JPH0126502B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for separating serum and blood clots, and more particularly to a method for separating serum and blood clots from a whole blood sample of a subject receiving heparin administration by centrifugation. In recent years, coupled with the remarkable progress in inspection technology,
Blood tests such as serum biochemical tests, serum immunological tests, and hematology tests have become widely used, and have come to greatly contribute to disease prevention and early diagnosis. Serum tests are the main body of blood tests, and the serum required for tests is usually obtained by coagulating blood collected in a blood test container and then centrifuging it to form blood clots with different specific gravities (fibrin and blood cells). separated from the mixed gel-like mass). After the blood is separated into serum and blood clots by centrifugation, the serum portion is sucked up with a pipette or collected by decantation. The serum portion is collected after waiting for the blood to coagulate, but the problem is that it takes a considerable amount of time for the blood to coagulate, making it impossible to perform tests quickly. It is a big problem that it takes time for blood to coagulate even in the blood of normal healthy people, but patients undergoing artificial dialysis or patients with thrombosis are given heparin to prevent blood clots. The blood of such patients contains a considerable concentration of heparin, making it difficult to separate and collect serum during clinical tests because blood coagulation is unlikely to occur. It is an object of the present invention to provide a method for significantly shortening the time required for blood coagulation even for such patient blood, and for satisfactorily separating serum components and blood clot components. The gist of the present invention is as follows: 1. In a method for separating blood into serum and blood clots by subjecting blood to centrifugation, in the blood in a blood test container, the formula (However, in the above formula, X is a halogen atom and x is a positive integer.) By adding hexadimethrine halide represented by Method 2 for separating serum and blood clots characterized by the presence of ( b) Formula (However, in the above formula, X is a halogen atom, x is a positive integer) Hexadimethrine halide and (b)
Selected from the group consisting of glass, silica, kaolin, celite and bentonite, with particle size
50μm or less, average particle size is 10μm or less, linseed oil absorption is 20-40ml/100g,
An adsorbent inorganic substance having a BET specific surface area value of 5000 to 30000 cm ² /g and a specific resistance value of 1×10 ¹⁰ Ω·cm or less, the ratio of (a) and (b) being 0.0005 to 10 parts by weight:
Add within the range of 1 part by weight, and the amount of (a) added should be in the range of 0.005 mg to 5 mg per ml of blood volume, so that hexadimethrine halide and adsorbent inorganic substances are present in the blood. Method 3 for separating serum and blood clots characterized by (However, in the above formula, X is a halogen atom, x is a positive integer) Hexadimethrine halide and (b)
2,2',4,4'-tetrahydroxybenzophenone is added in a ratio of 0.0005 to 10 parts by weight: 1 part by weight of (a) and (b), and ) is added in the range of 0.005 mg to 5 mg per ml of blood volume, and hexadimethrine halide and 2,2',4,4'-tetrahydroxybenzophenone are present in the blood. Method 4 for separating serum and blood clots In a method of separating blood into serum and blood clots by subjecting blood to centrifugation, the formula (a) is present in the blood in a blood test container. (However, in the above formula, X is a halogen atom, x is a positive integer) Hexadimethrine halide and (b)
Adding ellagic acid in a ratio of (a) and (b) within a range of 0.0003 to 6 parts by weight: 1 part by weight, and
Addition amount of (a) is 0.005 mg to 5 mg per ml of blood volume.
A method for separating serum and blood clots, characterized by the presence of hexadimethrine halide and ellagic acid in the blood. 5 In a method of separating blood into serum and blood clots by subjecting it to centrifugation, when blood in a blood test container contains the formula (a) (However, in the above formula, X is a halogen atom, x is a positive integer) Hexadimethrine halide and (b)
Epicatechin is added in a ratio of (a) to (b) of 0.0003 to 6 parts by weight: 1 part by weight, and the amount of (a) added is 0.005 mg to 5 mg per ml of blood volume. A method for separating blood serum and blood clots, characterized in that hexadimethrine halide and epicatechin are present in the blood. Next, the method of separating serum and blood clots of the present invention will be explained in more detail. A whole blood sample of a subject is subjected to a centrifugation operation to separate serum and blood clots, and the blood is placed in a test container and allowed to clot. However, even in normal healthy people, clotting takes time if left as is, but in patients undergoing artificial dialysis or patients with thrombosis, heparin is present in the blood because they are receiving heparin. death,
Due to its action, blood coagulation is less likely to occur. Therefore, in the present invention, hexadimethrine halide is present in the blood contained in the test container.
