JPH0249099B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a blood test container, and particularly to a vacuum blood collection tube used for collecting a whole blood sample from a subject and separating serum by centrifugation. (Prior Art) With the remarkable progress in testing technology, blood tests such as serum biochemical tests, serum immunological tests, and blood cell tests have become widely used and are useful for disease prevention and early diagnosis. Most blood tests are serum tests, and the serum required for the test is usually collected in a blood test container, coagulated, and then centrifuged.
It is separated from blood clots (gel-like masses mixed with fibrin and blood cells) that have different specific gravity. Blood has traditionally been collected from subjects using a syringe, but recently a vacuum blood collection tube has been used to collect blood. To collect blood using a vacuum blood collection tube, blood is collected into the vacuum blood collection tube under reduced pressure via a dedicated blood collection holder. Vacuum blood collection tubes are made of glass and synthetic resins such as polymethyl methacrylate.
However, even if blood is collected into vacuum blood collection tubes made of these materials, it takes a considerable amount of time for the blood to coagulate and separate into serum and blood clots, and the drawback is that the serum necessary for testing cannot be quickly obtained. has. This becomes a problem especially when there is a need to carry out an inspection urgently. Even with glass vacuum blood collection tubes, which are said to have a short blood coagulation time, it takes 40 to 60 minutes for the blood to coagulate after blood is collected.If a synthetic resin vacuum blood collection tube is used, it can be left for over 4 hours. It takes time. Furthermore, when a vacuum blood collection tube made of such a material is used, gel-like fibrin or blood clots tend to adhere firmly to the tube wall during blood coagulation, resulting in a reduction in the amount of serum collected. In addition, fibrin tends to remain in the serum, which causes problems in serum biochemical tests. Even when glass vacuum blood collection tubes, which are said to have relatively good serum separation, are used, the separation is extremely poor at low temperatures below 15°C, especially in the cold winter months. On the other hand, inorganic particles such as glass can be attached to the tube wall to shorten the blood coagulation time, or separation agents containing silicone oil, silicon compound powder, etc. can be dispensed into the blood collection tube to remove blood serum. Efforts have been made to improve separation. Such separating agents have thixotropic properties and are adjusted to have a specific gravity intermediate between serum and blood clots. Therefore, after the collected blood has coagulated, when the blood collection tube is centrifuged, the separating agent moves to the middle between the serum layer and the blood clot layer, allowing the serum layer to be separated. If it takes time to subject the thus separated serum to various tests, the blood collection tube is usually stored as is at a temperature of 4°C. However, the shelf life is relatively short, with glass blood collection tubes containing approximately
48 hours; even with polymethyl methacrylate vacuum blood collection tubes, it takes no more than 72 hours. (Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and its purpose is to coagulate a collected whole blood sample in a short time and to effectively coagulate it by centrifugation. An object of the present invention is to provide a vacuum blood collection tube that can separate serum and blood clots. Still another object of the present invention is to provide a vacuum blood collection tube that does not deteriorate the quality of serum even if it is stored as it is at low temperature for a long time after centrifugation, and is therefore effective as a serum storage container. It is about providing. (Means for Solving the Problems) The vacuum blood collection tube of the present invention has a bottomed tubular container whose inside can be evacuated and a sealable stopper that maintains the reduced pressure state of the container, and has an inner wall surface of the container. with a surfactant,
Linseed oil absorption is 20-40ml/100g, and BET
an adsorbent inorganic substance having a specific surface area value of 5,000 to 30,000 cm ² /g, the material of which is polyethylene terephthalate or polyethylene terephthalate copolymer, and the material of the sealable stopper is butyl rubber or chlorinated butyl rubber. Thereby, the above purpose is achieved. As the adsorbent inorganic substance applied to the inner wall surface of the vacuum blood collection tube of the present invention, fine powder of a water-insoluble inorganic substance that can be generally used as an adsorbent is used. Examples of adsorbent inorganic materials include glass, silica, kaolin, celite, and bentonite. The particle size is 50 μm or less, and the average particle size is 10 μm.
