JPH1146798A - Solution reagent for measuring blood coagulation time and dry composition for solution reagent for blood coagulation time - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject solution reagent high in storage stability and useful for clinical examinations, etc. by including tissue thromboplastin, a calcium ion-forming compound, a saccharide, a polyhydric alcohol, etc. in water or a bubber solution. SOLUTION: This solution reagent contains tissue thromboplastin, a calcium ion-forming compound (e.g. calcium dichloride), and a saccharide having three or more equatrial OH groups in the molecule (e.g. trehalose) and/or a polyhydric alcohol having three or more OH groups in the molecule (e.g. sorbitol) in water or a buffer solution. The solution reagent is high in storage stability, gives highly reliable measurement results also after long period storage, and can be used as a clinical examination reagent useful for diagnosing hepatic disorders, for controlling the coagulability of blood, etc. on clinical examinations, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a solution reagent for measuring blood coagulation time used in clinical tests.

[0002]

2. Description of the Related Art Blood coagulation ability is greatly affected by various diseases, anticoagulants or trauma. Therefore, blood coagulation ability is measured to obtain detailed information on the patient's condition. For example, since vitamin K is synthesized in the liver, if there is a disease in the liver, blood coagulation II, VII, IX, activated by the action of vitamin K
Factor X activity is reduced. Therefore, the blood coagulation ability is measured, the activity of these blood coagulation factors is examined, and liver damage can be known from the results. During surgery, a drug such as heparin is administered to reduce the blood coagulation ability to a certain range. At this time, the blood coagulation ability is measured to control the dose of heparin or the like.

[0003] Various methods have been used to measure the blood coagulation ability of humans and animals. For example, there are measurement of blood coagulation time, quantification of each blood coagulation factor using an antigen-antibody reaction, and the like. Among them, the measurement of blood coagulation time is widely used because of its simplicity.

[0004] The blood coagulation time is the time from the start of the blood coagulation reaction to the completion of the blood coagulation reaction.
Since the blood coagulation reaction is a cascade reaction in which reactions involving blood coagulation factors occur sequentially in many stages, if the concentration of a certain blood coagulation factor is low, the reaction involving that blood coagulation factor progresses slowly. The resulting blood clotting time is longer than normal. Therefore, by measuring the blood coagulation time and comparing it with the blood coagulation time of a normal person, an abnormality in the blood coagulation factor concentration in the blood can be known. For example, a substance or catalyst that triggers a certain blood coagulation reaction is reacted with a plasma sample or a whole blood sample (hereinafter, simply referred to as a blood sample) to be measured, and the blood coagulation time is measured. It is possible to know whether or not there is a shortage of a specific coagulation factor involved in the coagulation reaction. As test items for blood coagulation time using this measurement principle, prothrombin time (PT) for testing the normal / abnormality of the extrinsic coagulation pathway, and activated partial thromboplastin time (APTT) for testing the normal / abnormality of the intrinsic coagulation pathway And the like.

[0005] By the way, these conventionally used methods for measuring blood coagulation time are mainly methods using solution reagents, and these methods are still widely used at present. In this method, first, a solution reagent is prepared by adding distilled water or the like to a lyophilized reagent, and the solution reagent is prepared. The solution reagent is brought into contact with a blood sample to react. Is measured by measuring the time of blood coagulation.

Hereinafter, an example using the lyophilized reagent for PT measurement in the above method will be specifically described. The lyophilized reagent for PT measurement contains tissue thromboplastin and calcium salt as main components and is generally commercially available. In the measurement using the lyophilized reagent, first, a PT measurement solution reagent in which the lyophilized reagent was converted into an aqueous solution was prepared and heated, and then added to a preliminarily heated blood sample. Immediately after the addition, the change in turbidity or viscosity of the reaction system is measured (the turbidity and viscosity of the reaction system change when coagulation starts due to fibrin precipitation). The point at which a certain turbidity or viscosity change occurs is defined as the end point of the blood coagulation reaction, and the time from the addition of the blood sample to the end point is determined as the blood coagulation time. The blood coagulation time changes according to the activity of a coagulation factor involved in the PT reaction pathway (extrinsic coagulation pathway), and the longer the activity, the longer the blood coagulation time.

