Anti-freezing solution for time-temperature exposure visual indicator of biological sample

Abstract

Provided herein is an indicator system comprising permanganate, an organic reducing agent, an acid, and an inorganic perchlorate. Methods of using the indicator system to monitor the integrity of a biological sample are also provided. The systems and methods described herein can provide an individual aliquot level thaw status indicator that is easy to use for indicating time-temperature exposure of a biological sample.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 388,885, filed on July 13, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] A note about federally funded research

[0004] This invention was made with government support from the National Institutes of Health under Grant No. R21CA250999. The government has certain rights in this invention. Background Art

[0005] Clinical researchers face the problem that many bioanalytes of interest become unstable when biospecimens are thawed, improperly stored, or improperly handled. For example, the American Society of Clinical Oncology (ASCO) / College of American Pathologists (CAP) guidelines state that tissues collected for clinical HER2 testing in breast cancer must have a cold ischemic time of less than 1 hour before fixation. Estrogen receptor (ER) testing is more likely to produce false negative results when tissues are subjected to prolonged cold ischemia, which often occurs after surgical resection and biopsy. For plasma / serum and many types of tissue specimens to be frozen, the appropriate refrigeration temperature is well below the common laboratory freezer temperature of -20°C. Free prostate-specific antigen (PSA) in serum loses stability within 4 hours at 4°C. Cell-free DNA is unstable in plasma stored at 4°C for 24 hours. TIMP-1, VEGF, and VEGF-R in serum lose stability after approximately 3 months of storage at -20°C, but remain stable at -75°C. Every year, mishandling and inconsistencies in preanalytical sample handling and storage, including inconsistencies in biobanking practices, generate an unacceptably high number of costly false leads in biomedical research and compromise diagnostic integrity. Currently, there are few tools and widely accepted methods for evidence-based tracking of biospecimens exposed to thaw conditions.

[0006] Ex vivo biospecimen handling and storage conditions have the potential to affect the measurement of certain clinical biomarkers as much or more than the in vivo (clinical) conditions of interest. Therefore, the use of compromised biospecimens in diagnostic or biomedical research may directly lead to false conclusions. The danger of false findings / false conclusions is particularly great for non-targeted biomarker discovery studies, or in targeted analyses where the stability of one or more target analytes has not yet been validated. There are many pre-analytical variables (PAVs) that can compromise the integrity of tissues and biofluids collected for biomedical research. For frozen specimens, the most difficult PAV to control and track throughout the life of the archived sample is exposure to thawing conditions - especially those at the individual aliquot level. Clinical analytes of interest in all types of frozen biospecimens exhibit quantitative instability when the biospecimens are exposed to thawing conditions. Although this problem is sometimes overlooked or not taken seriously, more and more biomedical researchers are beginning to realize the potential disaster of using biospecimens with undocumented or questionable histories in research.

[0007] Common approaches to address this issue are to follow standard operating procedures (SOPs) and document deviations, or to implement evidence-based tracking of biospecimens exposed to thaw conditions. Unfortunately, there are few tools and no widely accepted methods for evidence-based biospecimen quality assurance (QA) or quality control (QC), other than careful adherence to SOPs and documentation, which would be ideal. In practice, biomedical researchers rarely employ any evidence-based QA / QC tools. Therefore, there is a need for easy-to-use, individual aliquot-level thaw status indicators for time-temperature exposure of biospecimens. Summary of the invention

[0008] In a first aspect, an indicator system is provided herein, comprising permanganate, an organic reducing agent (such as oxalic acid (salt)), an acid, an aqueous solvent, and an inorganic perchlorate, which is initially configured to prevent the reduction of the permanganate to Mn 2+ The mixture of permanganate, organic reducing agent, acid, aqueous solvent and inorganic perchlorate is a eutectic composition having a predetermined freezing point. At a temperature above the predetermined freezing point, the organic reducing agent causes the perchlorate to convert to Mn 2+ The reduction reaction occurs at a temperature lower than the predetermined freezing point, and the reduction reaction stops.

[0009] In some embodiments, the inorganic perchlorate is selected from the group consisting of sodium perchlorate (NaClO4), magnesium perchlorate (Mg(ClO4)2), and lithium perchlorate (LiClO4). In some embodiments, the eutectic composition comprises about 15 wt% to about 60 wt% of the inorganic perchlorate.

[0010] In some embodiments, the predetermined freezing point is below -10.0°C, including, for example, -18°C, -37°C, and -67°C.

[0011] In some embodiments, the predetermined freezing point is in the range of -16°C to -20°C (e.g., -18°C), and the eutectic composition comprises 20 wt% to 30 wt% (e.g., about 25 wt%) of LiClO4. In some embodiments, the predetermined freezing point is in the range of -35°C to -39°C (e.g., -37°C), and the eutectic composition comprises 50 wt% to 54 wt% (e.g., about 52 wt%) of NaClO4. In some embodiments, the predetermined freezing point is in the range of -65°C to -69°C (e.g., -67°C), and the eutectic composition comprises 42 wt% to 46 wt% (e.g., about 44 wt%) of Mg(ClO4)2.

[0012] In some embodiments, the eutectic composition comprises the same relative amounts of sodium perchlorate, magnesium perchlorate, or lithium perchlorate as used in the preceding paragraphs. For example, in some cases, the eutectic composition comprises 8.9 molal sodium perchlorate (moles of sodium perchlorate per kilogram of solvent). In some cases, the eutectic composition comprises 3.7 molal magnesium perchlorate. In some cases, the eutectic composition comprises 3.1 molal lithium perchlorate. In some embodiments, the eutectic composition comprises 0.5 mM to 5 mM permanganate. In some embodiments, the eutectic composition comprises 1 to 15 mM organic reducing agent. In some embodiments, the eutectic composition comprises 1 mM to 300 mM sulfuric acid or perchloric acid.

[0013] In some embodiments, the indicator system further comprises a multi-chamber storage vessel comprising a removable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the removable barrier is configured to prevent the permanganate and the organic reducing agent from mixing. In some embodiments, wherein the first chamber has permanganate, and wherein the second chamber has a solution comprising an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate.

[0014] In some embodiments, the indicator system further comprises a storage vessel, and the eutectic composition is built into a portion of the storage vessel. In some embodiments, the storage vessel is a multi-chamber storage vessel.

[0015] In a second aspect, a biological sample collection or storage system is provided herein, comprising a storage vessel (configured to hold a biological sample) and any indicator system described herein. In some embodiments, the biological sample collection or storage vessel comprises a recess configured to receive the indicator system and allow the indicator system to be observed without interfering with the biological sample.

[0016] In a third aspect, the present invention provides a method for monitoring biological sample storage, comprising: storing the biological sample in a biological sample storage vessel (e.g., configured to hold the biological sample therein); associating any indicator system described herein with the biological sample or a biological sample storage vessel (e.g., having a chamber configured to hold the biological sample); mixing permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate to form a eutectic composition; observing the color of the eutectic composition or measuring the absorbance at 525 nm; wherein a color change from pink to colorless or an absorbance less than 0.1 indicates that the biological sample is thawed, improperly stored, or improperly handled.

[0017] Advantageously, the present system can provide instant visual assessment of the integrity of biological samples at the individual aliquot level. Such systems are also designed to track limited exposure (several minutes to several hours) of biological samples at contraindicated temperatures. In addition, the indicators described herein can be customized for specific storage conditions and time-temperature exposure intervals. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A-1B A system with an indicator ( Figure 1A ) and a vessel for containing biological samples ( Figure 1B ) is an exemplary biological sample collection or storage system.

[0019] Figure 2A The time course of permanganate reduction of a 52% (w / w) NaClO4 system at 25°C monitored by absorbance at 525 nm is shown (n=6). The final concentrations of potassium permanganate, disodium oxalate and perchloric acid were 0.5 mM, 1.38 mM and 10 mM, respectively. Figure 2B Results are shown for an illustrative 52% (w / w) NaClO4 system eutectic frozen at -37°C and set to run at 25°C for 60 minutes (n=3 replicate overlays).

[0020] Figure 3 Shown is a run time versus temperature curve for a 52% (w / w) NaClO4 system that eutectic freezes at -37°C. The two digits at the end of each legend entry ("N-37C-xx") indicate the time, in minutes, that the reaction system was designed to run at 25°C.

