JPH0126505B2 - - 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 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. Even with glass blood test containers, which are said to have the shortest blood clotting time, it takes 40 to 60 minutes for blood to coagulate after blood is injected. It needs to be left for 4 hours before solidifying. Another problem with the conventional method of separating serum and blood clots is that coagulated whole blood is phase-separated into serum and blood clots with different specific gravities by means such as centrifugation, and pure serum for use in testing is obtained. When collecting blood serum, the separability of serum is generally poor. For this reason, it was necessary to use nerves to avoid sucking up red blood cells when pipetting it up, and great care was required to avoid red blood cells from getting into the decantation process, but in most cases blood cells There were drawbacks such as the contamination of the product, which affected clinical test values, and the need for recentrifugation. It is an object of the present invention to eliminate such drawbacks, to provide a method for significantly shortening the time required for blood coagulation, and for satisfactorily separating serum components and blood clot components. The gist of the present invention is to provide a method for separating blood into serum and blood clots by subjecting blood to a centrifugal separation operation. The hydrophilic substance is a modified silicone oil or polyhydric alcohol into which a polar group has been introduced. The above-mentioned water-soluble substance is composed of one or more types selected from partially esterified products of water-soluble polymer compounds, and the above-mentioned adsorbent inorganic substance is glass, silica, etc. , kaolin, celite, or bentonite, and has a linseed oil absorption of 20 to 40 ml/100 g,
The BET specific surface area value is 5000 to 30000 cm ² /g and the specific resistance value is 1 × 10 ¹⁰ Ω・cm or less, and the amount used is a hydrophilic substance of 1 × 10 ^-10 to 1 × 10 ^-2 g, water-soluble 1 x 10 ^-10 to 1 x 10 ^-2 g of substance, and 1 adsorbent inorganic substance
The invention consists in a method for separating blood serum and blood clots in an amount of ×10 ^-6 to 1 ×10 ^-2 g (all per ml of blood). Next, the method of separating serum and blood clots of the present invention will be explained in more detail. In order to centrifuge the subject's whole blood sample to separate serum and blood clots, the blood is placed in a test container.The test container is made of thermoplastic resin, thermosetting resin, etc. Any of resin, modified natural resin, glass, etc. can be used. By adding a composition consisting of a hydrophilic substance that has blood clot-removing properties and is poorly soluble or insoluble in water, a water-soluble substance, and an adsorbent inorganic substance to the blood placed in the test container, The composition is present in the blood. Examples of hydrophilic substances that have blood clot-removing properties and are poorly soluble or insoluble in water include silicone oils such as dimethylpolysiloxane, methylhydrodienepolysiloxane, and methylphenylpolysiloxane with hydroxyl groups, amino groups, carboxyl groups, Modified silicone oils into which polar groups such as epoxy groups and polyether groups have been introduced and partially esterified products of polyhydric alcohols can be used. The most suitable one is silicone oil into which a polar group has been introduced. Although the hydrophilic substance is not a surfactant, its presence in the container has the effect of preventing blood clots from adhering to the inner wall surface and peeling off the blood clots that try to adhere from the inner wall surface. In addition, when the composition is added to blood on the surface of a carrier such as polystyrene beads, it is possible to prevent coagulated blood from adhering to the carrier surface and prevent coagulated blood from forming when subjected to centrifugation. It has the function of allowing easy separation into serum and blood clots. As the water-soluble substance, water-soluble polymer compounds can be used, and the polymer compounds include polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, sodium polyacrylate, polyethyleneimine, sodium alginate, starch, bullulan, methyl cellulose, hydroxyethyl cellulose. , hydroxypropyl cellulose, carboxymethyl cellulose, cellulose acetate phthalate, gum arabic, gum tragando, locust bean gum, guar gum, pectin, carrageenan, furcerelan, tamarind seed polysaccharide, glue, gelatin, casein, and the like. Most preferably, polyvinylpyrrolidone, polyethylene oxide, etc. are used. These water-soluble substances have the function of preventing the hydrophilic substance from covering the surface of the adsorbent inorganic substance and reducing the blood coagulation promoting effect of the adsorbent inorganic substance, and improving the contact between the adsorbent inorganic substance and blood. . Examples of adsorbent inorganic substances include water-insoluble inorganic fine powders selected from glass, silica, kaolin, celite, and bentonite. In addition, the adsorbent inorganic substance has a particle size of 50μ or less,
It is preferable to use particles with 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 fine powder, and since the surface area is related to the extent of surface pores possessed by the adsorbent inorganic material, the surface pore size is determined by the oil absorption amount and specific surface area. You can know the extent of The adsorptive inorganic substance in the present invention has a linseed oil absorption of 20 to 40 ml/
100 g and a BET specific surface area value of 5000 to 30000 cm ² /g. Linseed oil absorption indicates a value measured in accordance with 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 layer 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 layer. It refers to the value calculated by multiplying by the average cross-sectional area of , and nitrogen gas, oxygen gas, argon gas, methane gas, etc. are used as the adsorption 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 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 very large, factor factor, prekallikrein,
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 of 5000.
