JPS6367861B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a blood test container, and more particularly to a bottomed tubular container, a so-called Spitz, used to separate serum 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). Conventional blood test containers are made of glass and synthetic resins such as polystyrene, polymethyl methacrylate, and polyethylene, but these generally have the following drawbacks. One is that it takes a considerable amount of time for blood to coagulate after it is injected into a blood test container, making it impossible to carry out tests quickly, especially when tests need to be carried out urgently. has become a problem. Even glass blood test containers, which are said to have the shortest blood clotting time, require 40 to 60 minutes for blood to coagulate after being injected, and synthetic resin blood test containers... It takes 4 hours or more for the blood to coagulate. Another drawback of conventional blood test containers is that they require a method such as centrifugation to phase-separate coagulated whole blood into serum and blood clots with different specific gravities to collect pure serum for use in tests. , serum separability is generally poor. In other words, gel-like fibrin or blood clots tend to adhere firmly to the tube wall, which causes the problem of drastically reducing the amount of serum collected.Furthermore, fibrin tends to remain in the serum, which makes it difficult to perform serum biochemical tests. There were problems such as causing problems. Even glass blood test containers, which are said to have relatively good serum separation, frequently suffer from the above-mentioned problems when used at low temperatures of 15° C. or lower, especially during winter. In order to eliminate the above-mentioned drawbacks, the present inventors investigated the structure of a substance that has the effect of promoting blood coagulation, and in particular, in order to most effectively activate blood coagulation factors, the inner wall surface of a blood test container As a result of intensive research into the structure of substances that should be present in the container, we found that when surfactants and adsorbent inorganic substances are present on the inner wall of the container, the time required for blood coagulation can be significantly shortened, and serum components and blood clot components can be reduced. The present inventors have discovered that good separation can be achieved and have completed the present invention. The gist of the present invention is that on the inner wall surface of the container,
(1) Nonionic surfactant is 1×10 ^-10 to 1×10 ^-3
g/cm ² and (2) linseed oil absorption 20-40ml/100g,
It has a BET specific surface area value of 5000 to 30000 cm ² /g, a specific resistance value of 1×10 ¹⁰ Ω・cm or less, is selected from the group consisting of glass, silica, kaolin, celite, and bentonite, and has a particle size of 50 μ or less. Adsorptive inorganic material with an average particle size of 10μ or less is 1×10 ^-6 ~
1×10 ⁻³ g/cm ² of the blood test container. Next, the blood test container of the present invention will be explained in more detail. In the present invention, any of thermoplastic resins, thermosetting resins, modified natural resins, and glass can be used as the material for the blood test container, that is, the container. Examples of thermoplastic resins include polyethylene, polypropylene, poly-4-methylpentene-1, polystyrene, polymethyl methacrylate, polyvinyl chloride, polyethylene terephthalate, polybutylene terephthalate, styrene-acrylonitrile copolymer, and styrene-butadiene copolymer. , styrene-isoprene copolymer, styrene-maleic anhydride copolymer, styrene-acrylic acid copolymer, styrene-methyl methacrylate copolymer, ethylene-propylene copolymer, ethylene-acrylic acid copolymer, ethylene- Acrylic ester copolymers, polyvinyl alcohol acetals, polyvinyl alcohol butyrals, etc., and as thermosetting resins, for example, unsaturated polyester resins, epoxy resins, epoxy-acrylate resins, etc. are used. As the modified natural resin, cellulose acetate, cellulose propionate, cellulose acetate butyrate, ethyl cellulose, ethyl chitin, etc. are used. As the glass, silicate glasses such as soda lime glass, phosphosilicate glass, and borosilicate glass, and quartz glass are used. A surfactant and an adsorbent inorganic substance are made to exist on the inner wall surface of the container. As the surfactant, nonionic surfactants are used, and among them, polyglycerin fatty acid ester, sorbitol fatty acid ester, polyether-modified silicone oil, etc. are suitable, and more specifically, for example, stearic acid polyglyceride. , oleic acid polyglyceride, sorbitan monostearate, sorbitan monooleate, polyethylene glycol-modified silicone oil, polypropylene glycol-modified silicone oil, and the like. Adsorptive inorganic substances include inorganic substances used as adsorbents, such as glass, silica,
Water-insoluble inorganic fine powders such as kaolin, celite, and bentonite fall under this category. In addition, the adsorbent inorganic substance has a particle size of 50μ or less,
Those with an average particle size of 10μ or less are used. 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 exert its blood coagulation promoting effect, it is necessary 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. The linseed oil absorption amount indicates the value measured in accordance with Japanese Industrial Standard K-5101.
