JP2008185564A - Blood coagulation test method and blood coagulation test device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily evaluating blood coagulation ability with a small amount of blood, and to provide a device for the method. <P>SOLUTION: The inspection method and device for blood coagulation ability are characterized by allowing invasion of blood by capillary phenomenon into the channel of microchips set on a board, and inspecting blood coagulation ability based on the invasion speed or the invasion distance of the blood in the channel. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a blood clotting ability testing method and a blood clotting ability testing apparatus for simply testing blood clotting ability, and more specifically, blood is allowed to enter a flow path provided in a microchip by capillary action. The present invention also relates to a method and an apparatus for simply examining blood coagulation ability based on the penetration speed or the penetration distance.

In order to examine blood coagulation abnormalities such as thrombotic diseases such as arteriosclerosis and hemophilia, blood coagulation ability is examined. As a method for examining the coagulation ability of blood, coagulation ability tests such as partially activated thromboplastin time, thromboplastin time, whole blood coagulation time, and thromboelastograph are performed.
However, these methods require a large amount of blood, and are expensive devices or methods that require human procedures to be examined. Blood is simpler, simpler, more expensive, and does not require expensive devices. There has been a demand for a method for examining the coagulation ability of the sucrose.

On the other hand, as a method for easily examining blood properties with a small amount of blood, blood collected from a blood inlet of a channel provided on a microchip is injected, and blood is measured from the channel by capillary action. The blood component is analyzed by reacting with a reagent for testing.
For example, Patent Document 1 proposes a method and apparatus for measuring blood glucose level by oxidizing glucose in blood using an enzyme such as glucose oxidase and oxygen in the air and measuring the hydrogen peroxide produced. Has been.

Patent Document 2 proposes a blood cell diagnostic system that measures the viscosity of blood such as mushy and smooth blood by a simple means.
However, these methods are methods for analyzing components in blood, and it has not been possible to test blood coagulation ability more simply with a simpler index.
Japanese Patent Laid-Open No. 8-294439 JP 2005-164296 A

  The present invention has been made in view of the above circumstances, and provides a method and apparatus for measuring blood coagulation ability more easily with less blood without requiring an expensive apparatus or a human procedure for testing. The issue is to provide.

In order to solve the above problems, the inventor of the present invention has earnestly studied and, as a result, obtained the knowledge that a microchip can be used to easily perform a test with a very small amount of blood. It was made.
That is, the present invention is a method for inspecting blood properties, for example, a microchip (hereinafter referred to as a microchip) in which a channel is provided by means such as forming a groove in a synthetic resin substrate and covering it with a flat lid. In some cases, blood is infiltrated into the flow channel by capillary action, and the blood coagulation ability is inspected based on the blood penetration speed or the penetration distance in the flow channel. Provide a method.
In the method for examining blood coagulation ability of the present invention, it is preferable to measure blood coagulation ability by infiltrating the anticoagulated blood into the flow channel after releasing or releasing the anticoagulation treatment. In addition, it is preferable to mix an agent that releases the anticoagulation treatment with an anticoagulation treatment agent and an agent that activates an intrinsic or extrinsic coagulation cascade and promotes blood coagulation, and enters the flow path.
Here, the anticoagulation treatment is preferably a treatment with citric acid, and it is preferable to release the anticoagulation treatment of the blood anticoagulated with citric acid by adding calcium. The anticoagulation treatment is preferably a treatment with heparin, and it is preferable to add heparinase to cancel the anticoagulation treatment of the blood anticoagulated with heparin.
And it is preferable to perform this test with the blood temperature kept constant.

In addition, the present invention allows blood to enter a flow path of a microchip having a flow path on a substrate by capillary action, and the blood coagulation ability based on the blood penetration speed or the penetration distance in the flow path. A test apparatus for blood coagulation, characterized in that a material that activates an intrinsic or extrinsic coagulation cascade to promote blood coagulation is used in at least a part of the flow path of the microchip. Providing equipment.
Here, the material that activates the intrinsic or extrinsic coagulation cascade to promote blood coagulation is preferably a material coated with glass or collagen.
In the blood coagulation ability test apparatus of the present invention, the flow path is preferably installed so as to meander, and an index indicating the evaluation of blood coagulation ability is preferably attached to the microchip. And it is preferable to have a heater for keeping the temperature of the blood to be tested constant.