It is required to have a surface area in the range ~30000 cm ² /g. In addition, the specific resistance value of the adsorptive inorganic material in the present invention is required to be 1×10 ¹⁰ Ω・cm or less, and optimally 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 specific resistance value for adsorptive inorganic substances maintains the integrity of the potential distribution between the protein and adsorbable inorganic substances,
It is presumed that this contributes to preventing changes in protein conformation. Adsorbent inorganic substances have a blood coagulation-promoting effect and speed up the coagulation rate, but on the other hand, the presence of adsorbent inorganic substances makes it easy for blood clots to adhere to the inner wall of the container, and even when coagulated blood is centrifuged, serum and blood cannot be separated. Although there was a possibility that phase separation from the mochi would be difficult, the presence of the hydrophilic substance improves the adhesion of the blood clot brought about by the adsorbent inorganic substance. In order to make the composition consisting of the hydrophilic substance, water-soluble substance, and adsorptive inorganic substance exist in the blood, it may be added directly to the blood in the test container, for example, or it may be added directly to the blood in the test container. It may also be added to the blood by adhering it to a carrier. The amount of the hydrophilic substance and water-soluble substance present in the blood is 1×10 ⁻¹⁰ g/ml or more, and the amount of adsorbable inorganic substance is 1×10 ⁻⁶ g/ml or more. However, if present in too large a quantity, it may have an adverse effect on blood test values, so the content of any component should be 10 ^-2 g/ml or less. Furthermore, it is preferable that the total amount of each component is 1×10 ⁻² g/ml or less. According to the method of the present invention, blood coagulation factors are activated, the time required for blood coagulation is significantly shortened, serum and blood clots are easily separated, and residual fibrin and blood are removed from the separated and collected serum. The problem of contamination with blood clot components is also resolved, and furthermore, the contraction of blood clot components progresses sufficiently, resulting in an increase in serum yield. Example 1 Polystyrene bead carrier 1 with an average particle size of 1.5 mm
Per kg, 0.1 g of alcohol-modified polydimethylsiloxane is used as a hydrophilic substance that has blood clot-removing properties and is poorly soluble or insoluble in water, 0.1 g of polyvinylpyrrolidone as a water-soluble substance, and fine silica powder (linseed oil) as an adsorbent inorganic substance. Oil absorption 30ml/100g, BET
Specific surface area value 12000cm ² /g, specific resistance value 2.6×10 ⁴ Ω・
cm, average particle size 5.5 μm) dispersed in a small amount of methyl alcohol was added and stirred to obtain a granular coagulation accelerator with each of these components adhered to the surface of the carrier. The amount of each component attached per 1 g of the above granular coagulation accelerator is 1 x 10 ^-4 g of alcohol-modified polydimethylsiloxane and 1 x 10 ^-4 g of polyvinylpyrrolidone.
