JPH0783920A - Blood-sampling tube for inspecting catechol amine - Google Patents

Abstract

PURPOSE:To eliminate the causes of error with a simple method and then accu rately detect catechol amine in serum and plasma by using a blood-sampling tube for inspecting catechol amine for serum where a reducer is sealed into the tube and for plasma where the reducer and a blood coagulation resistance agent are sealed. CONSTITUTION:A reducer is sealed into both blood-sampling tubes for serum and plasma to prevent oxidation breakdown of catechol amine at the moment when blood is sampled by the blood-sampling tubes. A reducer consisting of 0.1-500mg metasodium bisulfate, 1-500mg thiosodium sulfate, 0.1-500mg ascorbic acid, and 0.1-500mg glutathione per 1ml sampled blood is sealed into each blood-sampling tube. Also, the total amount of sealed reducer is increased or decreased when a plurality of reducers are used together. Further, a blood coagulation resistance agent is sealed into the blood-sampling tube for plasma in addition to the reducer. For example, coagulation resistance agents such as heparin, sodium citric acid, EDTA, and sodium fluoride are sealed independently or together.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a blood collection tube for precisely examining catecholamines in serum and plasma.

[0002]

BACKGROUND ART Adrenaline, noradrenaline and dopamine are known as catecholamines which are physiologically important components in the living body. Adrenaline is an adrenal medulla hormone, noradrenaline is a sympathomimetic transmitter, and dopamine is a precursor of noradrenaline and a central nervous system acting substance itself.
It plays an important role in the living body.

[0003] The above catecholamines normally exhibit physiological activities in various parts of the living body by circulating in blood, and the occurrence of catecholamines in the living body beyond a certain quantitative range correlates with functional changes. Therefore, it is possible to diagnose and prevent the disease by measuring the amount in blood, for example, pheochromocytoma, sympathoblastoma,
It is used in clinical settings for diagnosis of high blood pressure.

As a method for measuring the amount of catecholamine present in blood, a method is generally used in which blood is directly collected from a blood vessel and then analyzed and quantified by a predetermined method. In addition, as a method for collecting blood, a method using a blood collection tube has become popular today.

Although catecholamine is present in blood, its abundance is several pg to several hundred pg due to its functional properties.
It is an extremely small amount of / 1 ml, and a slight measurement error directly affects the diagnostic result. Furthermore, since catecholamines are easily oxidized and start to decompose in a neutral or alkaline solution from the moment of collection from blood (Yuichi Kumahara et al., “Biohormones”, Medical Institute, 481-501 (19).
77)), accurate measurement will be difficult unless some measure to prevent collapse is taken after blood collection.

For the purpose of solving this problem, a method of adding ascorbic acid or glutathione as a stabilizer of catecholamine to blood after blood collection is currently considered when measuring catecholamine concentration in blood (Cli).
n. Chem. , 27, 1983, (1981)). However, this method requires a great deal of time and labor for the preparation of the reagents in advance, so that when performing a large number of tests efficiently, it involves an extremely complicated operation, and in a short time from the blood collection to the stabilizer addition measure. However, it is impossible to prevent the catecholamine from collapsing, so that the measurement error cannot be eliminated and the reliability of the test result is poor.

[0007]

SUMMARY OF THE INVENTION In view of the above, the present invention provides a blood collection tube for a catecholamine test which is a simple operation capable of simultaneously testing a large number of samples and which prevents a measurement error from occurring. That is the purpose.

[0008]

[Means for Solving the Problems] The inventors of the present invention, while advancing research aiming at the above, focused on the oxidative degradation of catecholamines under the catalysis of heavy metals, and at the moment of blood collection by a blood collection tube. The inventors have found that if this oxidative disintegration can be prevented, it is suitable for the purpose, and that this disintegration inhibition can be carried out in the blood collection tube from which blood was collected, and the present invention has been completed.

