JPH08154697A - Container for blood test - Google Patents

Abstract

PURPOSE: To provide the subject container, comprising a blood coagulation accelerant containing a thrombin, etc., which is a blood coagulation accelerating component, an amine salt, etc., that is a heparin neutralizing component and an antifibrinolytic agent, etc., which is a blood clot stabilizing component in a bottomed tubular container and capable of coagulating even blood containing a heparin. CONSTITUTION: This container for blood tests is obtained by filling a blood coagulation accelerant containing thrombin or a thrombin-like serine protease as a blood coagulation accelerating component, an amine salt (e.g. hexadecyldimethylamine hydrochloride) and/or an organic compound having a quaternary nitrogen (e.g. dodecylmethylammonium chloride) as a heparin neutralizing component and antifibrinolytic or an antiplasmin agent (e.g. aprotinin) as a blood cot stabilizing component in the interior of a bottomed tubular container in the container for the blood tests employed in the field of clinical tests using a blood serum specimen. Thereby, the objective container for the blood tests, capable of coagulating blood containing a heparin in a short time and maintaining even the state after coagulation stable and useful for serum biochemical, seroimmunological tests, etc., is obtained.

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 the field of clinical test using a serum sample, and more particularly, a serum biochemical test and serum using the serum of a patient receiving heparin as a sample. The present invention relates to a blood test container that is particularly useful in immunological tests and the like.

[0002]

2. Description of the Related Art In recent years, blood tests such as a serum biochemical test, a serum immunological test, and a blood cell test have become widespread with the progress of testing techniques, and have greatly contributed to the prevention and early diagnosis of diseases. Of these, the serum test is the main component of the blood test, and the serum used for the test is usually centrifuged after coagulating the blood collected in a blood test container consisting of a bottomed tubular container. It is obtained by separating it from a blood clot with a high specific gravity (a gel-like aggregate in which fibrin and blood cells are mixed).

As a blood test container used for collecting blood, glass containers and synthetic resin containers such as polystyrene, polymethylmethacrylate, polyethylene and polyethylene terephthalate have been used. Has the following drawbacks. That is, after injecting blood into the blood test container, a considerable amount of time is required until coagulation, and serum cannot be obtained in a short time. Even a glass container, which is said to have the shortest blood coagulation time, requires 40 to 60 minutes from blood injection to coagulation, and this time is 4 hours or more in a synthetic resin container.

In the case of blood of a patient receiving heparin, such as a patient undergoing artificial dialysis or a thrombotic patient,
Yet another problem arises. Heparin binds to antithrombin III in blood and significantly inhibits the action of thrombin. Therefore, the conversion of fibrinogen to fibrin does not occur and, as a result, blood does not clot.

In order to solve these problems, Japanese Patent Laid-Open No. 63-63
No. 83,670, a cyclic organic compound (I) represented by the following general formula (I) as a blood coagulation accelerating component, and adjacent carbonyl groups in the formula are substantially on the same plane. A metal complex having a ligand as a ligand; an organic compound having an amine salt and / or quaternary nitrogen as a heparin neutralizing component; and a blood coagulation promoter containing an antifibrinolytic agent or an antiplasmin agent as a blood clot stabilizing component However, a blood test container characterized by being present inside has been proposed. In this blood test container, the amine salt or the organic compound having a quaternary nitrogen adsorbs and neutralizes heparin to inactivate it.
In addition, a metal complex having a cyclic organic compound (I) as a ligand activates blood coagulation factor XII in blood to coagulate blood, and an antifibrinolytic agent or antiplasmin agent dissolves a coagulated mass of fibrin. It is possible to secure the stability over time after solidification by preventing the above.

[0006]

Embedded image (Here, A represents a residue of a cyclic compound.)

[0007]

By the way, recently,
Advances in inspection technology have enabled faster inspections. Conventionally, it was usual to know the test result later when blood was collected for the test, but nowadays, blood is collected during the waiting time for the examination (about 30 to 60 minutes), It has become possible to carry out tests and to be able to know the results, and not only can important information be obtained at the time of examination, but more effective treatments can be performed earlier. Therefore, a blood test container for obtaining blood serum from heparin-administered patients has been required.