According to the blood coagulation ability test method of the present invention, blood is infiltrated into the flow path of the microchip provided with the flow path on the substrate by capillary action, and based on the blood infiltration speed or the infiltration distance in the flow path. Since blood coagulation ability can be examined, blood coagulation ability can be easily examined with a very small amount of blood, reducing the burden on the subject and reducing the subject's thrombotic disease and abnormal blood coagulation. The risk / presence and therapeutic effects of anticoagulants can be examined. In some cases, the test can be performed by the subject himself / herself.
In the blood coagulation ability test method of the present invention, after anticoagulation treatment is released, or after the anticoagulation treatment is released, the blood coagulation ability is measured by entering the flow path while releasing the anticoagulation treatment. The coagulation ability can be measured accurately even if it is not. Furthermore, if the anticoagulant-treated blood is mixed with an agent that releases the anticoagulant treatment and an agent that promotes blood coagulation by activating the intrinsic or extrinsic coagulation cascade and entering the flow path, the measurement time of the coagulation ability Can be shortened.
Here, when measuring the coagulation ability of blood subjected to anticoagulation treatment, it is preferable that citric acid and calcium are easily obtained if the blood is anticoagulated with citric acid or the anticoagulation treatment is canceled with calcium. Further, when blood is anticoagulated with heparin or anticoagulant is released with heparinase, a test reflecting the blood's original calcium concentration becomes possible.
When the test is performed with the blood temperature kept constant, the test can be performed more accurately.

The blood coagulation ability test apparatus of the present invention allows blood to enter the flow path of the microchip provided with the flow path on the substrate by capillary action, and the blood intrusion speed or infiltration distance in the flow path. Therefore, the blood coagulation ability can be easily examined with a very small amount of blood.
In the blood coagulation ability testing apparatus of the present invention, a material that activates the intrinsic or extrinsic coagulation cascade and promotes blood coagulation is used in at least a part of the flow path of the microchip. It is possible to shorten the measurement time of coagulation ability without mixing the reagent to be activated, and it is easy to obtain if the material that promotes blood coagulation is a material coated with glass or collagen.
Here, in the apparatus of the present invention, if the flow path is installed so as to meander, the apparatus can be further downsized.
Further, in the apparatus of the present invention, when an index indicating evaluation of blood properties is attached to the microchip, blood coagulation ability can be more easily evaluated.
Furthermore, when the apparatus of the present invention has a heater for keeping the temperature of blood to be inspected constant, more accurate inspection is possible.

Hereinafter, based on the best mode, a blood coagulation ability test method and test apparatus of the present invention will be described with reference to the drawings.
FIG. 1 is a conceptual diagram showing a first embodiment of the blood coagulation ability testing apparatus of the present invention.

As shown in FIG. 1, the blood coagulation ability testing apparatus 1 according to this embodiment includes a microchip including a blood inlet 10, a channel 11, an outlet 12, and a display unit 13.
When blood is injected into the blood inlet 10, blood enters the flow path 11 by capillary action, and the blood coagulation ability can be measured by measuring the penetration speed or the penetration distance at this time.

  The material that forms the microchip 1 may be any material that allows blood to enter the flow path 11 by capillary action and pass through the flow path 11. Such a material is preferably a hydrophilic material. Further, such a material is preferably a synthetic resin that can be molded at high speed and low cost by injection molding or the like. As such a synthetic resin, specifically, a polypropylene resin described in JP-A-2006-183060, a general formula XY (X: a polymer block incompatible with the polypropylene resin, Y: conjugate) And a hydrogenated derivative of a block copolymer represented by a diene elastomeric polymer block). Particularly preferred is a resin composition obtained by adding a hydrogenated polystyrene / vinyl-polyisoprene / polystyrene block copolymer to polypropylene (homopolymer or random copolymer). Even for hydrophobic resins, it is coated with a hydrophilic coating material that has anti-thrombogenicity or blood compatibility, such as surface treatment such as plasma, polyvinyl lactone amide (PVLA), or poly-2-methoxyethyl acrylate (PMEA). By making it hydrophilic, it can be used.

