JPH0244029B2 - - 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 of separating blood into serum and blood clots by subjecting it to centrifugation, after collecting blood in a blood test container, the blood has an anion exchange capacity. , a water-insoluble polymer having at least one group selected from the group consisting of a quaternary ammonium group, a tertiary amino group, a secondary amino group, and a primary amino group, and has a total ion exchange capacity of per gram of ion exchange resin, anion exchange resin in the range of 2.5 to 6.0 milliion equivalents per 10 c.c. of blood volume.
Method 2 for separating serum and blood clots, characterized by adding the anion exchange resin in the blood in an amount of 500 mg. Blood is centrifuged to separate serum and blood clots. In this method, after blood is collected in a blood test container, (a) quaternary ammonium groups, tertiary amino groups, secondary amino groups and It consists of a water-insoluble polymer having at least one group selected from the group consisting of primary amino groups, and has a total ion exchange capacity in the range of 2.5 to 6.0 milliion equivalents per gram of dry ion exchange resin. Anion exchange resin and (b) selected from the group consisting of glass, 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 μm. ~40ml/100g, BET specific surface area value is ~5000
30000cm ² /g, with a specific resistance value of 1×10 ¹⁰ Ω・cm or less, in which the ratio of (a) and (b) is 1.
Part by weight: Add within the range of 0.001 to 1.0 part by weight, and the amount of (a) added is 5 mg per 10 c.c. of blood volume.
Method 3 for separating serum from blood clots, characterized in that the anion exchange resin and the adsorbent inorganic substance are present in the blood, and the amount ranges from 500 mg to 500 mg. In this method, after blood is collected in a blood test container, (a) quaternary ammonium groups, tertiary amino groups, secondary It consists of a water-insoluble polymer having at least one group selected from the group consisting of amino groups and primary amino groups, and has a total ion exchange capacity of 2.5 to 6.0 milliion equivalents per gram of dry ion exchange resin. An anion exchange resin and (b) 2,2',4,4'-tetrahydroxybenzophenone in the range of (a) and (b) in a ratio of 1 part by weight: 0.0005 to 0.5 part by weight. The anion exchange resin and the 2,2',4,4'- Method 4 for separating serum and blood clots characterized by the presence of tetrahydroxybenzophenone In a method for separating blood into serum and blood clots by subjecting blood to centrifugation, (a) At least one selected from the group consisting of a quaternary ammonium group, a tertiary amino group, a secondary amino group, and a primary amino group, which has anion exchange ability. an anion exchange resin consisting of a water-insoluble polymer having one group, with a total ion exchange capacity in the range of 2.5 to 6.0 milliion equivalents per gram of dry ion exchange resin, and (b) ellagic acid; (a) and (b)
The ratio of (a) is 1 part by weight: 0.0005 to 0.5 part by weight, and the amount of (a) added is 1 part by weight.
A method for separating serum from blood clots 5, characterized in that the anion exchange resin and the ellagic acid are present in the blood in a range of 5 mg to 500 mg per cc. Blood is subjected to centrifugation. In the method of separating serum and blood clots, after blood is collected in a blood test container, (a) quaternary ammonium groups, tertiary amino groups, which have anion exchange ability, It consists of a water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group, and has a total ion exchange capacity of 2.5 to 6.0 per gram of dry ion exchange resin. An anion exchange resin and (b) epicatechin in the range of million ion equivalents are added to (a) and
The ratio of (b) is 1 part by weight: 0.0005 to 0.5 parts by weight, and the amount of (b) added is adjusted to the blood volume.
The present invention relates to a method for separating serum and blood clots, characterized in that the anion exchange resin and the epicatechin are present in the blood at doses ranging from 5 mg to 500 mg per 10 c.c. 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 and its effect is due to heparin administration. Less likely to cause blood clotting. Therefore, in the present invention, after blood is collected into a test container, an anion exchange resin is added to the blood. As the anion exchange resin, quaternary ammonium groups, tertiary amino groups, which have anion exchange ability,
A water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group is used. Examples of the anion exchange resin having a quaternary ammonium group include a modified styrene/divinylbenzene crosslinked copolymer having a trimethylbenzylammonium side chain or a dimethylhydroxyethylbenzylammonium side chain, and a modified product having an N-methylpyridine side chain. Modified copolymers of vinylpyridine and styrene or methyl methacrylate, cellulose derivatives having triethylaminoethyl side chains, N-triethyl chitosan, and the like are used. In these anion exchange resins having a quaternary ammonium group,
Since the class ammonium group exhibits cationic properties, it always bonds with a counter ion, which is an anion. As counter ions, halogen ions such as F ^- , Cl ^- , Br ^- , I ^-, etc.
