JPH0155416B2 - - 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 by coagulating the blood, 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 blood coagulation takes time 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 difficult 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 centrifuging blood, after collecting blood in a blood test container, the following formula is present in the blood: (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups, or ethyl groups, X is chlorine or bromine, and n is a positive integer.), and has an average molecular weight of 2000 to
350,000 polyamine sulfone, blood volume 10c.c.
A method for separating serum and blood clots, characterized by adding polyamine sulfone in the range of 0.005 mg to 5 mg per blood, to separate serum and blood clots. In the method of separating blood into a blood test container, after collecting blood into a blood test container, the formula (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups, or ethyl groups, X is chlorine or bromine, and n is a positive integer.), and has an average molecular weight of 2000 to
350,000 polyamine sulfone (a) and glass,
Selected from the group consisting of silica, kaolin, celite, and bentonite, with a particle size of 50 μm or less, an average particle size of 10 μm or less, and a linseed oil absorption of 20 to 40 ml/100 g, BET
An adsorbent inorganic substance (b) having a specific surface area value of 5000 to 30000 cm ² /g and a specific resistance value of 1×10 ¹⁰ Ω·cm or less, and the ratio of said (a) to said (b) is 0.0005 to 10% by weight. Department:
Polyamine sulfone and adsorptive inorganic substances are made to exist in blood by adding within the range of 1 part by weight, and by adjusting the amount of (a) added in the range of 0.005 mg to 5 mg per 10 c.c. of blood volume. Method 3 for separating serum and blood clots, characterized by In the formula (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups, or ethyl groups, X is chlorine or bromine, and n is a positive integer.), and has an average molecular weight of 2000 to
350,000 polyamine sulfone (a) and ellagic acid (b) in a ratio of 0.0003 to 6 parts by weight: 1 part by weight, and (stomach)
Addition amount of 0.005mg to 5mg per 10c.c. of blood volume
The present invention relates to a method for separating blood serum and blood clots, which is characterized in that polyamine sulfone and ellagic acid are present in 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, it takes time for the clot to clot if the plate is placed as is, but in the case of patients receiving artificial dialysis or patients with thrombosis, heparin is present in the blood because they are receiving heparin. Due to its presence, blood coagulation is less likely to occur. Therefore, in the present invention, after blood is collected into a test container, the polyamine sulfone in the blood is added. Polyamine sulfone is obtained by copolymerizing a diallylamine monomer and sulfur dioxide, and is represented by the following formula. (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups or ethyl groups, X is chlorine or bromine, and n is a positive integer.) In the present invention, the average molecular weight of the polyamine sulfone is 2000 to 35 A range of 10,000 is used. Polyamine sulfone is produced by combining a diallylamine monomer such as diallylamine, dimethallylamine, or a chlorinated or bromated derivative thereof, sulfur dioxide, water, methyl alcohol,
Dissolved in a solvent such as ethyl alcohol, dimethylformamide, dimethyl sulfoxide, etc., and using t-butyl hydroperoxide, azobisisobutyronitrile, ammonium persulfate, etc. as a polymerization catalyst,
It can be obtained by heating to about 30°C to carry out radical polymerization. Since catalyst fragments are attached to the ends of the polymer, for example, if t-butyl hydroperoxide is used as a catalyst, a t-butyloxy group will be attached to one end and a hydroxyl group will be attached to the other end. The optimal polyamine sulfone mentioned above is that R ₁ and R ₂ are hydrogen atoms, R ₃ and R ₄ are methyl groups, and
is a chlorine atom, and the average molecular weight is within the range of 2,000 to 350,000. When the polyamine sulfone added to the blood in the blood test container comes into contact with blood containing heparin, it quickly eliminates the effect of heparin and restores the normal coagulation function of the blood. Blood in a test container is coagulated within a short period of time, and after coagulation is completed, blood clot and serum are separated by means such as centrifugation, whereby serum can be easily collected. To explain this point in more detail, when normal blood comes into contact with the inner wall of the blood container, activation of factor Eventually, thrombin generated by activation of prothrombin acts on fibrinogen to form an insoluble fibrin network, and coagulation is completed. On the other hand, in blood to which heparin has been added, heparin acts cooperatively with antithrombin present in the blood, significantly inhibiting the action of thrombin. Heparin not only inhibits the action of thrombin but also
It is said to inhibit the effects of other coagulation factors, including factor XII. Therefore, by normal means, fibrinogen does not convert to fibrin in heparin-containing blood, and serum cannot be separated and collected because coagulation does not occur. It is thought that 1 to 10 units of heparin are present per 10 c.c. of blood in the blood of artificial dialysis patients or thrombosis patients receiving heparin administration. When polyamine sulfone is present in such heparin-containing blood, heparin is adsorbed and
Since it is removed from the blood, thrombin and other blood coagulation factors resume their normal functions. The appropriate amount of polyamine sulfone to add is blood volume 10
It ranges from 0.005mg to 5mg per cc. If the amount is less than this range, the effect of adsorbing heparin will be insufficient and sufficient blood coagulation will not be achieved. Moreover, if it exceeds this range, abnormal values may appear in clinical test values such as serum biochemical tests. One of the substances that synergistically exhibits a blood coagulation promoting effect when used in combination with polyamine sulfone 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. Furthermore, the adsorptive inorganic substance used has a particle size of 50 μm or less, and an average particle size of 10 μm or less. In particular, silica is an adsorbent inorganic material that is effective in shortening blood coagulation time.
