JPH10185910A - Plasma or serum separation filter and plasma or serum separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To quickly, efficiently and safely separate plasma and serum from a small amount of blood by filling a vessel with the three-dimensional porous matter of a specified average hole diameter so that the flow passage diameter and the flow passage length have a constant rate. SOLUTION: A disk-like vessel 4 having an inlet 1 in the upper face end and an outlet 2 in the center part of the bottom face is filled with a three-dimensional porous matter whose average hole diameter is 5-50μm and percentage of voids is 20-95%, and a plasma and serum separation filter is formed. The rate (L/D) of the flow passage length of plasma (the shorten distance up to the surface of the porous body 3 of the outlet 2 from the surface of the porous body 3 of the inlet 1 L to the flow passage diameter of blood (the circle-corresponding diameter of the surface area of the porous body 3 in contact with inflow blood) D is set 0.15-6. When a filter is constituted as described above, with regard to the moving speed of the porous matter 3, a plasma and serum component reaches the outlet 2 side faster than hemocyte component, because the speed of the plasma and serum component is faster than that of hemocyte. The time difference is utilized to separate plasma and serum from blood.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a filter for separating and recovering plasma or serum components from blood, and a method for separating plasma or serum. More specifically, when used in clinical tests, etc., plasma that can quickly obtain high-purity plasma or serum even with a small amount of blood, is easy to operate, has high safety, and is easy to manufacture. Alternatively, the present invention relates to a serum separation filter and a method for separating plasma or serum.

[0002]

2. Description of the Related Art A so-called biochemical test for measuring components in blood is widely used for diagnosis and follow-up of various diseases, and occupies an important position as a clinical test. The analysis technology has been remarkably advanced in recent years, and the development of various automatic analyzers has made it possible to analyze a large number of samples accurately and quickly.

However, in many fields of biochemical tests, serum or plasma is separated from blood in advance because the presence of blood cells such as red blood cells interferes with the test. for that reason,
Prior to the test, it is necessary to undergo a process of once coagulating blood collected from a patient or a subject and then centrifuging to obtain serum. Coagulation and centrifugation operations are time-consuming and not only hinder the shortening of clinical tests, but also require large centrifuges. In many cases, we ask external testers to provide the results, and it takes several days to obtain the test results. In addition, since the work of separating serum from blood still depends mostly on humans, workers touch the blood,
They are also at risk of infection.

[0004] As a means for solving the above problems, a technique generally called dry chemistry is known.
This is because, when a small amount of blood is dropped on a small plate composed of a serum separation layer composed of an ultrafine fiber filter such as glass fiber and a reaction layer located below the serum, the serum is separated by the serum separation layer and the lower layer is separated. Reaction and color development occur in the reaction layer, which is measured with a spectrophotometer. This dry chemistry is a simple method that does not use a liquid coloring reagent and does not require cumbersome serum collection by centrifugation.However, the measurable items are compared to general biochemical analysis and immunoassay using liquid reagents. The use of a single plate for one test item requires the use of a large number of plates for testing multiple items. However, it is not widely used because of its high cost.

As a method for obtaining plasma or serum without using centrifugation, Japanese Patent Application Laid-Open No. 53-72691 discloses a method for separating plasma from blood using a fine tubular filter element having one end closed as a filter medium. JP-A-60-1
No. 1166 proposes a method for separating plasma from blood using a filtration cartridge using a hollow fiber bundle. However, the former method has poor protein permeability and blood cells adhere to the filter surface,
Plasma filtration takes an extremely long time, and conversely, if the filtration pressure is increased to increase the filtration rate, there is a problem that red blood cells are lysed. Further, in the latter method using a filtration cartridge using a hollow fiber bundle, it is necessary to perform pretreatment by priming as preparation for plasma separation work, that is, work such as wetting the hollow fiber membrane with physiological saline or the like, There is a problem that the obtained plasma is diluted, and preparation work is more troublesome than plasma separation work itself.

In addition, since these membrane separation methods are based on the difference in molecular size between blood cells and plasma / serum, substances having relatively high molecular weight, such as proteins in blood, sufficiently pass through the membrane. However, there is a problem that the composition of each protein in the obtained plasma does not accurately reflect the composition of the protein in the original blood. Further, if the pore size of the membrane is too large, red blood cells are clogged, causing a problem of hemolysis, and have not been put to practical use.

Apart from the above, various techniques for separating serum or plasma for clinical tests using a fibrous filter have been proposed. Japanese Patent Application Laid-Open No. 4-208856 discloses a method for separating and recovering serum or plasma components comprising a glass fiber containing a polyacrylester derivative and polyethylene glycol and a lectin-impregnated layer. Also, Japanese Unexamined Patent Publication No.
Japanese Patent Laid-Open No. 196620/1992 discloses the above-mentioned JP-A-4-20885.
No. 6 discloses a serum / plasma separation instrument using a separation filter. Although these methods and instruments can collect serum or plasma for clinical tests without using centrifugation, the amount of serum and plasma obtained is 100%.
In addition, the time required for separation is about 2 minutes, which is shorter than that of centrifugation, but is not sufficient. Furthermore, these technologies use glass fiber as a separation material, and the electrolyte and the like are eluted from the glass fiber or adsorbed on the glass fiber, so that the concentration of the electrolyte, phosphorus, and lipid in the obtained plasma and serum can be separated. It has the disadvantage that it is very different from the previous blood. For this reason, these technologies have not yet become widespread.

Although it is different from the use for separating blood plasma and serum, for example, Japanese Patent Application Laid-Open No. 60-193468 discloses a leukocyte removal filter in which a plurality of microfiber nonwoven fabrics are stacked. Is now widespread. In addition, International Patent Application WO 89/230
No. 4, WO89 / 2305 or Japanese Unexamined Patent Application Publication No.
Japanese Patent Publication No. 168711 discloses a leukocyte removal filter using a porous body having a three-dimensional network structure having communicating pores. However, all of these technologies
It relates to a leukocyte removal filter, and does not give any information on plasma / serum separation for clinical tests.