However, the above-mentioned Japanese Patent Laid-Open No. 63-836.
Even when using the blood test container proposed in Japanese Patent Laid-Open No. 70-70, in the case of blood of a patient receiving heparin such as a patient undergoing artificial dialysis or a patient with thrombosis, the blood coagulation is about 40- There has been a demand for a blood test container that takes 60 minutes and coagulates blood more quickly. The present invention is to solve the above problems, the object is to be able to coagulate not only normal blood but also heparin-containing blood in a short time, and keep the state after coagulation stable. It is to provide a blood test container to be obtained.

[0009]

Means for Solving the Problems The present inventors have studied various factors related to blood coagulation, and as a result of earnest research, have found a substance capable of effectively solving the above problems and completed the present invention. The blood test container of the present invention comprises thrombin or a thrombin-like serine protease as a blood coagulation promoting component;
An organic compound having an amine salt and / or a quaternary nitrogen as a heparin neutralizing component; and a blood coagulation promoter containing an antifibrinolytic agent or an antiplasmin agent as a blood clot stabilizing component,
It is characterized by being present inside a bottomed tubular container.

The thrombin or thrombin-like serine protease used in the present invention functions to promote blood coagulation, and the thrombin-like serine protease is included in snake venom and the like. An adsorbing inorganic substance such as silica fine powder, which is a conventional blood coagulation promoter; glass; or a metal complex having the cyclic organic compound (I) as a ligand, is used in the initial stage of blood coagulation (blood coagulation). Factor XII), whereas thrombin or thrombin-like serine proteases affect the later stage of blood coagulation, where fibrinogen is converted to fibrin, resulting in faster blood clotting or clot formation.

The amine salt and / or the organic compound having a quaternary nitrogen used in the present invention acts as a heparin neutralizing agent which adsorbs and neutralizes heparin to inactivate it. The amine that constitutes the amine salt may be any of primary, secondary and tertiary amines, and the acid that constitutes the amine salt may be either an inorganic acid or an organic acid. Examples of the inorganic acid include hydrohalic acid such as hydrochloric acid, sulfuric acid, and sulfurous acid, and examples of the organic acid include formic acid and acetic acid. The organic residue of the amine salt is usually an acetyl group, but may be a hydrocarbon group containing a different element such as an imino group or an ether group. The amine salt may be an inner salt.

Specific examples of preferred amine salts include hexadecyldimethylamine hydrochloride represented by the formula (II) and tetradecyldi (aminoethyl) glycine represented by the formula (III).

[0013]

Embedded image

[0014]

Embedded image

An organic compound having a quaternary nitrogen is, for example, tetraalkylammonium. It may be a compound having an aryl group instead of an alkyl group, or a hydrocarbon group containing a different element such as an imino group or an ether group.
A preferred specific example is dodecyltrimethylammonium chloride represented by the formula (IV).

[0016]

[Chemical 4]

In addition to such a relatively low molecular weight compound, an organic polymer having a quaternary nitrogen can be used. Examples of such a polymer include a polycation having a repeating unit represented by the general formula (V).

[0018]

Embedded image (Here, R ^{1 to} R ⁴ are hydrogen or an alkyl group, X is a halogen group or an acid group, Y is an alkylene group or an -alkylene group —SO ₂ —, and the repeating number of the above unit is 5 to 200.
0. ) Among the compounds represented by (V), in particular (VI) formula or (VI)
Polycations having repeating units of the formula I) are preferred.

[0019]

[Chemical 6]

[0020]

[Chemical 7]

As the antifibrinolytic agent or antiplasmin agent used in the present invention, aprotinin, soybean trypsin inhibitor, ε-aminocaproic acid, p-aminomethylbenzoic acid, aminomethylcyclohexanecarboxylic acid, which have been conventionally used clinically, are used. And the like, and these are used alone or in appropriate combination.

In the present invention, when the amount of thrombin or thrombin-like serine protease decreases, blood coagulation becomes insufficient, and when the amount increases, the blood coagulation ability decreases, so 0.1 to 50 IU (unit: unit of blood) per ml. ) Is preferably used.

The amine salt and / or the organic compound having quaternary nitrogen is 10 ^-7 to 10 ^{-1 with} respect to ¹ ml of blood.
It is preferably used in a proportion of g and the antifibrinolytic or antiplasmin agents are preferably used in clinically recommended amounts. For example, aprotinin is about 100-60 per ml of blood.
The amount used is 0 KIU (unit), and soybean trypsin inhibitor is about 500 to 4000 IU per 1 ml of blood.
(Unit) used, ε-aminocaproic acid, p-
About aminomethylbenzoic acid and aminomethylcyclohexanecarboxylic acid, about 10 ^-8 to 10 per 1 ml of blood
^It is preferably used in a proportion of ^-2 g.

