Redox trapping useful for biospecimen preservation

Abstract

Provided are compositions and methods useful for redox quenching. Also provided is a method for biospecimen preservation, comprising combining the biospecimen with a redox quenching composition, to thereby quench and preserve the redox state of the biospecimen. Further provided is a method for detecting a redox state of a biospecimen, comprising combining the biospecimen with a redox quenching composition to form a quenched specimen; and detecting a redox sensor in the quenched specimen. Redox quenching compositions are also provided.

Description

[0001] Redox Trapping Useful for Biospecimen Preservation

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 584,565, filed September 22, 2023, and U.S. Provisional Patent Application No. 63 / 562,991, filed March 8, 2024, the disclosures of each of which are incorporated by reference herein in their entireties.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under Grant No. R21 GM144947- 01 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD

[0007] This invention relates to compositions and methods useful for biospecimen preservation.

[0008] BACKGROUND

[0009] Identifying the reduction-oxidation (redox) state in a biospecimen is useful in clinical settings for studying the pathogenesis and development of diseases or disorders, as well as to identify the appropriate timing for the administration of specific therapeutics in treating said diseases or disorders. For example, bacterial infections can both activate the immune system and induce mitochondria dysfunction, both of which are known to produce reactive oxygen and other oxidant species, cumulatively abbreviated here as ROS. Sepsis is similarly characterized by excessive levels of ROS due to the same reasons, and the detection of ROS and their reaction products with cellular molecules is one of the early indicators of the disease. The current best practice methods for detecting the redox state from patient samples utilize a blood sample due to its availability and ease of acquisition. However, from a redox perspective, blood is unstable and prone to oxidative damage due to the high iron content, which leads to oxidation via Fenton chemistry amongst other chemistries. Thus, fresh blood samples must be stored on ice and analyzed or processed for storage within a short (e.g., from minutes to one hour) window after sampling.

[0010] Clinical and pre-clinical research into the pathophysiology of infections and sepsis, as well as other diseases and disorders that revolve around the generation or effects of oxidation, also makes use of tissue specimens, which are similarly plagued by artifactual oxidation leading to inaccurate measurements of redox state but also of other molecular species. Repeated freeze / thaw cycles, for example, often degrade proteins due to the increased generation of ROS. Thus, novel compositions for, and methods of, quenching redox reactions in biospecimens are desirable to achieve preservation of the endogenous redox state and to improve the overall specimens' stability for accurate post-collection analytics and biomarker discovery.

[0011] SUMMARY

[0012] An aspect of the invention relates to a method for biospecimen preservation, comprising combining the biospecimen with a redox quenching composition, to thereby quench and preserve the redox state of the biospecimen. In some embodiments, the method includes collecting the biospecimen, wherein the biospecimen is added to the redox quenching composition during the collecting. In some embodiments, the method includes providing the biospecimen, and adding the redox quenching composition to the biospecimen.

[0013] Another aspect of the invention relates to a method for detecting a redox state of a biospecimen, comprising combining the biospecimen with a redox quenching composition to form a quenched specimen; and detecting a redox sensor in the quenched specimen. In some embodiments, the method includes collecting the biospecimen, wherein the biospecimen is added to the redox quenching composition during the collecting. In some embodiments, the method includes providing the biospecimen, and adding the redox quenching composition to the biospecimen.

[0014] In some embodiments, the redox quenching composition comprises one, two, three, or four or five or more of: clioquinol, deferrioxamine, bathophenanthrolinedisulfonate (BPDS), diethylenetriaminepentaacetic acid (DTP A), astaxanthin, butylated hydroxytoluene (BHT), a sulfenic acid reacting molecule (e.g., dimedone, 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD chloride), 2-nitro-5-thiobenzoate (TNB), P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate), a thiol -reactive agent (e.g., 4-(5-methanesulfonyl- [l,2,3,4]tetrazol-l-yl)-phenol (MSTP), 7V-ethylmaleimide (NEM), iodoacetamide and derivatives (IAM), methyl methanethiosulfonate (MMTS), dinitrofluorobenzene (DNFB), or monobromobimane (mBBr)), ethylenediaminetetraacetic acid (EDTA), and one or more protease inhibitors (e.g., a protease inhibitor cocktail, such as Pierce Protease Inhibitor).

[0015] In some embodiments, the redox quenching composition further comprises one or more additional redox altering compounds (e.g., ascorbic acid (vitamin C) and / or N-acetyl-para- aminophenol (acetaminophen)).

[0016] In some embodiments, the composition comprises the BHT, optionally in combination with astaxanthin and / or acetaminophen; the sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate); and the thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr).

[0017] In some embodiments, the redox quenching composition further comprises an alcohol (e.g., methanol and / or ethanol), a protein cross-linking reagent (e.g., polyethylene glycol and / or paraformaldehyde), and / or a phosphatase inhibitor (e.g., a phosphatase inhibitor cocktail, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, glycerophosphate, or any combination thereof). In particular embodiments, the biospecimen is a tissue sample. In some embodiments, the biospecimen is a blood sample.

[0018] In some embodiments, the redox quenching composition further comprises a y- glutamyl transferase inhibitor (e.g., 6-diazo-5-oxo-L-nor-Leucine and / or OU749) and / or a spin trap (e.g., 5,5-dimethyl-l -pyrroline N-oxide (DMPO)). In particular embodiments, the biospecimen is a blood sample.

[0019] In some embodiments, the biospecimen is a biological sample of a human or animal subject. In some embodiments, the subject is a healthy individual. In some embodiments, the subject has or is suspected of having an infectious disease, sepsis, sepsis shock, cancer, diabetes, obesity, and / or cardiovascular disease. In some embodiments, the method further comprises treating the subject with a therapeutic (e.g., a redox altering therapeutic or therapy).

[0020] Further provided is a redox quenching composition as described herein. In some embodiments, the composition is provided in a biological sample collection tube such as a blood sample collection tube.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 is a western blot image showing the monomer (i.e., reduced) and dimer (i.e., oxidized) forms of 2-cysteine peroxiredoxins (Prx) in red blood cell (RBC) fractions of blood samples from healthy donors after treatment with EDTA, the redox formulation without NEM (RM without X; i.e., RM), the redox formulation with NEM (RM with X; i.e., RMX), or NEM alone (X).

[0023] FIG. 2 is a graph showing the GSH (i.e., reduced glutathione) and GSSG (i.e., oxidized glutathione) levels in RBC fractions of blood samples from healthy donors after treatment with EDTA, RM, RMX, or NEM (X).

[0024] FIG. 3 is a western blot image and the corresponding bar graph quantifying the monomer and dimerized forms of Prx in 1.0 or 1.25 mL blood samples from healthy donors after treatment with heparin and LPS and then treated with or without an equal volume of RMX.

[0025] FIG. 4 is a schematic showing the experimental protocol for examining the stability of blood samples after repeat freeze / thaw cycle.