[0021] Figure 4The effect of multiple preliminary freeze-thaw cycles on the final reaction run time of a 25% (w / w) NaClO4 system, eutectic frozen at -37°C, nominally run at 25°C for 30 minutes without any freeze-thaw cycles is shown. Results for 1 to 6 freeze-thaw cycles (n=3) are shown.

[0022] Figure 5A The time course of permanganate reduction of a 44% (w / w) Mg(ClO4)2 system at 25°C monitored by absorbance at 525 nm is shown (n=6). The final concentrations of potassium permanganate, disodium oxalate and perchloric acid were 0.5 mM, 1.38 mM and 10 mM, respectively. Figure 5B Results are shown for an illustrative 44% (w / w) Mg(ClO4)2 system that was eutectic frozen at -67°C and designed to run at 25°C for 30 min without transition to a brown intermediate (n=3 replicates).

[0023] Figure 6 Shown is a plot of run time versus temperature for a 44% (w / w) Mg(ClO4)2 system that eutectically freezes at -67°C and does not experience a brown intermediate. The two digits at the end of each legend entry ("N-67C-xx") indicate the time, in minutes, that the reaction system was designed to run at 25°C.

[0024] Figure 7 Results are shown for an illustrative 25% (w / w) LiClO4 system eutectic frozen at -18°C and designed to run at 25°C for 60 minutes (n=3 replicate stacks).

[0025] Figure 8 Shown is a plot of run time versus temperature for a 25% (w / w) LiClO4 system that eutectically freezes at -18°C. The two digits at the end of each legend entry ("N-18C-xx") indicate the time, in minutes, that the reaction system was designed to run at 25°C.

[0026] Fig. 9 The effect of multiple preliminary freeze-thaw cycles on the final reaction run time of a 25% (w / w) LiClO4 system eutectically frozen at -18°C, nominally run at 25°C for 30 minutes without any freeze-thaw cycles, is shown.

[0027] Incorporation by Reference

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. DETAILED DESCRIPTION

[0029] The present disclosure describes colorimetric indicators for monitoring the handling and storage of biological specimens. A change in the color of the indicator indicates that the biological specimen has been thawed, improperly stored, or improperly handled. The indicator system described herein acts as a "clock" that slows down when the sample cools, stops when the sample is frozen, but restarts when the sample thaws. The indicator is not limited to a specific number of freeze / thaw cycles, but rather a measure of time above a predetermined freezing point threshold temperature.

[0030] Currently available indicators provide objective evidence that a biological specimen has been thawed, improperly stored, or improperly handled. Evidence-based biospecimen QA / QC tracking can be divided into two main forms: endogenous markers and exogenous indicators. The advantage of endogenous markers is that they do not need to be added to the sample at the time of collection, so they can be used retrospectively to analyze existing samples. However, endogenous markers have several inherent disadvantages: they may only be useful for specific biological specimens, testing requires consumption of some sample, have natural sample-to-sample variability, require instrumental analysis, and their use may be limited to specific time or temperature protocols. Therefore, there is a need for exogenous integrity markers that overcome these disadvantages shared by endogenous integrity markers.

[0031] In one aspect, the present disclosure provides an indicator system comprising permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate, wherein the indicator system is initially configured to prevent the reduction of the permanganate to Mn 2+ , wherein the mixture of permanganate, organic reducing agent, acid, aqueous solvent and inorganic perchlorate is a eutectic composition having a predetermined freezing point, wherein at a temperature above the predetermined freezing point, the organic reducing agent causes the permanganate to convert to Mn 2+ The reduction reaction occurs at a temperature lower than the predetermined freezing point, and the reduction reaction stops.

[0032] The exogenous indicators disclosed herein have many advantages: (1) the indicators are suitable for any type of tissue or fluid biological specimen (whether liquid or solid of anatomical origin); (2) the indicators eliminate the need for sample consumption or disturbance of the biological specimen during the integrity check process; (3) the indicators, with their bright pink indication, enable sample handlers to perform ultra-rapid visual inspections and make it impossible to overlook improperly handled samples; (4) the indicators can be based on inexpensive chemicals, thereby limiting implementation costs; (5) the indicators indicate that the biological specimen has been exposed to temperatures above a given temperature for longer than a strictly predefined time interval; and (6) the indicators eliminate the reliance on written handling / storage records, which can be a common source of false confidence in the integrity of archived biological specimens.

[0033] As used herein, "biological sample" refers to a material sample from a human, plant, animal, bacteria, fungus or other organism. The biological sample can be a biological fluid (e.g., blood, urine, semen, tears, plasma, sweat, bile, milk, cerebrospinal fluid, etc.), a tissue sample (e.g., a biopsy sample, muscle tissue, bone, bone marrow, etc.), a cell (e.g., a bacterial cell, a fungal cell, a stem cell, a blood cell, a bone marrow cell, etc.) or a biochemical material (e.g., DNA, RNA, protein, lysate, etc.).

[0034] As used herein, an indication that a biological sample has been "thawed, improperly stored, or improperly handled" generally means that the biological sample has been exposed to an undesirably high temperature or an undesirable time, beyond the appropriate safe storage and handling parameters for the relevant biological sample. Appropriately, the safe storage and handling parameters contribute to the stability of its molecular components. When a biological sample has been thawed, improperly stored, or improperly handled, the integrity of the biological sample may be compromised. As used herein, "compromised integrity" means that the biological sample may become unsuitable for diagnostic or analytical purposes (e.g., by loss or development of a particular quality or property). A biological sample may be unsuitable for diagnostic or analytical purposes because its molecular composition no longer reflects its original state.

[0035] The biological sample is stored adjacent to or near the indicator system described herein, and the indicator system is adjusted so that when the color of the indicator system changes from bright pink to colorless, it indicates that the biological sample has been thawed, improperly stored, or improperly handled in a form that may compromise the integrity of the biological sample. As used herein, "biological sample adjacent to...", "biological sample associated with...", and "biological sample located near..." are used interchangeably, referring to the close proximity and simultaneous handling of the biological sample and the indicator system, so that the biological sample and the indicator system are constantly and continuously subjected to the same storage conditions, the same temperature changes, and the same environmental conditions. When the biological sample is adjacent to the indicator system, associated with the indicator system, or located near the indicator system, they may be located in separate containers or vessels that are reversibly or irreversibly combined. In some embodiments, the biological sample can be associated with the indicator using tape, glue, rubber bands, paraffin film, plastic wrap, plastic wrap, aluminum or tin foil, paper wrap, etc. The biological sample and the indicator system can be placed in separate containers or vessels that are stored in the same box, container, freezer, or other suitable storage system.

[0036] In some embodiments, the biological sample is a biopsy sample. The biopsy sample can be associated with an indicator system immediately after excision from the subject, so that the integrity of the biopsy sample can be continuously monitored from excision to fixation. The biopsy sample is immediately associated with the indicator system described herein to ensure that the biopsy sample will not be improperly handled after excision and before fixation to prevent inaccurate subsequent test results due to improper handling and storage of the biopsy sample. The biopsy sample can be any sample collected from the subject for an appropriate purpose known in the art. In some embodiments, the biopsy sample is a tissue sample for cancer diagnosis.

[0037] In some embodiments, the biological specimen is part of a biobank or archive, where the long-term storage and processing of the biological specimen will be monitored. In the biobank or archive, the indicator system described herein can be adjacent to or associated with a single biological specimen or a group of biological specimens that are continuously processed simultaneously. Monitoring the biological specimen samples in the biobank or archive using the indicator system described herein can ensure the integrity of the standard operating procedures (SOPs) of the biological specimens over a long period of time and across multiple laboratories or responsible for maintaining the biobank or archive.

[0038] The indicator system consists of chemical components that, when mixed, produce a solution that is initially bright pink in color due to the reduction of permanganate to Mn 2+ The chemical components of the indicator system can be selected so that when the liquid is colorless or below a threshold absorbance value, the indicator and any biological specimen associated with it may have been thawed, improperly stored, or improperly handled. Indicators are designed to function at a variety of different temperatures or storage regimens associated with critical storage or handling parameters for various biological specimens.

