JPH10177029A - Specimen diluted solution - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable highly reliable measurements by using a specimen-diluted solution for measuring a composite coagulation factor, which is composed of an aqueous solution containing a chelating agent and a buffer solution. SOLUTION: Citric acid, sodium citrate, potassium citrate or the like is used as a chelating agent, and a concentration of the agent is set to be approximately 10-30mM when a concentration of calcium salt in a drying reagent used for measuring a coagulation time of blood is approximately 20mM. A buffer solution includes a buffering agent such as a GOOD buffering agent, trisaminomethane or the like, keeping a pH to be approximately 6-9 after specimen-diluted solution and a blood sample are mixed. The blood sample is diluted with the specimen-diluted solution and measured by the drying reagent. The ion concentration of the chelating agent originated from the blood sample is decreased, and an ion concentration of the chelating agent originated from the nearly constant specimen-diluted solution along influences a reaction, whereby a correlation to a conventional method is improved. Since ions of the chelating agent are included beforehand, excessive calcium ions are trapped and consequently, the concentration of calcium ions is maintained to be suitable for a blood coagulation reaction and measured values very little.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sample diluent used for measuring a blood coagulation time for measuring a complex coagulation factor used in a clinical test.

[0002]

2. Description of the Related Art Blood coagulation ability is greatly affected by various diseases, anticoagulants or trauma. For example, since vitamin K is synthesized in the liver, when the liver has a disease, the activity of blood coagulation factors II, VII, and X activated by the action of vitamin K decreases. 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 lower the blood coagulation ability to a certain range. At this time, the dose of heparin or the like is controlled by measuring the blood coagulation ability.

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.
The blood coagulation reaction is a cascade reaction in which reactions involving blood coagulation factors occur sequentially in many stages. When the concentration of a blood coagulation factor is low, the reaction involving the blood coagulation factor proceeds slowly. Accordingly, the obtained blood clotting time is delayed as compared with a normal case. 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 that triggers a blood coagulation reaction is reacted with a plasma sample or a whole blood sample (hereinafter referred to as a blood sample) to be measured, and the blood coagulation time is measured. You can know. Methods for measuring blood coagulation ability using this principle include the prothrombin time method (PT) and the activated partial thromboplastin time method (APTT).

[0005] Further, by reacting an adsorbed plasma from which a specific coagulation factor has been removed with a blood sample together with a substance or the like which triggers a coagulation reaction, and measuring the coagulation time, a coagulation factor which is not present in the adsorbed plasma is measured. There is also a measurement method in which only the amount is reflected in the blood clotting time. As a measurement item of blood coagulation ability using this method, there is a so-called complex coagulation factor measurement. In the measurement reagent used for the measurement,
It contains adsorbed plasma from which blood coagulation factors II, VII and X have been removed. Therefore, the deficiency of blood coagulation factors II, VII, and X in the blood can be determined by the measurement by prolonging the blood coagulation time. As described above, since hepatic dysfunction is associated with a decrease in the activity of blood coagulation factors II, VII, and X, hepatic dysfunction can be determined by measuring a complex clotting factor. This is one of the features of the complex coagulation factor measurement.

[0006] Incidentally, these conventionally used methods for measuring the blood coagulation time are mainly methods using a reagent solution, and are still widely used at present. In this method, first, a reagent solution is prepared by adding water or the like to a lyophilized reagent to prepare a reagent solution, the reagent solution is added to a blood sample and reacted, and a change in turbidity or a change in viscosity of the reaction solution is measured. This is a method for measuring blood coagulation time.

