contain about 0.60 mL to about 0.90 mL of the composition for a blood collection volume of about 4 mL to about 6 mL.

[0093] The blood sample collection tubes can be used to stabilize and preserve one or more of the following: proteins (e.g., prions), enzymes, antibodies, and biological materials that may or may not contain nucleic acids, including nucleic acids with and without post-translational modifications. For example, the tubes collect samples for analysis by one or more of surface flow cytometry, intracellular cytometry, ELISA-type assays, and mass spectrometry. The blood sample collection tubes can be used to stabilize and preserve extracellular vesicles, exosomes, ectosomes, and / or microvesicles. The blood sample collection tubes can be used to stabilize and preserve viruses. This may include inactivating the virus and quantifying the viral load. Viruses may be DNA / RNA, stranded / double-stranded, enveloped / non-enveloped. The blood sample collection tubes can be used to stabilize and preserve microorganisms, such as bacteria, molds, and yeasts. The blood sample collection tubes can be used to stabilize and preserve extracellular and / or intracellular parasites. Blood sample collection tubes can be used to stabilize and preserve exosomes, circulating tumor cells, other rare circulating cells, and tumor DNA.

[0094] The composition may be suitable for storing a blood sample for at least about 3 days. The composition may be suitable for storing a blood sample for at least about 7 days. The composition may be suitable for storing a blood sample for at least about 14 days. The composition may be suitable for storing a blood sample for at least about 30 days. The composition may be suitable for storing a blood sample for at least about 60 days. The composition may be suitable for storing a blood sample for at least about 90 days.

[0095] Synergy Between the Components of the Compositions of the Present Disclosure The relationship between the various components of the composition of the present disclosure and their effects on the various cells in blood is complex. To stabilize cfRNA, stabilization of both WBC and RBC is necessary, since each contains RNA that contaminates the acellular fraction. For example, without limitation, if there are too few components (e.g., IDU) that can release aldehydes in the composition, the stabilization of WBCs will not be affected, but if there are too many components (e.g., IDU) that can release aldehydes, then destabilization of RBCs will begin to occur. α-cyclodextrin helps stabilize white blood cells, but can damage red blood cells.

[0096] Thus, it is disclosed herein that various components of the composition of the present disclosure produce a synergistic effect, for example, in terms of stabilizing intracellular nucleic acid. Thus, in any of the embodiments of the aspects of the present disclosure, the polymer and components capable of releasing aldehyde provide a synergistic effect, for example, in terms of stabilizing intracellular nucleic acid. Thus, in an embodiment, α-cyclodextrin and IDU act synergistically, for example, to stabilize intracellular nucleic acid. In a further embodiment, α-cyclodextrin and DU act synergistically, for example, to stabilize intracellular nucleic acid. In an embodiment, PEG and IDU act synergistically, for example, to stabilize intracellular nucleic acid. In yet another embodiment, PEG and DU act synergistically, for example, to stabilize intracellular nucleic acid.

[0097] The compositions of the present disclosure facilitate stabilization of cfRNA, cfDNA, proteins and other cellular material within extracellular vesicles, genomic DNA and RNA as well as other RNA species (e.g., mRNA, miRNA, piRNA, siRNA, shRNA, etc.), allowing downstream isolation and examination of acellular and cellular material (e.g., DNA and RNA within white blood cells, circulating tumor cells (CTCs), circulating fetal cells, or other rare circulating cells). In further embodiments, the compositions of the present disclosure are useful for direct and indirect extracellular vesicle analysis.

[0098] In some embodiments, the cell is a blood cell. In further embodiments, the blood cell is a white blood cell (WBC) or a red blood cell (RBC). In some embodiments, the component capable of releasing aldehyde is diazolidinyl urea, imidazolidinyl urea, 1,3,5-tris(hydroxyethyl)-s-triazine, oxazolidine, 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-. 2,4-dione, quaternium-15, DMDM ​​hydantoin, 2-bromo-2-nitropropane-1,3-diol, 5-bromo-5-nitro-1,3-dioxane, tris(hydroxymethyl)nitromethane, hydroxymethylglycinate, polyquaternium, or combinations thereof. In any of the embodiments of the present disclosure, the component capable of releasing aldehyde is imidazolidinyl urea (IDU).