The blood test container of the present invention may be made of glass or resin.

In order to allow the blood coagulation promoter to exist inside the blood test container, a powdery one, or one dissolved or dispersed in an appropriate solvent is housed in the test container or in the test container. It can be applied to the wall.

Further, in order to prevent the high-concentration blood coagulation promoter from denaturing the protein component due to contact with blood, the blood coagulation promoter is carried on a carrier having a large specific surface area, and the carrier is stored in a test container. It is also possible to do so. The carrier is not particularly limited as long as it does not adversely affect the blood test and has a large specific surface area. For example,
Nonwoven fabric, woven fabric, resin beads and the like are preferably used. In order to carry the blood coagulation promoter on such a carrier,
For example, the solution or dispersion is applied to a carrier, or the carrier is dipped in the solution or dispersion to impregnate the carrier and then dried.
It is also possible to prepare an aqueous dispersion of a blood coagulation promoter containing an appropriate auxiliary agent such as gum arabic and rapidly freeze-dry this to obtain a blood coagulation promoter-supporting particulate material.

The blood test container of the present invention can also be used as a vacuum blood collection tube. The vacuum blood collection tube is manufactured by evacuating the inside of the test container containing the blood coagulation promoter and sealing it with a stopper having excellent sealing property, for example, a butyl rubber stopper.

[0028]

When blood containing heparin is collected in the blood test container of the present invention, heparin in the blood is adsorbed and neutralized by a neutralizing agent such as an amine salt and precipitates, so that heparin thrombin and factor XII are precipitated. There is no inhibitory effect on. Therefore, blood restores normal coagulation function. Furthermore, thrombin or thrombin-like serine protease activates fibrinogen in blood to coagulate blood. The time required for blood coagulation depends on the type of neutralizing agent in the blood coagulation promoter, the amount of blood coagulation promoter, the material of the container used, the amount of heparin in blood, etc. When used, it usually takes 5 to 15 minutes.

The antifibrinolytic agent or antiplasmin agent is
The fibrinolytic action of plasmin, which is antagonistically generated in the blood coagulation reaction process, is inhibited to ensure coagulation, and the coagulation state can be kept stable even after coagulation. Thus, not only normal blood but also blood containing heparin can be coagulated within a short time, and the coagulated state can be kept stable. Furthermore, since the serum and the blood clot are easily separated, the problem that the blood clot component is mixed in the separated and collected serum is solved. Since the degree of contraction of the blood clot component is sufficient, the serum yield is high.

[0030]

EXAMPLES Examples of the present invention will be described below. (Example 1) As a blood coagulation promoting component, thrombin (manufactured by Mochida Pharmaceutical Co., Ltd., trade name "Thrombin Mochida", 10,000I)
U / vial), a polycation [a compound represented by the formula (VII)] as a heparin neutralizing component, and a blood coagulation promoter comprising an aqueous dispersion containing aprotinin as a blood clot stabilizing component are impregnated into a polyester non-woven fabric, Allowed to dry thoroughly. The amount of each of the above components per 1 cm ^{2 of} non-woven fabric is 80
IU, 1.6 × 10 ⁻³ g, 2000 KIU. 1 cm ² of the non-woven fabric for 10 ml (φ16 mm × 100 m
m) A blood test container was manufactured by accommodating it in a polystyrene test tube.

10 I of heparin was added to the above blood test container.
Inject 8 ml of human fresh blood containing U / ml at the concentration of 3
After mixing by inversion, the mixture was left standing at room temperature of 23 to 25 ° C.
After the standing, the time required until the whole blood completely stopped flowing, that is, the blood coagulation time was measured. After the blood coagulated, it was centrifuged at 3000 rpm for 10 minutes to separate the serum and stored at room temperature of 23 to 25 ° C. The concentrations of fibrinogen and fibrin degradation products (hereinafter, fibrinogen and fibrin degradation products are referred to as FDP) in the obtained serum were measured 1 hour and 24 hours after the above end mixing of blood by inversion. The results are shown in Table 1.