[0039] The indicator system described herein utilizes permanganate reaction chemistry to control the reaction kinetics to synchronize with the lifetime of a specific biological specimen of interest under conditions of thawing, improper storage, or improper handling. - ) makes the indicator appear bright pink initially, and reacts with organic reducing agents (such as oxalate (C2O4 2- ) or its conjugate acid (H2C2O4)) to produce colorless manganese (Mn 2+ ), water and carbon dioxide (an example of such a reduction reaction is shown in Scheme 1). When the reaction composition is a liquid, the reaction is autocatalytic, and the initial bright pink color gradually fades and becomes colorless over time. When the reaction is cooled, the reaction rate slows down, and when the reaction freezes, the reaction essentially stops.

[0040] 2MnO4 - (Permanganate, bright pink) + 6H3O +(Hydroxy ion / acid) + 5H2C2O4 (oxalic acid) → 2Mn 2+ (colorless) + 14H2O + 10CO2 (gas, but remains dissolved)

[0041] Scheme 1. Permanganate to Mn 2+ An exemplary reduction of .

[0042] The actual reaction mechanism is complex and is carried out in many different steps that interact with each other, but the rate law and / or equilibrium constant of each step at room temperature (25°C) can be determined by known physical and analytical methods. For example, a computer-based mathematical simulation of the reaction was created using software (e.g., MatLab). By running the simulation under different reaction starting conditions, it was found that appropriate adjustment of the initial solution composition can make it possible to prepare solutions that fail at room temperature within a time range as short as a few seconds or as long as several hours, or to prepare solutions that fail for a longer time at a temperature of 0 to 25°C.

[0043] The fact that the reaction takes longer at lower temperatures is consistent with the fact that biological samples remain more stable at lower temperatures. Therefore, if the indicator is physically associated with the biological sample (e.g., by being stored in different subchambers of the same storage vessel), then keeping the biological sample at a lower temperature (e.g., on ice) as a preservative between the collection point and final archival storage will slow the deterioration of both the biological sample and the indicator. Similarly, accidental exposure to higher temperatures accelerates the deterioration of both the sample and the indicator.

[0044] In the disclosed system, oxalate is an exemplary organic reducing agent, although other organic reducing agents may also be used. Permanganate is able to oxidize carbon atoms if the carbon atoms contain sufficiently weak (or reactive) bonds, including (1) carbon atoms with π bonds, such as alkenes and alkynes; (2) carbon atoms with weak CH bonds, such as CH bonds in the α position of substituted aromatic rings and CH bonds in carbon atoms containing CO bonds, including alcohols and aldehydes; and (3) carbon atoms with particularly weak CC bonds, such as the CC bonds in ethylene glycol and the CC bonds next to aromatic rings and oxygen.

[0045] The composition and concentration of the chemical components can control the freezing point of the indicator system and control the reaction rate so that the reaction rate and freezing point are synchronized with the life of biological specimens that have been thawed, improperly stored, or mishandled. When the reaction composition changes from bright pink to colorless, the indicator indicates that the integrity of the biological specimen may have been compromised and is no longer suitable for its intended use.

[0046] The chemical components of the indicator system are selected so that their mixture forms a eutectic composition comprising permanganate, an organic reducing agent, an acid, a solvent (such as water) and an inorganic perchlorate. When the inorganic perchlorate is added to the eutectic composition, its concentration should maintain the eutectic nature of the composition and not significantly change the initial bright pink color of the composition.

[0047] As used herein, "eutectic composition" refers to a homogeneous composition that freezes and thaws at essentially a single temperature and does not partially freeze, partially thaw, or precipitate out of the composition. As used herein, "eutectic concentration" refers to the concentration at which a composition or compound freezes and thaws at essentially a single temperature and does not partially freeze or partially thaw, or the concentration at which a compound in a solution freezes and thaws at essentially a single temperature and does not partially freeze or partially thaw.

[0048] As used herein, "substantially a single temperature" means that all chemical components of the eutectic composition freeze or thaw within 2.0°C. Therefore, minor deviations in component concentrations from the ideal eutectic concentration are considered. In some embodiments, all components of the eutectic composition freeze or thaw within 1.0°C, 0.5°C, or 0.2°C. When the difference in freezing or thawing temperatures of the components is too large, kinetic control of the permanganate / oxalic acid reaction system may be lost.

[0049] Permanganate (MnO4 -) can be provided as any suitable permanganate known in the art. The permanganate in the eutectic composition is provided in a concentration suitable for producing an initial bright pink color detectable by the naked eye. The concentration of the permanganate can be about 0.1mM to about 10mM, including but not limited to about 0.1mM to about 8mM, about 0.1mM to about 5mM, about 0.2mM to about 10mM, about 0.2mM to about 8mM, about 0.2mM to about 5mM, about 0.5mM to about 10mM, about 0.5mM to about 8mM, about 0.5mM to about 5mM. In some embodiments, the eutectic composition comprises about 0.5mM to about 5mM of permanganate. For example, permanganate can be present at a concentration of about 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM or 10 mM. Suitable permanganates include, but are not limited to, ammonium permanganate (NH4MnO4), potassium permanganate (KMnO4), sodium permanganate (NaMnO4), magnesium permanganate (Mg(MnO4)2) and any suitable hydrate thereof. In some embodiments, permanganate is potassium permanganate. In some embodiments, permanganate is potassium permanganate at a concentration of about 0.1 mM to about 2 mM, including, for example, 0.25 mM, 0.5 mM, 0.75 mM and 1 mM. In some embodiments, the permanganate is sodium permanganate at a concentration of about 0.1 mM to about 2 mM, including, for example, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM.

[0050] The presence of permanganate in the eutectic composition produces an initial bright pink color. Typically, the bright pink color is visible to the naked eye, and the naked eye can detect and observe the transition from bright pink to colorless during the permanganate reduction process. As used herein, "bright pink" and "pink" are used interchangeably and refer to the color associated with the absorbance at 525nm. When measured using a spectrophotometer, the initial eutectic composition typically has an initial absorbance of about 0.5 to 1.5, but eutectic compositions with initial absorbances outside this range can also be used. When the permanganate in the eutectic composition is reduced, the color of the composition changes from pink to colorless, as seen by the naked eye. When measured using a spectrophotometer, the absorbance of the colorless composition at 525nm may be less than 0.1 (e.g., 0.1, 0.05, or 0). In some embodiments, the color observed by the naked eye changes from pink to colorless to indicate thawing, improper storage, or improper handling of the biological sample, while the persistent pink color observed by the naked eye indicates proper storage. In some embodiments, thawing, improper storage or improper handling of a biological sample is indicated by at least a 50% decrease in absorbance at 525 nm when measured by a spectrophotometer, while proper storage is indicated by a decrease in absorbance at 525 nm of less than 50% when measured by a spectrophotometer. In some embodiments, thawing, improper storage or improper handling of a biological sample is indicated by at least a 80%, 90%, 95% or 99% decrease in absorbance at 525 nm when measured by a spectrophotometer.

[0051] The organic reducing agent can be provided in any suitable form that allows permanganate reduction. The organic reducing agent can be present in a concentration of about 0.1 mM to about 100 mM, including but not limited to about 0.1 mM to about 50 mM, about 0.1 mM to about 25 mM, about 0.5 mM to about 100 mM, about 0.5 mM to about 50 mM, about 0.5 mM to about 25 mM, about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM and about 1 mM to about 15 mM. For example, the organic reducing agent can be present at a concentration of about 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM or 100 mM. In some embodiments, the concentration of the organic reducing agent is about 0.5 mM to about 20 mM. In certain other embodiments, the concentration of the organic reducing agent is from about 1 mM to about 15 mM, including, for example, 0.75 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM.

[0052] The oxalate in the eutectic composition can be provided in the form of oxalic acid or any suitable oxalate known in the art. The concentration of oxalate provided in the eutectic composition is suitable for autocatalytic reduction of permanganate to manganese. The concentration of oxalate in the reaction depends on the concentration of permanganate. Oxalic acid (salt) can be present at a concentration of about 0.1 mM to about 100 mM (e.g., 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM). Suitable oxalate compounds include, but are not limited to, oxalic acid (H2C2O4), sodium oxalate (Na2C2O4), potassium oxalate (K2C2O4), and magnesium oxalate (MgC2O4). In some embodiments, the oxalate is sodium oxalate at a concentration of about 0.5 mM to about 20 mM, including, for example, 0.75 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM. In some embodiments, the oxalate is potassium oxalate at a concentration of about 0.5 mM to about 20 mM, including, for example, 0.75 mM, 1 mM, 1.5 mM, 2 mM, and 2.5 mM.

