JP2000033081A - Vessel for blood inspection - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a vessel for blood inspection for shortening the pretreatment time of a specimen without exerting an adverse influence on biochemistry, serological inspection, etc., and without requiring excessive intimate mixing work. SOLUTION: A blood-coagulation X-th factor, a blood-coagulation Xa-th factor which is its active type, a compsn. contg. the blood-coagulation X-th factor or the compsn. contg. the blood-coagulation Xa-th factor is housed in this vessel for blood inspection. While the blood-coagulation X-th factor or the blood-coagulation Xa-th factor which is its active type may be used alone, the use as the compounded compsn. prepd. by using a polymer, such as vinyl pyrrolidone, vinyl alcohol or hydroxylalkyl methacrylate, at 0.001 to 10 mg per 1 mL blood in combination is more preferable in order to prevent the fixation of plasma components to the inside wall surface of the inspection vessel.

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 for a clinical blood test using serum obtained from coagulated blood as a sample.

[0002]

2. Description of the Related Art In recent years, there has been remarkable progress in analytical instruments used for blood tests, and the required sample amount has been greatly reduced, and the test time has been greatly reduced. For this reason, the ratio of the required time occupied by the sample pre-processing in the series of inspection processes from sample collection to sample pre-processing, analysis operation, and report of the result is so large that it cannot be ignored. Conventionally, when blood is taken out of an outpatient for a test, it takes more than a week to get a diagnosis from a doctor, receive a prescription, etc., and go to the hospital many times. However, as the concept of customer satisfaction has become widespread in hospital management, there is a need for outpatients to report test results to physicians while they are staying at the hospital and to make a diagnosis. It is growing rapidly. in this case,
The time allowed to complete all the steps of the test was about 30 minutes, and as a result, the time for sample pretreatment had to be reduced to about 10 minutes.

[0003] There are a great number of types of blood tests. Among them, in the pretreatment of a sample in a general hematology test, a blood glucose test, a coagulation function test, etc., an anticoagulant or a metabolic inhibitor is added to blood. , The pre-processing time is originally short and does not cause much problem. However,
In biochemical tests, serological tests, and the like, since serum is used as a sample, pretreatment of coagulating blood and collecting the serum by centrifugation must be performed.

[0004] Blood coagulates due to chemical reactions involving various types of proteolytic enzymes, phospholipids, calcium ions, and other proteins in a complex manner. The initiation of the coagulation reaction of blood collected in a test container is usually a contact reaction between blood coagulation factor XII and an extracorporeal foreign substance such as the inner wall surface of the test container, and the fibrinogen is converted into fibrin monomer from fibrinogen through a number of steps. It often takes more than one to two hours to complete the clotting, which leads to the formation of a red-black clot that undergoes conversion and blood cell components, ie, fibrin gel, and this long clotting time has become a major problem.

There have been many attempts to shorten blood coagulation, and it is common to add fine powders of silica-based minerals such as silica, kaolin and diatomaceous earth to blood to promote the activation of blood coagulation factor XII. is there. Coagulation time reduction rate is
Although it depends on the amount of the fine powder added, it is limited to about 15 minutes in consideration of hemolysis and other undesired side effects.
It takes up to about 25 minutes and does not satisfy the above requirements.

On the other hand, JP-A-58-1460, JP-A-60-106610, and JP-A-60-17495
No. 2 proposes to add thrombin which is a kind of blood coagulation factor or a thrombin-like enzyme derived from snake venom to blood to speed up coagulation. These enzymes play a role in converting fibrinogen, which is the final stage of the blood coagulation reaction, into fibrin, and in principle, can reduce the time from blood collection to serum collection to about 10 minutes. .

However, in order for the blood collection tube containing the enzymes that promote blood coagulation to function sufficiently, the blood and the drug containing the enzyme must be sufficiently mixed immediately after blood collection. If this is neglected, the coagulation time will be abnormally delayed, and a large amount of fibrin will precipitate in the supernatant after centrifugation.

[0008] This is because only the blood part which is in direct contact with thrombin or a thrombin-like enzyme derived from snake venom coagulates too rapidly, so that the fibrin formed or the three-dimensionally cross-linked fibrin gel surrounds these enzymes. This is considered to be because the enzyme prevents the enzyme from diffusing into the blood isolated outside the barrier wall, thereby halting the coagulation reaction thereafter.

Unfortunately, in a blood collection room of an actual hospital, it is busy to collect blood from many patients into many kinds of blood collection tubes at once, and complicated work such as thorough mixing immediately after blood collection is accepted. There are difficult things. As a result, the above-mentioned problem has become a reality, and has prevented the spread of such blood collection tubes.

