US20160324144A1 - Stabilization of whole blood at room temperature - Google Patents

Stabilization of whole blood at room temperature Download PDF

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US20160324144A1
US20160324144A1 US15/221,791 US201615221791A US2016324144A1 US 20160324144 A1 US20160324144 A1 US 20160324144A1 US 201615221791 A US201615221791 A US 201615221791A US 2016324144 A1 US2016324144 A1 US 2016324144A1
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antioxidant
composition
cells
mitochondria
nucleated cells
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Waltraud Ankenbauer
Ulrike Dietrich-Veenstra
Renate Kolb
Yvonne Maerz
Stephanie Wessner
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Roche Diagnostics Operations Inc
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Roche Diagnostics Operations Inc
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Assigned to ROCHE DIAGNOSTICS OPERATIONS, INC. reassignment ROCHE DIAGNOSTICS OPERATIONS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ROCHE DIAGNOSTICS GMBH
Assigned to ROCHE DIAGNOSTICS GMBH reassignment ROCHE DIAGNOSTICS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WESSNER, STEPHANIE, MAERZ, Yvonne, DIETRICH-VEENSTRA, Ulrike, Kolb, Renate, ANKENBAUER, WALTRAUD
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Publication of US20160324144A1 publication Critical patent/US20160324144A1/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

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  • the present disclosure pertains to compositions, uses, kits and methods useful in the stabilization of intact viable cells, particularly nucleated cells present in whole blood samples.
  • the present disclosure is directed to the stabilization of nucleated cells in a whole blood sample ex-vivo, effected by an additive being a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof.
  • the liquid composition further comprises a protease inhibitor.
  • a protease inhibitor for stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of the whole-blood sample ex-vivo, as well as kits including the composition for practicing said use. Additionally, specific methods are provided for stabilizing intact nucleated cells of a whole-blood sample ex-vivo.
  • Preservation of biological status or “stabilization” is understood as providing conditions under which changes concerning the composition and levels of biomolecules present in a living cell at the time of sampling are minimized while keeping the cell intact and viable.
  • biomolecules of interest are DNA, RNA and protein.
  • a first aspect reported herein is a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof.
  • a second aspect reported herein is the use of a composition as disclosed herein for stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of a whole-blood sample ex-vivo.
  • a third aspect reported herein is a kit of parts comprising a blood drawing container enclosing a liquid composition according to the disclosure herein, the kit further comprising packaging material, a label, and a user instruction sheet.
  • a fourth aspect reported herein is method for stabilizing intact nucleated cells of a whole-blood sample ex-vivo, the method comprising the steps of (a) providing the whole blood sample ex-vivo; (b) contacting and mixing the sample of step (a) with a composition according to the disclosure herein; (c) incubating the mixture obtained in step (b), thereby stabilizing intact nucleated cells of the ex-vivo whole-blood sample.
  • FIG. 1 Detection of MDA-MB-468 cells spiked into blood samples after storage for 48 hours at room temperature. Depicted is the percentage of MDA-MB-468 cells detected by immunostaining with Anti-CK5/8 antigens. “Box-and-whisker plot” indicating mean percentage of target cell retrieval by way of assay as described in Example 1.
  • 3rd box EDTA+Pefabloc® SC: MDA-MB-468 cells detected in blood sample stored in EDTA as anticoagulant and Pefabloc® SC as stabilizer.
  • 4th box-EDTA+SKQ1 MDA-MB-468 cells detected in blood sample stored in EDTA as anticoagulant and SKQ1 as stabilizer.
  • 6th box EDTA+glutathione: MDA-MB-468 cells detected in blood sample stored in EDTA as anticoagulant and glutathione as stabilizer.
  • FIG. 2 Detection of MDA-MB-468 cells spiked into blood samples after storage for 48 hours at room temperature. Depicted is the percentage of MDA-MB-468 cells detected by immunodetection with fluorescent anti-CK 5/8 antibodies. “Box plot” indicating mean percentage of target cell retrieval by way of the assay described in Example 2.
  • 3rd box CPDA+SS-31: MDA-MB-468 cells detected in blood sample stored in citrate, phosphate, dextrose, adenine and SS+31.
  • 4th box CPDA+glutathione: MDA-MB-468 cells detected in blood sample stored in citrate, phosphate, dextrose, adenine and glutathione.
  • FIG. 3 Influence of storage on DNA yield. Samples were stored in EDTA or EDTA supplemented with glutathione, SkQ1 and Pefabloc® SC (“Stabilizer 3”), as indicated in Example 3.