Porous silica containing 20% by weight or more exhibits excellent effects. Such adsorbent inorganic substances promote the activation of blood coagulation factors when they come into contact with blood,
It also has the effect of promoting coagulation of platelets. 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 degree of surface area of the adsorbent inorganic material, and since the surface area is related to the degree of surface pores of the adsorbent inorganic material, the surface pores are determined by the oil absorption amount and specific surface area. You can know the extent of The adsorbent 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
The amount of gas adsorbed on the surface of an adsorbent inorganic material,
This is the value calculated by calculating the amount of gas that cuts through the surface as a monomolecular layer from the equilibrium pressure at that time and the saturated vapor pressure of the adsorbed gas, and multiplying this by the average cross-sectional area of the adsorbed gas molecules. As the gas, nitrogen gas, oxygen gas, argon gas, methane gas, etc. are used. 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 XII, a contact factor, is activated, but for this to occur, three substances, factor XII, prekallikrein, and high-molecular-weight kininogen, must form a complex and be adsorbed on the surface of the foreign object. It is said that adsorption in the absence of one or two of these will not lead to activation. By the way, when an adsorbent inorganic substance is used in the hope of promoting blood coagulation, if the surface area is extremely large, factor In other words, by increasing the adsorption rate of polymeric kininogen,
The rate of complex formation among the three components necessary for activation of factor XII will be reduced, 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 of 5000.
Those having a surface area in the range ˜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 polyamine sulfone used is within the range of 0.0005 to 10 parts by weight per 1 part by weight of adsorbent inorganic material. Another substance that exhibits a synergistic blood coagulation promoting effect when used in combination with polyamine sulfone is ellagic acid. Elazinic acid is a substance with the following chemical structural formula. Elazinic acid is known as a substance that activates factor XII, which is one of the blood coagulation factors, but when used in combination with polyamine sulfone, it has an even better effect on blood coagulation than when used alone. It was confirmed that it has a promoting effect. The proportion of polyamine sulfone used is within the range of 0.0003 to 6 parts by weight per 1 part by weight of ellagic acid. In order to make the polyamine sulfone or the inorganic substance ellagic acid adsorbable with it present in the blood, it may be added directly to the blood in the test container, for example,
A dispersion in a dispersion medium such as water may be dropped into the blood, or a carrier having a specific gravity of about 1.04 to 1.06 may be attached to the blood. In the present invention, polyamine sulfone or an inorganic substance adsorbable thereto, and ellagic acid, are added to the blood after blood is collected in a blood test container. Due to the presence of ellagic acid, it not only has an excellent blood coagulation promoting effect on the serum of normal healthy people, but also helps patients receiving heparin, such as patients undergoing artificial dialysis or patients with thrombosis. Therefore, even in the case of blood that is difficult to coagulate, it has an excellent blood coagulation promoting effect, and when centrifuged, serum and blood clots are easily separated, and serum can be obtained in a good yield. . Furthermore, since polyamine sulfone has excellent heat resistance, it can withstand sterilization using an autoclave and does not affect blood test values even after sterilization. Example 1 Polyamine sulfone (molecular weight
2000 to 5000), 0.2% by weight aqueous solutions were prepared. 5 ml of fresh human blood containing 1.5 units of heparin per ml of blood in a 10 ml glass spittoon
Immediately after the injection, 0.05 ml of the above polyamine sulfone aqueous solution was added, mixed gently, and then allowed to stand at 23°C. Thereafter, the time required until the whole blood stopped flowing completely was measured as blood coagulation time, and blood coagulability was evaluated. Also, immediately after blood coagulation, 3000
Centrifugation was performed for 5 minutes at a rotational speed of revolutions per minute, and the state of serum separation was observed. In addition, the state of separation was observed again 24 hours later, and the presence or absence of precipitation on fibrin was examined. The state of serum separation was evaluated by the presence or absence of residual blood clot adhesion on the wall of the container in the serum layer and the presence or absence of fibrin network precipitation. Also, add 0.05 ml of the above polyamine sulfone aqueous solution to a 10 ml glass spittoon, and add this to 121