[0009]

As explained above, as a filter for separating plasma and serum components quickly, efficiently and safely from a small amount of blood for use in clinical examinations, it has excellent assemblability and sufficient performance. At present, there is no such thing.

An object of the present invention is to enable plasma, serum components having the same component composition as in blood to be quickly, simply and safely separated from blood without damaging blood cells in blood. Excellent in assemblability (easy to manufacture)
An object of the present invention is to provide a plasma / serum separation filter and a plasma / serum separation method.

[0011]

Means for Solving the Problems The present inventors have conducted various studies to solve the above-mentioned problems, and as a result, by using a three-dimensional porous body having communicating pores and controlling the diameter of the pores, the blood concentration in the blood is reduced. Can cause a difference in the moving speed of red blood cells and plasma / serum in the porous body, and adjust the ratio (L / D) of the blood flow path length (L) and the blood flow path diameter (D). By reducing the unevenness of the moving speed of the blood in the horizontal direction and widening the difference in the moving distance between the red blood cells and the plasma and serum caused by the difference in the moving speed, the plasma and serum components in the blood are separated from the blood cells. And found it possible to collect. Furthermore, by using a three-dimensional porous body, there is no need to stack or unravel a plurality of sheet-like bodies, it is easy to form and process into a desired filter shape, and further, channeling and clogging of blood. And that the occurrence of hemolysis can be prevented.

In the above description, leukocytes and platelets in blood have a property of adsorbing to a porous body (this property is known), and thus are adsorbed and removed when passing through the filter of the present invention. Next, erythrocytes do not have the property of adsorbing to the porous body, but by setting the pore diameter of the porous body, that is, the size of the channel, to be small, so-called steric hindrance occurs and the pores are deformed. While moving. In contrast, plasma and serum components are liquid, have no steric hindrance, and move through pores faster than red blood cells. In this way, there is a difference between the moving speeds of red blood cells and plasma / serum. Further, by increasing the L / D, the unevenness of the moving speed of blood in the horizontal direction can be reduced, and the difference in moving distance between red blood cells and plasma / serum can be increased.
In the serum, plasma and serum arrive at the porous body outlet first,
Only plasma and serum can be collected.

While the present invention aims to collect a small amount of plasma and serum in clinical tests and the like,
Since the porous material used as a leukocyte removal filter is intended to process a large amount of blood or red blood cell concentrate (usually about 100 to 500 ml), L / D is small to reduce clogging and passage resistance (ie, This is fundamentally different from that of the present invention in that it is designed to have a small thickness and a large diameter) and uses the principle of leukocyte removal by adsorption.

That is, according to the present invention, the average pore size is 5 to 50 μm.
Is loaded into a container having an inlet and an outlet so that the ratio (L / D) of the blood flow path length (L) to the blood flow path diameter (D) is 0.15 to 6 And a filter for separating plasma or serum.

The present invention also relates to the above-mentioned plasma or serum separation filter, wherein the porosity of the three-dimensional porous body is 20 to 95%; Is configured to supply blood over the entire circumference of the three-dimensional porous body, and the outlet of the container is configured so that plasma or serum flows out from the center of the disc-shaped three-dimensional porous body. The above-mentioned plasma or serum separating filter, wherein the hydrophilic agent is fixed to the three-dimensional porous body; the above-mentioned plasma or serum separating filter, wherein the hydrophilic agent is made of the three-dimensional porous body. The above-mentioned plasma or serum separation filter fixed on the surface; the above-mentioned plasma or serum separation filter, wherein the hydrophilic agent is polyvinylpyrrolidone

Furthermore, the present invention provides a hematocrit of 40
When 50 to 50% of fresh bovine blood is introduced from the inlet of the filter and a pressure difference of 0.4 to 0.6 kg / cm ² is applied between the inlet and outlet of the filter to collect plasma or serum, the collected plasma or serum The plasma or serum separation filter, wherein the volume of the liquid is 10 to 50% of the pore volume of the three-dimensional porous body;
50% of fresh bovine blood flows from the inlet of the filter and a pressure difference of 0.4 to 0.6 kg / cm ² is applied between the inlet and outlet of the filter to separate plasma or serum. When recovering an amount of plasma or serum corresponding to 10% of the pore volume, the concentration of the electrolyte in the separated plasma or serum is 90 times the concentration of the electrolyte in the plasma or serum obtained by centrifugation. The plasma or serum separation filter, characterized by having a hematocrit of 40 to 50% of fresh bovine blood flowing through the inlet of the filter, and
When a pressure difference of ² cm ² is applied between the inlet and the outlet of the filter to separate plasma or serum, and when the amount of plasma or serum corresponding to 10% of the pore volume of the three-dimensional porous body is recovered from the start of separation, the separated plasma or serum is removed. The present invention relates to the above-mentioned plasma or serum separation filter, wherein the protein concentration in the plasma or serum is 90 to 110% as compared with the protein concentration in the plasma or serum obtained by centrifugation.

Further, according to the present invention, the average pore diameter is 5 to 50 μm.
Is loaded into a container having an inlet and an outlet so that the ratio (L / D) of the blood flow path length (L) to the blood flow path diameter (D) is 0.15 to 6 A plasma or serum separation method, characterized in that a plasma or serum separation filter is used and the average linear velocity of blood is 1 to 300 cm / min; the pressure difference between the filter inlet and outlet is 0.01 to 5 kg / min. cm ² , wherein the blood plasma or serum is separated.

The three-dimensional porous body in the present invention is 1 m
m means a porous body having a thickness of not less than m. What is a porous body?
It refers to a sponge form in which both the matrix and the pores are continuous. Hereinafter, the three-dimensional porous body is simply referred to as a porous body.

The expression "the matrix is continuous" means that the matrix is continuous and integrated over the entire area of the porous body. The present invention does not include those in which the matrices are simply in contact with each other and entangled with each other, or those in which the matrices are partially fused together or where the matrices are adhered at several places by an adhesive or the like. However,
Even in the case of matrix-to-matrix point bonding, the matrix is substantially integrated over the entire porous body,
A matrix in which the matrices are line bonded and integrated is included in the present invention. In other words, a cotton-like material in which fibers are simply entangled or a nonwoven fabric in which a fiber material is partially welded or spot-bonded by an adhesive or the like is not included in the present invention. The present invention includes those in which two or more continuous beads or porous beads are thermally fused to each other.