The cross section of the flow path 11 is not particularly limited, and the width and diameter of the flow path 11 may be any size as long as blood can pass through the flow path 11 by capillary action, but if it is too thin, blood cells are clogged and are too thick. 10 to 500 μm is preferable, and 50 to 300 μm is more preferable, because it is difficult to make a difference when measuring the infiltration rate and the infiltration distance and evaluation of blood coagulation ability may be difficult. Similarly, the depth of the flow path 11 is preferably 10 to 500 μm, and more preferably 50 to 300 μm.
The shape of the flow path 11 may be linear or branched. Moreover, the shape which meanders as FIG. 1 shows from a viewpoint of lengthening the flow path 11 and achieving size reduction of the chip | tip 1 may be sufficient. Alternatively, it may be shaped like a record groove.
The inlet 10, the outlet 12, or the anticoagulant releasing agent addition port, which will be described later, may be a cross section of the channel 11 exposed on the end face of the chip 1, but as shown in FIG. It is preferable to have a structure having a dish-shaped recess that can open a hole larger than the cross section of 11 and temporarily store blood. In this case, it is preferable that a mortar-shaped depression is provided on the substrate so that blood can easily enter the flow path 11.
The flow path 11, blood inlet 10, outlet 12 and the like can be installed on the chip 1 according to a known method such as resin molding such as injection molding or excavation by laser light. When producing the same pattern in large quantities, resin molding such as injection molding is preferred.

In the present invention, the blood penetration distance means the penetration distance from the inlet 10 to the stop position when the blood stops in the flow path without reaching the outlet 12, and the blood penetration speed is Including the case where the blood reaches the discharge port 12 and the case where the blood does not reach, it means the amount of infiltration per unit time. The method and apparatus of the present invention evaluate blood coagulation ability based on these penetration distances and penetration speeds.
In the blood coagulation ability test apparatus of the present invention, at least a part of the chip 1 is preferably made of a transparent material so that the blood penetration speed or penetration distance can be visually confirmed and evaluated.
In the present invention, the blood coagulation ability is examined by utilizing the property that the flow path is narrowed in accordance with the aggregation of the invaded blood due to the activation of blood cells and the infiltration of blood is delayed. Therefore, it can be determined that the blood coagulation ability is high when the penetration speed is slow or the penetration distance is short. Conversely, when the infiltration rate is fast or the infiltration distance is long, it can be determined that the blood coagulation ability is low. By measuring the coagulation ability of blood, it is possible to examine a subject's risk of thrombotic diseases such as arteriosclerosis and the risk / presence of abnormal blood coagulation such as hemophilia, and the therapeutic effect of an anticoagulant.

In addition, in order to determine more simply, as shown in FIG. 1, an indicator 13 for visually determining blood coagulation ability may be provided on the chip 1.
Specifically, as the index 13, for example, a scale can be provided on the chip 1, and based on the scale, it can be determined that the coagulation ability is normal, needs attention, or is abnormal.
Alternatively, a mark such as “Normal”, “Caution required”, or “Abnormal” may be directly attached to the chip, or marks such as “O”, “Δ”, “X” may be added as shown in FIG. , ○ may be judged as normal, Δ as cautionary, and x as abnormal.

A very small amount of blood may be injected into the blood inlet 10, for example, 10 to 500 μl, preferably 50 to 100 μl. Whole blood, platelet-rich plasma, plasma or the like can be used as blood to be injected.
When measuring the coagulation ability of blood immediately after blood collection, it is not particularly necessary to carry out anticoagulation treatment. However, if it is necessary to take a period of time after blood collection until it is used for examination, it is preferable to subject the blood to anticoagulation treatment in advance. When examining the coagulation ability of the anticoagulated blood, immediately before the blood is injected into the blood inlet 10, the anticoagulant is mixed with the anticoagulated blood to release the anticoagulant, and immediately after that By injecting into the injection port 10 and entering the flow channel 11, the coagulation ability can be examined. Also in this case, it can be determined that the coagulation ability is high when the penetration distance is short, and conversely, it can be determined that the coagulation ability is low when the penetration distance is long. Also in this case, it is possible to attach the index 13 to the chip 1 and make a determination based on the index.
By measuring the coagulation ability of blood, it is possible to examine a subject's thrombotic disease, blood coagulation abnormality, therapeutic effect by an anticoagulant, and the like.

