JP3247070B2 - Blood test container and method for producing the same - Google Patents

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 field such as a serum biochemical test and a serum immunological test, and a method for producing the same .

[0002]

2. Description of the Related Art Blood tests are generally performed for the purpose of preventing and diagnosing diseases, but most of the blood tests are serum tests such as serum biochemical tests, serum immunological tests, and blood cell tests. The serum required for the test is usually obtained by coagulating blood collected in a blood test container, separating it from blood clots having different specific gravities by centrifugation, and using a pipette or decantation. Blood collected from a subject takes a relatively long time to coagulate, which is a problem when a patient must return to the hospital to know the result or when an urgent test needs to be performed. Conventionally, blood test containers used for collecting blood have been made of glass or synthetic resin such as polystyrene, polymethyl methacrylate, polyethylene, and polyethylene terephthalate, but the shortest glass container Takes 40 to 60 minutes, and takes 4 hours or more in a container made of synthetic resin. Therefore, conventionally, a coagulant capable of performing blood coagulation in a short time has been studied.

[0003] Blood clotting is initiated by the activation of blood coagulation factor XII and has been shown to have a complex pathway by which fibrinogen is eventually converted to fibrin after a number of reaction steps. JP-A-5-157747
The publication describes that as a component that promotes blood coagulation, an enzyme drug such as thrombin or a snake venom enzyme that promotes the reaction of converting fibrinogen, which is the final stage of the blood coagulation reaction system, into fibrin is used. . by the way,
In recent years, with the progress of inspection equipment, quicker inspection is desired. Conventionally, after blood collection, it takes 2
It usually takes more than one day, but it is desired that blood be collected and tested during a waiting time of about 30 to 60 minutes for a medical examination, and that results be obtained. The reason is not only that important information can be obtained at the time of consultation, but also that more effective treatment can be performed earlier. According to the method disclosed in the above publication, when a container made of synthetic resin is used, the coagulation time is greatly reduced, but most coagulate in about 5 minutes, but it takes about 10 minutes to actually completely coagulate. is there. For the above purpose, it is necessary to completely coagulate the blood within 5 minutes.
If centrifugation is performed before the blood has completely clotted, the coagulation reaction proceeds during centrifugation, and the generated fibrin remains in the serum, and the serum gels and cannot be removed from the test container. Can not be used for inspection. Therefore, it is impossible to obtain a serum sample within 10 minutes after the subsequent centrifugation operation.

[0004]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to provide a blood test container for coagulating blood in a short time and a method for producing the same.

[0005]

A blood test container according to the present invention.
Then, a specific hydrolytic enzyme and a specific inorganic substance
It is housed in a separated form inside the container.

[0006] The hydrolase is a protease,
In the peptide chain, those capable of hydrolyzing the bond between Arg and any amino acid residue and / or the bond between Lys and any amino acid residue are used.

Examples of the hydrolase include serine proteases such as trypsin, thrombin, and snake venom thrombin-like enzymes; thiol proteases such as cathepsin B and ficin; and metalloproteases such as kininase I. Particularly preferred are serine proteases. Used for When the amount of the above-mentioned hydrolase is small, blood coagulation time may be prolonged or coagulation may be incomplete. When the amount is large, the test value may be adversely affected. Is preferred, and 0.5 to
50 units are more preferred.

The above-mentioned inorganic substance has an effect of promoting the activation of a blood coagulation factor when it comes into contact with blood, and of promoting the aggregation of platelets. However, in order for the above-mentioned inorganic substance to effectively exert the blood coagulation accelerating action, the linseed oil absorption, the BET specific surface area value and the specific resistance value must be within specific ranges. The linseed oil absorption and the BET specific surface area value indicate the degree of the surface area of the inorganic substance, and the surface area is related to the degree of the surface pores of the inorganic substance. Therefore, the degree of the surface pores can be known from the oil absorption and the specific surface area. During blood coagulation, factor XII, a contact factor, is activated. For this purpose, three types of substances, factor XII, prekallikrein and high molecular weight kininogen, form a complex on the surface of the foreign substance and are adsorbed. It is said that adsorption in a state where one or two of these are missing does not lead to activation.