The expression "continuous pores" means that the pores communicate with each other over the entire area of the porous body. In other words, those obtained by bundling a plurality of straw-shaped materials such as hollow fibers, and those obtained by bundling and integrating them with an adhesive or heat fusion are only in communication with their respective holes, and are in communication with each other. Therefore, it is not included in the present invention.

As described above, the ultrafine fiber nonwoven fabric obtained by the melt blow method or the like is usually in the form of a sheet having a thickness of less than 1 mm, and cannot be said to be three-dimensional. Further, the fiber mass (web) formed by entanglement of the fibers is different from the porous body of the present invention because the matrix is not continuous. Also,
If the pores are not connected, blood cannot flow, which is obviously not suitable for the present invention.

Although the porous material in the present invention is not particularly limited, examples of the material include cellulose, cellulose acetate, polyurethane, polyacrylonitrile, polyvinyl acetal, polyvinyl formal, polyester, polyamide, polystyrene, polysulfone, and polyfluorocarbon. Vinyl chloride, polyvinylidene fluoride,
Polytrifluorochlorovinyl, vinylidene fluoride-tetrafluoroethylene copolymer, vinylidene fluoride-hexafluoropropylene copolymer, tetrafluoroethylene-propylene copolymer, tetrafluoroethylene-
Ethylene copolymers and the like are mentioned, and preferred are cellulose and polyvinyl formal. These can be used alone or in combination of two or more.

The porous body can be obtained by molding or processing the above-mentioned materials by a known method (for example, foaming, phase separation, etc.). For example, in a formalization reaction in which formaldehyde is bound to water-soluble polyvinyl alcohol using an acid as a catalyst, a pore-forming agent is added in advance, pores are formed, and after forming an insoluble porous substrate, the pore-forming agent is extracted. By doing so, a porous body can be obtained. Further, punching, shaving, and the like can be performed to obtain a desired shape.

The porous body has the following advantages as compared with the fiber material. 1) Good moldability (assembly of filter) into desired shape. This is because the porous body does not need to be overlapped or unraveled as in the case of a nonwoven fabric. In other words, in the case of fibers, there are cotton-like and non-woven fabrics, but cotton-like fibers are indefinite, and it is necessary to pack cotton into filter containers,
The nonwoven fabric fiber is sheet-shaped, and requires an operation of stacking a plurality of sheets. 2) It can have a uniform pore size and open pores, thereby preventing clogging and hemolysis. This is because, in the case of a porous body, pores are formed by a chemical reaction, and the pore size is determined by the chemical reaction. On the other hand, in the case of a fiber material, the pore size of the filter is determined by a physical factor (compression).
Hemolysis may occur in dense areas due to passage resistance.

The porous material can be used as it is, but it is also possible to modify the surface in order to further increase the affinity between blood and the porous material or to prevent protein adsorption. it can. The surface modification can be performed, for example, by fixing a hydrophilic agent to the porous body, in particular, by fixing a hydrophilic agent to the surface of the porous body.

Examples of the hydrophilicizing agent include polyethylene glycol, acrylic polymer, polyvinyl alcohol, ethylene-vinyl alcohol copolymer, polyvinyl pyrrolidone and the like. Polyvinylpyrrolidone is preferred because of its excellent affinity with the porous material.

The method for fixing the hydrophilizing agent to the porous body is not particularly limited, and any known method can be used, and the hydrophilizing agent can be physically or chemically fixed. By immobilizing the hydrophilizing agent on the surface of the porous body, the affinity between the porous body and blood increases, so that when blood is separated into blood cells and plasma, the passage resistance decreases and the separation speed can be increased. .

Here, polyvinylpyrrolidone will be described as an example for more specific description. For example, after immersing the porous body in an aqueous solution of polyvinylpyrrolidone as a hydrophilizing agent,
By taking out and drying, the hydrophilic agent can be easily and physically immobilized on the surface of the porous body. Further, by subjecting such a porous body in which polyvinylpyrrolidone is physically immobilized to the surface to heat treatment, radiation treatment, or the like, the polyvinylpyrrolidone can be easily crosslinked with each other. By cross-linking, the elution of polyvinylpyrrolidone into blood can be suppressed lower.

When performing the heat treatment, the method is not particularly limited. For example, a method of heating under pressure, such as an autoclave treatment, or a method of leaving it in a constant temperature bath may be used. Although the temperature of the heat treatment is not particularly limited, it is preferably 70 ° C. or higher, more preferably 100 ° C. or higher. The higher the heating temperature, the higher the efficiency of crosslinking. The upper limit temperature is preferably 200 ° C. or lower, although it depends on the properties of the porous body used and the heat resistance of polyvinylpyrrolidone itself.
150 ° C. or lower is more preferable. The heating time is preferably long for sufficient crosslinking, but is preferably 2 to 2 hours in view of the properties of the porous material used and the modification of polyvinylpyrrolidone itself.
0 minutes to 2 hours are preferred. In addition, crosslinking by heating,
It can be performed either in a state where the porous body is immersed in the hydrophilic agent solution (WET state) or in a state after immersion and dried (DRY state), and the polyvinylpyrrolidone is fixed to the porous body. Unreacted polyvinylpyrrolidone can be removed by washing with water or the like.

The method for fixing the hydrophilizing agent using radiation is not particularly limited. For example, γ-ray irradiation, electron beam irradiation, corona discharge and the like can be mentioned. Γ-ray irradiation is preferred from the viewpoint of the thickness that can be processed and the operability. The irradiation amount is not particularly limited as long as the irradiation amount is sufficient to crosslink polyvinylpyrrolidone. The irradiation amount is 10 to 50 KG as long as the porous body or polyvinylpyrrolidone itself is not denatured by irradiation.
y is preferred. Irradiation can be performed in a WET state or a DRY state. Unreacted polyvinylpyrrolidone can be removed by washing with water or the like.