[0053] The acid in the eutectic composition can be any suitable acid known in the art. The concentration of the acid provided in the eutectic composition is suitable for adjusting the reaction rate as needed and ensuring that the reaction remains autocatalytic when in the liquid state. The concentration of the acid can be from about 1 mM to about 5.0 M, including but not limited to from about 1 mM to about 2.0 M, from about 1 mM to about 1.0 mM, from about 1 mM to about 500 mM, from about 1 mM to about 300 mM, from about 1 mM to about 100 mM, from about 1 mM to about 50 mM, from about 1 mM to about 25 mM, from about 1 mM to about 15 mM, from about 1 mM to about 10 mM, and from about 1 mM to about 5 mM. For example, the acid can be present at a concentration of about 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 125 mM, 150 mM, 175 mM, 200 mM, 225 mM, 250 mM, 275 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 750 mM, 1.0 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, 4.5 M, or 5.0 M. In some embodiments, the acid concentration is 1 mM to 500 mM, including, for example, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 50 mM, 100 mM and 200 mM. Suitable acid can be selected from any known inorganic acid, including but not limited to sulfuric acid (H2SO4), hydrochloric acid (HCl), phosphoric acid (H3PO4) and perchloric acid (HClO4). In some embodiments, the acid is perchloric acid.

[0054] Aqueous solvent as used herein refers to a suitable solvent comprising water. Suitable aqueous solvents do not interfere with the chemical reactions, absorbance measurements or color detection described herein. In some embodiments, the aqueous solvent is water.

[0055] The eutectic composition also includes an inorganic perchlorate. The inorganic perchlorate is present in the eutectic composition at a concentration suitable for maintaining the eutectic properties of the entire composition. The inorganic perchlorate can be selected to adapt the predetermined freezing point to the required storage or processing temperature or close to the freezing point of the biological sample. In some embodiments, the inorganic perchlorate is present in a concentration of about 10% to about 60% by weight of the eutectic composition. The concentration of the inorganic perchlorate can be, for example, about 15% to about 60% by weight, about 20% to about 60% by weight, about 35% to about 60% by weight, or about 20% to about 55% by weight of the eutectic composition.

[0056] In some embodiments, the inorganic perchlorate comprises an alkali or alkaline earth metal cation and a perchlorate counter anion. Suitable inorganic perchlorates include, but are not limited to, sodium perchlorate (NaClO4), magnesium perchlorate (Mg(ClO4)2), and lithium perchlorate (LiClO4). Specifically, lithium perchlorate salt is an indicator having a melting point of -18°C and can be used for temporary storage of fresh frozen plasma. As a non-limiting example, the eutectic composition may comprise 20% to 30% by weight of LiClO4, 50% to 54% by weight of NaClO4, 42% to 46% by weight of MgMg(ClO4)2, or mixtures thereof.

[0057] It is worth noting that not all combinations of cations and perchlorate anions can be used as the inorganic perchlorate salts of the present invention. For example, some salts do not have eutectic points, but contain certain cations (e.g., Ca 2+ ) salts will form insoluble complexes with the oxalic acid (salt) reactant, thereby destroying the reaction system.

[0058] Perchlorate does not react with permanganate (e.g., cannot be oxidized by permanganate) and does not interfere with the visual indicator reaction. Therefore, compared with chloride-based inorganic salts, the system described herein can be designed to run for longer periods of time (e.g., run for more than one hour at room temperature, rather than a few minutes at room temperature). Chloride can be oxidized by permanganate, so if used, it will interfere with the reaction to a certain extent. Other inorganic salts with a eutectic freezing point from 0 to about -80°C can also be used in this system. However, it is worth noting that conventional organic antifreeze solutions (such as ethylene glycol or propylene glycol) are not suitable for this purpose because they tend to react with permanganate ions in a manner similar to oxalic acid.

[0059] The indicators described herein are suitable for use in applications where the desired storage and handling temperature of a biological sample is below a threshold of 0°C. "Desired storage and handling temperature" refers to a temperature at which a biological sample maintains its integrity when it is below the temperature. Below the desired storage and handling temperature, the above reaction system will freeze and stop indefinitely unless reheated. For the vast majority of biological molecules and biological samples, the lower the temperature, the better the stability. However, in some commercial applications (such as temporary storage of fresh frozen plasma), indicators that are active above 0°C (but stop below 0°C) may be useful (e.g., lithium perchlorate).

[0060] In some embodiments, the predetermined freezing point is selected to be within the operating temperature range of a freezer suitable for maintaining a biological specimen at an appropriate storage temperature, or within the temperature range for maintaining a biological specimen in a frozen state. However, appropriate long-term storage of many biological specimens (e.g., biobanks) requires that they be stored at -80°C or below (e.g., at liquid nitrogen vapor phase temperature, which is approximately -160°C). For example, plasma and serum contain so much protein that these liquid specimens will not actually freeze until the temperature reaches -30°C or lower. Therefore, storing plasma and serum at common research laboratory freezer temperatures of -20°C is unacceptable because biochemical reactions within the sample can continue at this temperature, causing the sample to fail. Therefore, a color-changing biological specimen integrity indicator that freezes at 0°C is useless for fully tracking the storage and handling conditions of archived plasma or serum samples. An ideal tracer should freeze at a temperature close to the freezing point of the specimen itself. If not, a conservative tracer should only stop at the appropriate archival storage temperature (or slightly above that temperature). To achieve this feature, the colorimetric indicator of the present disclosure is implemented in an antifreeze solution to allow the reaction to proceed normally.

[0061] In some embodiments, the predetermined freezing point is selected to indicate a malfunction of a freezer at a temperature suitable for maintaining a biological specimen at an appropriate storage and handling temperature or for maintaining a biological specimen in a frozen state.

[0062] By selecting the components, the predetermined freezing point can be tailored to the desired storage and handling temperature. The freezing point of the eutectic composition is specific to the combination of components, such as inorganic perchlorate, and the concentration of each component in the indicator system. The components of the indicator system can be selected based on the known eutectic temperature of the inorganic perchlorate so that the freezing point is close to or specific to the storage temperature of the biological sample of interest or the desired storage temperature. An exemplary embodiment includes 44% (w / w) magnesium perchlorate, which freezes eutectically at about -67°C. Another exemplary embodiment includes 52% (w / w) sodium perchlorate, which freezes eutectically at about -37°C. Yet another exemplary embodiment includes 25% (w / w) lithium perchlorate, which freezes eutectically at about -18°C. Other magnesium perchlorate, sodium perchlorate, and lithium perchlorate concentrations may result in partial freezing of the solution or partial precipitation of its salt before the entire solution becomes solid, thereby resulting in a loss of kinetic control of the permanganate / oxalic acid reaction system.

[0063] In some embodiments, the freezing point of the eutectic composition is the same as or close to the freezing point of the biological sample of interest. As used herein, "freezing point close to the freezing point of the biological sample" means that the freezing point is within 5°C of the freezing point of the biological sample. In some embodiments, the freezing point of the eutectic composition is within 2°C of the freezing point of the biological sample. In some embodiments, the freezing point of the eutectic composition is within 1°C of the freezing point of the biological sample.

[0064] In some embodiments, the predetermined freezing point can be below -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -35°C, -40°C, -45°C, -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, -80°C, -85°C, or -90°C. In some embodiments, the predetermined freezing point is below -10°C. In some embodiments, the predetermined freezing point is below -30°C. In some embodiments, the predetermined freezing point is below -60°C. In some embodiments, the predetermined freezing point is -18°C, -37°C, or -67°C.