  • FIG. 4 Blood from two blood donors was collected either in EDTA or in stabilizer as described in Example 5. Samples were spiked with MDA-MB-468 cells. Immediately and following a 72 hours long incubation at room temperature, the MDA-MB-468 cells were isolated and the nucleic acids were extracted. The set of nucleic acid parameters depicted in the figure was amplified by RT-PCR or PCR (depending on the template); changes of the respective nucleic acid levels were calculated using GCK DNA as endogenous reference. “Stabilizer 72 h” refers to the samples incubated for 72 hours with Stabilizer 5 as described in Example 5.
  • FIG. 5 Blood from 2 different donors was either stabilized with EDTA or with stabilizer.
  • MDA-MB-468 cells were spiked to the blood samples. The samples were stored for 48 hours at room temperature. MDA-MB-468 cells were enriched by erythrocyte lysis and immunomagnetic isolation using CD326 (EpCAM) Microparticles (see Example 7). The cells were cultured for more than 100 hrs.
  • the term “comprising” means that other steps and other components that do not affect the end result may be utilized.
  • the term “comprising” encompasses the expressions “consisting of,” and “consisting, essentially of”.
  • the use of singular identifiers such as “the,” “a,” or “an” is not intended to be limiting solely to the use of a single component, but may include multiple components.
  • a compound has the meaning of “one or more compound(s)”.
  • “plurality” is understood to mean more than one. For example, a plurality refers to at least two, three, four, five, or more.
  • the term “or” is understood to be inclusive.
  • the term “and/or” means one or all of the listed elements or a combination of any two or more of the listed elements. Ranges are used herein as a shorthand for describing each and every value that is within the range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Any value within the range can be selected as the terminus of the range. Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean.
  • room temperature means the ambient temperature of a typical laboratory, which is usually about 18° C. to 25° C. In a specific embodiment, room temperature is a temperature selected from the group consisting of 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., and 25° C.
  • a “purified” or “isolated” compound means the compound has been separated from the mixture in which it was formed. With respect to cells, a “purified” or “isolated” cell denotes one or more cells or a group of cells that were enriched or separated from a mixture of cells.
  • nucleated cells can be isolated from the mixture of cells in an ex vivo sample of whole blood by lysing the erythrocytes present in the sample and separating by centrifugation intact nucleated cells, discarding the supernatant and re-suspending the pelleted cells in a physiological buffer or in a cell culture medium.
  • a “liquid” composition is a composition which is present in the liquid state of aggregation.
  • the liquid composition as disclosed herein is an aqueous composition, a solution with water as solvent and a plurality of water-soluble compounds dissolved therein.
  • An “analyte” in general is a substance, specifically a cellular component, which is the target of an analytical process such as, but not limited to, a process for qualitative or quantitative detection of the substance.
  • a specific analyte for the purpose of the present disclosure is selected from DNA, RNA and protein, the latter comprising oligopeptides, polypeptides, and posttranslational modified derivatives thereof.
  • RNA needs to be reverse-translated to form cDNA; the cDNA reflecting the target RNA sequence can then be amplified and detected using a PCR-based DNA amplification technique.
  • Specific detection of a target protein is typically achieved making use of specific binders capable of binding to the target.
  • immunoassay detection methods are a non limiting example of this approach.
  • the present disclosure is specifically directed to stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of a whole-blood sample ex-vivo for a period of up to 72 hours (0 to 72 h), specifically 48 to 72 hours, the period counted from the time point of sampling the whole blood and contacting the whole blood with the stabilizing composition as disclosed herein.
  • the time between blood sampling and contacting the blood with the stabilizing composition is less than 30 seconds.
  • stabilizing includes the specific meaning of keeping intact the cell membranes of nucleated cells of the blood sample, and preserving and maintaining in the nucleated cells the cellular proteins and the cellular DNA as well as the cellular RNA and particularly the composition of cellular mRNA reflecting the gene expression status at the time of sampling; in addition, “stabilizing” encompasses providing an environment in which nucleated cells have a substantially reduced tendency to undergo necrosis or apoptosis, and in which the pattern of expressed proteins remains largely unchanged. The term “stabilizing” further includes supporting of vital cell functions, avoiding intoxication of cells and cell death, maintaining the nucleated cells in a resting but viable state.
  • citrate/citric acid refers to a mixture of trisodium citrate and citric acid.
  • Reference to Blood with EDTA as the sole anticoagulant means whole blood having an EDTA concentration of about 1.8 mg per ml of blood.
  • a first aspect as disclosed herein is a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof.
  • the analyte is selected from DNA, RNA and protein of intact nucleated cells of the whole-blood sample.
