JP2014197019A - Blood coagulation time extension reagent in blood coagulation reaction - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a reagent capable of sufficiently extending blood coagulation time, and correctly and sensitively implementing blood coagulation ability inspection without reducing an optical amount of change.SOLUTION: A blood coagulation ability measuring specimen diluting liquid contains a blood coagulation time extension reagent in blood coagulation reaction which assumes a benzamidine or its derivative to be an active ingredient.

Description

  The present invention relates to a blood coagulation ability measurement reagent typified by a fibrinogen measurement reagent, that is, a specimen dilution liquid for blood coagulation ability measurement using a blood coagulation time extending agent.

  The mechanism of blood coagulation is generally roughly divided into two systems. One is an endogenous system that starts with contact activation of blood coagulation factor XII with a foreign substance, and finally generates thrombin through a multistage reaction, and the other is blood coagulation by blood coagulation factor VII and tissue thromboplastin. It is an extrinsic system that begins with the activation of factor X and also generates thrombin (FIG. 1). In both systems, coagulation finally occurs as fibrinogen is converted into fibrin by the action of the generated thrombin. There are several blood coagulation ability tests using a blood coagulation activator in order to clarify the presence or absence of abnormalities in these blood coagulation mechanisms and the causes of the abnormalities, and they are widely used in clinical examinations.

Blood coagulation activators and blood coagulation tests using the same are as follows.
1) Blood coagulation test using thrombin Fibrinogen measurement, ATIII measurement, thrombin time measurement 2) Blood coagulation test using tissue thromboplastin Prothrombin time measurement, II, V, VII and X factor activity measurements using prothrombin time , Complex factor measurement (thrombotest, hepaplastin test, etc.)
3) Examination of blood coagulation ability using phospholipids Activity measurement of partial thromboplastin time measurement, activated partial thromboplastin time measurement, activated partial thromboplastin time measurement, activity of VIII, IX, XI, factor XII, prekallikrein, and high molecular weight kininogen , Snake venom time measurement, quantification of factor X using snake venom time measurement, lupus anticoagulant (LA) measurement using diluted snake venom time measurement, protein C activity measurement, thromboplastin production test any of the above 1) to 3) In this method, the time from the initiation of coagulation by mixing a reagent containing a blood coagulation activator or the like to a patient specimen until the fibrinogen is finally converted into fibrin and deposited is measured.

  Coagulation detection methods in blood coagulation ability tests can be broadly classified into mechanical detection methods and optical detection methods. The dynamic detection method is a method of monitoring a magnetic substance or the like put in a reaction solution by a magnetic force or the like and detecting that the viscosity becomes high due to solidification and the movement of the magnetic substance becomes slow. The optical detection method is a method for detecting that the reaction solution becomes white due to coagulation as a change amount of transmitted light or scattered light, and is most widely used because it is relatively simple. These are now generally detected by automated equipment. An example of the intensity change curve of the scattered light obtained by the scattered light detection method is shown in FIG. In the figure, point A indicates the point at which coagulation is induced by mixing a reagent containing a blood coagulation activator, and then fibrin precipitation starts through a multi-stage reaction. Change appears. When fibrinogen is consumed one after another and almost depleted in the reaction solution, the change in scattered light intensity disappears, the curve becomes flat like point C, and coagulation is completed. Based on such an intensity change curve of scattered light, the coagulation time is calculated by a known calculation parameter (Patent Document 1). Here, if the maximum value of the optical change amount (intensity change amount) of the scattered light is ΔH, it is obvious that the larger the ΔH, the sharper the coagulation detection and the more accurate detection becomes possible. Therefore, it is desirable that the reagent for measuring blood coagulation ability used has such a characteristic that the optical change amount is displayed large. However, if the change from point A to point C occurs in a very short time, the coagulation time cannot be measured with high accuracy. Therefore, a substance that extends the coagulation time is usually added to adjust the coagulation time to a desired length. As a blood coagulation time extender, Patent Document 2 exemplifies alkali metal or alkaline earth metal halide salts including sodium chloride, and Patent Document 3 exemplifies sodium propionate. However, such a conventional blood coagulation time prolonging agent has a problem in that it has a large negative effect of reducing ΔH depending on the addition amount at the same time. In other words, the amount of optical change after the addition of a reagent containing a blood coagulation activator cannot be obtained sufficiently, and there is a problem that measurement accuracy may be lowered. In the blood coagulation test, a multi-inspection curve created by plotting the measurement value of a sample prepared by serially diluting one type of calibration reference plasma with a sample diluent for the clotting time expressed in seconds. Based on the above, it is common to convert to a ratio (activity%) to normal activity. This is a major problem because it reduces the accuracy of the entire measurement.

  In order to solve the problems of these conventional methods, a method of adding a polymer substance such as polyethylene glycol, polyvinyl alcohol, and polymer polysaccharide has been proposed (Patent Document 4).

