WO2021067588A1 - Stabilization of total dna and rna complement and proteins from whole blood - Google Patents

Abstract

Matrices and methods of stabilizing and storing biological samples containing total DNA, total RNA, and proteins from whole blood. The present application in particular provides systems and methods for stabilizing the biological samples as raw samples, where the stabilization and storage occur before additional processing, isolation, and/or analytical steps have taken place.

Description

TITLE: STABILIZATION OF TOTAL DNA AND RNA COMPLEMENT AND PROTEINS FROM WHOLE BLOOD

CROSS-REFERENCE

This application is related to and claims priority under 35 U.S.C. § 119 to U.S. Provisional Application Ser. No. 62/908,906 filed on October 1, 2019 and entitled “STABILIZATION OF TOTAL DNA AND RNA COMPLEMENT AND PROTEINS FROM WHOLE BLOOD”; the entire contents of this patent application are hereby expressly incorporated herein by reference

GOVERNMENT SPONSORSHIP

This invention was made with government support under Grant Contract Number 12244564 awarded by the NIAID division of the NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to matrices and methods for stabilizing and storing biological samples containing total RNA, total DNA, and proteins in whole blood, particularly whole blood as dried blood spots (DBS) on paper, wherein the stabilization and storage occurs without requiring additional processing, isolation, and/or analytical steps.

BACKGROUND OF THE INVENTION

Many industries require effective methods and systems of stabilizing and storing fully intact nucleic acid sequences obtained from raw samples, such as genomic DNA, RNA, and proteins obtained from whole blood and plasma. For the pharmaceutical, medical, law enforcement, military, and other molecular research industries, it is highly desirable to store and have access to many biological samples containing nucleic acids. Stabilization and storage methods must maintain long-term sample integrity to prevent the loss of materials which are often irreplaceable or otherwise difficult to acquire. Further, to allow facilities to obtain and store a high volume of nucleic acids, such stabilization and storage means must be easily transportable and allow for a streamlined processing and handling of a high volume of samples, while not requiring complicated and expensive maintenance.

Existing methods of stabilizing and storing nucleic acids obtained from raw samples suffer from high cost and/or poor sample integrity. For example, the standard method for storage and preservation of RNA is at ultra-low temperatures, usually through the use of liquid nitrogen and/or freezers. However, shipping samples in this manner is expensive, hazardous, and often results in the samples being subject to high variations in temperature during the shipping process. Alternatively, some existing methods turn to desiccation. Although desiccated samples are less expensive to ship, desiccated samples require extensive laboratory preparation in order to stabilize the samples. This preparation is usually not feasible when the nucleic acids to be stabilized are in a raw sample, i.e. found in whole blood or plasma, as the sample must be stabilized and stored before nucleic acid isolation and additional processing.

It is a challenge to stabilize proteins (viral, eukaryotic, prokaryotic, etc.). The problem is further exacerbated in nucleic acids, which can degrade very quickly if stored in improper conditions. As a result, it is difficult to effectively stability DNA, including total DNA. RNA is especially labile; it can spontaneously degrade even in an aqueous medium. As a result, the storage of RNA — viral and total — poses a significant challenge beyond that of most nucleic acids. This has been discussed in the literature. For example, Garcia- Lerma et al of CDC describes the difficulties of storing viruses in dried plasma spots and dried blood spots at ambient. Garcia-Lerma et al. Rapid decline in the efficiency of HIV drug resistance genotyping from dried blood spots (DBS) and dried plasma spots (DPS) stored at 37C and high humidity , Journal of Antimicrobial Chemotherapy 64(1 ):33-6 (May 2009). Consequently, there is a need in the field to develop additional DNA, RNA, and protein storage materials and systems.

SUMMARY OF THE INVENTION

Therefore, it is a principal object, feature, and/or advantage of the present invention to provide systems and methods for the long-term preservation of nucleic acids from raw samples, such as DNA, RNA, and/or proteins, wherein the system preserves sample integrity at low cost under a variety of temperatures, humidity levels, and conditions.

Many industries require inexpensive, user-friendly, long-term storage systems for nucleic acids. Most biological and molecular research applications need to be able to store and analyze a high volume of samples, especially for high throughput screening/analysis. If the samples require complicated cooling or stabilization means, the systems are too costly. However, if the storage system requires too much labor or preparation, it is too time consuming and/or laborious to analyze a very high volume of samples. Similarly, in the contexts of epidemiology and laboratory disease testing, the loss of sample integrity during specimen transport or storage can lead to false-negative diagnostic results. Finally, in law enforcement and military applications, the loss of sample integrity can result in the loss of nucleic acid material that is irreplaceable, such as trace samples collected in the course of criminal investigation. Furthermore, in many applications and particularly in law enforcement, a high recovery of the sample material is critical, as even a 90% recovery may yield too little material to analyze.

Due to the high correlation with physical and immune conditions and other forms of identification, DNA, RNA and protein samples from blood are common examination targets for non-invasive physical tests and/or biomedical studies. However, it is challenging to provide effective stabilization and storage systems with respect to nucleic acids and proteins. Both can degrade very quickly. Aqueous RNA can be degraded by spontaneous phosphodiester bond cleavage as a result of acid or base catalyzed transesterification from the intramolecular nucleophilic attack of the 2' hydroxyl group on the phosphorous atom. Additionally, ribonuclease (RNases) which enzymatically degrade aqueous RNA are virtually ubiquitous in all cells, and pose a constant threat of contamination and degradation of purified RNA. Although DNA is somewhat more resistant than RNA, DNA quantity dramatically decreases due to WBC lysis.

These problems are particularly prescient for blood. Blood samples are often collected at one site and processed for isolation elsewhere. Under these circumstances, for example, the sample must typically be stabilized prior to shipping and purification. The first hurdle is that blood has a complex cellular composition, making it difficult to stabilize, particularly as whole blood. Any stabilization and storage system must account for the particular storage requirements of each component in the whole blood, e.g. DNA, RNA, proteins. Some existing storage systems combat the problem of complexity by requiring extraction, separation, and/or other processing prior to storage. Such solutions can be time-consuming, costly, and may still not result in a high recovery of relevant biological material.

The second hurdle is the instability of the proteins, DNA, and especially RNA in whole blood. Existing storage systems combat this problem by storing RNA at between -20 °C to -80 °C, or in liquid nitrogen to provide protection from degradative reactions. Significantly, existing methods cannot effectively stabilize and store RNA — especially RNA from whole blood or plasma — at room temperature. Further, existing low- temperature methods are extremely costly, as shipping RNA on dry is expensive, requires special handling, is subject to air travel regulations, is time sensitive, and requires a high cost of storage upon arrival to a destination in terms of the cost to run and maintain ultra- low temperature (ULT) freezers. Even when it is economically feasible to use cold temperature or liquid nitrogen storage, these methods are not failsafe. Power outages, natural disasters, shipping accidents, and machine malfunction, to name a few, have resulted in the loss of millions of dollars of biomolecular samples. Other available storage systems including dry storage and aqueous storage media generally require additional processing steps, both to prepare the sample for storage and to recover the sample from its storage state. These methods are often costly and/or time-consuming, reducing the feasibility of processing and handling a high volume of samples efficiently. Example storage conditions are discussed in further detail in Huang et al., The Effects of Storage Temperatures and Duration of Blood Samples on DNA and RNA Qualities , PlosOne, 1-13 (September 18, 2017), which is herein incorporated by reference in its entirety.

It is therefore an object of the present application to provide systems and methods for the inexpensive, long-term, and effective storage of DNA, RNA and proteins from whole blood at room temperature.

It is a further object of the present application to provide such systems and methods, wherein the systems and methods are capable of storing DNA, RNA and proteins from whole blood at room temperature.

