JP2000287954A - Container for blood examination - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a container for blood examination which easily obtains serum with little fibrin precipitation by coagulating blood in a shorter time without requiring complicated mixing work so much. SOLUTION: Housed in the body of a container are a blood coagulation promoter such as enzyme that enables hydrolysis of the bond of arginine in a peptide chain and an arbitrary amino acid residue and/or the bond of lysine and an arbitrary amino acid residue and a fibrin precipitation depressor comprising a blood coagulation XIII factor inhibitor and/or fibrin crosslinking inhibitor to suppress the precipitation of fibrin into serum during the centrifugal separation after the coagulation of blood.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood test container used in a clinical test using serum, such as a serum biochemical test or a serum immunological test.

[0002]

2. Description of the Related Art In recent years, with the progress of clinical testing techniques, blood tests such as serum biochemical tests, serum immunological tests, and blood cell tests have become widespread, and have greatly contributed to disease prevention and early diagnosis.

[0003] In the above serum test, it is necessary to separate serum from blood collected from a patient. In separating serum, first, blood is collected in a blood test container, coagulated, and then separated from the blood clot by centrifugation.

[0004] The separated serum is usually transferred to another container by pipetting or decanting. However, in recent years, in order to increase the test speed and to process a large number of samples, a method of dispensing serum directly from a blood test container to an automatic analyzer using an automatic dispenser has been widely used.

As a conventional blood test container, a glass container or a container made of a synthetic resin such as polystyrene, polymethyl methacrylate, or polyethylene has been used. However, these blood test containers have the following problems. was there.

[0006] First, after injecting blood into a blood test container, it takes a considerable amount of time to reach coagulation.
Therefore, when it was necessary to perform an urgent serum test, the request could not be met.

That is, even when a blood test container made of glass, which is considered to have the shortest blood coagulation time, requires 40 to 60 minutes from blood injection to coagulation. Furthermore, in a synthetic resin blood test container, a time of 4 hours or more was required until blood coagulation.

[0008] In order to solve the above problems, a method has been widely used in which fine particles such as silica, kaolin, and bentonite are introduced into a blood test container as a coagulation promoter.

Japanese Patent Application Laid-Open No. 5-157747 discloses a method of using an enzyme drug such as thrombin or a snake venom enzyme, which promotes the reaction of converting fibrinogen into fibrin, which is the final stage of the coagulation reaction, as a blood coagulation promoting component. Is disclosed.

In the method described in the above prior art, an enzymatic drug such as thrombin or snake venom is impregnated in a support such as a nonwoven fabric, filter paper or cloth and stored in the center of a bottomed blood test tube. ing. In this method, even when a tube made of a synthetic resin is used as a bottomed tube for a blood test, the coagulation time is greatly reduced, and coagulation is almost completed in about 5 minutes.

However, when an enzyme-based drug such as thrombin or snake venom enzyme is used, the coagulation reaction proceeds very quickly. If not sufficiently mixed immediately after blood collection, coagulation partially proceeds rapidly. Overall, the clotting time tended to be long. As a result, there is a problem that a large amount of fibrin precipitates in serum after centrifugation.
This is because the blood portion that has come into direct contact with thrombin or snake venom enzyme rapidly coagulates, and the resulting fibrin or three-dimensionally cross-linked fibrin gel surrounds the enzyme, and that between the enzyme and the uncoagulated blood portion. It is considered that this is because the above contact is suppressed, and the coagulation reaction thereafter becomes difficult to proceed.

However, in the case of urgency or when it is necessary to process a large number of samples, there is no time to sufficiently mix blood after collecting it in a blood test container. However, it is very difficult to use a method using an enzyme-based drug such as the above when a large amount of sample is processed or when an urgent case is required.

[0013]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned drawbacks of the prior art, simplifies the complicated work of mixing well immediately after blood collection, and allows blood to coagulate in a short time. Another object of the present invention is to provide a blood test container capable of reliably obtaining serum containing less fibrin.

[0014]

SUMMARY OF THE INVENTION The present invention has been made to achieve the above-mentioned object. In the blood test container according to the present invention, a blood coagulation accelerator and a blood coagulation accelerator are provided in the container body. A fibrin precipitation inhibitor which suppresses fibrin precipitation in serum during centrifugation is stored.

As the blood coagulation promoter, for example, an enzyme capable of hydrolyzing the bond between arginine and any amino acid residue in the peptide chain and / or the bond between lysine and any amino acid residue can be used. .

[0016] In a specific aspect of the present invention, as the fibrin precipitation inhibitor, a blood coagulation factor XIII inhibitor that inhibits a reaction involving blood coagulation factor XIII, and a fibrin crosslinking inhibitor that inhibits fibrin crosslinking. At least one of them is used.

