Preservation reagents for biological samples and use thereof

Abstract

The present invention relates to a reagent for stabilising a biological sample, comprising · a solution comprising o at least one amino acid, o at least one inorganic salt, o glucose and / or a protease inhibitor. The invention further relates to a method for stabilising a biological sample, comprising the steps · providing a reagent according to the first aspect of the invention, · contacting said biological sample with said reagent.

Description

[0001] Preservation reagents for biological samples and use thereof

[0002] Background

[0003] Follow a biopsy procedure, specimens can (a) be stabilized immediately for use in pathology (b) stored in a viability buffer at room temperature or cooled (c) and / or subjected to nanomechanical signature measurements, in viability buffer. In case (b) or (c), the sample moved from viability buffer to stabilization buffer after a nanomechanical measurement or after decision not to perform a nanomechanical measurement. Preservation of specimen viability is imperative to maintain the tissues nanomechanical properties throughout measurement and the prevent deterioration of the tissue which would affect routine pathology workflows.

[0004] Similar protocols have been disclosed in [1] and [2], using Ringer’s solution instead of the below described new disclosures.

[0005] Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to a reagent for stabilising a biological sample and a method of use thereof. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification.

[0006] Summary of the Invention

[0007] A first aspect of the invention relates to a reagent for stabilising a biological sample, comprising

[0008] • a solution comprising o at least one amino acid, o at least one inorganic salt, o glucose and / or a protease inhibitor.

[0009] A second aspect of the present invention relates to a method for stabilising a biological sample, comprising the steps

[0010] • providing a reagent according to the first aspect of the invention,

[0011] • contacting said biological sample with said reagent.

[0012] Terms and definitions

[0013] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

[0014] The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of” or “consisting of.”

[0015] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0016] Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0017] As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

[0018] “And / or” where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0019] The term additive in the context of the present specification relates to a compound or substance which enhances the properties of a reagent and thus protects a sample it is used on, e.g. it improves the samples viability. By using more than one additive, several effects can be taken advantage of.

[0020] The term protease inhibitor in the context of the present specification relates to a compound or substance which inhibits the activity of enzymes, in particular proteases. Inhibiting the activity of enzymes, in particular inhibiting proteases, increases the viability of tissue specimens since degradation of tissue components is slowed down.

[0021] The term nutrient in the context of the present specification relates to an organic or inorganic substance or compound that is absorbed by living organisms to sustain life and is processed in its metabolism. Nutrient increase the viability of tissue specimens because cells in the tissue can maintain their metabolism for longer periods of time, outside the human body.

[0022] The term anti-swelling agent in the context of the present specification relates to a substance or compound that prevents the swelling of endothelial cells. Suppression of swelling maintains the cells the cells normal nanomechanical properties and prevents them from bursting, which in turn compromises the viability of the tissue specimen.

[0023] The term dilution agent in the context of the present specification relates to a compound or substance that decreases the viscosity of a solution. Maintaining a viscosity close to the viscosity of water, through addition of a dilution agent, facilitates the measurement of nanomechanical properties, in contrast to high viscosity which hinders the measurement of nanomechanical properties.

[0024] The term anti-evaporation agent in the context of the present specification relates to a compound or substance that prevents water from evaporating from a solution. Reducing evaporation of buffer ensures that the indicated concentrations, supporting viability, can be maintained for a longer period of time.

[0025] Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

[0026] Detailed Description of the Invention

[0027] A first aspect of the invention relates to a reagent for stabilising a biological sample, comprising

[0028] • a solution comprising o at least one amino acid, o at least one inorganic salt, o glucose and / or a protease inhibitor.

[0029] Certain biological samples, specifically biological samples with a highly active cell metabolism require a preservation reagent including additives to stabilise the sample during its examination and maintain their viability to allow a reliable examination of said biological samples also over longer periods of time. In certain embodiments, the reagent for stabilising a biological sample further comprises at least one additive.

[0030] In certain embodiments, the at least one additive selected from the groups consisting of o a protease inhibitor and / or, o a nutrient and / or, o an anti-swelling agent and / or, o a dilution agent and / or o an anti-evaporation agent.

[0031] In certain embodiments, the at least one amino acid is selected from L-histidine and tryptophan.

[0032] In certain embodiments, the solution comprises two amino acids, and at least one inorganic salt.

[0033] In certain embodiments, the solution further comprises at least one organic salt.

[0034] In certain embodiments, a cation of the at least one inorganic salt is selected from the group consisting of Na+, K+, Ca2+and / or Mg2+.

[0035] In certain embodiments, a cation of the at least one organic salt is selected from the group consisting of Na+, K+, Ca2+and / or Mg2+.