By the way, when an inorganic substance is used in the expectation of a blood coagulation accelerating action, if the surface area is very large, Factor XII, prekallikrein, The proportion of high molecular weight kininogen will increase, in other words the proportion of the three complexes required for the activation of factor XII will decrease,
Rather, the blood coagulation promoting effect is reduced. Conversely, if the surface area of the inorganic substance is too small, the probability of adsorption of a coagulation factor will be small, and it will be impossible to expect a blood coagulation promoting action. Therefore, the inorganic substance used in the present invention is:
Linseed oil has an oil absorption of 20 to 40 ml / 100 g and a BET specific surface area of 5000 to 30000 cm ² / g.

The linseed oil absorption is JIS K-510.
Indicates a value measured according to 1. The BET specific surface area is determined from the amount of gas adsorbed on the surface of the inorganic substance, the equilibrium pressure at that time, the saturated vapor pressure of the adsorbed gas, and the amount of gas covering the surface as a monomolecular layer. It refers to a value calculated by multiplying the average cross-sectional area, and nitrogen gas, oxygen gas, argon gas, methane gas, or the like is used as the adsorption gas. According to this method, the surface area value including pores that cannot be measured by measuring the linseed oil absorption amount is measured. The specific resistance value is the reciprocal of the electric conductivity and is a value at normal temperature. It is presumed that the specific resistance value for the adsorptive inorganic substance maintains the consistency of the potential distribution between the protein and the adsorptive inorganic substance and contributes to preventing a change in protein conformation. The specific resistance of the inorganic substance used in the present invention is 1 × 10 ¹⁰ Ω · c
m, preferably 5 × 10 ⁴ Ω · cm.

Examples of the inorganic substance include inorganic substances used as an adsorbent, for example, water-insoluble inorganic fine powders such as glass, silica, kaolin, celite and bentonite. The particle size of the inorganic substance is 50 μm
Or less, and more preferably those having an average particle size of 10 μm or less. In particular, an inorganic substance effective for shortening the blood coagulation time is silica. In particular, porous silica containing at least 20% by weight of an amorphous component exhibits an excellent effect. When the amount of the inorganic substance is small, the blood coagulation time is prolonged or coagulation may be incomplete, and when the amount is large, the test value may be adversely affected. Therefore, 1 × 10 ⁻⁶ to 1 × per 1 ml of blood. 10 ⁻³ g is preferred, and 1
× 10 ^-5 to 1 × 10 ^-4 is more preferable.

Examples of the material of the tubular container include thermoplastic resins such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polymethyl methacrylate, and polyacrylonitrile; unsaturated polyester resins;
Thermosetting resins such as epoxy resins and epoxy-acrylate resins, modified natural resins such as cellulose acetate, cellulose propionate, ethyl cellulose, and ethyl chitin; silicate glass such as soda-lime glass, phosphosilicate glass, and borosilicate glass; and quartz glass And any of those containing these as a main component. A part of the outer surface of the tubular container may be colored for identification, and a scale for measuring a blood collection amount may be provided.

[0014] Oite the present invention, hydrolytic enzymes can be adsorbed to the inorganic substance, such procoagulant activity by not sufficiently diffuse into the blood is not reduced, the hydrolase and an inorganic material
There Ru housed in isolated form. The upper Symbol isolated form, after one inner surface of the container are stacked, one being laminated on a part of the other ones are stacked, or inner surface of the container,
It means that the other part is laminated on another part. Here, “one laminated” and “the other laminated” do not necessarily mean that the first laminated and the next laminated are completely two layers. The above form
The efficiency in the production, preferably <br/> physician in terms of ease and the like of the dispersion of the blood coagulation promoter when introduced blood mixing during inspection.

As a method of laminating the hydrolase and the inorganic substance on the inner surface of the container , one aqueous dispersion is applied to the inner surface of the container, dried, and then the other is coated and dried in the same manner. Such a method is related to the present invention.
This is a method for producing a blood test container. As a method of applying the aqueous solution or the aqueous dispersion to the inner surface of the container, the aqueous solution or the aqueous dispersion can be applied by spraying, or can be applied by dipping in the aqueous solution or the aqueous dispersion.

