WO2019230532A1

WO2019230532A1 - Blood sample preservative

Info

Publication number: WO2019230532A1
Application number: PCT/JP2019/020333
Authority: WO
Inventors: 泰之秋山; 篤史森本
Original assignee: 東ソー株式会社
Priority date: 2018-05-29
Filing date: 2019-05-22
Publication date: 2019-12-05
Also published as: JP7334468B2; JP2019207229A

Abstract

The present invention provides a method for producing a blood sample which can be stably preserved even when the blood sample is stored under vibrating condition and/or low temperature condition where the blood sample is not frozen. This method for producing a blood sample includes a target cell, an antiplatelet agent, and an anticoagulant.

Description

Blood sample preservative

The present invention relates to a method for preparing a blood sample that can be stored stably. In particular, the present invention relates to a method for preparing a blood sample that can be stably stored against vibrations and temperature changes in a room temperature environment where the blood sample does not freeze.

In recent years, cells have been selectively separated and collected from body fluids such as blood, tissue specimens obtained by suspending or dispersing tissues such as organs, or cell culture fluid, and basic research on the separated and collected cells. Researches are also being applied to clinical diagnosis and treatment. For example, tumor cells (Circulating Tumor Cell, hereinafter referred to as CTC) are collected from blood collected from cancer patients, and morphological analysis, tissue type analysis, and gene analysis are performed on the cells, and based on the knowledge obtained by these analyzes. Research is ongoing to determine treatment strategies.

However, it may take a long time to collect the blood from the cancer patient and to perform the above-described cell separation and recovery. If the time until separation / recovery is long, the cells contained in the solution deteriorate, and there is a risk that the shape of the cells will collapse and the nucleic acids and proteins in the cells accompanying them may be released. As a result, the recovery rate of the target cells contained in the solution may be reduced, and the analysis ability of nucleic acids and proteins contained in the target cells may be reduced.

In addition, if the location where the sample is collected is different from the location where the cells contained in the sample are separated, recovered, and detected, the sample is required to be transported. There is a possibility of an environment different from the temperature environment (around 25 ° C.) generally maintained in a facility that performs separation and collection and detection, and vibration is also applied. Therefore, the risk of deterioration of the cells contained in the solution is further increased.

As a preservative and method for preserving blood samples known so far, Patent Document 1 discloses a preservative containing a stabilizer containing a formaldehyde donor and an anticoagulant, and the preservation By adding an agent to a blood sample containing rare cells, separation and recovery of rare cells contained in the blood sample stored for a long period of time is realized. However, even when the method described in Patent Document 1 is used, if the above-described operation is performed when the blood sample is stored under vibration and / or at a low temperature at which the blood sample does not freeze (for example, 0 ° C. to 20 ° C.), high efficiency of the target cell is obtained. It was difficult to separate and recover.

Japanese National Table 2005-501236

An object of the present invention is to provide a blood sample that can be stably stored even if the blood sample is stored under vibration and / or at a low temperature at which the blood sample does not freeze, a method for preparing the same, and a preservative.

In order to solve the above-mentioned problems, the present inventors have intensively studied to arrive at the present invention.

That is, the present invention can be exemplified as follows.
[1]
A method for preparing a blood sample comprising a target cell, an antiplatelet agent and an anticoagulant.
[2]
The method according to [1], further comprising a formaldehyde donor compound.
[3]
The method according to [1] or [2], comprising a step of adding an antiplatelet agent and an anticoagulant to a blood sample and then storing at a low temperature.
[4]
The method according to any one of [1] to [3], further comprising a step of adding a hydrophilic polymer compound to the blood sample.
[5]
The method according to any one of [1] to [4], further comprising a step of adding a water-soluble vitamin E-like substance to a blood sample.
[6]
The method according to any one of [1] to [5], wherein the anticoagulant is a chelating agent.
[7]
The method according to any one of [1] to [6], wherein the antiplatelet agent is an inhibitor against platelet GPIIb / IIIa receptor.
[8]
Formaldehyde donor compounds are imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, bronopol (2-bromo-2-nitropropyne-1,3-diol), Diazolidinylurea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quaternium-15 (mesenamine 3-chloroallylochloride), sodium hydroxymethylglycine, methylol of amines and amides , A method according to any one of [1] to [7], which is one or more compounds selected from hydroxymethyl derivatives, methylol, and methenamine.
[9]
The method according to any one of [1] to [8], wherein the hydrophilic polymer compound is polyethylene glycol.
[10]
The method according to [3], wherein the low temperature is 0 ° C. or higher and lower than 25 ° C.
[11]
A method for detecting a target cell contained in a blood sample, comprising the following steps (1) to (3):
(1) A step of preparing a blood sample by the method according to any one of [1] to [10],
(2) a step of crushing or removing red blood cells contained in the obtained blood sample,
(3) A step of detecting target cells from the blood sample after performing the steps of (2) [12]
A method for collecting a target cell contained in a blood sample, wherein the target cell is detected by the method according to [11] and then the detected cell is collected.
[13]
A blood sample comprising a target cell, an antiplatelet agent, and an anticoagulant.
[14]
14. The blood sample of claim 13, further comprising a formaldehyde donor compound.
[15]
A blood sample preservative for cryopreservation, comprising an antiplatelet agent and an anticoagulant.
[16]
The blood sample preservative according to [15], further comprising a formaldehyde donor compound.

Hereinafter, the present invention will be described in detail.

In the present invention, a blood sample refers to a sample containing at least target cells and platelets and / or extracellular vesicles released from platelets. Specifically, blood (whole blood), diluted blood, serum, Samples such as plasma, spinal fluid, umbilical cord blood, component blood collection, samples that may contain blood-derived components such as urine, saliva, semen, feces, sputum, amniotic fluid, ascites, liver, lung, spleen, kidney, skin, Examples include a tissue suspension in which a piece of tissue such as a tumor or lymph node is suspended, or a fraction containing cells derived from a sample or tissue obtained by separation from the sample or tissue suspension described above. . Among these, as an example of a fraction containing a sample or tissue-derived cells, a fraction obtained by layering a sample or tissue suspension on a density gradient forming medium and then performing density gradient centrifugation can be mentioned. The target cells in the present invention are preferably cells that can exist in blood. The cells that can exist in blood include not only cells originally contained in blood but also cells that can be mixed into blood from other tissues. Examples of target cells in the present invention include tumor cells such as blood circulating tumor cells (CTC), circulating blood endothelial cells (CEC), circulating vascular endothelial cells (CEP), circulating fetal cells (CFC), and various stem cells. .

The blood sample according to an embodiment of the present invention preferably includes at least a target cell, an antiplatelet agent, and an anticoagulant. In the case where a blood sample is stored at around 25 ° C., which is a temperature environment for collecting the sample and separating / collecting or detecting a target cell contained in the sample, the blood sample is exemplified in Patent Document 1. Stable storage was possible by adding a preservative. However, when a blood sample added with a preservative exemplified in Patent Document 1 is stored at a low temperature (for example, around 15 ° C.) at which the sample does not freeze, a gel-like substance is generated, and the target cell contained in the sample Separation and recovery became difficult. Therefore, as a result of intensive studies, the present inventors have suppressed the generation of the gel-like substance by adding an antiplatelet agent and an anticoagulant to a blood sample, and the efficiency of separation and recovery of target cells contained in the sample is increased. I found it to improve.