JP-A-8-15263 Japanese Patent No. 2994557 Japanese Patent No. 3330685 Japanese Patent No. 3074611

However, the method of adding a polymer substance to the reagent increases the viscosity of the reagent itself, resulting in a problem that the sorting accuracy with a pipette or a reagent probe is lowered, and a sufficient turbidity difference between uncoagulated and coagulated can be obtained. Therefore, there is a problem that a sufficient amount of optical change cannot be obtained.
Accordingly, an object of the present invention is to provide a specimen diluent containing a blood coagulation time extending agent that does not reduce the optical change amount in the blood coagulation reaction. This can contribute to improving the accuracy of the blood coagulation test.

  Therefore, the present inventor has searched for a blood coagulation time prolonging agent that does not decrease the optical change amount using various compounds, and benzamidine or a derivative thereof has an action of prolonging the blood coagulation time, and the optical at the time of coagulation. It has been found that blood coagulation ability can be measured accurately and with high sensitivity if it is used as an additive for a specimen dilution liquid for blood coagulation ability measurement.

  That is, the present invention provides a specimen diluent for measuring blood coagulation ability, which contains a blood coagulation time prolonging agent in a blood coagulation reaction containing benzamidine or a derivative thereof as an active ingredient.

  Furthermore, the present invention provides a blood coagulation time extension in a blood coagulation reaction selected from the above-mentioned blood coagulation time extender and a conventional blood coagulation time extender, that is, an alkali metal or alkaline earth metal halide salt and propionate. It is intended to provide a method for adjusting an optical change amount in a blood coagulation reaction, characterized by using an agent together.

  When the specimen diluent containing the blood coagulation time-prolonging agent of the present invention is used, the optical change amount is difficult to decrease, and thus blood coagulation ability such as fibrinogen measurement, PT, and APTT can be accurately measured.

The mechanism of blood coagulation in clinical examination is shown schematically. An example of a scattered light intensity change curve obtained by the scattered light detection method when measuring blood coagulation ability is shown.

  The blood coagulation time extending agent of the present invention comprises benzamidine or a derivative thereof as an active ingredient. In the present specification, “benzamidine or a derivative thereof” is used as a general term for benzamidine and compounds having benzamidine as a mother nucleus. Examples of benzamidine or derivatives thereof include benzamidine, p-aminobenzamidine, m-aminobenzamidine, and salts thereof. Examples of the salt include acid addition salts, and examples of the acid addition salt include hydrochloride, sulfate, nitrate, phosphate, sulfamate and the like. Of the benzamidine or derivatives thereof, benzamidine or a salt thereof is preferably used, and benzamidine hydrochloride is particularly preferable.

  The blood coagulation time prolonging agent of the present invention has the effect of prolonging the blood coagulation time without reducing the change in turbidity of the reaction solution in the measurement of blood coagulation ability as observed with conventional blood coagulation time extending agents. This indicates that the formation state of the fibrin network, which is the final product of blood coagulation, is stronger than the conventional method. Therefore, the blood coagulation time extending agent of the present invention can be used for an optical detection method for directly detecting a change in turbidity, a mechanical detection method for monitoring the formation state of a fibrin network with a magnetic substance, and the like. It is preferred to use. Here, the optical detection method includes a scattered light amount detection method and a transmitted light amount detection method, and is preferably used for the scattered light amount detection method.

  The blood coagulation ability test to which the blood coagulation time extending agent of the present invention can be applied is based on the fact that fibrinogen is finally converted into fibrin from the initiation of coagulation by mixing a reagent containing a blood coagulation activator or the like with a patient sample. What is necessary is just to measure the time until precipitation. Examples include fibrinogen measurement, ATIII measurement, thrombin time measurement, prothrombin time measurement, II, V, VII, factor X activity measurement using prothrombin time, complex factor measurement (thrombotest, hepaplastin test, etc.), partial thromboplastin Time measurement, activated partial thromboplastin time measurement, activated partial thromboplastin time measurement using VIII, IX, XI, factor XII, prekallikrein, macromolecular kininogen activity measurement, snake venom time measurement, snake venom time measurement using factor X Quantification, lupus anticoagulant (LA) measurement using dilution snake venom time measurement, protein C activity measurement, thromboplastin production test, and other detection of abnormal coagulation.

  In the measurement of the blood coagulation ability test, a blood coagulation activator-containing reagent (for example, a thrombin-containing reagent in fibrinogen measurement) is added to a specimen, and the time until fibrin is precipitated is measured. The blood coagulation time prolonging agent of the present invention may be contained in a measurement system, and may be contained in a reagent containing a blood coagulation activator or the like, or may be contained in a sample diluent. It is preferable to make it contain. That is, the blood coagulation time prolonging agent of the present invention is preferably contained in a specimen diluent for fibrinogen measurement, for example.