g on a glass plate (approximately 250 x 250 x 5 mm), drop linseed oil little by little into the center of the sample from a burette, and mix the whole thing thoroughly with a spatula each time, until the sample suddenly becomes soft with a drop of linseed oil. The end point is just before it sticks to the glass plate, and the amount of linseed oil used up to that point is determined, and the number of ml of linseed oil per 100 g of sample is taken as the oil absorption amount of linseed oil. BET specific surface area values are Brunauer−Emmett,
This value is obtained from the multilayer adsorption theory proposed by John Teller, and the theory is published in the Journal of
American Chemical Society 60・309 (1938);
59, 2682 (1937), etc.
The BET specific surface area value is determined by calculating the amount of gas that will penetrate the surface as a monomolecular layer from the amount of gas adsorbed on the surface of the adsorbent inorganic material, the equilibrium at that time, and the saturated vapor pressure of the adsorbed gas. 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 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, factor ,
The adsorption rate of high-molecular-weight kininogen will increase, in other words, the rate of formation of the three-part complex required for activation of factor XII will decrease, and the blood coagulation promoting effect will be abolished. Become.
Conversely, if the surface area of the adsorbent inorganic material is too small,
The probability of adsorption of coagulation factors decreases, and a blood coagulation promotion effect cannot be expected. For this reason, the adsorbent inorganic substance in the present invention has a linseed oil absorption capacity.
20~40ml/100g, BET specific surface area value is 5000~
It is necessary to have a surface area in the range of 30000 cm ² /g. Further, the specific resistance value of the adsorptive inorganic material in the present invention is 1×10 ¹⁰ Ω・cm or less, more preferably 5×10 ⁴ Ω・cm.
cm or less is used. The specific resistance value is the reciprocal of electrical conductivity, and is the value at room temperature. When blood comes into contact with a foreign object, proteins such as albumin, globulin, and various blood coagulation factors immediately adsorb to the surface of the foreign object prior to the blood coagulation phenomenon, and the conformation change of the protein molecules at that time causes a change in the conformation of the protein molecules.
They have various effects on the subsequent biochemical reactions. In particular, the activation mechanism of blood coagulation factors, which is an enzymatic reaction, is greatly affected, and in some cases, the coagulation function is impaired. In addition, platelets adhering to adsorbed globulin and albumin that have undergone a large conformation change undergo abnormal melting and deformation, causing a phenomenon in which polymerized and precipitated fibrin chains strongly adhere to adsorbent inorganic materials. If the latter phenomenon occurs in a container intended for serum collection, even if centrifugation is performed, blood clots and serum will not be separated, making it impossible to achieve the purpose. Changes in protein conformation occur as a result of various interactions between adsorbent inorganics and proteins, such as hydrophobic interactions, hydrogen bonding interactions, and electrostatic interactions. This can be alleviated by using an adsorptive inorganic substance with relatively high conductivity. In other words, the polar groups of proteins induce a dipole moment group in the adsorbent inorganic substance with a corresponding distribution, but if the adsorbent inorganic substance is nonconductive, it is conductive The response becomes worse than that of the protein adsorbed on the surface, and the potential distribution of the protein adsorbed on the surface and the potential distribution of the adsorbing inorganic substance are inconsistent with each other, which causes local and non-uniform distortion of the protein. This leads to changes in conformation. Therefore, it is necessary for the adsorptive inorganic substance to have conductivity in order to maintain consistency in potential distribution between the protein and the adsorbent inorganic substance and to prevent changes in protein conformation. For this reason, the adsorptive inorganic substance in the present invention has a specific resistance value of 1×10 ¹⁰ Ω·cm or less. Adsorbable inorganic substances have a blood coagulation promoting effect by activating blood coagulation factors. However, when an adsorbent inorganic substance is present alone on the inner wall of the container, although the rate of blood coagulation is accelerated,
It also has the function of causing the blood produced by coagulation to adhere to the inner wall of the container, and even when subjected to centrifugation, it may be difficult to separate the coagulated blood into serum and blood clots. Red blood cells may be destroyed by the strong shear stress generated between them and the adsorbent inorganic material and dissolved into the serum. However, if a surfactant is present along with an adsorbent inorganic substance on the inner wall of the container, it is possible to prevent blood clots from adhering to the inner wall of the container and prevent hemolysis into serum during centrifugation. The amount of surfactant present on the inner wall of the container is 1×10 ^-10 to 1×10 ^-3 g/cm ²
