WO2008062520A1 - Drug efficacy testing method for antithrombotic agent - Google Patents

Drug efficacy testing method for antithrombotic agent Download PDF

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WO2008062520A1
WO2008062520A1 PCT/JP2006/323302 JP2006323302W WO2008062520A1 WO 2008062520 A1 WO2008062520 A1 WO 2008062520A1 JP 2006323302 W JP2006323302 W JP 2006323302W WO 2008062520 A1 WO2008062520 A1 WO 2008062520A1
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blood
solution
volume
aqueous solution
anticoagulant
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PCT/JP2006/323302
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French (fr)
Japanese (ja)
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Jun Kawasaki
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Jun Kawasaki
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/86Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors

Definitions

  • the present invention relates to an antithrombotic drug efficacy test method. More particularly, the present invention relates to an antithrombotic drug efficacy test method for quickly and easily determining the efficacy of an antithrombotic drug being administered for treatment.
  • Thromboembolism refers to a state in which blood has solidified in the heart or blood vessels. If blood is stopped by a thromboembolism, it will cause a lesion in the part nourished by the blood vessel, which can be fatal. Therefore, antithrombotic drugs are necessary to prevent thromboembolism.
  • antithrombotic drugs are necessary to prevent thromboembolism.
  • the present inventor has proposed a method for measuring the degree of blood clot formation on a thrombolast graph in the presence of an anticoagulant, when the platelet activity activator causes blood coagulation.
  • a patient who is receiving an antithrombotic drug is a system in which an anticoagulant such as henon or argatroban is added to a part of the collected blood (system X blood),
  • an anticoagulant such as henon or argatroban
  • a platelet activator such as adenosine diphosphate or collagen
  • thrombolasta taraf is measured at the same time, and R values of X system blood and Y system blood are compared. If the R value of the Y blood is significantly shorter than the R value of the X blood, it is determined that the efficacy of the antithrombotic therapy is not effective, and the R value of the Y blood is R of the X blood. If there is no significant difference from the value, the efficacy as antithrombotic therapy is judged to be effective.
  • Patent Document 1 Japanese Patent Application 2005—149183
  • the clotting time of the collected blood has the property that it varies depending on the person and the type and amount of the medicine taken by the same person.
  • the present invention is intended to provide an antithrombotic drug efficacy test method that is effective, reliably and rapidly implemented. That is, the present invention seeks to provide an antithrombotic drug efficacy test method for effectively, surely and quickly conducting various blood antithrombotic drug efficacy tests by shortening the time required for the preliminary test. It is. Furthermore, it is intended to provide an antithrombotic drug efficacy test method that can be easily, accurately, quickly and easily measured even by those who do not have specialized knowledge.
  • the gist of the present invention is indicated by itemized items as follows.
  • the amount of calcium chloride added to blood is the same for both X-system blood and Y-system blood, and the same amount of anticoagulant added to blood is the same for both X-system blood and Y-system blood.
  • Antithrombotic drug efficacy test method The amount of calcium chloride added to blood is the same for both X-system blood and Y-system blood, and the same amount of anticoagulant added to blood is the same for both X-system blood and Y-system blood. Antithrombotic drug efficacy test method.
  • antithrombotic drug efficacy test method wherein the anticoagulant solution is a heparin (undifferentiated heparin, low molecular heparin) aqueous solution or an argatroban aqueous solution.
  • the anticoagulant solution is a heparin (undifferentiated heparin, low molecular heparin) aqueous solution or an argatroban aqueous solution.
  • the final concentration containing the heparin aqueous solution is in the range of 0.1 Ol (UZmL) force and 0.1 (UZmL), and the final concentration containing the argatroban aqueous solution is from 0.1 (gZmL) to 5.0.
  • [5] The antithrombotic drug efficacy test method according to any one of [1] to [4], wherein the platelet activator solution is an adenosine diphosphate (ADP) aqueous solution or a collagen aqueous solution.
  • ADP adenosine diphosphate
  • the final concentration containing the adenosine diphosphate aqueous solution is 6. O / zM or more, and the final concentration containing the collagen aqueous solution is 1.0 gZmL or more. Antithrombotic drug efficacy test method.
  • the present invention relates to a system (X system blood) in which a salt calcium aqueous solution, an anticoagulant solution, and physiological saline are added to blood collected from a patient who has received antithrombotic drugs, and calcium chloride is added to the blood.
  • Antithrombotic drug efficacy test method for measuring the tropoelastograph and comparing the R values of X blood and Y blood in a system (Y blood) containing an aqueous solution, anticoagulant solution and platelet activator solution
  • Y blood containing an aqueous solution, anticoagulant solution and platelet activator solution
  • the antithrombotic drug efficacy test method As described above, it is possible to shorten the time required for the preliminary test and to effectively, quickly and accurately perform various blood antithrombotic drug efficacy tests. In particular, there is an effect that the time required for the antithrombotic drug efficacy test can be shortened. Furthermore, using the relationship between the ACT value obtained in advance and the final concentration of the anticoagulant solution to be added, even those who do not have specialized knowledge should conduct antithrombotic drug efficacy tests easily, accurately, quickly and simply. There is an effect that can be done.
  • clotting time is measured by other blood coagulometer.
  • Anticoagulation is performed in order to increase the clotting time of the blood (roughly synonymous with the R value on the thrombolast graph) to 30 to 40 minutes (thus suppressing thrombin partially or delaying production).
  • the amount of argatroban or heparin to be used as an agent is estimated and determined from the value of ACT from hemoclone (105 to 150 in normal adults) or the clotting time by other blood coagulometers. 2) Add reagent to start clotting of citrate blood sample on thrombolast graph.
  • the blood in the first channel (X system blood is a measurement channel), the blood is mixed with an aqueous solution of calcium chloride (sometimes abbreviated as chlorocalcium), an anticoagulant solution in an amount determined from the ACT value, Saline (hereinafter also referred to as raw food) is added, and in the second channel (channel for measuring Y-system blood), the blood is mixed with an aqueous solution of calcium chloride, an amount of anticoagulant solution determined from the ACT value, and platelets. Add a coagulant activator solution and measure the thrombolast graph at the same time.
  • R value a coagulant activator solution
  • the first channel is the measurement channel for X-system blood
  • the second channel is the channel for measuring Y-system blood
  • the second channel is the measurement channel for X-system blood
  • the first channel is for Y-system blood. It goes without saying that the channel can be measured.
  • 3) Comparing the R values of the first channel and the second channel if there is a significant difference, platelet stimulation in the second channel promoted clot formation in the second channel. It is determined that the platelet activity of blood is not greatly suppressed. If there is no significant difference, it is judged that platelet stimulation in the second channel cannot promote clot formation in the second channel, and the platelet activity of this blood is small and sufficiently suppressed.
  • the amount of argatroban aqueous solution or heparin aqueous solution used as the anticoagulant solution determined in 1) refer to the relationship between the ACT value obtained in advance and the optimum amount of argatroban aqueous solution or heparin aqueous solution to be added. And decide. Tables 11 and 12 show the ACT values and the empirically obtained optimal amounts (final concentrations) of argatroban or heparin to be added.
  • Reagents are also commonly referred to as The term “agent” is used as an anticoagulant, but when used as a reagent for antithrombotic drug efficacy test, the term “agent” is used.
  • the thrombolast graph is a device that observes the coagulation process of blood by the clot elasticity of whole blood.
  • Figure 1 shows a graph drawn with the clot elasticity on the vertical axis and the time change on the horizontal axis. Based on the measurement diagram shown in Fig. 1, the reaction time R value when the clot elasticity value width became 2 mm, the clot time K value and the clot elasticity value when the clot elasticity value width became 20 mm.
  • the maximum angle amplitude MA value and the angle ANG value when tangent is drawn in the graph of change in blood clot elasticity are obtained as numerical values.
  • the measurement operation itself is as simple as putting blood in a cuvette and starting the measurement.
  • the thrombolast graph used in the United States was a tronbolast graph C-TE G3000T manufactured by Moscope Co. and a Rotem GAMMA manufactured by Pentafuarm.
  • a test method using a thrombolast graph will be described.
  • the thrombolast graph to be used has at least a measuring section with 2 or more channels, and the measuring liquid volume is around 0.36 mL.
  • the amount of blood and reagents used in the antithrombotic drug efficacy test will be described.
  • the amount of reagent added to the blood is the specified volume ratio for both X-system blood and Y-system blood.
  • the salt and calcium added to blood has the same concentration and amount in both X and Y blood.
  • the anticoagulant solution is the same in both the X-type blood and the Y-type blood at the same concentration and the same amount.
  • the amount of X blood and Y blood used for the test is the same.
  • the calcium salt that can be stored in blood is 0.18 M calcium chloride aqueous solution (commercially available for intravenous injection) for 1 volume of anticoagulant solution and 1 volume of physiological saline for both system X blood and system Y blood.
  • the volume In the case of 20 mL containing 0.4 g of salty calcium; Otsuka Pharmaceutical, Fuso Pharmaceutical, Sanso, etc., the volume is 1 or more and 2 or less in this case (in this case, 0.18M calcium chloride aqueous solution
  • the volume can be any of 1.0, 1.5, 2.0, etc.), 0.2
  • the volume In the case of a 2M salt-calcium aqueous solution, the volume can be adjusted at a rate of 2, and in the case of a 0, 4M chloride chloride aqueous solution Capable of capacity 1
  • heparin undifferentiated heparin, low molecular weight heline
  • argatroban argatroban
  • Heparin or Argatroban final concentration force Heparin is 0. Ol (UZmL) force, etc. 0. l (UZmL) It is easy to mistake the number 1. Therefore, add the final concentration of argatroban from 0.1 (gZmL) to 5.0 (gZmL) so that the unit of volume is L). The actual amount to be added needs to be adjusted according to the length of the clotting time of the blood to be measured.
  • ADP adenosine diphosphate
  • collagen COL
  • adenosine diphosphate (ADP) or collagen is used as the platelet activator.
  • ADP can be adjusted so that its final concentration is 6 M or more.
  • Collagen (COL) should be prepared so that its final concentration is 1.0 g / mL or more. If the final concentration of ADP is at least 6 / zM or more, the thrombolast graph can be measured without problems. Similarly, if the final concentration of collagen is at least 1. O / z gZmL, the thrombolast graph can be measured without problems.
  • the maximum amount of ADP used is a final concentration of about M for collagen and about 10 gZmL for collagen.
  • the amount of reagent added to the cup of the thrombolast graph is such that the volume ratio of each reagent of aqueous chloride solution, aqueous solution of anticoagulant, and physiological saline is 1: 1: 1 in the system X blood. 30 / z L in the case of 1.5, 35 L in the case of 5: 1: 1, and 40 L in the case of 2: 1: 1.
  • the difference in the amount of addition is determined by the amount of calcium chloride in the reagent. For example, if the reagent mixing ratio is 1.3: 1: 1, the amount of reagent added is 33 / z. L.
  • actin can be calorieated as a blood coagulation promoter. If the amount of anticoagulant added increases, the measurement time of the thrombolast graph may become longer. In such a case, if actin is added, the thrombolast graph can be measured within an appropriate time.
  • the actin is preferably contained at a final concentration of 0.5 ng ZmL to 2.5 ng ZmL.
  • the amount of reagent such as a saline calcium aqueous solution, anticoagulant solution or physiological saline added to the blood is 10 L more than when no actin is added.
  • thrombolastograph There are two measuring instruments for the thrombolastograph, one from Haemoscope (Niles, IL, USA) and one from Rotem from Pentapharm (Munich, Germany). Blood volume is different. The total amount of Hemoscope thrombolast draft is specified as 0.36 mL, and when adding drugs, add 10 / z L per drug and determine the final concentration in it. Reduce the amount of blood to be added.
  • PENTAFU ARM Rotem has a constant blood volume of 0.30 mL, and as with calcium chloride, a 0.2 M aqueous solution of 20 mL of NaCl is recommended as in the case of the Thrombolastograph.
  • argatroban and collagen are each 10 L of calorie, and the total amount is 0.34 mL.
  • the maximum addition amount of this reagent is 40 L.
  • a maximum of 0.3 L is added. 4mL, minimum 0.33mL, no problem at all.
  • the maximum reagent volume of 40 L is added to 0.33 mL of blood, it becomes 0.37 mL, and the maximum volume of thrombolast graph is 0.36 mL.
  • the method is as follows: 0.1M of each of 18M salt ⁇ canorecum 5 L, 10 L, 20 L, and 30 L, 0.3 mL of whole blood and argatroban or heparin and collagen or ADP, which is a part of the reagent to be added this time.
  • the simulated dose was 10 L for each physiological saline, and a total of 20 L physiological saline was added to measure the degree of blood coagulation.
  • the time (minutes) at which the R point (the point indicating the start of solidification) appears on the thrombolast graph after the measurement was started was measured.
  • the R value is indicated by (mean person SD: mean person standard deviation). In the following examples, the R value is (mean It was shown by Shishi SD).
  • the optimum amount of 0.2M calcium chloride was examined in order to recalcified kennate blood.
  • the method is as follows: 0.2M calcium chloride 5 L, 10 L, 20 ⁇ L, 30 L, 0.3 mL whole blood and a dose simulating argatroban or henone and collagen or ADP as part of the reagent to be added this time
  • a total of 20 L of physiological saline was added to each physiological saline, and the degree of progress of blood coagulation was measured.
  • the time (minutes) at which point R (the point indicating the start of solidification) appears on the Trobolast graph after the measurement was started was measured.
  • the measurement results are shown in Table 2.
  • the R value is indicated by (mean SD: mean value standard deviation).
  • Table 2 shows that 0.2M calcium chloride 20 L is the optimum amount.
  • 0.2M salt calcium 5 L, 10 L is too little
  • 0.2 M calcium chloride 30 ⁇ L is too much
  • V deviation is also the point where
  • This example demonstrates the use of 0.4M calcium chloride to recalcified kennate blood. It is the Example which investigated the appropriate quantity.
  • the method is 0.4M calcium chloride 5 L, 10 / z LL, 15 ⁇ L, 20 / z L, 0.3 mL of whole blood and 1 part of the reagent added this time.
  • the amount of each physiological saline was 10 ⁇ L, and a total of 20 ⁇ L of physiological saline was added to simulate a collagen aqueous solution or an ADP aqueous solution, and the degree of progress of blood coagulation was measured.
  • the time (minutes) at which the R point (point indicating the start of solidification) appears on the thrombolast graph after the measurement was started was measured.
  • the measurement results are shown in Table 3. 0. 4M salt calcium 10 L has been shown to be optimal.
  • the 4M calcium chloride 5L was too little, and the 0.4M calcium chloride 15 ⁇ 20 ⁇ L was too much.
  • the amount of the calcium chloride aqueous solution added will be described based on an example in the case of following the blood addition method of Hemoscope Trombo Elastograph.
  • 20 ⁇ L of saline (saline) as the substitute dose of the reagent and the concentration and amount of salted calcium indicated in the table were added, and the total amount of blood was 0.36 mL. did.
  • Table 4 shows the results of 0.18M calcium chloride for recalcified kennate blood according to the blood scoring method of Hemoscope Thrombolastograph (method of adding whole blood to a total dose of 360 ⁇ ).
  • the result of the Example which investigated the optimal amount is shown.
  • the method is as follows: 0.1M salt ⁇ canoresum 5 L, 10 L, 20 L, L, each dose of a dose similar to argatroban or heparin and collagen or ADP as a part of the reagent Saline) 10 ⁇ L, total of 20 ⁇ L of raw food was added, and the blood volume was adjusted to 360 ⁇ L in total, and the degree of progress of blood coagulation was measured.
  • Example 1 was obtained by measuring the R value in accordance with the Rotem blood addition method of Pentafarm, and Example 2 was in accordance with the blood addition method of the Hemoscope thrombolast graph. R value was measured.
  • the addition amount of 0.18M calcium chloride aqueous solution is the optimum amount of 10 L and 20 L force
  • the addition amount of 0.2M calcium chloride aqueous solution is the optimum amount of 20 L. It has been shown that the optimum amount of 4M calcium chloride aqueous solution is 10 ⁇ L.
  • the amount of calcium chloride aqueous solution added to X-system blood and sputum system blood is 0.18M salt when mixing 1 volume of anticoagulant solution, 1 volume of physiological saline and calcium chloride aqueous solution. It can be seen that it is optimal to add 1 to 2 vol. Volume for aqueous calcium carbonate solution, 2 vol. For 0.2 wt. Calcium chloride aqueous solution, and 1 vol.
  • ADP adenosine diphosphate
  • collagen are used as platelet activator solutions in 4 ⁇ and 4 ⁇ g ZmL, respectively.
  • concentration 10 L (0. OlmL) and ADP (final concentrations of 4 ⁇ , 6 / ⁇ , 8 ⁇ ⁇ ⁇ ) with the final concentration of argatroban determined before this measurement in the presence of 0.2 M salt calcium 20 / zL ) And collagen (Col; final concentration of 0.5 ⁇ g / m 1. O The final concentration of mL 2.0 8 !
  • the concentration of the platelet activator solution added to the X-system blood and the Y-system blood is ADP (adenosine diphosphate) 6.
  • O / zgZmL It turns out that the above is the optimal amount.
  • Example 3 the optimum amount of actin (Actin from bovine muscle, A-3653, Sigma-Aldrich, St. Louis MO USA) as a blood coagulation promoter was examined.
  • actin Actin from bovine muscle, A-3653, Sigma-Aldrich, St. Louis MO USA
  • a rough optimum concentration of actin that promotes blood coagulation was determined by changing the final concentration from IngZmL to lpgZmL.
  • the R value was determined by a thromboelastograph. The results are shown in Table 9.
  • the final concentration of actin is 0.5 ngZmL (bottom stage) to 2.5 ng / mL (third stage), and the R value is significantly shorter than Control (without adding actin at all).
  • the final concentration of actin used to promote blood coagulation is 0.5 ngZmL (500 pgZmL) and 2.5 ng / mL.
  • Case 1 is a 71-year-old male with arteriosclerotic obstruction and renal failure who is taking 1 tablet (lOOmgZ day).
  • 1 volume of coagulant solution and 1 volume of platelet activator solution are stored.
  • Heparin at a final concentration of 0.lU/mL, argatroban is I got 3 M Caro.
  • the total liquid volume is 0.36 mL.
  • platelet aggregation caused by ADP shortens the R value by approximately 28%, and it can be determined that the current anti-platelet treatment of pretal lOOmgZ is insufficient.
  • a dose increase to 150mgZ is currently under consideration.
  • Case 2 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg / day).
  • Anticoagulant solution is added at a rate of 1 volume and platelet activator solution 1 volume.
  • Heparin had a final concentration of 0.1 lUZmL
  • argatroban had a final concentration of 0.3 / z gZmL
  • ADP had a final concentration of 8.3 M calories.
  • the total volume is 0.36 mL. In this case, the reduction is 10 15%, and it can be determined that there is no significant difference and effective treatment is being performed.
  • Case 3 is a 63-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg / day).
  • 2 volumes of 0.2M salt-calcium aqueous solution Add 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution to Y blood. It is added together.
  • Heparin at a final concentration of 0.1 lUZmL, argatroban at a final concentration of 1.2 g / mL, and ADP at a final concentration of 8.3 M calories.
  • the total liquid volume was 0.36 mL. In this case, the reduction is less than 10%, and it is judged that there is no significant difference, and effective treatment is considered.
  • Case 4 is a 54-year-old male with cerebral infarction taking 2 panalgin tablets (200 mgZ day).
  • 1 volume of coagulant solution and 1 volume of platelet activator solution are added.
  • the total volume is 0.36 mL. In this case, the reduction is 20% or less, and it is judged that there is no significant difference, and effective treatment is considered.
  • Case 5 is a 62-year-old male with atherosclerotic obstruction who is taking pretal 200 mg Z day.
  • 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added.
  • Heparin at a final concentration of 0.1 lUZmL
  • argatroban at a final concentration of 1.2 ⁇ g / mL
  • ADP at a final concentration of 8.3 M calories.
  • the total volume is 0.36 mL.
  • there is a shortening of 20% or less and there is a delay in coagulation due to platelet aggregation stimulation, which is evidence that platelet activity is often suppressed, and it can be judged that treatment is sufficient.
  • Panaldine 2 tablets (200mg / day)
  • Case 6 is a 66-year-old male with atherosclerotic obstruction who is taking pretal 200 mg Z day.
  • 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added.
  • Heparin was 0.2 UZmL and 0.1 LUZmL at the final concentration
  • argatroban was 0.3 / z gZmL and 0.2 / z gZmL at the final concentration
  • ADP was 8.3 M calorie at the final concentration.
  • the total volume is 0.36 mL.