The gist of the present invention is to enclose a reducing agent in the tube of a blood collection tube in advance for the purpose of stabilizing catecholamine before blood collection. Examples of the reducing agent used in the present invention include sodium metabisulfite, sodium thiosulfate, ascorbic acid, glutathione, dithiothreitol and the like. All of these are reagents widely used in the fields of biochemistry and clinical chemistry. In the present invention, these reducing agents may be used alone or in combination.

When sodium metabisulfite is used in the present invention, 0.1 to 500 m per 1 ml of blood collected.
g is enclosed in a blood collection tube. In the case of sodium thiosulfate, the same amount is 1 to 500 mg, in the case of ascorbic acid, the same amount is 0.1 to 500 mg, in the case of glutathione, the same amount is 0.1 to 500 mg, and in the case of glutathione, the same amount is 0. 5 to 500 mg, and in the case of dithiothreitol, 1 to 500 mg are sealed in the blood collection tube. If the encapsulation amount is smaller than the above, the stabilizing effect of catecholamine will be reduced, and even if it is larger than the above, more stabilizing effect cannot be expected and it will be wasted and it may affect the measurement result of catecholamine. Therefore, the enclosed amount is limited to the above range. In the present invention, when a plurality of the above reducing agents are used in combination,
The total amount of the enclosed amount can be appropriately increased or decreased for use.

Next, the present invention 2 will be described. When the blood collection tube of the present invention is applied for plasma, in addition to the above reducing agent,
A blood anticoagulant can be enclosed at the same time. Examples of such a blood anticoagulant include heparin, sodium citrate, EDTA, sodium fluoride and the like. These blood anticoagulants may be used alone or in combination.

In the present invention and the present invention 2, in addition to the reducing agent according to the present invention or the reducing agent according to the present invention 2 and a blood anticoagulant, ethylenediamine is used for the purpose of masking heavy metals that catalyze the oxidation of catecholamines. Tetraacetic acid (E
A chelating agent such as DTA) or ethylene glycol tetraacetic acid (EGTA) can be sealed in the tube at the same time. By thus simultaneously encapsulating the chelating agent, the effects of the present invention and the second invention can be further ensured.

When EDTA is used as described above, 0.01 to 10 EDTA · 2Na per 1 ml of blood is collected.
0 mg or 1 ml blood when using EGTA
0.01 to 200 mg per unit is enclosed in a blood collection tube together with the reducing agent. If the filling amount is smaller than the above, the effect of masking heavy metals is lost, and if the filling amount is larger than the above, coagulation of the sample is affected, so the amount is limited to the above range.

Usually, when a vacuum blood collection tube is used as a blood collection tube, the blood in the blood vessel is instantaneously sucked into the vacuum blood collection tube through the injection needle. When a vacuum blood collection tube is used as the blood collection tube of the present invention and the present invention 2, since the reducing agent exists in the blood collection tube, the blood sucked from the blood vessel is instantaneously mixed with the reducing agent, and the catecholamine It is possible to completely prevent oxidative degradation. Therefore, it is preferable that the blood collection tube is a vacuum blood collection tube used in the vacuum blood collection method.

The blood collection tube of the present invention and the second invention may have any structure as long as it is a commonly used blood collection tube. Usually, a glass tube or a plastic tube is sealed with a rubber stopper. Can be configured.

The reducing agent, the blood anticoagulant and the chelating agent according to the present invention and the second invention can be enclosed in a blood collection tube in the form of a solution or powder. In addition to the above reducing agent, blood anticoagulant, and chelating agent, a serum separating agent and the like can be simultaneously enclosed if necessary.

[0017]

EXAMPLES The present invention will be described in more detail with reference to test examples and examples.