It is a further object of the present application to provide systems and methods for the preservation of whole blood in a raw sample using a matrix ( e.g . a solid-state matrix) comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, and a cell separation reagent. In other embodiments, the composition is a matrix (e.g. a solid-state matrix) comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singlet oxygen quencher, an RNase inhibitor and a stabilizer. In some embodiments, the stabilizer is a cell separation reagent. In an embodiment, two metal chelators are used, and in further embodiments the two metal chelators comprise citric acid and an aminocarboxylate. In some embodiments, the hydroxyl radical scavenger comprises mannitol, a pH adjuster comprises lithium dodecyl sulfate and/or lithium hydroxide, the singlet oxygen quencher comprises cysteine, the RNase inhibitor comprises ATA, and the cell separation reagent comprises a polyethylene glycol.

According to an aspect of the present application, the composition stabilizes and stores a sample. In an embodiment, the sample is a sample of whole blood or plasma, and the sample is stored for at least five days. In an aspect, the sample is stored on a paper carrier as dried blood spots (DBS) or dried plasma spots (DPS). In a further aspect, the composition stabilizes and stores viral RNA at a temperature between about 20 °C and about 60 °C, including ambient temperature.

In another aspect, the present invention provides for a kit for stabilizing and storing whole blood samples, wherein the kit includes a composition comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singled oxygen quencher, an RNase inhibitor, and a cell separation reagent; one or more carriers; and one or more carriers. In in embodiment, the composition is combined with a sample comprising whole blood, the sample is then held in the one or more carriers, and the sample is sealed by the one or more closures. According to an aspect of the present application, the composition of the kit stabilizes whole blood for at least five days, and at an ambient temperature.

In an embodiment, the one or more carriers of the kit may comprise one or more vials, one or more wells, paper, and/or a cotton swab. The kit may further comprise an additional container for housing the composition and sample held in the one or more carriers and sealed by the one or more closures. In an aspect, the additional container comprises a box and/or an envelope. According to an embodiment, the kit may further comprise a pre-addressed mailing label.

In an aspect, methods of using the composition and/or kit are provided. In particular, the present application provides a method of using a kit for stabilizing and storing whole blood samples, the method comprising providing a composition comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singled oxygen quencher, an RNase inhibitor, and a cell separation reagent, wherein the composition stabilizes whole blood, including DNA, RNA and proteins, for at least five days, and wherein the composition stabilizes whole blood at an ambient temperature; collecting one or more raw samples; mixing the one or more raw samples with the composition in one or more carriers; and sealing the mixture in the carrier with closures.

In an aspect, the method further comprises the step of placing the sealed mixture in an additional container for housing the one or more carriers. The method may also comprise the step of adding protective materials to the additional container, wherein the protective materials comprise protective foam, packing peanuts, and/or shredded paper filler. Further, the method may also comprise the step of applying a pre-addressed mailing label to the additional container, and shipping the kit.

In a further aspect, the present application provides for a method of making and using the composition for stabilizing and storing whole blood. The method may comprise combining one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singlet oxygen quencher, an RNase inhibitor, and a cell separation reagent. The composition may be provided as a concentrate or diluted using a suitable solvent. In a further aspect, a method of using the composition is provided, the method comprising combining the composition with a sample to form a mixture and/or matrix (depending on the phase of the composition and sample), and placing the mixture and/or matrix into one or more carriers.

The composition may be provided as a liquid or a solid, and may be dehydrated or rehydrated as needed during the method of use. For example, the composition may be first provided as a liquid and subsequently dehydrated for the storage of a sample. The composition may be embedded in, saturated on, or may otherwise inundate a solid medium, such as paper or any other suitable matrix. In an aspect, the matrix and/or paper encapsulates, captures, and/or suspends the sample, facilitating stable storage of the sample.

In a further aspect, stabilization and storage of the sample as described herein occur before additional processing, isolation, and/or analytical steps have taken place, thus enabling the stabilization and storage of a raw sample. Additional aspects and details of the invention will be evident from the detailed description that follows. While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 depicts how paper is modified with base chemistries according to the disclosure to enhance the stabilization and storage of DNA, RNA, and proteins from whole blood.

Fig. 2 shows the TapeStation results identifying the number of copies of 18S rRNA found in a frozen blood sample, and treated and untreated TFN paper following

Fig. 3 depicts the results of stress-testing RNA treated with GTB formulations. Test RNA (16 ng) was added to a 6 mm punch of 903, and GTA or GTB-treated paper alone or in combination with 10 μL of whole blood (+B lanes) was added to the paper. All paper was incubated at high temperatures, was then extracted and analyzed for quality.

Fig. 4 shows the stabilization of total RNA in whole blood and plasma after incubation at 72 °C over a period of 88 hours.

Fig. 5 shows the antimicrobial activity of GTB-treated paper against Escherichia coli , Staphylococcus epidermidis and Aspergillus niger.

Fig. 6 shows the results of a time study of HIV stabilization on treated TFN paper compared to untreated TFN and 903 papers at 40 °C and a variety of humidity conditions.

Fig. 7 depicts the stabilization of zika virus in whole blood on 903 paper, untreated GenProtect paper, and treated GenProtect papers.

Fig. 8 shows yellow fever virus stabilization in whole blood on 903 paper, untreated GenProtect paper, and treated GenProtect papers.

Fig. 9 depicts the DNA isolated from GenProtect paper and whole blood.

Fig. 10 shows the average DNA concentration from samples taken from untreated and treated papers.

Various embodiments of the enzymatic detergent compositions, methods of use, and methods of manufacture are described herein. Reference to various embodiments does not limit the scope of the invention. Figures represented herein are not limitations to the various embodiments according to the invention and are presented for exemplary illustration of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The embodiments of this invention are not limited to particular systems and methods for stabilizing and storing raw samples containing nucleic acids, particularly whole blood and plasma samples, which can vary. It is further to be understood that all terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting in any manner or scope. For example, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” can include plural referents unless the content clearly indicates otherwise. Further, all units, prefixes, and symbols may be denoted in its SI accepted form.

Numeric ranges recited within the specification are inclusive of the numbers within the defined range. Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2¾, 3, 3.80, 4, and 5).

So that the present invention may be more readily understood, certain terms are first defined. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention pertain. Many methods and materials similar, modified, or equivalent to those described herein can be used in the practice of the embodiments of the present invention without undue experimentation, the preferred materials and methods are described herein. In describing and claiming the embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

The term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, concentration, density, distance, mass, pH, population, temperature, time, and volume. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities.

The term “actives” or “percent actives” or “percent by weight actives” or “actives concentration” are used interchangeably herein and refers to the concentration of those ingredients involved in cleaning expressed as a percentage minus inert ingredients such as water or salts.

The term “weight percent,” “wt.%,” “percent by weight,” “% by weight,” and variations thereof, as used herein, refer to the concentration of a substance as the weight of that substance divided by the total weight of the composition and multiplied by 100. It is understood that, as used here, “percent,” “%,” and the like are intended to be synonymous with “weight percent,” “wt.%,” etc.

The terms “nucleic acid,” “oligonucleotide” and “polynucleotide” may be used interchangeably and encompass DNA, RNA, cDNA, whether single stranded or double stranded, as well as chemical modifications thereof and artificial nucleic acids ( e.g PNA, LNA, etc.). The source of the nucleic acids may vary, including but not limited to RNA derived from whole blood and plasma, especially viral RNA.

The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.

The terms “residue” or “amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively “protein”). The amino acid may be a naturally occurring amino acid and, unless otherwise limited, may encompass known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The terms “matrix,” “dry state,” and “solid-state matrix” as used herein refer to cellulose paper that has been impregnated with the stabilizing solution according to the present application.