  • whole blood in all aspects and embodiments disclosed herein denotes blood that was directly collected from a vertebrate, specifically from a mammal, more specifically from a primate, even more specifically from a human, without any further treatment other than the blood drawing process. It is understood that the blood drawing process itself is not part of any of the procedures, uses and/or methods according to the present disclosure. Rather, the present disclosure relates to procedures, uses and methods based on the provision of a sample of whole blood “ex-vivo”, that is to say whole blood outside of and separate from the organism from which it was drawn. The whole blood ex vivo is provided as a “sample”, being understood as a measured amount of whole blood.
  • the composition as disclosed in all aspects and embodiments herein comprises an anticoagulant, specifically an anticoagulant selected from the group consisting of EDTA, citrate, heparin, hirudin, warfarin, and a mixture thereof.
  • Anticoagulant additives which at the same time permit integrity, that is to say the absence of lysis and the viability of nucleated cells are known to the art.
  • sodium citrate/citric acid is added to the whole blood sample to a final concentration of 11.7 mM and 2.1 mM, respectively.
  • the phosphate salt in the composition as disclosed in here effects the buffering of the whole blood sample at a physiological pH.
  • the phosphate salt is NaH 2 PO 4 , added to the whole blood sample to a final concentration of 2.1 mM in an even more specific embodiment.
  • Further additives are adenine and a cell-metabolizable sugar, in order to support cell integrity and viability.
  • adenine is added to the whole blood sample to a final concentration of 0.24 mM.
  • antioxidants are essential, in order to stabilize nucleated cells of a whole blood sample ex-vivo.
  • the observed beneficial effects of antioxidants point to reactive oxygen species as a possible cause for cell instability in the sample.
  • Reactive oxygen species are typically generated by NADPH oxidase, xanthine oxidase, the mitochondrial electron-transport chain, and dysfunctional endothelial nitric oxide synthase.
  • mitochondria produce substantial amounts of O 2 ⁇ at the mitochondrial electron-transport chain complexes I and III.
  • Complex I releases O 2 ⁇ into the mitochondrial matrix and is considered the main producer of O 2 ⁇ due to reverse electron flow from complex II under low-ADP conditions.
  • the matrix-localized mitochondrial superoxide dismutase 2 (SOD2) dismutates O 2 ⁇ to H 2 O 2 , which in turn is reduced to water by glutathione peroxidase or catalase.
  • SOD2 thus lies in the detoxification of O 2 ⁇ to prevent generation of ONOO ⁇ (peroxynitrite) and/or oxidative damage of mitochondrial electron-transport chain proteins and mitochondrial DNA, which may otherwise compromise mitochondrial function.
  • Mitochondria themselves are known to be sensitive to reactive oxygen species. Oxidative damage lowers their activity and may even increase their production of reactive oxygen species. Enhanced levels thereof are known to damage the mitochondrial DNA and to stimulate the release of mitochondrial apoptotic factors.
  • composition in all aspects and embodiments as disclosed in here comprises a mitochondria-targeted antioxidant.
  • An “antioxidant” in a generic sense is a molecule that is capable of inhibiting oxidation or reactions promoted by oxygen, reactive oxygen species, and peroxides. This includes the property of being capable of inhibiting and/or terminating a reaction of a free radical.
  • An antioxidant is typically a reducing agent which is capable of taking part in oxidation reactions as an electron donor, thus becoming oxidized itself.
  • An antioxidant according to the understanding as disclosed in here acts as a substitute oxidation target competing with cellular components which in the absence of the antioxidant would be oxidized and thereby compromised by oxidative damage.
  • a “mitochondria-targeted” antioxidant is an antioxidant which is typically applied extracellularly, which is capable of crossing the cellular membrane and capable of interacting with the mitochondrial membrane, in order to inhibit certain oxidation processes taking place in the mitochondrial compartment of the cell.
  • Specific examples for such cell-permeable antioxidants are SkQ1, MitoQ, SS-31, among others.
  • SkQ1 also known as a salt comprising 10-(6′-plastoquinonyl) decyltriphenylphosphonium, is known for its neuroprotective properties, among others. When applied to target cells extracellularly, SkQ1 has been shown to inhibit oxidative stress caused by reactive oxygen species in mitochondria.
  • MitoQ is a mixture of mitoquinol (reduced form) and mitoquinone (oxidized form), also known as (10-(2,5-dihydroxy-3,4-dimethoxy-6-methylphenyl)decyl) triphenylphosphonium and (10-(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dienyl)decyl) triphenylphosphonium.
  • MitoQ comprises a ubiquinol moiety which is covalently attached through an aliphatic carbon chain to a lipophilic triphenylphosphonium cation. The antioxidant reactions of extracellularly applied MitoQ predominantly occur within phospholipid bilayers.
  • the mitochondria targeted antioxidant is SkQ1.