Sterilize by autoclaving at ℃ for 20 minutes. 5 ml of the heparin-containing blood was injected into this, left to stand at 23°C, and blood coagulation, serum separation status, and presence or absence of fibrin precipitation after 24 hours were examined in the same manner as in Example 1, but without autoclave sterilization. There was no difference. Example 2 The blood coagulation time, serum separation state, and 24-hour test were carried out in the same manner as in Example 1, except that the polyamine sulfone represented by the formula in Example 1 had a molecular weight in the range of 170,000 to 230,000. The presence or absence of precipitation on the subsequent fibrin was examined. The results are shown in the column of Example 2 in Table 1. Example 3 Finely powdered silica (average particle size
5μm, linseed oil absorption 30ml/100g, BET specific surface area value 1.2×10 ⁴ cm ² /g, specific resistance value 2.6×10 ⁴ Ω・cm)
A dispersion of 2.0% by weight was prepared. In a polyethylene resin spittoon with a capacity of 10ml, 1
5 ml of fresh human blood containing heparin at a rate of 1.5 units per ml was collected, and immediately 0.05 ml of the polyamine sulfone aqueous solution in which silica was dispersed was added, mixed gently, and left at 23°C. In the same manner, blood coagulation time, serum separation status, and presence or absence of fibrin precipitation after 24 hours were examined. The results are shown in the column of Example 3 in Table 1. Example 4 A cellulose nonwoven fabric was impregnated with an aqueous solution of polyaminesulfone in which the finely powdered silica prepared in Example 3 was dispersed and dried to obtain a nonwoven fabric containing 0.1 mg of polyaminesulfone and 1 mg of finely powdered silica per 1 cm ² . In a polyethylene resin spittoon with a capacity of 10ml, 1
Collect 5 ml of fresh human blood containing heparin at a rate of 1.5 units per ml, immediately insert 1 cm ² of the nonwoven fabric,
The mixture was mixed gently and allowed to stand at 23°C, and the blood coagulability, serum separation state, and presence or absence of fibrin precipitation after 24 hours were examined in the same manner as in Example 1. Table 1 shows the results.
It is described in the column of Example 4. Example 5 A 2.0% by weight aqueous solution of polyamine sulfone having the same molecular weight of 2,000 to 5,000 as used in Example 1 was prepared by dispersing 2.0% by weight of ellagic acid. In a polyethylene resin spittoon with a capacity of 10ml, 1
Collect 5 ml of fresh human blood containing heparin at a rate of 1.5 units per ml, immediately add 0.05 ml of the polyamine sulfone aqueous solution in which ellagic acid is dispersed,
After gentle mixing, the mixture was left at 23° C., and the blood coagulation time, serum separation state, and presence or absence of fibrin precipitation after 24 hours were examined in the same manner as in Example 1. The results are shown in the column of Example 5 in Table 1. 【table】

Claims (1)

[Scope of Claims] 1. In a method of separating blood into serum and blood clots by centrifuging blood, after blood is collected in a blood test container, the following formula is present in the blood: (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups or ethyl groups, X is chlorine or bromine, and n is a positive integer.) Polyamine sulfone having an average molecular weight of 2,000 to 350,000 , per 10 c.c. of blood volume
By adding in the range of 0.005mg to 5mg,
A method for separating serum and blood clots, which is characterized by the presence of polyamine sulfone in blood. 2. In a method of separating blood into serum and blood clots by subjecting it to centrifugation, after blood is collected in a blood test container, the following formula is added to the blood: (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups or ethyl groups, X is chlorine or bromine, and n is a positive integer.) Polyamine sulfone having an average molecular weight of 2,000 to 350,000 (a), glass, silica,
It is selected from the group consisting of kaolin, celite, and bentonite, has a particle size of 50 μm or less, an average particle size of 10 μm or less, has a linseed oil absorption of 20 to 40 ml/100 g, and a BET specific surface area value of 5000 to 5000.
30000cm ² /g and an adsorbent inorganic substance (b) with a specific resistance value of 1×10 ¹⁰ Ω・cm or less, and the ratio of said (a) and said (b) is
0.0005 to 10 parts by weight: Add within the range of 1 part by weight, and the amount of (a) added is 0.005 per 10 c.c. of blood volume.
A method for separating blood serum and blood clots, characterized in that polyamine sulfone and an adsorbent inorganic substance are present in blood by controlling the amount of the polyamine sulfone to be in the range of 5 mg to 5 mg. 3. In a method of separating blood into serum and blood clots by subjecting it to centrifugation, after blood is collected into a blood test container, the following formula is added to the blood: (However, in the formula, R ₁ to R ₄ are hydrogen atoms, methyl groups or ethyl groups, X is chlorine or bromine, and n is a positive integer.) Polyamine sulfone with an average molecular weight of 2,000 to 350,000 (a) and ellagic acid (b) in a ratio of 0.0003 to 6 parts by weight: 1
The presence of polyamine sulfone and ellagic acid in the blood can be achieved by adding the polyamine sulfone and ellagic acid within the range of parts by weight, and by adjusting the amount of (a) added in the range of 0.005 mg to 5 mg per 10 c.c. of blood volume. Characteristic method for separating serum and blood clots.