The terms “stabilize” and “preserve” as used herein mean to render resistant to hydrolytic damage, oxidative damage, irreversible denaturation (unfolding or loss of secondary or tertiary structure), mechanical damage due to shearing or other force, and other damage. This resistance to damage also results in a retention of function and maintenance of integrity of a sample. Retention of function which is preserved and stabilized may include, without limitation, a pair of forward and reverse primers retaining their ability to prime amplification of a target polydeoxyribonucleotide or a target nucleic acid (e.g., genetic) locus; a reverse transcription primer retaining its ability to prime reverse transcription of a target polyribonucleotide; a biological sample retaining its biological activity or its function as an analyte in an assay, or components in the biological sample retaining their biological activity or their function as analytes in an assay; and bacterial cells retaining their infectivity in an appropriate medium (e.g., an agar medium or a fluid culture), or viral particles retaining their infectivity in an appropriate medium (e.g., a natural fluid or a laboratory cell culture).

As used herein, the terms “raw sample,” “raw material,” “whole sample” and “whole material” refer to a basic substance in its natural, modified, or semi-processed state wherein the material is not yet fully processed or prepared. The raw samples of the present application generally contain wholly or a high quantity of intact cells, i.e. cells that have not yet been intentionally lysed. Although some cells in a raw sample may be ruptured due to natural causes or the state of the sample upon collection, a raw sample according to the present application does not contain cells intentionally ruptured, or otherwise processed or prepared.

As used herein, the term “lysis” refers to the breaking down of the cell, often by viral, enzymatic, or osmotic reactions that comprises cell wall integrity. Cell lysis is used to break open cells to avoid shear forces that would otherwise denature or degrade sensitive proteins, DNA, RNA, and other components.

As used herein, the term “whole blood” means blood having none of the constituent components removed or intentionally separated. Whole blood contains, for example, red cells, white cells, and platelets suspended in blood plasma. Whole blood generally comprises approximately 55% plasma, 45% red blood cells, and <1% white blood cells and platelets. The whole blood may include components endemic to whole blood, and the whole blood may also include components nonnative to whole blood, including but limited viral, bacterial, pharmaceutical or other microorganism material such as HIV, hepatitis B, hepatitis C, etc. As used herein, the term “plasma” references the liquid portion of blood which, when part of whole blood, suspends red and white blood cells and platelets. Blood plasms generally contains about 92% water, 7% vital proteins (e.g. albumin, gamma globulin, and anti -hemophilic factor), and 1% mineral salts, sugars, fats, hormones and vitamins. The term “plasma” as used herein can refer to plasma occurring as part of whole blood, and/or it can refer to plasma separated from whole blood. The term “plasma” also encompasses all plasma derivatives, whether the derivatives occur within the plasma or have been separated from the plasma via fractionation. The plasma derivatives may be components endemic to plasma, including but not limited to Factor VIII Concentrate, Factor IX Concentrate, Anti- Inhibitor Coagulation Complex (AICC), Albumin, Immune Globulins, Anti-Thrombin III Concentrate, Alpha 1 -Proteinase Inhibitor Concentrate. The plasma derivatives may also be components nonnative to plasma, including but limited viral, bacterial, pharmaceutical or other microorganism material such as HIV, hepatitis B, hepatitis C, etc. Plasma may further include circulating RNA and other circulating genetic or other biomarker materials.

As used herein, the terms ambient temperature” or “room temperature” refers to a temperature range from about 18°C to about 27°C, or from about 20°C to about 25°C, or from about 22°C to about 40°C. In other embodiments, the term “ambient temperature” or “room temperature” refers to a temperature of about 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C or 27°C. In certain embodiments, the term “ambient temperature” or “room temperature” refers to a temperature of about 22°C,37°C, 39°C or 42°C. Compositions

The compositions of the present application may be used to stabilize and store one or more raw samples, particularly samples comprising whole blood. The compositions of the present application are capable of inhibiting and/or mitigating undesirable contact between the raw sample (and components therein) and various contaminants or potential sources of degradation.

In some embodiments, the compositions of the present application are inert with respect to the raw samples (and components therein). As used herein, “inert” means that the inorganic compound either does not bind to one or more types of samples or binds reversibly such that the raw samples are not degraded as a result of such binding. Further, in an embodiment, the compositions of the present application are inert with respect to one or more downstream methods that may be used to analyze the raw samples and components therein. In this context, “inert” means that the presence of the compositions of the present application together with a raw sample does not reduce the rate of the downstream methods of analysis by more than 50% and does not significantly reduce the fidelity of the method. Exemplary methods of analysis may include, without limitation, nucleic acid transcription and/or amplification ( e.g ., reverse transcription, PCR, real time PCR, etc.), endonuclease digestion (e.g., reactions involving type II endonucleases, such as EcoRI, BamHI, Hindlll, Notl, Smal, Bglll, etc.), cloning techniques (e.g, ligation), protein digestion (e.g, reactions involving proteinases such as proteinase K, trypsin, chymotrypsin, savinase, etc.), microarray analysis (e.g, of nucleic acids or proteins), immunoassays (e.g, immunoprecipitation, ELISA, etc.), mass spectroscopy, or any combination thereof. In certain embodiments, the inorganic compound is inert upon dilution (e.g, dilution by a factor of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more).

In an embodiment, the components in the composition of the present application may also be water soluble. As used herein in this context, “water soluble” means that the inorganic compound has a solubility in water, at 25 °C, of 1.0 mg/ml or greater. In certain embodiments, the inorganic compound has a solubility in water, at 25 °C, of at least 1.5 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml, 5.0 mg/ml, 7.5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 35 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, 200 mg/ml, or greater. In certain embodiments, the inorganic compound can be easily solubilized in water. For example, in certain embodiments, the inorganic compound can be solubilized in water, at 25 °C, in 75, 60, 50, 40, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer minutes. In other embodiments, the inorganic compound can be solubilized in water, at 25 °C, in 7, 6, 5, 4, 3, 2, 1.5, or fewer hours. In certain embodiments, the inorganic compound can be solubilized in water, at 25 °C, with or without the use of agitation (e.g, pipetting, shaking, or vortexing).

The compositions of the present application may comprise: one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singlet oxygen quencher, an RNase and/or DNase inhibitor, a cell separation reagent, and additional ingredients. pH Buffers/Adjusters

In some embodiments, the composition includes one or more pH buffers/adjusters.

The pH buffers/adjusters may be used to modify the pH of the composition and in doing so act as a precipitating agent. In some embodiments, the pH buffer is any of a large number of compounds known in the art for their ability to resist changes in the pH of a solution, such as an aqueous solution, in which the pH buffer is present. Selection of one or more particular pH buffers for inclusion in a stable storage composition may be done based on the present disclosure and according to routine practices in the art, and may be influenced by a variety of factors including the pH that is desirably to be maintained, the nature of the sample to be stabilized, the solvent conditions to be employed, the other components of the formulation to be used, and other criteria. For example, typically a pH buffer is employed at a pH that is within about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 pH unit of a proton dissociation constant (pKa) that is a characteristic of the buffer.

Examples of suitable pH buffers include, without limitation, pH buffers include citric acid, tartaric acid, malic acid, sulfosalicylic acid, sulfoisophthalic acid, oxalic acid, borate, CAPS (3 -(cyclohexylamino)- 1 -propanesul fonic acid), CAPSO (3- (cycl ohexyl amino)-2-hy droxy- 1 -propanesulfonic acid), EPPS (4-(2-hydroxy ethyl)- 1 - piperazinepropanesulfonic acid), HEPES (4-(2-hy draxyethyi)piperazine- 1 -ethanesuifonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), MOPSO (3-morpholino-2-hydroxypropanesulfonic acid), PIPES ( 1 ,4-piperazinediethanesuifonic acid), TAPS (N[tris(hydroxymethyl)methyl]- 3 -ami nopropanesulfoni c acid), TAPSO (2-hydroxy-3-[tris(hydroxymethyi)methylamino]- 1 -propanesulfonic acid), TES (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic arid), birine (N,N-bi s(2-hydroxy ethyl )gl y cine), tririne (N-

[tris(hydroxymethyl)methyI]glycine), tris (tris(hydroxymethyl)aminom ethane), bis-tris (2- [bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-l, 3-propanediol), 5-(4-dimethyl)amino benzylidene rhodanine, sulfosalicylic acid, lithium chloride, and lithium hydroxide, and/or lithium dodecyl sulfate.

