JPH0126024B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a separation composition used when subjecting a blood sample to centrifugation to separate its liquid components, serum or plasma, from solid components mainly composed of red blood cells. .

In recent years, testing of blood components in clinical laboratory departments has become extremely important, and the number of tests conducted has continued to increase. Among these, many biochemical tests use only serum or plasma as a sample, and it is necessary to separate serum or plasma from solid components as a preliminary operation for testing. For this reason, conventionally, serum or plasma has been collected by precipitating the blood cell portion by centrifugation and then sucking it up with a pipette. However, this method provides insufficient separation and is very time-consuming.Recently, various methods have been devised to perform this preparative separation operation easily and with high yield.

One method is to add a substance with a specific gravity intermediate between that of serum or plasma and the solid component into a blood sample, and use centrifugation to position the substance between the two to form a septum. . According to this method, it is possible to separate serum or plasma only by decantation, and it is possible to reduce time and labor, but on the other hand, it also has the following drawbacks.

That is, three types of forms of this material for forming partition walls have been proposed so far: a solid form, a sample form, and a mixture thereof.

Solid forms include, for example, polystyrene powder or pellet materials, but when these are used, their function as a barrier is insufficient, so blood cells may not be mixed into serum or plasma. It has various drawbacks, such as the fact that the partition wall itself is easily destroyed.

Also, acrylic polymers are known as liquid polymers, but in general, when trying to obtain a high viscosity liquid polymer from monomers such as acrylic polymers, it is necessary to control the reaction. Depending on the reaction method, there are many difficult manufacturing problems such as removal of impurities.
Moreover, liquid forms have the disadvantage that they are troublesome to transport and store before use because of their fluidity.

Furthermore, as mixtures of solid and liquid, thixotropic compositions such as silicone with silica powder added and modified liquid polybutadiene with alumina are known, but these generally deteriorate over time. The disadvantage is that the viscosity increases or decreases as the temperature increases. That is, if the viscosity increases, after storing the composition at the bottom of the spittoon for a long period of time, it is necessary to apply strong centrifugal force for a long time to move the composition to a position intermediate between the serum or plasma and the blood cells by centrifugation. time and sometimes 500 to 2000G, which is usually used in hospitals, testing centers, etc.
Even if a centrifugal force of However, after blood is added and centrifuged, when the spittoon is tilted to pour out the serum, the septum collapses, causing problems such as blood cells being mixed into the serum.

The present inventors have conducted extensive research in view of the current state of compositions for serum or plasma separation as described above.
It was discovered that by mixing a water-insoluble amine compound with a thixotropic agent and a viscous liquid, a composition that does not change in viscosity over time can be obtained, and based on this knowledge, it is possible to easily obtain a composition that does not change its viscosity over time. A composition for separating serum or plasma has been completed in which a stable partition wall is formed, serum or plasma can be easily and accurately separated, and at the same time, no special measures are required for transportation or storage.

The gist of the present invention is as follows: (1) 2 to 15 parts by weight of a thixotropic agent that is at least one selected from the group consisting of silica, glass, talc, and bentonite and has an average particle size of 1 mμ to 100μ; 20℃ selected from the group consisting of acrylic resin oligomers, epoxy modified products of animal and vegetable oils, and liquid chlorinated polybutenes.
Viscous liquid with a viscosity of 1000 cps or more
parts by weight and (3) 0.02 to 5 parts by weight of a water-insoluble amine compound having one or more alkyl groups having 8 or more carbon atoms in the molecule, and has a specific gravity of
1.03 to 1.08.

Next, the serum or plasma separation composition of the present invention will be explained in more detail.

The serum or plasma separation composition forms a partition wall that separates serum or plasma from solid components, and stabilizes the partition wall so that the serum or plasma and solid components do not mix after separation. This is required. For example, it is necessary to prevent the septum from flowing and collapsing when carrying the composition and test blood in a spittoon after performing a separation operation, or when tilting the spittoon to separate serum or plasma. There is. For this purpose, thixotropic agents are used.

Examples of thixotropic agents include inorganic powders such as silica, glass, talc, and bentonite, and surface-treated products of these that have been subjected to hydrophilic treatment or hydrophobic treatment by grafting or coupling reactions, and these may be used alone. or a mixture of two or more types.

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 used is 1 mμ to 100μ. 1m
If it is smaller than 100μ, it is difficult to handle, and when mixed with the viscous liquid described later, it tends to aggregate and form secondary particles, making it difficult to disperse uniformly. The dispersion stability of
This is because the separation composition as a whole lacks uniform fluidity.

