JP3061153B2 - Thromboplastin reagent - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a thromboplastin reagent for measuring the coagulation activity of a sample plasma using an optical measuring device, a viscosity detecting device, and the like. A thromboplastin reagent for measuring blood coagulation ability which contributes to the improvement of accuracy while making a sample having low coagulation activity possible by preparing a composition such that the amount of change in viscosity can be obtained. Things.

[0002]

2. Description of the Related Art The mechanism of blood coagulation will be described with reference to FIG. It is believed that the mechanism of blood coagulation usually occurs through two pathways. That is, one pathway is a pathway called extrinsic coagulation, in which coagulation factor VII is activated starting from tissue thromboplastin released from epidermal cells and the like, and coagulation is activated by the activated coagulation factor VII. This is the pathway by which factor X is activated, followed by activation of coagulation factors V and II, and finally coagulation by the conversion of fibrinogen to fibrin. the other one is,
This is a pathway called intrinsic coagulation, in which coagulation factor XII is activated by contact or the like to activate factor XI. Subsequently, the activated factor XI activates factor IX, and the activated factor IX is calcium ion / VIII
Activate factor X under the cooperative action of the factors. Thereafter, activation of factors V and II occurs, and finally, fibrinogen is converted into fibrin, which is a pathway by which coagulation occurs. As a method of detecting blood coagulation, a method of checking that the viscosity of a liquid increases as blood coagulates (viscosity detection method) and a method of detecting white turbidity as blood coagulates (turbidity detection method) , And a method in which these two are mixed. The stickiness detection method is to put a rod-like or spherical magnetic substance etc. into the plasma to be sampled,
In this method, when a coagulation detecting reagent is mixed, the movement of these magnetic substances and the like becomes slow due to coagulation. However, this viscosity detection method has a characteristic that the measurement result is greatly influenced by the state of formation of fibrin clot, which is the final product of blood coagulation (that is, the amount of fibrin or the hardness of the coagulation state). There is a fatal drawback that it cannot be detected unless the viscosity exceeds the value. Also, because it is a measurement principle that observes the movement of magnetic materials, etc.,
There is also a drawback that it is affected by the strength of magnetic materials and the like. The turbidity detection method is a method of performing coagulation measurement only by mixing a sample plasma and a coagulation reagent, and does not require the addition of a magnetic substance or the like. As a detection method, there is a transmitted light detection method or a scattered light detection method. In these detection methods, even when the amount of fibrinogen is small, it can be regarded as a change in the amount of transmitted light or a change in the amount of scattered light, so that there is no disadvantage as seen in the viscosity detection method. However, in any of the detection methods, it is natural that the more the amount of change in the amount of light, the more accurate the detection can be made. Therefore, the used coagulation reagent has such a characteristic that the amount of change in light is displayed large. Things are desirable.

FIG. 5 is a diagram for explaining a change in signal intensity obtained by the scattered light detection method. The results obtained by examining the process of plasma coagulation using an optical detection device (scattered light detection system) are shown. The point A in the figure is the time when the plasma and the coagulation reagent are mixed, and thereafter, the coagulation reaction proceeds in multiple stages, and appears as a change in scattered light due to the formation of stable fibrin (point B in the figure). As the formation of stable fibrin progresses, the change in scattered light increases, but most of the fibrinogen is consumed, the change in scattered light disappears, and the reaction is terminated (point C). The coagulation time is, for example, as disclosed in
As disclosed in JP-A-203959, the time T until the scattered light amount at the point B becomes 0% and the scattered light amount at the point C becomes 100% can be defined as 50% of the scattered light amount. ΔH is the amount of change in the amount of scattered light at the start and end of the coagulation reaction. To measure the pathway called extrinsic coagulation, the method called the quick one-step method is most common today. This measurement (test) is generally called PT (prothrombin time method). In addition to comprehensively measuring the activity of exogenous coagulation factors, the measurement of individual extrinsic coagulation factors using coagulation factor-deficient plasma is also possible. Is also possible. In addition, when an anticoagulant (such as warfarin) is being administered in the treatment of heart disease or the like, it is important as a measurement method capable of monitoring the effect of the anticoagulant.

[0004] Many of the reagents used in this method are prepared by crushing rabbit brain under acetone, dehydrating and drying, and then extracting tissue thromboplastin.
Since tissue thromboplastin has different reactivity and yield to exogenous coagulation factors depending on the extraction conditions, various extraction methods have been invented so far. For example, Ronald Bach et al.
J. Biological Chemistry, V
ol. 256, No. 16, (p8324-833
1), 1981) are surfactants (Triton X-100)
Is used to solubilize tissue thromboplastin to improve the yield. As a similar method, Japanese Patent Publication No. 63-56501 discloses the use of a surfactant of the cholic acid group at the time of tissue thromboplastin extraction, and the use of these surfactants is described in M. Hvatum, & H. Pr. P
rydz) (Boichimica et Boiph)
ysica Acta, Vol. 130 (p92-10
1), 1966) et al, and Gonmori Et and Takeda (Thromb
os. Haemostas. , Vol. 36 (p90 ~
103), 1976) et al. Report in detail in the literature a method for producing tissue thromboplastin. In addition, Tokunosho 5
JP-A-8-43080 discloses an invention in which a useful tissue thromboplastin is produced by maintaining the pH at the time of extraction on an alkaline side. Whichever extraction method is used, it is essential that the reagent used in the inspection method (prothrombin time method), which is called the one-step method of Quick, contains tissue thromboplastin. Must have uniform reactivity to extrinsic coagulation factors and have the effect of ultimately converting fibrinogen to fibrin.

[0005]

It is a proposition that a conventional blood coagulation reagent (hereinafter referred to as a PT reagent) has a more accurate reactivity to an extrinsic coagulation factor and finally forms fibrin. However, the display of the accurate measurement result was not so much omitted. For this reason, even if the test results regarding coagulation vary widely, it is in a situation where it has to be accepted. The problem to be solved by the present invention is
In order to display an accurate measurement result, the composition is to be adjusted so as to increase the amount of optical change. This makes it possible to measure a sample having a low coagulation activity, and contributes to improvement in accuracy of a normal sample. The present invention has been made in view of the above points,
It is an object of the present invention to provide a thromboplastin reagent that can increase the amount of optical change and the amount of change in viscosity.

[0006]

To achieve the above object, the present invention provides a thromboplastin reagent comprising a tissue thromboplastin-containing fraction extracted from an acetone-treated powder obtained by treating a mammalian tissue with acetone; The ion-containing thromboplastin reagent is characterized in that the electric conductivity is adjusted to 4.0 to 15.0 mS. Specifically, the thromboplastin reagent of the present invention is obtained by treating a tissue (a brain, a lung, a placenta, etc.) of a mammal (a human, a rabbit, a cow, a horse, a monkey, etc.) with an acetone, thereby obtaining an acetone-treated powder (hereinafter simply referred to as acetone). Powder), a tissue-thromboplastin-containing fraction is taken out of the acetone powder, and this fraction is adjusted to an electric conductivity of 4.0 to 15.0 mS.

An example of the manufacturing process will be described. I Tissue extraction For example, remove the rabbit brain. II Preparation of acetone powder The above rabbit brain is mixed and crushed with acetone. This is filtered and dried. III Collection of Tissue Thromboplastin-Containing Fraction The above acetone powder is dissolved in an acidic solution, mixed and stirred well, and then the floating fraction containing tissue thromboplastin is collected by centrifugation. IV solution preparation Adjust the above-mentioned tissue thromboplastin-containing fraction. V Lyophilization Lyophilize the above reagent. In addition, III is further divided into the following 2
By performing centrifugal separation in each step, a better fraction can be collected. III-1 Removal of blood components and contaminating proteins After the above-mentioned acetone powder is mixed with the liquid, centrifugation is performed to remove the floating fractions containing unnecessary components such as blood components and contaminating proteins, and collect sedimentation fractions. III-2 Collection of Tissue-Thromboplastin-Containing Fraction The above-mentioned sedimented fraction is mixed with an acidic solution, stirred, and then centrifuged to collect a floating fraction containing tissue thromboplastin. Further, by mixing acetone powder with an acidic liquid (for example, hydrochloric acid solution) in III-1, unnecessary components can be eluted in the liquid more, so that the removal rate of the unnecessary components is improved. Hemolyze). III-
In 2, the extraction rate of tissue thromboplastin is improved by mixing the precipitated fraction with sodium formate.

[0008] A liquid is mixed with the extracted tissue thromboplastin, and the electric conductivity of the liquid is adjusted to 4.0 to 15.0 mS.
(More preferably, 6.5 to 10.0 mS), a highly sensitive reagent can be obtained. Further, if the following conditions (1) and (2) are satisfied, the sensitivity can be further improved (the condition is best when the three conditions are satisfied). 1. pH 6.7-8.2 (more preferably 7.2-8.2)
7.5) 2. Osmotic pressure 100 to 700 mOsm / kgcm ² (more preferably 350 to 700 mOsm / kgcm ² ) An acid or alkali may be used for pH adjustment (for example, hydrochloric acid, sodium hydroxide). Further, an electrolyte may be used for adjusting the electric conductivity (for example, salt). To adjust the osmotic pressure, sugar or urea, which is a non-electrolytic water-soluble substance, may be used. The prepared thromboplastin reagent is preferably lyophilized so that long-term storage is possible. At the time of use, a predetermined amount of water is mixed with each of the dry powders to return to the original solution state before use.

As described above, in the present invention, a tissue-thromboplastin-containing fraction was obtained by dissolving an acetone-treated powder in water, mixing and stirring, and then centrifuging to obtain a floating fraction. It is desirable to use one. The tissue thromboplastin-containing fraction was prepared by dissolving the acetone-treated powder in hydrochloric acid water, collecting a sedimentable fraction containing no blood components or contaminating proteins by centrifugation, adding the liquid to the sedimented fraction, and stirring. It is preferably obtained by extracting a floating fraction generated by centrifugation. Alternatively, as the tissue thromboplastin-containing reagent, a prothrombin time reagent (PT reagent, a reagent for comprehensively measuring an extrinsic coagulation reaction in FIG. 6) is manufactured by DADE, USA and ORTHO.
, GD, Germany, Bering and BM. As reagents for measuring enzyme activity derived from vitamin K (reagents for measuring complex factor), thrombotest and hepaplastin test (both are trade names) from Eisai Co., Ltd., and complex factor H and complex factor T from International Reagents Co., Ltd.
(All are trade names) can be used.

[0010]

When the reagent is used, the freeze-dried one is dissolved in water and put back. By mixing a reagent containing tissue thromboplastin into plasma, coagulation factors in plasma are activated one after another,
Eventually, fibrinogen (fibrinogen) is converted to fibrin, and the coagulation reaction ends. In the final process,
First, a fibrin monomer is formed, and the monomers are polymerized one after another to form a polymer (fibrous), so that fibrin becomes fibrous. The fibrin fibers are entangled like a net, so that the mixture becomes turbid and gels.
The coagulation time can be measured by sequentially tracking the optical characteristics (eg, scattered light intensity) of the mixed solution from the reagent mixing. Since the reagent of the present invention has properly adjusted electric conductivity, the polymerization reaction from fibrin monomer to fibrin polymer is promoted, and the difference in optical properties between immediately after mixing the reagent (before coagulation) and at the end of coagulation is larger than before. Become. That is, high sensitivity is obtained. Further, by changing the pH and the osmotic pressure, the optical change amount changes. Therefore, by setting these values in an appropriate range, higher sensitivity can be achieved.

As described above, when the reagent of the present invention is mixed with the sample plasma, coagulation factors in the plasma are sequentially activated, and finally fibrinogen (fibrinogen) is converted into fibrin. At this time, for example, the clotting time is measured by optically detecting a change in turbidity (increase in turbidity), and as a result, the coagulation ability is calculated. In the final step of the coagulation reaction,
First, fibrinogen forms fibrin monomers. When it is polymerized spontaneously, fibers are formed, and the fibers are entangled to coagulate the mixed solution, increasing turbidity. If the change in turbidity is large, the accuracy and reproducibility of the measurement will be improved. Therefore, the present invention optimizes the condition of electric conductivity so that a larger change in turbidity occurs. Thereby, the polymerization reaction from the fibrin monomer to the fibrin polymer can be promoted, and the turbidity change can be increased.

[0012]

EXAMPLES The reagent used in the test method (prothrombin time method), which is called the one-step method of Quick, has a reactivity to an exogenous coagulation factor.
Incorporation of tissue thromboplastin is indispensable, and a reaction environment must be set so as to make the clot more robust so that the result of the reaction can be accurately measured. Therefore, in this example, highly reactive tissue thromboplastin was extracted from rabbit brain tissue by the following method. Anesthetize the rabbit using an anesthetic such as chloroform,
Blood is collected from the neck carotid artery. Next, a physiological saline solution is perfused from the cerebral artery to wash blood in the brain. After that, the skull is opened and the brain is removed. For 100 g of the rabbit brain obtained in this manner, 7
00 ml of cold acetone is mixed, crushed and stirred by a mixer, and dewatered and crushed. The ground material is filtered and then dried under vacuum. Hereinafter, this was mixed with acetone powder (A
P). Take 10 g of AP, mix in 100 ml of aqueous hydrochloric acid (0.02N), and centrifuge (3,000 rpm × 30).
Min) and collect the sedimentation fraction. The separated and removed floating fraction contains blood components and contaminating proteins. The collected precipitate fraction is mixed and stirred with 100 ml of sodium formate (0.05 N). Centrifugation (3,0
(00 rpm × 30 minutes) to collect the floating fraction containing tissue thromboplastin. A 50 mM buffer (phosphate buffer or barbital buffer or Tris buffer can also be used) for preserving the pH of the collected suspension containing about 100 ml of tissue thromboplastin, and a preservative ( 0.1% W / V sodium azide) and a stabilizer (0.1% calcium chloride) are mixed, and 0.5% (W / V) polyethylene glycol (molecular weight: 20 000) or 0.1% (W / V) dextran (molecular weight 200,000). Further, the pH was adjusted to 7.20 to 7.50 using 0.1 N sodium hydroxide, and the electric conductivity was adjusted to 6.5 to 1.
Mix salt so as to obtain 0.0mS, and adjust osmotic pressure to 350mS.
Glucose is mixed and adjusted to be ~ 700 mOsm / kgcm ² . The tissue thromboplastin-containing reagent thus obtained is lyophilized and stored. Next, the effect of removing the blood components and contaminating proteins by treating the acetone powder will be described. Dissolve the acetone powder in the liquid before extracting tissue thromboplastin,
In order to examine the effects of removing blood components and contaminating proteins, two samples were prepared, one that had been subjected to deproteinization treatment (the operation of washing the acetone powder with an acidic solution) and the other that had not been subjected to this treatment. Each measurement was performed 10 times using the reagents and compared. As a result, as shown in Table 1, the coagulation activity of the deproteinized PT reagent (without deproteinization) was higher than that of the PT reagent without deproteinization (without deproteinization) (in other words, as shown in Table 1). , The coagulation time was shortened) and the reproducibility was improved. From this, it can be seen that by performing the operation of washing the acetone powder with an acidic liquid before extracting tissue thromboplastin, the coagulation activity of the PT reagent is improved, and a highly accurate result can be obtained. .