[0026] FIG. 5 is a graph showing the vascular endothelial growth factor (VEGF) levels after various number of freeze / thaw (F / T) cycles in blood samples with either RMX or control treatments.

[0027] FIG. 6 is a schematic showing the experimental protocol for examining the clinical markers in a comprehensive metabolic panel (CMP) or a complete blood count (CBC) to compare RMX with the standard sodium fluoride / potassium oxalate (NaF / KOx) or lithium heparin (Li+Heparin) methodologies for sepsis patient samples.

[0028] FIG. 7 is a graph showing the lactate levels in blood samples stabilized with either NaF / KOx at room temperature (RT) or on ice compared to RMX at RT.

[0029] FIG. 8 is a schematic showing the experimental protocol for examining the lactate levels in blood samples using either an i-STAT device or liquid chromatography with tandem mass spectrometry (LC-MS / MS) to compare RMX and heparin vs. heparin alone for sepsis patient biological samples.

[0030] FIG. 9 is a graph showing the lactate levels in sepsis patient blood samples after treatment with either RMX and heparin vs. heparin as analyzed by i-STAT 30 minutes postcollection (A), LC-MS / MS 30 minutes post-collection (B), i-STAT 60 minutes post-collection (C), or LC-MS / MS 60 minutes post-collection (D).

[0031] FIG. 10 is a series of graphs showing the glucose, blood urea nitrogen (BUN), bilirubin, creatinine, alkaline phosphatase, and aspartate aminotransferase in sepsis patient blood samples after treatment with either RMX and heparin vs. heparin.

[0032] FIG. 11 is a Prx reducing western blot image showing the monomer and dimer forms of Prx and the lactate levels in sepsis patient blood samples after treatment with RMX, EDTA, or NaF / KOx. DETAILED DESCRIPTION

[0033] Embodiments of the present invention are now described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

[0034] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed singularly or in any combination.

[0035] Definitions

[0036] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0037] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0038] All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0039] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0040] As used herein, the transitional phrase “consisting essentially of’ is to be interpreted as encompassing the recited materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0041] The term “enhance” or “increase” refers to an increase in the specified parameter of at least about 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, 10-fold, 12-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or even 500-fold.

[0042] The term “inhibit” or “reduce” or grammatical variations thereof as used herein refers to a decrease or diminishment in the specified level or activity of at least about 15%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95% or more. In particular embodiments, the inhibition or reduction results in little or essentially no detectible activity (at most, an insignificant amount, e.g., less than about 10% or even 5%).

[0043] Compositions and Uses Thereof

[0044] As used herein, the term “redox quenching composition” refers to a composition that can reduce, inhibit, or prevent the formation of reactive oxidative species (ROS); prevent the propagation of metabolic and signaling reactions (e.g., accumulation of lactic acid); decrease or preserve the level of oxidants in a solution, composition, and / or biospecimen; inhibit the propagation of radical reactions (e.g., formation of lipid peroxyl radicals); and / or block or inhibit the reduction / oxidation reactive sites in proteins of a biospecimen.

[0045] In some embodiments, the redox quenching composition comprises one, two, three, or four or more of: clioquinol, deferrioxamine, BPDS, DTP A, astaxanthin, BHT, a sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate), a thiol -reactive agent (e.g., MSTP, NEM, LAM and derivatives, MMTS, DNFB, or mBBr), EDTA, and one or more protease inhibitors (e.g., a protease inhibitor cocktail; e.g., a Pierce Protease Inhibitor cocktail). In some embodiments, the clioquinol is present in a concentration from about 10 to about 500 pM (e.g., about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or about 500 pM), e.g., about 100 pM. In some embodiments, the deferrioxamine is present in a concentration from about 1 to about 50 pM (e.g., about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pM), e.g., about 10 pM. In some embodiments, the BPDS is present in a concentration from about 0.1 to about 5 mM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 mM), e.g., about 1 mM. In some embodiments, the DTPA is present in a concentration from about 0.1 to about 5 mM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 mM), e.g., about 1 mM. In some embodiments, the astaxanthin is present in a concentration from about 10 to about 250 pM (e.g., about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, or about 250 pM), e.g., about 50 pM. In some embodiments, the BHT is present in a concentration from about 10 to about 500 pM (e.g., about 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, 450, or about 500 pM), e.g., about 50 pM. In some embodiments, the sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, 0-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate) is present in a concentration from about 0.1 to about 5 mM (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 mM), e.g., about 1 mM. In some embodiments, the thiol-reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr) is present in a concentration from about 5 to about 100 mM (e.g., about 5, 10, 15, 20, 25, 30, 40, 45, 50, 60, 70, 80, 90, or about 100 mM), e.g., about 25 mM. In some embodiments, the EDTA is present in a concentration from about 0.5 to about 5 mg / mL (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or about 5 mg / mL), e.g., about 1.8 mg / mL. In some embodiments, the one or more protease inhibitors (e.g., protease inhibitor cocktail; e.g., Pierce Protease Inhibitor cocktail) is present in a concentration sufficient to reduce, inhibit, or prevent proteolysis of endogenous proteins in the biospecimen. In some embodiments, the protease inhibitor is Thermo™ Pierce Protease Inhibitor Mini Tablets (Catalog # A32953).

[0046] In some embodiments, the redox quenching composition further comprises an alcohol (e.g., methanol, ethanol, and / or isopropanol), a protein cross-linking reagent (e.g., polyethylene glycol, paraformaldehyde, disuccinimidyl glutarate, p-maleimidophenyl isocyanate, N-hydroxysuccinimide, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide, bissulfosuccinimidyl suberate, and / or dimethyl suberimidate), and / or a phosphatase inhibitor (e.g., a phosphatase inhibitor cocktail, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, glycerophosphate, or any combination thereof). In some embodiments, the alcohol is present in the redox quenching composition from about 1 or 5% to about 50, 70, 80 or 90% (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, or 90%) by volume of the redox quenching composition. In some embodiments, protein cross-linking reagent is present in the redox quenching composition from about 0.1 to about 20% (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20%) by volume of the redox quenching composition. In some embodiments, the phosphatase inhibitor is present in a concentration from about 1 to about 350 mM (e.g., about 1 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, or 350 mM). For example, in some embodiments, the sodium fluoride is present in a concentration from about 1 to about 25 mM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mM), the sodium orthovanadate is present in a concentration from about 1 to about 100 mM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM), the sodium pyrophosphate is present in a concentration from about 1 to about 100 mM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM), and / or the glycerophosphate is present in a concentration from about 1 to about 100 mM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM)). In some embodiments, the phosphatase inhibitor cocktail is Thermo™ Halt™ phosphatase inhibitor cocktail (Catalog # 78428) or Sigma Roche™ PhosSTOP™ (Catalog # 4906845001).