[0036] In certain embodiments, an anion of the at least one inorganic salt or the at least one organic salt is selected from Cl acetate, CO32; HCO3; ketoglutarate and glutarate.

[0037] In certain embodiments, an anion of the at least one inorganic salt is Cl’ or CO32; HCOs’.

[0038] In certain embodiments, an anion of the at least one organic salt is selected from the group consisting of acetate, ketoglutarate and glutarate.

[0039] In certain embodiments, the reagent for stabilising a biological sample comprises

[0040] • a solution comprising at least one amino acid, at least one inorganic salt,

[0041] • a protease inhibitor.

[0042] In certain embodiments, the protease inhibitor is selected from the group consisting of phenylmethylsulfonyl fluoride, alpha-1 antitrypsin, iodoacetamide, pepstatin, aprotinin, bestatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butan and leupeptin.

[0043] Protease inhibitors prevent the proteases from cleaving enzymes and subsequent degradation of proteins present in the biological sample and further inactivating functions of the biological sample. The addition of a protease inhibitor thus increases the viability of the biological sample over time, for example during the time until the biological sample is examined as well as for the duration of the examination.

[0044] In certain embodiments, the reagent for stabilising a biological sample comprises

[0045] • a solution comprising at least one amino acid, at least one inorganic salt,

[0046] • glucose.

[0047] The addition of glucose prolongs the cell viability outside the body. Such a prolonged cell viability is specifically advantageous in the measurement of cancer cells which have a faster metabolism than non-cancerous cells and thus require a higher amount of glucose for their viability in general and over time.

[0048] In certain embodiments, the reagent for stabilising a biological sample comprises

[0049] • a solution comprising at least one amino acid, at least one inorganic salt,

[0050] • glucose and / or a protease inhibitor.

[0051] • a nutrient.

[0052] In certain embodiments, the nutrient is selected from a carbohydrate.

[0053] Carbohydrates function as a nutrition agent for biological samples. The addition of carbohydrates to the preservation solution thus increases the viability of the biological sample.

[0054] In certain embodiments, the carbohydrate is selected from a disaccharide and / or a polyol and / or a monosaccharide, particularly wherein the carbohydrate is selected from a disaccharide and / or a monosaccharide, more particularly wherein the carbohydrate is a monosaccharide.

[0055] In certain embodiments, the carbohydrate is selected from the group consisting of glucose, galactose, fructose, mannitol and / or raffinose.

[0056] In case the reagent contains a solution comprising at least one amino acid, at least one inorganic salt, and glucose, and a nutrient is added as additive, the nutrient is selected from galactose, fructose, mannitol and / or raffinose. In certain embodiments, the carbohydrate is selected from the group consisting of glucose, galactose, fructose, and / or raffinose.

[0057] In certain embodiments, the carbohydrate is raffinose.

[0058] In certain embodiments, the reagent for stabilising a biological sample comprises

[0059] • a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, • an anti-swelling agent.

[0060] In certain embodiments, the anti-swelling agent is selected from polyethylene glycol, hydroxyethyl starch and raffinose.

[0061] The addition of an anti-swelling agent to a preservation agent for biological samples prevents the swelling of endothelial cells.

[0062] In certain embodiments, the reagent for stabilising a biological sample comprises

[0063] • a solution comprising at least one amino acid, at least one inorganic salt,

[0064] • glucose and / or a protease inhibitor,

[0065] • a dilution agent.

[0066] In certain embodiments, the dilution agent is selected from the group consisting of water and saline solution.

[0067] Maintaining a viscosity close to the viscosity of water, through addition of a dilution agent, facilitates the measurement of nanomechanical properties, in contrast to high viscosity which hinders the measurement of nanomechanical properties.

[0068] In certain embodiments, the reagent for stabilising a biological sample comprises

[0069] • a solution comprising at least one amino acid, at least one inorganic salt,

[0070] • glucose and / or a protease inhibitor,

[0071] • an anti-evaporation agent.

[0072] In certain embodiments, the anti-evaporation agent is selected from the group consisting of an alcohol, ethylene glycol and glycols.

[0073] The evaporation can be reduced by surrounding with or putting the specimen holder in proximity of a container of an aqueous solution. This locally increases the vapor pressure, reducing the evaporation of the specimen holders’ buffer itself.

[0074] The addition of an anti-evaporation agent reduces the evaporation of the reagent during the storage and the examination of the biological sample and thus allows for longer measurement times during the examination process.

[0075] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor and a nutrient.

[0076] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, and an anti-swelling agent. In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient and a dilution agent.