In a preferred embodiment, the pH buffer/adjuster is lithium hydroxide and/or lithium doecyl sulfate. Surprisingly, the present application has found that lithium, in particular lithium hydroxide and lithium dodecyl sulfate provide significantly improved stability of whole RNA and DNA in blood samples, particularly dried blood spots.

With the use of a pH buffer/adjuster, the compositions may have a pH of about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0. In an embodiment, the pH buffer/adjuster may be present in the composition in an amount of from about 0.1 mM (1.8 mg/dl) to about 10 mM (180 mg/dl), 10 mM (180 mg/dl) to 50 mM (900 mg/dl), 50 mM (900 mg/dl) to 100 mM (1800 mg/dl), 100 mM (1800 mg/dl) to about 300 mM (5400 mg/dl), preferably between about 150 mM (2700 mg/dl) to about 250 mM (4500 mg/dl), and more preferably between about 175 mM (3150 mg/dl) to about 225 mM 4050 mg/dl).

Metal Chelator

In some embodiments, the composition contains one or more metal chelators. In an embodiment, the composition contains two or more metal chelators. As used herein, a “metal chelator” is a compound that forms two or more bonds with a single metal ion. In certain embodiments, the one or more metal chelators chelate at least one type of metal ion selected from the group consisting of magnesium ions, chromium ions, manganese ions, iron ions, cobalt ions, nickel ions, copper ions, zinc ions, lead ions, or any combination thereof. In certain embodiments, the one or more metal chelators chelate at least one type of metal ion and inhibit metal-dependent reactions between such ions and raw sample present in the composition. In certain embodiments, the one or more metal chelators chelate at least one type of metal ion and prevent such ions from degrading the raw sample (i.e. cells, components within the cells such as nucleic acids, and other materials of the raw sample) present in the composition. In preferred embodiments, the one or more metal chelators chelate magnesium ions and/or manganese ions and inhibit metal dependent reactions between such ions and biomolecules present in the composition. In other preferred embodiments, the one or more metal chelators chelate magnesium ions and/or manganese ions and prevent such ions from degrading biomolecules present in the composition.

Examples of suitable metal chelators include without limitation boric acid, aurintricarboxylic acid (ATA) and salts thereof [e.g., triammonium aurintricarboxylate (aluminon)], borate, citric acid, citrate, salicylic acid, salicylate, l,2-bis(o- aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), glycoletherdiaminetetraacetic acid (GEDTA), N-(2- hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), 2,2'-bipyridine, o-phenanthroline, triethanolamine, and analogs, derivatives and salts thereof. In an embodiment, the composition is substantially free of boric acid.

The one or more metal chelators may be present in the composition from about 1.5 mM (27 mg/dl) to about 300 mM (5400 mg/dl), preferably between about 150 mM (2700 mg/dl) to about 250 mM (4500 mg/dl), preferably between about 160 mM (2889 mg/dl) to about 220 mM (3960 mg/dl), and more preferably between about 175 mM (3150 mg/dl) to about 200 mM (3600 mg/dl).

Hydroxyl Radical Scavenger/Oxygen Radical Scavenger

The composition may comprise a hydroxyl radical scavenger/oxygen radical scavenger. These scavengers are capable of inhibiting undesirable contact between the raw sample (and components therein) and various contaminants or potential sources of degradation. Hydroxy radical scavengers can in particular protect against the effects of oxygen.

Examples of suitable hydroxyl radical scavengers include, but are not limited to mannitol (including D-mannitol) and other sugar alcohols such as erythritol, sorbitol and xylitol, azides, cysteine, including L-cysteine, N- Acetyl Cysteine etc., lithium dodecyl sulfate (LiDS), dimethylsulfoxide, histidine, salicylic acid, salicylate, monosaccharides, disaccharides (e.g., cellobiose, lactose, maltose, sucrose, and trehalose), complex sugars, and analogs, derivatives and salts thereof.

Examples of suitable oxygen radical scavengers include, but are not limited to, sugar alcohols (e.g., erythritol, mannitol, sorbitol, and xylitol), monosaccharides (e.g., hexoses, allose, altrose, fructose, fucose, fuculose, galactose, glucose, gulose, idose, mannose, rhamnose, sorbose, tagatose, talose, pentoses, arabinose, lyxose, ribose, deoxyribose, ribulose, xylose, xylulose, tetroses, erythrose, erythrulose, and threose), disaccharides (e.g., cellobiose, lactose, maltose, sucrose, and trehalose), complex sugars (e.g., trisaccharides, kestose, isomaltotriose, maltotriose, maltotriulose, melezitose, nigerotriose, raflfmose, tetrasaccharides, stachyose, fructo-polysaccharides, galacto- polysaccharides, mannan-polysaccharides, gluco-polysaccharides, glycogen, starch, amylose, amylopectin, dextrin, cellulose, glucans, beta-glucans, dextran, fructans, inulin, glucosamine polysaccharides, chitin, aminoglycosides, apramycin, gentamycin, kanamycin, netilmicin, neomycin, paromomycin, streptomycin, tobramycin, glycosaminoglycans (mucopolysaccharides), chondroitin sulfate, dermatan sulfate, keratan sulfate, heparin, heparan sulfate, and hyaluronan), and analogs, derivatives and salts thereof.

The oxygen radical scavenger/hydroxyl radical scavenger may be present in the composition from about 100 mM (1800 mg/dl) to about 300 mM (5400 mg/dl), preferably between about 150 mM (2700 mg/dl) to about 250 mM (4500 mg/dl), and more preferably between about 175 mM (3150 mg/dl) to about 225 mM 4050 mg/dl). Singlet Oxygen Quencher

A singlet oxygen quencher is capable of inhibiting undesirable contact between the raw sample (and components therein) and various contaminants or potential sources of degradation. Singlet oxygen quenchers can in particular protect against the effects of oxygen.

Examples of suitable singlet oxygen quenchers include, but are not limited to, alkyl imidazoles ( e.g ., histidine, L-camosine, histamine, imidazole 4-acetic acid), indoles ( e.g ., tryptophan and derivatives thereof, such as N-acetyl-5-methoxytryptamine, N- acetyl serotonin, 6- methoxy-l,2,3,4-tetrahydro-beta-carboline), sulfur-containing amino acids (e.g., methionine, ethionine, djenkolic acid, lanthionine, N-formyl methionine, felinine, S-allyl cysteine, L-selenocysteine, S-[2-(4-pyridyl)ethy]-L-cysteine, S- diphenylmethyl-L-cysteine, S-trityl-homocysteine, L-cysteine, , N-acetyl-cysteine, S-ally- L-cysteine sulfoxide, S-aminoethyl-L-cysteine), phenolic compounds (e.g, tyrosine and derivatives thereof), aromatic acids (e.g, ascorbate, salicylic acid, and derivatives thereof), azides such as sodium azide, tocopherol and related vitamin E derivatives, and carotene and related vitamin A derivatives.

The singlet oxygen quencher may be present in the composition from about 100 mM (1800 mg/dl) to about 250 mM (4500 mg/dl), preferably between about 150 mM (2700 mg/dl) to about 225 mM (4050 mg/dl), and more preferably between about 175 mM (3150 mg/dl) to about 200 mM (3600 mg/dl).