The average pore size of the porous material used in the filter of the present invention needs to be 5 to 50 μm,
Preferably 8 to 30 μm, more preferably 10 to 20 μm
m. If the average pore size exceeds 50 μm, the resistance of red blood cells to pass through the pores (ie, deformation of the red blood cells and friction between the pore walls) decreases, and a sufficient difference in the moving speed between the red blood cells and the plasma / serum components occurs. And the separation becomes insufficient. If the average pore diameter is less than 5 μm, the blood flow path becomes too narrow, blood cells tend to be clogged, and the passage resistance of the filter becomes large, which is not preferable.

The average pore diameter in the present invention is the average of the pore diameters on the surface of the porous body or an arbitrary cross section. The average pore diameter is obtained by cutting the porous body in a direction perpendicular to the blood flow direction, taking an electron micrograph of the cross section, and determining the diameter of the pores within a certain area where at least 100 pores are present. Measure and determine by arithmetic mean. If the cross section of the pore is not circular, the cross section of the pore is determined by image processing or the like, and the equivalent diameter of the circle corresponding thereto is defined as the pore diameter. In this case, all the diameters mean diameters. The average pore diameter can be adjusted by changing the manufacturing conditions of the porous body.

Further, the pore diameter of the porous body of the present invention may be constant in the axial direction from the blood inlet to the permeate outlet, but the pore diameter is gradually reduced from the blood inlet to the permeate outlet. It is also possible to increase the efficiency of separating blood cell components and plasma / serum components near the permeate outlet. However, even in that case, the average pore diameter needs to be in the range of 5 to 50 μm.

In the present invention, a prefilter (for example, a porous material, cotton, non-woven fabric, or the like) is provided in the space between the plasma / serum separating porous material and the inner wall of the container.
It is also possible to install a mesh or the like. The average pore size of these prefilters is naturally larger than the average pore size of the above-mentioned porous body for plasma / serum separation. However, the average pore size of the entire filter does not take into account the average pore size of the prefilter. The average pore size in only the separation porous body portion is used.

The porosity of the porous material used in the filter of the present invention is preferably from 20 to 95%, more preferably from 30 to 90%. If the porosity is less than 20%, the bulk of the filter itself tends to increase, and the resistance to blood passage tends to increase. If the porosity is greater than 95%, the strength of the filter tends to decrease, and the assemblability tends to decrease.

The porosity means the ratio (%) of the volume of pores in the porous body to the entire volume of the porous body. Porosity is calculated as _{(1-r m / ρ)} × 100 (%).
Incidentally, r _m is the bulk density of the porous body, [rho is the density of the porous material.

The pore volume of the porous body can be calculated as (porous body volume × porosity) × 1/100. The porosity and the pore volume can be adjusted by changing the manufacturing conditions of the porous body.

In the present invention, the ratio (L / D) of the blood flow path length (L) to the blood flow path diameter (D) of the porous body loaded in the plasma / serum separation filter is 0.15 to 6. It is necessary to be 0.25-4, More preferably, it is 0.5-2. When L / D is less than 0.15, the blood flow path length is too short, the moving distance of each component in blood is short, and there is not much difference in the moving distance between blood cells and plasma / serum. Is large, so that the movement speed of each component in the blood becomes uneven in the horizontal direction.
Insufficient separation of serum components. When L / D is greater than 6, the separation efficiency is increased, but the moving distance is increased, so that the passage resistance when blood flows increases, and erythrocyte hemolysis is likely to occur.

In the present invention, the flow path length (L) is defined as the length from the surface of the porous body through which blood is supplied to the porous body to the surface of the porous body through which the permeate flowing toward the container outlet flows out of the porous body. It means the shortest distance. For example, when a conical porous body is used, the height of the cone is defined as the flow path length (L).

The diameter of the flow path (D) is used when the surface of the porous body through which blood is supplied to the porous body via the inlet of the container is circular. Is used. Depending on the shape of the porous body, for example, when the liquid is sent from the outer peripheral portion of the disc-shaped porous body toward the center, or when the liquid is sent from the bottom portion of the conical porous body toward the apex. In some cases, the equivalent circle diameter of the cross-sectional area of the blood flow channel changes depending on the location (according to the flow of the blood flow channel). It is defined as the circle equivalent diameter of the area of the surface of the porous body in contact. Also, the inflowing blood once accumulates in the space between the porous body and the inner wall of the container, and then is pressurized,
It is considered that the material is uniformly supplied to the surface of the porous body in an instant.

The flow path length (L) is preferably 5 to 100 m
m, more preferably 10 to 100 mm. If the flow path length is shorter than 5 mm, the separation of blood cells and plasma / serum tends to be insufficient. A longer flow path length is preferred because the separation efficiency is increased, but on the contrary, the passage resistance is increased and there is a risk of hemolysis. In addition, a larger filter is required, so that a larger amount of blood is required. Therefore, it is preferably 100 mm or less. The flow path diameter (D) is not uniquely determined because the optimum value changes depending on the change in the flow path length, but is preferably 2 to 100 mm, more preferably 5 to 100 mm.

In the present invention, as described above, the red blood cells and the plasma / serum are separated by utilizing the difference in the moving distance at the same point caused by the difference in the moving speed of the red blood cells and the plasma / serum. If the path length changes depending on the location of the porous body, the location where the plasma / serum reaches the outlet changes in the porous body, and the separation efficiency tends to decrease. For this reason, it is desirable that the blood flow path length be the same in all portions of the porous body.

The material of the container used for the filter of the present invention is not particularly limited. For example, polyethylenes, polypropylenes, nylons, polycarbonates, polystyrenes, polyesters, ABS (acrylonitrile-butadiene-styrene) resins, plastics such as acrylics, and the like are preferred from the viewpoints of workability, resistance to cracking, and light weight. When observing the internal state,
What is necessary is just to select a translucent material.