  • SkQ1 is added to the whole blood sample to a final concentration of 5-500 nM, advantageously to a final concentration of 50-250 nM, particularly about 100 nM.
  • the mitochondria targeted antioxidant is SS-31.
  • SS-31 is added to the whole blood sample to a final concentration of 0.1-50 ⁇ M, advantageously to a final concentration of 0.5-10 ⁇ M, particularly about 1 ⁇ M.
  • the antioxidants disclosed in here are applied in an aqueous solution
  • the antioxidants selected to practice all aspects and embodiments of the present disclosure are advantageously water soluble or solubilized in water, e.g. using a helper substance including, but not limited to a surfactant, a detergent and an emulsifier.
  • the antioxidant is a mixture of a first and a second antioxidant, wherein the first antioxidant is a mitochondria-targeted antioxidant, and the second antioxidant is an antioxidant other than a mitochondria-targeted antioxidant.
  • This group encompasses water-soluble molecules with a reducing activity, wherein the molecules are capable of undergoing oxidation outside of the mitochondrial compartment.
  • the second antioxidant in a further specific embodiment, is an antioxidant other than SkQ1, MitoQ, and SS-31.
  • the second antioxidant is selected from the group consisting of glutathione, acetylsalicylic acid, N-acetyl-5-aminosalicylic acid, N-acetylcysteine, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and a mixture thereof.
  • a protease inhibitor in the liquid composition in all aspects and embodiments as disclosed herein.
  • a specific embodiment in this regard includes an inhibitor of a serine protease.
  • a more specific embodiment in this regard includes an inhibitor of human neutrophil elastase.
  • a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo the liquid composition including a protease inhibitor selected from Sivelestat, AEBSF, and a mixture thereof.
  • the compound commercialized as “Sivelestat” or “ONO-5046”, also known as N- ⁇ 2-[( ⁇ 4-[(2,2-dimethylpropanoyl)oxy]phenyl ⁇ sulfonyl)amino]benzoyl ⁇ glycine is a selective neutrophil elastase inhibitor.
  • AEBSF is an abbreviation of 4-(2-Aminoethyl)benzenesulfonyl fluoride which a water soluble, and which is an irreversible serine protease inhibitor.
  • the hydrochloride is commercially available as Pefabloc® SC.
  • Pefabloc® SC is added to the whole blood sample to result in a final concentration of 0.05-1 mM, more specifically 0.1-0.5 mM, and even more specifically 0.2-0.3 mM, particularly about 0.25 mM.
  • Pefabloc® SC is a water-soluble serine protease inhibitor with a molecular weight of 239.5 Daltons, also known to the art as 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF).
  • AEBSF 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride
  • the compound is capable of inhibiting proteases like chymotrypsin, kallikrein, plasmin, thrombin, and trypsin.
  • the sugar is selected from glucose and fructose.
  • the glucose is added to the whole blood sample to a final concentration of 15-25 mM, advantageously to a final concentration of 19-21 mM, particularly about 20 mM.
  • a further aspect as disclosed in here is a kit of parts comprising a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo as disclosed in here, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, the liquid composition being enclosed in a blood drawing container, the kit further comprising packaging material, a label, and a user instruction sheet.
  • the liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo in all aspects and embodiments as disclosed in here is advantageously contained in a blood drawing container.
  • An example therefor is known to the art as a “vacutainer”.
  • a vacutainer is a blood collection tube provided as a sterile glass or plastic tube with a closure and a vacuum inside the tube facilitating the draw of a predetermined volume of liquid such as a whole blood sample from the vein of a subject.
  • a vacutainer tube according to the present disclosure contains a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo, in order to stabilize and preserve the whole blood sample prior to analytical testing. Tubes are available with or without a safety-engineered closure, with a variety of labeling options and closure colors as well as a range of draw volumes.
  • the vein is first punctured with a hypodermic needle which is carried in a translucent plastic holder.
  • the needle is double ended, the second shorter needle being shrouded for safety by the holder.
  • a Vacutainer test tube is pushed down into the holder, its rubber cap is pierced by the second needle and the pressure difference between the blood volume and the vacuum in the tube forces blood through the needle and into the tube.
  • the filled tube is then removed and another can be inserted and filled the same way. It is important to remove the tube before withdrawing the needle, as there may still be some suction left, causing pain upon withdrawal.
  • Another specific embodiment of all aspects as disclosed herein is enclosing a mixture of (a) a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo as disclosed herein, and (b) an amount of whole blood, i.e. the sample being a measured and/or predetermined amount of whole blood ex-vivo.
  • compositions for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo as disclosed in here, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, the use being for the purpose of stabilizing an analyte selected from DNA, RNA and protein in intact nucleated cells of a whole-blood sample ex-vivo.