[0099] In certain embodiments, the polymer is polyethylene glycol (PEG) (in various molecular weights as understood in the art), polyvinylpyrrolidone (PVP), magnesium gluconate, methylcellulose (MC), ethylcellulose (EC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), dextrin (in various molecular weights as understood in the art), dextran (in various molecular weights as understood in the art), polyethylene oxide, polyethyloxazoline, ficoll (in various molecular weights as understood in the art), α-cyclodextrin, β-cyclodextrin, Y-cyclodextrin, gelatin, sugars (such as, but not limited to, sugars), and the like. In any of the embodiments of the present disclosure, the polymer is α-cyclodextrin.

[0100] The concentration / amount of each component of the composition of the present disclosure is contemplated as follows: The component capable of releasing aldehyde (e.g., IDU or DU) is contemplated to be present in the composition at a concentration of about 10% to about 40%. The cyclodextrin or its functionalized derivative (e.g., α-cyclodextrin or its functionalized derivative) is contemplated to be present in the composition at a concentration of about 0.75% to about 4%. Functionalized derivatives of cyclodextrin are known in the art (e.g., Hanessian et al., J. Org. Chem. 60(15):4786-4797 (1995) and Zhou et al., Polymer Chemistry 1:1552-1559 (2010), each of which is incorporated herein by reference in its entirety).

[0101] With respect to anticoagulants, it is contemplated that citrate-based anticoagulants (e.g., ACD-A, ACD-B, or CPDA) will be present in a concentration of about 0.75% to about 4%. Non-citrate-based anticoagulants (e.g., EDTA) will be present in the composition in a concentration of about 0.05% to about 2%.

[0102] As used herein, the concentration or amount of a given component is described with respect to the concentration or amount of the component in the composition prior to the addition of the biological sample (e.g., blood). Thus, after the biological sample is added, the concentration or amount of each component described herein is expected to be about 20 times lower. By way of example, the amount of IDU present in the composition in a container (e.g., blood tube) prior to the addition of the biological sample may be about 10% to about 40% by volume. However, after the addition of the biological sample, the concentration of IDU in the container is expected to be about 0.5% to about 2% by volume. Thus, if the volume of the composition in the container prior to the addition of the biological sample is 0.2 ml, the amount of IDU in the container is about 20 μL to about 80 μL. After the addition of the biological sample, the amount of IDU in the container is about 1 μL to about 4 μL.

[0103] In any aspect or embodiment of the present disclosure, components with the same or similar properties may be substituted for each other.For example, in any aspect or embodiment of the present disclosure, DU (or any component that can release aldehyde) can be used instead of IDU.In any aspect or embodiment of the present disclosure, sugar substitutes (e.g., sucrose, mannitol, lactose, or trehalose) can be used instead of dextrose. EXAMPLES

[0104] The diagram provided here illustrates test data that helps identify components to minimize white blood cell lysis, minimize red blood cell lysis, and maintain stability of cell-free DNA, extracellular vesicles (and the RNA therein), and proteins.

[0105] FIG. 1 is a graph showing the change in red blood cell MCV using various anticoagulants. An increase in red blood cell mean cell volume indicates an increase (or a prediction of an increase) in red blood cell lysis. The graph shows results at three different storage temperatures from three different donors. Thus, it is preferred that anticoagulants that demonstrate the ability to maintain the mean cell volume remain similar to the mean cell volume at the time of sample collection.

[0106] Figure 2 is a graph showing exosome release during the use of various anticoagulants. Similar to the release of genomic DNA, the release of extracellular vesicles can pose a challenge to the effective isolation of RNA. As a result, an anticoagulant that maintains the release of exosomes and is similar to that used at the time of specimen collection is preferred.