It is preferable that If the amount of surfactant present on the inner wall of ^the container is less than 1×10 ^-10 g/cm ² , the prevention of blood clot adhesion to the inner wall of the container will not be sufficient; If it is larger than cm ² , there is a risk that the surfactant will be mixed into the serum and interfere with the serum test. Further, the amount of the adsorbent inorganic substance present on the inner wall surface of the container is preferably 1×10 ⁻⁶ to 1×10 ⁻³ g/cm ² . If the amount of adsorbable inorganic substances present on the inner wall surface of the container is less than 1×10 ^-6 g ^/ cm ² , sufficient activation effect on blood coagulation factors cannot be obtained; If the amount is more than ² , there is a risk that adsorbent inorganic substances may be mixed into the serum and inhibit the serum test. To obtain such a blood test container, for example, a surfactant and an adsorbent inorganic substance are dissolved and dispersed in a suitable binder or solvent and then sprayed or immersed on the inner wall of a prepared container. Just apply it. If the container is made of plastic, a surfactant is mixed in advance with the plastic as a molding material, and this is then molded using appropriate methods such as injection molding, blow molding, compression molding, transfer molding, vacuum molding, and casting molding. A container may be formed by a method and an adsorbent inorganic material dispersed in a suitable binder or solvent may be spray coated or dip coated thereon. In the blood test container thus obtained, blood coagulation factors are activated rapidly, the time required for blood coagulation is significantly shortened, and serum and blood clot can be easily separated. However, in many biochemical tests, only serum is used, and the serum is drawn out with a pipette after centrifugation using a blood test container, but the preparative separation operation is time-consuming. ,
It is not suitable for transportation as it is. For this purpose, a partition forming material may be present within the container. The septum-forming agent is a composition that has thixotropic properties and forms a septum by being positioned between serum and blood clots by centrifugation, and forms a septum so that the serum can be separated by decantation. It is a substance that forms As the thixotropic agent, inorganic powders such as silica, alumina, glass, talc, bentonite, titania, zirconium, asbestos, and carbon black, and organic powders such as styrene resins, acrylic resins, and vinyl chloride resins are preferably used. It will be done. Among these thixotropic agents, fine silica powder shows the best results. Silica fine powder is a fine powder whose main component is silicic acid anhydride, which has been subjected to hydrophobization treatment by grafting reaction or coupling reaction as necessary. I don't care if there is. The average particle size of these thixotropic agents is preferably 1 mμ to 100μ. 1
If it is smaller than mμ, it is difficult to handle, and when mixed with the viscous liquid described later, it tends to aggregate and form secondary particles, making uniform dispersion difficult. This is because the dispersion stability of the partition wall forming agent is poor and the partition forming agent as a whole lacks uniform fluidity. A viscous liquid is used to make the diaphragm-forming agent thixotropic by means of the thixotropic agent. The viscous liquid may have a strong interaction with the thixotropic agent, or may not have a strong interaction with the thixotropic agent, or may have good compatibility. Both may be used in combination. Having a strong interaction with a thixotropic agent is defined as mixing a certain thixotropic agent with a certain viscous liquid and uniformly dispersing it, then using a centrifugal separator with an arm length of 10 cm at a rotation speed of 4000 r.p.m. This refers to a case where no bias is observed in the distribution of the components of the mixture even after centrifugation for 30 minutes. The cause of such interaction is still not clear, but between materials with hydrophilic groups, it is mainly due to hydrogen bonding, and between materials without hydrophilic groups, it is due to cohesive force caused by the molecular structure. It is assumed that Viscous liquids that have a strong interaction with thixotropic agents include acid-modified products of liquid polymeric substances such as acrylic resin oligomers, polyester oligomers, liquid polyisoprene, liquid polybutene, and liquid polybutadiene, especially maleic acid-modified products, and Examples include animal and vegetable oils such as soybean oil, linseed oil, safflower oil, and fish oil, acid-modified products of the animal and vegetable oils, liquid polymer substances such as liquid polybutene and liquid polybutadiene, and