[0007]

By the way, if a solution reagent containing tissue thromboplastin and calcium salt, such as the above-mentioned solution reagent for PT measurement, is allowed to stand, precipitation occurs in several hours. When the blood coagulation time is measured using the solution reagent without resuspending the precipitate, the blood coagulation time is extended corresponding to the degree of sedimentation from the value that should be originally obtained. I understand. That is, it has been clarified that the performance of the reagent which has been conventionally used is deteriorated only by storing it in a stationary state, and the reagent cannot be stored for a long time.

[0008]

As described above, a clear correlation was found between the formation of a precipitate in the solution reagent for measuring blood coagulation time and the deterioration of the performance. Focusing on the stabilization of the suspension state of the reagents, intensive research was conducted on the method of stabilizing the suspension state of the solution reagent. As a result, they have found that the suspension state of the reagent can be stabilized by adding a specific amount of saccharide and / or polyhydric alcohol to the solution reagent containing tissue thromboplastin and calcium salt. Then, it was found that the performance of the solution reagent was stabilized for a long time, thereby completing the present invention.

That is, the present invention relates to a method for treating tissue thromboplastin, a calcium ion-forming compound in water or a buffer,
And a solution reagent for measuring blood coagulation time comprising a saccharide and / or a polyhydric alcohol. Preferably, the saccharide to be contained is a saccharide having three or more equatorial OH groups in one molecule. The solution reagent for measuring blood coagulation time is a polyhydric alcohol having three or more OH groups in one molecule.

Further, another invention is a dry composition for a solution reagent for measuring blood coagulation time, which comprises tissue thromboplastin, a calcium ion-forming compound, and a saccharide and / or a polyhydric alcohol, and preferably contains the composition. A solution reagent for measuring blood coagulation time, wherein the saccharide is a saccharide having three or more equatorial OH groups in one molecule or the polyhydric alcohol to be contained is a polyhydric alcohol having three or more OH groups in one molecule It is a dry composition for use.

In the solution reagent for measuring blood coagulation time of the present invention, 1) its viscosity is increased by containing a saccharide and / or a polyhydric alcohol, and collision between tissue thromboplastins in the reagent is suppressed, or 2). The moderate surfactant activity of saccharides and polyhydric alcohols suppresses the aggregation of tissue thromboplastin based on the affinity between hydrophobic regions, thereby suppressing the formation of aggregated tissue thromboplastin aggregates Seem.
Since saccharides and polyhydric alcohols do not inhibit the blood coagulation reaction, the solution reagent for measuring blood coagulation time of the present invention can provide highly reliable measurement results even after long-term storage (storage stability is low). Higher). In addition, saccharides having a large number of equatorial OH groups present in one molecule and polyhydric alcohols having a large number of OH groups in one molecule have high viscosity increasing effect and surface active effect. It seems that the above effect is particularly high when is added.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The tissue thromboplastin used in the present invention is a blood coagulation factor involved in the extrinsic coagulation pathway extracted from bovine brain, rabbit brain or the like with acetone or the like. Component. Tissue thromboplastin is a mixture of a plurality of proteins and lipid components, and exists in suspension in a solution reagent. The content (concentration) of tissue thromboplastin in the blood clotting time measuring solution reagent of the present invention is not particularly limited, and is generally 10 μg /
It is used at a concentration of 1 to 50 mg / ml from the point that the progress of the blood coagulation reaction is more stable.
Is preferred.

The calcium ion forming compound used in the present invention is a source of calcium ions essential for the blood coagulation reaction, and examples thereof include calcium salts such as calcium chloride and calcium lactate. The concentration of the calcium ion-forming compound in the solution reagent for measuring blood coagulation time of the present invention is not particularly limited, but is generally about 1 to 20 mM. The concentration is particularly preferably about 4 to 15 mM, because the progress of the blood coagulation reaction is more stable.

The saccharides used in the present invention include monosaccharides such as glucose, fructose, ribose, mannose and xylose, sucrose, trehalose, cellobiose,
Examples include disaccharides such as maltose, isomaltose, maltitol, palatinose, trehalose, and xylobiose, and polysaccharides such as oligosaccharides having a plurality of sugars such as trimaltose, glucosylsucrose, and oligoxylose, and dextran.

Among them, from the viewpoint of the high effect of the present invention,
Saccharides having three or more equatorial OH groups in one molecule can be suitably used.