[0107] Figure 3 is a graph showing plasma DNA concentration as a function of the percentage of preservative. An increase in DNA concentration indicates an increase in the amount of genomic DNA, which represents an increase in leukocyte lysis. Thus, a desirable amount of preservative maintains DNA concentration while avoiding excessive preservation and cross-linking that may damage DNA, extracellular vesicles, or proteins.

[0108] Figure 4A is a graph showing plasma RNA concentration as a function of the percentage of preservative. An increase in RNA concentration indicates an increase in the amount of released exosomes. Thus, a desirable amount of preservative maintains RNA concentration while avoiding excessive preservation and cross-linking that may damage DNA, extracellular vesicles, or proteins.

[0109] Figure 4B is a graph showing plasma RNA concentration as a function of the percentage of α-cyclodextrin. An increase in RNA concentration indicates an increase in the amount of released exosomes. Thus, a desirable amount of polysaccharide maintains RNA concentration while avoiding hemolysis.

[0110] Figure 5A is a graph showing ubiquitin B mRNA copy number as a function of the percentage of α-cyclodextrin. Increasing RNA concentration indicates increasing amounts of released exosomes. Thus, a desirable amount of polysaccharide maintains RNA concentration while avoiding hemolysis.

[0111] Figure 5B is a graph showing hemoglobin A mRNA copy number as a function of the percentage of α-cyclodextrin. An increase in RNA concentration indicates an increase in the amount of exosomes released. Thus, a desirable amount of polysaccharide maintains RNA concentration while avoiding hemolysis.

[0112] Synergistic Effects of the Components of the Compositions of the Present Disclosure The working hypothesis for the container (e.g., blood collection tube) containing the composition of the present disclosure was that cell-free RNA (cfRNA), including messenger RNA, microRNA, non-coding RNA, etc., is stable in plasma by encapsulation within extracellular vesicles (EVs). Thus, as aspects of the present disclosure are directed toward stabilizing the aspiration time concentration of cfRNA, the goal was to maintain the initial concentration of EVs. Experiments shown in Figures 6, 7, and 8 demonstrated that RNAxBCT stabilized the aspiration concentration of EVs and associated cfRNA for up to 7 days after blood collection. In the experiments, the functional components of RNAxBCT are the preservative imidazolidinyl urea (IDU) and α-cyclodextrin. The experiments were designed to test whether each of the aforementioned components would act alone (additive effects) or whether the combination would yield unexpected, more pronounced results (synergistic effects). As described below and shown in Figures 6-8, the combination demonstrated an unexpected synergistic effect.

[0113] Each preservative (i.e., IDU and α-cyclodextrin) was added alone (at the same concentrations as used in RNAx BCT) or together in a background of ACD-A, a basal anticoagulant. Samples were collected in each formulation-containing tube, and plasma was isolated immediately (day 0) or after 7 days of storage at room temperature (day 7). Cell-free RNA was purified from plasma samples using a commercially available QIAamp Circulating Nucleic Acid Isolation Kit and on-column DNase1 digestion, all in accordance with the manufacturer's recommendations (Qiagen). Purified cfRNA was then used for fluorescent analysis of total cfRNA according to the manufacturer's recommendations (Qubit HS RNA assay, Thermo-Fisher) or for droplet digital PCR (ddPCR, Bio-Rad QX200 workflow) using commercially available primer / probe sets for transcript-specific RNA expression.

[0114] As shown in Figure 6, total cfRNA, as measured by fluorometric analysis, increased nearly 60-fold over 7 days in non-stabilized ACD-A samples (n=3 independent donors). Adding IDU alone did not prevent the increase in cfRNA, whereas α-cyclodextrin alone limited the increase in cfRNA levels. However, combining IDU with α-cyclodextrin significantly blunted the increase in total cfRNA levels (only a 2-4 fold increase was observed).