Hereinafter, the reagent for measuring complex coagulation factor will be described in detail by way of example. The reagent for measuring complex coagulation factor contains tissue thromboplastin, bovine adsorbed plasma, and calcium salt as main components, and is generally commercially available as a lyophilized reagent. The method of use is as follows. First, a reagent solution for measuring a complex coagulation factor in which the lyophilized reagent is converted to an aqueous solution is prepared and preliminarily heated. Next, the reagent solution is added to the blood sample which has been preliminarily heated, and a change in turbidity or a change in viscosity is measured immediately after the addition. And
When coagulation begins due to fibrin precipitation, the turbidity and viscosity of the reaction system change. 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 clotting time thus obtained is the blood clotting time of the reagent for measuring a complex clotting factor. The blood coagulation time varies depending on the activity of the complex coagulation factor, and the lower the activity, the longer the time.

On the other hand, in recent years, a simple blood coagulation assay system using a dry reagent has been developed (Japanese Patent Laid-Open Publication No. 3-5040).
No. 76). The principle of the assay system is that a reagent in a dry state containing a reagent component for measuring blood coagulation time and magnetic particles (hereinafter also referred to as a dry reagent) is prepared, a blood sample is added to the dry reagent, and immediately thereafter, The magnetic particles are moved in a magnetic field composed of a combination of an oscillating magnetic field and a static magnetic field, and at the same time, a coagulation is detected by optically measuring a motion signal of the magnetic particles. The assay system has an advantage that preparation of a reagent solution for measuring blood coagulation time, preliminary heating, and the like, which are necessary in the conventional method for measuring blood coagulation time, are unnecessary.

The reagent component and the magnetic particles of the dry reagent for measuring blood coagulation time are usually filled in a dry state in a card-like reaction slide as shown in FIGS. In the measurement method, the dried reagent is set in a dedicated measurement device, and then a blood sample is dropped from the blood sample addition port 1 of the slide. When the dry reagent for measuring blood coagulation time is dissolved by the blood sample, the blood coagulation reaction starts, and the magnetic particles in the reagent also start to move. The movement of the magnetic particles is optically observed by a measuring device, and blood coagulation occurs when the movement signal attenuates by a fixed rate from the maximum value, or when the amount of change in the movement signal starts to change rapidly (inflection point). As the end point of the reaction, the time from the addition of the blood sample to the end point is measured as the blood clotting time (see FIG. 3).

The above publication discloses various dry reagents for measuring blood coagulation time using this system. or,
A dry reagent for measuring blood coagulation time for measuring complex coagulation factors is disclosed in JP-A-6-337267.

[0011]

Calcium ions are essential for the blood coagulation reaction. The blood sample for blood coagulation time measurement should contain citrate ion, a type of chelating agent for calcium ions, so that the blood coagulation reaction will not progress naturally and coagulate before the start of measurement. ing. Therefore, in general, the reagent for complex coagulation factor measurement contains calcium salt, which is an essential component of the blood coagulation reaction, at a sufficiently high concentration with respect to citrate ions in the blood sample, and the blood coagulation reaction proceeds stably during the measurement. It has been considered to be.

In the measurement using a reagent solution for measuring complex coagulation factors which has been conventionally used (hereinafter abbreviated as a conventional method), generally, 250 μl of a reagent solution is reacted with 30 μl of a blood sample. Therefore, at the time of measurement, the blood sample is diluted about 9 times. On the other hand, in the above-mentioned dry reagent for measuring blood coagulation time for measuring complex coagulation factors, the citrate ion concentration is about 9 times higher than the conventional method because the blood sample is reacted with the reagent as it is. Has become.
Therefore, in the dry reagent, a calcium salt is contained at a higher concentration in the reagent than in the conventional reagent. It is due to this effect that there is a reasonable correlation between the method using the dry reagent and the conventional method.

However, clinical practice has strongly demanded a method of using a reagent having the same composition as the dry reagent to further improve the correlation with the conventional method and increase the accuracy.
That is, it has been evaluated that the correlation between the current dry reagent and the conventional method is still incomplete.