[0047] In some embodiments, the redox quenching composition further comprises a y- glutamyl transferase inhibitor (e.g., 6-diazo-5-oxo-L-nor-Leucine (DON) and / or OU749) and / or a spin trap (e.g., DMPO, alpha-phenyl N-terti ary -butyl nitrone (PBN), and / or 3,5- dibromo-4-nitrosobenzenesulfonic acid (DBNBS)). In some embodiments, the y-glutamyl transferase inhibitor is present in a concentration from about 0.1 to about 100 pM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 pM). In some embodiments, the spin trap is present in a concentration from about 10 to about 500 mM (e.g., about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mM). In some embodiments, the spin trap is suitable for downstream applications (e.g., measurement of radical species by Electron Paramagnetic Resonance / EPR).

[0048] In some embodiments, the redox quenching composition further comprises one or more additional redox altering compounds (e.g., ascorbic acid and / or acetaminophen). In some embodiments, the additional redox altering compound is present in a concentration from about 1 to about 500 pM (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, or 500 pM).

[0049] In some embodiments, measuring the redox state of a biospecimen may include detecting a redox sensor. In some embodiments, the redox sensor is peroxiredoxin (PRX; Prxl- VI), reduced glutathione (GSH), oxidized glutathione (GSSG and other oxidized or alkylated species), reduced cysteine (Cys-SH), oxidized cysteine (Cys-S-S-Cys and other oxidized or alkylated species), the ratio of GSH / GSSG or Cys / CysSSCys, lipid peroxides or other oxidized lipid species and products (e.g., 4-hydroxy-2-nonenal (4-HNE), 4-hydroxy-2E-hexanal (4- HHE), and malondialdehyde (MDA)), oxidized nucleic acid species (e.g., 8-oxo-2’- deoxygunosine, 8-oxo-2’ -deoxyadenosine, FAPyGua, FAPyAde, 5,6-hydroxy-2’- deoxycytidine, 5-hydroxy-2’-deoxycytidine, 5, 6-hydroxy-2’ -deoxyuridine, 5-hydroxy-2’- deoxyuridine, 5,6-hydroxy-2’-deoxythymidine, and 5-hydroxymethyl-2’-deoxyuridine), carbohydrate species (e.g., advanced glycation end products (AGEs)) and / or oxidized low- density lipoproteins (LDL). The redox sensor may be detected by any method known in the art. In some embodiments, measuring the redox state of a biospecimen may include measuring the expression level of one or more genes and / or proteins in the biospecimen. In some embodiments, the one or more genes and / or proteins may include, but are not limited to, peroxiredoxins, NF-KB, superoxide dismutase (SOD), catalase, guaiacol peroxidase, dehydroascorbate reductase, ascorbate peroxidase, glutathione reductase, and the like.

[0050] As used herein, the terms “biospecimen” and “biological sample” refer to any sample obtained from a biological source including, but not limited to, an animal, plant, bacteria, cell culture, tissue culture, and organ culture. A biological sample may be obtained by any method known to those in the field. In some embodiments, a biological sample is obtained from an animal (e.g., a human or a non-human animal subject). In some embodiments, the biological sample is obtained from an adult or geriatric human subject. In some embodiments, a biological sample is a bodily fluid. In some embodiments, the biological sample is a tissue sample (e.g., an organ sample, a muscle sample, a skin sample, and / or a hair sample). In some embodiments, the tissue sample is a bone sample.

[0051] In some embodiments, the biological sample is a blood sample. In some embodiments, a blood sample as used herein comprises the one or more fractions of blood (e.g., plasma, serum, red blood cells (RBC), and / or peripheral blood mononuclear cells (PBMC) and their subtypes) isolated from a whole blood sample. The RBC fraction may be isolated from the whole blood sample by any method known to those in the field including, but not limited to, centrifugation. In some embodiments, an anticoagulant (e.g., an additional anticoagulant) is added to the blood sample. In some embodiments, the anticoagulant is heparin (e.g., sodium heparin or lithium heparin), EDTA (e.g., potassium EDTA or sodium EDTA), sodium fluoride / potassium oxalate (NaF / KOx), or citrate (e.g., sodium citrate, acid citrate dextrose, or citrate phosphate dextrose). In some embodiments, the blood sample is collected in a collection vessel (e.g., a collection tube; e.g., a test tube) that is coated in the anticoagulant. In some embodiments, the collection vessel that is coated in the anticoagulant is a K2EDTA collection tube. In some embodiments, the blood sample is collected into a vessel containing a redox quenching formulation of the present invention that comprises one or more anticoagulants. In some embodiments, the vessel is under negative pressure (e.g., a vacuum). In some embodiments, the blood sample is from about 0.1 to 10 mL (e.g., from about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL). In some embodiments, the biological sample is a blood sample and the clioquinol is present in an amount from about 5, 10 or 15 to about 40 or 50 pg per ImL of blood (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pg per ImL of blood), e.g., about 30.55 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the deferri oxamine is present in an amount from about 1, 2 or 3 to about 10, 15, 20 or 25 pg per ImL of blood (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or about 25 pg per ImL of blood), e.g., about 6.56 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the BPDS is present in an amount from about 250, 300, 350 or 400 to about 650, 700 or 750 pg per ImL of blood (e.g., about 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, or about 750 pg per ImL of blood), e.g., about 536.48 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the DTPA is present in an amount from about 100, 200 or 300 to about 500, 550 or 600 pg per ImL of blood (e.g., about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or about 600 pg per ImL of blood), e.g., about 393.5 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the astaxanthin is present in an amount from about 5, 10, 15 or 20 to about 40, 45 or 50 pg per ImL of blood (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or about 50 pg per ImL of blood), e.g., about 29.84 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the BHT is present in an amount from about 1, 3 or 5 to about 15, 20 or 25 pg per ImL of blood (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or about 25 pg per ImL of blood), e.g., about 11.02 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the sulfenic acid reacting molecule is dimedone and is present in an amount from about 50, 75 or 100 to about 200, 250 or 300 pg per ImL of blood (e.g., about 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or about 300 pg per ImL of blood), e.g., about 140.18 pg per ImL of blood. In some embodiments, the biological sample is a blood sample and the thiolreactive agent is NEM and is present in an amount from about 0.5, 1 or 1.5 to about 5, 8 or 10 mg per ImL of blood (e.g., about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or about 10 mg per ImL of blood), e.g., about 3.13 mg per ImL of blood. In some embodiments, the biological sample is a blood sample and the EDTA is present in an amount from about 0.25, 0.5 or 1 to about 2.5, 3, 4 or 5 mg per ImL of blood (e.g., about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 3.5, 4, 4.5, or about 5 mg per ImL of blood), e.g., about 1.8 mg per ImL of blood. In some embodiments, the biological sample is a blood sample and the protease inhibitor is Thermo Scientific™ Pierce Protease Inhibitor Mini Tablets (A32953) and is present in a lx dilution in the blood sample. Also provided is a blood collection tube comprising a composition as taught herein. The blood collection tube may be, for example, a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL or 6 mL tube, with the redox quenching composition provided therein for ease of use. In some embodiments, the blood collection tube is an evacuated tube (i.e., a vacuum collection tube).