[0065] The predetermined freezing point can be selected within different temperature ranges. In some embodiments, the predetermined freezing point can be 0°C to -15°C, 0°C to -18°C, 0°C to -20°C, 0°C to -25°C, 0°C to -30°C, 0°C to -35°C, 0°C to -40°C, 0°C to -45°C, 0°C to -50°C, 0°C to -55°C, 0°C to -60°C, 0°C to -65°C, 0°C to -70°C, 0°C to -75°C, 0°C to -80°C, 0°C to -85°C, -15°C to -30°C, -15°C to -35°C, -15°C to -40°C, -15°C to -45°C, -18°C to -30 ℃, -18 ℃ to -35 ℃, -18 ℃ to -40 ℃, -18 ℃ to -45 ℃, -20 ℃ to -30 ℃, -20 ℃ to -35 ℃, -20 ℃ to -40 ℃, -20 ℃ to -45 ℃, -25 ℃ to -30 ℃, -25 ℃ to -35 ℃, -25 ℃ to -40 ℃, -25 ℃ to -45 ℃, -35 ℃ to -80 ℃, -40 ℃ to -80 ℃, -45 ℃ to -80 ℃, -50 ℃ to -80 ℃, -35 ℃ to -85 ℃, -40 ℃ to -85 ℃, -45 ℃ to -85 ℃, or -50 ℃ to -85 ℃.

[0066] In some embodiments, the indicator system comprises 20 wt % to 55 wt % of an inorganic perchlorate, wherein the predetermined freezing point is below -15.0°C. As a non-limiting example, the predetermined freezing point may be -18.0°C, and the eutectic composition may comprise 20 wt % to 30 wt % LiClO4. As another non-limiting example, the predetermined freezing point may be -37.0°C, and the eutectic composition may comprise 50 wt % to 54 wt % NaClO4. As another non-limiting example, the predetermined freezing point may be -67.0°C, and the eutectic composition may comprise 42 wt % to 46 wt % Mg(ClO4)2.

[0067] The reaction rate of the liquid eutectic composition can be adjusted or regulated based on the compounds contained in the composition and their concentrations. In some embodiments, increasing the concentration of the acid increases the reaction rate. In some embodiments, decreasing the concentration of the acid decreases the reaction rate. In some embodiments, increasing the amount of Mn in the system 2+ to increase the reaction rate or reduce the initial concentration of Mn 2+ to reduce the initial concentration of the reaction.

[0068] The color transition of the system is nonlinear with time; specifically, it will remain the original bright pink until shortly before the end of the reaction, and then, as the reaction approaches the end point, it will rapidly transition from bright pink to clear. This is due to the autocatalytic nature of the reaction chemistry involved. In some cases (e.g., when magnesium perchlorate is used as the inorganic perchlorate), the system will transition from the original bright pink to brown and then clear.

[0069] Using different reaction starting conditions for simulation, components of the indicator system with tailored lifespans can be selected over different timeframes. As used herein, the "lifespan" of an indicator system is the length of time required for the indicator system to transition from an initially mixed eutectic composition to a state indicating thawing, improper storage, or improper handling at a specific temperature above a predetermined freezing point. In some embodiments, the indicator system has a lifespan of less than 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 60 minutes, 45 minutes, 30 minutes, 25 minutes, 20 minutes, 15 minutes, 10 minutes, 9 minutes, 8 minutes, 7 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute at a specific temperature above a predetermined freezing point.

[0070] In some embodiments, the chemical components of the indicator system are selected to have a lifetime of less than 120, 90, 60, 45, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute at 25°C. Indicator systems with a lifetime of less than 120 minutes at 25°C may be useful in a variety of applications (such as initial biological sample collection and pre-storage processing). Indicator systems with a lifetime of less than 1 minute at 25°C may be useful in a variety of applications (such as long-term storage) where storage temperatures above -30°C or thawing of biological samples is not appropriate.

[0071] In some embodiments, the chemical components of the indicator system are selected to have a lifetime of less than 24, 18, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 hour at 4° C. Indicator systems with a lifetime of less than 24 hours at 4° C. can be useful in a variety of applications (such as initial biological specimen collection and pre-storage processing).

[0072] In some embodiments, the chemical components of the indicator system are selected to have a lifetime of less than 60, 45, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minute at 0° C. Indicator systems with a lifetime of less than 60 minutes at 0° C. may be useful in a variety of applications (such as long-term storage) where brief storage or thawing of biological samples above -30° C. is permissible.

[0073] In some embodiments, the chemical components of the indicator system are selected to have a lifetime of less than 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 18 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, or 2 hours at -20°C. Indicator systems with a lifetime of less than 4 hours at -20°C may be suitable for a variety of applications (such as long-term storage) where storage temperatures above -30°C or thawing of biological samples is inappropriate. Indicator systems with a lifetime of 24 to 4 hours at -20°C may be suitable for a variety of applications (such as long-term storage) where short-term storage or thawing of biological samples above -30°C is allowed. Indicator systems with a lifetime of 7 to 1 day at -20°C may be suitable for a variety of applications (such as collecting or temporarily storing biological samples in places that lack refrigeration below -20°C).

[0074] A biological sample collection and storage system is also provided. The biological sample collection and storage system may include any indicator system and biological sample storage container described herein. As used herein, a "biological sample storage container" is a container configured to accommodate a biological sample. Suitably, the biological sample storage container is a well, a vial, a cup, a bag or other container suitable for storing the sample for temporary or long-term storage. As used herein, "temporary storage" refers to a storage time of several hours, several days or up to about a week, and "long-term storage" refers to a storage time of more than about a week, including a storage time of more than one month, more than one year or indefinite storage.

[0075] In some cases, the present indicator can be stored inside or on the outside of the cap of a flexible activated vial so that mishandled samples cannot be overlooked. For example, once the bright pink color turns clear (easily visible from the outside of the storage vial), this indicates that the total time-temperature range to which the specimen in the vial was exposed exceeds the permitted storage / handling parameters. This will facilitate empirical biospecimen storage and handling tracking during initial biospecimen handling and throughout the lifecycle of archived or biobank specimens.

[0076] The indicator system should first be configured to prevent the reduction of permanganate to Mn 2+ , but should also be configured to allow the user to prepare a mixture of permanganate, organic reducing agent, acid, aqueous solvent and perchlorate inorganic salt into a eutectic composition with a predetermined freezing point. This allows the user to initiate the reduction reaction at the point where monitoring of the biological sample is desired.

[0077] In some embodiments, the indicator system will be associated with the outside of the biological sample storage vessel and will never be in physical contact with the biological sample to be stored in the biological sample storage vessel. This allows the indicator system to be observed without disturbing the biological sample in the biological sample storage vessel.

[0078] In some embodiments, the indicator system may further include or be configured as a multi-chamber storage vessel that facilitates activation of the chemical reaction once the vessel is put into use (i.e., once the biological sample is placed therein). The multi-chamber storage vessel may include a removable barrier defining at least a first chamber and a second chamber. As used herein, a "removable barrier" is a barrier that is configured to be removable or lose structural integrity, thereby eliminating the distinction between the chambers. The chemical components (such as permanganate and the organic reducing agent) may be distributed between the two or more chambers in any suitable manner to prevent the reduction of the permanganate to Mn 2+ . In some embodiments, the multi-chamber storage vessel includes a detachable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the detachable barrier is configured to prevent the permanganate and the organic reducing agent from mixing. In some embodiments, the permanganate is contained in the first chamber, and the organic reducing agent is contained in the second chamber. In a specific embodiment, one chamber (e.g., the first chamber) contains permanganate, and another chamber (e.g., the second chamber) contains a solution comprising an organic reducing agent, an acid, and an aqueous solvent. Inorganic perchlorate can be contained with permanganate or a solution. In some embodiments, an inorganic perchlorate is contained with a solution. In some embodiments, wherein the first chamber has permanganate, and wherein the second chamber has a solution comprising an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate. The indicator system can be activated by bending, flexing, squeezing, breaking, folding, or destroying the detachable barrier, thereby allowing the chemical components in different chambers to mix.

[0079] In another aspect, the present disclosure provides a biological sample collection or storage system, comprising a biological sample storage vessel and an indicator system as described herein. As a non-limiting example, the indicator system may include a multi-chamber storage vessel, the multi-chamber storage vessel comprising a removable barrier, the removable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the removable barrier is configured to prevent the permanganate and the organic reducing agent from mixing.