As the shape of the porous body, for example, a cylindrical or prismatic shape, one end face (bottom face) to the opposite end face (bottom face)
In the case where blood flows in a shape such that the flow path of the blood flow and the area of the blood flow path are constant, such as in the case of flowing blood, when the blood flows from the outer periphery of the disk toward the center, and in the conical shape, from the bottom to the apex As in the case of flowing blood toward the blood flow, a shape or the like in which the blood flow area decreases as the blood flow proceeds, but is not limited thereto.

When the filter of the present invention is used, for example, for separating blood plasma and serum for clinical examination, it is desirable to reduce the amount of blood used. Therefore, the shape of the porous body () is more preferable.

The three-dimensional porous body has a disk shape, the inlet of the container is configured to supply blood over the entire circumference of the three-dimensional porous body, and the outlet of the container has a disk shape. A filter configured so that plasma or serum flows out from the center of the one-dimensional porous body has the highest separation efficiency and is particularly preferable.

In the plasma or serum separation filter of the present invention, the hematocrit allows fresh bovine blood of 40 to 50% to flow from the inlet of the filter to 0.4 to 0.6.
When a pressure difference of kg / cm ² is applied between the inlet and outlet of the filter to collect plasma or serum, the amount of plasma or serum obtained is preferably 10 to 50% of the pore volume of the porous body. More preferably, the amount of plasma or serum obtained is 15 to 50% of the pore volume of the porous body.

When the amount of plasma / serum obtained is less than 10% of the pore volume of the porous body, even if plasma / serum is obtained simply and with high safety, the amount of plasma / serum necessary for a clinical test is obtained. This requires a large amount of blood.

It is preferable that the amount of plasma / serum obtained is large because the amount of processed blood can be reduced accordingly. However, since the hematocrit of blood is usually 40 to 50%, theoretically, the maximum amount of plasma / serum collected is Is 50 of the processed blood volume
~ 60%. Of course, if blood with low hematocrit is used, the amount of plasma and serum collected is large, and if blood with high hematocrit is used, the amount of plasma and serum collected will be small,
In the present invention, blood having a normal hematocrit value is assumed.

The hematocrit is measured by a usual hematocrit measuring method, which is measured by centrifugation using a capillary for hematocrit.

The pressure difference between the inlet and the outlet of the filter may preferably be in the range of 0.01 to 5 kg / cm ² , as described later. The case where the pressure is 0.6 kg / cm ² is described. In addition, the method of giving a pressure difference between the entrance and the exit of the filter and the method of measuring the pressure difference will be described later.

Further, in the plasma or serum separation filter of the present invention, a fresh bovine blood having a hematocrit of 40 to 50% is introduced from the inlet of the filter to 0.4 to 50%.
When a pressure difference of 0.6 kg / cm ² is applied between the inlet and the outlet of the filter to separate plasma or serum, and from the start of separation, an amount of plasma or serum corresponding to 10% of the pore volume of the porous body is recovered. It is preferable that the concentration of the electrolyte in the separated plasma or serum is 90 to 110% as compared with the concentration of the electrolyte in the plasma or serum obtained by centrifugation.

It is preferable that the concentration of the electrolyte after the filter separation is within the range of 90 to 110% of the concentration of the electrolyte when centrifuged, in view of the measurement accuracy of the biochemical test, the reliability of the biochemical diagnosis, and the like. The conditions for centrifugation are as follows:
000 G for 10 minutes.

The electrolyte concentration is measured with an ion electrode of each electrolyte (ion).

Further, in the plasma or serum separation filter of the present invention, a fresh bovine blood having a hematocrit of 40% to 50% flows into the filter from the inlet of the filter by 0.4%.
When a pressure difference of ~ 0.6 kg / cm ² is applied between the inlet and outlet of the filter to separate plasma or serum and to recover an amount of plasma or serum equivalent to 10% of the pore volume of the porous material from the start of separation The protein concentration in the separated plasma or serum is preferably 90 to 110% as compared with the protein concentration in the plasma or serum obtained by centrifugation.

It is preferable that the protein concentration after the filter separation is in the range of 90 to 110% of the protein concentration when centrifuged, from the viewpoint of the measurement accuracy of the biochemical test, the reliability of the biochemical diagnosis, and the like. The conditions for centrifugation are as follows:
000 G for 10 minutes.

In the protein concentration, the total protein concentration is measured by the Burette method, and the albumin concentration is measured by the BCG (bromcresol green) method.

A plurality of filters of the present invention may be used by connecting them in series. For example, the filters may be connected in series to improve the separability between blood plasma components and blood cells, and may be connected in parallel to increase the amount of processing solution. Just connect them. Further, the present filter can be combined with a sampling container and used as an integral type. In this case, the present invention can be easily applied to an automatic analyzer or the like having a mechanism for automatically flowing a fixed amount of a blood sample into the plasma or serum separation filter unit of the present invention and automatically changing the filter. is there.

The method for separating plasma or serum of the present invention is characterized in that the filter (the three-dimensional porous body having an average pore diameter of 5 to 50 μm is formed by dividing the blood flow path length (L) with respect to the blood flow path diameter (D))
(A filter loaded into a container having an inlet and an outlet so that the ratio (L / D) is 0.15 to 6), and the average linear velocity of blood treatment is 1 to 300 cm / min. It is characterized by.

That is, the plasma using the filter of the present invention
In the serum separation method, the average processing linear velocity (linear velocity) of blood needs to be 1 to 300 cm / min, preferably 2 to 150 cm / min, more preferably 3 to 75 cm / min.
Minutes. When the average processing linear velocity of blood is less than 1 cm / min, the time for blood to move through the porous body is long,
It takes too much time to obtain plasma / serum, and the diffusion of each component during movement makes the separation of the plasma / serum component and the blood cell component insufficient. Average processing linear velocity of blood is 300c
When it is higher than m / min, the time for obtaining plasma / serum can be shortened, but the passage resistance increases and hemolysis may occur.