The above-mentioned blood coagulation factor XIII is known as a transglutaminase which forms a three-dimensional cross-link by an isopeptide bond between fibrin monomers generated when an enzyme such as thrombin acts on blood. By this three-dimensional cross-linking, fibrin becomes a strong gel state,
Constructs a fibrin gel, but does not require so urgent and cumbersome mixing even if enzymes that rapidly coagulate blood, such as thrombin and snake venom enzymes, are used by inhibiting blood coagulation factor XIII. In addition, it is possible to obtain serum in which fibrin precipitation hardly occurs.

Hereinafter, the present invention will be described in detail. In the present invention, the material constituting the container body of the blood test container is not particularly limited, and is conventionally used in blood test containers such as glass, thermoplastic resin, thermosetting resin, and modified natural resin. Materials can be used. Also, the shape of the container body is not particularly limited, and a bottomed cylindrical tube such as a test tube, or an appropriately shaped one such as a bottle can be used. The blood test container contains a blood coagulation accelerator and a fibrin precipitation inhibitor.

The blood coagulation promoter is not particularly limited, but can hydrolyze the bond between arginine and any amino acid residue and / or the bond between lysine and any amino acid residue in the peptide chain. Enzymes are used. Examples of such an enzyme include thrombin, serine proteases such as thrombin-like enzymes obtained from snake venom, thiol proteases such as cathepsin B and ficin, and metal proteases such as kininase I. In particular, serine proteases are preferable. Used for

The amount of the blood coagulation promoter is preferably 0.1 to 100 units per 1 mL of blood. If it is less than 0.1 unit, the blood coagulation time may be long or coagulation may be incomplete. If it exceeds 100 units, the test value may be adversely affected and the cost increases. More preferably, it is 0.5 to 50 units.

As a fibrin precipitation inhibitor, a blood coagulation factor XIII inhibitor and / or a ficin cross-linking inhibitor is used. Examples of the blood coagulation factor XIII inhibitor include substances such as maleimide, N-ethylmaleimide, p-chloromercuric benzoic acid, p-chloromercuric phenylsulfonic acid, and phenylmercuric, and derivatives thereof.

As the fibrin crosslinking inhibitor, a substance that inhibits the isopeptide bond to a glutamyl residue is used. Specifically, glycine derivatives such as glycine, glycylglycine, glycine methyl ester and glycine ethyl ester are used. And salts thereof, diamine compounds such as cadaverine and putrescine, and derivatives and salts thereof. These may be used alone,
More than one species may be used in combination.

The amount of the blood coagulation factor XIII inhibitor varies depending on its type.
Factor II inhibitory effect may not be sufficient, if too much,
The test value may be adversely affected. Therefore, one meter of blood
L, the amount of the blood coagulation factor XIII inhibitor is 0.0
The range is preferably 1 to 20 mg, more preferably 0.0
5-10 mg.

Since fibrin which has inhibited the reaction of blood coagulation factor XIII is susceptible to fibrinolysis by plasmin, an anti-fibrin solvent or an anti-plasmin agent may be added to the blood coagulation accelerator.

The type of anti-radiation solvent or anti-plasmin agent is not particularly limited, but t-4- (aminomethyl) -cyclohexanecarboxylic acid, ε-aminocaproic acid, p-aminomethylbenzoic acid, aprotinin, soybean Trypsin inhibitor and the like can be mentioned.
These may be used alone or in combination of two or more.

The anticoagulant or antiplasmin agent may be added to the blood coagulation accelerator in an amount that does not affect the clinical test using the obtained serum. For example, with respect to t-4- (aminomethyl) -cyclohexanecarboxylic acid, ε-aminocaproic acid, and p-aminomethylbenzoic acid, about 10 ⁻² to 10 ⁻⁸ g per 1 mL of blood.
In the case of aprotinin, about 100 to 600 KIU (unit) per 1 mL of blood, soybean trypsin inhibitor is about 500 to 4000 FU per 1 mL of blood.
(Unit) contained in the blood coagulation promoter.

The blood coagulation accelerator is stored in a blood test container, but the method is not particularly limited. For example, a method of applying a blood coagulation accelerator to a solution in the form of a solution and applying it to the inner wall of the container, drying it, storing it in a tube with powder or lump, holding it in a non-woven fabric, woven fabric, resin beads, etc. and storing it in the tube Examples thereof include a method, a method of containing a blood coagulation promoter in a solution in a tube, lyophilizing the solution, and a method of combining them.

By applying the blood clot adhesion preventing component to the inner wall of the container body, the blood clot detachability during centrifugation can be improved. Examples of the blood clot adhesion preventing component include hydrophilic substances that are hardly soluble or insoluble in water. For example, an aliphatic modified silicone oil such as dimethylpolysiloxane, an aromatic modified silicone oil such as methylphenylpolysiloxane, an alcohol modified silicone oil, a partially saponified polyvinyl alcohol, a copolymer of vinylpyrrolidone and vinyl acetate, etc. Can be mentioned. These may be used alone or in combination of two or more.