The antiplatelet agent contained in the blood sample according to one embodiment of the present invention refers to a substance that suppresses aggregation of platelets and extracellular vesicles released from platelets, blocks platelet ADP receptor, and platelets serotonin Examples include substances that directly affect platelet function and block platelet aggregation by blocking 2 receptors, inhibiting platelet cyclooxygenase, or blocking platelet GPIIb / IIIa receptors. Inhibitors that block platelet ADP receptor, block platelet serotonin 2 receptor, and inhibit platelet cyclooxygenase mainly act as a receptor on the platelet membrane surface or as a signal pathway inhibitor from the receptor, It is applied to suppress an increase in the calcium ion concentration in platelets that contributes to the expression of platelet GPIIb / IIIa receptor involved in platelet aggregation. On the other hand, an inhibitor for platelet GPIIb / IIIa receptor has a function of suppressing aggregation due to binding of platelet GPIIb / IIIa receptors expressed on the platelet membrane surface between platelets via von Willebrand factor, fibrinogen and the like. . Among them, since the calcium concentration in platelets increases by various routes, an inhibitor against platelet GPIIb / IIIa receptor that directly acts on platelet aggregation may be used as an antiplatelet agent. Examples of inhibitors for platelet GPIIb / IIIa receptors include tirofiban, abciximab, and eptifibatide. These may use only 1 type and may use 2 or more types together. When tirofiban, which is a platelet GPIIb / IIIa inhibitor, is used as an antiplatelet agent contained in the preservative of the present invention, the present invention is such that the final concentration of the blood sample after the preservative is 0.24 μg / mL or more. The preservative of the present invention is more preferable so that the final concentration is 24 μg / mL or more.

In the blood sample according to one embodiment of the present invention, an integrin inhibitor other than GPIIb / IIIa which is a kind of integrin expressed in platelets may be further added. The integrin inhibitor refers to a substance that suppresses aggregation of integrin-expressing cells and extracellular vesicles released from the cells, and includes substances that can bind to integrin present on the cell membrane surface. The type of integrin is not limited, but integrins are roughly classified into laminin binding integrin, collagen binding integrin, RGD sequence recognition integrin and LDV sequence recognition integrin, and when a substance capable of binding to each is added as an integrin inhibitor Good. For example, blood samples of cancer patients have many extracellular vesicles that possess RGD sequence-recognized integrin, such as integrin αvβ3, whose expression level is increased during angiogenesis or growth of cancer tissue. When inhibited, aggregation of the extracellular vesicle can be suppressed. As an inhibitor of RGD sequence recognition integrin, a peptide containing at least RGD as an amino acid sequence or a small molecule compound capable of binding to the integrin may be used.

As an example of the anticoagulant contained in the blood sample according to one embodiment of the present invention, it becomes a chelating agent that suppresses blood coagulation by coordinating calcium ions that cause blood coagulation, and causes blood coagulation. Examples include antithrombin agents including heparin that suppresses thrombin activity. Among them, chelating agents are preferable, and specific examples thereof include EDTA (ethylenediaminetetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), DCTA (1,2-diaminocyclohexanetetraacetic acid), EGTA (ethylene glycol bis-2-aminoethyl ether tetraacetic acid). ), Citric acid, oxalic acid, sodium fluoride, ACD (Acid Citrate Dextrose Solution). These may use only 1 type and may use 2 or more types together.

The blood sample according to one embodiment of the present invention may further contain a formaldehyde donor compound. A formaldehyde donor compound refers to a compound that itself does not act directly on cells, but can release formaldehyde by being decomposed to stabilize cells. Specific examples of formaldehyde donor compounds include imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, bronopol (2-bromo-2-nitropropyne-1,3- Diol), diazolidinyl urea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quaternium-15 (mesenamine 3-chloroallylochloride), hydroxymethylglycine sodium, amine, Examples include amide methylol, hydroxymethyl derivatives, methylol, methenamine, and paraformaldehyde. These may use only 1 type and may use 2 or more types together. Among them, a stabilizer capable of releasing formaldehyde more slowly than paraformaldehyde is preferable. Specifically, imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, bronopol ( 2-bromo-2-nitropropyne-1,3-diol), diazolidinyl urea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quaternium-15 (mesenamine 3) -Chloroallylochloride), sodium hydroxymethylglycine, methylols of amines and amides, hydroxymethyl derivatives, methylol, and methylenamine, but are not limited thereto. In particular, imidazolidinyl urea is one of the most preferred stabilizers.

The concentration of the formaldehyde donor compound contained in the preservative according to one embodiment of the present invention may be appropriately determined in consideration of the immobilization strength and the immobilization rate of the target cells contained in the blood sample possessed by the compound. . For example, when imidazolidinyl urea is used as the formaldehyde donor compound, the final concentration of the blood sample after addition of the preservative may be between 0.01% (w / v) and 10% (w / v). It is preferably between 25% (w / v) and 2% (w / v), more preferably between 0.3% (w / v) and 1.5% (w / v), 0.5 It is even more preferable when it is between 1% (w / v) and 1% (w / v).

The preservative according to one embodiment of the present invention may further contain a hydrophilic polymer compound in addition to the above-mentioned formaldehyde donor compound, antiplatelet agent and anticoagulant. The hydrophilic polymer compound is preferably a hydrophilic polymer having no charge, and examples thereof include polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, poly (hydroxyalkyl) methacrylate, polyacrylamide, a polymer having a phosphorylcholine group in the side chain, and many polymers. Examples thereof include saccharides and polypeptides, and polyethylene glycol is particularly preferred because of its high hydrophilicity. When polyethylene glycol is used as the hydrophilic polymer compound contained in the preservative of the present invention, the final concentration of the blood sample after addition of the preservative is 0.001% (w / v) to 10% (w / v). v), preferably between 0.01% (w / v) and 1% (w / v), preferably 0.01% (w / v) to 0.25% (w / v) ), More preferably 0.02% (w / v) to 0.2% (w / v). Furthermore, the molecular weight of polyethylene glycol contained in the preservative of the present invention may be an average molecular weight of 100 or more, preferably between 200 and 20,000, and more preferably between 600 and 2000. Hydrophilic polymer compounds are added to improve the stability of cells. However, by adding a hydrophilic polymer compound to a blood sample, the entropy in the sample is reduced, so that extracellular substances contained in the blood sample are reduced. Dispersion stability of particles containing vesicles is reduced, causing aggregation. Particularly at low temperatures, the entropy in the system is reduced, and aggregates containing gel-like substances are likely to be formed. Therefore, it is preferable to use a low molecular weight compound that can suppress a decrease in entropy due to the addition of a hydrophilic polymer compound. .

The osmotic pressure of the preservative according to one embodiment of the present invention may be adjusted to an arbitrary value in advance. By adjusting the osmotic pressure to be higher than the physiological osmotic pressure, the cells are contracted, so that the separation and concentration of the cells are performed when performing separation and concentration based on the difference in specific gravity between the cells and the solution, such as centrifugation after storage processing. This is good because it improves efficiency. When a preservative prepared at an osmotic pressure much higher than the physiological osmotic pressure is used, damage to the cells and collapse of the structure occur, so that the separation and concentration efficiency of the cells decreases. Here, the physiological osmotic pressure refers to an osmotic pressure of a solution (for example, a blood sample) containing target cells before adjusting the osmotic pressure, and is generally 260 mOsm / kg · H ₂ O to 300 mOsm / kg · Enter the range up to H ₂ O. The osmotic pressure of the blood sample to which the preservative is added may be in the range from 0 mOsm / kg · H ₂ O to 100 mOsm / kg · H ₂ O with respect to the physiological osmotic pressure, and from 0 mOsm / kg · H ₂ O to 60 mOsm. / kg · _H if still preferably be in the range of up to ₂ O, even more preferably be in the range of from 10mOsm / kg · _{H 2} O until 30mOsm / kg · _{H 2} O. For example, when the amount of preservative added to the blood sample is 150 μl / ml blood sample, the osmotic pressure of the preservative is from 0 mOsm / kg · H ₂ O to 900 mOsm / kg · H ₂ O with respect to the physiological osmotic pressure. The range of 0 mOsm / kg · H ₂ O to 600 mOsm / kg · H ₂ O is more preferable, and the range of 50 mOsm / kg · H ₂ O to 400 mOsm / kg · H ₂ O is more preferable. It is particularly preferable if it is present.