  • another aspect disclosed in here is a method for stabilizing intact nucleated cells of a whole-blood sample ex-vivo, the method comprising the steps of (a) providing the whole blood sample ex-vivo; (b) contacting and mixing the sample of step (a) with a liquid composition for stabilizing an analyte in intact nucleated cells of a whole-blood sample ex-vivo as disclosed in here, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant; (c) incubating the mixture obtained in step (b), thereby stabilizing intact nucleated cells of the ex-vivo whole-blood sample.
  • the whole blood sample contacted and mixed with the liquid composition as disclosed in here can be incubated at room temperature for a prolonged time interval, whereby the nucleated cells present in the whole blood sample are stabilized, as well as analytes in the cells, particularly DNA, RNA, and protein.
  • the step of incubating the mixture obtained in step (b) can be performed at room temperature for a time interval selected from the group consisting of 0 h-12 h, 0 h-24 h, 0 h-36 h, 0 h-48 h, 0 h-60 h, and 0 h-72 h, or even for longer, depending on the analyte to be detected thereafter.
  • the target cell for stabilization is a nucleated cell present in the ex-vivo sample of whole blood.
  • the nucleated cell is a nucleated cell of hematopoetic lineage or a nucleated cell of non-hematopoetic lineage.
  • a nucleated cell of hematopoetic lineage is selected from the group of cell types consisting of a megakaryocyte, a thrombocyte, a nucleated red blood cell, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a monocyte, a macrophage, a large granular lymphocyte, a small lymphocyte, a T lymphocyte, a B lymphocyte, a plasma cell, and a dendritic cell, or a precursor of any of the said cell types.
  • the nucleated cell is a nucleated cancer cell of hematopoetic lineage.
  • the target cell for stabilization is a nucleated cell present in the ex-vivo sample of whole blood, the nucleated cell being capable of undergoing two or more cell divisions thereby forming daughter cells.
  • the nucleated cell is capable of undergoing cell divisions not only in vivo but also ex-vivo in a culture medium.
  • the present disclosure provides liquid compositions for supporting and/or maintaining the viability of an intact nucleated target cell of a whole-blood sample ex-vivo, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof, wherein the intact nucleated target cell is capable of undergoing one or more cell divisions. More specifically, the nucleated target cell is capable of undergoing one or more cell divisions in a culture medium.
  • the skilled person is well aware of different culture media which support viability and cell division of mammalian cells, specifically of human cells, more specifically of cancer cells of human origin.
  • the culture media support cell viability and cell division in the absence of a composition for supporting and/or maintaining the viability of an intact nucleated target cell in an ex-vivo of whole-blood as disclosed in the present document.
  • the viability of the nucleated target cell is maintained in the ex-vivo whole-blood sample that is mixed with the stabilizing liquid composition as disclosed.
  • the nucleated target cell which is in a resting state can be isolated from the stabilized blood sample and be put into culture, whereby once removed from the stabilizers the nucleated cell becomes physiologically active again.
  • the resting state is reversed, even including the biological functions necessary for cell division.
  • the skilled person is now equipped with improved means to isolate a nucleated target cell from a sample of whole blood, wherein in a specific embodiment the target cell is capable of undergoing cell divisions ex vivo, and more specifically wherein the target cell is a cancer cell, e.g. but not limited to, a circulating tumor cell.
  • the target cell is a cancer cell, e.g. but not limited to, a circulating tumor cell.
  • a method to isolate and culture a nucleated target cell present in an ex-vivo sample of whole blood comprising the steps of (a) providing the ex-vivo whole blood sample; (b) contacting and mixing the sample of step (a) with a liquid composition, the composition being an aqueous solution, the solution comprising an anticoagulant, a phosphate salt, a cell-metabolizable sugar, adenine, and an antioxidant, wherein the antioxidant comprises a mitochondria-targeted antioxidant, preferably a mitochondria-targeted antioxidant selected from the group consisting of SkQ1, MitoQ, SS-31, and a mixture thereof; (c) incubating the mixture obtained in step (b), thereby stabilizing intact nucleated cells of the ex-vivo whole-blood sample; separating nucleated cells from the incubated mixture of step (c) and contacting the separated nucleated cells with a culture medium; and (d) incubating the nucleated cells in the
  • CTCs circulating tumor cells
  • the target cell for stabilization is a nucleated cancer cell present in the ex-vivo sample of whole blood.
  • the target cell for stabilization is a nucleated cell present in the ex-vivo sample of whole blood, wherein the individual from whom the sample of whole blood is obtained prior to stabilization (and detection) suffers from a cancer.