Thus, the present inventors have found that when a blood sample is diluted with an aqueous solution containing a buffer and then measured with a dry reagent, the concentration of citrate ion as a chelating agent in the reaction system is reduced. We thought that this problem could be solved because the reaction progressed in this state. However, as a result of the examination, the correlation with the conventional method was not improved, but the measured values varied greatly. That is, it was found that such a problem could not be solved simply by diluting the sample with an aqueous solution containing a buffer and using the diluted sample as a sample of a dry reagent.

[0015]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and the cause of the deviation from the measured value with the conventional method is that citrate ion, which is a chelating agent in each individual blood sample, It was found that it was related to the concentration diversity. It was also found that the cause of the large variation in the measured values was that the use of a diluted blood sample for the measurement resulted in an excessive calcium ion concentration in the reaction system, thereby inhibiting the blood coagulation reaction. And the inventors continued their research, and as a result, surprisingly,
The present inventors have found that such a problem can be solved by diluting a blood sample with a sample diluent comprising an aqueous solution containing a chelating agent and then measuring the dilution using a dry reagent for measuring blood coagulation time, and completed the present invention.

That is, the present invention is a sample diluent for measuring a complex coagulation factor comprising an aqueous solution containing a chelating agent.

Another aspect of the present invention is a sample diluent for measuring a complex coagulation factor comprising a buffer containing a chelating agent.

As the chelating agent contained in the sample diluent for complex coagulation factor measurement (hereinafter abbreviated as sample diluent) of the present invention, known chelating agents can be used. For example,
Citric acid, sodium citrate, potassium citrate, ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid disodium, ethylenediaminetetraacetic acid trisodium, ethylenediaminetetraacetic acid tetrasodium, ethylenediaminetetraacetic acid dipotassium, ethylenediaminetetraacetic acid dilithium, diaminopropanoltetraacetic acid and Alkali metal salts such as sodium salts and potassium salts, ethylenediamine diacetate and alkali metal salts such as sodium salts and potassium salts, ethylenediamine dipropionic acid and alkali metal salts such as sodium and potassium, glycol ether diamine tetraacetic acid and the like. Specific examples are given below. Sodium or potassium citrate is most preferred in view of solubility in water, reproducibility, and correlation with a conventional solution method. It is considered that the acid or the alkali metal salt thereof serving as the chelating agent acts in the ionic state (hereinafter, also referred to as chelating agent ion) in the sample diluent of the present invention.

The concentration of a suitable chelating agent cannot be limited because it depends on the concentration of the calcium salt contained in the dried reagent for measuring the blood coagulation time. For example, when the concentration of the calcium salt in the reagent is 20 mM, 10-30
A range of mM is preferred.

The buffer used in the present invention contains a buffer to keep the pH constant. In order for the blood coagulation reaction to proceed, it is desirable that the pH after mixing the sample diluent and the blood sample be between 6 and 9. Generally, 2- [4- (2-hydroxyethyl) -1 is used as a buffer.
-Piperazinyl] ethanesulfonic acid, 3- [4 [(2-
GOO such as hydroxyethyl) -1-piperazinyl] propanesulfonic acid and 2-morpholinoethanesulfonic acid
A series of compounds called D buffer, tris (hydroxymethyl) aminomethane and the like are preferably used. The concentration of the buffer is such that the pH can be kept constant even after being mixed with the blood sample and that the blood coagulation reaction is not significantly inhibited. If the suitable range is shown, it can be generally used in the range of 5 mM to 200 mM.
Further, the pH of the buffer solution can be suitably used generally in the range of 7 to 9.

An inorganic salt such as sodium chloride can be added to the sample diluent of the present invention in order to adjust the ionic strength, in addition to the above components. It is known that ionic strength affects the speed of the blood coagulation reaction. Since buffer concentration and chelating agent concentration also contribute to ionic strength,
The preferred concentration range of the inorganic salt is not particularly limited, but the sum of the buffer concentration, the chelating agent concentration and the inorganic salt concentration is 50 mM.
It can be suitably used in the range of up to 300 mM.