[0052] In some embodiments, the blood sample may be analyzed using any method known in the art including, but not limited to, technologies and analytical assays in research and clinical laboratories. Examples of clinical laboratory measurements include, but are not limited to, comprehensive metabolic panel (CMP) and / or a complete blood count (CBC). In some embodiments, the blood sample is analyzed for glucose, blood urea nitrogen (BUN), chloride, albumin, alanine transaminase, alkaline phosphatase, aspartate aminotransferase, white blood cells (WBC), hemoglobin (Hb), hematocrit, mean platelet volume (MPV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), RBC, neutrophil, lymphocyte, monocyte, eosinophil, basophil, lactate, creatinine, bilirubin, total protein, sodium potassium, calcium, and / or bicarbonate levels. Examples of research laboratory measurements include, but are not limited to, omics (e.g., proteomics, transcriptomics, genomics, metabolomics, metagenomics, phenomics, and / or epigenomics), targeted measurements of species of interest (e.g., ROS, nucleic acid species, metabolites, lipids, etc.), enzyme activity assays (e.g., peroxidase), imaging technologies (e.g., confocal microscopy), and the like.

[0053] In some embodiments, the blood sample may be treated with a redox altering compound, e.g., a compound that changes the redox state of a compositions by producing or reducing ROS by a method known to those in the art. In some embodiments, the redox altering compound is a redox quenching compound. In some embodiments, the redox altering compound is lipopolysaccharide (LPS) and / or hydrogen peroxide (H2O2).

[0054] In some embodiments, the biological sample is from a healthy subject. In some embodiments, the biological sample is from a subject having or suspected of having a disease or disorder. In some embodiments, the disease or disorder is associated with a change in the redox state in the subject and / or the biological sample. In some embodiments, the disease or disorder is an aging-associated disease or disorder. In some embodiments, the disease or disorder is sepsis, an infectious disease, cancer, diabetes (e.g., Type-2 diabetes), obesity, inflammation, hypertension, neurologic disease (e.g., Parkinson’s disease, Alzheimer's disease, amyotrophic lateral sclerosis, and / or multiple sclerosis), respiratory disease (e.g., chronic obstructive pulmonary disease and / or asthma) cardiovascular disease (e.g., congestive heart failure, atherosclerosis, myocardial infarction, stroke, and / or heart attack), and / or an aging- related disease (e.g., cancer, Alzheimer’s disease, Parkinson’s disease, hypertension, diabetes, arthritis, osteoarthritis, osteoporosis, atherosclerosis, nonalcoholic fatty liver disease, chronic obstructive pulmonary disease, macular degeneration, myocardial infarction, and / or stroke). "Subjects" are generally human subjects and include, but are not limited to, "patients." Subjects also include other animal subjects, particularly non-human mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g., rats and mice), lagomorphs, non-human primates, etc., for, e.g., veterinary medicine, laboratory research and / or pharmaceutical drug development purposes.

[0055] In some embodiments, redox quenching compositions of the present invention can be used to preserve the redox state of the biospecimen and / or reduce, inhibit or prevent degradation of the biological components (e.g., protein, nucleic acids, polysaccharides, and / or lipids) of the biospecimen. In some embodiments, the redox state of the biospecimen is preserved for about 5 to 60 minutes (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes); for about 1 to about 24 hours (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours); for about 1 to about 7 days (e.g., about 1, 2, 3, 4, 5, 6, or 7 days); for about 1 to about 4 weeks (e.g., about 1, 2, 3, or 4 weeks); or for about 1 to about 24 months (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months), or longer. In some embodiments, preserving the biospecimen includes preserving the biospecimen throughout at least one freeze-thaw cycle (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more freeze-thaw cycles). In some embodiments, preserving the redox state comprises preserving the redox state before, during, and / or after the administration of a redox altering therapy and / or a redox altering compound. In some embodiments, preserving the redox state comprises preserving the cysteine redox state before, during, and / or after the administration of a redox altering therapy and / or a redox altering compound. In some embodiments, the redox altering therapy comprises covalent inhibitors that target cysteine sites in proteins in the biospecimen.

[0056] Provided herein is a method for biospecimen preservation, comprising combining the biospecimen with a redox quenching composition, to thereby preserve the redox state and the stability of the biospecimen. In some embodiments, combining the biospecimen with the redox quenching composition comprises collecting the biospecimen, wherein the biospecimen is contacted to the redox quenching composition during the collecting. In some embodiments, combining the biospecimen with the redox quenching composition comprises providing the biospecimen and adding the redox quenching composition to the biospecimen, to thereby preserve the redox state of the biospecimen. In some embodiments, the biospecimen is a biological sample from a subject. In some embodiments, the combining is carried out in vitro (e.g., in a test tube).

[0057] Also provided herein is a method for detecting a redox state of a biospecimen, comprising combining the biospecimen with a redox quenching composition to form a quenched specimen, and detecting a redox sensor in the quenched specimen, to thereby detect the redox state of the biospecimen. In some embodiments, combining the biospecimen with the redox quenching composition comprises collecting the biospecimen, wherein the biospecimen is contacted to the redox quenching composition during the collecting to form the quenched specimen. In some embodiments, combining the biospecimen with the redox quenching composition comprises providing the biospecimen; adding the redox quenching composition to the biospecimen to form the quenched specimen; and detecting a redox sensor in the quenched specimen. In some embodiments, the biospecimen is a biological sample from a subject. In some embodiments, the subject has or is suspected of having a disease and / or a disorder. In some embodiments, the method further comprises treating the subject with an appropriate treatment regimen for the disease and / or disorder. In some embodiments, the appropriate treatment regimen is a redox altering therapy. In some embodiments, the redox altering therapy is acetaminophen, selenium, dietary supplements, ascorbic acid, tocopherols, retinoic acid, carotenoids, polyphenols, exercise, or combinations thereof. In some embodiments, the appropriate treatment regimen is one that reduces symptoms of the disease and / or disorder in the subject including, but not limited to, a non-steroidal anti-inflammatory (NSAID), an antitussive, an analgesic, an antihistamine, a decongestant, a cough suppressant, and combinations thereof.