and 5×10 ⁻⁴ g of fine silica powder. Next, prepare (a) a 10 ml glass test container, (b) a 10 ml polyethylene test container, and (c) a 10 ml polymethyl methacrylate test container, and add 8 ml of fresh human blood to each test container. Immediately after blood was collected, 1 g of the above-mentioned granular coagulation accelerator was added to each test container, and the test container was left at 20°C. The time required until the whole blood stopped flowing completely was measured as the blood coagulation time. Immediately after blood coagulation, centrifugation was performed at a rotational speed of 3000 rpm for 5 minutes, and 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 results are shown in the column of Example 1 in Table 1. Example 2 Sorbitan tristearate as a hydrophilic substance that has blood clot removal properties and is poorly soluble or insoluble in water
1.0g, polyvinylpyrrolidone as water-soluble substance
0.01 g, 1.0 g of the same fine silica powder as in Example 1 was used as the adsorbent inorganic substance, and the average particle size was 1.5 using a small amount of isopropyl alcohol as a dispersion medium.
The mixture was added to 1 kg of a polystyrene bead carrier of 1.0 mm in diameter and stirred to obtain a granular coagulation accelerator with each of the above-mentioned components adhered to the carrier surface. The amount of each component attached per gram of the above granular coagulation accelerator is sorbitan tristearate 1×10 ^-3
g, polyvinylpyrrolidone 1×10 ⁻⁵ g, and silica fine powder 1×10 ⁻³ g. Next, (a) 10ml glass test container, (b) 10ml
Prepare a test container made of polyethylene (1) and (c) a test container made of polymethyl methacrylate (10 ml), collect 8 ml of fresh human blood into each test container, and immediately add the granular coagulation accelerator to test container 1. True 1g
The mixture was added in portions and left at 20°C. Next, blood coagulation time, serum separation state, and serum yield were further evaluated in the same manner as in Example 1. The results are shown in the column of Example 2 in Table 1. Comparative Example 1 In Example 1, no hydrophilic substance or water-soluble substance was used, and only a coagulation accelerator in which fine silica powder was adhered to a polystyrene bead carrier was used. 8 ml of fresh human blood was placed in a 10 ml polymethyl methacrylate blood test container, 1 g of the coagulation promoter was added, and the blood coagulation time, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in the Comparative Example 1 column of Table 1. Comparative Example 2 In Example 1, no hydrophilic substance was used, but a coagulation accelerator in which polyvinylpyrrolidone and fine silica powder were adhered to a polystyrene bead carrier was used. 8 ml of fresh human blood was placed in a 10 ml polymethyl methacrylate blood test container, 1 g of the coagulation promoter was added, and the blood coagulation time, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in the Comparative Example 2 column of Table 1. Comparative Example 3 In Example 1, fine silica powder was not used, and a coagulation accelerator in which each component of alcohol-modified polydimethylsiloxane and polyvinylpyrrolidone was attached to a polystyrene bead carrier was used. 8 ml of fresh human blood was placed in a 10 ml polymethyl methacrylate blood test container, 1 g of the coagulation promoter was added, and the blood coagulation time, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in the Comparative Example 3 column of Table 1.

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Claims (1)

[Claims] 1 In a method of separating blood into serum and blood clots by centrifuging blood, the blood in the container contains a hydrophilic substance that has blood clot-removing properties and is sparingly soluble or insoluble in water, and a water-soluble substance. The composition is made to exist by adding a composition consisting of a hydrophilic substance and an adsorbent inorganic substance, and the hydrophilic substance is made of a modified silicone oil into which a polar group has been introduced or a partial esterified product of a polyhydric alcohol. The water-soluble substance is composed of one or more selected water-soluble polymer compounds, and the adsorbent inorganic substance is glass, silica, kaolin, celite, or One or more types selected from bentonite, linseed oil absorption 20-40ml/100g, BET specific surface area value
5,000 to 30,000cm ² /g and a specific resistance value of 1× ¹⁰ Ω・cm or less, and the amount used is 1× of the hydrophilic substance.
10 ^-10 〜1×10 ⁻² g, water-soluble substance 1×10 ⁻¹⁰ 〜1×
10 ^-2 g, and 1 x 10 ^-6 to 1 x 10 ^-2 g of adsorbent inorganic matter.
(both per ml of blood) A method for separating serum and blood clots.