The blood test container of the present invention may further contain a blood clot adhesion preventing component. This can prevent blood clot components after blood has clotted from adhering to the inner surface of the container, and the movement of the blood clot is not restricted during centrifugation,
Blood clot and serum can be separated well. As the blood clot adhesion preventing component, a hydrophilic substance that is hardly soluble or insoluble in water can be used. For example, an aliphatic modified silicone oil (for example, dimethylpolysiloxane and the like), an aromatic modified silicone oil (for example, methyl) Phenylpolysiloxane), partially saponified polyvinyl alcohol, and vinylpyrrolidone-vinyl acetate copolymer. When modified silicone oils are used, it is preferable to further add a water-soluble substance in order to prevent a decrease in blood coagulation accelerating performance due to covering of the surface of the inorganic substance.
Examples of the water-soluble substance include polyvinyl alcohol and polyvinyl pyrrolidone.

In order to prevent the blood clot from adhering to the inner surface of the container, it is necessary that the anti-clot adhesion component is in direct contact with the inner surface of the container and covers the entire inner surface. Therefore, when accommodating the blood coagulation promoter, the solution or dispersion of the blood coagulation promoter to be applied first among the hydrolytic enzymes and inorganic substances that are uniformly applied to the inner surface of the container in advance or applied to the inner surface. After mixing, they may be applied together. Further, it may be mixed with another solution or dispersion of the blood coagulation promoter and applied to the inner surface of the container.

The blood test container of the present invention may further contain a serum separating agent. The serum separating agent is stored in advance in the bottom of the container, moves between the clot and the serum by centrifugation after blood collection, and forms a partition to separate the serum. The serum separating agent is a gel-like substance having thixotropy.For example, to a synthetic resin having fluidity at room temperature, an additive such as a thixotropy-imparting agent, a specific gravity adjuster, and a viscosity adjuster are added and mixed. It can be obtained by: Examples of the synthetic resin include, for example, oligomers of dicyclopentadiene, and examples of the thixotropic agent include, for example, condensates of sorbitol and aromatic aldehydes, and polyoxyethylene polyoxyalkylene block copolymers. Examples of the agent include silica and the like, and examples of the viscosity modifier include phthalic acid ester and the like.

The blood test container of the present invention can be used not only as a normal blood test container but also as a vacuum blood collection tube. The vacuum blood collection tube is prepared by containing the above blood coagulation accelerator inside a normal blood collection tube made of glass or synthetic resin, exhausting the blood collection tube, and sealing it with a highly sealable stopper made of butyl rubber or the like.

[0020]

Next, embodiments of the present invention will be described. In Examples and Comparative Examples, the following were used as reagents, containers, and the like.・ Hydrolytic enzyme thrombin (trade name: Thrombin Mochida, manufactured by Mochida Pharmaceutical Co.) ・ Inorganic fine powder silica (trade name: Imsil A25, manufactured by Illinois Chemical Co.) Linseed oil absorption 30 ml / 100 g, BET specific surface area 12000 cm ² Specific resistance 2.5 × 10 ⁴ Ω · cm ・ Clot adhesion prevention agent vinylpyrrolidone-vinyl acetate copolymer (molar ratio 6: 4) (trade name: Lubiscol 64, manufactured by BASF) ・ Tubular container: polyethylene terephthalate Inner volume 10ml (16φ × 100mm) (Sekisui Chemical Co., Ltd.)