[0058] In some embodiments, the biospecimen is provided in a collection vessel (e.g., in a test tube, cell culture dish, vial, centrifuge tube, microcentrifuge tube, etc.) and the redox quenching composition is added to the collection vessel. In some embodiments, the redox quenching composition is provided in a collection vessel (e.g., in a test tube, cell culture dish, vial, centrifuge tube, microcentrifuge tube, etc.) and the biospecimen is added to the collection vessel. In some embodiments, the biospecimen is collected from a subject directly into the collection vessel comprising the redox quenching composition. In some embodiments, the biospecimen is a blood sample and the collection vessel is a blood collection tube comprising the redox quenching composition. In some embodiments, the blood collection tube is under negative pressure, i.e., a vacuum (e.g., a vacuum collection tube; e.g., a vacutainer). In some embodiments, the biospecimen is a tissue biopsy (e.g., a muscle, bone, and / or organ biopsy) and the collection vessel is a biopsy container. In some embodiments, the biospecimen is collected from the subject in a first collection vessel, and the combining of the biospecimen and the redox quenching composition is carried out in a second collection vessel.

[0059] In some embodiments, the composition is provided in a powder, a solution (liquid or frozen), or a freeze-dried form. In some embodiments, the composition is provided in a kit, e.g., in a prepackaged solution, powder, or freeze-dried form. In some embodiments, the kit may comprise separate components of the composition, e.g., the one, two, three, or four or more of: clioquinol, deferrioxamine, BPDS, DTP A, astaxanthin, BHT, sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkyne and alkenes, Wittig reagents, or an enolate), a thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr), EDTA, and one or more protease inhibitors (e.g., a protease inhibitor cocktail), which are then mixed to form the composition. In some embodiments, the components of the composition are mixed prior to combining with the biospecimen, e.g., from about 1 minute to about 60 minutes prior to combining with the biospecimen (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or about 60 minutes prior to combining with the biospecimen).

[0060] In some embodiments, the subject has or is suspected of having cancer and the appropriate treatment regimen is a treatment for cancer including, but not limited to, radiation therapy, chemotherapy (e.g., ROS altering chemotherapy, e.g., doxorubicin, daunorubicin, epirubicin, camptothecin, oxaliplatin, cisplatin, carboplatin, altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, temozolomide, thiotepa, trabectedin, irinotecan, topotecan, etoposide, teniposide, arsenic trioxide, arsenic hexoxide, and combinations thereof) , and / or cell therapy (e.g., B-cell, T-cell, or chimeric antigen receptor (CAR)-T cell therapy). In some embodiments, the cancer is lymphoma, a leukemia, a melanoma, a blastoma, a sarcoma, a myeloma, a glioma, and / or a germ cell tumor.

[0061] In some embodiments, the subject has or is suspected of having a viral infection and the appropriate treatment regimen is an antiviral (e.g., oseltamivir, acyclovir, remdesivir, nirmatrelvir, ritonavir, molnupiravir, chloroquine, hydroxychloroquine, azithromycin, abacavir, adefovir, umifenovir, emtricitabine / tenofovir alafenamide, ganciclovir, imiquimod, ibacitabine, nitazoxanide, and combinations thereof) and / or an antibody therapy (e.g., a monoclonal antibody therapy). In some embodiments, the viral infection is coronavirus infection, herpesvirus infection, adenovirus infection, Epstein-Barr virus infection, Ebola virus infection, human papilloma virus infection, cytomegalovirus infection, influenza virus infection, respiratory syncytial virus infection, rhinovirus infection, hepatovirus infection (e.g., hepatovirus A, hepatovirus B, hepatovirus C, hepatovirus D, and / or hepatovirus E), West Nile virus infection, alphavirus infection, coronavirus infection, and / or human immunodeficiency virus infection.

[0062] In some embodiments, the subject has or is suspected of having a cardiovascular disease and the appropriate treatment regimen is a beta blocker, a statin, an angiotensin II receptor blocker, an angiotensin-converting-enzyme (ACE) inhibitor, a calcium channel blocker, lifestyle changes (e.g., dietary changes and / or exercise), surgery (e.g., coronary artery bypass surgery, coronary stent placement, and / or coronary angioplasty), and combinations thereof. In some embodiments, the cardiovascular disease is coronary heart diseases, cardiomyopathy, hypertensive heart diseases, cardiac dysrhythmias, endocarditis, cardiomegaly, myocarditis, cerebrovascular disease, and / or peripheral arterial disease.

[0063] In some embodiments, the subject has or is suspected of having an aging-related disease and the appropriate treatment regimen is a beta blocker, a statin, an angiotensin II receptor blocker, an ACE inhibitor, a calcium channel blocker, lifestyle changes (e.g., dietary changes and / or exercise), surgery (e.g., coronary artery bypass surgery, coronary stent placement, and / or coronary angioplasty), radiation therapy, chemotherapy (e.g., ROS altering chemotherapy, e.g., doxorubicin, daunorubicin, epirubicin, camptothecin, oxaliplatin, cisplatin, carboplatin, altretamine, bendamustine, busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechlorethamine, melphalan, temozolomide, thiotepa, trabectedin, irinotecan, topotecan, etoposide, teniposide, arsenic trioxide, arsenic hexoxide, and combinations thereof), dietary supplements, ascorbic acid, an NSAID, an antibody (e.g., a monoclonal antibody), carbidopa-levodopa, a dopamine agonist, a monoamine oxidase-B inhibitor, a catechol-O-methyltransferase inhibitor, an N-methyl-D- aspartate antagonist, and / or a cholinesterase inhibitor.

[0064] In some embodiments, the aging-related disease is type-2 diabetes, Alzheimer’s disease, Parkinson’s disease, Huntington's diseases, amyotrophic lateral sclerosis, multiple system atrophy, obesity, increased cholesterol levels, hypertension, ocular disorders, cataracts, glaucoma, blood clotting disorders, arthritis, hearing loss, stroke, Lewy Body disease, osteoporosis, osteoarthritis, rheumatoid arthritis, intervertebral disc degeneration, chronic obstructive pulmonary disease, fatty liver disease, and / or nonalcoholic steatohepatitis.

[0065] In some embodiments, the subject has or is suspected of having an inflammatory and / or autoimmune disease and the appropriate treatment regimen is an analgesic, an antiinflammatory, a steroid, an immunosuppressant, cell therapy (e.g., B-cell or T-cell (e.g., CAR- T cell) therapy), a kinase inhibitor, and / or a cytokine inhibitor. In some embodiments, the inflammatory and / or autoimmune disease is psoriatic arthritis, rheumatoid arthritis, Sjogren’s syndrome, lupus (e.g., systemic lupus erythematosus), Crohn’s disease, celiac disease, ulcerative colitis, Graves’ disease, Hashimoto’s thyroiditis, Addison’s disease, dermatomyositis, psoriasis, Guillan-Barre syndrome, multiple sclerosis, myasthenia gravis, type-1 diabetes, vasculitis, pernicious anemia, inflammatory bowel disease, chronic inflammatory demyelinating polyneuropathy, and / or scleroderma.

[0066] In some embodiments, the subject has or is suspected of having a bacterial infection and / or sepsis and the appropriate treatment regimen is an antibiotic (e.g., imipenem, meropenem, tigecycline, piperacillin-tazobactam, ampicillin-sulbactam, moxifloxacin metronidazole, levofloxacin, aztreonam, cephalosporin, aminoglycoside, or combinations thereof).