The amount of the component for preventing blood clot adhesion is preferably 0.001 to 10 mg per 1 mL of blood. If the amount is less than 0.001 mg, a sufficient clot detachment function may not be exhibited, and if it exceeds 10 mg, the test value may be adversely affected. The blood clot adhesion preventing component is applied to a blood test container, and the method of applying the blood clot is not particularly limited. However, it is preferable to use a method of applying the solution in a solution and drying the solution. Alternatively, it may be mixed with the above-mentioned blood coagulation promoter and simultaneously applied to the inner wall of the blood test container.

Further, the blood test container according to the present invention may contain a serum separating agent. The serum separating agent is usually stored at the bottom of the container body. In this case, after the blood collected in the blood test container is coagulated and centrifuged, the serum separating agent moves between the blood clot and the serum, forming a partition, thereby forming the blood serum and the blood clot. Are separated.

Examples of the above-mentioned serum separating agent include known gel-like substances having thixotropic properties. For example, a synthetic resin having fluidity at room temperature, such as a thixotropic agent, a specific gravity adjusting agent and a viscosity adjusting agent. It is obtained by adding additives and mixing.

Examples of the synthetic resin include oligomers such as dicyclopentadiene, examples of the thixotropic agent include condensates of sorbitol and aromatic aldehydes, and examples of the specific gravity adjuster include: Examples of the viscosity modifier include silica and paraffin chloride. Examples of the viscosity modifier include phthalic acid ester and epoxidized soybean oil.

[0033]

Hereinafter, the present invention will be described in more detail by giving specific examples of the present invention. The present invention is not limited to the following examples.

(Example 1) 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation accelerator and blood coagulation XI
Physiological saline was added to 10 mg of maleimide as a factor II inhibitor to make a total volume of 2.0 mL. This solution was added in an amount of 40 μL to a polyethylene tube having an inner diameter of 16 mm and a length of 100 mm and dried to obtain a blood test container of Example 1.

Example 2 A blood test container was obtained in the same manner as in Example 1 except that the amount of maleimide used was changed from 10 mg to 40 mg.

(Example 3) 2000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter and 200 mg of glycine ethyl ester as a fibrin crosslinking inhibitor
Was added with physiological saline to make the total volume 2.0 mL. This solution was added to a polyethylene tube and dried in the same manner as in Example 1 to obtain a blood test container of Example 3.

Example 4 A blood test container was obtained in the same manner as in Example 3 except that the amount of glycine ethyl ester used was changed from 200 mg to 800 mg.

(Example 5) 8000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter and 100 m of glycine hydrochloride methyl ester as a fibrin crosslinking inhibitor
g and physiological saline and lithium hydroxide, pH =
A total 2.0 mL solution of 7.5 was obtained. This solution was added to a polyethylene tube in the same manner as in Example 1,
After drying, the blood test container of Example 5 was obtained.

Example 6 A blood test container was obtained in the same manner as in Example 5, except that the amount of glycine hydrochloride methyl ester used was changed from 100 mg to 400 mg.

Example 7 Physiological saline was added to 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter and 400 mg of glycylglycine as a fibrin crosslinking inhibitor to make a total volume of 2.0 mL. Thereafter, using this solution, a blood test container of Example 7 was obtained in the same manner as in Example 1.

Example 8 A blood test container was obtained in the same manner as in Example 7, except that the amount of glycylglycine used was changed from 400 mg to 1600 mg.

(Example 9) 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter and 400 mg of glycine hydrochloride ethyl ester as a fibrin crosslinking inhibitor
And added saline and lithium hydroxide, pH
Was adjusted to 7.5, and the total amount was 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

Example 10 A blood test container was obtained in the same manner as in Example 9, except that the amount of glycine hydrochloride ethyl ester used was changed from 400 mg to 800 mg.

(Example 11) 4000 units of snake venom thrombin-like enzyme (Batroxobin) (manufactured by American Diagnostica) as a blood coagulation promoter and glycine hydrochloride ethyl ester 800 as a fibrin crosslinking inhibitor
mg, and the pH value was adjusted to 7.5 by adding physiological saline and lithium hydroxide to obtain a solution having a total volume of 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

Example 12 A blood test container was obtained in the same manner as in Example 11, except that the amount of glycine hydrochloride ethyl ester was changed from 400 mg to 800 mg.

(Example 13) 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter, 40 mg of maleimide as a blood coagulation factor XIII inhibitor, and 20 m of alcohol-modified silicone oil as a clot release agent
g was added, and physiological saline was added to make a total volume of 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

(Example 14) 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter, and 200 g of glycine hydrochloride methyl ester as a fibrin crosslinking inhibitor
g, 20 mg of alcohol-modified silicone oil was added as a clot release agent, physiological saline and lithium hydroxide were added to adjust the pH to 7.5, and the total amount was adjusted to 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1 below.