  The blood coagulation activator-containing reagent only needs to be used for the blood coagulation ability test. Examples include a thrombin-containing reagent, a tissue thromboplastin-containing reagent, a phospholipid-containing reagent, and the like. Further, the specimen diluent may be any one that is used for the blood test ability test. For example, MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, etc. Good buffer solution, citrate buffer Liquid, phosphate buffer, acetate buffer, imidazole buffer, barbital buffer, physiological saline, water and the like.

The blood coagulation time extending agent of the present invention is preferably contained in the reaction solution so as to obtain a coagulation time that can be accurately measured in a blood coagulation test and to obtain a sufficient optical change amount. For example, in the case of fibrinogen measurement, when normal plasma (concentration of about 300 mg / dL) is measured, the reaction solution is such that a clotting time of 5 to 50 seconds, preferably 7 to 20 seconds, more preferably 9 to 15 seconds is obtained. It is preferable to contain in. When benzamidine or a salt thereof is contained in the sample diluent as a blood coagulation time extending agent, it is contained so as to be 0.1 to 500 mM, preferably 1 to 200 mM, more preferably 5 to 100 mM.
In the case of prothrombin time measurement, when normal plasma (activity of about 100%) is measured, the reaction solution has a clotting time of 9 to 60 seconds, preferably 9 to 30 seconds, more preferably 10 to 16 seconds. It is preferable to contain. In the activated partial thromboplastin time measurement, when normal plasma is measured, it may be contained in the reaction solution so that a clotting time of 15 to 80 seconds, preferably 15 to 60 seconds, more preferably 20 to 50 seconds can be obtained. preferable. Other blood coagulation ability tests may be included in the reaction solution so that a coagulation time suitable for the measurement method can be obtained.

  Further, the alkali metal or alkaline earth metal halide salt or propionate that can be used in combination with the blood coagulation time extending agent of the present invention is not particularly limited as long as it can prolong the blood coagulation time in the blood coagulation reaction. Examples of such salts include sodium salts, potassium salts, calcium salts, and magnesium salts. Preferably, sodium chloride, sodium bromide, sodium iodide, potassium chloride, potassium bromide, potassium iodide, calcium chloride, magnesium chloride, sodium propionate and the like can be mentioned. The concentration used can be appropriately set in consideration of the desired blood coagulation time and the amount of optical change in the blood coagulation reaction.

  In addition to the blood coagulation time-extending agent of the present invention, known polymer polysaccharides and synthetic polymers are included in the blood coagulation ability measurement reagent including the specimen diluent and the blood coagulation activator-containing reagent used for the blood coagulation ability test. Can be contained.

Specific examples of the high molecular weight polysaccharide include dextran such as dextran 40, dextran 70, dextran 200,000, dextran 500,000, and ficoll. These high molecular polysaccharides can be used alone or in combination of two or more. The amount of these polymeric polysaccharides added is preferably such that the concentration in the specimen diluent is 0.01 to 10% by weight / volume, more preferably 0.1 to 5 W / V%.
Specific examples of the synthetic polymers include polyvinyl alcohols such as polyvinyl alcohol 500, polyvinyl alcohol 1500, and polyvinyl alcohol 2000, polyethylene glycol 1500, polyethylene glycol 2000, polyethylene glycol 4000, polyethylene glycol 6000, polyethylene glycol 8000, polyethylene glycol 20000, and the like. And polyethylene glycol and polyvinyl pyrrolidone. These synthetic polymers can be used alone or in combination of two or more. The addition amount of these synthetic polymers is not particularly limited, but the concentration in the sample diluent is preferably 0.001 to 20 W / V%, more preferably 0.01 to 10 W / V%. .

  In addition to the blood coagulation time extending agent of the present invention, the reagent for measuring blood coagulation ability may also contain a buffer, calcium ion, an anticoagulant antagonist and the like.

  As a specific example of the buffer, a buffer having a buffer capacity in the range of pH 4 to 9 is appropriately selected and used. For example, good buffers such as MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, DIPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, citric acid, One or two or more types selected from phosphoric acid, acetic acid, imidazole, barbital, GTA and the like can be selected and used. The amount of the buffer added is not particularly limited as long as it has a buffering capacity, but the concentration in the blood coagulation ability measurement reagent is preferably 1 to 1000 mM, more preferably 10 mM. ~ 500 mM.

  As the calcium ions, water-soluble calcium compounds such as calcium chloride, calcium lactate, calcium gluconate, calcium glucuronate, calcium tartrate are used. These calcium compounds can be used alone or in combination of two or more. The amount of calcium compound used is not particularly limited as long as it can assist the coagulation reaction, but the concentration in the blood coagulation ability measurement reagent is preferably 5 mM to 100 mM, more preferably 10 mM to 50 mM. is there.