[0115] To determine the potential preference of IDU or α-cyclodextrin preservatives for various cell populations, ddPCR was utilized to determine transcript-specific changes in cfRNA levels. Hemoglobin alpha 1 and alpha 2 (HbA1 / 2) are expressed by red blood cells, and the total levels of these transcripts mirror that of total cfRNA (Figure 7). This was expected, given that globin transcripts are the major transcripts found in plasma. Next, we determined the levels of ubiquitin B, a gene expressed by all cells. In this regard, an effect on IDU was observed alone, whereas a combination of both IDU and α-cyclodextrin was required to almost completely stabilize ubiquitin B concentrations (Figure 8).

[0116] Overall, these data demonstrated an unexpected synergistic effect between components capable of releasing aldehydes (e.g., IDU) and α-cyclodextrin in stabilizing the aspiration time concentration of cfRNA.

[0117] As used herein, unless otherwise stated, the teachings contemplate that any member of a genus (list) may be excluded from the genus and / or that a member of a Markush group may be excluded from the group.

[0118] Unless otherwise stated, numerical values ​​set forth herein include all values ​​from the lower limit to the upper limit in increments of one unit, provided that there is at least two units between any lower and higher values. As an example, if a quantity of a component, property, or value of a process variable, such as temperature, pressure, time, etc., is stated to be, for example, 1 to 90, then values ​​in the intermediate ranges, preferably 20 to 80, more preferably 30 to 70, e.g. (15 to 85, 22 to 68, 43 to 51, 30 to 32, etc.), are intended to be within the teachings of this specification. Similarly, individual intermediate values ​​are also within the scope of the present teachings. For values ​​less than 1, one unit is considered to be 0.0001, 0.001, 0.01, or 0.1, as appropriate. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​listed are considered to be expressly set forth in this application in a similar manner. As will be apparent, comparative teachings of amounts expressed as weight / volume percentages of two or more components also encompass the relative weight ratios of the two or more components to one another, even if not explicitly stated. For example, if the teachings state 2% A and 5% B, the teachings also include a weight ratio of A:B of 2:5. Unless otherwise stated, all ranges include both endpoints and all numbers between the endpoints. The use of "about" or "approximately" in connection with a range applies to both ends of the range. Thus, "about 20 to 30" is intended to cover "about 20 to about 30," inclusive of at least the specified endpoint.

[0119] The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes.

[0120] The term "consisting essentially of" to describe a combination is intended to include the identified elements, components, ingredients, or steps, as well as other elements, components, ingredients, or steps that do not materially affect the basic and novel properties of the combination. Use of the terms "comprising" or "including" to describe a combination of elements, ingredients, ingredients, or steps herein also contemplates embodiments that consist essentially of the elements, ingredients, ingredients, or steps (i.e., the presence of any additional elements, ingredients, ingredients, or steps does not materially affect the properties and / or advantages derived from the teachings, or even consist of the elements, ingredients, ingredients, or steps).

[0121] A plurality of elements, components, ingredients, or steps may be provided by a single integrated element, component, ingredient, or step. Alternatively, a single integrated element, component, ingredient, ingredient, or step may be divided into separate elements, components, ingredients, ingredients, or steps. The disclosure of "a" or "one" describing an element, component, ingredient, or step is not intended to exclude additional elements, components, ingredients, or steps. All references herein to elements or metals belonging to a particular group refer to the Periodic Table of the Elements, published and copyrighted by CRC Press, Inc., 1989. References to one or more groups shall refer to one or more groups reflected in this Periodic Table of the Elements, using the IUPAC system for numbering the groups.

[0122] It is understood that the above description is intended to be illustrative and not limiting. Many embodiments and many applications other than the examples provided will be apparent to those skilled in the art upon reading the above description. The scope of the teachings should therefore not be determined with reference to the above description, but instead with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. Omission from the following claims of any aspect of the subject matter disclosed herein should not be construed as a disclaimer of such subject matter or as a failure by the inventors to consider such subject matter to be part of the subject matter of the disclosed invention.