epoxy-modified products of the above-mentioned animal and vegetable oils, which impart thixotropic properties. When the agent is an organic powder, examples thereof include oligomers of the same type as the thixotropic agent, such as styrene oligomers for polystyrene, and preferably have a viscosity of 1000 cps or more. When such a viscous liquid is used, the thixotropic agent and the viscous liquid will not separate. Viscous liquids that do not have strong interactions with thixotropic agents can also be used in the presence of water-insoluble amine compounds in the present invention. Examples of such viscous liquids include liquid polymer substances such as liquid paraffin, liquid polyisoprene, liquid polybutene, and liquid polybutadiene, styrene oligomers, and chlorinated products thereof; when the thixotropic agent is a styrene resin, Examples include liquid paraffin and its chlorinated products, and both preferably have a viscosity of 1000 cps or more. Furthermore, it is also possible to use a mixture of a viscous liquid that has a strong interaction with the thixotropic agent and a viscous liquid that has good compatibility therewith and does not have a strong interaction with the thixotropic agent. Good compatibility in this case means that no phase separation occurs even if both viscous liquids are mixed, uniformly dispersed, and then left at room temperature for one week. When using a mixture of a viscous liquid that has a strong interaction with the thixotropic agent and a viscous liquid that has good compatibility with the viscous liquid and does not have a strong interaction with the thixotropic agent, Excellent viscosity stability can be achieved. In this case, the mixing ratio of both viscous liquids is set within a suitable range taking into account the strength of their interaction with the thixotropic agent, but generally they have a strong interaction with the thixotropic agent. Per 100 parts by weight of the viscous liquid, 10 to 10 parts of the viscous liquid that does not have strong interaction with the thixotropic agent
200 parts by weight are used. However, the stability of the viscosity over time is significantly improved by using a water-insoluble amine compound. As water-insoluble amine compounds, those having one or more alkyl groups having 8 or more carbon atoms in the molecule are suitable, such as dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, dodecyldimethylamine, and tetradecyldimethylamine. , octadecyldimethylamine, polyoxyethylene octadecylamine, trioctylamine, etc. The above-mentioned amine compound is used because it has the property of being easily adsorbed on the surface of the thixotropic agent, and has an interaction with both the thixotropic agent and the viscous liquid, which reduces the viscosity over time. By working on the stability of In particular, amines having an alkyl group having 8 or more carbon atoms are suitable not only because they are highly water-insoluble and do not dissolve in separated serum or plasma, but also because they are suitable for use as thixotropic agents. This is because it is assumed that the long chain alkyl group of the amine compound adsorbed on the surface has a function of stabilizing the interaction between the thixotropic agents. By using the amine compound,
The stability of viscosity over time is significantly improved, and as a result, a composition for serum or plasma separation with excellent centrifugal separability and stability of partition walls can be obtained. The ratio of the thixotropic agent to the viscous liquid is 2 to 15 parts by weight of the thixotropic agent per 100 parts by weight of the viscous liquid, and if a water-insoluble amine compound is used, it is 0.02 to 5 parts by weight. It is preferable that there be one. The specific gravity of the partition wall forming agent is set to 1.03 to 1.08. This is because to form a septum, the specific gravity needs to be intermediate between that of serum and blood clot. specific gravity
1.03 to 1.08 is the specific gravity at room temperature, and the standard value is 20°C. When such a septum-forming agent is used, the serum and blood clot are separated by centrifugation, and a septum is formed at the interface between the serum and the blood clot, and once formed, the septum is stable and does not protect the container. Since it does not collapse even when tilted, serum can be easily separated by decantation or the like. Examples of the present invention are described below. Example 1 Per 100 parts by weight of polymethyl methacrylate,
Polyethylene glycol modified silicone oil 7
By injection molding the molding material with added weight part, the outer diameter is 15
A container with mmφ, inner diameter of 13 mmφ, and height of 100 mm was obtained. Apart from this, fine powdered silica (average particle size 4.0 μm,
Linseed oil absorption 30ml/100g, BET specific surface area value
^{A Freon} dispersion containing 0.2 wt. The amount of polyethylene glycol-modified silicone oil present on the inner wall of the container is 1×10 ^-5 g/cm ² ,
The amount of silica present was 1×10 ^-4 g/cm ² . 8 ml of fresh human blood in the blood test container obtained in this way.