An alkaline compound such as sodium hydroxide or potassium hydroxide or an acidic compound such as hydrochloric acid, sulfuric acid or phosphoric acid can be added to adjust the pH of the sample diluent to a desired value. The concentration and amount of the alkaline compound or acidic compound to be added vary depending on the concentration and type of the buffer, the desired pH and the like.

Further, a preservative such as sodium azide can be used to improve the stability of the sample diluent of the present invention during storage. As the concentration of the preservative, for example, when sodium azide is used, a concentration of 0.1 mM or less can be suitably used.

As a particularly preferred example of the sample diluent of the present invention, 30 mM 3- [4 [(2-
Hydroxyethyl) -1-piperazinyl] propanesulfonic acid, 120 mM sodium chloride, and 21 mM sodium citrate.

The method of preparing a sample diluent containing a chelating agent is not particularly limited, but a method of dissolving a predetermined amount of each of the above components in water such as distilled water or a buffer solution with stirring is generally used. It is commonly used. .

The method for diluting a blood sample using the sample diluent of the present invention is not particularly limited, and a known method can be used. Generally, a fixed amount of a blood sample and a sample diluent are placed in a test tube and mixed by shaking.

The preferred range of the dilution ratio when diluting a blood sample using the sample diluent of the present invention varies depending on the concentration of the chelating agent of the sample diluent used, but it cannot be limited. For example, when diluting a blood sample with a sample diluent containing 21 mM sodium citrate, a range of 2 to 6 times is preferable.

A dry reagent for measuring a blood coagulation time for measuring a complex coagulation factor can be produced by the method described in JP-A-6-337267. It is also possible to use commercially available reagents.

The measurement of the blood coagulation time with the dry reagent for measuring the blood coagulation time can be performed by detecting a change in viscosity or a change in turbidity. Preferably, a fixed amount of a blood sample is dropped on a dry reagent for measuring blood coagulation time, and immediately placed in a magnetic field composed of a combination of an oscillating magnetic field and a static magnetic field to move the magnetic particles, and at the same time, the movement signal of the magnetic particles is optically measured. It can be performed by a method of monitoring the change in time and detecting the change with time. Such a measurement can be performed using a commercially available blood coagulation time measuring device such as CG01 (A & T Co., Ltd.) or COAG1 (Wako Pure Chemical Industries, Ltd.).

When measuring the blood coagulation time by the above method, the end point of the blood coagulation reaction can be determined based on the change in the magnitude of the movement signal of the magnetic particles in the dry reagent for measuring the blood coagulation time. As the end point of the blood coagulation reaction, a point at which the rate of decay of the magnitude of the movement signal is maximized, or a point at which the magnitude of the movement signal is attenuated to an arbitrary ratio with respect to the maximum value, etc. Among them, the point at which the magnitude of the movement signal attenuates to an arbitrary ratio with respect to the maximum value is preferably used because the reproducibility of the obtained blood coagulation time is better.

The reason why the correlation with the conventional method can be improved by diluting a blood sample with the sample diluent of the present invention and then measuring with a dry reagent for measuring blood coagulation time is as follows. I guess.

By diluting the blood sample, the concentration of the chelator ion from the blood sample is reduced so as not to affect the blood coagulation reaction, and accordingly the diversity of the chelator ion concentration in each individual blood sample is reduced. Will be erased. In addition, as a result, only the concentration of the chelating agent ion derived from the sample diluent affects the reaction system, and almost the same concentration of the chelating agent ion exists in the reaction system regardless of the blood sample as in the conventional method. I think that's because I started to do it.

The reason why the measurement value of the blood sample is reduced by diluting the blood sample with the sample diluent of the present invention and then measuring the dilution using a dry reagent for measuring blood coagulation time is as follows. I guess.