Anticoagulant used for anticoagulation treatment includes citrate such as sodium or potassium citrate, oxalate such as sodium or potassium oxalate, ACD (Acid Citrate Dextrose), ethylenediaminetetraacetic acid (EDTA) salt, etc. Can be mentioned. Such an anticoagulant can be used as a solution such as a powder, a lyophilized product, or an aqueous solution. Among these, the existing 3.2% sodium citrate aqueous solution is preferable because it can be easily obtained. In this case, it is preferable to use 1 volume of anticoagulant for 9 volumes of blood.
In order to release the anticoagulation treatment by the anticoagulation treatment agent, an anticoagulation release agent such as calcium is added. In this case, an anticoagulant releasing agent is mixed with the anticoagulated blood immediately before being injected into the blood injection port 10 to release the anticoagulant treatment, and then enter the flow path 11. Alternatively, the anticoagulant releasing agent may be supplied from the anticoagulant releasing agent addition port 14 on the chip 1 using the chip 1 shown in FIG.

By the way, when calcium is used as an anticoagulant, it is impossible to perform a test reflecting the blood calcium concentration. Therefore, when a test reflecting the calcium concentration inherent in blood is performed, it is preferable to use an anticoagulant other than calcium. Examples of such anticoagulant include heparin, hirudin, hirulog (hirudin C-terminal region peptide), aprotinin, antithrombin antibody, thrombin aptamer, corn-derived trypsin inhibitor (1977. J. Biol. Chem 252.8105) and the like. Can be used. These inhibit the coagulation cascade by inhibiting blood coagulation factors and inhibit blood coagulation. Of these, heparin is preferred. When releasing the anticoagulation treatment with heparin, it can be released with heparinase or the like. Also in this case, an anticoagulant releasing agent is mixed with the anticoagulated blood immediately before being injected into the blood inlet 10 to release the anticoagulant treatment, and thereafter enter the flow path 11. Alternatively, the anticoagulant releasing agent may be supplied from the anticoagulant releasing agent addition port 14 on the chip 1 using the chip 1 shown in FIG.

  As a method for obtaining anticoagulated blood, anticoagulation is performed by a method in which an anticoagulant is added in advance to a syringe or a vacuum blood collection tube, or a method in which an anticoagulant is rapidly added to blood immediately after blood collection. Blood can be obtained. Further, after collecting blood with a vacuum blood collection tube containing heparin, an anticoagulant such as heparinase may be added to decompose heparin and replace with another anticoagulant. According to this method, blood subjected to anticoagulation treatment can be collected by freely selecting an anticoagulation treatment agent in accordance with the purpose of examining the coagulation ability of blood.

When examining the blood coagulation ability, an agent that activates the intrinsic blood coagulation cascade or the extrinsic blood coagulation cascade may be added to the blood in order to shorten the examination time.
In this case, it is preferable to use ellagic acid for activation of the intrinsic blood coagulation cascade and tissue thromboplastin for activation of the extrinsic coagulation cascade.

In particular, the blood coagulation ability test apparatus of the present invention uses a material that activates an intrinsic or extrinsic coagulation cascade in at least a part of the flow path 11 of the microchip 1 to promote blood coagulation. Preferably, at least a part of the chip 1, for example, a part in which a groove of the substrate is formed or a material in which a lid is coated with glass or collagen is used. Thereby, it is possible to shorten the measurement time of the coagulation ability without mixing a drug that activates blood coagulation.
Specifically, for example, the lid of the chip 1 is formed of a material such as glass or glass coated with collagen or plastic coated with collagen. Alternatively, the groove of the substrate of the chip 1 is formed of a material such as plastic coated with collagen. And the chip | tip 1 used as the flow path 11 is obtained by joining the lid | cover and a board | substrate and using the raw material which coated glass or collagen to at least one part.