[0010]

SUMMARY OF THE INVENTION The present invention has been made to solve the above problems, and has as its object to have no adverse effects on biochemistry, serologic tests, etc., and to avoid excessive mixing work. Another object of the present invention is to provide a blood test container for shortening the pretreatment time of a sample.

[0011]

SUMMARY OF THE INVENTION A blood test container according to the present invention comprises a blood coagulation factor X and its active form, a blood coagulation factor X.
A composition containing factor a, blood coagulation factor X, or a composition containing blood coagulation factor Xa is contained therein.

Conventionally, the above factors X and Xa themselves are known as proteolytic enzymes involved in the blood coagulation system, but are used in a blood test container to reduce the pretreatment time of a sample. It was never done. However, the present inventors have obtained a new finding that unlike the same blood coagulation factor, such as thrombin, is unlikely to cause an unexpected abnormal coagulation delay phenomenon, and reached the present invention.

In the present invention, the shape of the blood test container is not particularly limited, and a conventionally known one such as a bottomed tube or a bottle is used. In addition, the material of the blood test container is not particularly limited, and conventionally known materials such as various glasses and thermoplastic resins are used.

In the present invention, blood coagulation factor X or its active form, blood coagulation factor Xa, may be used alone, but it suppresses inactivation and prevents blood clot components from being contained in the test container. Polymers such as vinylpyrrolidone, vinyl alcohol and hydroxyalkyl methacrylate, and copolymers of these with vinyl acetate; polymers such as ethylene glycol and propylene glycol; and copolymers thereof, to prevent sticking to wall surfaces Alternatively, various water-soluble and hydrophilic polymer substances such as denatured cellulose and denatured silicone oil having polar groups introduced therein, such as alkoxide denatured, may be used in an amount of 0.001 mg to 10 mg per 1 mL of blood, and / or an amino group or a carboxyl group in one molecule. Group containing various amphoteric compounds, etc.
It is preferably used as a combined composition in which 1 mg to 10 mg are used in combination.

[0015] To be stored in a blood test container, a solution or suspension of the composition is dispensed to the bottom of the container or sprayed on the inner wall surface, and then freeze-dried or dried.
A known method such as vacuum drying at a temperature of not more than ℃ may be used. Also, in order to increase the contact area with blood, it is granulated into granules or applied to a carrier such as a nonwoven fabric or synthetic resin sheet or beads that are physically and chemically inert to blood. May be accommodated. In addition, the composition may be laminated and applied separately for each component, or may be granulated for each component.

The amount of blood coagulation factor X or its active form, blood coagulation factor Xa, is preferably 0.0001 μg or more per mL of blood. 0.0
If it is less than 001 μg, it is difficult to obtain a sufficient coagulation promoting effect. On the other hand, if it exceeds 1000 μg, the protein concentration in blood may be adversely affected. 0.0 in terms of cost and effectiveness
01 to 10 μg is more preferred. It is preferable to use factor Xa, which is an active form thereof, rather than factor X as a main component.

Blood coagulation factor X or blood coagulation factor X
Factor a is extracted from human or animal blood. Of the amino acid residues constituting these, only the portion related to the activity is selectively cut out, or a modified product or a derivative prepared by genetic engineering is used. No problem.

In addition to the present invention, fine powders such as silica-based minerals such as silica, kaolin and bentonite, and cis-type α-diketo compounds are added in an amount of 0.01 to 10 m / mL of blood.
When used together, the activation of blood coagulation factor XII, which acts at different stages of blood coagulation, is promoted, so that it has a synergistic effect in promoting the conversion of factor X to factor Xa, Activating to promote blood clot shrinkage is preferable because the serum separation efficiency can be further increased.

These activators of factor XII, when in direct contact with proteins such as factor X and factor Xa, adsorb them and change the tertiary structure of the factor to deactivate it. When housed in a test container, it may be dividedly applied to different portions of the factor X or factor Xa, for example, to different portions of the inner wall surface, or may be separately applied, Or when one is applied to the inner wall,
It is preferable to separate and arrange the other, for example, in the form of granules or by supporting it on a carrier.

In the present invention, when a water-soluble polymer containing a quaternary ammonium group in the molecule or a basic protein such as protamine sulfate is used in combination, even if the blood is in an anticoagulated state due to a heparin salt, it can be used in blood. Since heparin in the blood can be captured and inactivated, the blood coagulation power is recovered, and the coagulation promoting action of factor X or factor Xa is enhanced, which is preferable.