In order to cause the separating composition to become thixotropic by means of a thixotropic agent,
A viscous liquid is used.

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 means that a certain thixotropic agent is mixed with a certain viscous liquid, uniformly dispersed, and then mixed with a centrifuge 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. The cause of such interactions is not yet 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 forces caused by their molecular structures. It is assumed that this is the case.

Examples of viscous liquids that have a strong interaction with thixotropic agents include acrylic resin oligomers and epoxy modified products of animal and vegetable oils such as soybean oil, linseed oil, safflower oil, and fish oil.
℃) is 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 chlorinated polybutene, and those having a viscosity (at 20°C) of 1000 cps or more are used.

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. For every 100 parts by weight of the viscous liquid, 10 parts of the viscous liquid that does not have a strong interaction with the thixotropic agent is added.
~200 parts by weight are used.

However, it has been confirmed that the stability of 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 used, such as dodecylamine, tetradecylamine, hexadecylamine, octadecylamine,
These include dodecyldimethylamine, tetradecyldimethylamine, octadecyldimethylamine, and trioctylamine.

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. Excellent centrifugal separability is usually used in clinical tests.
Centrifugation is performed at about 500 to 2000G,
This means that it has excellent properties of being able to move with centrifugal force within this range and form partition walls. Furthermore, the expression "excellent partition wall stability" means that the partition walls formed by centrifugation have excellent strength and do not collapse even when the spittoon is tilted. If the viscosity increases over time, centrifugal separability will deteriorate if stored for a long time, and the normal centrifugal force of 500
At 2000G to 2000G, it becomes impossible to form a partition wall, and conversely, if the viscosity decreases over time, the stability of the partition wall will deteriorate if stored for a long time, and the partition wall will collapse even when the spittoon is tilted. According to the invention, such problems do not occur.

The proportions of the thixotropic agent, viscous liquid, and water-insoluble amine compound used are 2 to 15 parts by weight of the thixotropic agent and 0.02 to 5 parts by weight of the water-insoluble amine compound per 100 parts by weight of the viscous liquid. When used at such a ratio, partition walls can be formed in a short time by centrifugation.

The composition for serum or plasma separation in the present invention has a specific gravity of 1.03 to 1.08. This is because the specific gravity of the solid component is required to be intermediate between that of serum or plasma and that of the solid component in order to form the septum. A specific gravity of 1.03 to 1.08 is the specific gravity at room temperature, and the standard is 20
The value is in °C. The reason why specific gravity at room temperature is used as the standard is because clinical tests are usually conducted at room temperature. However, when the composition for serum or plasma separation of the present invention is used under temperature conditions other than room temperature, it may have a specific gravity of 1.03 to 1.08 in terms of specific gravity at room temperature. For example 40
If the specific gravity at 20° C. is 1.02 to 1.07, or even if the specific gravity at 0° C. is 1.04 to 1.09, the specific gravity at 20° C. is 1.03 to 1.08, which falls within the range of specific gravity in the present invention.

In order to separate serum or plasma from solid components such as blood cells using the separation composition of the present invention, it is sufficient to add the separation composition to sample blood and perform a centrifugation operation. Since a barrier wall is formed by the composition of the present invention at the interface between plasma and solid components such as blood cells, serum or plasma can be easily and accurately removed by decantation or the like.

According to the composition for serum or plasma separation of the present invention,
It has appropriate thixotropy and does not increase in viscosity over time, so it does not require particularly strong centrifugal force and can be easily used under normal centrifugation conditions even when used immediately after production or after long-term storage. A septum is formed within the blood collection tube, allowing serum or plasma to be easily and accurately taken out. In addition, since the viscosity does not decrease over time, the septum is stably formed even after long-term storage, and once the septum is formed, it will not collapse even if the blood collection tube is tilted. becomes.

Examples of the present invention will be described below.

Example 1 As a thixotropy agent, the average particle size was 10 mμ,
10 parts by weight of silica powder with a specific gravity of 2.2 at 20°C, 90 parts by weight of liquid chlorinated polybutene having a specific gravity of 1.00 at 20°C and a viscosity of 8000 cps, and 0.1 part by weight of octadecyldimethylamine were prepared.

Knead each of these ingredients with a three-roll kneader,
A separation composition having a specific gravity of 1.06 at 20°C was obtained.

Next, 1 g of the composition was injected into four glass Spitz tubes each having a capacity of 10 ml. Immediately after creating the composition,
Add sample blood to one of these bottles and after blood coagulation
When centrifuged at 2500 rpm (1160 G) for 3 minutes, the composition formed a partition between the serum and the blood clot, and the serum could be easily separated by decantation.

In addition, protein,
Biochemical serum tests were conducted on 32 items such as lipids, serum, enzymes, and inorganic ions when the composition was used as a serum separation agent and when it was not used. When these serum test values were compared, they were considered to be the same. Good results were obtained and it was found that the use of the composition had no effect on serum test values.

Further, an inverted test was conducted in which another spittoon containing only the above-mentioned composition was inverted and tilted at an angle of 60° with respect to a horizontal plane, but the composition did not flow. Furthermore, after storing the above composition at room temperature for 30 days, a similar test was conducted, but there was no formation of septa, no effect on serum test values, and no flow of the composition was observed in an inverted test.

Example 2 2 with a specific gravity of 1.0 and a viscosity of 25000 cps at 20°C
- 90 parts by weight of ethylhexyl acrylate oligomer, 10 parts by weight of silica powder with a specific gravity of 2.2 at 20°C, and 0.2 parts by weight of trioctylamine at 3 parts by weight.
The mixture was kneaded using this roll kneader to obtain a separation composition having a specific gravity of 1.06 at 20°C.

Next, 1 g of the composition was injected into a glass spittoon with a capacity of 10 ml, sample blood was added, and after blood coagulation, centrifugation was performed at 2500 rpm (1160 G) for 10 minutes. and formed a septum between the blood clots. In order to collect this serum, the spittoon was tilted, but the serum could be taken out without collapsing the septum. In addition, an inverted test was conducted in the same manner as in Example 1, but the composition did not flow.
Furthermore, after storing the above composition at room temperature for 30 days, a similar test was conducted, but there was no effect on the formation of partition walls, and no flow of the composition was observed in an inverted test.

Example 3 70 parts by weight of chlorinated polybutene with a specific gravity of 1.02 at 20°C and a viscosity of 10000 cps, and 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 silica powder with a specific gravity of 2.2 at 20°C, and 0.2 parts by weight of trioctylamine.
The mixture was kneaded using this roll kneader to obtain a separation composition having a specific gravity of 1.06 at 20°C.

Next, 1 g of the composition was injected into a glass spittoon with a capacity of 10 ml, sample blood was added, and after blood coagulation,
When centrifuged for 3 minutes at 2500 r.p.m. (1160 G), the composition formed a septum between the serum and the blood clot. In order to collect this serum, the spittoon was tilted, but the serum could be taken out without collapsing the septum. In addition, an inverted test was conducted in the same manner as in Example 1, but no flow of the composition occurred. Furthermore, after storing the composition at 40°C for 30 days,
A similar test was conducted, and it was found that there was no effect on the formation of partition walls, and there was no flow of the composition in an inverted test, indicating that the composition had excellent stability over time.

Comparative example 2 with a specific gravity of 1.0 and a viscosity of 25000 cps at 20℃
- 90 parts by weight of ethylhexyl acrylate oligomer and 10 parts by weight of silica powder with a specific gravity of 2.2 at 20°C are kneaded using a three-roll kneader,
A composition having a specific gravity of 1.06 at 20°C was obtained.

Next, 1 g of the composition was injected into a glass spittoon with a capacity of 10 ml, sample blood was added, and after blood coagulation,
When centrifuged at 2500 rpm (1160 G) for 10 minutes, the composition formed a barrier between the serum and the blood clot.

However, after storing the composition at room temperature for 30 days, the
When centrifugation was performed at m (1660G) and 3500rpm (2260G) for 10 minutes each, the sample did not move from the bottom of the spittoon, and no septum was formed.

Claims (1)

[Claims] 1 (1) 2 to 15 parts by weight of a thixotropic agent that is at least one selected from the group consisting of silica, glass, talc, and bentonite and has an average particle size of 1 mμ to 100μ; (2) acrylic resin oligomer;
The viscosity at 20℃ is selected from the group consisting of epoxy modified animal and vegetable oils and liquid chlorinated polybutene.
100 parts by weight of a viscous liquid of 1000 cps or more and (3)
For serum or plasma separation, comprising a mixture of 0.02 to 5 parts by weight of a water-insoluble amine compound having one or more alkyl groups having 8 or more carbon atoms in the molecule, and having a specific gravity of 1.03 to 1.08. Composition.