[0123] The description and figures presented herein are intended to acquaint those skilled in the art with the teachings, their principles, and their practical applications. Those skilled in the art may adapt and apply the teachings in their numerous forms, as may best suit the requirements of a particular application. Thus, the specific embodiments of the present teachings described are not intended to be exhaustive or limiting of the teachings. Thus, the scope of the teachings should not be determined with reference to the above description, but instead with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications and publications, are incorporated by reference for all purposes. Other combinations are possible, as may be gleaned from the following claims, which are also incorporated by reference into this written description.

Claims

1. (i) a component capable of releasing an aldehyde; (ii) an anticoagulant comprising citric acid, trisodium citrate, and dextrose; (iii) α-cyclodextrin or a functionalized derivative thereof; A composition comprising: A composition, wherein the component capable of releasing an aldehyde is imidazolidinyl urea, and the composition is in a volume of 0.02 mL to 2 mL in a tube.

2. (i) a component capable of releasing an aldehyde; (ii) an anticoagulant comprising citric acid, trisodium citrate, and dextrose; (iii) α-cyclodextrin or a functionalized derivative thereof; (iv) one or more polysaccharides, one or more polymeric components, or a combination thereof; A composition comprising: A composition, wherein the component capable of releasing an aldehyde is an imidazolidinyl urea.

3. The composition described in claim 2, wherein the one or more polysaccharides is starch, cellulose, or glycogen.

4. The composition described in claim 2, wherein the one or more polymer components is polyvinylpyrrolidone, polyethylene glycol, polyethylene oxide, or polyethyloxazoline.

5. (i) a component capable of releasing an aldehyde; (ii) an anticoagulant comprising citric acid, trisodium citrate, and dextrose; (iii) α-cyclodextrin or a functionalized derivative thereof; (iv) one or more transcription inhibitors, one or more enzyme inhibitors, one or more metabolic inhibitors, one or more protease inhibitors, one or more nuclease inhibitors, or a combination thereof; A composition comprising: A composition, wherein the component capable of releasing an aldehyde is an imidazolidinyl urea.

6. The composition of claim 5, wherein the one or more transcription inhibitors are actinomycin D, α-amanitin, triptolide, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, or flavopiridol.

7. The composition of claim 5, wherein the one or more enzyme inhibitors are diethyl pyrocarbonate, ethanol, aurintricarboxylic acid, glyceraldehyde, sodium fluoride, ethylenediaminetetraacetic acid, formamide, vanadyl-ribonucleoside complex, macaloid, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol, β-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphene hydrochloride, or a divalent cation.

8. The composition of claim 5, wherein the one or more metabolic inhibitors are glyceraldehyde, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, 1,3-bisphosphoglycerate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, glyceryl dihydroxyacetate, sodium fluoride, or K2C2O4.

9. The one or more protease inhibitors are selected from the group consisting of anti-pain, aprotinin, chymostatin, elastatinal, phenylmethylsulfonyl fluoride, APMSF, TLCK, TPCK, leupeptin, soybean trypsin inhibitor, indoleacetic acid, E-64, pepstatin, VdLPFFVdL, ethylenediaminetetraacetic acid, 1,10-phenanthroline, phosphoramon, amastatin, bestatin, diprotin A, diprotin B, alpha-2-macroglobulin, lima bean trypsin inhibitor, pancreatic protease inhibitor, egg white ovostatin, egg white The composition of claim 5, which is cystatin, doxycycline, sulfasalazine, curcumin, homocysteine, 6-aminocaproic acid, minacycline HCl, nicotinamide, chitosan, lysine, glyceraldehyde, phytic acid, b-cytoserol, C-AMP, polylysine low molecular weight, biochanin A, demeclocycline, chlortetracycline, oxytetracycline, cyclohexamide, rifampicin, soy milk, suramin, N-butyric acid, penicillamine, N-acetylcysteine, benzamidine, or AEBSF.