The preservative according to one embodiment of the present invention may further contain a water-soluble vitamin E-like substance. The water-soluble vitamin E-like substance is a water-soluble compound containing a functional group having an antioxidant action of vitamin E. In addition to the antioxidant action, the compound has a function as an inhibitor of cell apoptosis and neutrophil-specific cell death (netosis). Addition of this compound can suppress cell death of cells, especially leukocytes in blood samples, and can prevent damage to the substances that make up leukocytes, allowing specific binding to leukocytes It is possible to increase the efficiency of binding with various substances. Therefore, the separation efficiency of leukocytes from the blood sample is improved by binding magnetic particles that can specifically bind to the leukocytes. An example of a water-soluble vitamin E analog is Trolox. Trolox should be dissolved in an organic solvent such as DMSO and then added to the preservative. The volume of DMSO should be 10 μl / ml blood sample or less relative to the blood sample, and 4 μl / ml blood sample or less. If there is, it is preferable. The addition amount of Trolox may be 10 to 1000 μg / ml blood sample, preferably 20 to 300 μg / ml blood sample, and more preferably 100 to 250 μg / ml blood sample.

The preservative according to one embodiment of the present invention refers to a substance added to a blood sample. When the preservative of the present invention is added to a blood sample, all the substances constituting the preservative of the present invention may be added to the blood sample at one time, and some substances constituting the preservative of the present invention may be added. It is good also as an aspect which adds the remaining substance to the said blood sample after adding to a blood sample. In the latter case, the order of substances to be added is not limited, but if an anticoagulant is added to a blood sample in advance and then other constituents (at least a formaldehyde donor compound and an antiplatelet agent) are added to the blood sample, the blood sample It is preferable because coagulation can be suppressed. As an example of the operation of adding the above-mentioned anticoagulant to a blood sample, an aspect in which the anticoagulant is added to a blood sample by collecting blood in an anticoagulant-containing blood collection tube (EDTA blood collection tube, citrate blood collection tube, etc.) Can be given.

The storage temperature of a blood sample that has been stored by adding a preservative according to an embodiment of the present invention (hereinafter also simply referred to as a blood sample that has been stored) is such that the target cell is frozen and thawed when the sample is frozen. Non-specific adsorption, in which the labeling substance binds non-specifically to sites other than the target substance when labeling cells by denaturing the protein and other substances that compose the cell in a high-temperature environment while causing damage Therefore, it is preferable to store the blood sample at a temperature not lower than the temperature at which it does not freeze and lower than room temperature. Here, the low freezing temperature of the blood sample means near 0 ° C., and the room temperature means a temperature from 25 ° C. to 40 ° C. The blood sample to which the preservative of the present invention has been added is preferably stored in a temperature environment of 0 ° C. to 37 ° C., more preferably stored in a temperature environment of 0 ° C. to 20 ° C., and a temperature environment in the vicinity of 4 ° C. Even more preferred is storage.

The stored blood sample may be stored at rest or may be stored with vibration. The vibration includes rotational stirring, amplitude stirring, vertical reciprocating stirring by transportation, and irregular stirring.

When the target cells are separated, collected and detected from the stored blood sample, efficient separation and collection and clear detection of the target cells can be achieved by crushing or removing red blood cells contained in (or possibly contained in) the blood sample in advance. Good because you can. When erythrocytes are disrupted, they may be disrupted using ammonium chloride, a surfactant, a hypotonic solution, or the like. Among them, disruption using ammonium chloride is preferable because there is little damage to target cells. In addition, when removing red blood cells, the red blood cells may be removed by filtration using the difference in size between the red blood cells and the cells or by separation using the difference in specific gravity.

The target cells contained in the stored blood sample (or the blood sample obtained by crushing / removing the red blood cells described above) can be detected, for example, by applying to a slide and observing with a microscope or optical detector, or by flow cytometry. Or the like may be detected. When the target cell is detected by observation with a microscope or an optical detector, the suspension containing the cell is introduced into a cell holding means having a holding part capable of holding the cell, After holding the cells, the cells may be observed with a microscope or an optical detector. Examples of the holding part include a hole that can store the cells and a surface covered with a material (for example, poly-L-lysine) that can fix the cells. In addition, it is preferable that the size of the holding part is a size that can hold only one cell, since it is easy to collect and analyze specific cells (morphological analysis, tissue type analysis, gene analysis, etc.). In addition, when the cells are held in the holding part, it is preferable to use a dielectrophoretic force because the cells can be efficiently held in the holding part. When the dielectrophoretic force is used, specifically, the dielectrophoresis is generated by applying an alternating voltage, and the cells may be introduced into the holding portion. The AC voltage to be applied is preferably an AC voltage having a waveform in which charging and discharging of the cells in the holding unit are periodically repeated, the frequency is set between 100 kHz and 3 MHz, and the electric field strength is 1 × 10 ⁵ to 5 × 10. ^It is particularly preferable that it is between ⁵ V / m (see WO2011 / 149032 and JP2012-013549A).

Hereinafter, as an example of a method for detecting and collecting target cells contained in a blood sample using the preservative according to one embodiment of the present invention, a method for detecting and collecting tumor cells (CTC) contained in the blood sample However, the present invention is not limited to the content of the present description.

(1) Collect a blood sample from a patient suspected of having cancer. When collecting a blood sample, it is preferable to add an anticoagulant represented by a chelating agent such as citric acid or ethylenediaminetetraacetic acid (EDTA).

(2) A formaldehyde donor compound and an antiplatelet agent, which are preservatives for stabilizing CTC, are added to the blood sample (or diluted blood sample) collected in (1). In addition, when a blood sample is collected without adding an anticoagulant in (1), an anticoagulant is also added. At the time of the addition, all the substances constituting the preservative of the present invention may be added at once, or a solution containing each component may be added. Further, when polyethylene glycol is further added as a blood preservative, it is good because it contributes to the stabilization of the CTC form. Further, when a blood sample is collected in the state where the anticoagulant is added in (1) and a solution containing a formaldehyde donor compound and an antiplatelet agent is added to the blood sample in this step, the anticoagulant contained in the blood sample In order to maintain the concentration of anticoagulant, an anticoagulant may be added in this step. The amount of preservative added to the blood sample may be between 0.01 mL and 10 mL per mL of blood sample, and more preferably between 0.04 mL and 2 mL. A blood sample to which an aldehyde donor compound, an antiplatelet agent and an anticoagulant are added can be stably stored at a low temperature to a room temperature for at least 7 days.

(3) Red blood cells contained in a blood sample to which a blood preservative is added (a blood sample subjected to storage treatment) are disrupted using ammonium chloride. Red blood cell disruption with ammonium chloride is a disruption method that utilizes the difference in ion uptake between red blood cells and other cells, and is a preferred method for disrupting red blood cells because it can disrupt red blood cells while preventing damage to other cells. .

(4) After the erythrocyte crushing treatment, the blood component is removed by centrifugation, the CTC contained in the blood sample is pelletized, and then the CTC is suspended using an appropriate solution. The solution in which CTC is suspended may contain a protein to which a hydrophilic polymer is bound (for example, BSA to which polyethylene glycol is bound). The inclusion of the protein is preferable because the CTC recovery efficiency is improved. The concentration of the protein bound with the hydrophilic polymer may be between 0.01% (w / v) and 25% (w / v) as the final concentration of the protein in the suspension. % (W / v) to 5% (w / v) is preferable, and 0.05% (w / v) to 2% (w / v) is more preferable.

(5) The suspension containing CTC prepared in (4) is centrifuged again, and the pellet containing CTC is collected. If necessary, a step of suspending the collected pellet again in a solution containing a protein bound with a hydrophilic polymer and centrifuging it may be added.

(6) The cell suspension containing CTC obtained in (5) is introduced into a cell holding means having a holding part capable of holding the cells, and after holding the cells in the holding part, CTC contained in the blood sample is detected by observing with an optical detector or the like. CTC may be detected based on, for example, a difference in size and shape between CTC and bright cells (for example, blood components such as leukocytes) based on a bright field image. Cytokeratin (CK) or EpCAM (Epithelial Cell Adhesion) The cells may be stained with a labeled antibody against a protein expressed by CTC such as Molecular) and / or a labeled antibody against a protein expressed by contaminating cells (such as CD45 when the contaminating cells are leukocytes), and detection may be performed based on the staining result.

(7) Collect the CTC detected in (6) with the collecting means. An example of the collecting means is a means for collecting by suction discharge by a nozzle, and a specific example is an apparatus disclosed in Japanese Patent Application Laid-Open No. 2016-142616.