Incidentally, the average processing linear velocity of blood refers to the average velocity at which blood passes through the porous body. The average blood processing linear velocity is calculated by dividing the blood flow path length (L) by the time from when blood enters the porous body until the permeate flows out. For example, when liquid is sent from the disk-shaped outer peripheral portion toward the center (FIG. 1), the linear velocity varies depending on the location. In such a case, the average processing linear velocity is determined as described above. The average processing linear velocity of the blood can be adjusted by changing the porosity and the pore volume of the porous body, changing the applied pressure, and the like.

In the method for separating plasma / serum using the filter of the present invention, a pressure is applied to cause blood to pass through a porous body, and the difference between the moving speeds of blood cells and blood plasma / serum is utilized. Is a method of separating

The pressure difference between the inlet and the outlet of the filter cannot be unambiguously determined because it depends on the shape of the filter, the porosity of the porous body, and the average linear processing velocity of blood.
preferably kg / cm ^2, more preferably ^{0.05~3kg / cm 2, 0.1~1kg / cm} 2 is particularly preferred.

When the pressure difference is less than 0.01 kg / cm ^2, the load for blood to pass through the porous body is small. For example, when a highly hydrophobic material is used for the porous body, It cannot be sent into the body, it takes too much time until the average processing linear velocity decreases and plasma and serum are obtained, and there is no difference in the moving speed between plasma and serum and blood cells in the porous body, Plasma and serum tend to be insufficiently separated. When the pressure difference is larger than 5 kg / cm ² , the time for obtaining plasma / serum can be shortened, but the average processing linear velocity of blood is too fast, and the difference in permeation time between plasma / serum and blood cells is reduced. Due to the short length, the separation of plasma and serum becomes insufficient, the blood cell components are damaged to cause hemolysis, and plasma and serum cannot be collected, and the apparatus and the filter tend to be easily damaged.

Examples of a method of applying a pressure difference between the inlet and the outlet of the filter include a method of extruding blood with a pump or pressurized air from the inlet of the filter, a method of aspirating the filter at the outlet of the filter by reducing the pressure, or a method of adding the inlet. Examples of the method include a combination of pressure and outlet pressure reduction, but are not particularly limited. The pressure difference between the inlet and outlet of the filter is
Use the level gauge reading of the pump or suction device.

The blood applied to the present invention is not particularly limited, and any blood containing blood components can be used. The origin of blood may be anything, such as humans, cows, goats, dogs, rabbits and the like. Further, even if blood is used as it is, an additive such as an anticoagulant (eg, ACD solution (blood preservation solution), heparin, EDTA (ethylenediaminetetraacetic acid), etc.) or a hemagglutinating agent (eg, lectin, antibody, etc.) is added. May be used. Normally, when blood is left without adding additives or when a coagulant is added, fibrinogen in the blood changes to fibrin and blood coagulation proceeds, but these coagulable blood is used as it is. Alternatively, a product obtained by adding a process such as centrifugation to the product or a product obtained by adding a chemical process may be used.

Although plasma is obtained by removing only blood cell components from blood, in the present invention, it is assumed that blood cells do not substantially contain blood cell components. This is because even a plasma obtained by centrifuging blood cannot completely prevent a small amount of red blood cells, white blood cells, platelets or blood cell debris from being mixed. Here, the term "substantially free from blood cell components" refers to a substance obtained by removing 99.9% or more of blood cells in blood before separation. If blood cells are removed to such an extent, the effects of blood cells will not appear in the clinical laboratory data of the obtained plasma.

Serum corresponds to serum obtained by removing blood coagulation factors such as fibrinogen from plasma, but in the present invention, it is assumed that the serum does not substantially contain fibrinogen. Here, what does not substantially contain fibrinogen means:
It means that it is not detected by cellulose acetate electrophoresis.

[0069]

According to the present invention, the mechanism of separating the blood cell component and the plasma / serum component utilizes the difference in the moving speed of the two components in the pores of the porous material. Since blood and serum components move faster in the pores of the porous material than blood cells, blood is supplied to the porous material and a pressure difference is created. Arrives, and then the blood cell component arrives. By utilizing this time difference, plasma and serum can be obtained from blood.

That is, in the present invention, the greater the difference in the moving speed between the plasma / serum and the blood cells in the porous body, the larger the amount of plasma / serum obtained, but the final permeate outlet side Specifically, since blood cells penetrate, the concept is fundamentally different from conventional centrifugation using a specific gravity difference, membrane separation using the principle of sieving, leukocyte separation using the principle of adsorption, and the like.

Although gel chromatography, electrophoresis and the like are relatively similar in principle, the former uses the difference in the movable space in the gel, and the larger the molecular weight, the smaller the movable space. Differs in that it flows out earlier,
The latter is identical in that the difference in steric hindrance due to the gel structure is utilized, but differs in that the present invention moves the separation component by pressure, whereas in the electrophoresis, the separation component moves by Coulomb force.

As described above, unlike the centrifugal separation and the membrane separation, the present invention is not suitable for long-time processing or large-scale processing.
In a field such as a clinical test that requires a small amount of blood to be separated into plasma or serum in a short time, it can be said that this is an optimal means for separating plasma or serum. Furthermore, compared to the case of using a microfiber non-woven fabric, there is no need to stack or unravel the sheets, so it is excellent in assemblability and can be formed into a desired shape.
Since it has excellent processability and has uniform pores, it is possible to prevent performance degradation due to blood channeling and clogging and occurrence of hemolysis.

[0073]

EXAMPLES The present invention will now be described specifically with reference to examples, but the present invention is not limited thereto.