or OH ^- . Examples of anion exchange resins having a tertiary amino group include modified styrene/divinylbenzene crosslinked copolymers having dimethylbenzylamine side chains, acrylamide and divinylbenzene having N-3-dimethylaminotrimethyleneamide side chains; Crosslinked copolymers, copolymers of vinyl pyridine and styrene or methyl methacrylate, cellulose derivatives having diethylaminoethyl side chains, N-diethyl chitosan, and the like are used. As the anion exchange resin having a secondary amino group, for example, a modified styrene/divinylbenzene crosslinked copolymer having a methylbenzylamine side chain, monoethylaminoethyl cellulose, etc. are used. Examples of the anion exchange resin having a primary amino group include a modified styrene/divinylbenzene crosslinked copolymer having a benzylamine side chain, aminoethyl cellulose, P-aminobenzyl cellulose,
Chitosan etc. are used. The anion exchange resin used in the present invention may also have a mixed type of primary, secondary, and tertiary amine exchange group. Examples of such materials include modified styrene-divinylbenzene crosslinked copolymers having N-diethylaminobenzylamine side chains, guanidoethylcellulose, and the like. The anion exchange resin used in the present invention has a total ion exchange capacity of 2.5 to 6.0 milliion equivalents per gram of dry ion exchange resin, and particularly preferably 3.5 to 4.5 milliion equivalents. It has a quaternary ammonium group in the million ion equivalent range. Furthermore, it has been found that when a bromide ion is present as a counter ion to a quaternary ammonium group, more stable performance can be obtained than when other types of counter ions, such as chloride ions, are used. In other words, it was found that bromide ion as a counter ion has the effect of suppressing the destructive effect of ammonium groups on blood components, such as hemolytic effect, and does not impair the effect of efficiently supplementing heparin in the blood. When the anion exchange resin added to the blood in the blood test container comes into contact with blood containing heparin, it quickly eliminates the action of heparin and restores the normal coagulation function of the blood. Serum can be easily collected by coagulating blood in a blood test container within a short period of time and, after completion of coagulation, separating serum and blood clot by means such as centrifugation. 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 and significantly inhibits the action of thrombin. Heparin not only inhibits the action of thrombin but also
It is said to inhibit the effects of coagulation factor and other coagulation factors. Therefore, in normal means, fibrinogen does not convert to fibrin in heparin-containing blood, and since coagulation does not occur, serum cannot be separated and collected. 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 an anion exchange resin is present in such heparin-containing blood, heparin is adsorbed and removed from the blood, so that thrombin and other blood coagulation factors regain their normal functions. The appropriate amount of anion exchange resin added is 5 per 10c.c.
It ranges from mg to 500mg. If the amount is less than this range, the heparin adsorption effect will be insufficient,
Insufficient blood coagulation occurs. Moreover, if it exceeds this range, abnormal values may appear in clinical test values such as serum biochemical tests. The anion exchange resin in the present invention can be in any shape, including granules, sheets, and films. In the case of granular materials, those having a particle size of 20 microns to 5 mm can be used. In the above case, when the anion exchange resin is present 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 an anion exchange resin together 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 an anion exchange resin 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. Further, the adsorptive inorganic substance used has a particle size of 50 μm or less, and an average particle size of 10 μm or less.