[0080] Figure 1AAn exemplary multi-chamber storage vessel 10 is shown. The multi-chamber storage vessel may include an outer chamber 12 and an inner chamber 14 defined by a removable barrier. In an exemplary embodiment, permanganic acid / salt (such as permanganate) may be contained within the inner chamber 14, while an organic reducing agent may be contained in the outer chamber 12. The outer chamber 12 may be surrounded by a flexible material that may be manipulated without losing structural integrity. In contrast, the inner chamber 14 may be surrounded by a rigid or brittle material that may not withstand such manipulation. Such an indicator will cause the chemical components to mix when the indicator is bent, flexed, folded, or broken. Although Figure 1A The exemplary embodiment shown in shows an inner chamber 14 within an outer chamber 12, but the chambers may be configured in other arrangements (such as side-by-side chambers).

[0081] In some embodiments, the biological specimen storage vessel may be configured to be associated with an indicator system. Figure 1B An exemplary biological specimen collection vessel 20 is shown in FIG. 1 , which has a recess 22 that allows the biological specimen collection vessel to be associated with an indicator system, such as Figure 1A The indicator system shown. The recess 22 can be configured to reversibly or irreversibly associate with the indicator system on the outer surface of the biological sample collection vessel 20. This allows the indicator system to be observed without disturbing the biological sample. In some embodiments, the biological sample collection or storage system herein includes a recess that is configured to receive the indicator system and allows the indicator system to be observed without disturbing the biological sample.

[0082] In some embodiments, the biological sample storage vessel and the indicator system are incorporated into a portion of the biological sample storage vessel. For example, the indicator system can be incorporated into a lid, a wall, a parallel vertical or horizontal chamber, a single well of a multi-well plate, or a plurality of wells of a multi-well plate, each well also being suitable for holding and storing a biological sample.

[0083] In some embodiments, the indicator system can be incorporated into a package for surrounding a biological sample storage vessel. For example, the indicator system can be incorporated into a box or bag suitable for surrounding a biological sample storage vessel.

[0084] This system provides a quality control (QC) tool for empirically tracking exposure of biospecimens to thaw conditions or other unacceptable time-temperature intervals. Specifically, it is a liquid-based color change system that tracks exposure of biospecimens at the level of individual aliquots at time-temperature intervals that exceed predefined limits and is intended as an empirical monitor of total exposure of biospecimens to thaw conditions. It can be implemented during initial processing or during long-term storage of biospecimens in a biobank. If deployed appropriately, these indicators can ensure the integrity of precious research biospecimens throughout their lifecycle, i.e., from point of collection to long-term biobanking.

[0085] In certain embodiments, the indicator liquid is designed to be activated and stored in a transparent subchamber of a biological specimen storage and / or processing vessel. The indicator liquid includes a bright pink solution that, once activated, remains bright pink for a predefined but customizable period of time at a specific temperature before becoming colorless. Typical expiration times span from a few minutes to a few hours. If the temperature rises, the solution will become colorless faster; if the temperature drops, it will take longer for the solution to change color. If the solution is frozen, it will remain pink indefinitely (i.e., the "clock" stops, but if it is thawed again, the "clock" restarts and the solution will eventually become colorless).

[0086] Useful solutions with freezing points may include 0° C., -18° C., -37° C., or -67° C. Solutions with freezing points below 0° C. may be used to empirically track improper biological specimen storage, such as storing plasma or serum at -20° C. (not completely frozen) instead of -80° C. In some embodiments, the inorganic perchlorate of the indicator system of the biological specimen collection or storage system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30° C.

[0087] In another aspect, provided herein is a kit comprising an indicator system as described herein and a storage vessel incorporating the indicator system and useful for storing a biological sample. In some embodiments, the kit comprises separate solutions for each indicator system component.

[0088] In another aspect, the present invention provides a method for monitoring the storage or processing of a biological sample, comprising: storing the biological sample in a biological sample storage vessel; associating the indicator system described herein with the biological sample or the biological sample storage vessel; mixing permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate to form a eutectic composition; observing the color of the eutectic composition or measuring the absorbance at 525 nm; wherein a color change from pink to colorless or an absorbance less than 0.1 indicates that the biological sample has been thawed, improperly stored, or improperly handled.

[0089] In various embodiments, the present disclosure provides a method for monitoring the integrity of a biological sample, comprising the following steps: obtaining an indicator system as described herein, whose predetermined freezing point is close to the freezing point of the biological sample or close to the storage or processing temperature of the biological sample; storing the biological sample near the indicator system and processing the biological sample and the indicator system simultaneously; and observing the color of the indicator system. A change in color from pink to colorless indicates a loss of integrity of the biological sample due to thawing, improper handling, or improper storage. Thus, a user can store the biological sample in a biological sample storage vessel, and the indicator system can be associated with the biological sample or the biological sample storage vessel. When it is necessary to start monitoring the storage or processing of the biological sample, permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate can be mixed to form a eutectic composition. After mixing, the indicator system will display a bright pink color and initiate a permanganate reduction reaction. When an indicator system with a multi-chamber storage vessel is used, mixing of the chemical components can be achieved by removing a removable barrier separating the permanganate from the organic reducing agent. As a non-limiting example, the mixing step may include removing a removable barrier defining a first chamber of the multi-chamber storage vessel having the permanganate therein and a second chamber of the multi-chamber storage vessel having the organic reducing agent, acid, aqueous solvent, and inorganic perchlorate therein.

[0090] In some embodiments, the inorganic perchlorate of the indicator system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30.0°C. In some embodiments, the color change can be observed with the naked eye. In some embodiments, the color change is observed by measuring the absorbance of the indicator system at 525nm. When the absorbance of the indicator system at 525nm is less than 0.1, less than 0.05 or 0, the integrity of the biological specimen is lost due to thawing, improper handling or improper storage. In some embodiments, the indicator system is in a well of a multi-well plate or in a cuvette to facilitate easy measurement of absorbance.

[0091] Elimination of endogenous Mn 2+ :Mn 2+ Can be present in the reaction system as an autocatalytic contaminant originating from purchased chemicals. For some target indicator solutions, this can cause the reaction system to react faster than expected. 2+ The substances can be removed by using inorganic cation exchange resins to bind multivalent metal cations. For example, pretreated KMnO4 is expected to minimize the amount of Mn in the starting reaction system. 2+ Alternatively, all labware can be pre-cleaned with ultrapure 2% nitric acid, passed through the stock solution with ozone, and / or treated with persulfate or sodium bismuthate to remove all Mn 2+ Stoichiometrically oxidized to MnO4 -Consuming this contaminant is important as it will allow the reaction to proceed longer while also minimizing the formation of MnO2 which can lead to a brown product.

[0092] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the invention belongs. All definitions as defined and used herein should be understood to take precedence over dictionary definitions, definitions in documents incorporated by reference, and / or the ordinary meanings of the defined terms.

[0093] All references, patents, and patent applications disclosed herein are hereby incorporated by reference into the subject matter for which they are cited, and in some cases may be incorporated in their entirety.

[0094] Throughout the specification and claims of this application, unless explicitly stated to the contrary, the indefinite article "a" or "an" should be understood to mean "at least one".

[0095] The phrase "and / or" used in the specification and claims should be understood to mean "one or both" of the elements so combined, i.e., elements that exist in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" elements so combined. In addition to the elements specifically specified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to the specifically specified elements. Thus, as a non-limiting example, when "A and / or B" is used with open language (such as "comprising"), in one embodiment, it may refer to only A (optionally including elements other than B); in another embodiment, it may refer to only B (optionally including elements other than A); in yet another embodiment, it may refer to both A and B (optionally including other elements); and so on.

[0096] "Or" as used in this specification and claims should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, that is, including at least one of a certain number or series of elements, but also including more than one, and also including other items that are optional and not listed. Only when the opposite term such as "only one of them..." or "exactly one..." or "consisting of..." is used in the claims, it means that exactly one of several elements or a series of elements is included. In general, as used herein, "or" can only be interpreted as indicating mutually exclusive parallel selections (i.e., "one or the other but not both") when it is preceded and followed by exclusive words such as "any one", "one of (these)", "only one of (these)" or "exactly one of (these)". "Substantially consisting of..." when used in the claims should be understood as its ordinary meaning in the field of patent law.