Example 1 A plasma / serum separation filter as shown in FIG. 1 was prepared [FIG. 1 (b) is a view of the filter of FIG. In FIG. 1B, the inside is shown by being partially broken, and the porous body 3 is hatched. That is, a hole having a diameter of 1.0 mm is provided at the end of the upper surface of the container as the inlet 1, a hole having a diameter of 1.0 mm is provided at the center of the bottom as the outlet 2, and a diameter of 52.0 mm inside the container.
In a 2.0 mm-thick acrylic disc-shaped container 4, a polyvinyl formal sponge having an average pore diameter of 12 μm and a porosity of 86% as a porous body 3 having a diameter of 50.0 mm and a thickness of 2.0 was used.
1.0 m between the inner peripheral surface of the container and
m. The pore volume of the porous body at the time of filling was 3.4 ml. The cross-sectional area of the blood inlet part of the container (corresponding to the side area of the disc-shaped porous body) is 2 mm x 50 m
m × π = 100πmm ² , the equivalent circle diameter is 20 mm, and the blood flow path length is 25 mm (half of the diameter of the porous body 50 mm), so that L / D is 25/20 (= 1.25).
It is.

An ACD solution as an anticoagulant was added to the blood, and bovine blood having a hematocrit of 45% was fed from the outer periphery of the container to the inside at a pressure of 0.5 kg / cm ² . Blood passes through the porous body in a horizontal direction (perpendicular to the side surface of the porous body) with respect to the bottom surface of the porous body from the outer periphery of the porous body toward the center in the container, and after about 5 seconds. A permeate was obtained from the outlet. The permeate started to be mixed with blood cells after about 6 seconds. The permeated liquid without blood cell contamination could be collected for 5 to 1 second after pressurization, and the total amount was 0.7 ml. At this time, the average processing linear velocity in the porous body determined from the collected permeate was 30.
cm / min.

The hemoglobin concentration of the obtained permeate was 3 m
Hemolysis was not observed below g / dl. In addition, all red blood cells, white blood cells, and platelets measured by a hemacytometer were below the detection limit. Biochemical analysis value of the obtained permeate [measured by a biochemical automatic analyzer (manufactured by Hitachi); in this apparatus, the total protein concentration is the Burette method, the albumin concentration is the BCG method, and the electrolyte concentration is each electrolyte (ion). Is shown in Table 2, but no significant difference was observed with the analysis value of plasma obtained by centrifuging the same blood. Also,
When the concentration of fibrinogen in the obtained permeate was determined by cellulose acetate electrophoresis, it was reduced to about 50% of the concentration in plasma obtained by centrifugation, but was not completely removed. Further, when left overnight, no precipitation of fibrin was observed.

Example 2 The same filter as used in Example 1 was used. Without adding an anticoagulant to this blood, human blood having a hematocrit of 47% immediately after blood collection was supplied at a pressure of 0.5 kg / cm ² . Blood flows from the outer periphery to the center in the container,
After passing through the porous body in the horizontal direction to the bottom surface of the porous body (the direction perpendicular to the side surface of the porous body), a permeate was obtained from the outlet after about 5 seconds. The permeate started to be mixed with blood cells after about 7 seconds. The permeated liquid without blood cell contamination could be collected for 5 to 2 seconds after pressurization, and the total amount was 1.0 ml. At this time, the average processing linear velocity in the porous body determined from the collected permeate was 3
It was 0 cm / min.

The hemoglobin concentration of the obtained permeate was 3 m
Hemolysis was not observed below g / dl. In addition, all red blood cells, white blood cells, and platelets measured by a hemacytometer were below the detection limit. Table 2 shows the biochemical analysis values of the obtained permeate.
As shown in the figure, there was no significant difference from the analysis value of serum obtained by centrifuging the same blood after coagulation. In addition, fibrinogen was not detected in the obtained permeate, and the obtained permeate was serum instead of plasma. The concentration of fibrinogen in the plasma obtained by centrifuging blood from the same person to which heparin was added as an anticoagulant was 220 mg / dl.

Although the experiment was carried out in the same manner as in Example 1 except for the blood used, a difference was observed in the amount of permeate that could be collected because the size of red blood cells was different between human and cow. it is conceivable that. Further, it is considered that fibrinogen was completely removed because the anticoagulant was not added and fibrinogen was adsorbed to the filter.

Example 3 An experiment was conducted in the same manner as in Example 1 except that cellulose having an average pore diameter of 10 μm and a porosity of 83% was used as a porous body. As a result, a permeate free of blood cells was obtained for about 4 seconds from about 15 seconds later, and the amount was 0.8 ml. At this time, the average processing linear velocity of the blood in the porous body was 10 cm /
Minutes. The hemoglobin concentration of the obtained permeate was 3 m
Hemolysis was not observed below g / dl. In addition, all red blood cells, white blood cells, and platelets measured by a hemacytometer were below the detection limit. Table 2 shows the biochemical analysis values of the obtained permeate.
As shown in the figure, there was no significant difference from the analysis value of the plasma obtained by centrifuging the same blood. Further, the fibrinogen concentration in the obtained permeate was reduced to 15% of the plasma obtained by centrifugation.

Comparative Example 1 An experiment was conducted in the same manner as in Example 1, except that cellulose having an average pore diameter of 2 μm and a porosity of 80% was used as a porous body. As a result, the filter was clogged, and a permeate could not be obtained. This is considered to be because the average pore diameter was 2 μm and the pore diameter was too small, so that the blood cell component clogged the pores.

Comparative Example 2 An experiment was conducted in the same manner as in Example 1 except that a polyvinyl formal sponge having an average pore diameter of 60 μm and a porosity of 91% was used as a porous body. As a result, the permeate from the filter contained erythrocytes from the beginning, and plasma / serum could not be separated. It has an average pore size of 60 μm,
It is considered that there was no sufficient difference in the movement speed between red blood cells and plasma / serum, and separation was not possible.

Comparative Example 3 A plasma / serum separation filter as shown in FIG. 2 was prepared [FIG. 2 (b) is a view of the filter of FIG. In FIG. 2 (b), the inside is shown partially broken, and the porous body 3 is hatched.) That is, in the container 4, the effective cross-sectional shape is 50.0 mm, the thickness is 5 mm, and the average pore diameter is 1 as the porous body 3.
A polyvinyl formal sponge having a porosity of 2 μm and a porosity of 86% was filled. L / D at this time is 5/50 (= 0.1)
It is. Using this filter, bovine blood of 45% hematocrit to which ACD solution was added was applied at a pressure of 0.5 kg / cm ^2.
Through the filter. As a result, a permeate was obtained after 10 seconds, but this permeate contained erythrocytes from the beginning, and plasma / serum could not be separated. The cause is L
It is considered that / D was 0.1, the moving distance of blood was short, and the separation of red blood cells and plasma / serum was not sufficiently performed.