Hexadimethrine halide is N, N, N', N'-
It is a polymer of tetramethylhexamethylene diamine, trimethylene dihalide, and trimethylene halide, and is represented by the following formula. (However, in the above formula, X is a halogen atom, and x is a positive integer.) In the above formula, X is preferably bromine, iodine, etc. However, when X is bromine, Polyprene (trade name) is a commercially available product. In addition to being used as a heparin neutralizing substance, it is also used as a peptide degradation promoting substance to determine the amino acid sequence in polypeptides. By adding hexadimethrine halide to the blood contained in the container, the heparin-containing blood quickly loses the effect of heparin and restores the blood coagulation function. Heparin exists in the blood of dialysis patients and thrombosis patients receiving heparin administration at a rate of 1 to 10 units per 10 c.c. of blood, but when hexadimethrine halide is present in the blood, this Since heparin is adsorbed to hexadimethrine halide and removed from the blood, other blood coagulation factors including trompin regain their normal functions. The appropriate amount of hexadimethrine halide is
A range of 0.005 mg to 5 mg per ml of blood is preferred; if the amount is less than 0.005 mg, the heparin neutralizing effect will be insufficient and sufficient blood coagulation will not be obtained. In addition, if the amount exceeds 5 mg, abnormal values tend to appear in clinical test values such as serum biochemical tests. In the above case, when hexadimethrine halide is allowed to exist alone in the blood, a significant blood coagulation promoting effect is observed. However, it is possible to further improve the blood coagulation promoting effect by using hexadimethrine halide in combination with an adsorbent inorganic substance, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid or epicatechin. can. One of the substances that synergistically exhibits a blood coagulation promoting effect when used in combination with hexadimethrine halide is an adsorbent inorganic substance. Adsorptive inorganic substances include inorganic substances used as adsorbents, such as glass, silica,
Selected from water-insoluble inorganic fine powders such as kaolin, celite, and bentonite. In addition, the adsorbent inorganic substance has a particle size of 50 μm or less,
Those with an average particle size of 10 μm or less are used. In particular, silica is an adsorbent inorganic material that is effective in shortening blood coagulation time.
Porous silica containing at least % by weight exhibits excellent effects. Such adsorbent inorganic substances have the effect of promoting activation of blood coagulation factors and aggregation of platelets when they come into contact with blood. However, in order for the adsorbent inorganic substance to effectively exhibit a blood coagulation promoting effect, it is preferable that the linseed oil absorption amount, BET specific surface area value, and specific resistance value exist within certain ranges. The linseed oil absorption amount and BET specific surface area value represent the extent of the surface area of the adsorbent inorganic material, and since the surface area is related to the extent of surface pores possessed by the adsorbent inorganic material, the extent of surface pores is determined by the oil absorption amount and specific surface area. You can know. The adsorptive inorganic material in the present invention has a linseed oil absorption of 20 to 40 ml/100 g,
Those having a BET specific surface area value of 5,000 to 30,000 cm ² /g are used. Linseed oil absorption indicates a value measured in accordance with Japanese Industrial Standard K-5101. The BET specific surface area value is calculated from the amount of gas adsorbed on the surface of an adsorbent inorganic material, the equilibrium pressure at that time, and the saturated vapor pressure of the adsorbed gas to determine the amount of gas that will penetrate the surface as a monomolecular layer. It refers to a value calculated by multiplying the average cross-sectional area of gas molecules, and nitrogen gas, oxygen gas, argon gas, methane gas, etc. are used as the adsorbed gas. According to this method, the surface area value including pores, which cannot be measured by measuring linseed oil absorption, can be measured. During blood coagulation, factor factor, or contact factor, is activated, and for this purpose, three substances, factor factor, prekallikrein, and high-molecular-weight kininogen, form a complex and are adsorbed on the surface of a foreign object. are necessary, and it is said that adsorption without one or two of these will not lead to activation. By the way, when an adsorbent inorganic substance is used with the expectation of promoting blood coagulation, if the surface area is very large, factor factor, prekallikrein, and The adsorption rate of high-molecular-weight kininogen will increase, in other words, the rate of complex formation of the three components necessary for activation of factor will decrease, and the blood coagulation promoting effect will be abolished. . On the other hand, if the surface area of the adsorbent inorganic material is too small, the probability of adsorption of coagulation factors will be low, making it impossible to expect a blood coagulation promoting effect.
For this reason, the adsorbent inorganic material in the present invention has a linseed oil absorption of 20 to 40 ml/100 g and a BET specific surface area value of 20 to 40 ml/100 g.