  • the target cell is a nucleated cell of non-hematopoetic lineage, particularly a cancer cell of non-hematopoetic lineage.
  • the cancer cell is a circulating tumor cell, specifically a cell shedded from a solid tumor into the circulation, the circulation including lymph and blood.
  • the circulating tumor cell is selected from a solid tumor of cells selected from the group consisting of epithelial, connective tissue, bone, cartilage, muscle, and nerve cells.
  • Circulating tumor cells are specific nucleated cells the present disclosure as presented here aims to make detectable in whole blood, by stabilizing the cancer cells in the whole blood over a period of time, preferably at room temperature.
  • Sample 1 contained EDTA as anticoagulant only and no additive
  • sample 2 to 6 were supplemented with Sivelestat (10 ⁇ g/ml), Pefabloc® SC (1 mM), SkQ1 (100 nM), Mito Q (100 nM) and glutathione (3 mM), respectively.
  • Nucleated cells were enriched by erythrocytes lysis.
  • a 50 ⁇ l aliquot of peach sample was mixed with 200 ⁇ l of lysis buffer (100 mM NH 4 Cl, 5 mM Hepes, 0.5 mM KHCO 3 , 0.1 mM EDTA) and incubated for 10 min at room temperature. After centrifugation at 200 ⁇ g for 15 min, the supernatant was removed and the pellet was resuspended in 250 ⁇ l of lysis buffer. After centrifugation at 200 ⁇ g for 15 min, the supernatant was removed and the pellet was resuspended in 1,000 ⁇ l of PBS, 0.3 mM EDTA. The cells were sedimented by centrifugation at 200 ⁇ g for 15 min. The pellet was resuspended in PBS, 0.3 mM EDTA and the volume of the samples adjusted to 500 ⁇ l.
  • a volume of 50 ⁇ l of the cell suspension was spotted on a glass slide and air-dried.
  • DAPI-containing mounting medium was added, the spots covered with cover slips and after a further incubation for 20 min analyzed on a Zeiss Axio Observer Microscope. The total number of nucleated cells and the CK 5/8 positive cells were analyzed with the Assay Builder Software from Zeiss.
  • FIG. 1 The result is shown in FIG. 1 .
  • the samples containing protease inhibitors like Sivelestat (inhibitor of neutrophilic elastase) and Pefabloc SC (serine protease inhibitor) showed higher signals than the control sample without additive.
  • Particular positive effects were observed with SkQ1 and MitoQ, antioxidants targeted to mitochondria.
  • a further positive effect was observed with the antioxidant glutathione.
  • FIG. 1 illustrates the detection of MDA-MB-468 cells spiked into blood samples after storage for 48 hours at room temperature. Depicted is the percentage of MDA-MB-468 cells detected by immunostaining with Anti-CK 5/8 antigens.
  • MDA-MB-468 cells were spiked into blood samples obtained from healthy individuals to result in a concentration of 100,000 spiked cells/ml blood. The samples were processed either immediately or after storage at room temperature for 48 hours.
  • Sample 1 contained EDTA as sole anticoagulant and no further additive
  • Sample 3 was additionally supplemented with the mitochondria targeted antioxidant SS-31 (1 ⁇ M), sample 4 with glutathione (0.75 mM). Nucleated cells were enriched by erythrocytes lysis and processed as described in Example 1.
  • FIG. 2 illustrates the detection of MDA-MB-468 cells spiked into blood samples after storage for 48 hours at room temperature. Depicted is the percentage of MDA-MB-468 cells detected by immunodetection with fluorescent Anti-CK5/8 antibodies.
  • nucleated cells were enriched by erythrocyte lysis. Aliquots of 350 ⁇ l sample were mixed with 1,000 ⁇ l of lysis buffer (80 mM NH 4 Cl, 10 mM Hepes buffer, 0.1 mM EDTA) and incubated for 10 min at room temperature. After centrifugation at 300 ⁇ g for 15 min, the supernatant was removed and the pellet was resuspended in 1,000 ⁇ l of lysis buffer. After centrifugation at 300 ⁇ g for 15 min, the supernatant was removed and the pellet was resuspended in 1,000 ⁇ l of PBS. The cells were sedimented by centrifugation at 300 ⁇ g for 15 min.
  • lysis buffer 80 mM NH 4 Cl, 10 mM Hepes buffer, 0.1 mM EDTA
  • the pellet was resuspended in PBS and the DNA isolated with High Pure Template Preparation Kit (Roche Applied Science, Cat. 11 796 828 001) according to the protocol recommended by the manufacturer.
  • the DNA was eluted with 100 ⁇ l elution buffer from the HighPure columns.