It is preferable that it is below. In particular, when silica powder is used as the adsorbent inorganic material, blood coagulates in a short time. In particular, porous silica containing 20% by weight or more of amorphous components exhibits excellent effects. The adsorbent inorganic substance has the effect of promoting activation of blood coagulation factors and promoting aggregation of platelets when it comes into contact with blood. The speed of blood coagulation varies depending on the surface area and shape of the adsorbent inorganic material. By the way, when blood coagulates, a complex is first formed by three substances in the blood, factor factor (contact factor), prekallikrein, and polymeric kininogen, and is adsorbed onto the surface of a foreign substance. Then,
Factor factor is activated and blood clotting begins. Therefore, if the surface area of the adsorbent inorganic material is too small, the complex will be difficult to adsorb, resulting in a slow blood coagulation rate. Conversely, even if the surface area of the adsorbent inorganic material is too large, factor factor, prekallikrein, and high-molecular-weight kininogen will be adsorbed without complete complex formation, and therefore factor factor will not be activated, resulting in blood coagulation. speed becomes slower. As described above, the adsorptive inorganic substance needs to have an appropriate surface area and surface shape. The surface shape of adsorbent inorganic materials, such as surface area and surface pore diameter, can be expressed by linseed oil absorption and BET specific surface area. The linseed oil absorption amount of the adsorbent inorganic substance is measured according to Japanese Industrial Standard K-5101. First, 1 to 5 g of adsorbent inorganic material is placed on a glass plate (approximately
250 x 250 x 5 mm), drop a small amount of linseed oil into the center of the sample from a biuret, and mix the whole thing thoroughly with a spatula each time, so that the sample suddenly becomes soft with one drop of linseed oil and sticks to the glass plate. The previous point is the end point. Calculate the number of milliliters of linseed oil required to soften 100 g of the sample from the amount of linseed oil used, and use this as the linseed oil absorption amount. The BET specific surface area value is 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 Chemistry Society 60, 309 (1938);
59, 2682 (1937), etc.
The BET specific surface area value is calculated from the amount of gas adsorbed on the surface of an adsorbent inorganic material, the equilibrium pressure at that time, and the saturated vapor pressure of the adsorbed gas, and calculates the amount of gas that can cover the surface as a monomolecular layer, and then calculates the amount of gas that can cover the surface as a monomolecular layer. This is a value calculated by multiplying by the average cross-sectional area of the molecule. As the adsorption gas, nitrogen gas, oxygen gas, argon gas, methane gas, etc. are used. According to this method,
Surface area values including pores, which cannot be determined by linseed oil absorption, can be measured. The adsorbent inorganic material applied to the inner wall surface of the vacuum blood collection tube of the present invention has a linseed oil absorption amount of 20 to 40ml/100ml.
g, BET specific surface area value is 5000 to 30000 cm ² /g. Generally, when the collected blood comes into contact with a foreign object,
Prior to the blood coagulation phenomenon, proteins such as albumin, globulin, and various blood coagulation factors are immediately adsorbed to the surface of a foreign object. At that time, the protein molecule may undergo a change in conformation. In particular, since the activation mechanism of blood coagulation factors is an oxygen reaction, it is greatly affected, and in some cases, the blood coagulation function is impaired. In addition, platelets adhering to adsorbed globulin or albumin that have undergone large conformation changes undergo abnormal melting and deformation, and a phenomenon occurs in which the polymerized and precipitated fibrin chains strongly adhere to adsorbent inorganic materials. If such a phenomenon occurs in a blood collection tube, blood clots and serum will not be separated even if centrifugation is performed. Changes in protein conformation occur due to interactions between adsorbent inorganic substances and protein molecules, such as hydrophobic interactions, hydrogen bonding interactions, and electrostatic interactions. Among these, the influence of electrostatic interactions between adsorbent inorganic substances and protein molecules is relatively large. When a protein molecule approaches an adsorbent inorganic substance, a dipole moment group having a distribution corresponding to the polar groups of the protein molecule is induced in the adsorbent inorganic substance. If the adsorptive inorganic substance is non-conductive, even if a dipole moment group is induced, the potential distribution of the protein molecule and the potential distribution of the adsorbent inorganic substance will lack consistency with each other. As a result, protein molecules are locally distorted, resulting in a change in conformation. However, when the adsorptive inorganic material has conductivity, the consistency of the potential distribution between the protein molecule and the adsorptive inorganic material is maintained, and changes in protein conformation can be prevented. The adsorptive inorganic substance in the present invention has relatively high conductivity,
Its specific resistance value is 1×10 ¹⁰ Ω·cm or less. Therefore, the protein molecules do not change their conformation, and the blood coagulation function does not deteriorate or the separation property deteriorates. The amount of adsorbent inorganic material applied to the inner wall of the container is 1×
10 ⁻⁶ to 1×10 ⁻³ g/cm ² . If the amount is too low, a sufficient activation effect on blood coagulation factors will not be obtained, and if the amount is excessive, adsorbent inorganic substances may be mixed into the serum, which may interfere with serum tests. In addition to adsorbent inorganic substances, a surfactant is applied to the inner wall of the vacuum blood collection tube. The surfactant has the effect of preventing blood clots from adhering to the inner wall of the container and preventing hemolysis into serum during centrifugation. As the surfactant, a nonionic surfactant is used. In particular, fatty acid esters of polyglycerin such as stearic acid polyglyceride and oleic acid polyglyceride; fatty acid esters of sorbitan such as sorbitan monostearate and sorbitan monooleate; polyethers such as polyethylene glycol-modified silicone oil and polypropylene glycol-modified silicone oil. Modified silicone oil is preferably used. The amount of surfactant applied to the inner wall of the container is 1×
10 ⁻¹⁰ to 1×10 ⁻³ /cm ² . If it is too small, the effect of preventing blood clots from adhering to the inner wall of the container will not be sufficient, and if it is too large, the surfactant may be mixed into the serum and interfere with serum testing. If no surfactant is added, the rate of blood coagulation will be accelerated due to adsorbent inorganic substances, but since adsorbent inorganic substances have the effect of causing blood clots formed by coagulation to adhere to the inner wall of the container, centrifugation However, it is difficult to separate coagulated blood into serum and blood clots. Furthermore, red blood cells may be destroyed due to the strong shear stress generated between the blood clot and the adsorbent inorganic material on the inner wall surface of the container during centrifugation, and hemoglobin may dissolve into the serum. A septum-forming agent is further provided inside the vacuum blood collection tube of the present invention. The partition forming agent contains a thixotropic agent and a viscous liquid, and has thixotropic properties. The septum-forming agent is located between serum and blood clot when centrifuged, and has the function of forming septa. In many biochemical tests, only serum is used, and serum is collected using a blood test container with a pipette after centrifugation. can be fractionated by decantation. Therefore, the fractionation operation does not take much time, and blood clot components are not mixed into the serum by moving the container after centrifugation. Thixotropic agents for partition wall forming agents include inorganic powders such as silica, alumina, glass, talc, kaolin, bentonite, titania, zirconium, asbestos, and carbon black, as well as styrene resins, acrylic resins, and vinyl chloride resins. Contains organic powder. Among these thixotropic agents, fine silica powder is preferably used. Fine silica powder is a fine powder containing silicic anhydride as a main component, which has been subjected to hydrophobization treatment by grafting reaction or coupling reaction as necessary. The average particle size of the thixotropic agent is 10 ^-3 μm to 100 μm. If it is smaller than 10 ^-3 μm, it is difficult to handle, and when mixed with the viscous liquid described below, it tends to aggregate and form secondary particles and is not uniformly dispersed. If it is larger than 100 μm, the dispersion stability in a viscous liquid will be poor, and the partition forming agent as a whole will lack uniform fluidity. The specific surface area of the thixotropic agent is 10 to 500
m ² /g. When the specific surface area is within this range, excellent thixotropy can be obtained. When the specific surface area is smaller than 10 m ² /g, if the thixotropic agent is an inorganic fine powder, it becomes difficult to mix with the viscous liquid and tends to cause sedimentation. Those having a specific surface area of more than 500 m ² /g tend to aggregate and are not uniformly dispersed in a viscous liquid. The viscous liquid may or may not have a strong interaction with the thixotropic agent. Here, "having a strong interaction with the thixotropic agent" means that the thixotropic agent is mixed with a viscous liquid and dispersed uniformly, and then the thixotropic agent is mixed with a viscous liquid and then used in a centrifugal separator with an arm length of 10 cm at a rotation speed of 4000 rpm for 30 minutes. This refers to a case where no bias is observed in the distribution of the components of the mixture even after centrifugation for a minute. The cause of such interactions is 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 Examples of viscous liquids that have a strong interaction with thixotropic agents include acid-modified liquid polymer substances such as acrylic resin oligomers, polyester oligomers, liquid polyisoprene, liquid polybutene, and polybutadiene; maleic acid-modified substances; Examples include animal and vegetable oils such as soybean oil, linseed oil, safflower oil, and fish oil; acid-modified products of these animal and vegetable oils; liquid polymeric substances such as liquid poplybutene and liquid polybutadiene; and epoxy-modified products of the above-mentioned animal and vegetable oils. When the thixotropy imparting agent is an organic powder, an oligomer of the same type as the thixotropy imparting agent is preferably used, such as a styrene oligomer for polystyrene. The viscosity of the viscous liquid that has a strong interaction with the thixotropic agent is preferably 200 cps or more. When a viscous liquid having such a viscosity 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 the water-insoluble amine compounds described below. 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, liquid paraffin or its chlorinated product is used. The viscosity of these viscous liquids is
It is preferable that it is 1000 cps or more. When the viscous liquid that has a strong interaction with the thixotropic agent and the viscous liquid that does not have a strong interaction with the thixotropic agent have good compatibility, a mixture thereof can also be used. Here, "having good compatibility" refers to a state in which no phase separation occurs even if both viscous liquids are mixed and uniformly dispersed and then left at room temperature for one week. Use of these mixtures provides excellent viscosity stability over time. The mixing ratio is appropriately set in consideration of the strength of interaction with each thixotropic agent. Generally, 10 to 600 parts by weight of a viscous liquid that does not have a strong interaction with a thixotropic agent is mixed with 100 parts by weight of a viscous liquid that has a strong interaction with a thixotropic agent. When the partition-forming agent contains a water-insoluble amine compound, the stability of the viscosity of the partition-forming agent over time is further improved. As the water-insoluble amine compound, one having one or more alkyl groups having 8 or more carbon atoms in the molecule is suitable. These include, for example, dodecylamine, tetradecylamine, hexadecylamine, octadecylamine, dodecyldimethylamine, tetradecyldimethylamine, octadecyldimethylamine, polyoxyethylene octadecylamine, trioctylamine. Water-insoluble amine compounds have the property of being easily adsorbed on the surface of the thixotropic agent and also interact with viscous liquids, so by adding them, the viscosity of the partition forming agent can be stabilized over time. gender is ensured. In particular, amines having an alkyl group having 8 or more carbon atoms are highly water-insoluble and have the property of not being dissolved in separated serum or plasma. Furthermore, when adsorbed onto the surface of the thixotropy-imparting agent, the long-chain alkyl group of the amine compound is considered to have the function of stabilizing the interaction between the thixotropy-imparting agents. Therefore, the viscosity of the partition forming agent is extremely stable over time, and as a result, centrifugal separability and partition wall stability are excellent. In the partition forming agent, the thixotropic agent is contained in a proportion of 2 to 15 parts by weight per 100 parts by weight of the viscous liquid. Water-insoluble amine compounds are viscous liquids
It is added in a proportion of 0.02 to 5 parts by weight per 100 parts by weight. The specific gravity of the partition wall forming agent obtained in this way is 1.03 to 1.03 at room temperature, typically at 20°C.
It is 1.08. This corresponds to a value approximately intermediate between the specific gravity of serum and that of blood clots. Polyethylene terephthalate having the following structural formula is used as the material for the bottomed tubular container of the vacuum blood collection tube of the present invention. Polyethylene terephthalate has high crystallinity, so to suppress this, a polyethylene terephthalate copolymer is used in which a crystallization inhibitor such as 1,4-cyclohexanedimethanol is copolymerized at a proportion of 1 to 50% by weight. sell. Polyethylene terephthalate has excellent gas barrier properties and can maintain a predetermined degree of reduced pressure. It also has much better impact resistance than glass or polymethyl methacrylate, and there is no risk of breakage due to reduced pressure. Regarding disposal after use, incineration, which cannot be done with glass vacuum blood collection tubes, becomes possible. To obtain the vacuum blood collection tube of the present invention, first, an adsorbent inorganic substance, a surfactant, and a partition forming agent are applied to the inner wall surface of the bottomed tubular container. Specifically, for example, the above-mentioned adsorbent inorganic substance or the like is dissolved or decomposed in a suitable binder or solvent, and the solution is spray coated or dip coated onto the inner wall surface. A surfactant is mixed in advance with polyethylene terephthalate pellets, which is then molded into a container using an appropriate molding method such as injection molding, blow molding, compression molding, transfer molding, vacuum forming, or casting molding. The adsorbent inorganic material dispersed in a suitable binder or solvent may be spray coated or dip coated. The septum-forming agent may be dispensed into the container from the beginning, or may be added when the collected blood is coagulated and then centrifuged. A sealable stopper for maintaining the vacuum state of a vacuum blood collection tube is obtained by vulcanization molding of butyl rubber or chlorinated butyl rubber in a conventional manner. To reduce the pressure inside the blood collection tube, it is sufficient to seal the blood collection tube with the rubber stopper in the reduced pressure container. (Function) Blood is collected from the subject into the thus obtained vacuum blood collection tube whose interior is in a reduced pressure state, and when left at room temperature, the blood coagulates in about 20 to 30 minutes. When this is centrifuged, it is separated into serum and blood clots.
The material for the container of the vacuum blood collection tube of the present invention is polyethylene terephthalate or polyethylene terephthalate copolymer, which has low hydrophilicity. An adsorption layer of water molecules is not formed on the inner wall surface in contact with the inner wall surface.
Therefore, the serum separating agent and the inner wall surface of the blood collection tube come into close contact, completely separating serum and blood clots. Therefore, it is considered that inorganic ions from the blood clot do not enter the serum layer via the water molecule adsorption layer on the inner wall. Therefore, serum separated by centrifugation can be stored at low temperature for a long time. Using the vacuum blood collection tube of the present invention, it is possible to store it at 4°C for more than 340 hours. This corresponds to approximately 7 times as much as when using a glass blood collection tube. On the other hand, when a conventional vacuum blood collection tube into which a serum separation agent is dispensed is used, an adsorption layer of water molecules is formed, so the serum separation agent does not adhere firmly to the inner wall of the blood collection tube, resulting in a poor blocking effect. It is thought that inorganic ions and the like diffuse from the blood clot into the serum layer. As a result, separated serum cannot be stored for a long time. As described above, by using the vacuum blood collection tube made of the specific material of the present invention, the collected whole blood sample can be coagulated in a short time, and serum and blood clots can be effectively separated by centrifugation. After separation,
It is possible to use the blood collection tube as it is as a serum storage container. (Example) The present invention will be described below with reference to Examples. Example 1 Polyethylene glycol modified silicone oil and fine silica powder (average particle size 4.0 μm; linseed oil absorption 30 μ/100 g; BET specific surface area value 1.2×10 ⁴
cm ² /g; specific resistance value 2.6×10 ⁴ Ω・cm), respectively 0.5
A Freon dispersion was prepared in a proportion of % by weight. This dispersion was coated on the inner wall of a 10 ml spittoon made of polyethylene terephthalate.
After thoroughly drying the tube, the inside was evacuated and the tube was sealed with a butyl rubber stopper to prepare a vacuum blood collection tube. The degree of internal vacuum was set so that the amount of blood collected was 6 ml. After collecting fresh human blood with the vacuum blood collection tube, 20
The tube was left at ℃ and the time required for the whole blood to stop flowing completely was measured. Immediately after blood clots
Centrifugation was performed for 5 minutes at a rotation speed of 3000 revolutions/minute, and the state of serum separation was observed. The results are shown in Table 1. As is clear from Table 1, when the obtained vacuum blood collection tube was used, blood coagulation was extremely rapid and the serum separation state was also good. Example 2 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 hydrophobized finely powdered silica, and 0.2 parts by weight of trioctylamine were kneaded in a three-roll kneader to form a partition forming agent with a specific gravity of 1.06 at 20°C. I got it. 1 g of this septum-forming agent was injected into a spittoon coated with the same Freon dispersion as in Example 1, the degree of vacuum inside was set so that the amount of blood collected was 6 ml, and the tube was sealed with a butyl rubber stopper. After blood was collected using the thus obtained vacuum blood collection tube, blood coagulation and serum separation state were observed in the same manner as in Example 1. The results are shown in Table 1. Next, immediately after centrifugation to observe the performance as a serum storage container,
Serum was collected on the 3rd, 7th, and 14th days after being cooled and stored at 4°C, and LDH and K, which are listed as biochemical test items, were measured. The results are shown in Table 2. As is clear from the results in Table 1, the obtained vacuum blood collection tube showed rapid blood coagulation and good serum separation. Furthermore, as is clear from the results in Table 2, LDH and K showed stable values, making it a good serum storage container. Comparative Example 1 Blood was collected using a commercially available 6 ml glass vacuum blood collection tube, and blood coagulation and serum separation status were observed in the same manner as in Example 1. The results are shown in Table 1.
Next, the performance as a serum storage container was tested in the same manner as in Example 2. The results are shown in Table 2. [Table] [Table] (Effects of the Invention) According to the present invention, it is possible to coagulate a collected whole blood sample in a short time and to effectively separate serum and blood clots by centrifugation. A vacuum blood collection tube can be obtained. Such a vacuum blood collection tube is also effective as a storage container for serum after centrifugation, and the obtained serum can be stored for a long time at low temperature.

Claims (1)

[Scope of Claims] 1. The container has a bottomed tubular container whose inside can be evacuated and a stopper that can be tightly plugged to maintain the reduced pressure state of the container, and a surfactant and a linseed oil absorption amount are provided on the inner wall surface of the container. 20〜
40ml/100g, and BET specific surface area value is 5000~
30000 cm ² /g of adsorbent inorganic material, the material of the tubular container is polyethylene terephthalate or polyethylene terephthalate copolymer, and the material of the sealable stopper is butyl rubber or chlorinated butyl rubber. Blood vessels. 2 The specific resistance value of the adsorptive inorganic substance is 1× ¹⁰ Ω・
The vacuum blood collection tube according to claim 1, which has a diameter of less than cm. 3. The vacuum blood collection tube according to claim 1, wherein the surfactant is a nonionic surfactant. 4 The surfactant is 1×10 ^-10 to 1×10 ^-3 g/
Vacuum blood collection tube according to claim 1, given in cm ² . 5 The adsorbent inorganic substance is 1×10 ^-6 to 1×10 ^-3 g/
Vacuum blood collection tube according to claim 1, given in cm ² . 6. The vacuum blood collection tube according to claim 1, wherein a septum-forming agent is provided in the container. 7. The vacuum blood collection tube according to claim 6, wherein the septum-forming agent contains a thixotropic agent and a viscous liquid. 8. The vacuum blood collection tube according to claim 6, wherein the septum-forming agent contains a thixotropic agent, a viscous liquid, and a water-insoluble amine.