Those having a surface area in the range of 5000 to 30000 cm ² /g are used. Further, in the present invention, the adsorptive inorganic material has a specific resistance value of 1×10 ¹⁰ Ω·cm or less, and most preferably 5×10 ⁴ Ω·cm or less. The specific resistance value is the reciprocal of electrical conductivity, and is the value at room temperature. The proportion of adsorbent inorganic material and hexadimethrine halide used is within the range of 0.0005 to 10 parts by weight of hexadimethrine halide per 1 part by weight of adsorbent inorganic material. Another substance that synergistically exerts a procoagulant effect when used in combination with hexadimethrine halide is 2,2',4,4'-tetrahydroxybenzophenone. 2,2',4,4'-tetrahydroxybenzophenone has the following chemical structural formula. 2,2',4,4'-tetrahydroxybenzophenone has a melting point of 195℃ and a solubility in solvents of 30℃.
0.1% by weight for water, 50% by weight for methanol, 40% by weight for ethanol, and 10% by weight for a 1:1 water-ethanol solution. Further, the maximum absorption wavelength position is 345 mμ, and the color value (Gardner) is No. 8 in a 1% by weight methanol solution. The ratio of 2,2',4,4'-tetrahydroxybenzophenone and hexadimethrine halide used is 1 part by weight of 2,2',4,4'-tetrahydroxybenzophenone and hexadimethrine halide.
The range is 0.0005 to 10 parts by weight. Another substance that exhibits a synergistic procoagulant effect when used in combination with hexadimethrine halide is ellagic acid. Elazinic acid is a substance with the following chemical structural formula. Elazinic acid is known as a substance that activates factor factor, which is one of the blood coagulation factors, but when used in combination with hexadimethrine halide, it improves blood flow even better than when used alone. It was confirmed that it has a coagulation promoting effect. The ratio of ellagic acid and hexadimethrine halide used is within the range of 0.0003 to 6 parts by weight of hexadimethrine halide per 1 part by weight of ellagic acid. Yet another substance that synergistically exerts a procoagulant effect when used in combination with hexadimethrine halide is epicatechin. Epicatechin is a substance with the following chemical structural formula. Epicatechin is a colorless columnar crystal with a decomposition temperature of 237
~238℃, the maximum absorption wavelength position is 282mμ, and it is a substance that is hardly soluble in water. The ratio of epicatechin and hexadimethrine halide to be used is within the range of 0.0003 to 6 parts by weight of hexadimethrine halide per 1 part by weight of epicatechin. In order to make hexadimethrine halide or an inorganic substance adsorbable with it, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin present in the blood, for example, in the blood in the test container, It may be added directly to the blood, or it may be dispersed in a dispersion medium such as water and dropped into the blood.
A substance attached to a carrier of about 1.04 to 1.06 may be added to blood. In the present invention, hexadimethrine halide or an inorganic substance adsorbable thereto, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin are added to blood in a blood test container. The presence of hexadimethrine halide or its adsorbable inorganic substances, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin in the blood of normal healthy people Not only does it have an excellent effect on promoting blood coagulation, but it also has an excellent effect on blood that is difficult to coagulate due to heparin administration, such as in patients receiving artificial dialysis or patients with thrombosis. It has a blood coagulation promoting effect, and when centrifuged, serum and blood clots are easily separated, and serum can be obtained in good yield. Example 1 Bottomed outer diameter 15m/m, inner diameter 13m/m, height
A 100m/m glass tube was prepared. In addition, 100 mg of hexadimethrine bromide dissolved in 5 ml of physiological saline was prepared. After injecting 5 ml of fresh human blood containing 5 units of heparin per 5 ml of blood into a glass tube, immediately add 0.25 ml of the above solution of hexadimethrine bromide and leave at 20°C until the whole blood has completely flowed. The time required for it to disappear was measured as blood coagulation time, and blood coagulability was evaluated. Also, immediately after blood coagulation
Centrifugation was performed for 5 minutes at a rotational speed of 3000 revolutions per minute, the state of serum separation was observed, and the supernatant serum was collected with a pipette, and the amount was taken as the serum yield.