  • the clotting time on the platelet aggregation stimulation side is long. The results show that the therapeutic effect is sufficient.
  • Case 7 is a 65-year-old male with cerebral infarction.
  • Add 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to system X blood, and 1 volume of 0.18M aqueous calcium chloride solution to system Y blood, anticoagulant 1 volume of drug solution and 1 volume of platelet activator solution are stored.
  • Argatroban has a final concentration of 4.0.
  • the total volume is 0.36 mL. In this case, the reduction is 20% or less, and it can be judged that there is no significant difference, and the treatment is sufficient.
  • Case 8 is a 58-year-old male with cerebral infarction who is taking Biaspilin lOOmgZ day.
  • Add 0.1 volume of 18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M saline calcium solution to Y blood, anticoagulant Add 1 volume of solution and 1 volume of platelet activator solution! /
  • ACT value of 190 seconds heparin had a final concentration of 0.02 U / mL, argatroban had a final concentration of 0.75 gZmL, and collagen had a final concentration of 10 / gZmL.
  • Total liquid volume is 0. 36 mL.
  • Clinical trial example 9 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg Z day). Add 2 volumes of 0.2M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution It is added together.
  • heparin was added at final concentrations of 0.03 U / mL and 0.02 UZmL
  • argatroban was added at final concentrations of 0.3 and 0.5 ⁇ g ZmL
  • collagen was added at a final concentration of 10 gZmL.
  • the total volume is 0.36 mL.
  • shortening of 20% or more was observed, and the antiplatelet action of pretal is considered insufficient.
  • Clinical trial example 10 was a 58-year-old male with a cerebral infarction. This is an example. Add 2 volumes of 0.2M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X-system blood, and 2 volumes of 0.2M calcium chloride solution to Y-system blood. Add 1 volume of anticoagulant solution and 1 volume of platelet activator solution! Heparin was at a final concentration of 0.1 lU / mL, argatroban was at a final concentration of 1.2 g / mL, and collagen was at a final concentration of 8.3 / zg / mL. The total volume is 0.36 mL. In this case, the amount of argatroban is too small to be determined by argatroban, and heparin 20
  • Panaldine treatment is considered inadequate due to a reduction of more than%. After this, the R value of the thrombolast graph 7 months later was shown after changing to nospirin lOOmg / day. In any case, the treatment effect of bias pilin is considered to be insufficient with a shortening of 20% or more.
  • Case 11 was a 75-year-old male with atherosclerotic obstruction who was taking Pretal 200 mgZ and Epadele 900 mgZ.
  • 1 volume of 0.4M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to system X blood, and 1 volume of 0.4M saline calcium calcium solution is added to system Y blood.
  • 1 volume of anticoagulant solution and 1 volume of platelet activator solution are stored.
  • heparin has a final concentration of 0.07 UZmL
  • argatropane has a final concentration of 3.0 gZmL
  • collagen has a final concentration.
  • 10 / z gZmL The total volume is 0.36 mL.
  • Clinical trial example 12 is a 77-year-old male with atherosclerotic obstruction, taking pretal 200 mg Z day and Dorner 60 mg Z day. Add 1 volume of 0.4M saline-calcium water solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to system X blood, and 1 volume of 0.4M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution. For an ACT value of 111 seconds, heparin was at a final concentration of 0.03 and 0.06 UZmL, argatroban was at a final concentration of 3.0 gZmL, and collagen was at a final concentration of 10 g / mL.
  • the total volume is 0.36 mL.
  • Treatment with pretanol 200 mg / l and donoren 60 mg Z days is inadequate.
  • the R value of the thrombolast graph after 2 months is shown.
  • the power of the Arg3.0 data alone is insufficient.
  • the other three data show that all three are sufficient for this treatment.
  • Case 13 is a 59-year-old female with arteriosclerotic obstruction and taking pretal 200 mg Z day.
  • 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M aqueous solution of sodium chloride is added to Y-system blood.
  • 1 volume of coagulant solution and 1 volume of platelet activator solution are added.
  • ACT value of 193 seconds heparin at a final concentration of 0.03 UZmL, argatro The final concentration of van was 1.5 g / mL, and the final concentration of collagen was 10 g / mL.
  • the total volume is 0.36 mL. All show a reduction of 20% or more, and pretal 200mgZday is not adequately treated.
  • Case 14 was a 66-year-old male with atherosclerotic obstruction who was taking pretal 200 mgZ and ⁇ farin 2 mgZ.
  • 2 volumes of 0.2M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to system X blood, and 2 volumes of 0.2M aqueous calcium chloride solution are anticoagulated to system Y blood.
  • 1 volume of drug solution and 1 volume of platelet active solution are stored.
  • heparin was added at a final concentration of 0.03 UZmL, argatroban at a final concentration of 0.25 ⁇ g ZmL, and collagen at a final concentration of 1 mg / z gZmL.
  • the total volume is 0.36 mL.
  • Antiplatelet therapy is considered inadequate [Table 22]
  • Case 15 was a 62-year-old male with atherosclerotic obstruction who was taking pretal 200 mg / day. 2 volumes of 0.2M aqueous calcium chloride solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to the X-system blood, and 2 volumes of 0.2M calcium chloride solution are added to the Y-system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added.
  • ACT value of 179 seconds heparin had a final concentration of 0.06 UZmL, argatroban had a final concentration of 1.5 ⁇ gZmL, and collagen had a final concentration of 10 ⁇ gZmL. The total volume is 0.36 mL. Heparin 0.06 is the only treatment that has been shown to be sufficient, but all three other argatroban measurements have been shown to be inadequate. Insufficient. [0063] [Table 23] Clinical trial 15
  • Clinical trial example 16 is a 62-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z day. 2 volumes of 0.2M aqueous calcium chloride solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to the X-system blood, and 2 volumes of 0.2M calcium chloride solution are added to the Y-system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. For an ACT value of 190 seconds, heparin was added at a final concentration of 0.03 and 0.04 UZmL, argatroban was added at a final concentration of 0.75 and 1.
  • Case 17 was a 68-year-old male with atherosclerotic obstruction who was taking pretal 200 mg Z day.
  • ACT value of 190 seconds heparin was added at a final concentration of 0.1 lUZmL, argatroban was added at a final concentration of 0.4 // gZmL, and ADP was added at a final concentration of 8.3 / M.
  • the total liquid volume is 0.36 mL. All showed a reduction of 20% or more, and pretal 200 mg / day was judged to be inadequate.
  • Clinical trial example 18 is a 70-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z day. 2 volumes of 0.2M saline-calcium aqueous solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to system X blood, and 0.2M calcium chloride aqueous solution is added to system Y blood. 2 volumes, 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. For ACT value of 199 seconds, heparin is added at a final concentration of 0.03 and 0.05 U / mL, argatroban is added at a final concentration of 1.0 and 2.
  • Case 19 is a case of a 65-year-old female cerebral infarction who is taking Panalgin 200 mg Z day.
  • the total liquid volume is 0.36 mL.
  • Heparin in (1) shows that only the thing stretched with 0.1 is sufficient for treatment, V, but the results for the other two heparins and argatroban are insufficient. Therefore, treatment with panalgin 200mgZ is considered inadequate.
  • Case 20 was a 68-year-old female antiplatelet drug (?), With normal platelet function. 1 volume of 0.18M saline-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y-system blood. Add 1 volume of anticoagulant solution and 1 volume of platelet activator solution. For ACT values of 178 seconds, argatroban was added at final concentrations of 1.0, 3.0 and 4.0 / X g / mL, and collagen was added at final concentrations of ⁇ / z gZmL. The total volume is 0.36 mL.
  • the amount of argatroban to be added is 2 to 3 times the amount of argatropan or heparin obtained from the case of kaolin (-) trial.
  • the platelet function that is not suppressed can be determined sufficiently by the kaolin-added card.
  • Case 21 was a 78-year-old female with antiplatelet drug (?) And normal platelet function.
  • 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y blood.
  • argatroban was added at final concentrations of 0.25, 0.5, 1.0 and 1.5 g / mL, and collagen was added at a final concentration of 10 gZmL. The total volume is 0.36 mL.
  • the R value can be reduced to 30 to 40 minutes if 2 to 3 times the amount of argatroban to be added obtained from ACT in the Kaolin (1) trial is given to the Kaolin (+) trial. Also this power Pop and pin is used for the second time, and measurement is uneven.
  • Clinical trial 22 was a 78-year-old female antiplatelet drug (?), With normal platelet function. 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y blood. Add 1 volume of coagulant solution and 1 volume of platelet activator solution! For ACT values of 176 seconds, argatropan was adjusted to 1.0, 2.0 and 2.5 g / mL at final concentrations, and collagen was adjusted to 10 gZmL at final concentrations. The total volume is 0.36 mL. Again, in the case of kaolin trials, the R-value can be almost certainly set to 30 to 40 minutes with an amount 2 to 3 times the amount of argatroban determined by ACT. It is also possible to determine uncontrolled platelets.
  • Clinical trial example 23 was a 77-year-old male with atherosclerotic obstruction who was taking Pretal 200 mg Z day and Dorner 60 mg Z day. Add 1 volume of 0.18M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M calcium chloride aqueous solution to Y blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are stored. Argatroban has a final concentration of 0.5, 1.5 and 2.0 for an ACT value of 203 seconds
  • the total volume is 0.36 mL.
  • the measurement is sometimes unstable.
  • the antiplatelet treatment in this trial is inadequate.
  • Clinical trial example 24 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg Zday). Add 0.1 volume of 18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M calcium chloride aqueous solution, anticoagulant to Y blood. 1 volume of liquid and 1 volume of platelet activator liquid are added. For ACT values of 176 seconds, argatroban was given a final concentration of 1.5 and 4. O / z gZmL and collagen was given a final concentration of 10 gZmL. The total volume is 0.36 mL. Both kaolin (?) And kaolin (+) indicate that the platelet function is suppressed and that the difference is. Antiplatelet therapy is inadequate.
  • Case 25 was a 44-year-old male antiplatelet drug (?) In which the coagulation function was normal. Add 1 volume of 0.18M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and 1 volume of 0.18M salt calcium aqueous solution to system Y blood. One volume of anticoagulant solution and one volume of platelet activator solution are added. In addition to actin, argatroban was added at a final concentration of 1.0 and 2.5 g / mL and collagen was added at a final concentration of 10 gZmL for an ACT value of 185 seconds. The total volume is 0.36 mL. Platelet function is thought to have declined.
  • Case 26 is a case of a 46-year-old male antiplatelet drug (?) With normal clotting ability (aPTT, PT are normal).
  • aPTT normal clotting ability
  • 1 volume of drug solution and 1 volume of platelet activator solution are added.
  • argatroban was added at a final concentration of 1.5 and 2.5 ⁇ mL and collagen at a final concentration of ⁇ / z gZmL against an ACT value of 167 seconds.
  • the total volume is 0.36 mL. Even though the coagulation ability is normal, the platelet function is considered to have decreased.
  • Clinical trial 27 is a 48-year-old male antiplatelet drug (?) In which blood coagulation ability is normal. 1 volume of 0.4M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to the X system blood, and 1 volume of 0.4M salt calcium aqueous solution is added to the Y system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. In addition to actin, argatroban was added at a final concentration of 2.0 and 3.5 gZmL and collagen was added at a final concentration of 10 gZmL for an ACT value of 158 seconds. The total volume is 0.36 mL. In this case, platelet function is considered normal. The measurement of lactin (+) is unstable.
  • Clinical trial example 28 is a 67-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z ⁇ .
  • 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M aqueous solution of sodium chloride is added to Y-system blood.
  • 1 volume of coagulant solution and 1 volume of platelet activator solution are added.
  • argatroban was added at 1.0 and 2.
  • C ⁇ gZmL and collagen at 10 gZmL at a final concentration for an ACT value of 181 seconds. The total volume is 0.36 mL.
  • the addition of actin shows a value closer to the measured value without the use of the coagulation promoter (the first measured value). It is considered that the measurement ability with the addition of actin is superior to the value with kaolin.
  • the power that pretal treatment is inadequate
  • the power that can be found with the addition of actin Kaolin treatment has shown that the treatment effect of pretal is sufficient.
  • the present invention is a test method for conducting an antithrombotic drug efficacy test appropriately, quickly and simply. Through the antithrombotic drug efficacy test method of the present invention, it greatly contributes to the medical, medical and pharmaceutical industries.
  • FIG. 1 is a diagram for explaining thrombolast graph measurement.

Abstract

It is intended to provide a testing method for conducting a drug efficacy test for an antithrombotic agent rapidly and simply. It is a drug efficacy testing method for an antithrombotic agent in which thromboelastograph is measured in a system in which an aqueous solution of calcium chloride, an anticoagulant solution and physiological saline are added to blood collected from a patient administered with the antithrombotic agent (X system blood) and a system in which an aqueous solution of calcium chloride, an anticoagulant solution and a platelet activator solution are added to the blood (Y system blood) and R values of the X system blood and the Y system blood are compared, characterized in that the same amount of calcium chloride and the same kind and the same amount of the anticoagulant are added to the X system blood and the Y system blood, respectively.

Description

明 細 書  Specification
抗血栓薬薬効試験方法  Antithrombotic drug efficacy test method
技術分野  Technical field
[0001] 本発明は、抗血栓薬薬効試験方法に関するものである。更に詳しくは、治療に投 与中の抗血栓薬の薬効を迅速かつ簡易に判定するための抗血栓薬薬効試験方法 に関する。  [0001] The present invention relates to an antithrombotic drug efficacy test method. More particularly, the present invention relates to an antithrombotic drug efficacy test method for quickly and easily determining the efficacy of an antithrombotic drug being administered for treatment.
背景技術  Background art
[0002] 血栓塞栓は、心臓又は血管の中で血液が固まった状態のことをいう。血栓塞栓によ り血液が止まると、その血管で養われている部分に病変を生じ、致命傷になり得るの で、血栓塞栓を防止するため抗血栓薬が必要なものとなっている。一方、抗血栓薬、 抗血小板作用薬の薬効の判定に関しては、血液中にトロンビンという強大な血小板 活性賦活剤が存在するので、元来、抗血小板作用剤の薬効を明確に判定することが 困難で、有効な判定手段が無 、のが実情であった。  [0002] Thromboembolism refers to a state in which blood has solidified in the heart or blood vessels. If blood is stopped by a thromboembolism, it will cause a lesion in the part nourished by the blood vessel, which can be fatal. Therefore, antithrombotic drugs are necessary to prevent thromboembolism. On the other hand, regarding the determination of the efficacy of antithrombotic drugs and antiplatelet agonists, it is difficult to clearly determine the efficacy of antiplatelet agonists from the beginning because there is a strong platelet activity activator called thrombin in the blood. In fact, there was no effective judgment means.
[0003] 本発明者は、抗凝固剤の存在下にトロンボェラストグラフ上で、血餅形成の度合い を測定する方法が、血小板活性賦活剤が血液凝固促進の原因になっている場合、 抗血小板作用剤がどのように血液凝固の促進を阻害するのかを検定、試験する方法 として適して!/ヽるのではな!/ヽかと考えて、抗血栓薬薬効試験法を完成させ特許出願( 特願 2005— 149183 (特許文献 1) )した。その要旨は、抗血栓薬の投与を受けてい る患者力 採取した血液の一部にへノ^ン又はアルガトロバン等の抗凝固剤を加え た系(X系血液)、前記血液の一部に前記抗凝固剤及びアデノシン二リン酸又はコラ 一ゲン等の血小板活性剤を加えた系(Y系血液)にっき、同時にトロンボェラストダラ フを測定し、 X系血液及び Y系血液の R値を比較して、 Y系血液の R値が X系血液の R値に比し、有意に短縮した場合、抗血栓療法として薬効は有効でないと判定し、 Y 系血液の R値が X系血液の R値と有意差がなければ抗血栓療法としての薬効は有効 であると判定するものである。  [0003] The present inventor has proposed a method for measuring the degree of blood clot formation on a thrombolast graph in the presence of an anticoagulant, when the platelet activity activator causes blood coagulation. Appropriate as a method to test and test how platelet agonists inhibit the promotion of blood clotting! Japanese Patent Application No. 2005-149183 (Patent Document 1)). The gist of the system is that a patient who is receiving an antithrombotic drug is a system in which an anticoagulant such as henon or argatroban is added to a part of the collected blood (system X blood), In a system to which an anticoagulant and a platelet activator such as adenosine diphosphate or collagen are added (Y system blood), thrombolasta taraf is measured at the same time, and R values of X system blood and Y system blood are compared. If the R value of the Y blood is significantly shorter than the R value of the X blood, it is determined that the efficacy of the antithrombotic therapy is not effective, and the R value of the Y blood is R of the X blood. If there is no significant difference from the value, the efficacy as antithrombotic therapy is judged to be effective.
[0004] 特許文献 1 :特願 2005— 149183 [0004] Patent Document 1: Japanese Patent Application 2005—149183
発明の開示 発明が解決しょうとする課題 Disclosure of the invention Problems to be solved by the invention
[0005] 採取した血液の凝固時間は人により、また同一人でも摂取している医薬の種類や 量によって異なるという性質をもっている。従来の方法では、このような多種多様な血 液につ!、て抗血栓薬薬効試験を行うに際しては、事前に血液の凝固時間等を把握 しておく必要があり、この事前試験のために相当の時間を要していた。本発明は、有 効、確実且つ迅速に実施するための、抗血栓薬薬効試験方法を提供しょうとするも のである。即ち、本発明は、事前試験に要する時間を短縮して、多種多様な血液の 抗血栓薬薬効試験を有効、確実且つ迅速に実施するための抗血栓薬薬効試験方 法を提供しょうとするものである。更に、専門的な知識を有しない者でも、容易、正確 、迅速かつ簡便に測定可能な抗血栓薬薬効試験方法を提供しょうとするものである  [0005] The clotting time of the collected blood has the property that it varies depending on the person and the type and amount of the medicine taken by the same person. In the conventional method, it is necessary to know the blood coagulation time in advance when conducting antithrombotic drug efficacy tests for such a wide variety of blood solutions. It took a considerable amount of time. The present invention is intended to provide an antithrombotic drug efficacy test method that is effective, reliably and rapidly implemented. That is, the present invention seeks to provide an antithrombotic drug efficacy test method for effectively, surely and quickly conducting various blood antithrombotic drug efficacy tests by shortening the time required for the preliminary test. It is. Furthermore, it is intended to provide an antithrombotic drug efficacy test method that can be easily, accurately, quickly and easily measured even by those who do not have specialized knowledge.
課題を解決するための手段 Means for solving the problem
[0006] 本発明の要旨を箇条書きで示すと、以下のようになる。 [0006] The gist of the present invention is indicated by itemized items as follows.
[1]抗血栓薬の投与を受けている患者力 採取した血液に塩ィ匕カルシウム水溶液、 抗凝固剤液及び生理食塩水を加えた系(X系血液)、前記血液に塩化カルシウム水 溶液、抗凝固剤液及び血小板活性剤液を加えた系(Y系血液)にっき、トロンボエラ ストグラフを測定し、 X系血液及び Y系血液の R値を比較する抗血栓薬薬効試験方 法であって、  [1] The power of patients receiving antithrombotic drugs A system in which a salted calcium aqueous solution, an anticoagulant solution and physiological saline are added to collected blood (system X blood), a calcium chloride solution in the blood, An antithrombotic drug efficacy test method for measuring the thromboelastograph in a system (Y blood) to which an anticoagulant solution and a platelet activator solution are added, and comparing the R values of the X system blood and the Y system blood,
血液に加える塩化カルシウムは、 X系血液、 Y系血液ともに、同量であり、同じく血液 に加える抗凝固剤は、 X系血液、 Y系血液ともに、同種のもの同量であることを特徴と する抗血栓薬薬効試験方法。  The amount of calcium chloride added to blood is the same for both X-system blood and Y-system blood, and the same amount of anticoagulant added to blood is the same for both X-system blood and Y-system blood. Antithrombotic drug efficacy test method.
[2]前記抗凝固剤液がへパリン (未分化へパリン、低分子へパリン)水溶液又はアル ガトロバンの水溶液であることを特徴とする [1]に記載の抗血栓薬薬効試験方法。  [2] The antithrombotic drug efficacy test method according to [1], wherein the anticoagulant solution is a heparin (undifferentiated heparin, low molecular heparin) aqueous solution or an argatroban aqueous solution.
[3]前記へパリン水溶液を含有させる終濃度が 0. Ol (UZmL)力ら 0. 1 (UZmL) の範囲であり、アルガトロバン水溶液を含有させる終濃度が 0. 1 ( gZmL)から 5. 0 ( μ g/mL)の範囲であることを特徴とする [2]に記載の抗血栓薬薬効試験方法。  [3] The final concentration containing the heparin aqueous solution is in the range of 0.1 Ol (UZmL) force and 0.1 (UZmL), and the final concentration containing the argatroban aqueous solution is from 0.1 (gZmL) to 5.0. The antithrombotic drug efficacy test method according to [2], which is in a range of (μg / mL).
[4]前記へパリン水溶液、アルガトロバン水溶液を含有させる終濃度を、予め経験的 に求めた ACT値とカ卩えるべきへパリン水溶液、アルガトロバン水溶液の終濃度の関 係から決定し、該決定した終濃度でへパリン水溶液、アルガトロバン水溶液を含有さ せることを特徴とする [3]に記載の抗血栓薬薬効試験方法。 [4] The relationship between the final concentration of the heparin aqueous solution and argatroban aqueous solution, the ACT value obtained empirically in advance, and the final concentration of the heparin aqueous solution and argatroban aqueous solution to be obtained. The antithrombotic drug efficacy test method according to [3], wherein a heparin aqueous solution and an argatroban aqueous solution are contained at a final concentration determined from the person in charge.
[5]前記血小板活性剤液がアデノシン二リン酸 (ADP)水溶液又はコラーゲン水溶 液であることを特徴とする [ 1 ]から [4]の 、ずれかに記載の抗血栓薬薬効試験方法。 [5] The antithrombotic drug efficacy test method according to any one of [1] to [4], wherein the platelet activator solution is an adenosine diphosphate (ADP) aqueous solution or a collagen aqueous solution.
[6]前記アデノシン二リン酸水溶液を含有させる終濃度が 6. O /z M以上、前記コラー ゲン水溶液を含有させる終濃度が 1. 0 gZmL以上であることを特徴とする [5]に 記載の抗血栓薬薬効試験方法。 [6] The final concentration containing the adenosine diphosphate aqueous solution is 6. O / zM or more, and the final concentration containing the collagen aqueous solution is 1.0 gZmL or more. Antithrombotic drug efficacy test method.
[7]X系血液において、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の容量 比を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の添カロ 量は Z = 1の場合は 30 L、 Z = 2の場合は 40 Lとし、 1 < Zく 2の場合は「30 + (4 0- 30) X (Z—l)」 Lであり、 Y系血液においては、塩化カルシウム水溶液、抗凝 固剤液及び血小板活性剤液の各試薬の容量比を Z : 1: 1とするとき、塩化カルシウム 水溶液、抗凝固剤液及び血小板活性剤液の添加量は Z= lの場合は 30 レ Z = 2 の場合は 40 Lとし、 1 <2< 2の場合は「30+ (40— 30) X (Z— 1)」 Lであること を特徴とする [ 1]から [7]の ヽずれかに記載の抗血栓薬薬効試験方法。  [7] When the volume ratio of calcium chloride aqueous solution, anticoagulant solution, and physiological saline is Z: 1: 1 in the blood of system X, the amount of calories added to the calcium chloride aqueous solution, anticoagulant solution, and physiological saline is 30 L if Z = 1, 40 L if Z = 2, and if 1 <Z 2 then "30 + (4 0-30) X (Z—l)" L, Y blood When the volume ratio of each reagent of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution is Z: 1: 1, the amount of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution added is 30 Z for Z = l 40 L for Z = 2 and 1 <2 <2, "30+ (40—30) X (Z—1)” L The antithrombotic drug efficacy test method according to any one of [1] to [7].
[8]前記 X系血液及び Y系血液に更に血液凝固促進剤としてァクチン水溶液を終濃 度 0. 5ngZmLから 2. 5ngZmLの範囲で含有させることを特徴とする請求項 1から [6]に記載の抗血栓薬薬効試験方法。 [8] The method according to any one of [1] to [6], wherein the X-system blood and the Y-system blood further contain an aqueous actin solution as a blood coagulation promoter in a final concentration range of 0.5 ngZmL to 2.5 ngZmL. Antithrombotic drug efficacy test method.
[9]X系血液において、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の容量 比を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の添カロ 量は Z = 1の場合は 40 L、 Z = 2の場合は 50 Lとし、 1 < Zく 2の場合は「40 + (4 0- 30) X (Z—l)」 /z Lであり、  [9] When the volume ratio of calcium chloride aqueous solution, anticoagulant solution, and physiological saline is Z: 1: 1 in the blood of system X, the amount of calories added to the calcium chloride aqueous solution, anticoagulant solution, and physiological saline is 40 L if Z = 1, 50 L if Z = 2, and if 1 <Z 2 then "40 + (4 0-30) X (Z—l)" / z L,
Y系血液においては、塩化カルシウム水溶液、抗凝固剤液及び血小板活性剤液の 容量比を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び血小板活性剤 液の添力卩量は Z= lの場合は 40 L、Z = 2の場合は 50 Lとし、 1 <Zく 2の場合は 「40+ (40- 30) X (Z— 1)」 Lであることを特徴とする [8]に記載の抗血栓薬薬効 試験方法。  In Y-system blood, when the volume ratio of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution is Z: 1: 1, the added amount of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution Is 40 L when Z = l, 50 L when Z = 2, and `` 40+ (40-30) X (Z—1) '' L when 1 <Z Yes The antithrombotic drug efficacy test method according to [8].
[10]X系血液において、 0. 18M塩ィ匕カルシウム水溶液を 1容量〜 2容量、抗凝固 剤液を 1容量及び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 1 8M塩化カルシウム水溶液を 1容量〜 2容量、抗凝固剤液を 1容量及び血小板活性 剤液を 1容量の割合で含有させることを特徴とする [ 1]から [9]の 、ずれかに記載の 抗血栓薬薬効試験方法。 [10] In X-system blood, 0.1 to 2 volumes of 0.1M aqueous calcium chloride solution, anticoagulant 1 volume of drug solution and 1 volume of physiological saline, and in Y-system blood, 0.1 to 2 volumes of 0.1M aqueous 8M calcium chloride solution, 1 volume of anticoagulant solution, and platelet activator solution The antithrombotic drug efficacy test method according to any one of [1] to [9], wherein the antithrombotic drug efficacy test is characterized in that it is contained in a proportion of 1 volume.
[11]X系血液において、 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固剤液を 1容 量及び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 2M塩化カル シゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液を 1容量の割合で 含有させることを特徴とする [ 1]から [9]の ヽずれかに記載の抗血栓薬薬効試験方 法。  [11] In X-system blood, contain 2 volumes of 0.2M saline-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline, and 0.2M chloride in Y-system blood. The antithrombotic drug efficacy according to any one of [1] to [9], characterized by containing 2 volumes of aqueous calcium solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution Test method.
[12]X系血液において、 0. 4M塩ィ匕カルシウム水溶液を 1容量、抗凝固剤液を 1容 量及び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 4M塩化カル シゥム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液を 1容量の割合で 含有させることを特徴とする [ 1]から [9]の ヽずれかに記載の抗血栓薬薬効試験方 法。  [12] In X-system blood, contain 0.4 volume of 0.4M saline-calcium aqueous solution, 1 volume of anticoagulant solution, and 1 volume of physiological saline. The antithrombotic drug efficacy according to any one of [1] to [9], characterized by containing 1 volume of calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution Test method.
発明の効果 The invention's effect
本発明は、抗血栓薬の投与を受けて 、る患者力 採取した血液に塩ィ匕カルシウム 水溶液、抗凝固剤液及び生理食塩水を加えた系(X系血液)、前記血液に塩化カル シゥム水溶液、抗凝固剤液及び血小板活性剤液をカ卩えた系(Y系血液)にっき、トロ ンボエラストグラフを測定し、 X系血液及び Y系血液の R値を比較する抗血栓薬薬効 試験方法であって、 X系血液及び Y系血液に添加されて 、る塩ィ匕カルシウムは同量 、同抗凝固剤は同種のもの同量であることを特徴としている。抗血栓薬薬効試験方 法を上記のように構成することにより、事前試験に要する時間を短縮し、多種多様な 血液の抗血栓薬薬効試験を有効、迅速且つ正確に実施できるという効果を奏する。 特に、抗血栓薬薬効試験に要する時間を短くできるという効果を奏する。更に、予め 経験的に得た ACT値と加えるべき抗凝固剤液の終濃度の関係を用い、専門的知識 を有しない者でも、容易、正確、迅速かつ簡便に抗血栓薬薬効試験を行うことができ るという効果を奏する。  The present invention relates to a system (X system blood) in which a salt calcium aqueous solution, an anticoagulant solution, and physiological saline are added to blood collected from a patient who has received antithrombotic drugs, and calcium chloride is added to the blood. Antithrombotic drug efficacy test method for measuring the tropoelastograph and comparing the R values of X blood and Y blood in a system (Y blood) containing an aqueous solution, anticoagulant solution and platelet activator solution However, when added to X-system blood and Y-system blood, it is characterized by the same amount of calcium salt and the same amount of the same anticoagulant. By configuring the antithrombotic drug efficacy test method as described above, it is possible to shorten the time required for the preliminary test and to effectively, quickly and accurately perform various blood antithrombotic drug efficacy tests. In particular, there is an effect that the time required for the antithrombotic drug efficacy test can be shortened. Furthermore, using the relationship between the ACT value obtained in advance and the final concentration of the anticoagulant solution to be added, even those who do not have specialized knowledge should conduct antithrombotic drug efficacy tests easily, accurately, quickly and simply. There is an effect that can be done.
発明を実施するための最良の形態 以下、本発明を実施形態に基づ!/ヽて説明する。まず、抗血栓薬薬効試験法の手順 につ 、て説明する。 1)抗血栓薬とくに抗血小板薬を服用して 、る患者の血液をタエ ン酸サンプルとして採取する。その際、如何ほどの凝固時間を有する血液かを、判別 するために米国 ITC社製 Hemochron (へモクロン)により Activated CoagulationBEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described based on embodiments. First, the procedure of the antithrombotic drug efficacy test method is explained. 1) Taking an antithrombotic drug, especially an antiplatelet drug, and collecting the blood of the patient as a taenic acid sample. At that time, in order to determine how long the blood has clotting time, activated coagulation by Hemochron made by ITC in the US
Time (ACT)を測定する力、他の血液凝固計により凝固時間を測定する。その血 液の凝固時間(トロンボェラストグラフ上の R値とほぼ同義)を 30〜40分にひきのば す (トロンビンを一部抑制するか、生成を遅らせる意味を有する)ために、抗凝固剤と して使用するアルガトロバン又はへパリンの量をへモクロンからの ACTの数値(正常 成人では 105〜150)又は他の血液凝固計による凝固時間から推察し、決定する。 2 )トロンボェラストグラフ上にて、クェン酸血液サンプルの凝固をスタートさせるために 、試薬を添加する。即ち、第 1チャンネル (X系血液を測定チャンネル)においては、 該血液に、塩ィ匕カルシウム(以下、塩カルと省略することがある)水溶液、 ACT値から 決定した量の抗凝固剤液、生理食塩水(以下、生食とも称する)を加え、第 2チャンネ ル (Y系血液を測定するチャンネル)においては、該血液に、塩化カルシウム水溶液 、 ACT値から決定した量の抗凝固剤液、血小板凝集賦活剤液を加えて、それぞれ 同時にトロンボェラストグラフを測定する。ここで特に R値に注目する。尚、第 1チャン ネルを X系血液の測定チャンネルとし、第 2チャンネルを Y系血液を測定するチャン ネルとした力 第 2チャンネルを X系血液の測定チャンネルとし、第 1チャンネルを Y 系血液の測定するチャンネルとすることもできることは言うまでもない。 3)第 1チャンネ ル及び第 2チャンネルの R値を比較して、有意差があれば、第 2チャンネルでの血小 板刺激が第 2チャンネルでの凝血塊形成を促進したことになり、この血液の血小板活 性は大きぐ抑制されていないと判定する。有意差が無ければ、第 2チャンネルでの 血小板刺激が第 2チャンネルでの凝血塊形成を促進できず、この血液の血小板活性 は小さぐ十分に抑制されていると判定する。尚、 1)で決定する抗凝固剤液として使 用するアルガトロバン水溶液又はへパリン水溶液の量は、予め経験的に求めた ACT 値と添加すべきアルガトロバン水溶液又はへパリン水溶液の至適量の関係を参照し て決定する。 ACT値と添加すべきアルガトロバン又はへパリンの至適量(終濃度)を 経験的に求めたものを、表 11及び表 12に示した。また、試薬類は一般的に称する場 合は、抗凝固剤のように剤なる文言としたが、抗血栓薬薬効試験用試薬として使用 する場合は、抗凝固剤液のように剤液と液なる文言を付した。 Force to measure Time (ACT), clotting time is measured by other blood coagulometer. Anticoagulation is performed in order to increase the clotting time of the blood (roughly synonymous with the R value on the thrombolast graph) to 30 to 40 minutes (thus suppressing thrombin partially or delaying production). The amount of argatroban or heparin to be used as an agent is estimated and determined from the value of ACT from hemoclone (105 to 150 in normal adults) or the clotting time by other blood coagulometers. 2) Add reagent to start clotting of citrate blood sample on thrombolast graph. That is, in the first channel (X system blood is a measurement channel), the blood is mixed with an aqueous solution of calcium chloride (sometimes abbreviated as chlorocalcium), an anticoagulant solution in an amount determined from the ACT value, Saline (hereinafter also referred to as raw food) is added, and in the second channel (channel for measuring Y-system blood), the blood is mixed with an aqueous solution of calcium chloride, an amount of anticoagulant solution determined from the ACT value, and platelets. Add a coagulant activator solution and measure the thrombolast graph at the same time. Here we pay particular attention to the R value. Note that the first channel is the measurement channel for X-system blood, the second channel is the channel for measuring Y-system blood, the second channel is the measurement channel for X-system blood, and the first channel is for Y-system blood. It goes without saying that the channel can be measured. 3) Comparing the R values of the first channel and the second channel, if there is a significant difference, platelet stimulation in the second channel promoted clot formation in the second channel. It is determined that the platelet activity of blood is not greatly suppressed. If there is no significant difference, it is judged that platelet stimulation in the second channel cannot promote clot formation in the second channel, and the platelet activity of this blood is small and sufficiently suppressed. For the amount of argatroban aqueous solution or heparin aqueous solution used as the anticoagulant solution determined in 1), refer to the relationship between the ACT value obtained in advance and the optimum amount of argatroban aqueous solution or heparin aqueous solution to be added. And decide. Tables 11 and 12 show the ACT values and the empirically obtained optimal amounts (final concentrations) of argatroban or heparin to be added. Reagents are also commonly referred to as The term “agent” is used as an anticoagulant, but when used as a reagent for antithrombotic drug efficacy test, the term “agent” is used.
[表 11]  [Table 11]
[0010] [表 12] [0010] [Table 12]
[0011] トロンボェラストグラフについて簡単に説明する。トロンボェラストグラフは、血液の 凝固過程を全血での血塊弾性でみる装置である。血塊弾性値を縦軸にとりその時間 変化を横軸にとってグラフを描いたものを図 1に示した。図 1のような測定図に基づい て、血塊弾性値の巾が 2mmになった時点を反応時間 R値、血塊弾性値の巾が 20m mになった時点を凝固時間 K値、血塊弾性値の巾の最大振幅 MA値及び血塊弾性 の変化のグラフで接線を引いたときの角度 ANG値等が数値として得られる。測定の 操作自体は、血液をキュベットに入れて、測定を開始するという簡単なものとなってい る。尚、トロンボェラストグラフは米国へモスコープ社製トロンボェラストグラフ C—TE G3000T型機及びペンタフアーム社製ローテム GAMMA—を使用した。 [0011] The thrombolast graph will be briefly described. The thrombolast graph is a device that observes the coagulation process of blood by the clot elasticity of whole blood. Figure 1 shows a graph drawn with the clot elasticity on the vertical axis and the time change on the horizontal axis. Based on the measurement diagram shown in Fig. 1, the reaction time R value when the clot elasticity value width became 2 mm, the clot time K value and the clot elasticity value when the clot elasticity value width became 20 mm. The maximum angle amplitude MA value and the angle ANG value when tangent is drawn in the graph of change in blood clot elasticity are obtained as numerical values. The measurement operation itself is as simple as putting blood in a cuvette and starting the measurement. The In addition, the thrombolast graph used in the United States was a tronbolast graph C-TE G3000T manufactured by Moscope Co. and a Rotem GAMMA manufactured by Pentafuarm.
[0012] トロンボェラストグラフを使用する試験法について説明する。 1)測定にはトロンボェ ラストグラフを用いる。使用するトロンボェラストグラフは、少なくとも測定部が 2チャン ネル以上を有するもので、測定液量は 0. 36mL前後である。 2)抗血栓薬を投与され ている患者の血液を、凝固検査用採血管に採取する。通常、凝固検査用採血管に は、凝血が始まらないようにクェン酸が混入されている。 3)トロンボェラストグラフの第 1チャンネルに、塩化カルシウム水溶液、血液、抗凝固剤液及び生理食塩水を加え る。この際、塩ィ匕カルシウムは、採血管内に混入していたクェン酸によって不活性ィ匕 されたカルシウムを再活性ィ匕させ、凝血を開始するためのものである。 4)トロンボエラ ストグラフの第 2チャンネルに、塩化カルシウム水溶液、血液、抗凝固剤液及び血小 板活性剤液を加える。 5)第 1チャンネルと第 2チャンネルを同時にトロンボエラストグ ラフで測定し、トロンボェラストグラフ上の R値を比較する。 6)第 2チャンネルの R値が 第 1チャンネルの R値に比し、有意に短縮した場合、投与中の抗血栓薬による薬効は 有効でな!ヽと判断される。 7)第 2チャンネルの R値が第 1チャンネルの R値と有意差 カ い場合、投与中の抗血栓薬による薬効は有効と判断される。  [0012] A test method using a thrombolast graph will be described. 1) Use thrombolast graph for measurement. The thrombolast graph to be used has at least a measuring section with 2 or more channels, and the measuring liquid volume is around 0.36 mL. 2) Collect blood from a patient receiving antithrombotic drugs into a blood collection tube for coagulation tests. Normally, clotting acid is mixed in the blood collection tube for coagulation test so that the blood clot does not start. 3) Add calcium chloride aqueous solution, blood, anticoagulant solution and physiological saline to the first channel of the thrombolast graph. At this time, salt calcium is used to reactivate calcium that has been inactivated by citrate mixed in the blood collection tube and to start clotting. 4) Add calcium chloride aqueous solution, blood, anticoagulant solution and platelet activator solution to the second channel of the thromboelastograph. 5) Measure the first channel and the second channel at the same time with the thromboelast graph, and compare the R values on the thrombolast graph. 6) If the R value of the second channel is significantly shorter than the R value of the first channel, the efficacy of the antithrombotic drug during administration is judged to be effective! 7) If the R value of the second channel is significantly different from the R value of the first channel, the efficacy of the antithrombotic drug during administration is judged to be effective.
[0013] 抗血栓薬薬効試験に使用する血液及び試薬の液量について、説明する。血液に 加える試薬の量は、 X系血液、 Y系血液ともに、規定の容量比である。血液に加える 塩ィ匕カルシウムは、 X系血液、 Y系血液ともに、その濃度と量は同一である。抗凝固 剤液も、 X系血液、 Y系血液ともに、同じ種類のもの同濃度で同量である。試験に使 用する X系血液の量と Y系血液の量は同じである。血液にカ卩える塩ィ匕カルシウムは、 X系血液、 Y系血液ともに、抗凝固剤液 1容量、生理食塩水 1容量に対して、 0. 18 M塩化カルシウム水溶液 (静注用として巿販されて ヽる 20mL内に塩ィ匕カルシウム 0 . 4g含むもの;大塚製薬、扶桑薬品、三晃など)の場合容量 1以上 2以下の割合でカロ え(この場合、 0. 18M塩化カルシウム水溶液の容量は 1. 0、 1. 5、 2. 0など何れで もよい)、 0. 2M塩ィ匕カルシウム水溶液の場合は容量 2の割合でカ卩え、 0、 4M塩化力 ルシゥム水溶液の場合は容量 1の割合でカ卩える。  [0013] The amount of blood and reagents used in the antithrombotic drug efficacy test will be described. The amount of reagent added to the blood is the specified volume ratio for both X-system blood and Y-system blood. The salt and calcium added to blood has the same concentration and amount in both X and Y blood. The anticoagulant solution is the same in both the X-type blood and the Y-type blood at the same concentration and the same amount. The amount of X blood and Y blood used for the test is the same. The calcium salt that can be stored in blood is 0.18 M calcium chloride aqueous solution (commercially available for intravenous injection) for 1 volume of anticoagulant solution and 1 volume of physiological saline for both system X blood and system Y blood. In the case of 20 mL containing 0.4 g of salty calcium; Otsuka Pharmaceutical, Fuso Pharmaceutical, Sanso, etc., the volume is 1 or more and 2 or less in this case (in this case, 0.18M calcium chloride aqueous solution The volume can be any of 1.0, 1.5, 2.0, etc.), 0.2 In the case of a 2M salt-calcium aqueous solution, the volume can be adjusted at a rate of 2, and in the case of a 0, 4M chloride chloride aqueous solution Capable of capacity 1
[0014] 抗凝固剤は、へパリン (未分化へパリン、低分子へノ リン)又はアルガトロバンを用 いる(いずれも凝固因子 Ila抑制作用を有すもの)。へパリン又はアルガトロバンの終 濃度力 へパリンは 0. Ol (UZmL)力ら 0. l (UZmL) (容量の単位であるリットルは 通常 Lの筆記体の小文字が使用される力 Lの小文字 1は数字の 1と間違いやすいの で、ここでは容量の単位リットルは Lで示した)になるように、アルガトロバンの終濃度 が 0. 1 ( gZmL)から 5. 0 ( gZmL)になるように加える。そして、実際に添加す る量は、測定対象者血液の凝固時間の長さにより調節する必要があり、抗血栓薬服 用者の特殊な血液に対処するため、濃度の異なる多種類の抗凝固剤液を作成して おくのが好ましい。前もって用意しておく濃度としては、へパリンでは、 0. 01、 0. 02 、 0. 03、 0. 04、 0. 05、 0. 06、 0. 07、 0. 08、 0. 09、 0. 1の 10種類の各濃度(単 位はいずれも UZmL)の試薬を作成し用意しておくのがよい。アルガトロバンでは、 0. 1、 0. 3、 0. 5、 0. 75、 1. 0、 1. 5、 2. 0、 2. 5、 3. 0、 3. 5、 4. 0、 4. 5、 5. 0の 1 3種類の濃度(単位は 、ずれも μ g/mL)の試薬を作成し用意しておくのがよ!、。 [0014] As the anticoagulant, heparin (undifferentiated heparin, low molecular weight heline) or argatroban is used. (Both have a coagulation factor Ila inhibitory effect). Heparin or Argatroban final concentration force Heparin is 0. Ol (UZmL) force, etc. 0. l (UZmL) It is easy to mistake the number 1. Therefore, add the final concentration of argatroban from 0.1 (gZmL) to 5.0 (gZmL) so that the unit of volume is L). The actual amount to be added needs to be adjusted according to the length of the clotting time of the blood to be measured. To deal with the special blood of the antithrombotic drug users, various types of anticoagulants with different concentrations are used. It is preferable to prepare a chemical solution. Concentrations to be prepared in advance for heparin are: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0 It is advisable to prepare and prepare reagents for each of the 10 concentrations in 1 (all units are UZmL). In Argatroban, 0.1, 0.3, 0.5, 0.75, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 Prepare and prepare reagents with three concentrations of 5.0 (unit: μg / mL)!
[0015] 血小板活性剤は、アデノシン二リン酸 (ADP)又はコラーゲンを用いる。 ADPは、そ の終濃度が 6 M以上になるようにカ卩える。また、コラーゲン (COL)は、その終濃度 が 1. 0 g/mL以上になるようにカ卩える。 ADPは、少なくとも、終濃度 6 /z M以上カロ えれば、トロンボェラストグラフの測定は問題なく行うことができる。同様に、コラーゲン も、少なくとも、終濃度 1. O /z gZmL以上カ卩えれば、トロンボェラストグラフの測定は 問題なく行うことができる。それぞれ使用量の上限値は特に無いが、実際上は、使用 量として、 ADPでは終濃度 M程度、コラーゲンでは終濃度 10 gZmL程度が 最大添加量となる。 [0015] As the platelet activator, adenosine diphosphate (ADP) or collagen is used. ADP can be adjusted so that its final concentration is 6 M or more. Collagen (COL) should be prepared so that its final concentration is 1.0 g / mL or more. If the final concentration of ADP is at least 6 / zM or more, the thrombolast graph can be measured without problems. Similarly, if the final concentration of collagen is at least 1. O / z gZmL, the thrombolast graph can be measured without problems. There is no particular upper limit for the amount used, but in practice, the maximum amount of ADP used is a final concentration of about M for collagen and about 10 gZmL for collagen.
[0016] トロンボェラストグラフのカップ内への試薬添カ卩量は、 X系血液においては、塩化力 ルシゥム水溶液、抗凝固剤液、生理食塩水の各試薬の容量比が 1: 1: 1の場合は 30 /z L、 1. 5 : 1 : 1の場合 35 L、 2 : 1: 1の場合 40 Lである。この添カ卩量のちがいは 試薬に占める塩ィ匕カルシウムの容量によって決定するものであり、例えば、試薬の混 合比が仮に 1. 3 : 1 : 1の場合添加する試薬量は 33 /z Lとなる。これを一般化すると、 塩化カルシウム水溶液、抗凝固剤液、生理食塩水の各試薬の容量比が Z: 1: 1の場 合、試薬添力卩量は Z= lの場合は 30 L、Z = 2の場合は 40 Lとし、 1 <Z< 2の場 合は「30+ (40- 30) X (Z— 1)」 /z Lになる。 Y系血液においては、塩化カルシウム 水溶液、抗凝固剤液及び血小板活性剤液の各試薬の容量比を Z : 1: 1とするとき、 塩化カルシウム水溶液、抗凝固剤液及び血小板活性剤液の添加量は Z= 1の場合 は 30 : L、 Z = 2の場合は 40 Lとし、 1 <2< 2の場合は「30+ (40— 30) X (Z— 1 )」;z Lになる。 [0016] The amount of reagent added to the cup of the thrombolast graph is such that the volume ratio of each reagent of aqueous chloride solution, aqueous solution of anticoagulant, and physiological saline is 1: 1: 1 in the system X blood. 30 / z L in the case of 1.5, 35 L in the case of 5: 1: 1, and 40 L in the case of 2: 1: 1. The difference in the amount of addition is determined by the amount of calcium chloride in the reagent. For example, if the reagent mixing ratio is 1.3: 1: 1, the amount of reagent added is 33 / z. L. Generalizing this, if the volume ratio of each reagent of calcium chloride aqueous solution, anticoagulant solution, and physiological saline is Z: 1: 1, the reagent loading is 30 L when Z = l, Z When L = 2, it is 40 L, and when 1 <Z <2, it becomes “30+ (40-30) X (Z— 1)” / z L. In Y-system blood, calcium chloride When the volume ratio of each reagent of the aqueous solution, anticoagulant solution and platelet activator solution is Z: 1: 1, the addition amount of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution is Z = 1 30: L, Z = 2 is 40 L, 1 <2 <2, "30+ (40-30) X (Z-1)";
[0017] トロンボェラストグラフの測定を安定に行うために、血液凝固促進剤としてァクチン をカロえることができる。抗凝固剤の添加量が多くなつた場合、トロンボェラストグラフの 測定時間が長くなることがある。このような場合、ァクチンを添加しておくと、適当な時 間内でトロンボェラストグラフの測定を行うことができる。ァクチンは終濃度で 0. 5ng ZmL〜2. 5ngZmL含有させるのが好ましい。また、ァクチンを加える場合は、血液 に加える塩ィ匕カルシウム水溶液、抗凝固剤液、生理食塩水等の試薬の添加量は、ァ クチンをカ卩えない場合よりも 10 L多くなる。  [0017] In order to stably measure the thrombolast graph, actin can be calorieated as a blood coagulation promoter. If the amount of anticoagulant added increases, the measurement time of the thrombolast graph may become longer. In such a case, if actin is added, the thrombolast graph can be measured within an appropriate time. The actin is preferably contained at a final concentration of 0.5 ng ZmL to 2.5 ng ZmL. In addition, when adding actin, the amount of reagent such as a saline calcium aqueous solution, anticoagulant solution or physiological saline added to the blood is 10 L more than when no actin is added.
[0018] トロンボェラストグラフの測定器には、 Haemoscope 社(Niles、 IL、 USA)のトロ ンボエラストグラフと Pentapharm 社(ミュンヘン、ドイツ)のローテムの 2社の測定器 があり、それぞれで使用する血液量が異なる。へモスコープ社のトロンボェラストダラ フは、全体量を 0. 36mLと規定し、薬剤を添加する場合、 1薬剤につき 10 /z Lとして 、その中に終濃度を定めて添加する。そして加える血液量はその分、減らして添加す る。例えば、抗凝固剤液と血小板賦活剤液を添加する場合、それぞれ 10 L、合計 20 Lが薬剤としてカ卩えられ、さらに、へモスコープ社が推奨する 0. 2M塩ィ匕カルシ ゥム水溶液 20 Lが加わるために、血液量は 0. 36mL力ら 40 Lを減じた 0. 32m Lとなる。ペンタフアーム社のローテムにおいては、添カ卩する血液量は 0. 30mLと常 に一定であり、塩化カルシウムとしてはトロンボェラストグラフと同様に 0. 2M塩化力 ルシゥム水溶液 20 Lを推奨しており、さらに今回の場合アルガトロバンとコラーゲン 力 それぞれ 10 Lカロわり、全体として 0. 34mLとなる。  [0018] There are two measuring instruments for the thrombolastograph, one from Haemoscope (Niles, IL, USA) and one from Rotem from Pentapharm (Munich, Germany). Blood volume is different. The total amount of Hemoscope thrombolast draft is specified as 0.36 mL, and when adding drugs, add 10 / z L per drug and determine the final concentration in it. Reduce the amount of blood to be added. For example, when an anticoagulant solution and a platelet activator solution are added, 10 L each, 20 L in total, can be stored as the drug, and in addition, the 0.2 M salt aqueous calcium solution recommended by Hemoscope 20 Because L is added, the blood volume becomes 0.32 mL, which is 0.36 mL force minus 40 L. PENTAFU ARM Rotem has a constant blood volume of 0.30 mL, and as with calcium chloride, a 0.2 M aqueous solution of 20 mL of NaCl is recommended as in the case of the Thrombolastograph. Furthermore, in this case, argatroban and collagen are each 10 L of calorie, and the total amount is 0.34 mL.
[0019] ペンタフアーム社のローテムの血液添加法に従う場合、本試薬の最大添加量は塩 化カルシウムを含めて 40 Lであり、これに常に 0. 3mLの血液をカ卩えると最大 0. 3 4mL、最低 0. 33mLとなり全く問題はない。次に、へモスコープ社のトロンボェラスト グラフの血液添加法に従う場合、血液 0. 33mLに試薬最大量 40 Lを添加すると 0 . 37mLとなり、トロンボェラストグラフの最大容量 0. 36mLを 10 /z L超えてしまうが、 ローテムにおいてローテム用に巿販しているある 1種類の試薬と塩ィ匕カルシウムをカロ えると 0. 375mLになるものがあること、およびトロンボェラストグラフの全体量を 0. 3 8mLにして測定したデータを発表して 、る研究を散見できることなどから、この全体 量でも正確なデータを得ることが可能と考えられることから、抗血栓薬薬効試験にお V、て添加する血液量を規定するにあたり、以上の 2社の血液添加法の違 、に鑑み、 この 2社の方法を合わせて採用して、この試験についての血液添力卩量は 0. 30mLか ら 0. 33mLまでの量を添カ卩する。 [0019] When following the Pentafarm Rotem blood addition method, the maximum addition amount of this reagent, including calcium chloride, is 40 L. When 0.3 mL of blood is constantly added to this, a maximum of 0.3 L is added. 4mL, minimum 0.33mL, no problem at all. Next, according to the blood addition method of the Hemoscope thrombolast graph, when the maximum reagent volume of 40 L is added to 0.33 mL of blood, it becomes 0.37 mL, and the maximum volume of thrombolast graph is 0.36 mL. L is exceeded, Measured with one reagent sold for rotem at Rotem and a salt-calcium content of 0.375 mL and a total thrombolast graph volume of 0.3 8 mL The amount of blood to be added in the antithrombotic drug efficacy test is prescribed because it is considered that accurate data can be obtained even with this total amount. In consideration of the difference in the blood addition method of the above two companies, the two companies' methods were adopted in combination, and the amount of blood force added for this test was from 0.30 mL to 0.33 mL. I will add it.
[0020] カオリン塗布カップ、ピンを含むカオリン製剤またはセライト (Celite)バイアルまたは セライト製剤の使用は、カオリンおよびセライトの同量、同濃度など同じ条件下で可能 であるにとどまらず、むしろ推奨される。更に、血液凝固促進剤 (ァクチン)を含有する 試薬の使用及びカオリン塗布カップ、ピンなど血液凝固促進剤の使用は、安定した 測定を可能にするという特徴がある。 [0020] Use of kaolin-coated cups, pins containing kaolin formulations or Celite vials or celite formulations is not only possible under the same conditions, such as the same amount and concentration of kaolin and celite, but rather recommended . Furthermore, the use of a reagent containing a blood coagulation promoter (actin) and the use of a blood coagulation promoter such as a kaolin-applied cup or pin are characterized by enabling stable measurement.
実施例 1  Example 1
[0021] 塩化カルシウム水溶液の添カ卩量について、先ず、ペンタフアーム社のローテムの血 液添加法に従った場合の実施例に基づいて説明する。  [0021] The amount of the calcium chloride aqueous solution added will be described based on an example in the case of following the Rotem blood addition method of Pentafarm.
[0. 18M塩化カルシウム水溶液]  [0. 18M calcium chloride aqueous solution]
ペンタフアーム社のローテムの血液添カ卩法に従って、血液量 0. 3mLに、本試薬の 代用量として生食 20 /z Lをカ卩え、更に 0. 18M塩ィ匕カルシウムを所定量カ卩えた場合 の R値を測定した。 R値は、(mean士 SD)で示した。 R値の測定結果は、表 1に示し た。即ち、クェン酸血液を recalcified (タエン酸によってカルシウムが抑制され、フィ ブリンがブロックされて、凝固が始まらなくなっているところに、再びカルシウムを与え て、凝固をスタートさせる)するために 0. 18M塩化カルシウムの至適量を調べた実施 ί列である。方法は 0. 18M塩ィ匕カノレシゥム 5 L、 10 L、 20 L、 30 Lのそれぞ れに、全血液 0. 3mLと今回加える試薬の 1部であるアルガトロバン又はへパリン及 びコラーゲン又は ADPに模した用量を各生理食塩水 10 L、計 20 Lの生理食塩 水を加えて血液凝固が進行する具合を測定した。測定を始めてからトロンボェラスト グラフ上の R点 (凝固の始まりを示すポイント)が現れる時間(分)を測定した。 R値は、 (mean士 SD :平均値士標準偏差)で示した。以下の、実施例にても、 R値は (mean 士 SD)で示した。表 1に示したトロンボェラストグラフの測定結果から、 0. 18M塩化 カルシウムを使用する場合、添加量は 10 μ Lと 20 μ Lが至適量であることがわかる。 0. 18M塩化カルシウム 5 Lでは少なすぎ、 0.18M塩化カルシウム 30 Lでは多 すぎて、 ヽずれも凝固が始まるポイントである R点は遅くなつて 、る。 According to Pentafarm's Rotem blood supplementation method, 0.3 mL of blood was added to 20 mL of raw food as a substitute for this reagent, and 0.18 M salt calcium was added to a predetermined amount. The R value was measured. The R value is indicated by (mean person SD). The measurement results of R value are shown in Table 1. That is, 0.18M chloride is used to recalcified citrate blood (where calcium is suppressed by taenoic acid and fibrin is blocked and coagulation does not start, and calcium is given again to start coagulation). It is the implementation column which investigated the optimal amount of calcium. The method is as follows: 0.1M of each of 18M salt ノ canorecum 5 L, 10 L, 20 L, and 30 L, 0.3 mL of whole blood and argatroban or heparin and collagen or ADP, which is a part of the reagent to be added this time. The simulated dose was 10 L for each physiological saline, and a total of 20 L physiological saline was added to measure the degree of blood coagulation. The time (minutes) at which the R point (the point indicating the start of solidification) appears on the thrombolast graph after the measurement was started was measured. The R value is indicated by (mean person SD: mean person standard deviation). In the following examples, the R value is (mean It was shown by Shishi SD). From the results of the thrombolast graph shown in Table 1, it can be seen that when 0.18M calcium chloride is used, the optimal additions are 10 μL and 20 μL. 0. 18M calcium chloride 5 L is too little, 0.18M calcium chloride 30 L is too much.
[0022] [表 1] [0022] [Table 1]
* : 10〃 Lと有意差 (pく 0. 05), † : 20 Lと有意差 (pく 0. 05)  *: Significant difference from 10〃L (p <0. 05), †: Significant difference from 20 L (p <0. 05)
統計は 0 n e— wa y AN OVAと F i s h e rの多重比較法を用いた。  For the statistics, a multiple comparison method of 0 n e-wa y AN OVA and F i s she r was used.
[0023] [0.20M塩化カルシウム水溶液] [0023] [0.20M calcium chloride aqueous solution]
この実施例は、クェン酸血液を recalcifiedするために 0.2M塩化カルシウムの至 適量を調べた実施例である。方法は 0.2M塩化カルシウム 5 L、 10 L、 20 μ L、 30 Lのそれぞれに、全血 0.3mLと今回加える試薬の 1部であるアルガトロバン又 はへノ^ン及びコラーゲン又は ADPに模した用量を各生理食塩水 10 レ計 20 Lの生理食塩水を加えて、血液凝固が進行する具合を測定した。測定を始めてからト ロンボェラストグラフ上の R点 (凝固の始まりを示すポイント)が現れる時間(分)を測定 した。測定結果は表 2に示した。 R値は、(mean士 SD:平均値士標準偏差)で示した 。表 2から、 0.2M塩化カルシウム 20 Lが至適量であることがわかる。 0.2M塩ィ匕 カルシウム 5 L、 10 Lでは少なすぎ、 0.2M塩化カルシウム 30 μ Lでは多すぎて 、 Vヽずれも凝固が始まるポイントである R点が遅くなつた。  In this example, the optimum amount of 0.2M calcium chloride was examined in order to recalcified kennate blood. The method is as follows: 0.2M calcium chloride 5 L, 10 L, 20 μL, 30 L, 0.3 mL whole blood and a dose simulating argatroban or henone and collagen or ADP as part of the reagent to be added this time A total of 20 L of physiological saline was added to each physiological saline, and the degree of progress of blood coagulation was measured. The time (minutes) at which point R (the point indicating the start of solidification) appears on the Trobolast graph after the measurement was started was measured. The measurement results are shown in Table 2. The R value is indicated by (mean SD: mean value standard deviation). Table 2 shows that 0.2M calcium chloride 20 L is the optimum amount. 0.2M salt calcium 5 L, 10 L is too little, 0.2 M calcium chloride 30 μL is too much, V deviation is also the point where coagulation begins, R point is slow.
[0024] [表 2]  [0024] [Table 2]
* : 20 / Lと有意差 (p< 0. 05)  *: Significant difference from 20 / L (p <0. 05)
統計は On e -wa y ANOVAと F i s h e rの多重比較法を用いた。  For statistics, the multiple comparison method of On e -wa y ANOVA and F i sh er was used.
[0.40M塩化カルシウム水溶液] [0.40M calcium chloride aqueous solution]
この実施例は、クェン酸血液を recalcifiedするために 0.4M塩化カルシウムの至 適量を調べた実施例である。方法は 0. 4M塩化カルシウム 5 L、 10 /z LL、 15 μ L· 、 20 /z Lのそれぞれに、全血 0. 3mLと今回加える試薬の 1部であるアルガトロバン 水溶液又はへノ リン水溶液及びコラーゲン水溶液又は ADP水溶液に模した用量を 各生理食塩水 10 μ L、計 20 μ Lの生理食塩水を加えて、血液凝固が進行する具合 を測定した。測定を始めてからトロンボェラストグラフ上の R点 (凝固の始まりを示すポ イント)が現れる時間(分)を測定した。測定結果は表 3に示した。 0. 4M塩ィ匕カルシ ゥム 10 Lが至適量であることが示されている。 0. 4M塩化カルシウム 5 Lでは少 なすぎ、 0. 4M塩化カルシウム 15 μ 20 μ Lでは多すぎて、いずれも凝固が始まる ポイントである R点が遅くなつた。 This example demonstrates the use of 0.4M calcium chloride to recalcified kennate blood. It is the Example which investigated the appropriate quantity. The method is 0.4M calcium chloride 5 L, 10 / z LL, 15 μL, 20 / z L, 0.3 mL of whole blood and 1 part of the reagent added this time. The amount of each physiological saline was 10 μL, and a total of 20 μL of physiological saline was added to simulate a collagen aqueous solution or an ADP aqueous solution, and the degree of progress of blood coagulation was measured. The time (minutes) at which the R point (point indicating the start of solidification) appears on the thrombolast graph after the measurement was started was measured. The measurement results are shown in Table 3. 0. 4M salt calcium 10 L has been shown to be optimal. The 4M calcium chloride 5L was too little, and the 0.4M calcium chloride 15µ20µL was too much.
[0026] [表 3] [0026] [Table 3]
* : 2 0 Lと有意差 (p < 0 . 0 5 )  *: Significantly different from 20 L (p <0. 0 5)
統計は 0 n e _ w a y A N O V Aと F i s h e rの多重比較法を用いた。 実施例 2  The statistics used the multiple comparison method of 0ne_wayANOVA and Fisher. Example 2
[0027] 塩化カルシウム水溶液の添カ卩量について、次に、へモスコープ社トロンボエラストグ ラフの血液添加法に従った場合の実施例に基づいて説明する。本実施例では、試 薬の代用量として生食(生理食塩水) 20 μ Lと表に表示した塩ィ匕カルシウムの濃度と 量を加え、血液は全体で 0. 36mLになるように添カ卩した。  [0027] The amount of the calcium chloride aqueous solution added will be described based on an example in the case of following the blood addition method of Hemoscope Trombo Elastograph. In this example, 20 μL of saline (saline) as the substitute dose of the reagent and the concentration and amount of salted calcium indicated in the table were added, and the total amount of blood was 0.36 mL. did.
[0. 18M塩化カルシウム水溶液]  [0. 18M calcium chloride aqueous solution]
表 4は、へモスコープ社トロンボェラストグラフの血液添カ卩法(全用量が 360 μ に なるように全血を添加する方法)に従って、クェン酸血液を recalcifiedするために 0. 18M塩化カルシウムの至適量を調べた実施例の結果を示している。方法は 0. 18M 塩ィ匕カノレシゥム 5 L、 10 L、 20 L、 Lのそれぞれに、今回カロえる試薬の 1 部であるアルガトロバン又はへパリン及びコラーゲン又は ADPに模した用量を各生 食(生理食塩水) 10 μ L、計 20 μ Lの生食をカ卩えて、さらに合計して 360 μ Lになるよ うに血液量を調整して、血液凝固が進行する具合を測定した。測定を始めてからトロ ンボエラストグラフ上の R点が現れる時間(分)を測定した。 0. 18M塩ィ匕カルシウム 1 0 μ Lと 20 μ Lが至適量であることが示されている。 0.18M塩化カルシウム 5 μ で は少なすぎ、 0.18M塩化カルシウム 30 Lでは濃すぎて、いずれも凝固が始まるポ イントである R点が遅くなつて 、る。 Table 4 shows the results of 0.18M calcium chloride for recalcified kennate blood according to the blood scoring method of Hemoscope Thrombolastograph (method of adding whole blood to a total dose of 360 μ). The result of the Example which investigated the optimal amount is shown. The method is as follows: 0.1M salt 匕 canoresum 5 L, 10 L, 20 L, L, each dose of a dose similar to argatroban or heparin and collagen or ADP as a part of the reagent Saline) 10 μL, total of 20 μL of raw food was added, and the blood volume was adjusted to 360 μL in total, and the degree of progress of blood coagulation was measured. Toro from the beginning of measurement The time (minutes) at which the R point appears on the lumboelast graph was measured. 0. 18M salt calcium 10 μL and 20 μL have been shown to be optimal. 0.18M calcium chloride 5μ is too little, 0.18M calcium chloride 30L is too thick, and the R point, which is the point at which coagulation begins, is slow.
[表 4]  [Table 4]
* 1 0〃 Lと有意差 (pく 0. 05), † : 20 Lと有意差 (pく 0. 05) 統計は On e -wa y ANOVAと F i s h e rの多重比較法を用いた。  * 1 0 〃 L and significant difference (p <0. 05), †: 20 L and significant difference (p <0.05) The statistics used the multiple comparison method of On e -way ANOVA and F i s she r.
[0029] [0.2M塩化カルシウム水溶液] [0029] [0.2M calcium chloride aqueous solution]
クェン酸血液を recalcifiedするために 0.2M塩化カルシウムの至適量を調べた実 施 ί列である。方法は 0.2Μ塩ィ匕カノレシゥム 5 L、 10 L、 20 L、 30 Lのそれぞ れに、今回加える試薬の 1部であるアルガトロバンまたはへパリン、及びコラーゲン又 は ADPに模した用量を各生食(生理食塩水) 10 L計 20 Lの生食を加えて、さら に合計して 360 Lになるように血液量を調整して、血液凝固が進行する具合を測定 した。測定を始めてからトロンボェラストグラフ上の R点が現れる時間(分)を測定した 。結果は表 5に示した。 0.2M塩化カルシウム 20 Lが至適量であることが示されて いる。 0.2M塩化カルシウム 5 L、 10 Lでは少なすぎ、 0.2M塩化カルシウム 30 μ Lでは濃すぎて、いずれも凝固が始まるポイントである R点が遅くなつている。  This is a series of experiments in which the optimal amount of 0.2M calcium chloride was investigated to recalcified kennate blood. The method is as follows: 0.2 mg salt, 5 liters, 10 liters, 20 liters, and 30 liters, each dose of argatroban or heparin, which is a part of the reagent added, and collagen or ADP. (Saline) 10 L total 20 L of raw food was added, and the blood volume was adjusted to 360 L in total, and the degree of progress of blood coagulation was measured. The time (minutes) at which the point R on the thrombolast graph appears after the measurement was started was measured. The results are shown in Table 5. An optimal amount of 20 L 0.2M calcium chloride has been shown. 0.2M calcium chloride 5 L and 10 L are too little, and 0.2M calcium chloride 30 μL is too thick.
[0030] [表 5]  [0030] [Table 5]
* : 20 ; Lと有意差 (pく 0. 0 5) † : 30 Lと有意差 (pく 0. 05)  *: 20; Significant difference from L (p <0. 0 5) †: Significant difference from 30 L (p <0. 05)
統計は On e -wa y ANOVAと F i s h e rの多重比較法を用いた。  For statistics, the multiple comparison method of On e -wa y ANOVA and F i sh er was used.
[0.4M塩化カルシウム水溶液] [0.4M calcium chloride aqueous solution]
クェン酸血液を recalcifiedするために 0.4M塩化カルシウムの至適量を調べた実 施 ί列である。方法は 0.4Μ塩ィ匕カノレシゥム 5 L、 10 L、 15 L、 20 Lのそれぞ れに、今回加える試薬の 1部であるアルガトロバン又はへパリン、及びコラーゲン又は ADPに模した用量を各生食 10 レ計 20 /z Lの生食をカ卩えて、更に合計して 360 Lになるように血液量を調整して、血液凝固が進行する具合を測定した。測定を始 めてからトロンボェラストグラフ上の R点が現れる時間(分)を測定した。結果は、表 6 に示した。 0. 4M塩化カルシウム 10 Lが至適量であることが示されている。 0. 4M 塩化カルシウム 5 μ Lでは少なすぎ、 0. 2Μ塩化カルシウム 15 μ 20 μ Lでは濃す ぎて、 、ずれも凝固が始まるポイントである R点が遅くなつて 、る。 This is a series of experiments in which the optimum amount of 0.4M calcium chloride was investigated to recalcified kennate blood. The method is 0.4 Μ salt candy canopy 5 L, 10 L, 15 L, 20 L respectively In addition, a dose simulating that of argatroban or heparin, which is one part of the reagent added this time, and collagen or ADP is added to each raw meal, a total of 20 / z L of raw food, so that the total is 360 L. The amount of blood was adjusted to measure the degree of blood coagulation. After starting the measurement, the time (minutes) at which the R point on the thrombolast graph appears was measured. The results are shown in Table 6. 0. It has been shown that 10 L of 4M calcium chloride is the optimum amount. 0.4 M calcium chloride is too little at 5 μL, and 0.2 μ calcium chloride at 15 μ20 μL is too concentrated.
[0032] [表 6] [0032] [Table 6]
* : 2 0 Lと有意差 (p < 0 . 0 5 )  *: Significantly different from 20 L (p <0. 0 5)
統計は O n e - w a y A N O V Aと F i s h e rの多重比較法を用いた。  For the statistics, a multiple comparison method of One-way ANOVA and Fisher was used.
[0033] 以上、実施例 1はペンタフアーム社のローテムの血液添加法に従って R値を測定し たものであり、実施例 2はへモスコープ社トロンボェラストグラフの血液添カ卩法に従つ て R値を測定したものである。いずれの測定においても、 0. 18M塩化カルシウム水 溶液の添加量は、 10 L及び 20 L力至適量であり、 0. 2M塩化カルシウム水溶液 の添加量は、 20 Lが至適量であり、 0. 4M塩化カルシウム水溶液の添加量は、 10 μ Lが至適量であることが示されている。この結果から、 X系血液、 Υ系血液に添加す る塩化カルシウム水溶液の量が、抗凝固剤液を 1容量、生理食塩水を 1容量及び塩 化カルシウム水溶液を混合させる場合、 0. 18M塩ィ匕カルシウム水溶液では 1容量 以上 2容量以下、 0. 2Μ塩化カルシウム水溶液では 2容量、 0. 4Μ塩化カルシウム 水溶液を 1容量添加するのが至適量であることがわかる。即ち、 X系血液においては 、塩化カルシウム水溶液、抗凝固剤液、生理食塩水の混合比が、 0. 18M塩ィ匕カル シゥム水溶液の場合、 0. 18M塩ィ匕カルシウム水溶液 1容量以上 2容量以下、抗凝 固剤液 1容量、生理食塩水 1容量(1〜2: 1 : 1)となること、 0. 2Μ塩ィ匕カルシウム水 溶液の場合は、 0. 2Μ塩ィ匕カルシウム水溶液 2容量、抗凝固剤液 1容量、生理食塩 水 1容量(2 : 1 : 1)となること、 0. 4Μ塩化カルシウム水溶液の場合は、 0. 4Μ塩化力 ルシゥム水溶液 1容量、抗凝固剤液 1容量、生理食塩水 1容量(1 :1:1)となること、 Y 系血液においては、塩化カルシウム水溶液、抗凝固剤液、血小板活性賦活剤液の 混合比が、 0.18M塩化カルシウム水溶液の場合、 0. 18M塩化カルシウム水溶液 1 容量以上 2容量以下、抗凝固剤液 1容量、血小板活性賦活剤液 1容量(1 2: 1: 1) となること、 0.2M塩化カルシウム水溶液の場合は、 0.2M塩化カルシウム水溶液 2 容量、抗凝固剤液 1容量、血小板活性賦活剤液 1容量(2: 1: 1)となること、 0.4M塩 化カルシウム水溶液の場合は、 0.4M塩ィ匕カルシウム水溶液 1容量、抗凝固剤液 1 容量、血小板活性賦活剤液 1容量(1 :1:1)となることがわ力る。 [0033] As described above, Example 1 was obtained by measuring the R value in accordance with the Rotem blood addition method of Pentafarm, and Example 2 was in accordance with the blood addition method of the Hemoscope thrombolast graph. R value was measured. In any measurement, the addition amount of 0.18M calcium chloride aqueous solution is the optimum amount of 10 L and 20 L force, and the addition amount of 0.2M calcium chloride aqueous solution is the optimum amount of 20 L. It has been shown that the optimum amount of 4M calcium chloride aqueous solution is 10 μL. From this result, it can be seen that the amount of calcium chloride aqueous solution added to X-system blood and sputum system blood is 0.18M salt when mixing 1 volume of anticoagulant solution, 1 volume of physiological saline and calcium chloride aqueous solution. It can be seen that it is optimal to add 1 to 2 vol. Volume for aqueous calcium carbonate solution, 2 vol. For 0.2 wt. Calcium chloride aqueous solution, and 1 vol. That is, in X-system blood, when the mixing ratio of calcium chloride aqueous solution, anticoagulant solution, and physiological saline is 0.18M salt calcium aqueous solution, 0.18M salt calcium aqueous solution 1 volume or more 2 volumes Hereafter, 1 volume of anti-coagulant solution and 1 volume of physiological saline (1-2: 1: 1), in the case of 0.2% salty calcium aqueous solution, 0.2% salty calcium aqueous solution 2 Capacity, 1 volume of anticoagulant solution, 1 volume of physiological saline (2: 1: 1), 0.4Μ chloride power in the case of 0.4Μ calcium chloride aqueous solution 1 volume of Lucium aqueous solution, 1 volume of anticoagulant solution, 1 volume of physiological saline (1: 1: 1), Y blood is mixed with calcium chloride aqueous solution, anticoagulant solution, platelet activator solution When the ratio is 0.18M calcium chloride aqueous solution, 0.18M calcium chloride aqueous solution 1 volume or more and 2 volumes or less, anticoagulant solution 1 volume, platelet activity activator solution 1 volume (1 2: 1: 1), In the case of 0.2M calcium chloride aqueous solution, 0.2M calcium chloride aqueous solution 2 volumes, anticoagulant solution 1 volume, platelet activity activator solution 1 volume (2: 1: 1), 0.4M calcium chloride aqueous solution Is 1 volume of 0.4M saline-calcium aqueous solution, 1 volume of anticoagulant solution, and 1 volume (1: 1: 1) of platelet activity activator solution.
実施例 3  Example 3
[0034] [血小板賦活剤]  [0034] [Platelet activator]
一般的常識として、 ADP (アデノシン二リン酸)とコラーゲンが血小板賦活剤液とし て使用されるのは、それぞれ 4 μ Μと 4 μ gZmLである。し力し、アルガトロバンやへ ノ リンでトロンビンの生成を抑えたり、トロンビンを抑制しておいて、血小板凝集を AD pやコラーゲンで刺激して、血液凝固を促進させるためには、それぞれ如何ほどの濃 度が必要なのかを調べた。 0.2M塩ィ匕カルシウム 20 /zLの存在下、この測定前に決 定したアルガトロバンの終濃度を持つ 10 L(0. OlmL)と ADP (終濃度として、 4 μ Μ, 6/ζΜ, 8 Μ)及びコラーゲン(Col;終濃度として、 0.5 μ g/m 1. O mL 2.0 8 !11 を各10 1^(0. OlmL)にそれぞれの終濃度を定め、さらに血 液量は 0.32mLとし、全体量を 0.36mLとしてトロンボェラストグラフを測定した。コ ントロールは全血 0.32mL、生食 20 L Argはこの測定前に得たへモクロンの AC T値力も決定した終濃度をもつアルガトロバン 10 レ生食 10 Lに全血 0.31mL が添加されている。測定値は、それぞれ表 7及び表 8に示した。測定値は、平均値士 標準偏差で示している。測定値は、健康な成人 10人について得られたものである。 As common general knowledge, ADP (adenosine diphosphate) and collagen are used as platelet activator solutions in 4 μΜ and 4 μg ZmL, respectively. However, in order to suppress thrombin generation with argatroban or helin, or suppress thrombin and stimulate platelet aggregation with AD p or collagen to promote blood coagulation, We investigated whether the concentration was necessary. 10 L (0. OlmL) and ADP (final concentrations of 4 μΜ, 6 / ζΜ, 8 を 持 つ) with the final concentration of argatroban determined before this measurement in the presence of 0.2 M salt calcium 20 / zL ) And collagen (Col; final concentration of 0.5 μg / m 1. O The final concentration of mL 2.0 8 ! 11 was determined for each 10 1 ^ (0. OlmL), the blood volume was 0.32 mL, and the total volume was 0.36 mL, and the thrombolast graph was measured. The control is 0.32 mL of whole blood, and 20 L Arg of raw food is 0.31 mL of whole blood added to 10 L of raw argatroban 10 L with the final concentration that also determined the ACT value of the hemoclone obtained before this measurement. The measured values are shown in Table 7 and Table 8, respectively. The measured values are shown as mean value standard deviation. Measurements were taken for 10 healthy adults.
[0035] [表 7]  [0035] [Table 7]
* : コントロールと差 (pく 0. 05) † :アルガトロパンと差 (pく 0. 05)  *: Control and difference (p 0. 05) †: Argatropan and difference (p 0. 05)
統計は 0 n e— way AN0VAと F i s h e rの多重比較法を用いた。 [0036] 表 7から、 2列目のアルガトロバンのみで平均が 36. 8分に延長した R値力 3列目 の ADP4 /Z Mの終濃度が加わっても、平均 28. 3分と、 R点は有意に短縮せず、血 液凝固が促進されることはない。しかし 4列目に見られるように、 ADP6 /Z Mの終濃度 が加わることにより、平均 26. 0分に、また 5列目で見られるように、 ADP8 μ Μの終 濃度が加わると、平均 24. 1分にそれぞれ有意に短縮している。アルガトロバンでトロ ンビンを抑えた場合、 ADPは終濃度 6 Μ以上の濃度で、血液凝固の促進させ得る ことを示してる。 The statistics used were 0 ne-way AN0VA and Fisher's multiple comparison method. [0036] From Table 7, the average R value was extended to 36.8 minutes with only the second row of argatroban. Even if the final concentration of ADP4 / ZM in the third row was added, the average was 28.3 minutes, and the R point Does not significantly shorten and does not promote blood clotting. However, as seen in the fourth row, the addition of the final concentration of ADP6 / ZM results in an average of 26.0 minutes, and as seen in the fifth row, the addition of ADP8 μΜ final concentration averages 24 Each is significantly shortened to 1 minute. When thrombin is suppressed with argatroban, ADP can promote blood coagulation at a final concentration of 6% or more.
[0037] [表 8]  [0037] [Table 8]
* :コントロールと差 (pく 0 . 0 5 ) † :アルガトロバンと差 (pく 0 . 0 5 )  *: Difference from control (p <0.05) †: Difference from argatroban (p <0.05)
統計は O n e - w a y A N O V Aと F i s h e rの多重比較法を用いた。  For the statistics, a multiple comparison method of One-way ANOVA and Fisher was used.
[0038] 表 8から、 2列目のアルガトロバンのみで平均が 36. 9分に延長した R値力 3列目 のコラーゲン 0. 5 gZmLの終濃度が加わっても、平均 31. 4分と、 R点は有意に短 縮せず、血液凝固が促進されることはない。しかし 4列目に見られるように、コラーゲ ン 1. Ο /z gZmLの終濃度が加わることにより、平均 27. 2分に、また 5列目で見られ るように、コラーゲン 2. Ο /z gZmLの終濃度が加わると、平均 23. 2分にそれぞれ有 意に短縮している。アルガトロバンでトロンビンを抑えた場合、コラーゲンは終濃度 1. 0 gZmL以上の濃度で、血液凝固を促進させ得ることを示している。この測定で、 全体量を 0. 36mLとしてへモスコープ社のトロンボェラストグラフに沿った方法で測 定したが、ペンタフアーム社のローテムに沿った方法では添加する血液は 0. 3mLで あり、この例より使用する血液量は少ない。一方試薬量は同量である。したがって、ぺ ンタファーム社ローテムの測定法に沿った測定結果は、この実施例より、試薬の影響 が強く出る可能性が高いと考えることが出来る。たとえばペンタフアーム社のローテム に沿った血液量で、へモスコープ社トロンボェラストグラフで有意差が出な 、コラーゲ ン 0. 5 /z gZmLで、 R値を有意に短縮する結果が出る可能性が高い。しかし、上記 の表に提示している結果は、 "いずれの方法においても、 R値を有意に短縮する結果 を引き起こす血小板凝集賦活剤液の濃度"を見出したものであると考えることが出来 る。ペンタフアーム社ローテムで有意差が出たモノでも、へモスコープ社トロンボエラ ストグラフで有意差が出ない場合は、有意差を引き起こす血小板賦活剤として採用 することは出来ず、結論として上記のデータを採用するのが妥当と考えられる。 [0038] From Table 8, the average of R value force extended to 36.9 minutes with only the second row of argatroban, even if the final concentration of 0.5 gZmL of collagen in the third row was added, the average was 31.4 minutes. The R point is not significantly shortened and does not promote blood clotting. However, as seen in the 4th row, the addition of collagen 1. z / z gZmL resulted in an average of 27.2 min and collagen 2. Ο / z as seen in the 5th row. When the final concentration of gZmL is added, it is significantly shortened to an average of 23.2 minutes respectively. When thrombin is suppressed with argatroban, collagen has been shown to promote blood coagulation at a final concentration of 1.0 gZmL or higher. In this measurement, the total volume was 0.36 mL, and it was measured by the method according to the Hemoscope Trombolast graph, but the method added according to the Pentafarm Rotem method was 0.3 mL of blood added. Less blood is used than in the examples. On the other hand, the reagent amount is the same. Therefore, it can be considered that there is a higher possibility that the influence of the reagent is stronger than the measurement result of the measurement result according to the measurement method of the Pentafarm Rotem. For example, with the blood volume along the Pentafarm Rotem, there is a possibility that the Collagen 0.5 / z gZmL will result in a significantly shortened R value, with no significant difference on the Hemoscope thrombolast graph. high. However, the results presented in the table above can be thought of as finding “the concentration of the platelet aggregation activator solution that causes the result to significantly shorten the R value in any method”. The Even if there is a significant difference in the Pentaffarm Rotem, if there is no significant difference in the Hemoscope Thromboelastograph, it cannot be adopted as a platelet activator that causes a significant difference, and the above data is used as a conclusion. Is considered reasonable.
[0039] 以上、表 7及び表 8から、 X系血液、 Y系血液に添加する血小板活性剤液の濃度が 、 ADP (アデノシン二リン酸) 6. O/zM以上又はコラーゲン 1. O/zgZmL以上が至 適量であることがわかる。  [0039] As described above, from Table 7 and Table 8, the concentration of the platelet activator solution added to the X-system blood and the Y-system blood is ADP (adenosine diphosphate) 6. O / zM or higher or collagen 1. O / zgZmL It turns out that the above is the optimal amount.
実施例 4  Example 4
[0040] [血液凝固促進剤としてァクチン]  [0040] [actin as blood coagulation promoter]
次に、実施例 3において、血液凝固促進剤としてァクチン (Actin from bovine muscle、 A— 3653、 Sigma— Aldrich、 St. Louis MO USA)の添カ卩至適量を 調べた。先ず、血液凝固を促進するァクチンのおおまかな至適濃度を、終濃度を In gZmLから lpgZmLに変化させて求めた。実施例 1、 2に準じて、トロンボエラストグ ラフにより R値を求めた。その結果を表 9に示した。  Next, in Example 3, the optimum amount of actin (Actin from bovine muscle, A-3653, Sigma-Aldrich, St. Louis MO USA) as a blood coagulation promoter was examined. First, a rough optimum concentration of actin that promotes blood coagulation was determined by changing the final concentration from IngZmL to lpgZmL. In accordance with Examples 1 and 2, the R value was determined by a thromboelastograph. The results are shown in Table 9.
[0041] [表 9] ァクチン終濃度 (n g/p g) R m n ) コントロ-ル 生食 1 0 L +全血 0. 3 3 r nL 4- 0. 2 M塩カル 2 0 L 1 2. 2 ± 2 4 ァクチン (終濃度 1 0 n g/mL) 1 0 /i L +全血 0. 3 3 r nL - 0. 2 M塩カル 2 0 z L 1 2. 0土 1 9 ァクチン (終濃度 1 n g/mL) 1 0 w L +全血 0. 3 3 r nL + 0. 2 M塩カル 2 0 ^ L 1 0. 4土 1 6 * ァクチン (終濃度 1 0 O p gZmL) 1 0 / +全血0. 3 3 r nL + 0. 2 M塩カル 2 0 1 0. 8 ± 2 1 ァクチン (終濃度 1 0 p g/mL) 1 0 L +全血 0. 3 3 r nL + 0. 2 M塩カル 2 0 μ L 1 0. 5士 1 9 ァクチン (終濃度 1 p g/mL) 1 0 L +全血 0. 3 3 r nL + 0. 2 M塩カル 2 0 μ L 1 0. 5土 1 4 [0041] [Table 9] Final concentration of actin (ng / pg) R mn) Control raw diet 10 L + whole blood 0.3 3 r nL 4- 0. 2 M salt calcium 2 0 L 1 2. 2 ± 2 4 Actin (final concentration 10 ng / mL) 10 0 / i L + whole blood 0.3 3 r nL-0. 2 M salt 2 0 z L 1 2.0 soil 1 9 actin (final concentration 1 ng / mL) 1 0 w L + whole blood 0. 3 3 r nL + 0. 2 M salt 2 0 ^ L 1 0. 4 Sat 1 6 * actin (final concentration 10 O p gZmL) 1 0 / + total Blood 0.3 3 r nL + 0.2 M salt 2 0 1 0. 8 ± 2 1 Actin (final concentration 10 pg / mL) 1 0 L + whole blood 0.3 3 r nL + 0.2 M Salt Cal 2 0 μL 1 0. 5 people 1 9 Factin (final concentration 1 pg / mL) 1 0 L + Whole Blood 0.3 3 r nL + 0.2 M Salin 2 0 μ L 1 0. 5 Sat 14
* : コントロールと差 (p < 0. 0 5) *: Control and difference (p <0. 0 5)
統計は 0 n e— wa y ANOVAと F i s h e rの多重比較法を用いた。  For the statistics, a multiple comparison method of 0 n e- wa y ANOVA and F i sh er was used.
[0042] 表 9から、添加する血液凝固促進剤としてのァクチンの終濃度が IngZmLのとき( 二段目)、血液凝固の開始点 (R点)が最も早く現れて 、て、ァクチンを添加しな 、コ ントロールの 12.2分より 1.8分早い 10.4分となっていることがわかる。次に、ァクチ ンの血液凝固促進の至適濃度を IngZmL近辺で、 0.5ngZmL単位ずつ変化させ て更に細かぐァクチンの血液凝固促進作用における至適濃度の解明を行った。そ の結果を表 10に示した。表 10から、ァクチンの終濃度が 0.5 ngZmL (最下段)か ら 2.5 ng/mL (三段目 )で Control (ァクチンを全く添カ卩しな 、もの)に比べ、有意 に R値が短くなつていることがわかる。以上の、表 9、表 10の結果から、血液凝固促進 の為に使用するァクチンの終濃度は 0.5ngZmL(500pgZmL)力ら 2.5ng/mL が血液凝固促進剤として使用する終濃度として至適濃度であることがわ力る c [0042] From Table 9, when the final concentration of actin as a blood coagulation promoter to be added is IngZmL (second stage), the start point (R point) of blood coagulation appears the earliest, and actin is added. It can be seen that 10.4 minutes is 1.8 minutes earlier than 12.2 minutes of control. Next, the optimum concentration of lactin for promoting blood coagulation was clarified by changing the optimum concentration of lactine for promoting blood coagulation in increments of 0.5 ngZmL in the vicinity of IngZmL. The results are shown in Table 10. From Table 10, the final concentration of actin is 0.5 ngZmL (bottom stage) to 2.5 ng / mL (third stage), and the R value is significantly shorter than Control (without adding actin at all). You can see that Based on the results in Tables 9 and 10 above, the final concentration of actin used to promote blood coagulation is 0.5 ngZmL (500 pgZmL) and 2.5 ng / mL. Optimal concentration at which it is I Chikararu c but as the final concentration to be used as a blood coagulation promoter
[表 10]  [Table 10]
* : コントロールと差 ( p < 0 . 0 5 )  *: Control and difference (p <0. 0 5)
統計は O n e - w a y A N O V Aと F i s h e rの多重比較法を用いた。 実施例 5  For the statistics, a multiple comparison method of One-way ANOVA and Fisher was used. Example 5
以下に、実際に本発明の試薬で R値を測定した結果を示す。  The results of actually measuring the R value with the reagent of the present invention are shown below.
治験例 1は、 71才、男性、動脈硬化性閉塞症および腎不全のケースで、プレター ル 1錠(lOOmgZ日)内服している事例である。 X系血液に 0. 2M塩化カルシウム水 溶液を 2容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血 液に 0. 2M塩ィヒカルシウム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤 液 1容量の割合でカ卩えている。へパリンは終濃度で 0. lU/mL,アルガトロバンは 3 Mカロえた。全液量は 0. 36mL である。この場合は、 ADPによって血小板凝集を起こすことで、 R値がほぼ 28%短縮 し、現在行われているプレタール lOOmgZ日の抗血小板治療は不十分と判定でき る。現在 150mgZ日への増量を検討中である。 Case 1 is a 71-year-old male with arteriosclerotic obstruction and renal failure who is taking 1 tablet (lOOmgZ day). Add 2 volumes of 0.2M calcium chloride solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M aqueous calcium chloride solution to system Y blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are stored. Heparin at a final concentration of 0.lU/mL, argatroban is I got 3 M Caro. The total liquid volume is 0.36 mL. In this case, platelet aggregation caused by ADP shortens the R value by approximately 28%, and it can be determined that the current anti-platelet treatment of pretal lOOmgZ is insufficient. A dose increase to 150mgZ is currently under consideration.
治験例 2は、 69才、男性、動脈硬化性閉塞症のケースで、プレタール 2錠(200mg /日)内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、 抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩 化カルシウム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割 合で加えている。へパリンは終濃度で 0. lUZmL、アルガトロバンは終濃度で 0. 3 /z gZmLまた ADPは終濃度で 8. 3 Mカロえた。全液量は 0. 36mLである。この場 合は、 10 15%の短縮であり、有意差なしと判定し、有効な治療が行われてると判 定できる。  Case 2 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg / day). Add 2 volumes of 0.2M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M calcium chloride aqueous solution to system Y blood. Anticoagulant solution is added at a rate of 1 volume and platelet activator solution 1 volume. Heparin had a final concentration of 0.1 lUZmL, argatroban had a final concentration of 0.3 / z gZmL, and ADP had a final concentration of 8.3 M calories. The total volume is 0.36 mL. In this case, the reduction is 10 15%, and it can be determined that there is no significant difference and effective treatment is being performed.
治験例 3は、 63才、男性、動脈硬化性閉塞症のケースで、プレタール 2錠(200mg /日)内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、 抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩 化カルシウム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割 合で加えている。へパリンは終濃度で 0. lUZmL、アルガトロバンは終濃度で 1. 2 g/mLまた ADPは終濃度で 8. 3 Mカロえた。全液量は 0. 36mLであった。この 場合は、 10%以下の短縮であり、有意差なしと判定し、有効な治療が行われてると考 えられる。 Case 3 is a 63-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg / day). 2 volumes of 0.2M salt-calcium aqueous solution Add 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution to Y blood. It is added together. Heparin at a final concentration of 0.1 lUZmL, argatroban at a final concentration of 1.2 g / mL, and ADP at a final concentration of 8.3 M calories. The total liquid volume was 0.36 mL. In this case, the reduction is less than 10%, and it is judged that there is no significant difference, and effective treatment is considered.
治験例 4は、 54才、男性、脳梗塞のケースで、パナルジン 2錠(200mgZ日)内服 している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固剤液 を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシウム 水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合でくわえて いる。へパリンは終濃度で 0. lUZmL、アルガトロバンは終濃度で 1. 2 /z gZmLま たコラーゲンは終濃度で 10 g/mLカ卩えた。全液量は 0. 36mLである。この場合 は、 20%以下の短縮であり、有意差なしと判定し、有効な治療が行われてると考えら れる。  Case 4 is a 54-year-old male with cerebral infarction taking 2 panalgin tablets (200 mgZ day). Add 2 volumes of 0.2M saline-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and add 2 volumes of 0.2M calcium chloride aqueous solution to Y blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are added. Heparin at a final concentration of 0.1 lUZmL, argatroban at a final concentration of 1.2 / z gZmL, or collagen at a final concentration of 10 g / mL. The total volume is 0.36 mL. In this case, the reduction is 20% or less, and it is judged that there is no significant difference, and effective treatment is considered.
治験例 5は、 62才、男性、動脈硬化性閉塞症のケースで、プレタール 200mgZ日 内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固 剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシ ゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加え ている。へパリンは終濃度で 0. lUZmL、アルガトロバンは終濃度で 1. 2 μ g/mL また ADPは終濃度で 8. 3 Mカロえた。全液量は 0. 36mLである。この場合は、 20 %以下の短縮および、よく血小板活性が抑制されて ヽる証拠の血小板凝集刺激によ る凝固の遅れがあり、治療は十分と判定できる。  Case 5 is a 62-year-old male with atherosclerotic obstruction who is taking pretal 200 mg Z day. Add 2 volumes of 0.2M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and 2 volumes of 0.2M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. Heparin at a final concentration of 0.1 lUZmL, argatroban at a final concentration of 1.2 μg / mL, and ADP at a final concentration of 8.3 M calories. The total volume is 0.36 mL. In this case, there is a shortening of 20% or less, and there is a delay in coagulation due to platelet aggregation stimulation, which is evidence that platelet activity is often suppressed, and it can be judged that treatment is sufficient.
[表 13] 治験例 1: [Table 13] Case 1:
プレタール 1錠(lOOmg "日)  1 tablet pretal (lOOmg "day")
治験例 2:  Case 2:
プレタール 2錠(200mg/日)  2 tablets of pretal (200mg / day)
治験例 3:  Case Study 3:
プレタール 2錠(200mg/日)  2 tablets of pretal (200mg / day)
4] 治験例 4 : Four] Case Study 4:
パナルジン 2錠(200mg/日) Panaldine 2 tablets (200mg / day)
治験例 6は、 66才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ日 内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固 剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシ ゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加え ている。へパリンは終濃度で 0. 2UZmL及び 0. lUZmL、アルガトロバンは終濃度 で 0. 3 /z gZmL及び 0. 2 /z gZmLまた ADPは終濃度で 8. 3 Mカロえた。全液量 は 0. 36mLである。すべての測定で、血小板凝集刺激側の凝固時間が長い。治療 効果は十分との結果がみられる。 [0047] [表 15] 治験例 6. Case 6 is a 66-year-old male with atherosclerotic obstruction who is taking pretal 200 mg Z day. Add 2 volumes of 0.2M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and 2 volumes of 0.2M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. Heparin was 0.2 UZmL and 0.1 LUZmL at the final concentration, argatroban was 0.3 / z gZmL and 0.2 / z gZmL at the final concentration, and ADP was 8.3 M calorie at the final concentration. The total volume is 0.36 mL. In all measurements, the clotting time on the platelet aggregation stimulation side is long. The results show that the therapeutic effect is sufficient. [0047] [Table 15] Clinical trial 6.
プレターノレ 200mg/日  Pretanore 200mg / day
実施例 6 Example 6
[0048] トロンボェラストグラフ測定前にへモクロンによる ACT (正常成人 105〜 150)を測 定し、加える抗凝固薬のアルガトロパンまたはへパリンの量を決定する事例について 説明する。  [0048] We will explain an example of measuring ACT (normal adults 105-150) with hemocron before determining the amount of argatropane or heparin to be added, before measuring the thrombolastograph.
治験例 7は、 65才、男性、脳梗塞のケースで、 (1)バイアスピリン lOOmgZ日内服 して 10日目のトロンボェラストグラフ測定した R値を示している。 X系血液に 0. 18M 塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割 合で加え、 Y系血液に 0. 18M塩ィヒカルシウム水溶液を 1容量、抗凝固剤液を 1容量 及び血小板活性剤液 1容量の割合でカ卩えている。 ACT値 201秒に対して、アルガト ロバンは終濃度で 4. 0 全 液量は 0. 36mLである。この場合、 20%以下の短縮であり、有意差なしと判定し、治 療は十分と判定できる。 (2)次に、ノ ィアスピリン lOOmgZ曰内服をヮーフアリン 3mg /日に変更した後 1日目に測定したトロンボェラストグラフの R値を示す。 ACT値 20 2秒に対して、アルガトロバンは終濃度で 2. 5 μ gZmLまたコラーゲンは終濃度で 1 0 μ gZmL加えた。全液量は 0. 36mLである。この場合は、 20%以下の短縮であり 、有意差なしと判定し、血小板抑制作用のないヮーフアリンに変更したが、まだノィァ スピリンの血小板抑制作用が残っている。と考えられる。(3)更に、ヮーフアリン 3mg /日内服を一週間続けた後 5mgZ日に変更 (増量)して 7日目に測定したトロンボェ ラストグラフの R値を示す。 ACT値 213秒に対して、へパリンは終濃度で 0. 07 /ζ Μ、 アルガトロバンは終濃度で 1. 0 μ gZmLまたコラーゲンは終濃度で 10 μ gZmLカロ えた。全液量は 0. 36mLである。何れも 50%以上の短縮を認め、血小板の抑制は 見られない。以前処方していたバイアスピリンの影響は全く見えず、血小板抑制作用 のないヮーフアリンの影響で凝固時間が 213秒と延長している。 Case 7 is a 65-year-old male with cerebral infarction. (1) Thrombolast graph of R measured on the 10th day after oral administration of biaspiline lOOmgZ. Add 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to system X blood, and 1 volume of 0.18M aqueous calcium chloride solution to system Y blood, anticoagulant 1 volume of drug solution and 1 volume of platelet activator solution are stored. For an ACT value of 201 seconds, Argatroban has a final concentration of 4.0. The total volume is 0.36 mL. In this case, the reduction is 20% or less, and it can be judged that there is no significant difference, and the treatment is sufficient. (2) Next, the R value of the thrombolast graph measured on the first day after changing the oral dose of neurospirin lOOmgZ ヮ farin 3 mg / day is shown. ACT value 20 For 2 seconds, argatroban was added at a final concentration of 2.5 μgZmL, and collagen was added at a final concentration of 10 μgZmL. The total volume is 0.36 mL. In this case, it was shortened by 20% or less, and it was determined that there was no significant difference, and it was changed to フ phalin having no platelet inhibitory effect, but the platelet inhibitory effect of neurospirin still remains. it is conceivable that. (3) Furthermore, the R value of the thrombolast graph measured on the 7th day after changing to 5mgZ (increase) after one week of oral administration of cephalin 3mg / day is shown. For the ACT value of 213 seconds, heparin had a final concentration of 0.07 / ζ Μ, argatroban had a final concentration of 1.0 μgZmL, and collagen had a final concentration of 10 μgZmL. The total volume is 0.36 mL. In both cases, a reduction of 50% or more was observed, and platelet suppression was not observed. The effect of the previously prescribed bias pilin is not visible at all, and the coagulation time is extended to 213 seconds due to the effect of フ farin, which has no platelet inhibitory action.
[表 16] [Table 16]
治験例 7 Case Study 7
(1)バイアスピリン lOOmg/日内服して 10日目 (1) 10 days after taking Biaspilin lOOmg / day
(2) バイアスピリン lOOmg/日内服をヮーフアリン 3mg/日に変更 1 日目 (2) Biaspilin lOOmg / day changed to ヮ -farin 3mg / day Day 1
(3) 一週間続けたヮ一フアリン 3mg/日を 5mg/日に変更(増量)して 7日目(3) change the one week Wa one Fuarin 3m g / day was continued to 5mg / day (increase) for 7 days
治験例 8は、 58才、男性、脳梗塞のケースで、バイアスピリン lOOmgZ日内服して いる事例である。 X系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 18M塩ィヒカルシウム 水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて!/、 る。 ACT値 190秒に対して、へパリンは終濃度で 0. 02U/mL、アルガトロバンは終 濃度で 0. 75 gZmLまたコラーゲンは終濃度で 10/ gZmLカ卩えた。全液量は 0. 36mLである。この場合、判定不能、おそらくアルガトロバンによる抗凝固によって出 た結果を選ぶ方が正し 、確率が高 、。 1ヶ月後にトロンボェラストグラフの R値を測定 した。 V、ずれも 20%以上の短縮でバイアスピリンの効果は不十分である。 Case 8 is a 58-year-old male with cerebral infarction who is taking Biaspilin lOOmgZ day. Add 0.1 volume of 18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M saline calcium solution to Y blood, anticoagulant Add 1 volume of solution and 1 volume of platelet activator solution! / For an ACT value of 190 seconds, heparin had a final concentration of 0.02 U / mL, argatroban had a final concentration of 0.75 gZmL, and collagen had a final concentration of 10 / gZmL. Total liquid volume is 0. 36 mL. In this case, it is not possible to make a decision, and it is probably better to select the result obtained by anticoagulation with argatroban, and the probability is high. One month later, the R value of the thrombolast graph was measured. V and deviation are shortened by 20% or more, and the effect of bias pilin is insufficient.
[0051] [表 17] 治験例 8. [0051] [Table 17] Clinical trial example 8.
バイアスピリン lOOmg/日  Bias pilin lOOmg / day
[0052] 治験例 9は、 69才、男性、動脈硬化性閉塞症のケースで、プレタール 2錠(200mg Z日)内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、 抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩 化カルシウム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割 合で加えている。 ACT値 170秒に対して、へパリンは終濃度で 0. 03U/mL及び 0 . 02UZmL、アルガトロバンは終濃度で 0. 3及び 0. 5 μ gZmLまたコラーゲンは終 濃度で 10 gZmL加えた。全液量は 0. 36mLである。何れも 20%以上の短縮が みられ、プレタールの抗血小板作用は不十分と考えられる。プレタール 2錠(200mg /日)内服を 3ヶ月間継続した後のトロンボェラストグラフの R値から、やはり 20%以 上の短縮がみられ、プレタールの抗血小板作用は不十分と考えられる。 [0052] Clinical trial example 9 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg Z day). Add 2 volumes of 0.2M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 2 volumes of 0.2M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution It is added together. For an ACT value of 170 seconds, heparin was added at final concentrations of 0.03 U / mL and 0.02 UZmL, argatroban was added at final concentrations of 0.3 and 0.5 μg ZmL, and collagen was added at a final concentration of 10 gZmL. The total volume is 0.36 mL. In both cases, shortening of 20% or more was observed, and the antiplatelet action of pretal is considered insufficient. From the R value of the thrombolast graph after taking Pretal 2 tablets (200 mg / day) for 3 months, there is still a reduction of more than 20%, indicating that the anti-platelet effect of Pretal is insufficient.
[0053] [表 18] 治験例 9 [0053] [Table 18] Clinical trial example 9
プレタール 2錠(200mg/ Θ )  2 tablets of pretal (200mg / Θ)
[0054] 治験例 10は、 58才、男性、脳梗塞のケースで、パナルジン 200mgZ日内服して いる事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固剤液を 1 容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシウム水 溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて!/ヽる 。へパリンは終濃度で 0. lU/mL,アルガトロバンは終濃度で 1. 2 g/mLまたコ ラーゲンは終濃度で 8. 3 /z g/mLカ卩えた。全液量は 0. 36mLである。この場合は、 アルガトロバンの量が少なすぎてアルガトロバンでは判定不能であり、へパリンで 20[0054] Clinical trial example 10 was a 58-year-old male with a cerebral infarction. This is an example. Add 2 volumes of 0.2M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X-system blood, and 2 volumes of 0.2M calcium chloride solution to Y-system blood. Add 1 volume of anticoagulant solution and 1 volume of platelet activator solution! Heparin was at a final concentration of 0.1 lU / mL, argatroban was at a final concentration of 1.2 g / mL, and collagen was at a final concentration of 8.3 / zg / mL. The total volume is 0.36 mL. In this case, the amount of argatroban is too small to be determined by argatroban, and heparin 20
%以上の短縮の故に、パナルジン治療は不十分と考えられる。これ以後、ノ ィァスピ リン lOOmg/日に変更し 7ヶ月後のトロンボェラストグラフの R値を示した。何れも、 2 0%以上の短縮で、バイアスピリンの治療効果は不十分と考えられる。 Panaldine treatment is considered inadequate due to a reduction of more than%. After this, the R value of the thrombolast graph 7 months later was shown after changing to nospirin lOOmg / day. In any case, the treatment effect of bias pilin is considered to be insufficient with a shortening of 20% or more.
[表 19] [Table 19]
治験例 10 Clinical trial 10
パナルジン 200mg/日 Panardine 200mg / day
これ以後バイアスピリン 100mg/dayに治療を変更し、 7ヶ月後 治験例 11は、 75才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ 日とェパデール 900mgZ日を内服している事例である。 X系血液に 0. 4M塩化カル シゥム水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え 、 Y系血液に 0. 4M塩ィ匕カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び血小 板活性剤液 1容量の割合でカ卩えている。 ACT値 181秒に対して、へパリンは終濃度 で 0. 07UZmL、アルガトロパンは終濃度で 3. 0 gZmLまたコラーゲンは終濃度 で 10 /z gZmLカロえた。全液量は 0. 36mLである。プレタール 200mgとエノ Thereafter, the treatment was changed to Biaspilin 100 mg / day, and 7 months later Case 11 was a 75-year-old male with atherosclerotic obstruction who was taking Pretal 200 mgZ and Epadele 900 mgZ. 1 volume of 0.4M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to system X blood, and 1 volume of 0.4M saline calcium calcium solution is added to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are stored. For ACT value of 181 seconds, heparin has a final concentration of 0.07 UZmL, argatropane has a final concentration of 3.0 gZmL, and collagen has a final concentration. At 10 / z gZmL. The total volume is 0.36 mL. Pretal 200mg and Eno
900mgの治療は不十分である。  900mg treatment is inadequate.
[0057] [表 20] 治験例 11 [0057] [Table 20] Clinical trial example 11
プレタール 200mg/日 +ェパデール 900mg/ 0  Pre-tar 200mg / day + Epadale 900mg / 0
[0058] 治験例 12は、 77才男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ日 とドルナー 60mgZ日内服している事例である。 X系血液に 0. 4M塩ィ匕カルシウム水 溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血 液に 0. 4M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤 液 1容量の割合でカ卩えている。 ACT値 111秒に対して、へパリンは終濃度で 0. 03 及び 0. 06UZmL、アルガトロバンは終濃度で 3. 0 gZmLまたコラーゲンは終濃 度で 10 g/mLカロえた。全液量は 0. 36mLである。プレターノレ 200mg/曰とドノレ ナー 60mgZ日の治療は不十分である。 2ヶ月後のトロンボェラストグラフの R値を示 した。 Arg3. 0のデータのみが不十分となっている力 あとの 3つのデータは、 3つと もすベてこの治療で十分との結果が出て 、る。 [0058] Clinical trial example 12 is a 77-year-old male with atherosclerotic obstruction, taking pretal 200 mg Z day and Dorner 60 mg Z day. Add 1 volume of 0.4M saline-calcium water solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to system X blood, and 1 volume of 0.4M calcium chloride aqueous solution to system Y blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution. For an ACT value of 111 seconds, heparin was at a final concentration of 0.03 and 0.06 UZmL, argatroban was at a final concentration of 3.0 gZmL, and collagen was at a final concentration of 10 g / mL. The total volume is 0.36 mL. Treatment with pretanol 200 mg / l and donoren 60 mg Z days is inadequate. The R value of the thrombolast graph after 2 months is shown. The power of the Arg3.0 data alone is insufficient. The other three data show that all three are sufficient for this treatment.
[0059] [表 21] 治験例 12 [0059] [Table 21] Clinical trial 12
プレターノレ 200maZ日 +ドルナー 60ma 日 Prenoreno 200maZ + Dorner 60ma
治験例 13は、 59才 女性 動脈硬化性閉塞症のケースで、プレタール 200mgZ日 内服している事例である。 X系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝 固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 18M塩化力 ルシゥム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で 加えている。 ACT値 193秒に対して、へパリンは終濃度で 0. 03UZmL、アルガトロ バンは終濃度で 1. 5 g/mLまたコラーゲンは終濃度で 10 g/mLカ卩えた。全液 量は 0. 36mLである。何れも 20%以上の短縮を示しており、プレタール 200mgZd ayの治療は不十分である。 Case 13 is a 59-year-old female with arteriosclerotic obstruction and taking pretal 200 mg Z day. 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M aqueous solution of sodium chloride is added to Y-system blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are added. For ACT value of 193 seconds, heparin at a final concentration of 0.03 UZmL, argatro The final concentration of van was 1.5 g / mL, and the final concentration of collagen was 10 g / mL. The total volume is 0.36 mL. All show a reduction of 20% or more, and pretal 200mgZday is not adequately treated.
治験例 14は、 66才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ 日とヮーフアリン 2mgZ日内服している事例である。 X系血液に 0. 2M塩化カルシゥ ム水溶液を 2容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y 系血液に 0. 2M塩ィヒカルシウム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活 性剤液 1容量の割合でカ卩えている。 ACT値 209秒に対して、へパリンは終濃度で 0. 03UZmL、アルガトロバンは終濃度で 0. 25 μ gZmLまたコラーゲンは終濃度で 1 Ο /z gZmLカ卩えた。全液量は 0. 36mLである。抗血小板療法は不十分と考えられる [表 22] Case 14 was a 66-year-old male with atherosclerotic obstruction who was taking pretal 200 mgZ and フ farin 2 mgZ. 2 volumes of 0.2M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to system X blood, and 2 volumes of 0.2M aqueous calcium chloride solution are anticoagulated to system Y blood. 1 volume of drug solution and 1 volume of platelet active solution are stored. For an ACT value of 209 seconds, heparin was added at a final concentration of 0.03 UZmL, argatroban at a final concentration of 0.25 μg ZmL, and collagen at a final concentration of 1 mg / z gZmL. The total volume is 0.36 mL. Antiplatelet therapy is considered inadequate [Table 22]
治験例 13 Case Study 13
プレタール 200mg/日  Pre-tar 200mg / day
治験例 15は、 62才 男性 動脈硬化性閉塞症のケースで、プレタール 200mg/ 日内服している事例である。 X系血液に 0. 2M塩ィヒカルシウム水溶液を 2容量、抗凝 固剤液を 1容量及び生理食塩水を 1容量の割合でカ卩え、 Y系血液に 0. 2M塩化カル シゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加 えている。 ACT値 179秒に対して、へパリンは終濃度で 0. 06UZmL、アルガトロバ ンは終濃度で 1. 5 μ gZmLまたコラーゲンは終濃度で 10 μ gZmLカ卩えた。全液量 は 0. 36mLである。へパリン 0. 06で伸ばしたモノだけが治療は十分との結果が出て いるが、他のアルガトロバンの全ての 3つの測定が不十分との結果が出ており、プレ タール 200mgZ日の治療は不十分と考えられる。 [0063] [表 23] 治験例 15 Case 15 was a 62-year-old male with atherosclerotic obstruction who was taking pretal 200 mg / day. 2 volumes of 0.2M aqueous calcium chloride solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to the X-system blood, and 2 volumes of 0.2M calcium chloride solution are added to the Y-system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. For an ACT value of 179 seconds, heparin had a final concentration of 0.06 UZmL, argatroban had a final concentration of 1.5 μgZmL, and collagen had a final concentration of 10 μgZmL. The total volume is 0.36 mL. Heparin 0.06 is the only treatment that has been shown to be sufficient, but all three other argatroban measurements have been shown to be inadequate. Insufficient. [0063] [Table 23] Clinical trial 15
プレターノレ 200mg/日  Pretanore 200mg / day
[0064] 治験例 16は、 62才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ 日内服している事例である。 X系血液に 0. 2M塩ィヒカルシウム水溶液を 2容量、抗凝 固剤液を 1容量及び生理食塩水を 1容量の割合でカ卩え、 Y系血液に 0. 2M塩化カル シゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加 えている。 ACT値 190秒に対して、へパリンは終濃度で 0. 03及び 0. 04UZmL、 アルガトロバンは終濃度で 0. 75及び 1. O /z gZmLまたコラーゲンは終濃度で 10 gZmL加えた。全液量は 0. 36mLである。 HepO. 04で伸ばしたモノだけが治療は 十分との結果が出ている力 他のへパリンおよびアルガトロバンの全ての 3つの測定 が不十分との結果が出ており、プレタール 200mgZdayの治療は不十分と考えられ る。 [0064] Clinical trial example 16 is a 62-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z day. 2 volumes of 0.2M aqueous calcium chloride solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to the X-system blood, and 2 volumes of 0.2M calcium chloride solution are added to the Y-system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. For an ACT value of 190 seconds, heparin was added at a final concentration of 0.03 and 0.04 UZmL, argatroban was added at a final concentration of 0.75 and 1. O / z gZmL, and collagen was added at a final concentration of 10 gZmL. The total volume is 0.36 mL. The power that only the thing stretched with HepO. 04 has shown that the treatment is sufficient The result that all three measurements of other heparin and argatroban are inadequate, the treatment of pre-tar 200mgZday is insufficient it is conceivable that.
治験例 17は、 68才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ日 内服している事例である。 X系血液に 0. 2M塩化カルシウム水溶液を 2容量、抗凝固 剤液を 1容量及び生理食塩水を 1容量の割合でカ卩え、 Y系血液に 0. 2M塩ィ匕カルシ ゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加え ている。 ACT値 190秒に対して、へパリンは終濃度で 0. lUZmL、アルガトロバン は終濃度で 0. 4// gZmLまた ADPは終濃度で 8. 3 / M加えた。全液量は 0. 36m Lである。何れも 20%以上の短縮を示し、プレタール 200mg/日の治療は不十分と 判定される。 Case 17 was a 68-year-old male with atherosclerotic obstruction who was taking pretal 200 mg Z day. Add 2 volumes of 0.2M calcium chloride aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and add 0.2M salt calcium aqueous solution to system Y blood. Add 2 volumes, 1 volume of anticoagulant solution and 1 volume of platelet activator solution ing. For an ACT value of 190 seconds, heparin was added at a final concentration of 0.1 lUZmL, argatroban was added at a final concentration of 0.4 // gZmL, and ADP was added at a final concentration of 8.3 / M. The total liquid volume is 0.36 mL. All showed a reduction of 20% or more, and pretal 200 mg / day was judged to be inadequate.
[表 24] 治験例 16 [Table 24] Clinical trial cases 16
プレターノレ 200mg/日  Pretanore 200mg / day
治験例 17 Clinical trial 17
プレタ一ゾレ 200mg/日  Pret zole 200mg / day
[0066] 治験例 18は、 70才 男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ 日内服している事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝 固剤液を 1容量及び生理食塩水を 1容量の割合でカ卩え、 Y系血液に 0. 2M塩化カル シゥム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加 えている。 ACT値 199秒に対して、へパリンは終濃度で 0. 03及び 0. 05U/mL, アルガトロバンは終濃度で 1. 0及び 2. Ο /z gZmLまたコラーゲンは終濃度で 10 g /mL加えた。全液量は 0. 36mLである。へパリン 0. 05で伸ばしたモノだけが治療 は不十分との結果が出ている力 他のへパリン及びアルガトロバンの全ての 3つの測 定が十分との結果が出ており、プレタール 200mgZ日の治療は十分と考えられる。 [0066] Clinical trial example 18 is a 70-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z day. 2 volumes of 0.2M saline-calcium aqueous solution, 1 volume of anti-coagulant solution and 1 volume of physiological saline are added to system X blood, and 0.2M calcium chloride aqueous solution is added to system Y blood. 2 volumes, 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. For ACT value of 199 seconds, heparin is added at a final concentration of 0.03 and 0.05 U / mL, argatroban is added at a final concentration of 1.0 and 2. Ο / z gZmL, and collagen is added at a final concentration of 10 g / mL. It was. The total volume is 0.36 mL. Heparin is the force that only the thing stretched with 0.05 is showing that the treatment is inadequate The result that all three measurements of other heparin and argatroban are sufficient, the pre-tar 200mgZ day Treatment is considered sufficient.
[0067] [表 25] 治験例 18  [0067] [Table 25] Clinical trial 18
プレターノレ 200mg/日  Pretanore 200mg / day
治験例 19は、 65才 女性 脳梗塞のケースで、パナルジン 200mgZ日内服して いる事例である。 X系血液に 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固剤液を 1 容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシウム水 溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて ヽる 。 ACT値 199秒に対して、へパリンは終濃度で 0. lU/mL、アルガトロバンは終濃 度で 1. 2 // g/mLまたコラーゲンは終濃度で 10 // g/mL加えた。全液量は 0. 36 mLである。(1)でのへパリン 0. 1で伸ばしたモノだけが治療は十分との結果が出て V、るが、他の 2つのへパリンおよびアルガトロバンの 3つの測定が不十分との結果が 出ており、パナルジン 200mgZ日の治療は不十分と考えられる。 Case 19 is a case of a 65-year-old female cerebral infarction who is taking Panalgin 200 mg Z day. Add 2 volumes of 0.2M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X-system blood, and 2 volumes of 0.2M calcium chloride solution to Y-system blood. Add 1 volume of anticoagulant solution and 1 volume of platelet activator solution. ACT value of 199 seconds, heparin at final concentration of 0.1 lU / mL, argatroban at final concentration At a rate of 1.2 // g / mL and collagen was added at a final concentration of 10 // g / mL. The total liquid volume is 0.36 mL. Heparin in (1) shows that only the thing stretched with 0.1 is sufficient for treatment, V, but the results for the other two heparins and argatroban are insufficient. Therefore, treatment with panalgin 200mgZ is considered inadequate.
[0069] [表 26] 治験例 19 [0069] [Table 26] Clinical trial example 19
パナルジン 200mg/日  Panardine 200mg / day
実施例 7 Example 7
[0070] 以後は、カオリン塗布カップ、ピン使用による治験例である。  [0070] The following are clinical trials using kaolin-coated cups and pins.
治験例 20は、 68才 女性 抗血小板薬 (?)、血小板機能は正常のケースである。 X 系血液に 0. 18M塩ィ匕カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食 塩水を 1容量の割合でカ卩え、 Y系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗 凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて 、る。 ACT値 178秒 に対して、アルガトロバンは終濃度で 1. 0、 3. 0及び 4. 0 /X g/mLまたコラーゲンは 終濃度で ΙΟ /z gZmL加えた。全液量は 0. 36mLである。この結果から、カオリン治 験例においては、 ACT力 得られた添加すべきアルガトロバン量は、カオリン(―)治 験例の場合に ACT力 得られるアルガトロパン量またはへパリン量の、 2〜3倍量を 与えることにより、 R値を 30〜40分にすることの可能性を示唆している。また、抑制さ れていない血小板機能がカオリン添カ卩によっても充分この結果力も判定できる。 Case 20 was a 68-year-old female antiplatelet drug (?), With normal platelet function. 1 volume of 0.18M saline-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y-system blood. Add 1 volume of anticoagulant solution and 1 volume of platelet activator solution. For ACT values of 178 seconds, argatroban was added at final concentrations of 1.0, 3.0 and 4.0 / X g / mL, and collagen was added at final concentrations of ΙΟ / z gZmL. The total volume is 0.36 mL. Based on this result, in the case of kaolin trial, the amount of argatroban to be added is 2 to 3 times the amount of argatropan or heparin obtained from the case of kaolin (-) trial. The This suggests that the R value could be 30-40 minutes. In addition, the platelet function that is not suppressed can be determined sufficiently by the kaolin-added card.
[表 27] 治験例 20 [Table 27] Clinical trial cases 20
血小板機能は正常  Normal platelet function
治験例 21は、 78才女性 抗血小板薬 (?)、血小板機能は正常のケースである。 X 系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食 塩水を 1容量の割合でカ卩え、 Y系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗 凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて 、る。 ACT値 213秒 に対して、アルガトロバンは終濃度で 0. 25、 0. 5、 1. 0及び 1. 5 g/mLまたコラ 一ゲンは終濃度で 10 gZmL加えた。全液量は 0. 36mLである。カオリン(一)治 験例における ACTから得られる添加すべきアルガトロバン量の 2〜3倍をカオリン(+ )治験例に与えれば、 R値を 30〜40分に出来ることがほぼ確実である。また、この力 ップ&ピンは 2度目の使用で、測定にムラが認められる, Case 21 was a 78-year-old female with antiplatelet drug (?) And normal platelet function. 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y blood. Add 1 volume of coagulant solution and 1 volume of platelet activator solution. For the ACT value of 213 seconds, argatroban was added at final concentrations of 0.25, 0.5, 1.0 and 1.5 g / mL, and collagen was added at a final concentration of 10 gZmL. The total volume is 0.36 mL. It is almost certain that the R value can be reduced to 30 to 40 minutes if 2 to 3 times the amount of argatroban to be added obtained from ACT in the Kaolin (1) trial is given to the Kaolin (+) trial. Also this power Pop and pin is used for the second time, and measurement is uneven.
[0073] [表 28] 治験例 21 [0073] [Table 28] Clinical trial example 21
血小板機能は正常  Normal platelet function
[0074] 治験例 22は、 78才 女性 抗血小板薬 (?)、血小板機能は正常のケースである。 X 系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食 塩水を 1容量の割合でカ卩え、 Y系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗 凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えて!/、る。 ACT値 176秒 に対して、アルガトロパンは終濃度で 1. 0、 2. 0及び 2. 5 g/mLまたコラーゲンは 終濃度で 10 gZmLカ卩えた。全液量は 0. 36mLである。やはり、カオリン治験例で は、 ACTによる表力 決定されるアルガトロバン量の 2〜3倍の量で、ほぼ確実に 30 〜40分に R値にすることが出来る。また抑制されていない血小板を判定することも、 おおむね可能である。 [0074] Clinical trial 22 was a 78-year-old female antiplatelet drug (?), With normal platelet function. 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X blood, and 1 volume of 0.18M calcium chloride aqueous solution is added to Y blood. Add 1 volume of coagulant solution and 1 volume of platelet activator solution! For ACT values of 176 seconds, argatropan was adjusted to 1.0, 2.0 and 2.5 g / mL at final concentrations, and collagen was adjusted to 10 gZmL at final concentrations. The total volume is 0.36 mL. Again, in the case of kaolin trials, the R-value can be almost certainly set to 30 to 40 minutes with an amount 2 to 3 times the amount of argatroban determined by ACT. It is also possible to determine uncontrolled platelets.