Test Example 0 to 500 mg was added to 5 ml of pooled plasma collected beforehand to remove endogenous dopamine by leaving it at room temperature for 72 hours.
As a catecholamine stabilizer, sodium metabisulfate, sodium thiosulfate, ascorbic acid, glutathione, and dithiothreitol were added and mixed so as to have a concentration of / ml. To 5 ml of this catecholamine stabilizer-added pool plasma, dopamine hydrochloride solution (15 ng /
0.1 ml of 1 / 1000N hydrochloric acid solution) was added to prepare dopamine-added plasma having a concentration of 300 pg / ml. After 1 ml of this dopamine-added plasma was prepared and left at room temperature for 60 minutes, the dopamine concentration (DA) remaining without being decomposed was measured by the catecholamine measurement system SS.
It was measured using LC-20 (manufactured by Sekisui Chemical Co., Ltd.) to examine the stabilizing effect of each reagent on a sample of known concentration. Table 1
The measurement results of the dopamine concentration are shown in. 300 mg in the table
Values above / ml are due to errors in preparation and measurement of dopamine-added plasma.

[0019]

[Table 1]

As can be seen from the above results, each of the reagents used has a catecholamine stabilizing effect, and the effective concentration range is 0.1 mg to 500 mg of sodium metabisulfite and 1 m of sodium thiosulfate per 1 ml of blood.
g-500 mg, ascorbic acid 0.1 mg-500 m
g, glutathione 0.5 mg to 500 mg, and dithiothreitol 1 mg to 500 mg.

Example 1 A vacuum blood collection tube containing ascorbic acid was prepared by adding 5 mg of ascorbic acid to a 7 ml glass blood collection tube and using a rubber stopper as a closing material and vacuum stoppering under vacuum. Using this blood collection tube, 3 ml of blood was collected from a healthy male person three times for the same person, and after standing at room temperature for 60 minutes, 120 minutes, and 240 minutes, 1 ml of serum was collected by centrifugation. The noradrenaline concentration (NA) in 1 ml of this serum was catecholamine measuring system SSL
It measured using C-20 (made by Sekisui Chemical Co., Ltd.). The results are shown in Table 2.

Comparative Example 1 The procedure of Example 1 was repeated except that ascorbic acid was not added. The results are shown in Table 2.

[0023]

[Table 2]

[0024] Usually, when separating serum, it is necessary to take a sufficiently long time to form a strong blood clot so that blood cell components are not mixed in the serum layer. As can be seen from Table 2, ascorbic acid had the effect of stabilizing noradrenaline even when left at room temperature for 240 minutes. The present invention is extremely useful for measuring catecholamine concentration in serum.

Example 2 To a 7 ml plastic blood collection tube, 3 mg of sodium metabisulfite, 0.1 mg of EDTA and 5 units of heparin were added, and a rubber stopper was used as a vacuum stopper plugging material. A vacuum blood collection tube containing sodium sulfite, EDTA and heparin was prepared. Using this blood collection tube, 5 ml of blood was collected from a healthy man and immediately 2 ml of plasma was collected by centrifugation. The concentration of noradrenaline (NA), adrenaline (AD) and dopamine (DA) in 1 ml of this plasma was measured by a fully automatic catecholamine analyzer H.
It measured using LC-8030 (made by Tosoh Corporation). The measurement results are shown in Table 3.

Comparative Example 2 Example 2 was repeated except that sodium metabisulfite and EDTA were not added. The measurement results are shown in Table 3.

[0027]

[Table 3]

In Comparative Example 2, measurement was performed immediately after blood collection, but dopamine was already undetectable due to decomposition. In addition, it was confirmed that the measured values of the other two components were lower than those of the system containing the stabilizer. The present invention is extremely useful for improving the sensitivity of test values.

[0029]

Industrial Applicability When the vacuum blood collection tube of the present invention is used, catecholamine is stabilized at the same time as blood collection, so that catecholamine does not disintegrate, so that the sensitivity of catecholamine measurement can be increased and the reproducibility of measurement can be enhanced. In addition, the labor of adding a stabilizing reagent after blood collection, which has been conventionally performed, can be omitted, so that the examination efficiency can be improved.

Claims (2)

[Claims] 1. A blood collection tube for a serum catecholamine test, characterized in that a reducing agent is enclosed in the tube. 2. A blood collection tube for plasma catecholamine test, characterized in that a reducing agent and a blood anticoagulant are enclosed in the tube.