[0077] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor a nutrient and an anti-evaporation agent.

[0078] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, a protease inhibitor and an anti-swelling agent.

[0079] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, an anti-swelling agent and a dilution agent.

[0080] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, an anti-swelling agent and an anti-evaporation agent.

[0081] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, an dilution agent and an anti-evaporation agent.

[0082] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, and an anti-swelling agent.

[0083] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, and a dilution agent.

[0084] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, and an anti-evaporation agent.

[0085] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, an anti-swelling agent and a dilution agent.

[0086] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, an anti-swelling agent and an anti-evaporation agent. In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a dilution agent and an anti-evaporation agent.

[0087] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, an anti-swelling agent and a dilution agent.

[0088] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, an anti-swelling agent and an anti-evaporation agent.

[0089] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a dilution agent and an anti-evaporation agent.

[0090] In certain embodiments, the reagent for stabilising a biological sample comprises a solution comprising at least one amino acid, at least one inorganic salt, glucose and / or a protease inhibitor, a nutrient, an anti-swelling agent, a dilution agent and an anti-evaporation agent.

[0091] In certain embodiments, the viscosity of the reagent is less than 1.07 q in mPa s at 25°C.

[0092] The lower the viscosity the better a biological sample is measurable in atomic force microscopy.

[0093] In certain embodiments, the reagent is optically transparent. Optical transparency allows for the biological sample being examined using a light source, in particular a laser, at a constant power.

[0094] In certain embodiments, the reagent is optically transparent at a wavelength in the range of 750 to 100 nm.

[0095] In certain embodiments, the reagent is optically transparent at a wavelength in the range of 800 to 900 nm.

[0096] In certain embodiments, the reagent is optically transparent at a wavelength in the range of 810 to 850 nm.

[0097] The sample is measured using a light source being directed at the sample. It is thus important that the solution is optically transparent at the wavelength the light source is used at. Only then can a reliable an correct measurement of the sample take place.

[0098] In certain embodiments, the reagent is free of particles. A particle free solution allows for a correct, sensitive and reliable measurement using a light source. The light source does not lose intensity or is diverted and the results show no scattering effects.

[0099] In certain embodiments, the biology sample is an ex-vivo biological sample.

[0100] In certain embodiments, the biological sample is a tissue sample.

[0101] In certain embodiments, the biological sample is a tissue sample, wherein the tissue sample is an ex-vivo tissue sample.

[0102] In certain embodiments, the biological sample is a neoplasm.

[0103] In certain embodiments, the biological sample is a neoplasm, wherein the neoplasm is an ex vivo sample.

[0104] In certain embodiments, the biological sample is a biopsy sample.

[0105] A second aspect of the present invention relates to a method for stabilising a biological sample, comprising the steps

[0106] • providing a reagent according to the first aspect of the invention,

[0107] • contacting said biological sample with said reagent.

[0108] The embodiments of the first aspect of the invention defining the reagent also define the reagent according to the first aspect of the invention used in the second aspect of the invention.

[0109] In certain embodiments, the biology sample is an ex-vivo biological sample.

[0110] In certain embodiments, the biological sample is a tissue sample.

[0111] In certain embodiments, the biological sample is a tissue sample, wherein the tissue sample is an ex-vivo tissue sample.

[0112] In certain embodiments, the biological sample is a neoplasm.

[0113] In certain embodiments, the biological sample is a neoplasm, wherein the neoplasm is an ex vivo sample.

[0114] In certain embodiments, the biological sample is a biopsy sample.

[0115] In certain embodiments, the method is a method for stabilising an ex-vivo biological sample, comprising the steps

[0116] • providing a reagent according to the first aspect of the invention,

[0117] • contacting said ex-vivo biological sample with said reagent.

[0118] In certain embodiments, the method is a method for stabilising a biopsy sample, comprising the steps providing a reagent according to the first aspect of the invention,

[0119] • contacting said biopsy sample with said reagent.

[0120] Examples

[0121] Example 1: Reagents (Viability Buffer)

[0122] Basic reagent:

[0123] The basic reagent without an additive is prepared as an aqueous solution in double-distilled water of:

[0124] Sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2-Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L.

[0125] Reagent with a nutrient:

[0126] The reagent is prepared as an aqueous solution in double-distilled water of: the basic reagent containing: sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2- Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L. and is supplemented with 5 wt% of glucose.

[0127] Reagent with a nutrient:

[0128] The reagent is prepared as an aqueous solution in double-distilled water of: the basic reagent containing: sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2- Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L. and is supplemented with 5 mmol / L adenosine.

[0129] Reagent with a protease inhibitor:

[0130] The reagent is prepared as an aqueous solution in double-distilled water of: the basic reagent containing: sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2- Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L. and is supplemented with protease inhibitor cocktail P1860 or protease inhibitor cocktail complete or complete Mini as per manufacturer’s instructions. Reagent with an anti-swelling agent:

[0131] The reagent is prepared as an aqueous solution in double-distilled water of: the basic reagent containing: sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2- Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L. and is supplemented with 195 mmol / L glucose.

[0132] Reagent with an anti-swelling agent:

[0133] The reagent is prepared as an aqueous solution in double-distilled water of: the basic reagent containing: sodium chloride: 15 mmol / L; Potassium Chloride: 9 mmol / L; Magnesium Chloride: 4 mmol / L; Calcium Chloride: 0.015 mmol / L; Potassium hydrogen 2- Ketoglutarate: 1 mmol / L; Histidine: 198 mmol / L; Mannitol: 30 mmol / L; Tryptophan: 2 mmol / L. and is supplemented with 0.03 mmol / L polyethylenglycol.

[0134] Example 2: Method for stabilizing a biological sample

[0135] A biological sample placed in a viability buffer in a specimen holder, is transferred after the measurement from the specimen holder into a 10% formaldehyde solution or Bouin solution to stabilise the sample.

[0136] Alternatively, the viability buffer inside the specimen holder is replaced during the measurement through a 10% formaldehyde solution or Bouin solution to stabilise the sample.

[0137] The method has shown no visible changes with pathological tissue evaluability in breast tissue and lung tissue.

[0138] The addition of a protease inhibitor as additive to the viability buffer has shown an improved tissue stability in pancreas tissue.

[0139] Cited prior art documents:

[0140] [1] Plodinec, Marija, et al. "The nanomechanical signature of breast cancer." Nature nanotechnology 7.11 (2012): 757-765.

[0141] [2] Plodinec, Marija, and Roderick YH Lim. "Nanomechanical characterization of living mammary tissues by atomic force microscopy." Mammary Stem Cells: Methods and Protocols (2015): 231-246.

[0142] All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

Claims

Claims1. A reagent for stabilising a biological sample, comprising• a solution comprising o at least one amino acid, o at least one inorganic salt, o glucose and / or a protease inhibitor.

2. The reagent for stabilising a biological sample, wherein the solution further comprises at least one additive.

3. The reagent according to claim 2, wherein the at least one additive is selected from o a nutrient and / or, o an anti-swelling agent and / or, o a dilution agent and / or o an anti-evaporation agent.

4. The reagent according to any of the preceding claims, wherein the at least one amino acid is selected from L-histidine and tryptophan.

5. The reagent according to any of the preceding claims, wherein the reagent further comprises at least one organic salt.

6. The reagent according to any of the preceding claims, wherein the cation of the at least one inorganic salt or the at least one organic salt is selected from the group consisting of Na+, K+, Ca2+and / or Mg2+.

7. The reagent according to any of the preceding claims, wherein an anion of the at least one inorganic salt or the at least one organic salt is selected from Cl; acetate, CO32HCO3 ketoglutarate and glutarate.

8. The reagent according to any of the preceding claims, wherein the protease inhibitor is selected from the group consisting of phenylmethylsulfonyl fluoride, alpha- 1 antitrypsin, iodoacetamide, pepstatin, aprotinin, bestatin, trans-Epoxysuccinyl-L-leucylamido(4- guanidino)butan or leupeptin or 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride.

9. The reagent according to claim 3, wherein the nutrient is selected from the group consisting of carbohydrate, in particular wherein the carbohydrate is selected from a disaccharide and / or a polyol and / or a monosaccharide, particularly wherein the carbohydrate is selected from a disaccharide and / or a monosaccharide.

10. The reagent according to claim 9 and 10, wherein the carbohydrate is selected from the group consisting of galactose, fructose, mannitol and / or raffinose, particularly wherein the carbohydrate is selected from the group consisting of galactose, fructose, and / or raffinose.

11. The reagent according to claim 3, wherein the anti-swelling agent is selected from polyethylene glycol, hydroxyethyl starch, glucose, and raffinose.

12. The reagent according to any of the preceding claims, wherein the reagent is optically transparent.

13. The reagent according to any of the preceding claims, wherein the viscosity of the reagent is less than 1.07 q in mPa s at 25°C.

14. The reagent according to any of the preceding claims, wherein the biological sample is a tissue sample, in particular, wherein the biological sample is a neoplasm.

15. A method for stabilising a biological sample, particularly for stabilising a biological sample, comprising the steps• providing a reagent according to claims 1 to 14, contacting said biological sample with said reagent.