[0013]

[Table 1]

In order to examine the effect of mixing a high molecular substance with a reagent, a tissue thromboplastin-containing PT was used.
A mixture of a polymer with a reagent and a PT reagent without any mixture were prepared, and their coagulation curves were compared. As a result, as shown in FIG.
The amount of change in the scattered light of the reagent was larger than that of the PT reagent not mixed with the reagent. (△ H> △ H ₁ ) This indicates that the coagulation reaction can be analyzed more precisely by mixing the PT reagent with a polymer substance. In addition, polyethylene glycol having a molecular weight of 20,000 was used as the high molecular substance, and the amount added in the case of mixing was 0.5 W / V%. ΔH indicates the amount of change in scattered light when a polymer substance is added, and ΔH ₁ indicates the amount of change in scattered light when no polymer substance is added.

FIG. 2 shows that the electric conductivity is 4.0 to 15.0 ms.
This shows the effect of the adjustment. In order to examine the effect of adjusting the electric conductivity in the PT reagent to 4.0 to 15.0 mS, salt was added to the tissue thromboplastin extract and the PT was adjusted so that the electric conductivity became 2.0 to 20.0 mS. Reagents were prepared, coagulation measurement of plasma was performed using those PT reagents, and the average value of the amount of change in scattered light at that time (average value measured 10 times each) was compared. As a result, as shown in FIG. 2, although the change amount of the scattered light tends to decrease as the electric conductivity increases,
There was a tendency to be stable between 4.0 and 15.0 mS. From this, the electric conductivity in the PT reagent was set to 4.
It can be seen that by adjusting the amount to 0 to 15.0 mS, the amount of change in scattered light can be maintained at a high level, and a stable amount of change in scattered light can be obtained with respect to a change in electrical conductivity due to the sample plasma. .

FIG. 3 shows the effect of adjusting the pH to 6.7-8.2, preferably 7.2-7.5. To examine the effect of adjusting the pH in the PT reagent to 6.7 to 8.2, preferably 7.2 to 7.5, 1N hydrochloric acid and 1N sodium hydroxide were added to the tissue thromboplastin extract. PH of 6.7 to
A PT reagent was prepared in the form of 8.5, and a coagulation measurement of plasma was performed using those PT reagents, and the average value of the amount of change in scattered light at that time (average value when measured 10 times each)
Were compared. As a result, as shown in FIG.
In the range of pH 7.5 to 7.5, the amount of change in scattered light tends to be relatively high, but in the range of pH 7.5 to 8.2, the amount of change in scattered light tends to decrease as the pH increases.
At 8.2 or more, the amount of change in scattered light tended to be lower and more stable. From this, the pH in the PT reagent was adjusted to 6.7.
To 8.2, preferably 7.2 to 7.5, while maintaining a high amount of change in scattered light, and
It can be seen that a stable amount of change in scattered light can be obtained even with a change in pH due to the sample plasma.

FIG. 4 shows an osmotic pressure of 100 to 700 mOsm / kg.
cm ² , preferably from 350 to 700 mOsm / kgcm ² , showing the effect of adjustment. The osmotic pressure in the PT reagent should be 100-700 mOsm / kgcm ² , preferably 3
In order to examine the effect of adjusting the adjustment to 50 to 700 mOsm / kgcm ² , glucose was mixed with the tissue thromboplastin extract to reduce the osmotic pressure in the PT reagent by 50.
A reagent adjusted to ９００900 mOsm / kgcm ² was prepared, a coagulation measurement of plasma was performed using those PT reagents, and the average value of the amount of change in scattered light at that time (average value when measured 10 times each) was determined. Compared. As a result, as shown in FIG. 4, the amount of change in scattered light tends to decrease with an increase in osmotic pressure, but when the osmotic pressure is 100 to 700 mOsm / kgcm ² , particularly when the osmotic pressure is between 350 to 700 mOsm / kgcm ² , The amount of change in scattered light tended to be stable. From this, by adjusting the osmotic pressure in the PT reagent to 100 to 700 mOsm / kgcm ² , preferably to 350 to 700 mOsm / kgcm ² , while maintaining a high amount of change in scattered light, and It can be seen that a stable change in scattered light can be obtained even with a change in osmotic pressure due to the sample plasma.