Example 1 0.5% by weight of vinylpyrrolidone-vinyl acetate copolymer
0.25 g of fine powdered silica was uniformly dispersed in the aqueous solution to make the total amount 10 g. 0.025 g of this liquid was uniformly sprayed on the inner surface of the tubular container and air-dried. Next, 10,000 units of thrombin was dissolved in a 0.5% by weight aqueous solution of a vinylpyrrolidone-vinyl acetate copolymer to give a total amount of 3.12.
5 g. 0.025 g of this solution was uniformly sprayed on the inner surface of the tubular container sprayed with the fine powdered silica, and air-dried to prepare a blood test container. Blood in this container
When 8 ml of liquid is collected, the amount of thrombin is 1 ml of blood
10 units per unit, the amount of fine powder silica is 1 ml of blood
7.8 × 10 ⁻⁵ g. 8 ml of healthy human blood
Blood samples are collected in a liquid test container and blood clots from the end of collection.
The time it took to run was measured. Blood test for coagulation
Even if the blood container is tilted, the top surface of the blood does not move and
Even when blood did not flow out. Then clotted blood
The solution is centrifuged at 1300G (2500 rpm) for 5 minutes at 25 ° C.
Separation of the heart and visualization of the separated serum for the presence of fibrin
Observed. Table 1 shows the results.

(Example 2 ) A blood test container was prepared in the same manner as in Example 1 except that the amount of thrombin in Example 1 was changed from 10,000 units to 5000 units, and the amount of fine powder silica was changed from 0.25 g to 0.12 g. Produced. When 8 ml of blood is collected in this container, the amount of thrombin is 5 units per ml of blood, and the amount of fine powder silica is 3.8 × 10 ⁻⁵ g per ml of blood. Example 1 below
The clotting time was measured and the presence or absence of fibrin was visually observed in the same manner as described above. Table 1 shows the results.

Comparative Example 1 0.5% by weight of vinylpyrrolidone-vinyl acetate copolymer
0.25 g of fine powdered silica was uniformly dispersed in the aqueous solution to make the total amount 10 g. 0.025 g of this liquid was uniformly sprayed on the inner surface of the tubular container and air-dried to prepare a blood test container. When 8 ml of blood is collected in this container, the amount of the fine powdered silica is 7.8 × 10 ⁻⁵ g per ml of blood. Thereafter, the coagulation time was measured and the presence or absence of fibrin was visually observed in the same manner as in Example 1. Table 1 shows the results.

Comparative Example 2 0.5% by weight of vinylpyrrolidone-vinyl acetate copolymer
10,000 units of thrombin was dissolved in the aqueous solution to make a total amount of 3.125 g. 0.025 g of this liquid was uniformly sprayed on the inner surface of the tubular container and air-dried to prepare a blood test container. When 8 ml of blood is collected in this container, the amount of thrombin is 10 units per ml of blood. Thereafter, the coagulation time was measured and the presence or absence of fibrin was visually observed in the same manner as in Example 1. Table 1 shows the results.

[0027]

[Table 1]

[0028]

The blood test container of the present invention is as described above, and blood can be coagulated in a short time by using a hydrolase and a specific inorganic substance in combination.
Furthermore, since the coagulation state is kept stable and the separation of serum and blood clot is facilitated, fibrin and blood clot components are not mixed in the separated and collected serum. Since the degree of contraction of the blood clot component is sufficient, the serum yield is also high. Further, since the hydrolase and the inorganic substance are separated, the stability of the blood coagulation accelerator is maintained, and the blood coagulation ability does not decrease.

Continuation of front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/48 A61B 5/15

Claims (2)

(57) [Claims] 1. A hydrolase capable of hydrolyzing a bond between Arg and any amino acid residue in a peptide chain and / or a bond between Lys and any amino acid residue, and a linseed oil absorption of 20 to 40 ml / 100 g, BET specific surface area is 500
0-30000 cm ² / g, specific resistance value is 1 × 10 ¹⁰ Ω ·
cm or less of inorganic matter is separated inside the bottomed tubular container.
A blood test container stored in the form
One of the hydrolase and the above inorganic substance is the inner surface of the tubular container
After the other is laminated, the other is laminated on the inner surface of the tubular container
A blood test container characterized in that: 2. Production of the blood test container according to claim 1.
A method, wherein the hydrolase and the inorganic substance are:
Apply and dry one aqueous solution or aqueous dispersion on the inner surface of the tubular container
After that, the other aqueous solution or aqueous dispersion is applied to the inner surface of the tubular container.
Manufacture of blood test containers characterized by being applied and dried
Construction method.