Further, in order to activate other coagulation factors or platelets, a small amount of thrombin may be used in addition to the present invention. A small amount of thrombin is used for blood coagulation factor V and VIII
In order to activate the factor, the coagulation time can be reduced and the serum separation efficiency can be increased by using the factor Xa in combination with the above-mentioned silica or kaolin. When the amount of such thrombin used is less than 0.01 unit per 1 mL of blood, the effect of addition is not seen, and when it exceeds 5.0 units, a fibrin gel having a barrier property is formed immediately after the reaction between thrombin and blood. Since diffusion of factor Xa is prevented, 0.0
1 to 5.0 units are preferable, and 0.1 to 2.0 units are more preferable.

[0022]

The blood test container of the present invention contains blood coagulation factor X, its active form, blood coagulation factor Xa, a composition containing blood coagulation factor X, or a composition containing blood coagulation factor Xa. Is housed inside. Factor Xa is an enzyme that acts before the stage in which thrombin is involved in the blood coagulation reaction system, and when these and a composition containing them diffuse into collected blood, they are originally contained in the blood. Prothrombin is converted to activated thrombin. The thrombin generated in the blood converts fibrinogen originally contained in the blood into fibrin monomer as described above, and finally generates a fibrin gel in which blood cell components are entangled, thereby completing coagulation. I do. That is, the Xth
Factor a is not involved in the final coagulation reaction that converts fibrinogen to fibrin, so unlike thrombin or a thrombin-like enzyme derived from snake venom, a barrier fibrin gel that surrounds factor Xa at the moment of contact with blood. It is thought that the abnormal coagulation delay phenomenon is unlikely to occur because it does not form and can diffuse throughout the surrounding blood.

The factor X may be a factor XII or a factor IX.
After being converted to factor Xa via activation of the factor,
As in the case of factor Xa, a barrier fibrin gel surrounding factor X is not formed at the moment of contact with blood, as in the case of factor Xa. Conceivable. Therefore, it is released from the heavy overturning operation immediately after blood collection, and a sufficient effect can be achieved by a slight overturning operation of about 1 to 2 times.

[0024]

Next, embodiments of the present invention will be described. Example 1 Human-derived blood coagulation factor Xa (manufactured by SERBIO) at 10 μg / mL, polyvinylpyrrolidone (manufactured by Wako Pure Chemical Industries) at 2% weight / volume (W / V)%, polyethylene oxide-modified silicone oil ( An aqueous solution was prepared so as to have a concentration of 0.1 W / V%. Spray 25 μl of this solution onto the inner wall surface of a 5 mL polyethylene terephthalate blood collection tube,
Vacuum drying was performed at 0 ° C. to prepare a blood test container of the present invention. The blood concentration of factor Xa at the time of 4 mL blood collection was 0.0
6 μg / mL. 4 mL of fresh blood collected from a Japanese white rabbit is dispensed into the above container, mixed twice by inversion, and allowed to stand at room temperature of about 20 ° C. As observed. Further, about 5 minutes after blood collection, the blood was centrifuged at about 1800 G for 5 minutes, and the state of serum separation such as hemolysis and fibrin precipitation in the supernatant was observed. The above results are shown in Table 1, where clear coagulation behavior was observed and the serum separation state after centrifugation was also very good. In addition, serum was sampled, and the influence of typical biochemical tests and serological test items on test values was evaluated. The results are shown in Table 2. As compared with Comparative Example 2, the same results were obtained.

Example 2 A blood test container was prepared and evaluated in the same manner as in Example 1, except that the concentration of blood coagulation factor Xa in the aqueous solution was 1.7 μg / mL. The blood concentration of factor Xa at the time of collecting 4 mL of blood is 0.01 μg / mL. The results are shown in Table 1, where clear coagulation behavior was observed and the serum separation state after centrifugation was very good. In addition, Table 2 shows the results of evaluation of the influence on the test values, and results comparable to Comparative Example 2 were obtained.