(Example 2) As a blood coagulation promoting component, thrombin-like serine protease (manufactured by American Diagnostica Inc., trade name "Batroxobin 100B")
u ") is 1500 IU / ml, tetradecyldi (aminoethyl) glycine is 2.0 wt% as a heparin neutralizing component, and aprotinin is 40,000 K as a blood clot stabilizing component.
A blood coagulation accelerating agent consisting of a physiological saline dispersion containing IU / ml was prepared. 50 μl of the obtained blood coagulation promoter was placed in a 10 ml (φ16 mm × 100 mm) polystyrene test tube and dried to manufacture a blood test container.

Blood coagulation time and FDP concentration were measured in the same manner as in Example 1 using the above blood test container, and the results are shown in Table 1.

Example 3 As a blood coagulation promoting component, thrombin (manufactured by Mochida Pharmaceutical Co., Ltd., trade name “Thrombin Mochida”,
10000 IU / vial), 1500 IU, 0.4 g of hexadecyldimethylamine hydrochloride as a heparin-neutralizing component and 50 mg of aminomethylcyclohexanecarboxylic acid as a blood clot-stable component are prepared, and a polystyrene bead carrier having an average particle diameter of 1.2 mm is prepared. 1 kg was thoroughly mixed with a small amount of ethanol as a dispersion aid, and then dried,
A polystyrene bead carrier with a blood coagulation promoter attached was obtained. 1.3 IU of thrombin, 3.3 × 10 ⁻⁴ g of hexadecyldimethylamine hydrochloride and 3.3 × 10 ⁻⁵ g of aminomethylcyclohexanecarboxylic acid adhered to 1 g of the obtained polystyrene bead carrier. 1.2 g of the bead carrier was housed in a polystyrene test tube for 10 ml (φ16 mm × 100 mm) to manufacture a blood test container.

Blood coagulation time and FDP concentration were measured in the same manner as in Example 1 using the above blood test container, and the results are shown in Table 1.

Example 4 As a blood coagulation promoting component, thrombin (manufactured by Mochida Pharmaceutical Co., Ltd., trade name “Thrombin Mochida”,
10000 IU / vial) 1500 IU / ml, polycation [compound represented by formula (VI)] 2.0 wt% as a heparin neutralizing component, and ε- as a blood clot stable component.
A blood coagulation promoting agent was prepared from a physiological saline dispersion containing 0.4% by weight of aminocaproic acid. Using the obtained blood coagulation promoter, a blood test container was manufactured in the same manner as in Example 2, and the obtained blood test container was used in the same manner as in Example 1 to perform blood coagulation time and FDP concentration. Was measured and the results are shown in Table 1.

Comparative Example 2.0% by weight of an iron complex of n-propyl oxide gallate as a blood coagulation promoting component, 2.0% by weight of tetradecyldi (aminoethyl) glycine as a heparin neutralizing component and a blood clot. A blood coagulation promoter comprising a physiological saline dispersion containing aprotinin as a stable component at a concentration of 40,000 KIU / ml was prepared. Using the obtained blood coagulation promoter, a blood test container was manufactured in the same manner as in Example 2, and the obtained blood test container was used in the same manner as in Example 1 to perform blood coagulation time and FDP concentration. Was measured and the results are shown in Table 1.

[0038]

[Table 1]

From Table 1, the FDP concentration of Examples 1 to 4 and Comparative Example was 2.5 μ after 1 hour and 24 hours.
It is g / ml or less, which means that the coagulated mass of fibrin is not dissolved and the stability over time after coagulation is secured.

[0040]

The configuration of the blood test container of the present invention is as described above, and not only normal blood but also blood containing heparin can be coagulated within a short time (5 to 15 minutes). The state after coagulation can be kept stable. Furthermore, since the serum and the blood clot are easily separated, the problem that the blood clot component is mixed in the separated and collected serum is solved. Since the degree of contraction of the blood clot component is sufficient, the serum yield is high. Therefore,
INDUSTRIAL APPLICABILITY The blood test container of the present invention is useful in the field of clinical tests using a serum sample, particularly in a serum test using the serum of a patient receiving heparin administration as a sample.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/86

Claims (1)

[Claims] 1. A thrombin or thrombin-like serine protease as a blood coagulation-promoting component; an organic compound having an amine salt and / or quaternary nitrogen as a heparin-neutralizing component; A blood test container, wherein a blood coagulation promoter containing a plasmin agent is present inside a bottomed tubular container.