The present invention is characterized by a method for preparing a blood sample containing at least an antiplatelet agent and an anticoagulant. According to the present invention, a blood sample can be stably stored even if the prepared blood sample is stored under low temperature conditions where vibration and / or the blood sample does not freeze. In addition, the blood sample of the present invention can be stably stored even in a room temperature environment. Therefore, the method for storing a blood sample of the present invention is particularly useful when transporting a blood sample whose vibration or storage temperature environment changes. The preservation method of the present invention is also useful when the amount of target cells contained in a blood sample is very small. Furthermore, the method of the present invention is useful for detecting cells with high accuracy because it can suppress non-specific binding when cells are labeled at low temperatures.

As an example, by applying the present invention to the separation and collection of tumor cells (CTC) contained in blood, CTC can be separated and collected stably and efficiently, so that the amount of blood collected can be reduced and the burden on the patient is reduced. Can be reduced. In addition, when the diagnosis of cancer is performed based on the presence of CTC, the reliability of the determination result of the presence or absence of CTC is improved, so that cancer can be diagnosed with high accuracy.

It is a figure which shows an example of the cell holding | maintenance apparatus which can be utilized with the detection method of this invention. It is a front view of the apparatus shown in FIG.

Hereinafter, the present invention will be described in more detail using examples and comparative examples, but the present invention is not limited to these examples.

Example 1
(1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5000, one end of which is a methoxy group and the other end is an N-hydroxysuccinimide ester group, and bovine serum albumin (BSA) (300 mg, 0.3 mg). 3 mmol) was dissolved in sodium hydrogen carbonate buffer (0.1 M, 15 mL), and the solution was stirred at around 25 ° C. for 3 hours to prepare BSA to which polyethylene glycol was bound (PEG-BSA). In the preparation, the molar ratio of mPEG-NHS to BSA (mPEG-NHS / BSA) was set to 2. After the preparation, the solution was replaced with pure water for 3 days using a dialysis membrane having a molecular weight cut off of 10,000.

(2) 2.3 g of imidazolidinyl urea, 2.3 g of polyethylene glycol (PEG) having a molecular weight of 6000, and 30 mg of ethylenediaminetetraacetic acid (EDTA) were dissolved in ultrapure water so as to be 30 mL as a solution.

(3) Human lung cancer cells (PC9 cells) were cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 37 ° C. for 24 to 96 hours in a 5% CO ₂ environment, and then 0.25% Cells were detached from the medium using trypsin / 1 mM EDTA and labeled with a fluorescent dye (CFSE, manufactured by Dojindo Laboratories). The target cells were fluorescently labeled PC9 cells.

(4) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in (2) above (3) ) And about 100 PC9 cells that were fluorescently labeled and 240 μL of 0.5 mg / mL tirofiban aqueous solution were added, and the resulting solution was used as a diluted blood sample.

(5) The diluted blood sample was left at 25 ° C. or 15 ° C. for 5 days.

(6) Of the diluted blood sample after standing, 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium bicarbonate are added to the suspension corresponding to 3 mL of blood. After measuring up to 90 mL with the lysed blood, it was centrifuged at 900 × g for 10 minutes at 25 ° C. Red blood cells are destroyed by this operation, and observation of the separated and recovered PC9 cells is improved.

(7) After removing the supernatant after centrifugation, the pellet containing PC9 cells was added to PBS containing PEG-BSA (0.1% (w / v) as BSA) prepared by the method described in (1) ( Resuspended in 30 mL of phosphate buffered saline).

(8) The resuspension is centrifuged at 600 × g for 5 minutes at 25 ° C., the supernatant is removed, anti-CD45 antibody-modified magnetic particles (Dynabeads CD45, manufactured by Thermo Fisher Science) are added, and the magnet is attached. Used to remove leukocytes expressing CD45.

(9) A suspension containing PC9 cells from which leukocytes have been removed in (8) was added with PEG-BSA (0.1% (w / v) as BSA) and 280 mM sucrose prepared by the method described in (1). Resuspend in 30 mL of the containing solution.

(10) The resuspension was centrifuged at 600 × g for 5 minutes at 25 ° C., the supernatant was removed, and the pellet containing PC9 cells was again added to PEG-BSA (0.1% (w / v)) and 30 mL of a solution containing 300 mM sucrose. This operation is an operation for removing blood components including platelets and concentrating target PC9 cells.

(11) After the resuspension was centrifuged at 600 × g for 5 minutes at 25 ° C., the suspension containing PC9 cells from which the supernatant was removed was introduced into the cell holding device 100 shown in FIGS. By applying an AC voltage (1 MHz, 20 Vp-p) from the generator 50 to the

electrode substrates

31 and 32 for 3 minutes, the PC9 cells were held in the holding unit 60 of the device. The cell holding device 100 used in this example includes an insulator 12 having a plurality of through-holes 12a having a diameter of 30 μm, a light-shielding chromium film (light-shielding member 11) having a plurality of through-holes 11a having a diameter of 30 μm, and an electrode substrate 31. Are provided in close contact with each other in the order of the insulator 12-the light shielding member 11-the electrode substrate 31, and further a 1 mm thick spacer 20 having a sample inlet 21, a discharge outlet 22 and a through-hole 23 on the upper surface of the insulator 12. The electrode substrate 32 is provided in close contact with the upper surface of the spacer 20. The holding hole 60 capable of holding the cell 70 having a diameter of 30 μm and a depth of 30 μm is formed by the through holes 11a / 12a and the electrode substrate 31.

(12) The number of PC9 cells held in the holding unit 60 was measured, and the recovery rate was calculated by dividing by the number of PC9 cells added in (4).

Comparative Example 1
(1) After collecting 5 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), about 9 PC9 cells fluorescently labeled in Example 1 (3) 100 pieces were added, and the resulting solution was used as a diluted blood sample.

(2) After leaving the diluted blood sample at 25 ° C. for 2 days, the PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

Comparative Example 2
(1) After 5 mL of blood was collected from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in Example 1 (2) was collected into the blood collection tube. In addition, about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(2) The diluted blood sample was allowed to stand at 25 ° C. or 15 ° C. for 2 days, and then PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

Table 1 summarizes the results of the recovery rates in Example 1 and Comparative Examples 1 and 2. PC9 cells when using a blood sample (Example 1) added with a formaldehyde donor compound (imidazolidinyl urea), an antiplatelet agent (tirofiban) and an anticoagulant (EDTA) and stored for 5 days in the step of separating and recovering the target cell The recovery rate was 89.1% at a storage temperature of 25 ° C. and 85.4% at a storage temperature of 15 ° C., showing a high recovery rate without being affected by the storage temperature. On the other hand, the recovery rate of PC9 cells when using a blood sample (Comparative Example 1) stored at 25 ° C. with the addition of an anticoagulant and without the addition of formaldehyde donor compound and antiplatelet was only left for 2 days. Compared with Example 1 of 33.0%, it was significantly reduced. In addition, the recovery rate of PC9 cells when using a blood sample (Comparative Example 2) that was stored for 2 days with the addition of a formaldehyde donor compound and an anticoagulant but no antiplatelet agent was 76.7 at a storage temperature of 25 ° C. %, Which was higher than that of Comparative Example 1, but was hardly recovered at 1.5% at a storage temperature of 15 ° C. In Comparative Example 2, a gel-like substance is generated in the blood sample after being stored at 15 ° C. for 2 days, and cells enter the gel-like substance during erythrocyte crushing treatment or solution replacement operation with ammonium chloride. It is thought that cell recovery was hindered. On the other hand, in the blood sample (Example 1) to which a formaldehyde donor compound, an antiplatelet agent and an anticoagulant were added and stored, the generation of gel-like substances was greatly suppressed, and as a result, the recovery rate of PC9 cells was improved. It is thought that.

Example 2
(1) After collecting 4 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.6 mL of the solution prepared in Example 1 (2) into the blood collection tube About 100 PC9 cells fluorescently labeled in Example 1 (3) and 192 μL of 0.5 mg / mL tirofiban aqueous solution were added, and the resulting solution was used as a diluted blood sample.