The state of serum separation was evaluated by the presence or absence of residual blood clot adhesion on the vessel wall in the serum layer and the presence or absence of fibrin network precipitation. As is clear from the results in the Example 1 column of Table 1, serum could be easily collected from heparin-containing blood in a short time by the method of the present invention. In addition, in order to examine whether the presence of hexadimethrine bromide affected blood test values, a serum biochemical test (28 items) was conducted using the serum obtained above. A significant difference test was conducted on the test values between the case where hexadimethrine bromide was not used and the above case (n=5), and the presence or absence of a difference was tested at a significance level of 5%. As a result, it was confirmed that the test values were not affected at all. Example 2 In Example 1, in addition to 100 mg of hexadimethrine bromide, natural amorphous assilicic acid fine powder (average particle size 2 μm, linseed oil absorption 30 ml/100 g, BET specific surface area value 1.5 × 10 ^{4 cm2} /
Example 1 was carried out in the same manner as in Example 1, except that 500 mg of the test sample was dissolved and dispersed in 5 ml of physiological saline, and a polyethylene container was used ^as the test container. Blood coagulability, serum separation status, and serum yield of heparin-containing blood were evaluated. The results are as shown in the column of Example 2 in Table 1, and the coagulation of heparin-containing blood was significantly promoted, and the serum separation state was also very good. Examples 3 to 5 Instead of the adsorptive inorganic substance in Example 2, 2,2',4,4'-tetrahydroxybenzophenone (Example 3) was used as a substance added to heparin-containing blood in combination with hexadimethrine bromide. ), ellagic acid (Example 4), epicatechin (Example 5)
It was used. In these cases, the dosage is 400 mg of 2,2',4,4'-tetrahydroxybenzophenone, 300 mg of ellagic acid, 400 mg of epicatechin, and 50 mg of physiological saline per 100 mg of hexadimethrine bromide.
ml and dispersed. Next, in the same manner as in Example 1, the blood coagulability, serum separation state, and serum yield of the heparin-containing blood placed in a polyethylene test container were evaluated. The results are shown in the columns of Examples 3-5. When using 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin as substances used in combination with hexadimethrine bromide, they significantly accelerate coagulation of heparin-containing blood and separate blood serum. It was in extremely good condition. Comparative Example 1 Heparin-containing blood was placed in the same glass tube as in Example 1 in the same manner as in Example 1, and
Although it was left for 10 hours without adding hexadimethrine bromide, blood coagulation was not completed and serum separation was impossible. 【table】

Claims (1)

[Scope of Claims] 1. In a method for separating blood into serum and blood clots by subjecting it to centrifugation, in the blood in a blood test container, the formula (However, in the above formula, X is a halogen atom, and x is a positive integer.) By adding hexadimethrine halide in the range of 0.005 mg to 5 mg per ml of blood, hexadimethrine halide is present in the blood. A method for separating serum and blood clots. 2 In a method of separating blood into serum and blood clots by subjecting it to centrifugation, when blood in a blood test container contains the formula (a) (However, in the above formula, X is a halogen atom and x is a positive integer.) Hexadimethrine halide represented by and has an average particle size of 10 μm or less,
Linseed oil absorption is 20~40ml/100g, BET specific surface area is 5000~ ^30000cm2 /g, specific resistance is ^1x1010
Add an adsorbent inorganic substance with an adsorption capacity of Ω・cm or less in a ratio of 0.0005 to 10 parts by weight: 1 part by weight of (a) and (b), and add the amount of (a) to blood. per 1ml amount
A method for separating blood serum and blood clots, characterized in that hexadimethrine halide and an adsorbent inorganic substance are present in the blood in a range of 0.005 mg to 5 mg. 3 In a method of separating blood into serum and blood clots by subjecting it to centrifugation, when blood in a blood test container contains the formula (a) (However, in the above formula, X is a halogen atom, x is a positive integer) Hexadimethrine halide and (b)
2,2',4,4'-tetrahydroxybenzophenone, the ratio of (a) and (b) being 0.0005 to 10 parts by weight:
Add within the range of 1 part by weight, and the amount of (a) added is in the range of 0.005 mg to 5 mg per 1 ml of blood volume,
Hexadimethrine halide and 2,2′ in the blood.
A method for separating serum and blood clots, characterized by the presence of 4,4'-tetrahydroxybenzophenone. 4 In a method of separating blood into serum and blood clots by subjecting it to centrifugation, when blood in a blood test container contains the formula (a) (However, in the above formula, X is a halogen atom; 1 part by weight, and the amount of (a) added is in the range of 0.005 mg to 5 mg per 1 ml of blood volume, so that hexadimethrine halide and ellagic acid are present in the blood. Characteristic method for separating serum and blood clots. 5 In a method of separating blood into serum and blood clots by subjecting it to centrifugation, when blood in a blood test container contains the formula (a) (However, in the above formula, X is a halogen atom, and x is a positive integer.) Hexadimethrine halide and (b) epicatechin expressed by
Part by weight: Add within the range of 1 part by weight, and (a)
A method for separating serum and blood clots, characterized in that the amount of hexadimethrine halide and epicatechin added is in the range of 0.005 mg to 5 mg per ml of blood volume, and hexadimethrine halide and epicatechin are allowed to exist in the blood.