  When the flow path 11 is formed using glass, an effect close to that obtained when an agent that activates intrinsic blood coagulation such as ellagic acid is mixed by blood flowing while contacting the glass is obtained. When the flow path 11 is formed using a collagen-coated material, platelets and erythrocytes adhere to collagen in a short time and are activated, and blood clotting is caused on the cell surface by a phospholipid membrane or tissue factor. Promoted.

  In addition, blood that has been anticoagulated with citric acid or heparin is allowed to enter the chip 1 in which the channel 11 is formed using collagen-coated material or glass, without releasing the anticoagulation treatment, so that platelets and leukocytes It is possible to inspect the adhesive function. Also in this case, the flow path 11 is narrowed in accordance with the aggregation due to the activation of blood cells, and the blood infiltration is delayed. Therefore, it is possible to inspect using the blood infiltration speed as an index.

In addition, in order to test more accurately, it is preferable to measure the temperature of the blood to be tested at a constant temperature, and in order to perform the measurement under conditions closer to the living body, the blood is kept at 30 to 40 ° C, particularly preferably 37 ° C. It is preferable to measure in the state kept. In order to perform measurement while keeping the temperature of blood to be inspected constant, the whole blood coagulation ability inspection apparatus including ancillary equipment may be warmed up and measured, or only the microchip 1 may be warmed up. Specifically, a mode in which the microchip 1 is placed in a thermostat and measurement, a mode in which the microchip 1 is placed on a thermostat plate, and the like are exemplified.

FIG. 2 shows a second embodiment of the method and apparatus for blood coagulation test of the present invention.
In this embodiment, the flow path 11 is formed in a Y shape, and the anticoagulated blood is injected into the blood inlet 10. On the other hand, an anticoagulant releasing agent is added to the anticoagulant releasing agent addition port 14 and the blood coagulating ability is measured by allowing the anticoagulant releasing agent to enter the channel 11 while releasing the anticoagulant treatment of blood.
Also in this case, the above-mentioned anticoagulant and anticoagulant releasing agent can be used. Also in this case, it can be determined that the coagulation ability is high when the penetration distance is short, and conversely, it can be determined that the coagulation ability is low when the penetration distance is long. Also in this case, an index can be attached to the chip 1 and determination can be made based on the index.
By measuring the coagulation ability of blood, it is possible to examine a subject's thrombotic disease, blood coagulation abnormality, therapeutic effect by an anticoagulant, and the like.

In addition, an antithrombotic agent is simultaneously added to the anticoagulant releasing agent addition port 14 together with an anticoagulant releasing agent to activate an intrinsic or extrinsic coagulation cascade to promote blood coagulation, thereby evaluating the efficacy of the antithrombotic agent. It can also be done.
In FIG. 2, the flow path is formed in a Y-shape, and the blood flow path after mixing with the anticoagulant release agent is a straight line. However, the flow path 11 in this portion is meandered as in FIG. May be.
In addition, although the display part is abbreviate | omitted in FIG. 2, it cannot be overemphasized that a display part may be provided also in this aspect.

  Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Example 1]
6 μl of the following blood coagulation initiator A as a blood coagulation release agent was added to 300 μl of whole blood anticoagulated with a 3.2% sodium citrate aqueous solution so that the volume was 10% by volume with respect to 90% by volume of whole blood. Thirty seconds after the addition of the coagulation release agent, 50 μl was injected into a microchip natural flow type II (product name) manufactured by Ritchell with a flow path shown in FIG. The arrival time to each point (index) is shown in A column of Table 1 in seconds.
Blood coagulation initiator A; solution obtained by mixing 60 μl of 2M calcium chloride solution with 100 μl of in-tem reagent (Pentapharm; composition ellagic acid + rabbit brain-derived phospholipid)

[Example 2]
The same test as in Example 1 was performed, except that palin was added to the unfractionated 0.5 units / ml of the anticoagulated whole blood used in Example 1, and then coagulation initiator A was added. . The penetration time to each point is shown in column B of Table 1 in seconds.