Inhibitors

Depending on the components of interest within the raw sample, the composition may comprise one or more RNase and/or DNase inhibitors. Suitable inhibitors may include, without limitation, aurintricarboxylic acid (ATA) and salts thereof [e.g., triammonium aurintricarboxylate (aluminon)], boric acid, borate, citric acid, citrate, salicylic acid, salicylate, l,2-bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid (BAPTA), diethylene triamine pentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA), ethylene glycol tetraacetic acid (EGTA), glycoletherdiaminetetraacetic acid (GEDTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (HEDTA), nitrilotriacetic acid (NTA), 2,2'-bipyridine, o-phenanthroline, triethanolamine, mammalian ribonuclease inhibitor proteins [e.g., porcine ribonuclease inhibitor and human ribonuclease inhibitor (e.g., human placenta ribonuclease inhibitor and recombinant human ribonuclease inhibitor adenosine 5'- pyrophosphate, 2'-cytidine monophosphate free acid (2'-CMP), 5'-diphosphoadenosine 3 '-phosphate (ppA-3'-p), 5'-diphosphoadenosine 2'- phosphate (ppA-2'-p), leucine, oligovinysulfonic acid, poly(aspartic acid), tyrosine- glutamic acid polymer, 5'-phospho-2'-deoxyuridine 3 '-pyrophosphate P' 5 '-ester with adenosine 3 '-phosphate (pdUppAp), and analogs, derivatives and salts thereof.

The RNase and/or DNase inhibitors may be present in the composition from about 0.1 mM (1.8 mg/dl) to about 10 mM (180 mg/dl), preferably between about 0.5 mM (9 mg/dl) to about 7 mM (126 mg/dl), and more preferably between about 1 mM (18 mg/dl) to about 5 mM (90 mg/dl).

Stabilizers

In some embodiments, the composition comprises one or more stabilizers. In an embodiment, the composition comprises two or more stabilizers. As used herein, a “stabilizer” is any agent capable of protecting nucleic acids, particularly nucleic acids occurring in a raw sample, from damage during storage. This may include without limitation, for example circulating RNA, viral RNA, DNA, and others.

In a preferred embodiment the stabilizer comprises a cell separation reagent. In a preferred embodiment, the cell separation reagent is polyethylene glycol. Suitable examples of cell separation reagents include, without limitation, polyethylene glycol 200 (PEG 200), polyethylene glycol 300 (PEG 300), polyethylene glycol 400 (PEG 400), polyethylene glycol 540 (PEG 540), polyethylene glycol 600 (PEG 600), polyethylene glycol 1000 (PEG 1000), polyethylene glycol 1450 (PEG 1450), polyethylene glycol 3350 (PEG 3350), polyethylene glycol 4000 (PEG 4000), polyethylene glycol 4600 (PEG 4600), polyethylene glycol 8000 (PEG 8000), Carbowax MPEG 350, Carbowax MPEG 550, Carbowax MPEG 750, and others. The stabilizer may be present in the composition from about 35 wt.% to about 65 wt.%, preferably between about 40 wt.% to about 60 wt.%, and more preferably between about 45 wt.% to about 55 wt.%.

Additional Ingredients

In some embodiments, the compositions can optionally contain one or more additional ingredients. For example, an antimicrobial agent, an organic or inorganic dye, a plasticizer, a preservative, a reducing agent, a hydroperoxide removing agent, a detergent, a buffering agent, a pH adjuster, an excipient, a bulking agent, a dispersion agent, a solubilizer, a solidification aid, or a combination thereof.

Antimicrobial Agent

The composition may further comprise a microcidal or antimicrobial agent. As used herein, an “antimicrobial agent” is any compound that slows or stops the growth of a microorganism. In certain embodiments, the inorganic compound kills one or more microbial organism, such as a bacterium, protist, and/or fungus. In certain embodiments, the inorganic compound inhibits the growth of one or more microbial organism, such as a bacterium, protist, virus, or fungus. Suitable antimicrobial agents may include, without limitation, penicillin, cephalosporin, ampicillin, amoxycillin, aztreonam, clavulanic acid, imipenem, streptomycin, gentamycin, vancomycin, clindamycin, polymyxin, erythromycin, bacitracin, amphotericin, nystatin, rifampicin, tetracycline, chlortetracycline, doxycycline, chloramphenicol, ammolfme, butenafme, naftifme, terbinafme, ketoconazole, fluconazole, elubiol, econazole, econaxole, itraconazole, isoconazole, imidazole, miconazole, sulconazole, clotrimazole, enilconazole, oxiconazole, tioconazole, terconazole, butoconazole, thiabendazole, voriconazole, saperconazole, sertaconazole, fenticonazole, posaconazole, bifonazole, flutrimazole, nystatin, pimaricin, amphotericin B, flucytosine, natamycin, tolnaftate, mafenide, dapsone, caspofungin, actofunicone, griseofulvin, potassium iodide, Gentian Violet, ciclopirox, ciclopirox olamine, haloprogin, silver sulfadiazine, undecylenate, undecylenic acid, undecylenic alkanolamide, Carbol- Fuchsin, nevirapine, delavirdine, efavirenz, saquinavir, ritonavir, indinavir, nelfmavir, amprenavir, zidovudine (AZT), stavudine (d4T), lamivudine (3TC), didanosine (DDI), zalcitabine (ddC), abacavir, acyclovir, penciclovir, valacyclovir, ganciclovir, Rutin, Tannic acid, Direct Red 80, Purpurin compounds and analogs, derivatives and salts thereof. Plasticizer

The composition may additional comprise a plasticizer. As used herein, a “plasticizer” is any agent capable of facilitating or improving the storage function of a dry- state matrix. Thus, in certain embodiments, the plasticizer improves the mechanical properties of a dry-state matrix. In certain embodiments, the plasticizer improves the durability, including resistance to vibrational and other damage, of a dry-state matrix. In certain embodiments, the plasticizer facilitates the reversible dissociation between inorganic compounds and raw sample upon re-hydration of a dry-state matrix. In other embodiments, the plasticizer facilitates the reversible dissociation between stabilizers and raw sample upon re-hydration of a dry-state matrix.

Suitable plasticizers may include polyols such as long-chain polyols, short-chain polyols, and sugars. The plasticizer may include, without limitation, polyvinyl alcohol, polyserine, monosaccharides, disaccharides, complex sugars, ethylene glycol, 1-3 propane diol, glycerol, butane triol (e.g., n-butane triol or isobutane triol), erythritol, pentane triol (e.g, n- pentane triol or isopentane triol), pentane tetraol (e.g, n-pentane tetraol, isopentane tetraol), pentaerythritol, xylitol, sorbitol and mannitol.

Preservatives

The composition may further comprise preservatives used to further prevent the degradation of and damage to the raw sample (and components therein).

Reducing Agents

The composition may additional comprise a reducing agent. Examples of suitable reducing agents include, but are not limited to, cysteine and mercaptoethylene. Examples of metal chelators include, but are not limited to, EDTA, EGTA, o-phenanthroline, dithionite, dithioerythritol, dithiothreitol (DTT), dysteine, 2-mercaptoethanol, mercaptoethylene, bisulfite, sodium metabi sulfite, pyrosulfite, pentaerythritol, thioglycolic acid, citrate, urea, uric acid, vitamin C, vitamin E, superoxide dismutases, and analogs, derivatives and salts thereof.

Hydroperoxide Removing Agents

The composition may further comprise a hydroperoxide removing agent. Examples of suitable hydroperoxide removing agents include, but are not limited to, catalase, pyruvate, glutathione, and glutathione peroxidases. Raw Sample Material

The raw sample according to the present application generally contains wholly or a high quantity of intact cells, i.e. cells that have not yet been intentionally lysed. Although some cells in a raw sample may be ruptured due to natural causes or the state of the sample upon collection, a raw sample according to the present application does not contain cells intentionally ruptured, or otherwise processed or prepared.

The source of the raw sample may comprise, without limitation, a biological fluid, a biological suspension, a fluid aspirate, blood, plasma, serum, lymph, cerebrospinal fluid, gastric fluid, bile, perspiration, ocular fluid, tears, oral fluid, sputum, saliva, a buccal sample, a tonsil sample, a nasal sample, mucus, a nasopharyngeal sample, semen, urine, a vaginal sample, a cervical sample, a rectal sample, a fecal sample, a wound or purulent sample, hair, a tissue, a tissue homogenate, cells, a cellular lysate, a tissue or cell biopsy, skin cells, tumor or cancer cells, a microbe, a pathogen, a bacterium, a fungus, a protozoan or a virus, or any combination thereof. Preferably the raw sample comprises DNA, RNA, and/or proteins, including nucleic acids such as single- stranded and double-stranded polynucleotides containing RNA nucleotides and/or DNA nucleotides. In a further preferred embodiment, the raw sample comprises one or more nucleic acid types according to the Table 1 below.