[0018]

The thromboplastin reagent containing the tissue thromboplastin of the present invention has the same reactivity to extrinsic coagulation factors as the conventional tissue thromboplastin reagent and the electric conductivity conditions are adjusted appropriately. The state of formation of fibrin clot, which is the final product of blood coagulation, is strong. Therefore, in a measuring instrument based on the viscosity detection method, the detection range can be expanded even when the amount of fibrinogen is small, and the disadvantage of being affected by the strength of a magnetic substance or the like can be solved. In the turbidity detection method, the amount of change in transmitted light or scattered light increases, and more accurate detection can be performed. Although the above has been described in the field of blood coagulation, the method and reagents for preparing a coagulation reaction environment and realizing the reaction are not limited to the reagents used in the prothrombin time shown by the present invention, and use tissue thromboplastin. Applies to all reagents. As a reagent using tissue thromboplastin, in addition to a reagent used for prothrombin time (PT), it can be applied to a coagulation activity of a protein derived from vitamin K, for example, a hepaplastin test, a thrombotest and the like.

[Brief description of the drawings]

FIG. 1 is a graph showing the relationship between the amount of scattered light and time of the thromboplastin reagent of the present invention and a conventional thromboplastin reagent.

FIG. 2 is a graph showing the relationship between electric conductivity and the amount of change in scattered light.

FIG. 3 is a graph showing the relationship between pH and the amount of change in scattered light.

FIG. 4 is a graph showing the relationship between the osmotic pressure and the amount of change in scattered light.

FIG. 5 is a graph showing a relationship between a change in a signal obtained by a scattered light detection method and blood coagulation.

FIG. 6 is a block diagram showing a mechanism of blood coagulation.

Continuation of the front page (56) References JP-A-58-154515 (JP, A) JP-A-57-178161 (JP, A) JP-A-4-183400 (JP, A) JP-A-61-283869 (JP) JP-A-62-250364 (JP, A) JP-A-50-85393 (JP, A) JP-A-60-230066 (JP, A) JP-A-59-187800 (JP, A) 5-207897 (JP, A) JP-A-57-182168 (JP, A) JP-B-41-10034 (JP, B1) JP-T-61-502908 (JP, A) (58) Fields investigated (Int. Cl. ^7, DB name) G01N 33/86 G01N 21/17

Claims (6)

(57) [Claims] The present invention relates to a tissue thromboplastin-containing fraction and a calcium ion-containing thromboplastin reagent extracted from an acetone-treated powder obtained by treating a mammalian tissue with acetone, and has an electrical conductivity of 4.0 to 15.0 mS. A thromboplastin reagent, which is adjusted. 2. The tissue thromboplastin-containing fraction is characterized by being obtained by dissolving an acetone-treated powder in water, mixing and stirring, and centrifuging to remove a floating fraction produced. The thromboplastin reagent according to claim 1. 3. A tissue thromboplastin-containing fraction is obtained by dissolving an acetone-treated powder in aqueous hydrochloric acid, collecting a sedimentable fraction containing no blood components or contaminating proteins by centrifugation, and adding a liquid to the sedimented fraction. The thromboplastin reagent according to claim 1, which is obtained by removing a floating fraction generated by centrifugation after stirring. 4. The thromboplastin reagent according to claim 1, wherein the pH is adjusted to 6.7 to 8.2. 5. The thromboplastin reagent according to claim 1, wherein the osmotic pressure is adjusted to 100 to 700 mOsm / kgcm ² . 6. The thromboplastin reagent according to claim 1, wherein the reagent is freeze-dried after completion of the preparation.