An adsorbent inorganic material that is particularly effective in shortening blood coagulation time is silica, and porous silica containing 20% by weight or more of amorphous components is particularly effective. 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 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 mg,
Those having a BET specific surface area value of 5,000 to 30,000 cm ² /g are used. Linseed oil absorption is Japanese Industrial Standard K
−5101. The BET specific surface area value is calculated by calculating the amount of gas that covers the surface as a monomolecular weight from the amount of gas adsorbed on the surface of the adsorbent inorganic material, the equilibrium pressure at that time, and the saturated vapor pressure of the adsorbed gas, and then calculating the amount of gas that covers the surface as a monomolecular weight. It refers to the value calculated by multiplying the average cross-sectional area, and nitrogen gas, oxygen gas, argon gas, methane gas, etc. are used as the adsorption gas. And according to this method,
Surface area values including pores that cannot be measured by measuring linseed oil absorption are measured. During blood coagulation, factor XII, or contact factor, is activated, and for this purpose, three substances, factor XII, prekallikrein, and polymeric kininogen, form a complex and are adsorbed on the surface of the foreign object. 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 in the hope of promoting blood coagulation, if the surface area is extremely large,
The adsorption rate of factor The rate of complex formation 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 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 ratio of the anion exchange resin and adsorbent inorganic substance used is within the range of 0.001 to 1.0 parts by weight of the adsorbent inorganic substance per 1 part by weight of the anion exchange resin. Other substances that synergistically exhibit a blood coagulation promoting effect when used in combination with anion exchange resins are 2, 2', 4,
4'-tetrahydroxybenzophenone. 2,2',4,4'-tetrahydroxybenzophenone has the following chemical structure. 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 anion exchange resin and 2,2',4,4'-tetrahydroxybenzophenone is 2,2',4,4'-tetrahydroxybenzophenone per 1 part by weight of anion exchange resin. It is within the range of 0.0005 to 0.5 part by weight. Another substance that exhibits a synergistic blood coagulation promoting effect when used in combination with an anion exchange resin is ellagic acid. Elazinic acid is a substance with the following chemical structural formula. Elazinic acid is one of the blood clotting factors, factor XII.
Although it is known as a substance that activates factors, it was confirmed that when used in combination with an anion exchange resin, it has a more excellent blood coagulation promoting effect than when used alone. The ratio of ellagic acid to the anion exchange resin is 0.0005 to 0.5 per 1 part by weight of the anion exchange resin.
It is considered to be within the range of parts by weight. Yet another substance that synergistically exhibits a blood coagulation promoting effect when used in combination with an anion exchange resin 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 the anion exchange resin and epicatechin used is within the range of 0.0005 to 0.5 parts by weight of epicatechin per 1 part by weight of the anion exchange resin. anion exchange resin or an adsorbent inorganic substance,
In order to make 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin present in the blood, they may be added directly to the blood in a test container, or they may be added to water, etc. You can drop it into the blood by dispersing it in a dispersion medium of
It may be attached to a carrier of about 1.06 and added to blood. In the present invention, an anion exchange resin or an inorganic substance adsorbable thereto, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin are added to blood in a blood test container. Due to the presence of anion exchange resins or inorganic substances adsorbable with them, 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin in the blood, serum levels of normal healthy people increase. Not only does it have an excellent effect on promoting blood coagulation, but it also has an excellent effect on patients undergoing artificial dialysis and patients with thrombosis.
Even in the case of blood that is difficult to coagulate due to heparin administration, it has an excellent blood coagulation promoting effect, and when centrifuged, serum and blood clots are easily separated, and serum Obtained in good yield. Example 1 Bottomed outer diameter 15 m/m, inner diameter 13 m/m, height 100
A glass tube of m/m was prepared. In addition, 5 g of an anion exchange resin in the form of 400 mesh particles, which is a modified styrene/divinylbenzene crosslinked copolymer having trimethylbenzylammonium side chains, was dispersed in 25 ml of physiological saline. The anion exchange resin used here was a chlorine salt type having chlorine ions as counter ions, and had an ion exchange capacity of 3.5 milliequivalents per 1 g of dry weight. After injecting 5 ml of fresh human blood containing 5 units of heparin per 5 ml of blood into a glass test container, immediately add 0.25 ml of anion exchange resin dispersion and leave it at 20°C to complete whole blood. The time required for the blood to stop flowing is measured as the blood coagulation time.
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 using the method of the present invention. In addition, in order to investigate whether the presence of the anion exchange resin has an effect on blood test values,
Serum biochemical tests using the serum obtained above (28
Item) was implemented. A significant difference test was performed on the test values between the case where the anion exchange resin was not used and the above case (n=5), and the presence or absence of a difference at a significance level of 5% was tested. As a result, it was confirmed that the test values were not affected at all. Example 2 In Example 1, in addition to the anion exchange resin, natural amorphous assilicic acid fine powder (average particle size 5 μm, linseed oil absorption 30 ml/100 g,
BET specific surface area value 1.2×10 ⁴ cm ² /g, specific resistance value 2.6×
10 ⁴ Ω・cm), 4 g of anion exchange resin,
Blood coagulation, serum separation state, and serum yield were conducted in the same manner as in Example 1, except that 0.25 ml of a dispersion of 1 g of adsorbent inorganic material dispersed in physiological saline was added to 5 ml of heparin-containing blood in a polyethylene test container. was 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 In place of the adsorbent inorganic substance in Example 2, 2,2',4,4'-tetrahydroxybenzophenone (ex. Example 3), ellagic acid (Example 4), and epicatechin (Example 5) were used. The amounts used in these cases were 4 g of anion exchange resin, 0.8 g of 2,2',4,4'-tetrahydroxybenzophenone, 0.2 g of ellagic acid, and 0.8 g of epicatechin. Next, in the same manner as in Example 1, the blood coagulability, serum separation state, and serum yield of the heparin-containing blood in the polyethylene test container were evaluated. The results are shown in the columns of Examples 3-5. When 2,2',4,4'-tetrahydroxybenzophenone, ellagic acid, and epicatechin are used in combination with the anion exchange resin, they significantly promote coagulation of heparin-containing blood and also improve serum separation. 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 anion exchange resin, blood coagulation was not completed and serum separation was impossible.