The term "equatorial OH group" as used herein means an OH group arranged in a direction substantially parallel to the surface of the cyclic sugar molecule. In addition, saccharides generally exist as a mixture of a plurality of structural isomers, and the structural isomers can be mutually converted. Therefore, even if the same saccharide is used, the number of equatorial OH groups depends on the formed structural isomer. May be different.
Therefore, in such a case, the number of equatorial OH groups present in one molecule of the saccharide is determined by taking the average number in consideration of the abundance ratio of all possible structural isomers of the saccharide, and calculating the average number. Is the number of equatorial OH groups in one molecule. That is, when the saccharide has a structural isomer, each equatorial position O of each structural isomer
The sum of the product of the number of H groups and the abundance is determined, and the sum is used as the number of equatorial OH groups in one molecule.

For example, when the saccharide is glucose, α
The abundances of the type structural isomer and the β type structural isomer, and the number of OH groups at the equatorial position are 40% and 60%, respectively.
And the number of equatorial OH groups per glucose is 4 × 0.4 + 5 × 0.6.
= 4.6.

Examples of the saccharide having three or more equatorial OH groups in one molecule include glucose, mannose,
Sucrose, trehalose, oligoxylose, dextran and the like.

The polyhydric alcohol used in the present invention is not particularly limited as long as it has a plurality of OH groups in one molecule, and ethylene glycol, glycerol, sorbitol and the like can be used. In view of the above, it is preferable to use a polyhydric alcohol having three or more OH groups in one molecule. Examples of such polyhydric alcohols include glycerol and sorbitol.

The sugars and polyhydric alcohols may be used alone or as a mixture of a plurality of them.

The concentration of the saccharide and / or polyhydric alcohol in the blood clotting time measuring solution reagent of the present invention is not particularly limited, and may be appropriately determined depending on the types and amounts of the contained tissue thromboplastin and calcium ion-forming substance. In general, it can be suitably used in the range of about 5 to 40% by weight. In particular, when a plurality of saccharides and polyhydric alcohols are used as a mixture, the total concentration can be suitably used in the range of about 5 to 40% by weight, and the more preferable concentration range is about 10 to 30% by weight. %.

The solution reagent for measuring blood coagulation time of the present invention comprises:
The tissue or thromboplastin, the calcium ion-forming compound, and the saccharide and / or polyhydric alcohol are contained in water or buffer, but the water or buffer used at this time is not particularly limited. As the water, it is preferable to use distilled water or ion-exchanged water. When a buffer is used, a buffer may be added to the water. Buffers that can be used at this time include 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid (HEPES) and 3- [4- (2-hydroxyethyl) -1
-Piperazinyl] propanesulfonic acid (EPPS)
Od's buffers, tris (hydroxymethyl) aminomethane and the like are mentioned as examples. The concentration of the buffer is
The concentration is not particularly limited as long as it is a concentration that can sufficiently maintain the pH when mixed with the blood sample, and does not significantly inhibit the blood coagulation reaction, but it is generally preferable to use it in a range of 1 to 50 mM. It is.

Generally, in order for the blood coagulation reaction to proceed smoothly, it is desirable that the water or the buffer is a buffer having a pH of about 6 to 9; The buffer and the pH adjuster may be added to water to adjust the pH to about 6 to 9. Examples of the pH adjusting agent that can be used at this time include alkaline compounds such as sodium hydroxide and potassium hydroxide, or hydrochloric acid, sulfuric acid,
Examples include acidic compounds such as phosphoric acid. The amount of these pH adjusters to be added may be appropriately determined according to the concentration and type of the buffer, the desired pH and the like.

Further, an inorganic salt for controlling the speed of a blood coagulation reaction can be added to the solution reagent for measuring a blood coagulation time of the present invention. Examples of the inorganic salt include sodium salts such as sodium chloride, sodium bromide and sodium thiocyanate, and potassium salts such as potassium chloride. The concentration of the inorganic salt is not particularly limited, but is generally used in the range of 10 to 500 mM.

The method for preparing the solution reagent for measuring blood coagulation time of the present invention is not particularly limited, and a known method can be used. Generally, a predetermined amount of a calcium ion-forming compound and a saccharide and / or a polyhydric alcohol are added to a buffer solution, and the mixture is dissolved by stirring with a magnetic stirrer or the like. A method in which tissue thromboplastin is added to a lysate and then stirred for about 1 hour, and a predetermined amount of saccharide and / or polyhydric alcohol is contained in a freeze-dried tissue thromboplastin containing a commercially available calcium ion-forming compound. And a method of adding and mixing distilled water or ion-exchanged water to the dry composition for a solution reagent for measuring blood coagulation time of the present invention described below.