(Comparative Example 1) Physiological saline was added to 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation promoter to make the total volume 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

(Comparative Example 2) The amount of thrombin used was 400
A blood test container was obtained in the same manner as in Comparative Example 1, except that the unit was changed from 0 units to 8000 units.

Comparative Example 3 To 4000 units of a snake venom (Batroxobin) thrombin-like enzyme (manufactured by Nasca) as a blood coagulation promoter, physiological saline was added to make a total amount of 2.0 units.
mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

Comparative Example 4 Thrombin (Mochida Pharmaceutical Co., Ltd.) 4000 units as a blood coagulation promoter, blood coagulation XI
Compounding 3.0 mg of maleimide as a factor II inhibitor,
Physiological saline was added to these to make the total volume 2.0 mL.
Using this solution, a blood test container was obtained in the same manner as in Example 1.

(Comparative Example 5) 4000 units of thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) as a blood coagulation accelerator and 3.0 mg of glycine hydrochloride methyl ester as a fibrin crosslinking inhibitor
And add saline and lithium hydroxide to pH
Was adjusted to 7.5, and the total amount was adjusted to 2.0 mL. Using this solution, a blood test container was obtained in the same manner as in Example 1.

[Evaluation] Examples 1 to 4 obtained as described above
Using the blood test containers of Example 14 and Comparative Examples 1 to 5, 8 mL of human fresh blood was added to each blood test container, mixed only gently once, and allowed to stand for 5 minutes. After 5 minutes, 3000 per minute
Centrifugation was performed for 5 minutes by rotation, and the state of the serum after separation was visually confirmed. The results are shown in Table 1 below.

[0054]

[Table 1]

After the above centrifugation for 5 minutes, Examples 1-1
In all of the blood test containers of Comparative Example 1 and Comparative Examples 1 to 5, coagulation was completed, but in Comparative Examples 1 to 5, fibrin was precipitated in serum. That is, it is considered that the strong crosslinked fibrin hinders the diffusion of the coagulation component, and a partially uncoagulated portion remains.

On the other hand, in Examples 1 to 14, no fibrin was precipitated in the serum after separation.

[0057]

As described above, in the blood test container according to the present invention, since the blood coagulation accelerator and the fibrin precipitation inhibitor are contained in the container body, blood coagulation proceeds rapidly after blood collection. In addition, the blood coagulation time can be shortened, and at the time of coagulation, the precipitation of fibrin into the serum is suppressed by the fibrin precipitation inhibitor, so that serum containing almost no fibrin contamination can be obtained.

Therefore, serum can be obtained in a short time, so that it can be used in an urgent medical setting, and fibrin hardly precipitates, so that highly reliable test results can be obtained. Become.

In the present invention, the blood coagulation component is composed of an enzyme capable of hydrolyzing the bond between arginine and any amino acid residue in the peptide chain and / or the bond between lysine and any amino acid residue. In such a case, blood coagulation proceeds more rapidly. Therefore, the time required to obtain serum can be shortened, and the addition of the above-mentioned fibrin precipitation inhibitor not only suppresses the precipitation of fibrin, but also does not require sufficient mixing immediately after blood collection.

When a blood coagulation factor XIII inhibitor and / or a fibrin cross-linking inhibitor is used as a fibrin precipitation inhibitor, a reaction involving the blood coagulation factor XIII inhibitor is inhibited, or a fibrin cross-linking inhibitor is used. Inhibits fibrin cross-linking, so that the deposition of fibrin in serum can be effectively suppressed.

Claims (5)

[Claims] 1. A blood container comprising a blood coagulation promoter and a fibrin precipitation inhibitor which suppresses fibrin from being precipitated in serum during centrifugation after blood coagulation. Inspection container. 2. The blood coagulation promoter is an enzyme capable of hydrolyzing a bond between arginine and any amino acid residue and / or a bond between lysine and any amino acid residue in a peptide chain. The blood test container according to the above. 3. The blood test container according to claim 1, wherein the fibrin precipitation inhibitor is a blood coagulation factor XIII inhibitor and / or a fibrin crosslinking inhibitor. 4. The method according to claim 1, wherein the blood coagulation factor XIII inhibitor is maleimide, N-ethylmaleimide, p-chloromercuric benzoic acid, p-chloromercuric phenylsulfonic acid and phenylmercuric acid, and derivatives or salts thereof. The blood test container according to claim 3, which is at least one selected from the group consisting of: 5. The blood test container according to claim 3, wherein the fibrin crosslinking inhibitor is at least one selected from the group consisting of glycine, a diamine compound, a derivative thereof, and a salt thereof.