(2) The diluted blood sample was transported back and forth over a distance of about 1000 km from Kanagawa Prefecture to Yamaguchi Prefecture. The diluted blood sample was vibrated by the transportation, and the temperature of the diluted blood sample was a minimum of 7.6 ° C., a maximum of 26.8 ° C., and an average of 14.5 ° C.

(3) After storing the diluted blood sample for 5 days including the transportation of (2), the PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

Example 3
After collecting 4 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.6 mL of the solution prepared in Example 1 (2) was collected into the blood collection tube. About 100 PC9 cells fluorescently labeled with 1 (3), 192 μL of 0.5 mg / mL tirofiban aqueous solution, and 74 μL of 3.6 mg / mL heparin PBS solution were added, and the resulting solution was used as a diluted blood sample. PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 2.

Comparative Example 3
After collecting 10 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 1.5 mL of the solution prepared in Example 1 (2) was added to the blood collection tube, and PC9 cells were separated and recovered and the recovery rate calculated in the same manner as in Example 2 except that about 100 PC9 cells fluorescently labeled in Example 1 (3) were added and the resulting solution was used as a diluted blood sample. Was done.

Table 2 summarizes the results of recoveries in Examples 2 and 3 and Comparative Example 3. PC9 cell recovery rate when using blood samples (Examples 2 and 3) stored with added formaldehyde donor compound (imidazolidinyl urea), antiplatelet agent (tirofiban) and anticoagulant in the target cell separation and recovery step 70.5% (Example 2) and 37.1% (Example 3) and blood samples stored with the addition of formaldehyde donor compound and anticoagulant but no antiplatelet agent (Comparative Example 3) Compared to when 3.6 was used (3.6%). Therefore, by adding a formaldehyde donor compound, an antiplatelet agent and an anticoagulant to the blood sample, target cells contained in the blood sample due to vibration during transport and temperature change (from 7.6 to 26.8 ° C.) It can be seen that the impact on recovery can be suppressed. In addition, in the blood sample (Example 3) to which EDTA and heparin were added as an anticoagulant, the recovery rate of PC9 cells was reduced as compared with the blood sample (Example 2) to which only EDTA was added as an anticoagulant (Example). Example 2: 70.5%, Example 3: 37.1%) Therefore, when adding an anticoagulant, it is preferable to add only a substance (chelating agent) that can chelate calcium ions in a blood sample such as EDTA. I understand that. In addition, the blood sample preserve | saved on the conditions of the comparative example 3 produced | generated many gel-like substances compared with the blood sample preserve | saved at 15 degreeC in the comparative example 2. On the other hand, in the blood samples (Examples 2 and 3) to which a formaldehyde donor compound, an antiplatelet agent and an anticoagulant were added and stored, the generation of gel-like substances was suppressed, and as a result, the recovery rate of PC9 cells was improved. It is thought that.

Example 4
(1) Dissolve 2.3 g of imidazolidinyl urea, 2.3 g of PEG with a molecular weight of 6000, 30 mg of EDTA, and tirofiban of any weight shown in the following (a) to (e) with ultrapure water so as to be 30 mL as a solution. did.
(A) 9.6 mg
(B) 4.8 mg
(C) 2.4 mg
(D) 0.12 mg
(E) 0.048 mg
(2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (e) 0.75 mL of either solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(3) The diluted blood sample was allowed to stand at 15 ° C. for 5 days, and then PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Examples 1 (6) to (12).
Comparative Example 4
PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 4 except that tirofiban was not added in Example 4 (1).

Table 3 summarizes the results of the recovery rates in Example 4 and Comparative Example 4. Increasing the amount of tirofiban from 0.048 mg to 9.6 mg improved the cancer cell recovery rate from 7.4% to 85.2%. In the blood sample after the storage treatment, the generation of gel-like substance can be confirmed when tirofiban is added in an amount of 0.048 mg to 0.12 mg, and the occurrence of a gel-like substance can be confirmed at 2.4 mg, but 4.8 mg or more of tirofiban can be confirmed. Since the formation of the gel-like substance was not confirmed at the added amount, it is considered that the formation of the gel-like substance is a factor in reducing the cancer cell recovery rate. Although the formation of a gel-like substance was observed when the amount of tirofiban added was 0.048 mg, the recovery rate of cancer cells showed a higher value compared to the case where tirofiban was not added (0%). However, it can be seen that the recovery rate of cancer cells is improved by adding tirofiban. Furthermore, when the amount of tirofiban added 4.8 mg (70.8%) in which the formation of a gel-like substance was not confirmed and the recovery rate of cancer cells of 9.6 mg were compared, the higher the amount added, the higher the recovery rate. It can be seen that tirofiban contributes to the improvement of the recovery rate in addition to the suppression of the formation of the gel-like substance.

Example 5
(1) 2.3 g of imidazolidinyl urea, 0.23 g of PEG having a molecular weight of 2000, 30 mg of EDTA, and any of the antiplatelet agents shown in the following (a) to (c) were dissolved in PBS so as to be 30 mL as a solution.
(A) Tirofiban 9.6 mg
(B) eptifibatide 4mg
(C) eptifibatide 0.4mg
(2) After 5 ml of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (c) 0.75 mL of either solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(3) The diluted blood sample was allowed to stand at 4 ° C. for 2 days, and then PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Examples 1 (6) to (12).
Comparative Example 5
PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 5 except that no antiplatelet agent was added in Example 5 (1).

Table 4 summarizes the results of the recovery rates in Example 5 and Comparative Example 5. By adding tirofiban and eptifibatide, which are antiplatelet agents, the cancer cell recovery rate was improved to about 85% as compared with the case of no addition (62.9%). This indicates that tirofiban and eptifibatide are both effective in improving the cancer cell recovery rate.

Example 6
(1) 2.3 g of imidazolidinyl urea, 0.23 g of PEG having a molecular weight of 6000, 30 mg of EDTA, 9.6 mg of tirofiban, and any amount of formalin shown in the following (a) to (c) to be 30 mL as a solution, Dissolved in ultrapure water.
(A) 56 μl
(B) 5.6 μl
(C) Not added (2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who had obtained informed consent, the blood was prepared in (1) above (a) ) To (c) 0.75 mL of any solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(3) After leaving the diluted blood sample at 4 ° C. for 2 days, the PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

The results of the recovery rate in Example 6 are shown in Table 5. The recovery rates of (a) and (b) with added formalin decreased to 17.1% and 69.1%, respectively, compared to the recovery rate (73.6%) of (c) without formalin. . From this, it can be seen that the recovery rate of cancer cells decreases when formaldehyde is added to the preservative.

Example 7
(1) 2.3 g of imidazolidinyl urea, 30 mg of EDTA, 9.6 mg of tirofiban, and any amount of PEG having a molecular weight of 6000 shown in (a) to (f) below in ultrapure water so as to be 30 mL as a solution. Dissolved.
(A) 2.3 g
(B) 1.15 g
(C) 0.575 g
(D) 0.23 g
(E) 0.023 g
(F) Not added (2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood was prepared in (1) above (a). ) To (f) 0.75 mL of any solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

Table 6 shows the results of the recovery rate in Example 7. In (a) and (b) to which PEG was added at a high concentration, the gel-like substance was formed and the cancer cell recovery rate was 10% or less. On the other hand, in (c) and (d) in which the amount of PEG added was smaller than that in (b), no gel-like substance was formed, and the cancer cell recovery rates were 66.7% and 78.6%, respectively. Furthermore, in (e) where the amount of PEG added is smaller than that in (d), the gel-like substance is not formed, but the cancer cell recovery rate is 19.9%, which is lower than in (c) and (d). In (f) in which PEG was not added, although a gel-like substance was not formed, the cancer cell recovery rate was 8.5%, which was a low value compared to (e). From this result, it can be seen that the addition of PEG is involved in the formation of a gel-like substance, and the addition of a high concentration reduces the recovery rate of cancer cells, but on the other hand, if the addition amount of PEG is small or not added, the gel It can also be seen that the recovery rate of cancer cells is reduced due to a factor different from the formation of. PEG plays the role of a cell stabilizer, and it is considered that the storage stability of the cells is increased and the cancer cell recovery rate is improved by adding a certain amount.