  From Table 1, it can be seen that the blood (B) of Example 2, which was anticoagulated with heparin and suppressed coagulation ability, had a shorter infiltration time and a low coagulation ability because the time to reach point 8 was shorter. . For example, if the test is stopped in 5 minutes, the blood (A) of Example 1 reaches only between points 6 and 7, whereas the blood (B) of Example 2 is between points 7 and 8. It is possible to visually observe the state reached. In this way, it can be seen that the blood (B) of Example 2 has a lower coagulation ability without using a measuring jig.

  As described above, the present invention has been described based on the preferred embodiments. However, the present invention is not limited to the above-described embodiments and embodiments, and various modifications can be made without departing from the scope of the present invention. It is. For example, the blood coagulation ability measuring apparatus of the present invention comprises a microchip, but other accessories such as jigs and apparatuses for assisting blood and various drug injection and blood discharge, or the infiltration speed and infiltration distance. It may be provided with a jig, a device, or the like for assisting measurement.

  The apparatus and method of the present invention can easily measure the blood coagulation ability of a subject, and can confirm the risk and presence or absence of a thrombotic disease or blood coagulation abnormality in a subject, as well as the therapeutic effect of an anticoagulant. It can be suitably used for simple evaluation.

It is a conceptual diagram which shows the coagulation ability test | inspection apparatus of the blood which concerns on the 1st form example of this invention. It is a conceptual diagram which shows the blood coagulation ability test | inspection apparatus based on the 2nd form example of this invention. It is the figure which simplified and showed the flow path of the coagulation ability test | inspection apparatus of the blood which concerns on Example 2. FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Test apparatus (microchip) of blood coagulation ability, 10 ... Blood injection port, 11 ... Flow path, 12 ... Discharge port, 13 ... Display part, 14 ... Anticoagulation release agent addition port.

Claims (13)

A blood characterized in that blood is infiltrated into a channel of a microchip provided with a channel on a substrate by capillary action, and the blood coagulation ability is inspected on the basis of the blood infiltration speed or the infiltration distance in the channel. Inspection method of coagulation ability. The blood coagulation ability test method according to claim 1, wherein the blood coagulation ability is measured by allowing the blood subjected to anticoagulation treatment to enter the flow path after releasing the anticoagulation treatment or while releasing the anticoagulation treatment. The blood coagulation according to claim 1, wherein the anticoagulant-treated blood is mixed with an agent that releases the anticoagulation treatment and an agent that activates an intrinsic or extrinsic coagulation cascade to promote blood coagulation, and enters the channel. Noh inspection method. The blood coagulation ability test method according to claim 2 or 3, wherein the anticoagulation treatment is treatment with citric acid. The method for examining blood coagulation ability according to claim 4, wherein the anticoagulation treatment with citric acid is canceled by adding calcium. The blood coagulation ability test method according to claim 2 or 3, wherein the anticoagulation treatment is treatment with heparin. The method for examining blood coagulation ability according to claim 6, wherein the anticoagulation treatment with heparin is canceled by adding heparinase. The blood coagulation ability test method according to claim 1, wherein the test is performed in a state where the blood temperature is kept constant. An inspection device that infiltrates blood into the flow path of a microchip provided with a flow path on a substrate by capillary action and inspects the blood coagulation ability based on the blood penetration speed or penetration distance in the flow path. A blood coagulation ability testing apparatus, wherein a material that activates an intrinsic or extrinsic coagulation cascade to promote blood coagulation is used in at least a part of the flow path of the microchip. The blood coagulation ability test apparatus according to claim 9, wherein the material that activates the intrinsic or extrinsic coagulation cascade to promote blood coagulation is a material coated with glass or collagen. The blood coagulation ability testing apparatus according to claim 9 or 10, wherein the flow path is provided to meander. The blood coagulation ability test apparatus according to any one of claims 9 to 11, wherein an index indicating evaluation of blood coagulation ability is attached to the microchip. The blood coagulation ability testing apparatus according to any one of claims 9 to 12, further comprising a heater for maintaining a constant temperature of blood to be examined.

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