[0097] As used herein, "approximately" or "about" in relation to a quantity is generally considered to include quantities within 5% of that quantity in either direction (greater or less than that quantity), unless otherwise specified or it is obvious from the context that it is otherwise (unless the number exceeds 100% of possible values). When a range is mentioned, the range is inclusive unless otherwise specified or it is obvious from the context that it is otherwise.

[0098] The present invention has been described in terms of one or more preferred embodiments, but it should be understood that many equivalents, alternatives, variations and modifications besides those explicitly described are possible and within the scope of the present invention.

[0099] Example

[0100] This article describes color-change based indicator fluids designed for a variety of unique biospecimen handling / storage requirements. The internal chemical reaction “clock” running these indicators slows as the sample cools and stops indefinitely when the sample is frozen—but restarts when the sample thaws, regardless of the number of freeze-thaw cycles. A subset of these indicators do not freeze until -18°C, -37°C, or -67°C, thus facilitating tracking of nominally very low but inappropriate storage temperature exposures in a way that is difficult to ignore by anyone handling the sample. Such indicators can be used for processing tracking during biospecimen storage and initial biospecimen handling, as well as for empirical tracking of the integrity of archived or biobanked specimens throughout their lifecycle (i.e., within an archived biospecimen collection or biobank).

[0101] Specifically, the examples described herein relate to a bright pink liquid solution that visibly loses color and turns to a clear liquid when exposed to time and temperature conditions exceeding certain design thresholds. Under "appropriate" sample storage conditions (however defined), a specific indicator solution composition can be defined, wherein the indicator solution composition remains bright pink below a specified freezing point, but turns completely colorless when cumulatively exposed to time-temperature conditions exceeding allowable storage and handling requirements. The time required for the solution to turn to a clear color when the solution is above a predefined temperature limit (i.e., the design freezing point of the indicator solution) depends on the exact solution composition - but can also be flexibly adjusted depending on the purpose and requirements of tracking biological samples.

[0102] Example 1 contains a eutectic composition of NaClO4 as an antifreeze salt.

[0103] The freezing point of the eutectic composition of the indicator system is -37°C and contains about 52% by weight of NaClO4. The final concentrations of potassium permanganate, disodium oxalate and perchloric acid in the indicator system are 0.5 mM, 1.38 mM and 10 mM, respectively. The reaction time course of the eutectic composition containing NaClO4 as an antifreeze salt was studied. Extended reaction times (e.g., one hour or longer) were achieved at room temperature. The permanganate reduction time course of the 52% (w / w) NaClO4 system at 25°C was monitored by measuring the absorbance at 525 nm (n=6) ( Figure 2A Additional tests (n=3) were performed on systems designed to operate at 25°C for 60 minutes ( Figure 2B Table 1 shows the run time reproducibility of a 52% (w / w) NaClO4 system eutectic frozen at -37°C and designed to run for 60 minutes at 25°C (n = 3 replicates per day x 6 days).

[0104] The operating time and temperature curves of a 52% (w / w) NaClO4 system were studied, which was eutectic frozen at -37°C ( Figure 3 In addition, the effect of multiple initial freeze-thaw cycles on the final reaction run time of a 25% (w / w) NaClO4 system, which was eutectic frozen at -37 °C ( Figure 4 ). The system was nominally run at 25°C for 30 minutes without any freeze-thaw cycles. The reaction was immediately flash frozen in a dry ice / isopropanol bath. Each initial freeze-thaw cycle then consisted of removing the time-temperature indicator system from the -80°C freezer, completely thawing it in a room temperature water bath, and then immediately returning it to the -80°C freezer. Typically, the total time in the liquid state outside the -80°C freezer was 350-450 seconds.

[0105] Example 2 contains a eutectic composition of Mg(ClO4)2 as an antifreeze salt.

[0106] The freezing point of the eutectic composition of the indicator system is -67°C and contains about 44% by weight of Mg(ClO4)2. The final concentrations of potassium permanganate, disodium oxalate and perchloric acid in the indicator system are 0.5 mM, 1.38 mM and 10 mM, respectively. The reaction time course of the eutectic composition containing Mg(ClO4)2 as an antifreeze salt was studied. Extended reaction times (e.g., one hour or more) were achieved at room temperature. The permanganate reduction time course of the 44% (w / w) Mg(ClO4)2 system at 25°C was monitored by measuring the absorbance at 525 nm (n=6) ( Figure 5A Additional testing (n=3) was performed on a system designed to operate at 25°C for 30 minutes without transition to the brown intermediate ( Figure 5BTable 2 shows the run time reproducibility of a 44% (w / w) Mg(ClO4)2 system that was eutectic frozen at -67°C and designed to run at 25°C for 30 minutes without brown intermediate transition (n = 3 replicates per day x 6 days).

[0107] The operating time and temperature curve of a 44% (w / w) Mg(ClO4)2 system was studied. The system freezes eutectically at -67°C without the appearance of a brown intermediate ( Figure 6 ).

[0108] Example 3 Eutectic composition containing LiClO4 as antifreeze salt.

[0109] The freezing point of the eutectic composition of the indicator system is -18°C and contains about 25% by weight of LiClO4. The final concentrations of potassium permanganate, disodium oxalate and perchloric acid in the indicator system are 0.5 mM, 1.38 mM and 5 mM, respectively. The reaction time course of the eutectic composition containing LiClO4 as an antifreeze salt was studied. By measuring the absorbance at 525 nm ( Figure 7 ) Monitoring of the permanganate reduction time course at 25°C for an illustrative 25% (w / w) LiClO4 system eutectically frozen at -18°C designed to run for 60 minutes at 25°C (n=3). Table 3 shows the run time reproducibility of a 25% (w / w) LiClO4 system eutectically frozen at -18°C and designed to run for 60 minutes at 25°C (n=3 replicates per day x 6 days).

[0110] The operating time and temperature curves of a 25% (w / w) LiClO4 system were studied, which was eutectic frozen at -18°C ( Figure 8 In addition, the effect of multiple initial freeze-thaw cycles on the final reaction run time of a 25% (w / w) LiClO4 system, which was eutectic frozen at -18 °C ( Fig. 9 ). The system was nominally run at 25°C for 30 minutes without any freeze-thaw cycles. The reaction was immediately flash frozen in a dry ice / isopropanol bath. Each initial freeze-thaw cycle then consisted of removing the time-temperature indicator system from the -80°C freezer, completely thawing it in a room temperature water bath, and then immediately returning it to the -80°C freezer. Typically, the total time in the liquid state outside the -80°C freezer was 350-450 seconds.

[0111] Table 1. Run time reproducibility of 52% (w / w) NaClO4 system

[0112]

[0113]

[0114] Total average of all runs: 60.11

[0115] Total standard deviation (SD) for all runs: 2.52

[0116] Total coefficient of variation (CV) %: 4.19

[0117] % deviation from 60-minute average: 3.39

[0118] Table 2. Run time reproducibility of 44% (w / w) Mg(ClO4)2 system

[0119]

[0120]

[0121] Total average of all runs: 30.43

[0122] Total standard deviation (SD) for all runs: 1.07

[0123] Total coefficient of variation (CV) %: 3.51

[0124] % deviation from 30-minute average: 3.06

[0125] Table 3. Run time reproducibility of 25% (w / w) LiClO4 system

[0126]

[0127]

[0128] Total average of all runs: 63.22

[0129] Total standard deviation (SD) for all runs: 2.01

[0130] Total coefficient of variation (CV) %: 3.18

[0131] % deviation from 60-minute average: 5.76

[0132] For the sake of completeness, various aspects of the disclosure are listed in the following numbered clauses:

[0133] Item 1. An indicator system comprising permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate, which is initially configured to prevent the reduction of the permanganate to Mn 2+ ,

[0134] wherein the mixture of the permanganate, the organic reducing agent, the acid, the aqueous solvent and the inorganic perchlorate is a eutectic composition having a predetermined freezing point, and

[0135] The organic reducing agent causes the permanganate to convert to Mn at a temperature above a predetermined freezing point. 2+ The reduction reaction occurs at a temperature lower than a predetermined freezing point, and the reduction reaction stops.