As an antagonist of the anticoagulant, protamine, polybrene (hexadimethrine bromide), or the like is used. These anticoagulant antagonists can be used alone or in combination of two or more. The amount of the anticoagulant antagonist used is not particularly limited as long as it is sufficient to neutralize the anticoagulant contained in the sample plasma, such as heparin, but the concentration in the reagent for measuring blood coagulation ability is 10%. It is preferably ⁻⁵ to 10 ⁻² W / V%, more preferably 5 × 10 ^{−4 to} 5 × 10 ⁻³ W / V%.

The reagent for measuring blood coagulation ability may be a liquid product, a frozen product or a dried product, and the dried product is dissolved by adding purified water or a buffer solution.
An appropriate preservative may be added to the reagent for measuring blood coagulation ability. As the preservative, one or a combination of two or more of ciprofloxacin, propionic acid, sodium benzoate, sodium azide, and procrine 300 can be selected and used. Moreover, you may include general stabilizers, such as salts, such as sodium chloride, an amino acid, and sugar, as needed.
The concentration described above describes the concentration in the solution product, but the dried product describes the concentration when dissolved in water or a buffer solution at the time of use.

  In order to measure blood coagulation ability using a sample diluent containing a blood coagulation time extending agent in the blood coagulation reaction of the present invention, a conventional method may be followed. Taking fibrinogen measurement as an example, the standard solution is diluted 5 times, 10 times, and 20 times with the sample diluent (dilution ratio can be adjusted as appropriate). Next, 2 volumes of each diluted standard solution is heated at 37 ° C. for 3 minutes, 1 volume of thrombin reagent previously heated to 37 ° C. is added, and the coagulation time is measured. Next, the measured values of each diluted standard solution are plotted on a graph. A plasma sample is diluted 10-fold with a sample diluent (dilution ratio can be adjusted as appropriate), the diluted sample is measured in the same manner, and the concentration can be obtained from the graph based on the obtained clotting time. it can.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1
(Confirmation of prolongation of clotting time)
When the fibrinogen concentration in a plasma sample is measured by the thrombin time method, generally, thrombin is added to plasma, the clotting time is measured, and the concentration is obtained from a calibration curve using a standard solution with a known fibrinogen concentration. As a procedure, 90 μL of a sample diluent is added to and mixed with 10 μL of sample plasma, 50 μL of a thrombin-containing reagent is added as a blood coagulation activator-containing reagent, and the clotting time is measured.

  In this example, commercially available normal plasma (Coagtrol N, manufactured by Sysmex) is used as a plasma sample, Coagpia Fbg thrombin reagent (manufactured by Sekisui Medical) is used as a thrombin-containing reagent, and a fully automatic blood clotting time analyzer as a clotting time measuring device. Core Grex 800 (manufactured by Sysmex) was used. This instrument employs a scattered light detection method, and can acquire data on change in scattered light intensity over time as well as coagulation time.

  When HEPES buffer (pH 7.5) was used as the sample diluent, commercially available normal plasma coagulated in a few seconds (4.6 seconds). As a blood coagulation time extender, alkali metal halide salts (sodium bromide, potassium bromide, potassium chloride, lithium chloride, and sodium chloride) or benzamidine hydrochloride, which is a conventional method, are added to HEPES buffer, and blood is added. The amount of optical change (the amount of change in scattered light intensity) in the coagulation time and the blood coagulation reaction was measured. As a result, as shown in Table 1, benzamidine hydrochloride has a coagulation time extending action equivalent to that of the conventional method, and the decrease in optical change is extremely small.

Example 2
(Adjustment of optical change in blood coagulation reaction)
Example 1 using a specimen diluent (HEPES buffer pH 7.5) in which the concentration of sodium benzamide, a conventional blood coagulation time extender, and benzamidine hydrochloride, which is a blood coagulation time extender of the present invention, was adjusted. Normal commercial plasma was measured as described above. As a result, as shown in Table 2, it can be seen that the maximum value (ΔH) of the optical change amount (scattering light intensity change amount) of the scattered light that can obtain the same coagulation time can be appropriately adjusted to a desired size.

Claims (3)

  A specimen diluent for measuring blood coagulation ability, comprising a blood coagulation time prolonging agent in a blood coagulation reaction containing benzamidine or a derivative thereof as an active ingredient.   The sample diluent for measuring blood coagulation ability according to claim 1, which is a sample diluent for measuring fibrinogen.   Combined use of a blood coagulation time extender in a blood coagulation reaction containing benzamidine or a derivative thereof as an active ingredient and a blood coagulation time extender in a blood coagulation reaction selected from alkali metal or alkaline earth metal halide salts and propionate A method for adjusting an optical change amount in a blood coagulation reaction.