[0067] In some embodiments, the methods of the present invention are carried out in vitro. In some embodiments, the methods of the present invention further comprise freezing the biospecimen and further optionally thawing the biospecimen.

[0068] The present invention is explained in greater detail in the following non-limiting examples.

[0069] EXAMPLES

[0070] Analysis of the redox state of a biological sample can help to advance clinical research in various redox-related diseases such as sepsis, reduce pre-analytical variability for personalized medicine by preventing ex vivo metabolism and / or sample degradation, lead to the discovery of new biomarkers to aid in the staging of patients, and aid in the development of new treatment regimens of redox therapeutics. For example, blood is one of the most commonly used biospecimens for diagnostic use and long-term storage for biomarker discovery. However, blood is highly unstable and prone to oxidative damage. Successful quenching of the redox state of a biospecimen may entail preventing artifactual ROS formation (e.g., through Fenton reactions) during the collection and storage, stopping the propagation of single electron / radical reaction (e.g., liquid peroxidation), and blocking reactive centers in proteins and small molecules.

[0071] Detecting the redox state of a biospecimen can be achieved using one or more redox sensors. For example, 2-cysteine peroxiredoxin proteins (PrxI-IV) are highly abundant, are recycled through the NADPH system, and dimerize when oxidized so can be detected via western blot. Glutathione is another highly abundant protein that is recycled through NADPH and glutathione reductase, while the levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) can be quantified via mass spectrometry.

[0072] We developed a redox formulation (abbreviated here as RMX) to trap the endogenous redox state, ensure redox quenching in a variable volume of a biospecimen (e.g., blood), produce higher quality specimens which are exposed to repeated freeze / thaw cycles, and test RMX in stabilizing biospecimens from patients with sepsis to improve analysis and storage. An example of the RMX composition is shown in Table 1. The formulation was dried under nitrogen gas (N2). Table 1. RMX composition.

[0073] Efficient Trapping of Endogenous Redox State

[0074] To determine the time frame during which RMX provides redox stability, blood samples were collected from healthy donors in K2EDTA tubes to prevent coagulation, and then immediately transferred to tubes containing the redox formulation without NEM (RM without X), the redox formulation with NEM (RM with X), or NEM alone (X). The red blood cell (RBC) fraction was isolated, and the redox state was tested at 15, 30, or 120 minutes after treatment by both western blot using an antibody that recognizes Prxl, II, and IV (Fig. 1) and mass spectrometry analysis of glutathione species (Fig. 2).

[0075] Both measuring methods demonstrated that RM with NEM and NEM alone produced sufficient stability in the redox state across all time points by increasing the stability of reduced monomeric Prx and by increasing the abundance of reduced glutathione (GSH) species, thus in both cases preventing artifactual oxidation of these species post-collection. Inclusion of other components in the RMX composition provides additional quenching of other reactive species beyond the sulfur-containing species. For example, def erri oxamine can bind excess iron and prevent ROS generation (HO*) through the Fenton reaction.

[0076] To determine the redox quenching capacity under conditions of increased oxidative burden, blood samples were collected from healthy donors into Heparin tubes to prevent coagulation, and then the redox state was altered using either lipopolysaccharide (LPS, 1 pg / mL), 100 pM H2O2, or 1 mM H2O2. The samples were treated with the redox formulation without NEM (RM without X), the redox formulation with NEM (RM with X), or NEM alone (X). RMX was capable of quenching Prx oxidation (more monomeric reduced Prx detected) in both the control vehicle and H2O2 treated blood. Further, RMX treatment was able to conserve the higher Prx oxidation state of the LPS treated sample, while the standard collection methodology (e.g., heparin alone) could not. RMX decreased the GSSG levels across the control and pro-oxidant conditions, resulting in increased GSH / GSSG ratios.

[0077] Quantitative Redox Quenching of a Variable Volume of Blood

[0078] As sample volumes can be variable during the blood draw, we examined the ability of RMX to quench various volumes of blood. Blood was collected from healthy donors in Heparin tubes as anticoagulant and the samples (1 or 1.25 mL) were transferred immediately to tubes containing the amount of RMX aimed for redox quenching of 1 mL blood volume. Prx protein oxidation states did not vary significantly between the 1 mL and 1.25 mL samples treated with RMX, indicating that this formulation (e.g., ingredients and their respective amounts) is capable of quenching higher sample volumes in cases where the phlebotomist draws more than the specified volume for the respective blood collection tube (Fig. 3). Stability of Blood Specimens with Repeat Freeze / Thaw Cycles

[0079] Often times, blood specimens undergo processing during testing to isolate various fractions or multiple freeze / thaw cycles during storage for retesting, which can alter the redox state of the sample and thus impact therapeutic efficacy. Accordingly, we developed a testing methodology for blood samples outlined in Fig. 4. As shown, blood samples are collected in heparin tubes to prevent coagulation and then transferred into either a control tube or a tube containing lipopolysaccharide (LPS, 1 pg / mL) to mimic bacterial infection. These are then transferred again into tubes containing either RMX or empty tubes.13C-labeled GSH was added to each tube as internal control and incubated for 30 minutes, and one aliquot was taken as the TO time point. The remaining aliquots were frozen at -80 °C. Samples were stored for 1, 3, 6, and 12 months at -80 °C. During storage, the samples underwent 1 or 4 thaw and refreeze events. At each thaw / freeze session samples were saved for molecular analysis. As seen in Fig. 5, RMX treatment significantly improved the VEGF protein stability levels after 1 month particularly with increasing thaw / freeze sessions. This effect was more evident with LPS treatment, which increases VEGF expression levels.

[0080] Clinical Diagnostic Measurements in Sepsis Patients

[0081] The exacerbated inflammatory response and mitochondrial dysfunction that underlie sepsis are known to be correlated with the increased ROS both globally in circulation and more locally in the affected organ and / or tissue. This oxidative stress can then further affect cellular metabolism and deteriorate the endothelium causing more cellular dysfunction. The ROS that arise in sepsis accelerate the progression of the disease, causing organ failure and septic shock. For clinical diagnosis, reducing any metabolic changes including those related to redox metabolism after blood collection allows providers to accurately perform clinical laboratory measurements to inform on the course of treatment. Lactic acid is a critical clinical metabolite used for sepsis treatment decisions. Current methodologies dictate that blood samples be taken in standard sodium fluoride / potassium oxalate (NaF / KOx) tubes, stored on ice, and need to be run within an hour of collection. However, the samples are commonly not stored properly on ice and there are often delays in processing, resulting in overestimation of lactic acid measurements. Efficient trapping of the redox state and metabolism in clinical samples of sepsis patients would improve the accuracy of diagnosis of the progression of the disease, allowing for the delivery of more appropriate treatment regimens. Accordingly, we collected blood specimens from 24 patients admitted to, or who were planned to be admitted to, the intensive care unit with a known or suspected sepsis infection as determined by culture analysis. Patients with hospital presentation greater than 72 hours or who declined consent were not entered into the study. Blood samples were collected thus far from 24 patients at one time point in an approximately 10 mL aliquot to analyze either by comprehensive metabolic panel (CMP), complete blood count (CBC), or lactate measurements. These blood samples were distributed among tubes containing RMX or other standard clinical tubes for blood sample analysis, such as heparin lithium (Li+Heparin; green top, standard for CMP analysis), K2EDTA (purple top, standard for CBC profiling), or NaF / KOx (gray top, standard for lactic acid) according to the experimental design in Fig. 6.