  • the primer and probe sequences used were: t(14/18) forward primer acctgaggagacggtgac (SEQ ID NO:1); t(14/18) reverse primer tggggttttgacctttagaga (SEQ ID NO:2); t(14/18)-FAM detection probe 6FAM-ctctgggtgggtctgtgttgaaaca-BHQ2 (sequence is SEQ ID NO:3).
  • Blood samples from breast cancer patients containing circulating tumor cells were collected either in citrate (11.7 mM)/citric acid (2.1 mM), NaH 2 PO 4 (2.1 mM), Dextrose (20.1 mM), Adenine (0.24 mM) and SkQ1 (100 nM), designated as “Stabilizer 4”, or in CellSave (Veridex Cell Save Preservative tube, Cat. No. 7900005). Samples #59, #60, #62, #63, #58 were stored for 48 hours at room temperature, samples #55, #56 and #57 were stored for 72 hours at room temperature. Nucleated cells were enriched by erythrocyte lysis.
  • RNA samples 2 ⁇ l aliquots from the isolated RNAs was reverse transcribed using the Transcriptor first strand cDNA Synthesis kit (Roche Applied Science, Cat. No. 04897030001) in reaction volumes of 20 ⁇ l. Aliquots of 5 ⁇ l cDNA were amplified with the LightCycler 480 Probes Master in a LightCycler 480 instrument. The primers and probes that were used are described in Table 1.
  • RNA target forward primer reverse primer detection probe E- 5′-GGT TAA GCA CAA 5′-CAC CTG ACC CTT 5′-CAC AGT CAC TGA Cadherin CAG CAA CA-3′; GTA CGT-3′; CAC CAA CGA TAA SEQ ID NO: 4 SEQ ID NO: 5 TCC TCC GA-3′ SEQ ID NO: 6 ⁇ cMyc 5′-CCC CTG GTG 5′-CTC ATC TTC TTG 5′-ACA CCG CCC ACC CTC CAT GAG GA-3′; TTC CTC CTC AGA-3′; ACC AGC AGC G-3′ SEQ ID NO: 7 SEQ ID NO: 8 SEQ ID NO: 9 ⁇ EpCam 5′-GTT TGC GGA 5′-AGG ATT CAC CTT 5′-TGA GAA TAA TGT CTG CAC TTC AG-3′; TAA CAT CTT TTT-3′; TAT CAC TAT TGA SEQ ID NO: 10 SEQ ID NO: 5
  • RT reverse transcription
  • DNA generation is monitored at each cycle of PCR.
  • the threshold cycle The amplification cycle during which the fluorescence becomes measurable against background is called the threshold cycle or “crossing point”.
  • Results are shown in Tables 2a-2d.
  • the first line indicates the sample number and the respective storage time at room temperature.
  • the second line specifies the type of stabilizer reagent.
  • RT-PCR was performed in duplicates, indicated as “Test A” and “Test B”.
  • the numbers in the table are the crossing points obtained after real-time RT-PCR. “neg” indicates negative results.
  • RNA molecules indicated above are known to be specific for certain types of circulating tumor cells.
  • the RNAs coding for E-Cadherin, EpCam, Muc 1, cyclin, CAV and CK18 surprisingly remained detectable in the samples preserved with Stabilizer 4.
  • the cMyc RNA which was also expressed in leukocytes was detectable with the stabilizer reagent CellSave as well, but with a strong shift in crossing points. This indicated that the respective RNA level was decreased by several orders of magnitude.
  • RNAs PPIA, TBP, beta2m and GAPDH used as controls were detectable in all samples.
  • RNA in blood samples stored for 48 hours or 72 hours can be preserved in the Stabilizer 4 reagent.
  • MDA-MB-468 cells were spiked into blood samples obtained from two healthy individuals at 14.4 ⁇ 10 6 cells/ml. From each donor four samples were prepared. Sample 1 and 2 contained EDTA as anticoagulant only and no additive, sample 3 and 4 were stabilized with citrate (11.7 mM), citric acid (2.1 mM), NaH 2 PO 4 (2.1 mM), Dextrose (20.1 mM), Adenine (0.24 mM), Pefabloc® SC (0.25 mM), SS-31 (1 ⁇ M) and acetylsalicylic acid (1 mM) as stabilizer (“Stabilizer 5”). Aliquots from these samples were processed immediately after set up, other aliquots were processed after storage at room temperature for 72 hours. MDA-MB-468 cells were isolated from blood by capturing with BreastSelectBeads from Adnagen Cat. No.: T1-508 with 1,000 ⁇ l aliquots of sample material according to the protocol recommended by the manufacturer.
  • RNA was transcribed into cDNA using Transcriptor First Strand cDNA Synthesis Kit Cat. No: 04897030001 in reactions of 70 ⁇ l volume, each reaction containing 12.5 ⁇ l of isolated nucleic acid.