Table 1

In an embodiment, the raw sample is contained within and/or bound by the dry state matrix of the present application. In some embodiments, at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the raw sample by mass is contained within and/or bound by the dry state matrix of the present application. The raw sample contained within and/or bound by the composition of the present application may be stored in a closed container ( e.g ., a capped tube, vial or well) at a temperature from about -80 °C to about 40 °C for at least about 1 day, 3 days, week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months,

4 months, 1 year, 1.5 years or 2 years.

Surprisingly, raw samples stored and preserved according to the present application are highly resistant to hydrolytic damage, oxidative damage, denaturation (e.g., irreversible unfolding or irreversible loss of secondary structure or tertiary structure), and other mechanical damage. Further, unexpectedly, the raw samples stored and preserved according to the present application have a high retention of function/activity, and demonstrate this retention of activity for up to 2 years.

Form of the Composition

In an embodiment, the composition is a dry state, such as a dry state matrix. In an embodiment, the components of the composition concentrate upon drying and form a crystalline or paracrystalline structure. In certain embodiments, the composition does not form a glass structure upon drying. As used herein, the term “glass structure” refers to a solid-state structure in which the molecules comprising the glass structure display only short-range order, rather than extended-range crystalline order with respect to one another. In certain embodiments, the components of the composition are capable of co-localization with the raw sample. For example, in certain embodiments, the matrix formed by the components of the composition concentrates upon drying and forms a crystalline or paracrystalline state in direct contact with the cells of the raw sample.

In an embodiment, the composition may be provided as a powder, tablet, pill, or may be carried by a solid support, such as a cotton swab, a filter paper, or a sponge. The composition may also be contained in any suitable container. In a preferred embodiment, the composition and raw sample are carrier by paper, and are stabilized in the form of dried blood spots (DBS) and/or dried plasma spots (DPS). The composition may be directly added to a raw sample (or vice versa), raw sample/liquid mixture, or present in a collection vessel prior to collection of the raw sample or raw sample/liquid mixture. In some embodiments, the composition added to a raw sample, raw sample/liquid mixture, or other type of raw sample fully solidifies. In some embodiments, composition together with raw sample is fully solidified into a matrix. In other embodiments, the composition added to a raw sample, raw sample/liquid mixture, or other type of raw sample only solidifies partially. The partially solidified composition together with raw sample may form a matrix.

In another embodiment, the composition may be delivered in pre-measured aliquots loaded into sample collection vessels and/or wells, to which an appropriate volume of the raw sample may be added. In such a circumstance, the collection vessels and/or wells are agitated to aid in the even distribution and dispersal of both the composition of the present application and the raw sample.

In a further embodiment, a vial for collecting raw samples can be supplied with pre-measured aliquots of the composition of the present application; an appropriate volume of the raw sample may be subsequently added. Much like the collection vessels and/or wells, the vial is then agitated.

In a still further embodiment, the composition of the present application is provided as part of a kit for collecting samples. The kit may comprise a composition according to the present application, a raw sample, a carrier comprising a container or solid support for the composition and raw sample, and instructions for using the kit for the stabilization and storage of a given raw sample. The kits according to the present application may be adapted for shipment by mail. For example, in addition to the composition, raw sample, carrier, and instructions, the kit may comprise closures for closing/sealing the carrier from contamination (such as tape, a sealable bag, a cap, a stopper, or other sealant material), an additional container (comprising a box, flexible pouch, envelope, etc.) for receiving and transporting the carrier, a pre-addressed mailing label, and a protective or cushioning material such as protective foam, packing peanuts, and/or shredded paper filler, etc. Significantly, the system of the present application effectively stabilizes raw samples such that the samples do not need to be refrigerated or frozen during shipping or storage. Methods of Preparation, Storing, and Preserving

In an embodiment, the compositions of the present application can be prepared by mixing one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singlet oxygen quencher, and an inhibitor together with a cell separation reagent, and transferring the resulting mixture to a carrier.

In an embodiment, a raw sample may be stabilized and stored at room temperature for up to 2 years by providing the composition of the present application, collecting one or more raw samples, mixing the one or more raw samples with the composition of the present application, and optionally allowing the mixture to dry. In some embodiments, the mixture will form a matrix. The mixture may be wholly solid, or solid in part.

In a further embodiment, after stabilization and storage for a desired period of time, the raw sample bound in/by the composition of the present application may be rehydrated by the addition of an aqueous solution (e.g., water or an aqueous buffer) shortly before the composition is to be used in a biochemical reaction (e.g., PCR) or an analysis (e.g., an immunoassay).

In an embodiment, the compositions of the present application as provided in a kit may be used by providing the composition of the present application in a carrier, collecting one or more raw samples, mixing the one or more raw samples with the composition in a carrier, sealing the mixture in the carrier with closures, placing the sealed mixture in an additional container, adding protective materials to the additional container, and applying a pre-addressed mailing label to the additional container.

In a still further embodiment, the composition of the present application may be used as part of automated and/or high throughput preparation, stabilization, and storage of raw samples.

EXAMPLES

Embodiments of the present invention are further defined in the following non limiting Examples. It should be understood that these Examples, while indicating certain embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the embodiments of the invention to adapt it to various usages and conditions. Thus, various modifications of the embodiments of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

EXAMPLE 1 Stabilization of viral RNA from Whole Blood To evaluate the stabilization and storage capabilities of viral RNA, paper was treated with several solutions. Initial stock solutions 1-3 were prepared as shown in Tables 2-4 below, with solution 3 including lithium. An ATA solution was added separately (as Reagent B) to stock solution 1. The solutions were added to paper at a load of 95 gm/m² to 188 gm/m². To treat the papers with the solution on paper treated with just the base chemistry, 25 μL of solution was spotted on the paper before spotting the whole blood. The solution spotted on paper was dried at either room temperature or at temperatures of up to 95 °C. The paper was then spotted with 50μL to 150 μL of whole blood. The paper was then dried overnight at room temperature, or up to 30 minutes at 72 °C, or up to 10 minutes at 95 °C.

Table 2. GTA Solution 1

Table 3. GTA Solution 2

Table 4. GTA Solution 3

EXAMPLE 2. Stabilization of Total RNA from Whole Blood

To further evaluate the ability of the invention to stabilize whole RNA, solutions were prepared according to Tables 5 and 7-8 below. The stock solution was prepared by combining components into a first set of reagents (Reagent A) and heating to 60°C. Then, additional components were combined into a second reagent mixture (Reagent B). The two reagents were combined to form the stock solution. A further enhancement solution was also evaluated. The enhancement solution was prepared as shown in Table 6. The solutions and blood were applied to paper using the methods of Example 1, wherein the stock solution was loaded on the paper in an amount of approximately 120 gm/m². The enhancement solution was further used for spotting the paper before spotting the blood, and 25 μL was loaded onto the paper per 50 μL to 200 μL of whole blood.

Table 5. GTB Formulation 1

Table 6. GTB Enhancement Formulation 1

Table 7. GTB Formulation 2

Table 8. GTB Formulation 3

EXAMPLE 3. Stabilization of HIV Virus in Whole Blood & Plasma

Further formulations were developed to stabilize HIV virus in both whole blood and plasma. Using GenProtect paper, punches of 6 mm of treated TFN paper were pressed into 384 well Genplates to provide a uniform area of paper to contain the liquid sample. Each well of the GenPlate was spotted with 10μL of blood mixed with 6,600 copies of HIV virus. GenPlates were incubated at 72 °C for 3 hours. The higher temperatures accelerated the degradation of HIV RNA, allowing faster test results (in 3 hours) and easy identification of successful chemistries. After 3 hours at 72 °C four punches of dried blood spots (DBS) were extracted and sent to Quest for HIV quantification by COB AS® Amplicor test. The results of this testing are shown in Table 9 below.

Table 9. Recovery of HIV RNA

Surprisingly, the formulations of the application containing a lithium component provided improved stabilization of HIV RNA. Storage upon the paper alone had 20%- 50% recovery of HIV RNA. In comparison, the formulations of the invention provided a surprising improvement, showing between 80%-100% recovery of HIV RNA.

EXAMPLE 4. RNA Heat Stress Screening Further analysis was done to assess RNA quality when stored using the systems and chemistries disclosed herein. As a measure of RNA quality, the Agilent TapeStation (TS) RNA Integrity Number (RIN) of 18s and 28s rRNA extracted from the DBS as well as a 450bp long PCR product of 18s rRNA were considered. Often, 18s rRNA is a better degradation indicator than 28s rRNA. We now have qRT-PCR assays for 18s rRNA of 914bp, 750bp, 705bp, 430bp, 255bp and 117bp (overlapping assays within the amplicons). These varied amplicon lengths will help precisely determine the extent of 18S RNA degradation/stabilization. As 18s rRNA is l,800bp in length, if the large 900bp fragment amplifies then it is assumed that >99% protection of this RNA. If the large 900bp fragment fails to amplify or amplifies weakly, the fragments lower in size (750bp and smaller) will then be used in sequential PCRs until failure of the 117bp amplicon would indicate the 18s RNA is degraded >87%. A standard curve using the 914bp amplified product was developed to quantify the 18s rRNA by qRT-PCR. Although the 18S RNA cannot consistently be visualized using a gel (due to the background of total RNA) it is possible to get excellent protection of the 18s rRNA as measured by qRT-PCR. Further discussion of methods for assigning integrity values to RNA is found in Schroeder et al., The RIN: An RNA Integrity Number for Assigning Integrity Values to RNA Measurements , BMC MOL Bio. 7:3, 1-14 (2006), which is herein incorporated by reference in its entirety.

10 μL of whole blood was spiked with HIV and spotted on both untreated and treated TFN paper. The DBS samples in untreated and treated TFN were aged for three days at 72 °C (equivalent to 64 days at ambient temperature). Total RNA was extracted both from untreated and treated DBS, and also from a frozen whole blood sample. All RNA was treated with Dnase I to remove any carryover 18S DNA. As shown in Figure 4, quantification by qRT-PCR of the 914bp amplicon for the 18S rRNA in frozen blood samples and the DBS on untreated TFN paper both yielded 100 copies of 18S rRNA. In stark contrast, there were 10,000 copies of 18S rRNA in DBS on the treated paper. Thus, papers treated with the chemistries of the invention gave 2-logs more RNA compared to untreated papers.

Further analysis was conducted to assess the extent to which GTB-treated paper stabilized high molecular weight RNA doped into blood. The RNA heat stress test was again conducted to assign RNA Integrity Numbers. Prototype GTB treated Ahlstrom- Munksjo papers and untreated Paper type 1 filter paper were treated with whole blood which had been doped with internal RNA standards (rRNA surrogate: 1065 and 2340 bp RNA) and an encapsidated control virus particle (icosahedral positive-sense single stranded RNA virus). After drying, the DBS were stressed at 72 °C for approximately 48 hours. The results are shown in Figure 3. As shown in Figure 3, GTB-treated filter paper stabilized total RNA over 48 hours at 72 °C and had an RNA Integrity Number (RIN) score of 8.4 without blood and 6.3 in a dried blood spot, compared with RIN 9.3 for the RNA starting material. By comparison Paper type 1 paper stabilized the total RNA for only 6 hours at 72 °C.

Figure 3 therefore demonstrates a surprising improvement in paper-based nucleic acid field collection. What is shown is that after prolonged exposure at a temperature of 72 °C, the RNA complement of a dried blood spot remained nearly intact over the lkb-2kb domains tested via Agilent bioanalyzer CE (RIN=6.4). There are no known existing methods of RNA stabilization in dried blood which comes close to the observed level of protection afforded by GTB at the extreme temperatures deployed in the stress testing. EXAMPLE 5. Stabilization of Total RNA in Whole Blood and Plasma

Chemistries developed for viral RNA stabilization work well with HIV virus in whole blood & likely plasma. Encapsidated HIV RNA is much more resistant to degradation than naked viral or cellular (18s & 28s rRNA) RNA. To develop chemistries needed to stabilize total RNA large amounts of RNA are needed for testing. If the total RNA was isolated from whole blood, it is still difficult to maintain the quality of the total RNA as the extracted RNA would degrade rapidly even when frozen at -80 °C. For the purpose of testing, Surrogate RNA (a commercial RNA plasmid system called pGEM express positive control template (T7 RNA polymerase) manufactured by Promega) was employed.

Total RNA was spotted onto paper treated with the GTB formulations (described above as GTB Formulations 1-3 in Tables 6-8) containing a lithium component, according to the process described in Example 1. The RNA-spotted paper was incubated for 2, 24, and 88 hours at 72 °C (equivalent to about 117 days at ambient temperature). The samples were then analyzed by Agilent TS as described in Example 4. Each test was run in duplicate at each time point.

The results are shown in Figure 4. According to Figure 4, lanes 1, 2, 9, 10, 14, 15, and 16 are total RNA with Chemistry 1, and have approximately 77% RNA remaining at 88 hours. Lanes 3, 4, 11, 12, 17, 18, and 19 are total RNA with Chemistry 2, and having about 75% RNA remaining at 88 hours. Lanes 5 an d8 are untreated paper spotted with total RNA, and shows total degradation of RNA at 24 hours under the high temperature conditions. Lanes 7, 13, and 21 are RNA kept at -80 °C, and function as a control. Finally, lanes 6 and 20 have no RNA extracts, and function as a blank, illustrating there is little chemistry background contributing to the results in the treated lanes.

EXAMPLE 6 Antimicrobial Activity of Treated Filter Paper

Formulations containing a lithium component provide improved stabilization of rRNA in papers treated with either a formulation without a lithium component (see Example 1) and a formulation with a lithium component (see Example 1). GTA (FI) and GTB (FII) provide effective stabilization for about 1 year at 25 °C compared to untreated Paper type 1 paper cards. rRNA of a RIN of 9.8 was spotted on the papers and challenged at 72 °C for 14 days to accelerate the ageing of the paper samples. rRNA in equal volumes was spotted on all 3 paper types before challenging. All control sample kept at -80 °C (OT in Figure 5), freshly spotted rRNA samples and the 72 °C and ambient aged samples were extracted using the TriBD organic phase separation method.

The results of assessing antimicrobial activity is shown in Figure 5. Figure 5 shows that GTB chemistry has pronounced antimicrobial activity relative to the Paper type 1 filter paper and the GenTegra GT-A chemistry coated on Ahl strom -Munksjo TFN-D1 filter paper. The ability of GT-B to provide for substantial antimicrobial activity against Escherichia coli , Staphylococcus epidermidis and Aspergillus niger , in ASTM tests performed by Ahl strom -Munksjo, is a major finding and could be a defining attribute of GT-B, especially when international DBS shipping is required per APHIS-USDA standards.

EXAMPLE 7 Increased Recovery of Viral Nucleic Acids from Paper Using the surrogate RNA, recovery from paper treated with the GTB formulations (see Tables 6-8) of the application containing a lithium component was further improved by incorporating chemistries in paper that facilitate release of RNA from TFN upon hydration. Different extraction chemistries & amounts of polymer carrier were compared in order to assess which best facilitate RNA recovery from TFN. 6mm TFN spots punched in 384 plates were treated with the solutions and were spotted with 10μL of 16ng/μL of the surrogate. Two spots for each treated TFN paper were extracted with optimal extraction method. The RNA recoveries from the spots were quantified by Qubit fluorescent method. The results are shown in Table 10 below.

Table 10

Previously it was routine to recover approximately 50% of RNA from treated papers. As shown in Table 10, however, recovery was increased from about 50% to greater than 90%, which is a substantial and surprising improvement.

EXAMPLE 8. Stress Test of Dried Blood Spots on GT-Cards Under Additional

Stress

Samples may inadvertently be subject to a variety of storage conditions. As a result, a successful product should provide stability under a wide range of environmental temperatures and humidity. Dried blood spots were applied to untreated and GTA-treated paper according to the methods of Example 1 at a relative humidity (RH) of >30% at 40 °C, mimicking conditions in the tropics. Dried blood spots stored at >80% RH were compared to dried blood spots stored at 40 °C with approximately 30% RH. RNA from 4 blood spots per time point were extracted at times of 1 and 5 days. The extracted viral RNA was tested at 1 and 5 days by COB AS HIV-1 assay. Experimental controls were duplicate DBS on GT-Cards kept at -80C & 23C. The results are shown in Figure 6, which demonstrates improved storage capabilities of the treated TFN paper, even under high humidity conditions. The same stress test was performed with 2 Flaviviruses - Zika Virus and Yellow

Fever virus. The Flavivirus was spiked in whole blood before spotting on paper. For this study, GP paper spotted with 25 μL of a 50% solution of GTA or GTB enhancement solution was spotted on the paper and dried at 72C for 30 minutes. 100μL of whole blood spiked with Zika or Yellow Fever virus was spotted on the cards and dried overnight at ambient temperature and humidity in a BSL 2 hood. The papers were stressed at 40 °C and 80% humidity mimicking tropical conditions for up to 14 days. Controls stored at ambient

(25 °C) were tested at day 2 and day 8. Additional controls of whole blood spotted on the paper were placed at -80 °C as control samples.

Viral RNA from all of the samples were isolated using the QIAamp Viral RNA kit. qPCR assays for a lkb fragment of both the Flavivirus (Zika and Yellow Fever) were optimized to a short (~80bp) fragment of the viral RNA’s. Titration of the RNA standard for Zika show that the lkb Zika qRT-PCR assay has similar outcome to the ~80bp short assay. Quantity of RNA computed from the qRT-PCR for both the short and long assay compare to the expected theoretical amount of Zika RNA. The results are shown in Tables 11 and 12 below, as well as Figures 7-11.

Table 11. !

Table 12.

GP papers supplemented with enhancement solution added to the paper were compared to paper not supplemented with the enhancement solution and plain Paper type 1 paper before spotting with whole blood spiked with the virus and stressed at 40C and 80% humidity. The papers were stressed without any desiccant for protection against damage from hydrolysis and oxidation of the nucleic acid. As shown in Figures 7-8, papers treated with enhancement solutions gave a favorable outcome for both Yellow fever and Zika compared to papers that were not spotted with enhancement solution.

EXMAPLE 9 Stabilization of Entire Nucleic Acid Component and Protein Content

The GenProtect formulation stabilizes the entire nucleic acid component of the whole blood and the protein content of whole blood too (data pending). 40uL of whole blood spotted on GenProtect RNA paper supplemented with GTA solution and PAPER

TYPE II paper and 20uL of whole blood spotted on microsampling tips (VAMS) was dried at ambient in a BSL II hood overnight. The DNA was isolated from the entire spot with GenSolve DNA kit (GenTegra) from all of the paper types and microsampling tips. The DNA from 200uL of whole blood was isolated with Qiagen Blood kit for DNA from whole blood. The results are shown in Figures 9-10.

As shown in Figures 9-10, the amount of DNA released from GenProtect RNA paper was comparable to amount of DNA in the whole blood (Post Hoc analysis of Tukey test). DNA isolated from the GenProtect and PAPER TYPE II cards and microsampling tips visualized on a 2% agarose gel stained with ethidium bromide show no appreciable difference between whole blood controls and all three types of DBS samples used. DNA extracted from the microsampling tips gave a lower yield than GenProtect and PAPER TYPE II cards but there is no apparent difference in the quality of the DNA visualized on the gel. Lanes 1 to 14 are GenProtect samples. Lane 16 is PAPER TYPE II, Lane 17 is microsampling blood DNA sample, Lane 18 is DNA extracted from whole blood

The inventions being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the inventions and all such modifications are intended to be included within the scope of the following claims.

The above specification provides a description of the manufacture and use of the disclosed compositions and methods. Since many embodiments can be made without departing from the spirit and scope of the invention, the invention resides in the claims.

Claims

CLAIMS What is claimed is:

1. A composition for stabilizing and storing a biological sample comprising: one or more metal chelators; a pH adjuster; a hydroxyl radical scavenger; a singlet oxygen quencher; an inhibitor; and a cell separation reagent.

2. The composition of claim 1, wherein the biological sample is whole blood, and wherein the whole blood contains DNA, RNA, and/or proteins.

3. The composition of any one of claims 1-2, wherein the pH adjuster comprises lithium hydroxide and/or lithium dodecyl sulfate.

4. The composition of any one of claims 1-3, wherein the one or more metal chelators comprise citric acid, ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and/or ethylenediaminetetraacetic acid.

5. The composition of any one of claims 1-4, wherein the hydroxyl radical scavenger comprises D-mannitol.

6. The composition of any one of claims 1-5, wherein the singlet oxygen quencher comprises L-cysteine, and/or D-acetyl cysteine.

7. The composition of any one of claims 1-6, wherein the inhibitor comprises aurintricarboxylic acid ammonium salt.

8. The composition of any one of claims 1-7, wherein the cell separation reagent comprises a polyethylene glycol.

9. The composition of any one of claims 1-8, further comprising a reducing agent.

10. The composition of claim 9, wherein the reducing agent is urea.

11. The composition of any one of claims 1-10, wherein the composition stabilizes and stores whole DNA, RNA and/or proteins from whole blood for at least one year.

12. The composition of any one of claims 1-11, wherein the composition stabilizes and stores a biological sample at a temperature between about 20 °C and about 72 °C.

13. The composition of claim 12, wherein the biological sample is stored on a paper carrier as dried blood spots (DBS) or dried plasma spots (DPS).

14. A kit for stabilizing and storing a biological sample comprising: a composition comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singled oxygen quencher, an inhibitor, and a cell separation reagent; one or more carriers; and one or more closures; wherein the composition is combined with a biological sample comprising whole blood held in the one or more carriers, and sealed by the one or more closures; and wherein the composition stabilizes the biological sample for at least one year, and wherein the composition stabilizes the biological sample at an ambient temperature.

15. The kit of claim 14, wherein the one or more carriers comprises one or more vials, one or more wells, paper, and/or a cotton swab.

16. The kit of any one of claims 14-15, further comprising an additional container for housing the composition and sample held in the one or more carriers and sealed by the one or more closures.

17. The kit of claim 16 wherein the additional container comprises a box and/or an envelope.

18. The kit of claim 17, further comprising a pre-addressed mailing label.

19. A method of using a kit for stabilizing and storing a biological sample comprising: providing a composition comprising one or more metal chelators, a pH adjuster, a hydroxyl radical scavenger, a singled oxygen quencher, an inhibitor, and a cell separation reagent, wherein the composition stabilizes the biological sample for at least one year, and wherein the composition stabilizes the biological sample at an ambient temperature; collecting one or more raw samples; mixing the one or more raw samples with the composition in one or more carriers; and sealing the mixture in the carrier with closures.

20. The method of claim 19, further comprising the step of placing the sealed mixture in an additional container for housing the one or more carriers.

21. The method of any one of claims 19-20, further comprising the step of adding protective materials to the additional container, wherein the protective materials comprise protective foam, packing peanuts, and/or shredded paper filler

22. The method of claim 21, further comprising the step of applying a pre-addressed mailing label to the additional container.