Example 3 Blood coagulation factor Xa was 10 μm
g / mL, vinylpyrrolidone / vinyl acetate copolymer (BA
An aqueous solution was prepared so that 1 W / V% of SF) and 1 W / V% of aminomethylcyclohexanecarboxylic acid (manufactured by Aldrich). This solution (25 μl) was spray-coated on the inner wall surface of a polyethylene terephthalate (5 mL) blood collection tube, and dried at about 40 ° C. under vacuum. No. X at 4mL blood sampling
The blood concentration of factor a is 0.06 μg / mL. 5 W / V% of finely divided silica (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.1% of a nonionic surfactant (manufactured by Asahi Denka Co., Ltd.) composed of an ethylene oxide / propylene oxide block copolymer.
A methanol solution was prepared so as to have a W / V%, and 5 mL of this solution was applied to 500 g of polystyrene beads of about 1 mmφ (manufactured by Sekisui Chemical Co., Ltd.) and dried to prepare a solution. Thereafter, 0.2 g of the silica-coated beads were housed in the blood collection tube described above to prepare a blood test container of the present invention, and the evaluation was performed in the same manner as in Example 1. The results are shown in Table 1, where clear coagulation behavior was observed and clot retraction was also good. Also, the serum separation state after centrifugation was very good. Further, the results of evaluation of the influence on the test values are shown in Table 2. As a result, a result comparable to that of Comparative Example 2 was obtained.

Example 4 A blood test container was prepared in the same manner as in Example 1 except that thrombin was added to the aqueous solution of Example 1 so as to be 160 units / mL. Was evaluated. The blood concentration of factor Xa at the time of collecting 4 mL of blood is 0.06 μg / mL, and the blood concentration of thrombin is 1.0 unit / mL. The results are shown in Table 1, where clear coagulation behavior was observed and the serum separation state after centrifugation was very good.

Example 5 A blood test container was prepared in the same manner as in Example 1 except that thrombin was added to the aqueous solution of Example 1 so as to be 80 units / mL. Was evaluated. The blood concentration of factor Xa at the time of collecting 4 mL of blood is 0.06 μg / mL, and the blood concentration of thrombin is 0.5 units / mL. The results are shown in Table 1, where clear coagulation behavior was observed and the serum separation state after centrifugation was very good.

Comparative Example 1 Example 1 was repeated except that thrombin (manufactured by Mochida Pharmaceutical Co., Ltd.) was used at a concentration of 1600 units / mL in place of blood coagulation factor Xa in an aqueous solution to be spray-coated on a blood collection tube. In the same manner as described above, a blood test container was prepared and evaluated. The blood concentration of thrombin at the time of collecting 4 mL of blood is 10 units / mL. The results are shown in Table 1. Although the blood fluidity disappeared relatively quickly, the clot was weak and the coagulation behavior was not clear. In addition, fibrin was precipitated in the supernatant after centrifugation, and its volume occupied half of the supernatant layer. Table 2 shows the results of evaluation of the effect on test values.
As shown in the above, results comparable to those of Comparative Example 2 were obtained.

(Comparative Example 2) 4 mL of fresh blood collected from a Japanese white rabbit was dispensed into two clean 5 mL hard glass blood collection tubes each of which was mixed twice by inversion.
It was left at room temperature of 0 ° C. One of them was evaluated in the same manner as in Example 1. The results are shown in Table 1. The coagulation could not be completed by the standing time until the centrifugation, and a large amount of fibrin gel precipitated in the supernatant layer. After confirming complete coagulation after 2 hours, the other one was centrifuged at about 1800 G for 5 minutes to collect serum, and a typical biochemical test, serology was performed. Inspection items were measured and used as reference values for evaluating the effects on inspection values of other examples.
The results are shown in Table 2.

Comparative Example 3 A blood test container was prepared and evaluated in the same manner as in Comparative Example 1, except that the thrombin concentration in the aqueous solution was 160 units / mL. The blood concentration of thrombin at the time of collecting 4 mL of blood is 1.0 unit / mL. The results are shown in Table 1. Although the blood fluidity disappeared relatively quickly, the clot was weak and the coagulation behavior was not clear. Further, fibrin was precipitated in the supernatant after centrifugation, and its volume occupied 2/3 of the supernatant layer.

[0032]

[Table 1]

[0033]

[Table 2]

[0034]

The blood test container of the present invention is as described above, has no adverse effect on biochemistry, serological tests, etc., and differs from a blood collection tube containing thrombin or a thrombin-like enzyme derived from snake venom. The time required for sample pretreatment can be reduced without the need for intensive mixing work, which can further contribute to outpatient customer satisfaction.

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Hironobu Ishikawa 4560, Kaiseicho, Shinnanyo-shi, Yamaguchi Prefecture Sekisui Chemical Co., Ltd. F-term (reference) 2G045 AA01 BB29 BB38 CA25 HA06 4C038 UA02 UC04

Claims (1)

[Claims] 1. A blood coagulation factor X, its active form, blood coagulation factor Xa, a composition containing blood coagulation factor X, or a composition containing blood coagulation factor Xa is contained therein. A blood test container, characterized in that: