JP2002221519A - Anticoagulant and anticoagulant treating method using it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an anticoagulant and an anticoagulant treating method capable of being used for blood cell count inspection or biochemical inspection. SOLUTION: This anticoagulant is composed of a serine protease inhibitor and is used for the blood cell count inspection or the biochemical inspection. The anticoagulant treating method executes an anticoagulant treatment to a blood specimen by use of the anticoagulant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anticoagulant and a method for treating an anticoagulant which can be used in a blood count test or a biochemical test.

[0002]

2. Description of the Related Art At present, different anticoagulants are used for blood tests depending on the purpose of the test. Generally, EDTA salts are often used in blood cell count tests, and anticoagulants are not used or heparin is used in biochemical tests.

[0003] The reason why anticoagulants are properly used in this way is that there are measurement items that are adversely affected by the use of each anticoagulant. For example, when EDTA salt is used in a biochemical test, the chelating action affects the enzyme reaction system and electrolyte items, and shows a low value. On the other hand, when heparin is used in a blood count test, platelets aggregate and the platelet count decreases. It also causes an apparent increase in white blood cells. Therefore, unless an appropriate anticoagulant is used according to the type of test,
An accurate test result cannot be obtained, which may lead to a misdiagnosis.

[0004]

A blood test involves collecting blood in a blood collection tube (about 2 to about 5 ml per tube) containing several different anticoagulants for a plurality of test items. Is usually done well. However, an increase in the amount of blood to be collected is troublesome and burdens the patient whose blood is to be collected.

[0005] Also, preparing blood collection tubes with different anticoagulants for each test item leads to an increase in test cost. Further, if the number of blood collection tubes is large, there is a possibility that the sample may be mistaken. Therefore, an anticoagulant capable of performing a plurality of blood tests with one blood collection tube is desired. That is, an object of the present invention is to provide an anticoagulant that can be used for both a blood cell count test and a biochemical test, and a test method using the same.

[0006]

DISCLOSURE OF THE INVENTION The present inventors aim at a simple method and to reduce the burden on a patient, in other words, to carry out both a blood cell count test and a biochemical test with one sample. And diligent research on anticoagulants. That is, mesyl which has been used as a therapeutic drug for pancreatitis and generalized intravascular coagulation (DIC) and an anticoagulant for perfused blood during extracorporeal blood circulation in patients with bleeding lesions or bleeding tendency Using acid nafamostat (hereinafter referred to as “Fusan”), we tried to convert the counting measurement of white blood cells and red blood cells and the qualitative and quantitative measurement of biological components such as total protein and total cholesterol to completely different purposes. And surprisingly,
The present inventors have found that it is possible to perform counting measurement of white blood cells and red blood cells and qualitative and quantitative measurement of biological components such as total protein and total cholesterol without causing various adverse effects on normally expected measurement, and have completed the present invention. .

[0007] That is, it is an anticoagulant comprising a serine protease inhibitor and used for both a blood cell count test and a biochemical test.

Further, the serine protease inhibitor is preferably Fusan.

Further, there is provided an anticoagulant treatment method for subjecting a blood sample to an anticoagulant treatment using an anticoagulant used for a blood cell counting test or a biochemical test comprising a serine protease inhibitor. .

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION As the anticoagulant of the present invention, it is most preferable to use the serine protease inhibitor Fusan.

The anticoagulant of the present invention requires a sufficient amount to prevent coagulation of the blood sample and a sufficient amount to prevent aggregation, deformation and adhesion of platelets in the blood sample. It is necessary to use. Therefore, it is preferable that the final concentration of Fusan be 3 × 10 ⁻⁴ M or more.

In the blood test method of the present invention, first,
A serine protease inhibitor is added to the blood sample. Here, the blood sample means a sample collected by peripheral blood apheresis of mammals, especially humans. Further, a serine protease inhibitor may be added to a blood sample, or a serine protease inhibitor may be dissolved in an appropriate solvent and used by dispensing into a test tube or the like for collecting a blood sample. After pouring and drying, the blood sample may be added. Temperature at the time of addition is not particularly limited, for example,
Room temperature or so can be mentioned. Examples of the drying method include freeze-drying, vacuum drying, hot-air drying, and spray-drying. Freeze-drying and spray-drying are preferred, and freeze-drying is most preferred in terms of solubility. Freeze-drying is a method in which a solution is frozen and then dried in a vacuum to convert it into a solid.

[0013] In addition, if only the fusan is dried, when it comes into contact with blood, the local concentration of the fusan becomes high, and there is a possibility that the fusocytes may be lysed (hemolysis). In order to prevent hemolysis, it is necessary to lyse blood immediately upon contact with blood. Therefore, it is preferable to add polyvinyl alcohol (PVA) having an action of protecting blood cells and polyethylene glycol (PEG) to enhance solubility.

Further, the concentration of PVA is 0.2 to 5.0.
(W / v)%, PEG is preferably used at 0.5 to 5.0 (w / v)%.

[0015] The blood sample obtained by the anticoagulant of the present invention may be used first for a blood cell count test, and then centrifuged to collect plasma for use in a biochemical test. May be divided into two, and one may be used for a blood cell count test and the other may be used for a biochemical test. If the amount of the sample used for the biochemical test does not affect the blood cell count test, centrifuge the obtained blood sample, collect the required amount of plasma, and conduct the biochemical test. After that, the remaining blood sample may be stirred to perform a blood cell count test. In particular, it is preferable to perform a biochemical test after the blood cell count test or to separately perform the blood cell count test and the biochemical test.
It is also possible to perform a blood cell count test and a biochemical test at the same time. It is to be noted that the blood cell count test and the biochemical test are suitably performed at a temperature of about room temperature, but can be adjusted as appropriate according to the equipment to be used.

The counting of leukocytes in the blood cell test method of the present invention means not only the total number of leukocytes but also lymphocytes, monocytes, granulocytes, (neutrophils, eosinophils and basophils) contained therein. And the like to determine the count value of each leukocyte and its ratio.

The counting of erythrocytes means not only the counting of erythrocytes but also the measurement of hemoglobin concentration and hematocrit value and the erythrocyte constants (mean erythrocyte volume (MCV), mean erythrocyte hemoglobin amount (MC
H), average erythrocyte hemoglobin concentration (MCHC)), reticulocyte count and its ratio, and the like.

Further, the counting of platelets includes obtaining not only the number of platelets but also the average platelet volume and the ratio of large platelets.

The method of counting blood cells may be any of a manual method and a method using a blood cell counter. In addition, there are an electric resistance type, an optical type, and the like in the blood cell counter, and any measurement principle may be used.

The biochemical tests include, for example, so-called blood tests such as a nitrogen-containing component test, a metal test, a plasma protein-related test, a collagen reaction, a vital pigment test, an enzyme-related test, a lipid test, an electrolyte test, and a glucose metabolism test. It means a test for qualitatively and quantitatively determining various components by a biochemical method.

Specifically, creatine phosphokinase (CPK), blood urea nitrogen, (BUN), urate dehydrogenase (LDH), alkaline phosphatase (ALP), GOT, GPT, triglyceride (TG),
Uric acid (UA), Na, K, Cl, total cholesterol (TCHO),
High density lipoprotein (HDLC), total protein (TP), albumin (ALB), amylase (AMYL), γ-glutamine transpeptidase (GGT), creatinine (CRE), total bilirubin (TBIL), C-reactive protein ( (CRP) and other tests. These measurements were performed using the creatine phosphate substrate / diaphorase / formazan dye method, urease / BCG indicator method, lactate substrate / diaphorase / formazan dye method, p-nitrophenyl phosphate substrate method, uricase / POD method, BCG indicator method, etc. It can be appropriately selected from known methods. The biochemical test may be any of a manual method and an automatic measuring method as long as these general biochemical test items can be measured.

[0022]

EXAMPLES Hereinafter, the anticoagulant and the method for treating the anticoagulant of the present invention will be described with reference to examples.

First, the collected blood has a final concentration of 0.5% (w / v), polyvinyl alcohol (PVA: Wako Pure Chemical Co., Ltd., degree of polymerization 500), polyethylene glycol (PEG: Wako Pure Chemical Co., Ltd.). Manufactured, average molecular weight 200) 1.0
(W / v)%, Fusan (manufactured by Torii Pharmaceutical Co., Ltd.), which is a serine protease inhibitor, was prepared so as to have a solution having the concentration shown in Table 1, and dispensed into blood collection tubes, followed by freeze-drying. Then, the appropriate concentration was examined.

The mechanism of action of a serine protease inhibitor is to inhibit serine proteases such as thrombin and factor Xa by acylation or sulfonylation, thereby suppressing blood coagulation and further suppressing arachidonic acid releasing action, Has the function of suppressing coagulation.

The above blood samples were subjected to a blood cell count test (white blood cell count (WBC) and each white blood cell ratio (neutrophil ratio (NEUT%), lymphocyte ratio (LYMPH%), monocyte ratio (MONO%), eosinophil ratio (EOSI%), basophil ratio (BASO%)), red blood cell count (RB
C), hemoglobin concentration (Hgb), hematocrit (Hc
t), MCV, MCH, MCHC, and platelet count (PLT)) (device used: SE-9000; Sysmex Corporation). Table 1 shows the results of the average values of the measured PLTs of the three samples. EDTA-2K was used as a control.

[0026]

[Table 1]

As shown in Table 1, the concentration of Fusan was 3%.
Coagulation becomes impossible at × 10 ^-7 M, but PLT increases as the concentration increases, and when the concentration is 3 × 10 ^-4 M, EDTA-2K, which has been conventionally used for blood cell count tests, It showed equivalent performance, and it was confirmed that platelets were not aggregated.

From the results of the appropriate concentration examined above, 3 × 1
Blood collection tube containing 0 ^-4 M Fusan (PVA 0.5%, PEG
Lyophilized product containing 1.0%)
After 4 and 8 hours, a blood cell count test was performed.
-9000; Sysmex Corporation). EDTA-2K was used as a control for a blood cell count test. Table 2 shows the results of the average value of the blood cell count of Fusan for the three healthy subjects, and Table 3 shows the results of the average value of the blood cell count of the control.

[0029]

[Table 2]

[0030]

[Table 3]

In the PLT item, those using the anticoagulant of the present invention are EDTA which has been conventionally used for hemocytometry.
It showed the same performance as -2K, and it was confirmed that platelets were not aggregated. Similarly, the other blood cell count test items showed the same performance as the conventional product.

Next, the blood collection tube after the blood cell count test was centrifuged, and the obtained plasma was subjected to BUN, UA, CRE, TCH using the FDC-3000 series (Fuji Film Co., Ltd.).
O, TP, ALB, TBIL, HDLC, CRP, GGT, GOT, GPT, CPK, L
Measurements were made on biochemical test items of DH, ALP, AMYL, TG, Na, K, and Cl. Serum and heparinized plasma were used as controls for biochemical tests. The results of the biochemical tests are shown in FIGS.

As shown in FIGS. 1 to 10, the samples using the anticoagulant of the present invention showed the same behavior between samples as serum or heparin-treated plasma, and the biochemical test items were measured. It was confirmed that it was possible.

[0034]

According to the present invention, since the same anticoagulant can be used for both a blood cell count test and a biochemical test, the number of blood collection tubes can be reduced. Thereby, the labor and cost of blood collection can be reduced, and the burden on the patient can be further reduced. In addition, since a blood cell count test and a biochemical test can be performed with a single blood collection tube, it is possible to connect each fully automatic device with a sample transport device to achieve systemization.

[0035]

[Brief description of the drawings]

FIG. 1 is a graph showing measurement results of biochemical test items, BUN, and UA when using Fusan as an anticoagulant.

FIG. 2 is a graph showing measurement results of biochemical test items, CRE, and TCHO when using Fusan as an anticoagulant.

FIG. 3 is a graph showing measurement results of biochemical test items, TP, and ALB when using Fusan as an anticoagulant.

FIG. 4 is a graph showing measurement results of biochemical test items, TBIL, and HDLC when using Fusan as an anticoagulant.

FIG. 5 is a graph showing measurement results of biochemical test items, CRP, and GGT when using Fusan as an anticoagulant.

FIG. 6 is a graph showing measurement results of biochemical test items, GOT, and GPT when using Fusan as an anticoagulant.

FIG. 7 is a graph showing measurement results of biochemical test items, CPK, and LDH when Fusan is used as an anticoagulant.

FIG. 8 is a graph showing measurement results of biochemical test items, ALP, and AML when using Fusan as an anticoagulant.

FIG. 9 is a graph showing measurement results of biochemical test items, TG, and Na when Fusan is used as an anticoagulant.

FIG. 10 is a graph showing the measurement results of biochemical test items, K, and Cl when using Fusan as an anticoagulant.

Claims (6)

[Claims] 1. An anticoagulant comprising a serine protease inhibitor and used for a blood cell count test or a biochemical test. 2. The anticoagulant according to claim 1, wherein the serine protease inhibitor is nafamostat mesilate. 3. The anticoagulant according to claim 2, wherein the final concentration of nafamostat mesilate is 3 × 10 ⁻⁴ M or more. 4. An anticoagulant treatment method in which a blood sample is treated with an anticoagulant using an anticoagulant for a blood cell count test or a biochemical test, which is composed of a serine protease inhibitor. 5. The method according to claim 4, wherein the serine protease inhibitor is nafamostat mesilate. 6. The method for treating an anticoagulant according to claim 5, wherein the final concentration of nafamostat mesilate is 3 × 10 ⁻⁴ M or more.