As described in the section of the problem to be solved by the invention, in order to enable measurement without diluting a blood sample, a dry reagent for measuring a blood coagulation time for measuring a complex coagulation factor is highly required. Concentration of calcium salt is included. When a blood sample is diluted with a diluent containing no chelating agent ion, the concentration of the chelating agent ion in the blood sample is reduced by the dilution, so that calcium ions in the reaction system are in an excessive state. As a result, the blood coagulation reaction is inhibited, and the blood coagulation time is prolonged. Therefore, the dispersion of the measured values is large. However, when a blood sample is diluted with the sample diluent of the present invention, excess calcium ions are trapped because chelating agent ions are contained in advance,
The calcium ion concentration is an appropriate concentration for the progress of the blood coagulation reaction. Therefore, it is considered that the dispersion of the measured values is reduced.

[0035]

The present invention will be described in more detail with reference to the following Examples, which by no means limit the scope of the present invention.

Example 1 Simultaneous reproducibility when measurement was performed using the sample diluent of the present invention A dispensing reagent solution having the composition shown in Table 1 was prepared, and the reaction was performed in the form of a reaction as shown in FIGS. The slides were dispensed in 22 μl aliquots. The dispensed reaction slide was freeze-dried to obtain a dry reagent for blood coagulation time measurement (hereinafter abbreviated as TB reagent) for complex coagulation factor measurement. The freezing method used was a method of freezing in a freezer at −40 ° C. for a day and night, and the freeze-drying was performed by gradually increasing the temperature in a vacuum from −40 ° C. to 40 ° C. in 45 hours.

[0037]

[Table 1]

Next, a sample diluent 1 and a sample diluent 2 of the present invention having the compositions shown in Table 2 were prepared.

[0039]

[Table 2]

A blood sample (human plasma) was diluted three-fold using each sample diluent.

As a blood sample, "standard plasma normal range Eisai" (manufactured by Eisai Co., Ltd.) was used. Using a TB reagent and each diluted blood sample (hereinafter, abbreviated as diluted plasma), blood coagulation time measurement apparatus CG01 (manufactured by A & T Co., Ltd.) was used to perform 10 consecutive measurements. The measurement was carried out by setting the TB reagent in the CG01 measuring section according to the instruction manual and adding 25 μl of the diluted plasma to the TB reagent.

The blood coagulation time was calculated by analyzing the time-dependent change (hereinafter abbreviated as a waveform) of the movement signal of the magnetic particles obtained after adding the diluted plasma to the TB reagent.
More specifically, the unstable portion of the waveform immediately after the addition of the diluted plasma (25 seconds after the addition of the diluted plasma) is deleted, and the point at which the waveform has attenuated 5% from the maximum point is the end point of the blood coagulation reaction. And the time from the addition of the diluted plasma to the end point was defined as the blood coagulation time.

Table 3 shows the average value and the CV value of the measurement results of the respective blood coagulation times.

[0044]

[Table 3]

As shown in Table 3, it was found that very good reproducibility was exhibited. FIG. 4 shows a waveform when the diluted plasma was measured using the sample diluent 2.

Comparative Example 1 Simultaneous Reproducibility when Measurement was Performed Using a Diluent Different from the Present Invention A TB reagent was prepared in the same manner as in Example 1. Next, aqueous solution A was prepared with the composition shown in Table 4.

[0047]

[Table 4]

The blood sample (human plasma) was diluted three-fold using the aqueous solution A. In addition, a blood sample (human plasma) was diluted three times with distilled water.

As a blood sample, "standard plasma normal range Eisai" (manufactured by Eisai Co., Ltd.) was used.

Simultaneous reproducibility was examined in the same manner as in Example 1 using each of the diluted blood samples. Table 5 shows the average value and CV value obtained in each measurement.

[0051]

[Table 5]

As shown in Table 5, the results were clearly inferior to those of Example 1.