[0136] Item 2. The indicator system of Item 1, comprising 0.5 mM to 5 mM permanganate, 1 mM to 15 mM organic reducing agent, and 1 mM to 300 mM acid.

[0137] Clause 3. The indicator system of any one of clauses 1 to 2, wherein the inorganic perchlorate comprises NaClO 4 , Mg(ClO 4 ) 2 , or LiClO 4 .

[0138] Clause 4. The indicator system of any one of clauses 1 to 3, wherein the indicator system comprises 15 wt% to 60 wt% of an inorganic perchlorate.

[0139] Clause 5. The indicator system according to any one of clauses 1 to 4, wherein the predetermined freezing point is below -10.0°C.

[0140] Clause 6. The indicator system of any one of clauses 1 to 5, wherein the indicator system comprises 20 wt% to 55 wt% of an inorganic perchlorate, and wherein the predetermined freezing point is below -15.0°C.

[0141] Clause 7. The indicator system of any one of clauses 1 to 6, wherein the predetermined freezing point is -18.0°C and the eutectic composition comprises 20 wt% to 30 wt% LiClO4.

[0142] Clause 8. The indicator system of any one of clauses 1 to 6, wherein the predetermined freezing point is -37.0°C and the eutectic composition comprises 50 wt% to 54 wt% NaClO4.

[0143] Clause 9. The indicator system of any one of clauses 1 to 6, wherein the predetermined freezing point is -67.0°C and the eutectic composition comprises 42 wt% to 46 wt% Mg(ClO4)2.

[0144] Clause 10. The indicator system of any one of Clauses 1 to 9, wherein the permanganate is potassium permanganate.

[0145] Clause 11. The indicator system according to any one of clauses 1 to 10, wherein the organic reducing agent is oxalic acid (salt).

[0146] Clause 12. The indicator system of any one of clauses 1 to 11, wherein the acid is perchloric acid.

[0147] Clause 13. The indicator system according to any one of clauses 1 to 12, wherein the aqueous solvent is water.

[0148] Clause 14. The indicator system of any one of Clauses 1 to 13, wherein the eutectic composition is pink when mixed, and wherein the absorbance of the composition measured at 525 nm decreases within a predetermined period of time.

[0149] Item 15. An indicator system according to any one of items 1 to 14, further comprising a multi-chamber storage vessel, the multi-chamber storage vessel comprising a removable barrier, the removable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the removable barrier is configured to prevent mixing of the permanganate and the organic reducing agent.

[0150] Clause 16. The indicator system of Clause 15, wherein the first chamber has permanganate and the second chamber has a solution comprising an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate.

[0151] Item 17. A biological sample collection or storage system comprising a biological sample storage vessel and the indicator system according to Item 1.

[0152] Clause 18. A biological specimen collection or storage system according to Clause 17, wherein the inorganic perchlorate of the indicator system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30°C.

[0153] Clause 19. The biological specimen collection or storage system of any one of Clauses 17 to 18, comprising a recess configured to receive the indicator system and allow viewing of the indicator system without disturbing the biological specimen.

[0154] Clause 20. A biological specimen collection or storage system according to any one of clauses 17 to 19, wherein the indicator system further comprises a multi-chamber storage vessel, the multi-chamber storage vessel comprising a removable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the removable barrier is configured to prevent mixing of the permanganate and the organic reducing agent.

[0155] Clause 21. A method of monitoring storage or processing of a biological sample, comprising:

[0156] storing the biological sample in a biological sample storage container;

[0157] Associating the indicator system according to clause 1 with a biological sample or a biological sample storage container;

[0158] mixing a permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate to form a eutectic composition;

[0159] Observe the color of the co-crystal composition or measure the absorbance at 525 nm; wherein a color change from pink to colorless or an absorbance less than 0.1 indicates that the biological sample has been thawed, improperly stored, or improperly handled.

[0160] Clause 22. The method of clause 21, wherein the inorganic perchlorate of the indicator system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30.0°C.

[0161] Clause 23. A method according to any one of clauses 21 to 22, wherein the mixing step comprises removing a removable barrier defining a first chamber of a multi-chamber storage vessel having permanganate therein and a second chamber of a multi-chamber storage vessel having an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate therein.

Claims

1. An indicator system comprising permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate, which is initially configured to prevent the reduction of the permanganate to Mn 2+ , wherein the mixture of the permanganate, the organic reducing agent, the acid, the aqueous solvent and the inorganic perchlorate is a eutectic composition having a predetermined freezing point, and The organic reducing agent causes the permanganate to convert to Mn at a temperature above a predetermined freezing point. 2+ The reduction reaction occurs at a temperature lower than a predetermined freezing point, and the reduction reaction stops. 2 . The indicator system according to claim 1 , comprising 0.5 mM to 5 mM permanganate, 1 mM to 15 mM organic reducing agent and 1 mM to 300 mM acid.

3. The indicator system of claim 1, wherein the inorganic perchlorate comprises NaClO4, Mg(ClO4)2, or LiClO4.

4. The indicator system of claim 1, wherein the indicator system comprises 15 wt% to 60 wt% inorganic perchlorate.

5. The indicator system of claim 1, wherein the predetermined freezing point is below -10.0°C.

6. The indicator system of claim 1, wherein the indicator system comprises 20 wt% to 55 wt% inorganic perchlorate, and wherein the predetermined freezing point is below -15.0°C.

7. The indicator system of claim 1, wherein the predetermined freezing point is -18.0°C and the eutectic composition comprises 20 wt% to 30 wt% LiClO4.

8. The indicator system of claim 1, wherein the predetermined freezing point is -37.0°C and the eutectic composition comprises 50 wt% to 54 wt% NaClO4.

9. The indicator system of claim 1, wherein the predetermined freezing point is -67.0°C and the eutectic composition comprises 42 wt% to 46 wt% Mg(ClO4)2.

10. The indicator system of claim 1, wherein the permanganate is potassium permanganate.

11. The indicator system of claim 1, wherein the organic reducing agent is oxalate.

12. The indicator system of claim 1, wherein the acid is perchloric acid.

13. The indicator system of claim 1, wherein the aqueous solvent is water.

14. The indicator system of claim 1, wherein the eutectic composition is pink when mixed, and wherein the absorbance of the composition measured at 525 nm decreases over a predetermined period of time.

15. The indicator system of claim 1 , further comprising a multi-chamber storage vessel comprising a removable barrier defining a first chamber having permanganate therein and a second chamber having an organic reducing agent therein, wherein the removable barrier is configured to prevent mixing of the permanganate and the organic reducing agent.

16. The indicator system of claim 15, wherein the first chamber contains permanganate and the second chamber contains a solution comprising an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate. 17 . A biological sample collection or storage system, comprising a biological sample storage vessel and the indicator system according to claim 1 .

18. The biological specimen collection or storage system of claim 17, wherein the inorganic perchlorate of the indicator system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30°C.

19. The biological specimen collection or storage system of claim 17, comprising a recess configured to receive the indicator system and allow viewing of the indicator system without disturbing the biological specimen.

20. A biological specimen collection or storage system according to claim 17, wherein the indicator system further comprises a multi-chamber storage vessel, the multi-chamber storage vessel comprising a removable barrier, the barrier defining a first chamber having permanganate and a second chamber having an organic reducing agent, wherein the removable barrier is configured to prevent mixing of the permanganate and the organic reducing agent.

21. A method for monitoring storage or processing of a biological sample, comprising: storing the biological sample in a biological sample storage container; Associating the indicator system according to claim 1 with a biological sample or a biological sample storage vessel; mixing a permanganate, an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate to form a eutectic composition; Observe the color of the eutectic composition or measure the absorbance at 525 nm; wherein a color change from pink to colorless or an absorbance less than 0.1 indicates that the biological sample has been thawed, improperly stored, or improperly handled.

22. The method of claim 21, wherein the inorganic perchlorate of the indicator system comprises NaClO4 or Mg(ClO4)2, and the predetermined freezing point is below -30.0°C.

23. The method of claim 21, wherein the mixing step comprises removing a removable barrier defining a first chamber of the multi-chamber storage vessel having permanganate therein and a second chamber of the multi-chamber storage vessel having an organic reducing agent, an acid, an aqueous solvent, and an inorganic perchlorate therein.

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