10. The composition of claim 5, wherein the one or more nuclease inhibitors are diethyl pyrocarbonate, ethanol, aurintricarboxylic acid, formamide, vanadyl-ribonucleoside complex, macaloid, ethylenediaminetetraacetic acid, proteinase K, heparin, hydroxylamine-oxygen-cupric ion, bentonite, ammonium sulfate, dithiothreitol, beta-mercaptoethanol, cysteine, dithioerythritol, tris(2-carboxyethyl)phosphene hydrochloride, or a divalent cation.

11. A composition described in any one of claims 2 to 10, wherein the composition is in an amount of 0.02 mL to 2 mL in a tube.

12. The composition of any one of claims 1, 2 or 5, wherein the anticoagulant is anticoagulant citrate dextrose-A, anticoagulant citrate dextrose-B, or citrate-phosphate-dextrose-adenine.

13. The composition of any one of claims 1, 2 or 5, wherein the dextrose is present at a concentration of 2% to 20%.

14. The composition described in claim 13, wherein the citrate ion concentration is 200 mM to 500 mM.

15. The composition of claim 14, wherein the citric acid is present at a concentration of 0.5% to 4%.

16. The composition of claim 15, wherein the trisodium citrate is present at a concentration of 3% to 15%.

17. A composition according to any one of claims 1, 2 or 5, wherein the component capable of releasing the aldehyde is present in a concentration of from 10% to 40%.

18. The composition of claim 17, wherein the α-cyclodextrin is present at a concentration of 0.75% to 4%.

19. The composition described in claim 18, wherein the composition is for maintaining the stability of a biological sample, and the stability of the sample is maintained for at least one day in a temperature range of 2°C to 37°C.

20. The composition described in claim 18, wherein the composition is for maintaining the stability of a biological sample, and the stability of the sample is maintained for 1 to 8 days in a temperature range of 2°C to 37°C.

21. A method for treating a single blood sample with a composition according to any one of claims 1 to 20, comprising: contacting the single blood sample with the composition; and isolating one or more of DNA, RNA, extracellular vesicles, circulating tumor cells, circulating rare cells, or proteins from the single blood sample.

22. Use of a composition described in any one of claims 1 to 20 for isolating both DNA and RNA from a blood sample.

23. Use of a composition described in any one of claims 1 to 20 for isolating extracellular vesicles from a blood sample.

24. Use of a composition described in any one of claims 1 to 20 for isolating circulating tumor cells or tumor debris.

25. A method for inhibiting lysis of a cell, comprising contacting the cell with a composition described in any one of claims 1 to 20, wherein the inhibition prevents release of nucleic acids and / or extracellular vesicles from the cell.

26. The method of claim 25, wherein the cell is a white blood cell.

27. The method of claim 25, wherein the cells are red blood cells.

28. The composition of any one of claims 25 to 27, wherein the nucleic acid is cell-free RNA, cell-free DNA, cellular RNA, or cellular DNA.

29. The method of claim 25, wherein dissolution is inhibited for at least 24 hours.

30. The method of claim 29, wherein dissolution is inhibited for at least 2 days, at least 3 days, or at least 4 days.

31. The method of claim 25, further comprising maintaining the cells for at least 24 hours and then isolating the nucleic acid from the cells.

32. The method of claim 31, wherein the cells are maintained at room temperature.

33. Use of a composition according to any one of claims 1 to 20 in stabilizing a cell, comprising: The use comprises contacting the cell with the composition, wherein the contacting prevents release of nucleic acids and / or extracellular vesicles from the cell.

34. The use of claim 33, wherein the cells are white blood cells.

35. The use of claim 33, wherein the cells are red blood cells.

36. The use of any one of claims 33 to 35, wherein the nucleic acid is cell-free RNA, cell-free DNA, cellular RNA, or cellular DNA.

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