Comparative Example 2 Simultaneous Reproducibility when Plasma Samples were Measured Undiluted A TB reagent was prepared in the same manner as in Example 1. For blood samples, “Standard plasma normal range Eisai” (Eisai Co., Ltd.)
(Manufactured by KK)

Using the TB reagent and the plasma, blood coagulation time was measured continuously 10 times with a blood coagulation time measuring device CG01 (manufactured by A & T Corporation). The measurement method is T according to the instruction manual.
Reagent B was set in the CG01 measuring section, and the plasma was added by adding 25 μl of the plasma to the TB reagent. Thereafter, the simultaneous reproducibility was examined in the same manner as in Example 1. Table 6 shows the average value and CV value obtained by the measurement.

[0055]

[Table 6]

As shown in Table 6, the results were clearly inferior to those of Example 1. FIG. 5 shows the waveform when the plasma was measured without dilution.

Example 2 Correlation with the Conventional Method Using 92 human plasma samples as blood samples, the results of measurement of complex coagulation factors by the conventional method and the dilution of each human plasma three times with the sample diluent of the present invention were performed. The correlation with the result measured with the TB reagent of Example 1 was examined. In addition, the correlation between the results of measurement of complex coagulation factors by the conventional method using the same 92 human plasma samples and the results of undiluted measurement of each human plasma using the TB reagent of Example 1 was also investigated. The effect was confirmed by comparing with the correlation of the above. Table 7 shows the sample diluent of the present invention used in this study.

[0058]

[Table 7]

The measurement method using the TB reagent and the blood clotting time calculation method were performed in the same manner as in Example 1. In addition, the conventional method employed in this example is a method using “T Kokusai” as a reagent.
(Manufactured by Kokusai Reagent Co., Ltd.), KC-10A as a measuring device
(Manufactured by Amelung Co., Ltd.) and the measurement was performed according to the respective instruction manuals.

FIG. 6 shows the correlation between the result of the measurement of the complex coagulation factor by the conventional method and the result of the measurement of the human plasma undiluted with the TB reagent. FIG. 7 shows the result of the measurement of the complex coagulation factor by the conventional method. TB with human plasma diluted 3 times with the sample diluent of the present invention
The correlation diagram with the result measured with the reagent was shown.

It is apparent from the comparison between the two methods that the correlation with the conventional method is better when the dilution is performed three times with the diluent of the present invention and the measurement is performed.

[0062]

The blood coagulation used in a measurement method that does not dilute the blood sample by diluting the blood sample using the sample diluent of the present invention and measuring the blood coagulation time with the dry reagent for measuring the blood coagulation time. Less variation in blood coagulation time measurement dry reagent with the same composition as the time measurement dry reagent,
Reliable measurement can be performed.

Therefore, there is a great feature that the same blood sample need not be measured a plurality of times to increase the reliability of the measured value. This can make a significant contribution to improving the reliability of clinical tests and reducing costs, and the industrial significance of the present invention is extremely large.

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of a reaction slide of the present invention.

FIG. 2 is a diagram showing an example of the structure of the reaction slide of the present invention.

FIG. 3 shows a change in the kinetic signal intensity of magnetic particles when a blood sample is added to a dry reagent for measuring blood coagulation time.

FIG. 4 is a diagram showing a time-dependent change of a movement signal when a blood sample is diluted with a sample diluent of the present invention and measurement is performed ten times.

FIG. 5 is a diagram showing a time-dependent change of a movement signal when a blood sample is measured 10 times without dilution.

FIG. 6 is a correlation diagram in which the measured values obtained by the conventional method are plotted on the X axis, and the measured values obtained by measuring human plasma undiluted with CG01 are plotted on the Y axis.

FIG. 7 is a correlation diagram prepared by taking measured values by the conventional method on the X-axis and diluting human plasma with the sample diluent of the present invention and measuring by CG01 on the Y-axis.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Blood sample addition port 2 Reagent filling part 3 Reagent addition port 11 Transparent resin plate 12 Spacer 13 White substrate

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

Claims (2)

[Claims] 1. A sample diluent for measuring a complex coagulation factor, comprising an aqueous solution containing a chelating agent. 2. A sample diluent for measuring a complex coagulation factor, comprising a buffer containing a chelating agent.