[0082] Lactate levels were used as the primary means of analyzing the value of RMX in quenching metabolism in the blood samples as many clinicians use lactate levels to determine which sepsis treatment methods to apply. As shown in Fig. 7, RMX prevented pre-analytical accumulation of lactic acid in the blood sample after collection. RMX at room temperature performed as well as NaF / KOx on ice, and was superior to NaF / KOx when both specimens were kept at room temperature. Lactic acid levels analyzed using the i-STAT, a point of care blood analyzer, and liquid chromatography with tandem mass spectrometry (LC-MS / MS) further demonstrated that RMX was able to reduce post-collection accumulation of lactate levels in blood samples for up to 60 minutes (Figs. 8-9). Further clinical analyses demonstrated that RMX did not interfere with the currently standard clinical protocols for measuring glucose, blood urea nitrogen (BUN), chloride, albumin, and alanine transaminase levels in the CMP test; and the white blood cell (WBC), hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet, neutrophil, lymphocyte, and monocyte levels in the CBC test (Fig. 10). By comparison, RMX performed better than the currently used methodologies in stabilizing the lactate, creatinine, and bilirubin levels.

[0083] Importantly, RMX was able to capture the Prx redox state in sepsis patients, which is not possible with NaF / KOx. Using patients 14, 15, and 17 as examples, RMX trapped the monomeric reduced Prx, which had heterogenous expression among all patients (Fig. 11). Moreover, patient 15 had higher levels of lipid peroxidation of polyunsaturated fatty acids (PUFAs), which was quenched by RMX. Tissue and Bone Preservation

[0084] In addition to preserving blood samples, the compositions described herein can also be used for preserving and stabilizing the redox state in tissue and bone samples. For such applications, additional reagents can be included such as methanol, ethanol, polyethylene glycol (PEG), paraformaldehyde or other protein crosslinkers, and / or phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, sodium pyrophosphate, P-glycerophosphate, and / or phosphatase inhibitor cocktails such as Thermo Halt Phosphatase Inhibitor cocktail).

[0085] The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

What is claimed is:

1. A method for biospecimen preservation, comprising: combining a biospecimen with a redox quenching composition, to thereby preserve the redox state of the biospecimen.

2. The method of claim 1, wherein the combining comprises: collecting the biospecimen, wherein the biospecimen is contacted to the redox quenching composition during the collecting.

3. The method of claim 1, wherein the combining comprises: providing the biospecimen; and adding the redox quenching composition to the biospecimen.

4. The method of any one of claims 1-3, wherein the redox quenching composition comprises two, three, four, or five or more of: clioquinol, deferrioxamine, bathophenanthrolinedisulfonate (BPDS), diethylenetriaminepentaacetic acid (DTPA), astaxanthin, butylated hydroxytoluene (BHT), a sulfenic acid reacting molecule (e.g., dimedone, 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD chloride), 2-nitro-5-thiobenzoate (TNB), P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate), a thiolreactive agent (e.g., 4-(5-methanesulfonyl-[l,2,3,4]tetrazol-l-yl)-phenol (MSTP), N- ethylmaleimide (NEM), iodoacetamide and derivatives (IAM), methyl methanethiosulfonate (MMTS), dinitrofluorobenzene (DNFB), or monobromobimane (mBBr), an anticoagulant (e.g., ethylenediaminetetraacetic acid (EDTA)), and one or more protease inhibitors (e.g., a Pierce Protease Inhibitor cocktail).

5. The method of claim 4, wherein the redox quenching composition comprises: BHT, optionally in combination with astaxanthin and / or acetaminophen; a sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate); and a thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr).

6. The method of claim 4 or claim 5, wherein, upon combining, and when present: the clioquinol is in a concentration from about 10 to about 500 pM; deferrioxamine is in a concentration from about 1 to about 50 pM;BPDS is in a concentration from about 0.1 to about 5 mM;DTPA is in a concentration from about 0.1 to about 5 mM; astaxanthin is in a concentration from about 10 to about 250 pM;BHT is in a concentration from about 10 to about 500 pM; the sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate) is in a concentration from about 0.1 to about 5 mM; the thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr) is in a concentration from about 5 to about 100 mM; the anticoagulant (e.g., EDTA) is in a concentration from about 0.5 to about 5 mg / mL; and / or the protease inhibitors (e.g., Pierce Protease Inhibitor) is in a concentration sufficient to reduce, inhibit or prevent proteolysis of endogenous proteins in the biospecimen.

7. The method of any one of claims 1-6, wherein the biospecimen is a biological sample of a human or non-human animal subject.

8. The method of claim 7, wherein the biological sample is a blood sample.

9. The method of claim 8, wherein the blood sample comprises the red blood cell (RBC) fraction, the serum fraction, and / or the PBMC and PBMC subtype fraction.

10. The method of claim 9, wherein the blood sample comprises an additional anticoagulant (e.g., heparin, EDTA, or potassium oxalate (KOx) (e.g., NaF / KOx)).

11. The method of any one of the preceding claims, wherein the combining is carried out in vitro.

12. The method of claim 11, wherein the combining is carried out in a test tube comprising the redox quenching composition, wherein the redox quenching composition is optionally provided in a solution (liquid or frozen), powder, or freeze-dried form.

13. The method of any one of the preceding claims, wherein the method further comprises freezing the biospecimen.

14. The method of claim 13, wherein the method further comprises thawing the biospecimen, and optionally wherein the method comprises two or more freeze / thaw cycles.

15. The method of claim 7, wherein the biological sample is a bone sample or a tissue sample (e.g., a muscle, organ, and / or tumor sample).

16. The method of claim 15, wherein the redox quenching composition further comprises an alcohol (e.g., methanol, ethanol, and / or propanol), a protein cross-linking reagent (e.g., polyethylene glycol and / or paraformaldehyde), and / or a phosphatase inhibitor (e.g., a phosphatase inhibitor cocktail, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, glycerophosphate, or any combination thereof).

17. The method of claim 16, wherein: the alcohol is present in the redox quenching composition from about 1 to about 90% by volume of the redox quenching composition; protein cross-linking reagent is present in the redox quenching composition from about 0.1 to about 20% by volume of the redox quenching composition; and / or the phosphatase inhibitor is present in a concentration from about 1 to about 100, 200 or 350 mM.