  • PCR was performed with the with the LightCycler 480 Probes Master in a LightCycler 480 instrument in 20 ⁇ l reactions containing 5 ⁇ l of the cDNA samples. Initial denaturation was for 10 min at 95° C., 50 cycles with 10 seconds denaturation at 95° C. and annealing/elongation at 58° C. for 1 min.
  • Detection format Dual color hydrolysis Probe UPL (Universal Probe Library, Roche Diagnostics GmbH Mannheim, Germany) probe as indicated below.
  • Table 3 describes the primer/probes for the RNA/DNA targets analysed.
  • the relative quantification method of Livak and Schmittgen was used (Livak K. J. and Schmittgen T. D., Methods 2001, 25(4) 402-408).
  • the GCK target was amplified from the DNA present in the total nucleic acid preparation.
  • FIG. 4 show a strong decline in RNA levels from cells isolated from blood stored in EDTA. When the cells were isolated from blood stored in Stabilizer 5, very little influence on the RNA was observed. The expression pattern was only slightly affected during storage.
  • MDA-MB-468 cells were spiked into blood samples obtained from healthy individuals to result in a final concentration of 300,000 spiked cells/ml blood. The samples were processed either immediately or after storage at room temperature for 48 hours. Sample 1 contained EDTA as anticoagulant only and no further additive, sample 2 contained citrate (11.7 mM)/citric acid (2.1 mM), NaH 2 PO 4 (2.1 mM), Dextrose (20.1 mM), Adenine (0.24 mM), Pefabloc® SC (0.25 mM), SS-31 (1 ⁇ M) and acetylsalicylic acid (1 mM) as stabilizer (“Stabilizer 5”, see Example 5).
  • nucleated cells were enriched by erythrocyte lysis.
  • Aliquots of 1 ml blood were mixed with 4 ml of lysis buffer (80 mM NH 4 Cl, 10 mM Hepes buffer, 0.1 mM EDTA) and incubated for 10 min at room temperature. After centrifugation at 200 ⁇ g for 15 min, the supernatant was removed and the pellet was resuspended in 2 ml of PBS/3 mM EDTA. The cells were sedimented by centrifugation at 200 ⁇ g for 15 min. The supernatant was removed and the cell pellets resuspended in 300 ⁇ l of PBS supplemented with 2 mM EDTA and 0.5% BSA.
  • lysis buffer 80 mM NH 4 Cl, 10 mM Hepes buffer, 0.1 mM EDTA
  • MDA-MB-468 cells were isolated using CD326 (EpCAM) Microparticles from Miltenyi (Cat No 130-061-101), MS columns and the MACS separator system (Milteny Cat. Nos. 130-042-201, 130-042-102) making use of the protocol recommended by the manufacturer.
  • CD326 EpCAM
  • Miltenyi Cat No 130-061-101
  • MS columns MS columns
  • MACS separator system Milteny Cat. Nos. 130-042-201, 130-042-102
  • the enriched cells were suspended in 300 ⁇ l of RPMI medium.
  • the viability of the cells was tested with the Trypan blue exclusion method. 10 ⁇ l of each sample was mixed with 10 ⁇ l of Trypan blue (Sigma-Aldrich T8154). The number of MDA-MB-468 cells excluding the dye was counted in a Neubauer chamber. The yield of viable cells was calculated using the cell number spiked into the blood samples as 100%.
  • Stabilizer 5 can preserve a significant number of cells. When just EDTA is used as a stabilizing agent the cell viability is negatively affected, to a surprisingly significant extent.
  • MDA-MB-468 cells treated and isolated as described in Example 6 were seeded into the wells of an E-Plate 96-well device (Roche Applied Science, Cat. No. 06472451001, Roche Diagnostics GmbH Mannheim, Germany) for the Real-Time Cell Analyzer (RTCA) MP (xCELLigence system). 50 ⁇ l aliquots of cells were seeded into wells containing 50 ⁇ l of RPMI medium. Duplicates were used for analysis. The E-Plate was incubated over night at 37° C. in an incubator to allow attachment of MDA-MB-468 cells to the bottom of the E-Plate. In order to remove contaminating blood derived cells the medium was removed and cells attached to the culture plate were washed 3 times with PBS. 150 ⁇ l of fresh RPMI medium was added per well and the E-Plate transferred to the Real-Time Cell Analyzer. Cell index was measured once per hour for 101 hours.
  • RTCA Real-Time Cell Analyzer
  • FIG. 5 Cell growth can be observed with cells isolated from blood stored for 48 hours in stabilizer described in this invention.

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