Example 8
(1) 2.3 g of imidazolidinyl urea, 30 mg of EDTA, 9.6 mg of tirofiban, and 2.3 g of PEG having a molecular weight of 6000 were dissolved in ultrapure water so as to be 30 mL as a solution.

(2) After collecting 5 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in (1) above, About 100 PC9 cells fluorescently labeled with 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(3) The diluted blood sample is allowed to stand at 4, 10, 25, and 37 ° C. for 5 days, and then PC9 cells are separated and recovered and the recovery rate is calculated in the same manner as in Examples 1 (6) to (12). It was.

Example 9
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 6000 0.23 g of PEG were dissolved in ultrapure water so as to be 30 mL as a solution.

(3) The diluted blood samples were allowed to stand at 4 and 25 ° C. for 2 days, respectively, and PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Examples 1 (6) to (12).

Table 7 summarizes the results of the recovery rates in Examples 8 and 9. When the PEG addition amount is 2.3 g, no gel-like substance is formed at a storage temperature of 10 to 37 ° C., and at a storage temperature of 10 ° C., the cancer cell recovery rate is 62.8%, and recovery is performed at storage temperatures of 25 ° C. and 37 ° C. It was confirmed that although it was slightly lower than the rate of 89%, it could be recovered. Gel formation was confirmed at a storage temperature of 4 ° C., and the cancer cell recovery rate was 2.3%, which was lower than the recovery rate at a storage temperature of 10 ° C. or higher. On the other hand, when the amount of PEG added was 0.23 g, the recovery rate was about 80% at a storage temperature of 4 ° C. to 25 ° C., and it was found that it can be stably recovered regardless of the temperature.

Example 10
(1) Dissolve imidazolidinylurea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and PEG 0.23 g of any molecular weight shown in (a) to (d) below in ultrapure water so that the solution is 30 mL. did.
(A) Molecular weight 2000
(B) Molecular weight 4000
(C) Molecular weight 6000
(D) Molecular weight 20000
(2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (d) 0.75 mL of either solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

The results of the recovery rate in Example 10 are shown in Table 8. As a result of lowering the molecular weight of PEG to 20000, 6000, 4000, 2000, the cancer cell recovery rates are improved to 66.0%, 73.6%, 74.5%, and 82.7%, respectively. became. The formation of a gel-like substance was not confirmed at any molecular weight, and it was considered that the recovery rate of cancer cells was improved as a result of the improvement of cell stability by PEG.

Example 11
(1) Dissolve 30 mg of EDTA, 9.6 mg of tirofiban, 0.23 g of PEG with a molecular weight of 2000, and any amount of imidazolidinyl urea shown in the following (a) to (c) in ultrapure water so that the solution becomes 30 mL. did.
(A) 1.15 g
(B) 2.3 g
(C) 4.6 g
(2) After 5 ml of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (c) 0.75 mL of either solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

Table 9 shows the results of the recovery rate in Example 11. When the amount of imidazolidinyl urea added is (a) and (b), the cancer cell recovery rate is about 60%, which is higher than the high concentration (c) cancer cell recovery rate of 18.2%. It was. From this result, it can be seen that when the amount of imidazolidinyl urea added is large, the recovery rate of cancer cells decreases.

Example 12
(1) 2.3 g of imidazolidinyl urea, 30 mg of EDTA, 9.6 mg of tirofiban, and 0.23 g of PEG having a molecular weight of 2000 were dissolved in any of the solvents shown in the following (a) to (d) so as to be 30 mL as a solution. .
(A) Ultrapure water (b) PBS
(C) PBS in which 0.9% sodium chloride (NaCl) is dissolved
(D) PBS in which 1.8% NaCl is dissolved
(2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (d) 0.75 mL of either solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

The results of the recovery rate in Example 12 are shown in Table 10. The cancer cell recovery rate was improved from 83.9% to 94.0% by changing the solvent from ultrapure water to PBS. On the other hand, when a solution obtained by further dissolving 0.9% and 1.8% NaCl in PBS was used as a preservative solvent, the cancer cell recovery was reduced to 52.8% and 9.2%, respectively. From this result, the cancer cell recovery rate is improved and the osmotic pressure is higher in (b) which is hypertonic than (a) which is close to the osmotic pressure of blood (about 280 mOsm / kg · H ₂ O). In (c) and (d), since the osmotic pressure in the cytoplasm is largely different, it is considered that the recovery rate was lowered by partially damaging the cells.

Example 13
(1) 30 mg of EDTA, 9.6 mg of tirofiban, and 0.23 g of PEG having a molecular weight of 2000 were dissolved in PBS so as to be 30 mL as a solution.

(3) The diluted blood sample was allowed to stand at 4 ° C. for 2 days, and then PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Examples 1 (6) to (12).
Comparative Example 6
PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 13, except that tirofiban was not added in Example 13 (1).

Table 11 summarizes the results of recovery rates in Example 13 and Comparative Example 6. When the formaldehyde donor was not added, it decreased to 66.4% compared with the recovery rate (94.0%) of Example 12 (b) to which the formaldehyde donor was added. This shows that the storage stability of the cells is improved by adding a formaldehyde donor. On the other hand, when no formaldehyde donor was added and no antiplatelet agent was added, the cancer cell recovery rate (66.4%) when antiplatelet agent was added was 3.2% higher. Furthermore, the formation of a gel-like substance was confirmed. From this result, it can be seen that the addition of an antiplatelet agent suppresses the formation of a gel-like substance and improves the recovery rate of cancer cells even when no formaldehyde donor is added.

Reference example 1
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg or no addition, and molecular weight 2000 PEG 0.23 g were dissolved in PBS so as to be 30 mL as a solution.
(2) After collecting 5 mL of blood from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), the blood diluted with 5 mL of PBS is 300 × g for 10 minutes at 25 ° C. Centrifuged. 1 mL of supernatant plasma was collected in an Eppendorf tube, 0.15 mL of the solution prepared in (1) was added to the Eppendorf tube, and the resulting solution was used as a diluted blood sample.
(3) After the diluted blood sample was allowed to stand at 4 ° C. for 2 days, the presence or absence of formation of a gel-like substance was observed.

The formation of a gel-like substance was not confirmed in the solution to which tirofiban was added, whereas the formation of a gel-like substance was confirmed from the solution to which tirofiban was not added. The formation of a gel-like substance was confirmed despite the fact that most of the cells and platelets were not contained in plasma, indicating that the gel-like substance is not an aggregate of cells and platelets.

Example 14
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in PBS so as to be 30 mL as a solution.

(2) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo), the blood collection tube is subjected to any of the following (a) or (b) The solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.
(A) 0.75 mL of the solution prepared in (1)
(B) A mixed solution of a solution prepared by dissolving 0.75 mL of the solution prepared in (1) and 0.575 mg of Trolox in 11.5 μL of DMSO (3) After leaving the diluted blood sample at 4 ° C. for 2 days, Example 1 (6 ) To (11), the cells contained in the suspension were held in the holding unit 60. (4) The number of PC9 cells and white blood cells held in the holding unit 60 were counted and counted. Was divided by the number of PC9 cells added in (2) to calculate the recovery rate, and the leukocyte residual rate was calculated by dividing the counted number of leukocytes by the number of leukocytes contained in the blood of (2).

The results of the recovery rate in Example 14 are shown in Table 12. When Trolox was added, the recovery rate of leukocytes decreased from 7.4% to 3.8%, compared to the non-trolox-added cancer cell recovery rate of 88% in both cases. did. A decrease in the leukocyte residual rate means that the concentration of cancer cells after blood treatment is improved. Since Trolox also has the effect of suppressing leukocyte cell death, the protein on the surface of leukocyte cell membrane is retained by improving the storage stability of leukocytes in the treatment process with magnetic particles that bind to leukocytes and remove leukocytes. Therefore, it is considered that leukocytes could be removed because the magnetic particles could be combined with leukocytes with high efficiency.

Example 15
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in ultrapure water so as to be 30 mL as a solution.