[Table] Example 1 Not completed

Claims (1)

[Scope of Claims] 1. 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 blood has an anion exchange capacity. , quaternary ammonium group, tertiary amino group, 2
The water-insoluble polymer has at least one group selected from the group consisting of primary amino groups and primary amino groups, and has a total ion exchange capacity of 2.5 to 6.0 per gram of dry ion exchange resin.
Serum, characterized in that the anion exchange resin is present in the blood by adding an anion exchange resin in the range of milliion equivalents in the range of 5 mg to 500 mg per 10 c.c. of blood volume. and how to separate it from blood clots. 2. In a method of separating blood into serum and blood clots by subjecting it to a centrifugal separation operation, after blood is collected into a blood test container, (a) class ammonium group, tertiary amino group,
It consists of a water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group, and has a total ion exchange capacity of 2.5 to 1 gram per gram of dry ion exchange resin.
anion exchange resin in the range of 6.0 milliion equivalents and (b) selected from the group consisting of glass, silica, kaolin, celite and bentonite, with a particle size of 50μ
m or less, the average particle size is 10 μm or less, the linseed oil absorption is 20 to 40 ml/100 g, BET
Specific surface area value is 5000-30000cm ² /g, specific resistance value is 1
×10 Adding an adsorbent inorganic substance having a resistance of ¹⁰ Ω・cm or less in a ratio of (a) and (b) within the range of 1 part by weight: 0.001 to 1.0 part by weight, and addition of (a) Blood volume 10c.c.
A method for separating serum from blood clots, characterized in that the anion exchange resin and the adsorbent inorganic substance are present in the blood in a range of 5 mg to 500 mg per blood clot. 3. In a method of separating blood into serum and blood clots by centrifuging blood, after collecting blood in a blood test container, (a) a class ammonium group, tertiary amino group,
It consists of a water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group, and has a total ion exchange capacity of 2.5 to 1 gram per gram of dry ion exchange resin.
An anion exchange resin in the range of 6.0 million ion equivalents and (b) 2,2',4,4'-tetrahydroxybenzophenone, the ratio of (a) and (b) being 1 part by weight: 0.0005
The anion exchange resin and the 2,
A method for separating blood serum and blood clots, characterized in that 2',4,4'-tetrahydroxybenzophenone is present. 4. 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, (a) class ammonium group, tertiary amino group,
It consists of a water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group, and has a total ion exchange capacity of 2.5 to 1 gram per gram of dry ion exchange resin.
An anion exchange resin and (b) ellagic acid having a range of 6.0 million ion equivalents are added in a ratio of (a) and (b) within the range of 1 part by weight: 0.0005 to 0.5 part by weight, and ( Addition amount of b) is 5 mg or more per 10 c.c. of blood volume.
A method for separating serum and blood clots, characterized in that the anion exchange resin and the ellagic acid are present in the blood in an amount in the range of 500 mg. 5. 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, (a) class ammonium group, tertiary amino group,
It consists of a water-insoluble polymer having at least one group selected from the group consisting of a secondary amino group and a primary amino group, and has a total ion exchange capacity of 2.5 to 1 gram per gram of dry ion exchange resin.
An anion exchange resin and (b) epicatechin having a range of 6.0 million ion equivalents are added in a ratio of (a) and (b) within a range of 1 part by weight: 0.0005 to 0.5 part by weight, and ( Addition amount of b) is 5 mg or more per 10 c.c. of blood volume.
A method for separating serum and blood clots, characterized in that the anion exchange resin and the epicatechin are present in the blood in a range of 500 mg.