[0075] [表 29] 治験例 22 [0075] [Table 29] Clinical trial 22
血小板機能は正常  Normal platelet function
[0076] 治験例 23は、 77才男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ日 とドルナー 60mgZ日内服している事例である。 X系血液に 0. 18M塩ィ匕カルシウム 水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系 血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活 性剤液 1容量の割合でカ卩えている。 ACT値 203秒に対して、アルガトロバンは終濃 度で 0. 5、 1. 5及び 2. 0 [0076] Clinical trial example 23 was a 77-year-old male with atherosclerotic obstruction who was taking Pretal 200 mg Z day and Dorner 60 mg Z day. Add 1 volume of 0.18M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M calcium chloride aqueous solution to Y blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are stored. Argatroban has a final concentration of 0.5, 1.5 and 2.0 for an ACT value of 203 seconds
全液量は 0. 36mLである。カオリン使用において、ときどき測定が不安定になる典型 である。この治験例の抗血小板治療は不十分である。  The total volume is 0.36 mL. When using kaolin, the measurement is sometimes unstable. The antiplatelet treatment in this trial is inadequate.
[0077] [表 30] 治験例 23 [0077] [Table 30] Clinical trial 23
プレタール 200mqZ日 +ドルナー eOmaZ日  Pre-tar 200mqZ days + Dorner eOmaZ days
[0078] 治験例 24は、 69才、男性、動脈硬化性閉塞症のケースで、プレタール 2錠(200m gZ日 )内服している事例である。 X系血液に 0. 18M塩化カルシウム水溶液を 1容量 、抗凝固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 18M 塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の 割合で加えている。 ACT値 176秒に対して、アルガトロバンは終濃度で 1. 5及び 4. O /z gZmLまたコラーゲンは終濃度で 10 gZmLカ卩えた。全液量は 0. 36mLであ る。カオリン (?)もカオリン(+ )もレ、ずれも血小板機能は抑えられてレ、なレ、ことをしめし ている。抗血小板治療は不十分である。 [0078] Clinical trial example 24 is a 69-year-old male with arteriosclerotic obstruction and taking 2 pretal tablets (200 mg Zday). Add 0.1 volume of 18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 1 volume of 0.18M calcium chloride aqueous solution, anticoagulant to Y blood. 1 volume of liquid and 1 volume of platelet activator liquid are added. For ACT values of 176 seconds, argatroban was given a final concentration of 1.5 and 4. O / z gZmL and collagen was given a final concentration of 10 gZmL. The total volume is 0.36 mL. Both kaolin (?) And kaolin (+) indicate that the platelet function is suppressed and that the difference is. Antiplatelet therapy is inadequate.
[0079] [表 31] 治験例 24 [0079] [Table 31] Clinical trial cases 24
プレタール 2錠(200mg/日) 2 tablets of pretal (200mg / day)
実施例 8  Example 8
[0080] これ以後 4例はァクチン終濃度 IngZmL含有の試薬を使用して測定された治験例 であり、ァクチンを含有しない試薬及びカオリン塗布カップ &ピンでの測定も同時に 行っている。  [0080] Since then, 4 cases are clinical trial cases measured using a reagent containing a final concentration of actin, IngZmL, and the measurement using a reagent not containing actin and a kaolin-coated cup and pin is simultaneously performed.
治験例 25は、 44才男性 抗血小板薬 (?)凝固機能は正常のケースである。 X系血 液に 0. 18M塩ィ匕カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水 を 1容量の割合で加え、 Y系血液に 0. 18M塩ィヒカルシウム水溶液を 1容量、抗凝固 剤液を 1容量及び血小板活性剤液 1容量の割合で加えている。ァクチン以外に、 AC T値 185秒に対して、アルガトロバンは終濃度で 1. 0、及び 2. 5 g/mLまたコラー ゲンは終濃度で 10 gZmLカ卩えた。全液量は 0. 36mLである。血小板機能は低下 していると考えられる。  Case 25 was a 44-year-old male antiplatelet drug (?) In which the coagulation function was normal. Add 1 volume of 0.18M saline-calcium aqueous solution to system X blood, 1 volume of anticoagulant solution and 1 volume of physiological saline, and 1 volume of 0.18M salt calcium aqueous solution to system Y blood. One volume of anticoagulant solution and one volume of platelet activator solution are added. In addition to actin, argatroban was added at a final concentration of 1.0 and 2.5 g / mL and collagen was added at a final concentration of 10 gZmL for an ACT value of 185 seconds. The total volume is 0.36 mL. Platelet function is thought to have declined.
[0081] [表 32] [0081] [Table 32]
治験例 25 Clinical trial 25
凝固機能は正常  Normal coagulation function
[0082] 治験例 26は、 46才男性 抗血小板薬 (?)凝固能は正常 (aPTT, PTは正常)のケー スである。 X系血液に 0. 2M塩ィヒカルシゥム水溶液を 2容量、抗凝固剤液を 1容量及 び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 2M塩化カルシウム水溶液を 2 容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で加えている。ァクチ ン以外に、 ACT値 167秒に対して、アルガトロバンは終濃度で 1. 5、及び 2. 5 μ mLまたコラーゲンは終濃度で ΙΟ /z gZmLカ卩えた。全液量は 0. 36mLである。凝固 能は正常でも血小板機能は低下していると考えられる。 [0082] Case 26 is a case of a 46-year-old male antiplatelet drug (?) With normal clotting ability (aPTT, PT are normal). Add 2 volumes of 0.2M saline aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline to X blood, and 2 volumes of 0.2M calcium chloride aqueous solution, anticoagulation to Y blood. 1 volume of drug solution and 1 volume of platelet activator solution are added. In addition to the ACT value, argatroban was added at a final concentration of 1.5 and 2.5 μmL and collagen at a final concentration of ΙΟ / z gZmL against an ACT value of 167 seconds. The total volume is 0.36 mL. Even though the coagulation ability is normal, the platelet function is considered to have decreased.
[0083] [表 33] [0083] [Table 33]
治験例 26 Case Study 26
凝固能は正常 (aPTT,PTは正常)  Normal clotting ability (aPTT, PT are normal)
[0084] 治験例 27は、 48才男性 抗血小板薬 (?)血液凝固能は正常のケースである。 X系 血液に 0. 4M塩化カルシウム水溶液を 1容量、抗凝固剤液を 1容量及び生理食塩水 を 1容量の割合でカ卩え、 Y系血液に 0. 4M塩ィ匕カルシウム水溶液を 1容量、抗凝固 剤液を 1容量及び血小板活性剤液 1容量の割合で加えている。ァクチン以外に、 AC T値 158秒に対して、アルガトロバンは終濃度で 2. 0、及び 3. 5 gZmLまたコラー ゲンは終濃度で 10 gZmLカ卩えた。全液量は 0. 36mLである。この場合は、血小 板機能は正常と考えられる。ァクチン ( + )の測定が不安定である。 [0084] Clinical trial 27 is a 48-year-old male antiplatelet drug (?) In which blood coagulation ability is normal. 1 volume of 0.4M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to the X system blood, and 1 volume of 0.4M salt calcium aqueous solution is added to the Y system blood. 1 volume of anticoagulant solution and 1 volume of platelet activator solution are added. In addition to actin, argatroban was added at a final concentration of 2.0 and 3.5 gZmL and collagen was added at a final concentration of 10 gZmL for an ACT value of 158 seconds. The total volume is 0.36 mL. In this case, platelet function is considered normal. The measurement of lactin (+) is unstable.
[0085] [表 34] [0085] [Table 34]
治験例 27 Clinical trial 27
血液凝固能は正常  Normal blood clotting ability
[0086] 治験例 28は、 67才男性 動脈硬化性閉塞症のケースで、プレタール 200mgZ曰 内服している事例である。 X系血液に 0. 18M塩化カルシウム水溶液を 1容量、抗凝 固剤液を 1容量及び生理食塩水を 1容量の割合で加え、 Y系血液に 0. 18M塩化力 ルシゥム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液 1容量の割合で 加えている。ァクチン以外に、 ACT値 181秒に対して、アルガトロバンは終濃度で 1. 0、及び 2. C^ gZmLまたコラーゲンは終濃度で 10 gZmL加えた。全液量は 0. 36mLである。カオリン(+ )の測定値よりァクチン添加の方が凝固促進剤非使用の 測定値 (最初の測定値)に近い値を示している。ァクチン添加での測定力 カオリン 使用での値より優れていると考えられる。プレタールの治療は不十分であること力 ァ クチン添加では判明する力 カオリン処理ではプレタールの治療効果は十分との結 果が出ている。 [0086] Clinical trial example 28 is a 67-year-old male with atherosclerotic obstruction and taking pretal 200 mg Z 曰. 1 volume of 0.18M calcium chloride aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are added to X-system blood, and 1 volume of 0.18M aqueous solution of sodium chloride is added to Y-system blood. 1 volume of coagulant solution and 1 volume of platelet activator solution are added. In addition to ACTIN, argatroban was added at 1.0 and 2. C ^ gZmL and collagen at 10 gZmL at a final concentration for an ACT value of 181 seconds. The total volume is 0.36 mL. Compared with the measured value of kaolin (+), the addition of actin shows a value closer to the measured value without the use of the coagulation promoter (the first measured value). It is considered that the measurement ability with the addition of actin is superior to the value with kaolin. The power that pretal treatment is inadequate The power that can be found with the addition of actin Kaolin treatment has shown that the treatment effect of pretal is sufficient.
[0087] [表 35] 治験例 28 [0087] [Table 35] Clinical trial 28
プレターノレ 200mg/日 Pretanore 200mg / day
産業上の利用可能性 Industrial applicability
[0088] 本発明は、抗血栓薬薬効試験を適正、迅速かつ簡便に行うための試験方法である 。本発明の抗血栓薬薬効試験法を通して、医学、医療業界、薬品業界に大きく貢献 するものである。  [0088] The present invention is a test method for conducting an antithrombotic drug efficacy test appropriately, quickly and simply. Through the antithrombotic drug efficacy test method of the present invention, it greatly contributes to the medical, medical and pharmaceutical industries.
図面の簡単な説明  Brief Description of Drawings
[0089] [図 1]トロンボェラストグラフ測定を説明する図である [0089] FIG. 1 is a diagram for explaining thrombolast graph measurement.
符号の説明  Explanation of symbols
[0090] R 血塊弾性値の巾が 2mmになった時点  [0090] R When the width of clot elasticity reaches 2mm
K 血塊弹'性値の巾が 20mmになった時点  K When the width of the clot's sex value reaches 20mm
MA 血塊弾性値の巾の最大振幅 MA値  MA Maximum amplitude of clot elasticity value MA value
ANG 血塊弾性の変化のグラフで接線を引 、たときの角度  ANG Angle when a tangent line is drawn on the change graph of blood clot elasticity

Claims

請求の範囲 The scope of the claims
[1] 抗血栓薬の投与を受けている患者力 採取した血液に塩ィ匕カルシウム水溶液、抗 凝固剤液及び生理食塩水を加えた系(X系血液)、前記血液に塩化カルシウム水溶 液、抗凝固剤液及び血小板活性剤液を加えた系(Y系血液)にっき、トロンボェラスト グラフを測定し、 X系血液及び Y系血液の R値を比較する抗血栓薬薬効試験方法で あって、  [1] Power of patients receiving antithrombotic drugs A system (X blood) in which a salted calcium aqueous solution, an anticoagulant solution and physiological saline are added to the collected blood, and a calcium chloride aqueous solution in the blood, It is an antithrombotic drug efficacy test method that measures the thrombolast graph in a system (Y blood) to which an anticoagulant solution and platelet activator solution are added, and compares the R values of the X blood and Y blood. ,
血液に加える塩化カルシウムは、 X系血液、 Y系血液ともに、同量であり、同じく血液 に加える抗凝固剤は、 X系血液、 Y系血液ともに、同種のもの同量であることを特徴と する抗血栓薬薬効試験方法。  The amount of calcium chloride added to blood is the same for both X-system blood and Y-system blood, and the same amount of anticoagulant added to blood is the same for both X-system blood and Y-system blood. Antithrombotic drug efficacy test method.
[2] 前記抗凝固剤液がへパリン (未分化へパリン、低分子へパリン)水溶液又はアルガ トロバンの水溶液であることを特徴とする請求項 1に記載の抗血栓薬薬効試験方法。  [2] The antithrombotic drug efficacy test method according to [1], wherein the anticoagulant solution is a heparin (undifferentiated heparin, low molecular heparin) aqueous solution or an argatroban aqueous solution.
[3] 前記へパリン水溶液を含有させる終濃度が 0. 01 (U/mL)力 0. 1 (UZmL)の 範囲であり、アルガトロバン水溶液を含有させる終濃度が 0. 1 ( g/mL)から 5. 0 ( μ g/mL)の範囲であることを特徴とする請求項 2に記載の抗血栓薬薬効試験方法  [3] The final concentration containing the heparin aqueous solution is in the range of 0.01 (U / mL) force 0.1 (UZmL), and the final concentration containing the argatroban aqueous solution is 0.1 (g / mL). 5. The antithrombotic drug efficacy test method according to claim 2, which is in the range of 0 (μg / mL).
[4] 前記へパリン水溶液、アルガトロバン水溶液を含有させる終濃度を、予め経験的に 求めた ACT値と加えるべきへパリン水溶液、アルガトロバン水溶液の終濃度の関係 カゝら決定し、該決定した終濃度でへパリン水溶液、アルガトロバン水溶液を含有させ ることを特徴とする請求項 3に記載の抗血栓薬薬効試験方法。 [4] The final concentration of the heparin aqueous solution and the argatroban aqueous solution is determined from the empirically obtained ACT value in advance and the final concentration of the heparin aqueous solution and the argatroban aqueous solution to be added. 4. The antithrombotic drug efficacy test method according to claim 3, wherein a heparin aqueous solution and an argatroban aqueous solution are contained.
[5] 前記血小板活性剤液がアデノシン二リン酸 (ADP)水溶液又はコラーゲン水溶液 であることを特徴とする請求項 1から請求項 4のいずれかに記載の抗血栓薬薬効試 験方法。  5. The antithrombotic drug efficacy test method according to any one of claims 1 to 4, wherein the platelet activator solution is an adenosine diphosphate (ADP) aqueous solution or a collagen aqueous solution.
[6] 前記アデノシン二リン酸水溶液を含有させる終濃度が 6. 0 μ Μ以上、前記コラーゲ ン水溶液を含有させる終濃度が 1. 0 gZmL以上であることを特徴とする請求項 5 に記載の抗血栓薬薬効試験方法。  [6] The final concentration containing the adenosine diphosphate aqueous solution is 6.0 μΜ or more, and the final concentration containing the collagen aqueous solution is 1.0 gZmL or more. Antithrombotic drug efficacy test method.
[7] X系血液にお!ヽて、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の容量比 を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の添加量 は Z = 1の場合は 30 L、 Z = 2の場合は 40 Lとし、 1 < Zく 2の場合は「30 + (40 - 30) X (Z—l)」 Lであり、 Y系血液においては、塩化カルシウム水溶液、抗凝固 剤液及び血小板活性剤液の各試薬の容量比を Ζ : 1: 1とするとき、塩化カルシウム水 溶液、抗凝固剤液及び血小板活性剤液の添加量は Z= lの場合は 30 レ Ζ = 2の 場合は 40 Lとし、 1 <2< 2の場合は「30+ (40— 30) X (Ζ— 1)」 Lであることを 特徴とする請求項 1から請求項 7のいずれかに記載の抗血栓薬薬効試験方法。 [7] For system X blood, when the volume ratio of calcium chloride aqueous solution, anticoagulant solution and physiological saline is Z: 1: 1, calcium chloride aqueous solution, anticoagulant solution and physiological saline The amount added is 30 L when Z = 1, 40 L when Z = 2, and 30 + (40 when 1 <Z -30) X (Z-l) ”L, and in Y blood, when the volume ratio of each reagent of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution is Ζ: 1: 1, The amount of calcium water solution, anticoagulant solution, and platelet activator solution added is 30 L for Z = l, 40 L for 2 = 2, and 30 + (40—30 for 1 <2 <2. The method of testing an antithrombotic drug efficacy according to any one of claims 1 to 7, wherein X (Ζ- 1) "L.
[8] 前記 X系血液及び Υ系血液に更に血液凝固促進剤としてァクチン水溶液を終濃度 0. 5ngZmLから 2. 5ngZmLの範囲で含有させることを特徴とする請求項 1から請 求項 6に記載の抗血栓薬薬効試験方法。  [8] The X-type blood and the phlegm blood further contain an aqueous solution of actin as a blood coagulation promoter in a final concentration range of 0.5 ngZmL to 2.5 ngZmL. Antithrombotic drug efficacy test method.
[9] X系血液にお!、て、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の容量比 を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び生理食塩水の添加量 は Z = 1の場合は 40 L、 Z = 2の場合は 50 Lとし、 1 < Zく 2の場合は「40 + (40 - 30) X (Z—l)」 /z Lであり、  [9] For system X blood! When the volume ratio of calcium chloride aqueous solution, anticoagulant solution and physiological saline is Z: 1: 1, calcium chloride aqueous solution, anticoagulant solution and physiological saline The amount added is 40 L when Z = 1, 50 L when Z = 2, and is 40 + (40-30) X (Z-l) "/ z L when 1 <Z ,
Y系血液においては、塩化カルシウム水溶液、抗凝固剤液及び血小板活性剤液の 容量比を Z : 1: 1とするとき、塩化カルシウム水溶液、抗凝固剤液及び血小板活性剤 液の添力卩量は Z= lの場合は 40 L、Z = 2の場合は 50 Lとし、 1 <Zく 2の場合は 「40+ (40- 30) X (Z— 1)」 Lであることを特徴とする請求項 8に記載の抗血栓薬 薬効試験方法。  In Y-system blood, when the volume ratio of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution is Z: 1: 1, the added amount of calcium chloride aqueous solution, anticoagulant solution and platelet activator solution Is 40 L when Z = l, 50 L when Z = 2, and `` 40+ (40-30) X (Z—1) '' L when 1 <Z The antithrombotic drug efficacy test method according to claim 8.
[10] X系血液において、 0. 18M塩ィ匕カルシウム水溶液を 1容量〜 2容量、抗凝固剤液 を 1容量及び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 18M 塩ィ匕カルシウム水溶液を 1容量〜 2容量、抗凝固剤液を 1容量及び血小板活性剤液 を 1容量の割合で含有させることを特徴とする請求項 1から請求項 9のいずれかに記 載の抗血栓薬薬効試験方法。  [10] In X-system blood, 0.1 to 2 volumes of 0.1M aqueous calcium chloride solution, 1 volume of anticoagulant solution and 1 volume of physiological saline are contained, and in Y-system blood, 0. The composition according to any one of claims 1 to 9, characterized in that it contains 1 to 2 volumes of 18M aqueous solution of calcium chloride salt, 1 volume of anticoagulant solution and 1 volume of platelet activator solution. The antithrombotic drug efficacy test method listed.
[11] X系血液において、 0. 2M塩ィ匕カルシウム水溶液を 2容量、抗凝固剤液を 1容量及 び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 2M塩ィ匕カルシゥ ム水溶液を 2容量、抗凝固剤液を 1容量及び血小板活性剤液を 1容量の割合で含有 させることを特徴とする請求項 1から請求項 9のいずれかに記載の抗血栓薬薬効試 験方法。  [11] In system X blood, contain 0.2 volume of 0.2M salt-calcium aqueous solution, 1 volume of anticoagulant solution and 1 volume of physiological saline, and in system Y blood, 0.2M salt 10. The antithrombotic agent according to any one of claims 1 to 9, comprising 2 volumes of aqueous calcium solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution. Medicinal efficacy test method.
[12] X系血液において、 0. 4M塩ィ匕カルシウム水溶液を 1容量、抗凝固剤液を 1容量及 び生理食塩水を 1容量の割合で含有させ、 Y系血液において、 0. 4M塩ィ匕カルシゥ ム水溶液を 1容量、抗凝固剤液を 1容量及び血小板活性剤液を 1容量の割合で含有 させることを特徴とする請求項 1から請求項 9のいずれかに記載の抗血栓薬薬効試 験方法。 [12] In system X blood, 1 volume of 0.4M saline-calcium aqueous solution and 1 volume of anticoagulant solution 1 volume of normal saline and 1 volume of 0.4M saline solution, 1 volume of anticoagulant solution and 1 volume of platelet activator solution 10. The method for testing the efficacy of an antithrombotic drug according to any one of claims 1 to 9, wherein:
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