(2) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), the blood collection tube is subjected to any of the following (a) to (c) The solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.
(A) 0.75 mL of the solution prepared in (1)
(B) Mixed solution of 0.75 mL of the solution prepared in (1) and 11.5 μL of DMSO (c) Mixed solution of 0.75 mL of the solution prepared in (1) and 46 μL of DMSO (3) Diluted blood sample at 4 ° C. for 2 days After standing, PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

The results of the recovery rate in Example 15 are shown in Table 13. When DMSO was added to blood, it decreased to 6.6% in (c) compared with 62.8% in (b), compared to the cancer cell recovery rate without addition (72.8%). % And no addition were almost the same. From this result, it can be seen that adding a small amount of DMSO to blood does not affect the cancer cell recovery rate.

Example 16
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in ultrapure water so as to be 30 mL as a solution.

(2) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), the blood collection tube is subjected to any of the following (a) to (c) The solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.
(A) 0.75 mL of the solution prepared in (1) and a solution obtained by dissolving 0.144 mg of Trolox in 11.5 μL of DMSO (b) 0.75 mL of the solution prepared in (1) and 0.575 mg of Trolox Mixed solution of solution dissolved in 11.5 μL of DMSO (c) 0.75 mL of solution prepared in (1) and mixed solution of 1.15 mg of Trolox dissolved in 11.5 μL of DMSO (3) Diluted blood sample at 4 ° C. After leaving for 2 days, PC9 cells were separated and recovered and the recovery rate was calculated in the same manner as in Example 1 (6) to (12).

Table 14 shows the results of the recovery rates in Examples 15 (a) and 16. Trolox was added in the range of 0.144 mg to 1.15 mg, but the cancer cell recovery rate was 69.1% to 76.7% compared with the case without addition (72.8%). I couldn't. This result shows that the addition of Trolox does not affect the cancer cell recovery rate.

Example 17
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in ultrapure water so as to be 30 mL as a solution.

(2) After collecting 5 mL of blood from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in (1) and ascorbine are collected into the blood collection tube. A mixed solution of a solution obtained by dissolving 0.1 mg of acid in 10 μL of ultrapure water and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

When ascorbic acid as an antioxidant was added, the cancer cell recovery rate was 12.9%, compared with the result (72.8%) in which the antioxidant of Example 15 (a) was not added, Decreased significantly. From this result, it can be seen that among the antioxidants, ascorbic acid is not suitable as a blood preservative because it causes a decrease in the recovery rate of cancer cells.

Example 18
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in PBS so as to be 30 mL as a solution.

(2) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), the blood collection tube is subjected to any of the following (a) to (c) The solution and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.
(A) 0.75 mL of the solution prepared in (1) (blood preservative)
(B) 0.75 mL of the solution prepared in (1) and 0.75 mL of the solution prepared in solution (c) (1) within 10 minutes after mixing 0.75 mL of the solution prepared in (1) and 0.575 mg of Trolox in 11.5 μL of DMSO A solution of 0.575 mg of Rox dissolved in 11.5 μL of DMSO for 4 weeks after mixing. (3) After leaving the diluted blood sample at 4 ° C. for 2 days, the same method as in Example 1 (6) to (11), (4) The number of PC9 cells and leukocytes held in the holding unit 60 were counted, and the counted number of PC9 cells was the number of PC9 cells added in (2). Then, the recovery rate was calculated, and the leukocyte residual rate was calculated by dividing the counted leukocyte count by the leukocyte count contained in the blood of (2).

The results of the recovery rate in Example 18 are shown in Table 15. The cancer cell recovery rate and leukocyte residual rate when using a solution that had passed 4 weeks after adding Trolox to the blood preservative were 90% higher than when using the solution within 10 minutes. The residual rate was 2.6 to 2.9%, showing almost no change. On the other hand, compared with the condition without Trolox addition, there is no change in the cancer cell recovery rate, but the leukocyte residual rate has decreased from 5.4% in the non-addition condition. It can be seen that the effect of improving leukocyte removal efficiency by the blood pretreatment is maintained even after a long time has elapsed since the addition of Trolox.

Example 19
(1) Human lung cancer cells (PC9 cells) are cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) at 37 ° C. for 24 to 96 hours in a 5% CO ₂ environment, and then 0.25% Cells were detached from the culture medium using trypsin / 1 mM EDTA. The peeled PC9 cells were used as target cells.

(2) 2.3 g of imidazolidinyl urea, 30 mg of EDTA, 9.6 mg of tirofiban, and 0.23 g of PEG with a molecular weight of 2000 were dissolved in PBS so as to be 30 mL as a solution.

(3) After 5 ml of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in (2) and Toro are collected into the blood collection tube. A mixed solution of 0.575 mg of Rox dissolved in 11.5 μL of DMSO and about 100 PC9 cells detached in (1) were added, and the resulting solution was used as a diluted blood sample.

(4) After the diluted blood sample was allowed to stand at 4 ° C. for 7 days, the cells contained in the suspension were held in the holding unit 60 in the same manner as in Example 1 (6) to (11) (5) While applying an AC voltage under the conditions of Example 1 (11), a 300 mM mannitol aqueous solution containing 0.01 (w / v)% poly-L-lysine was introduced, allowed to stand for 3 minutes, and then the AC voltage was applied. Was stopped and the aqueous solution was removed by suction.
(6) An aqueous solution containing 50% (v / v) ethanol and 1% (w / v) formaldehyde (hereinafter referred to as “cell membrane permeation reagent”) is introduced and allowed to stand for 10 minutes to permeate the cell membrane. The cells containing CTC in the holding part were sampled.
(7) The cell membrane permeation reagent was removed by suction, and PBS was introduced to wash the remaining cell membrane permeation reagent.
(8) Introduction of an aqueous solution (hereinafter referred to as a labeling reagent) containing a fluorescently labeled antibody capable of specifically binding to proteins inside and outside the cell membrane and a fluorescent reagent (DAPI: 4 ′, 6-diamidino-2-phenylindole) for labeling the cell nucleus And left to stand for 30 minutes. As the labeled antibody, an antibody against CD45 expressed on the surface of leukocytes and an antibody against cytokeratin (CK) expressed in the cytoplasm of PC9 cells are used.
(9) The labeling reagent was removed by suction, and the remaining labeling reagent was removed by introducing PBS.
(10) After placing the cell holding means containing the PC9 cells labeled in (9) on the stage of the fluorescence microscope, the whole holding part is imaged in order to observe all the cells captured in the plurality of holding holes. It was. A fluorescent microscope (IX83; manufactured by Olympus) equipped with a computer-controlled electric stage and a CMOS camera (ORCA-Flash 4.0; manufactured by Hamamatsu Photonics) was used. LabVIEW (manufactured by National Instruments) was used as image acquisition and analysis software.
(11) Among the cells imaged in (10), the cells are stained with DAPI indicating that they have a cell nucleus (DAPI positive), and are not stained with an antibody against CD45 expressed in leukocytes (CD45 negative) ), Cells stained with an antibody against CK (CK positive) were detected as target cells (PC9 cells).

(12) The number of detected PC9 cells was counted, and the detection rate was calculated by dividing by the number of PC9 cells added in (3).
The detection rate of PC9 cells was 87.9%, and no change was seen even when compared with the recovery rate of 90.3% of PC9 cells in Example 18 (b), which was not labeled with an antibody. This result shows that the antigen expressed in PC9 cells is stably retained and preserved even after 7 days of storage, and it can be seen that the target cells can be labeled with high precision even when labeling with an antibody or the like. .
Example 20
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 9.6 mg, and molecular weight 2000 PEG 0.23 g were dissolved in PBS so as to be 30 mL as a solution.

(2) After 5 mL of blood is collected from a healthy person who has obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), 0.75 mL of the solution prepared in (1) and Toro are collected into the blood collection tube. A mixed solution of 0.575 mg of Rox dissolved in 11.5 μL of DMSO and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution was used as a diluted blood sample.

(3) The diluted blood sample is allowed to stand at ice-cooled (0 ° C.), 4, 25, and 37 ° C. for 2 days, and then separated and collected and recovered by the same method as in Examples 1 (6) to (12). The rate was calculated.