The solution reagent for measuring blood coagulation time of the present invention comprises:
A freeze-drying protective agent or the like is added thereto, and the mixture is freeze-dried to obtain a dry composition for a solution reagent for measuring a blood coagulation time, which can be stored in a dry state. Examples of the lyophilized protective agent that can be used at this time include amino acids such as bovine serum albumin, glycine, and glycylglycine. The amount of the lyophilized protective agent is not particularly limited, but is generally in the range of about 0.1 to 10% by weight when dissolved in distilled water or the like and used as the solution reagent for measuring blood coagulation time of the present invention. It is preferred that

The method for preparing the dry composition for a blood coagulation time measuring solution reagent of the present invention is not particularly limited. For example, a method in which the above-mentioned freeze-drying protective agent is added to the blood coagulation time measuring solution reagent of the present invention. Is dispensed into vials and freeze-dried after freezing.

The method of measuring blood coagulation time using the solution reagent for measuring blood coagulation time of the present invention is the same as that of the conventional method for measuring blood coagulation time except that the reagent used is the solution reagent for measuring blood coagulation time of the present invention. There is no particular difference from the measurement method using the solution reagent for use.

When the blood coagulation time is measured using the dry composition for measuring a blood coagulation time of the present invention, a predetermined amount of distilled water or the like is added to the dry composition to prepare a suspension. As a solution reagent solution, the measurement may be performed in the same manner as when a conventional solution reagent for measuring blood coagulation time is used using the solution reagent solution. For example, in a test tube, to a predetermined amount of a blood sample for measurement heated to 37 ° C., a predetermined amount of a solution coagulation time measuring solution reagent of the present invention preliminarily heated to 37 ° C. is added and mixed. The change is measured by monitoring the change in viscosity or turbidity and measuring the time until the turbidity or viscosity changes by a certain amount.

As a measuring device capable of measuring the blood coagulation time, commercially available KC-10A (Amerung), C
A-5000 (Toa Medical Electronics Co., Ltd.) and the like.

[0031]

EXAMPLES The present invention will be further described with reference to examples, but the present invention is not limited to these examples.

Examples 1 to 6 A storage stability test was carried out using the solution reagent for measuring blood coagulation time of the present invention.

First, 10 ml each of the solution reagents for measuring blood coagulation time of the present invention having the compositions shown in Tables 1 and 2 were prepared.

[0034]

[Table 1]

[0035]

[Table 2]

The reagents were prepared by adding and mixing the above components to 30 mM HEPES buffer (manufactured by Dojindo Chemical Co., Ltd.) in all of Examples 1 to 6, and then adjusting the pH to 7.35.

Each of the prepared solution reagents for measuring blood coagulation time was stored at 4 ° C. in a container with a lid. Immediately after the start of the test, 0.3 ml of the upper part of the solution reagent was sampled every other week, and a turbidity test and a reagent performance evaluation test were performed by the following methods.

[Turbidity test] Taking advantage of the fact that the turbidity at the upper part of the solution reagent is reduced by the occurrence of precipitation, 0.05 ml of the sampled solution reagent for measuring blood coagulation time and 0.35 ml of water are mixed. The measurement was performed by measuring the absorbance of the mixture at a wavelength of 660 nm.

[Reagent performance evaluation test] To 0.1 ml of commercially available control plasma (Dade CITROL-1), 0.2 ml of a sample reagent for measuring blood coagulation time was added.
The measurement was performed by measuring the blood coagulation time. The blood coagulation time was measured using KC-10A (manufactured by Amelung). The measurement was performed according to the instruction manual.

FIG. 1 shows the results of the turbidity test when a saccharide was added, and FIG. 2 shows the results of the turbidity test when a polyhydric alcohol was added. The results of the evaluation of the reagent performance are shown in FIG. 3, and the results of the evaluation of the reagent performance in the case of adding the polyvalent alcohol are shown in FIG.

As apparent from FIGS. 1 to 4, although the turbidity slightly increased depending on the type of sugar or polyhydric alcohol, the blood coagulation time was maintained substantially the same over 9 weeks. It was confirmed that the solution reagent for measuring blood coagulation time of the present invention had good storage stability over a long period of time.

The number of OH groups at the average equatorial position of the sugar contained and the OH of the polyvalent alcohol contained
It was also confirmed that the greater the number of groups, the higher the stability.