After injecting the sample, the sample was left at 20°C, and the time required for the whole blood to stop flowing completely was measured as the blood coagulation time, and blood coagulability was evaluated. Immediately after blood coagulation, centrifugation was performed at a rotational speed of 3000 rpm for 5 minutes, 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. As is clear from the results in the Example 1 column of Table 1, blood coagulation was extremely rapid in the obtained blood test container, and the serum separation state was also good. Example 2 A blood test container was obtained in the same manner as in Example 1 except that polypropylene was used instead of polymethyl methacrylate. Next, using this blood test container, blood coagulability, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in the column of Example 2 in Table 1. Example 3 A 10ml glass blood test container was prepared, and 0.1% by weight of sorbitan monooleate and 1.0% by weight of the same finely powdered silica as used in Example 1 were added to it.
An isopropyl alcohol dispersion containing dissolved and dispersed was applied. The amount of sorbitan monooleate present is 1×10 ^-5 g/cm ² and the amount of silica present is 1
×10 ^-4 g/cm ² . Next, using this blood test container, blood coagulability, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in the column of Example 2 in Table 1. As is clear from these results, blood coagulation in the obtained blood test container was extremely rapid, and the serum separation state was also good. Example 4 70 parts by weight of chlorinated polybutene with a specific gravity of 1.02 at 20°C and a viscosity of 10000 cps, with a specific gravity of 1.02 at 20°C.
21 parts by weight of epoxidized soybean oil with a viscosity of 1.0 and 1700 cps, 9 parts by weight of the same finely powdered silica as in Example 1, and 0.2 parts by weight of trioctylamine were kneaded in a three-roll kneader, and then mixed at 20°C. A partition forming agent with a specific gravity of 1.06 was obtained. 1 g of this partition forming agent was poured into a container obtained in the same manner as in Example 1. Using the blood test container thus obtained, blood coagulation, serum separation state, and serum yield were evaluated in the same manner as in Example 1. The results are shown in Table 1 for Example 3.
Shown in the column. Incidentally, the serum was collected by decantation, and the serum could be taken out without collapsing the septum. Comparative Example 1 Using a 10 ml polypropylene container, the blood coagulability, 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. As shown in these results, the blood coagulation time was long, the serum separation state was poor, and the serum yield was low. 【table】

Claims (1)

[Claims] 1. On the inner wall of the container, (1) nonionic surfactant
×10 ^-10 to 1 × 10 ^-3 g/cm ² and (2) linseed oil absorption
20~40ml/100g, BET specific surface area value is 5000~
30000cm ² /g, specific resistance value is 1×10 ¹⁰ Ω・cm or less, selected from the group consisting of glass, silica, kaolin, celite, and bentonite, and has a particle size of
1. A blood test container, characterized in that an adsorbent inorganic substance having a particle size of 50 μm or less and an average particle size of 10 μm or less is present in an amount of 1×10 ⁻⁶ to 1×10 ⁻³ g/cm ² .