18. The method of any one of the preceding claims, wherein the redox quenching composition further comprises a y-glutamyl transferase inhibitor (e.g., 6-diazo-5-oxo-L-nor- Leucine and / or OU749) and / or a spin trap (e.g., 5,5-dimethyl-l -pyrroline N-oxide (DMPO)).

19. The method of claim 18, wherein the y-glutamyl transferase inhibitor (e.g., 6-diazo-5- oxo-L-nor-Leucine and / or OU749) is present in a concentration from about 0.1 to about 100pM; and the spin trap (e.g., DMPO) is present in a concentration from about 10 to about 500 mM.

20. The method of any one of the preceding claims, wherein the redox quenching composition further comprises one or more additional redox altering compounds (e.g., ascorbic acid and / or acetaminophen).

21. The method of claim 20, wherein the one or more additional redox altering compounds is present in a concentration from about 1 to about 500 pM.

22. A method for detecting a redox state of a biospecimen, comprising: combining the biospecimen with a redox quenching composition to form a quenched specimen, and detecting a redox sensor in the quenched specimen.

23. The method of claim 22, wherein the combining comprises: collecting the biospecimen, wherein the biospecimen is contacted to the redox quenching composition during the collecting to form the quenched specimen.

24. The method of claim 22, wherein the combining comprises: providing the biospecimen, and adding the redox quenching composition to the biospecimen.

25. The method of claim 22, wherein the redox quenching composition comprises one, two, three, or four or more of: clioquinol, deferrioxamine, BPDS, DTP A, astaxanthin, BHT, a sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate), a thiol -reactive agent (e.g., MSTP, NEM, LAM, MMTS, DNFB, or mBBr), EDTA, and a protease inhibitor (e.g., Pierce Protease Inhibitor).

26. The method of claim 25, wherein: the clioquinol is present in a concentration from about 10 to about 500 pM; deferrioxamine is present in a concentration from about 1 to about 50 pM;BPDS is present in a concentration from about 0.1 to about 5 mM;DTPA is present in a concentration from about 0.1 to about 5 mM;Astaxanthin is present in a concentration from about 10 to about 250 pM;BHT is present in a concentration from about 10 to about 500 pM;Dimedone is present in a concentration from about 0.1 to about 5 mM;NEM is present in a concentration from about 5 to about 100 mM;EDTA is present in a concentration from about 0.5 to about 5 mg / mL; and / or the protease inhibitor (e.g., Pierce Protease Inhibitor) is present in a concentration sufficient to reduce, inhibit or prevent proteolysis of endogenous protein in the biospecimen.

27. The method of any one of claims 22-26, wherein the redox sensor comprises a 2- cysteine peroxiredoxin (PRX).

28. The method of any one of claims 22-27, wherein the redox sensor comprises a reduced glutathione (GSH) and / or an oxidized glutathione (GSSG) (e.g., a GSH / GSSG ratio).

29. The method of any one of claims 22-28, wherein the biospecimen is a biological sample of a human or non-human animal subject.

30. The method of claim 29, wherein the biological sample is a blood sample.

31. The method of 30, wherein the blood sample comprises the RBC fraction.

32. The method of claim 30 or claim 31, wherein the blood sample comprises an anticoagulant.

33. The method of claim 32, wherein the anticoagulant is heparin, EDTA, or NaF / KOx.

34. The method of any one of claims 22-33, wherein the combining is carried out in vitro (e.g., in a test tube).

35. The method of claim 34, wherein the combining is carried out in a test tube and the test tube is under negative pressure (e.g., a vacuum).

36. The method of claim 35, wherein the test tube contains the redox quenching composition.

37. The method of claim 36, wherein the biospecimen in a blood sample and is added directly to the test tube from a subject.

38. The method of any one of claims 22-37, wherein the biospecimen is from a subject that has or is suspected of having sepsis.

39. The method of claim 38, wherein the method further comprises treating the subject with an antibiotic therapy.

40. The method of any one of claims 22-39, wherein the biospecimen is from a subject that has or is suspected of having an infectious disease, cancer, diabetes, obesity, cardiovascular disease, or an aging-related disease.

41. The method of claim 40, wherein the method further comprises treating the subj ect with a redox altering therapy.

42. The method of any one of claims 22-41, wherein the biospecimen is from an adult or geriatric subject.

43. The method of claim 29, wherein the biological sample is a tissue sample (e.g., a bone tissue sample).

44. The method of claim 43, wherein the redox quenching composition further comprises an alcohol (e.g., methanol, ethanol, and / or propanol), a protein cross-linking reagent (e.g., polyethylene glycol and / or paraformaldehyde), and / or a phosphatase inhibitor (e.g., a phosphatase inhibitor cocktail, sodium fluoride, sodium orthovanadate, sodium pyrophosphate, glycerophosphate, or any combination thereof).

45. The method of claim 44, wherein: the alcohol is present in the redox quenching composition from about 1 to about 90% by volume of the redox quenching composition;the protein cross-linking reagent is present in the redox quenching composition from about 0.1 to about 20% by volume of the redox quenching composition; and / or the phosphatase inhibitor is present in a concentration from about 1 to about 350 mM.

46. The method of any one of claims 22-45, wherein the redox quenching composition further comprises a y-glutamyl transferase inhibitor (e.g., 6-diazo-5-oxo-L-nor-Leucine and / or OU749) and a spin trap (e.g., DMPO).

47. The method of claim 46, wherein the y-glutamyl transferase inhibitor (e.g., 6-diazo-5- oxo-L-nor-Leucine and / or OU749) is present in a concentration from about 0.1 to about 100 pM; and the spin trap (e.g., DMPO) is present in a concentration from about 10 to about 500 mM.

48. The method of any one of claims 22-47, wherein the redox quenching composition further comprises one or more additional redox altering compounds (e.g., ascorbic acid and / or acetaminophen).

49. The method of claim 48, wherein the one or more additional redox altering compounds is present in a concentration from about 1 to about 500 pM.

50. A redox quenching composition comprising two, three, four, or five or more of: clioquinol, deferrioxamine, BPDS, DTP A, astaxanthin, BHT, a sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate), a thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr), EDTA, and a protease inhibitor (e.g., Pierce Protease Inhibitor).

51. The redox quenching composition of claim 50, wherein said composition comprises: the BHT, optionally in combination with astaxanthin and / or acetaminophen; the sulfenic acid reacting molecule (e.g., dimedone, NBD chloride, TNB, P-ketoesters, strained alkynes and alkenes, Wittig reagents, or an enolate); and the thiol -reactive agent (e.g., MSTP, NEM, IAM, MMTS, DNFB, or mBBr).

52. A blood collection tube comprising the composition of claim 50 or claim 51.

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