Comparative Example 1 A solution reagent having the same composition as the solution reagent for measuring blood coagulation time of Example 1 was prepared except that no ribose was added, and the same test as in Example 1 was performed.

FIG. 5 shows the result of the turbidity test of Comparative Example 1, and FIG. 6 shows the result of the reagent performance evaluation test. In Comparative Example 1, precipitation occurred already in the first week, and the turbidity was reduced because the suspension could not be maintained. In addition, the blood coagulation time increased with the number of storage days, and the reagent performance was significantly deteriorated. Results of FIGS. 1-4 and FIG.
It is clear from the comparison with the results of Examples 1 to 6 that Examples 1 to 6 have better results.

Example 7: Storage stability test of a solution reagent obtained by regenerating a dried composition for a solution reagent for blood coagulation time measurement A final concentration of 1% was added to the solution reagent for blood coagulation time measurement of Example 3.
And add bovine serum albumin (Sigma)
Dissolve and add 10 ml of this to a vial bottle.
After freezing for one day in a freezer, freeze-drying was performed to prepare a dry composition for a solution reagent for measuring blood coagulation time. In addition, freeze-drying is carried out in a vacuum state from -40 ° C to 40 ° C.
The temperature was raised stepwise in 8 hours.

Next, 10 ml of water was added to the prepared dry composition for blood coagulation time measuring solution reagent to form a suspended reagent solution. The same test as in Example 3 was performed on the reagent solution. A 9-week storage stability test was performed in the same manner as in Example 3.

FIG. 7 shows the results of the turbidity test, and FIG. 8 shows the reagent performance evaluation test. It was shown that the reagent solution obtained by regenerating the dried composition for a blood coagulation time measuring solution reagent had long-term suspension stability and stability of the reagent performance.

[0048]

The solution reagent for measuring blood coagulation time of the present invention has excellent long-term stability of reagent performance. The conventional solution-type reagent has a disadvantage that the reagent performance is gradually deteriorated only by standing and stored, and the reagent can be used for only a few days even when resuspended. As a result, the performance deteriorates without using the reagent,
They were often discarded. The present invention solves such disadvantages of the conventional reagent, and has a great feature that it can greatly contribute to cost reduction of a clinical test.

Conventionally, a new solution-type reagent had to be prepared and used every few days, and the performance of the reagent solution was different due to variations in the reagent preparation operation. There was also a problem that it fluctuated. The solution reagent for measuring blood coagulation time of the present invention also solves this problem, and has a feature that measurement with little variation can be performed for a long period of time. Therefore, it also contributes greatly to the accuracy of clinical tests. Thus, the industrial value of the present invention is very large.

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of turbidity tests of Examples 1 to 3.

FIG. 2 is a diagram showing the results of turbidity tests of Examples 4 to 6.

FIG. 3 is a diagram showing the results of reagent performance evaluation tests of Examples 1 to 3.

FIG. 4 is a diagram showing the results of reagent performance evaluation tests of Examples 4 to 6.

FIG. 5 is a diagram showing the results of a turbidity test of Comparative Example 1.

FIG. 6 is a diagram showing the results of a reagent performance evaluation test of Comparative Example 1.

FIG. 7 is a diagram showing the results of a turbidity test of Example 7.

FIG. 8 is a diagram showing the results of a reagent performance evaluation test of Example 7.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Yoshinori Yoshimura Inventor 1-1, Mikage-cho, Tokuyama-shi, Yamaguchi Pref.

Claims (4)

[Claims] 1. Tissue thromboplastin, a calcium ion-forming compound, and a saccharide and / or
Alternatively, a solution reagent for measuring blood coagulation time comprising a polyhydric alcohol. 2. The method according to claim 1, wherein the saccharide is OH at the equatorial position in one molecule.
The solution reagent for measuring blood coagulation time according to claim 1, which is a saccharide having three or more groups, and wherein the polyhydric alcohol is a polyhydric alcohol having three or more OH groups in one molecule. 3. A dry composition for a solution reagent for measuring blood coagulation time, comprising tissue thromboplastin, a calcium ion-forming compound, and a saccharide and / or a polyhydric alcohol. 4. A saccharide having an equatorial OH in one molecule.
The dry composition for a solution reagent for measuring blood coagulation time according to claim 3, wherein the saccharide is a saccharide having three or more groups, and the polyhydric alcohol is a polyhydric alcohol having three or more OH groups in one molecule.