The results of the recovery rate in Example 20 are shown in Table 16. No gel-like substance is formed at a storage temperature of about 0 to 37 ° C., and the cancer cell recovery rate is 72.6% to 80.5%, indicating that it can be stably recovered regardless of the storage temperature.

Example 21
(1) PC9 cells were not added in Example 19 (3), and the storage conditions of Example 19 (4) were left at ice-cooled (0 ° C.), 4, 15, 20, 25, and 37 ° C. for 2 days. Otherwise, the labeled cells were imaged in the same manner as in Example 19 (2) to (10).

(2) The cells imaged in (1) are stained with DAPI indicating that they have cell nuclei (DAPI positive) and stained with an antibody against CD45 expressed in leukocytes (CD45 positive) ) Cells were detected as white blood cells.

(3) For the detected leukocytes, the degree of labeling to the leukocytes due to non-specific adsorption of the CK antibody is detected, and the luminance obtained by the fluorescent dye modified with the CK antibody is detected in 256 gradations, and about 300 Average CK antibody brightness was determined for individual leukocytes.

The results of the recovery rate in Example 21 are shown in Table 17. The luminance due to CK antibody binding to leukocytes is about 10 at a storage temperature of about 0 ° C. to 4 ° C., whereas the luminance is about 13 at 10 ° C. to 20 ° C. and the luminance at 25 ° C. to 37 ° C. As a result, the binding of CK antibody to leukocytes increased with increasing storage temperature. Since leukocytes do not express CK antigen, an increase in the binding amount of CK antibody to leukocytes is an increase in the non-specific adsorption amount of the antibody. Specific and nonspecific binding of antibodies, as non-specific adsorption to cells can be reduced at storage temperatures (<25 ° C) lower than typical laboratory temperatures (about 25 ° C). And the detection accuracy of specific cells can be improved.

Example 22
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 19.2 mg, and molecular weight 2000 PEG 0.23 g were dissolved in PBS so as to be 30 mL as a solution.

The cancer cell recovery rate of Example 22 to which 19.2 mg of tirofiban was added was 90.7%, compared with the cancer cell recovery rate (90.3%) of Example 18 (b) to which 9.6 mg of tirofiban was added. As a result, no gel-like substance was formed. From this result, it can be seen that the addition of a high concentration of tirofiban does not affect the cancer cell recovery rate and the formation of a gel-like substance.
Example 23
(1) imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 19.2 mg, and PEG 0.23 g or (d) ethylene glycol 0.23 g of any molecular weight shown in the following (a) to (c) as a solution in 30 mL So that it was dissolved in PBS.
(A) Molecular weight 1540
(B) Molecular weight 600
(C) Molecular weight 200
(2) After 5 mL of blood was collected from an EDTA-2K blood collection tube (VP-DK050K, Terumo) from a healthy person who obtained informed consent, the blood collection tube was prepared in (1) (a) to (d) A mixed solution of 0.75 mL of any solution and 0.575 mg of Trolox dissolved in 11.5 μL of DMSO and about 100 PC9 cells fluorescently labeled in Example 1 (3) were added, and the resulting solution Was used as a diluted blood sample.

The results of the recovery rate in Example 23 are shown in Table 18. By changing the molecular weight of PEG to 1540, 600, 200 and ethylene glycol and additives, the cancer cell recovery rates were 92.0%, 90.3%, 53.8%, and 46.0%, respectively. Compared with the cancer cell recovery rate (90.3%) under the condition where molecular weight of PEG of 18 (b) was added, molecular weight of 1540 and 600 of PEG did not change. On the other hand, a decrease in the cancer cell recovery rate was confirmed with a molecular weight of PEG of 200 and ethylene glycol. PEG is added to improve the stability of the cells. However, when the molecular weight of PEG is less than 600 and ethylene glycol reduces the effect of maintaining the stability, it is considered that the cancer cell recovery rate has decreased.

Example 24
(1) Imidazolidinyl urea 2.3 g, EDTA 30 mg, tirofiban 19.2 mg, molecular weight 2000 PEG 0.23 g, and peptide 13.3 mg containing the following RGD sequence were dissolved in PBS so as to be 30 mL as a solution.
(A) Peptide of GRGDNP sequence (b) Cilengitide which is a peptide of cyclic RGD sequence
(C) Not added (2) After collecting 5 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, Terumo), the solution prepared in (1) above was collected into the blood collection tube 0 A mixed solution of .75 mL and 0.575 mg of Trolox dissolved in 11.5 μL of DMSO was added, and the resulting solution was used as a diluted blood sample.

(3) The diluted blood sample was allowed to stand at 4 ° C. for 2 days, and after confirming the fluidity of the blood sample, hemolysis was performed in the same manner as in Example 1 (6) to confirm the presence or absence of gel formation.

As a result of performing Example 24, the fluidity of the blood sample decreased and the increase in viscosity was confirmed in (a) and (b) in which the RGD peptide was added compared to (c) in which no RGD peptide was added. It was. On the other hand, gel formation after hemolysis was not confirmed under any of the conditions (a) to (c). From this result, it can be seen that the addition of the RGD peptide inhibits the function of the integrin capable of binding to the peptide and changes the physical properties of the blood sample.

Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention.

It should be noted that the entire content of the specification, claims, drawings and abstract of Japanese Patent Application No. 2018-102432 filed on May 29, 2018 is cited here as the disclosure of the specification of the present invention. Incorporated.

DESCRIPTION OF SYMBOLS 100: Cell holding | maintenance apparatus 11: Light-shielding member 12: Insulator 11a * 12a: Through-hole 20: Spacer 21: Inlet 22: Outlet 23: Through-hole 31/32: Electrode substrate 40: Conductor 50: Signal generator 60: Holding unit 70: cell

Claims

A method for preparing a blood sample comprising a target cell, an antiplatelet agent and an anticoagulant.
The method of claim 1 further comprising a formaldehyde donor compound.
The method according to claim 1, comprising a step of adding the antiplatelet agent and the anticoagulant to the blood sample and then storing at a low temperature.
The method according to any one of claims 1 to 3, further comprising the step of adding a hydrophilic polymer compound to the blood sample.
The method according to any one of claims 1 to 4, further comprising the step of adding a water-soluble vitamin E analog to a blood sample.
The method according to any one of claims 1 to 5, wherein the anticoagulant is a chelating agent.
The method according to any one of claims 1 to 6, wherein the antiplatelet agent is an inhibitor against platelet GPIIb / IIIa receptor.
Formaldehyde donor compounds are imidazolidinyl urea, benzyl hemiformal (phenylmethoxymethanol), 5-bromo-5-nitro-1,3-dioxane, bronopol (2-bromo-2-nitropropyne-1,3-diol), Diazolidinyl urea, DMDM hydantoin (1,3-dimethylol-5,5-dimethylhydantoin), mesenamine (hexamethylenetetramine), quaternium-15 (mesenamine 3-chloroallylochloride), sodium hydroxymethylglycine, methylol of amines and amides The method according to any one of claims 1 to 7, which is one or more compounds selected from hydroxymethyl derivatives, methylol, and metheneamine.
The method according to any one of claims 1 to 8, wherein the hydrophilic polymer compound is polyethylene glycol.
The method according to claim 3, wherein the low temperature is 0 ° C or higher and lower than 25 ° C.
A method for detecting a target cell contained in a blood sample, comprising the following steps (1) to (3):
(1) a step of preparing a blood sample by the method according to any one of claims 1 to 10,
(2) a step of crushing or removing red blood cells contained in the obtained blood sample,
(3) Detecting the target cell from the blood sample after performing the step (2)
A method for collecting a target cell contained in a blood sample, wherein the target cell is detected by the method according to claim 11 and then the detected cell is collected.
A blood sample comprising a target cell, an antiplatelet agent, and an anticoagulant.
14. The blood sample of claim 13, further comprising a formaldehyde donor compound.
A blood sample preservative for cryopreservation, comprising an antiplatelet agent and an anticoagulant.
The blood sample preservative of claim 15, further comprising a formaldehyde donor compound.