Comparative Example 4 A plasma / serum separation filter as shown in FIG. 3 was prepared [FIG. 3 (b) is a view of the filter of FIG. In FIG. 3 (b), the inside is shown partially broken, and the porous body 3 is hatched.) That is, the container 4 was filled with a polyvinyl formal sponge having an effective sectional shape of 10.0 mm, a thickness of 100.0 mm, an average pore diameter of 12 μm, and a porosity of 86% as the porous body 3. L / D at this time is 100/10
(= 10). Using this filter, hematocrit 45% bovine blood to which ACD solution was added was applied at a pressure of 0.5k.
g / cm ² through the filter. As a result, a permeate was obtained after 5 minutes, but the obtained permeate was hemolyzed, and the hemoglobin concentration was 50 mg / dl. It is considered that the cause is that L / D is as large as 10, the resistance of blood to pass through the porous body increases, and red blood cells are destroyed.

The methods for measuring the physical properties in the above Examples and Comparative Examples are described below. Average pore size The porous body was cut in a direction perpendicular to the blood flow direction, and an electron micrograph of the cross section was taken.
The diameter of the pores in a certain area where 00 pores are present was measured and determined by arithmetic averaging. When the shape of the pore cross section is not circular, the pore cross section is obtained by image processing or the like,
The equivalent diameter of the circle corresponding thereto was defined as the pore diameter (diameter).

[0086] was calculated as Porosity _{(1-r m / ρ)} × 100 (%). Incidentally, r _m is the bulk density of the porous body, [rho is the density of the porous material. Pore volume Calculated as (porous body volume × porosity) × 1/100.

Average Processed Linear Velocity The blood flow path length (L) was calculated by dividing the time from when blood entered the porous body to when the permeate flowed out. Pressure difference The indicated value of the level gauge of the pump or suction device was used.

Table 1 shows the conditions and the like in the above Examples and Comparative Examples.
Shown in Table 2 shows the biochemical analysis values of the permeate obtained in Examples 1 to 3 above.

[0089]

[Table 1]

[0090]

[Table 2]

[0091]

According to the plasma / serum separation filter of the present invention, high-purity plasma / serum can be obtained quickly even with a small amount of blood, assemblability and operability are simple, and safety is improved. It is possible to perform high plasma / serum separation. That is, if the plasma / serum separation filter of the present invention is used, it is possible to quickly, easily, and safely remove blood cell components from a test solution containing blood components in other studies such as clinical tests. Yes, and it is possible to separate and remove cells in the cell culture.

[Brief description of the drawings]

FIG. 1 is a view showing one embodiment of a plasma / serum separation filter of the present invention.

FIG. 2 is a diagram showing an example of a filter of Comparative Example 3.

FIG. 3 is a diagram showing an example of a filter of Comparative Example 4.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Inlet 2 Outlet 3 Porous body 4 Container

 ──────────────────────────────────────────────────の Continued on the front page (72) Takashi Hayashi 2-1-1 Katata, Otsu City, Shiga Prefecture, Toyobo Co., Ltd.

Claims (11)

[Claims] 1. A three-dimensional porous body having an average pore diameter of 5 to 50 μm has a blood flow path length (L) with respect to a blood flow path diameter (D).
A plasma or serum separation filter, which is loaded in a container having an inlet and an outlet so that the ratio (L / D) of the filter is 0.15 to 6. 2. The porosity of the three-dimensional porous body is 20 to 95%.
The plasma or serum separation filter according to claim 1, wherein: 3. The three-dimensional porous body has a disk shape, the container inlet is configured to supply blood over the entire circumference of the three-dimensional porous body, and the container outlet has a disk shape. The plasma or serum separation filter according to claim 1 or 2, wherein the filter is configured so that plasma or serum flows out from the center of the one-dimensional porous body. 4. The plasma or serum separation filter according to claim 1, wherein a hydrophilic agent is fixed to the three-dimensional porous body. 5. The plasma or serum separation filter according to claim 4, wherein the hydrophilizing agent is fixed on the surface of the three-dimensional porous body. 6. The plasma or serum separation filter according to claim 4, wherein the hydrophilizing agent is polyvinylpyrrolidone. 7. A fresh bovine blood having a hematocrit of 40 to 50% is introduced from an inlet of the filter to obtain a 0.4 to 0.
When a pressure difference of 6 kg / cm ² is applied between the inlet and outlet of the filter to collect plasma or serum, the volume of the collected plasma or serum is 10 to 5 times the pore volume of the three-dimensional porous body.
The plasma or serum separation filter according to any one of claims 1 to 6, which is 0%. 8. A fresh bovine blood having a hematocrit of 40 to 50% is introduced from an inlet of the filter to a 0.4 to 0.5%.
When a pressure difference of 6 kg / cm ² is applied between the inlet and outlet of the filter to separate plasma or serum, and when the amount of plasma or serum corresponding to 10% of the pore volume of the three-dimensional porous body is recovered from the start of the separation, 8. The method according to claim 1, wherein the concentration of the electrolyte in the separated plasma or serum is 90 to 110% as compared with the concentration of the electrolyte in the plasma or serum obtained by centrifugation. Plasma or serum separation filter. 9. A filter having a hematocrit of 40-50% of fresh bovine blood flowing through the inlet of the filter, and having a hematocrit of 0.4-50%.
When a pressure difference of 6 kg / cm ² is applied between the inlet and outlet of the filter to separate plasma or serum, and when the amount of plasma or serum corresponding to 10% of the pore volume of the three-dimensional porous body is recovered from the start of the separation, The protein concentration in the separated plasma or serum is 90 to 110% as compared with the protein concentration in the plasma or serum obtained by centrifugation. Plasma or serum separation filter. 10. A three-dimensional porous body having an average pore diameter of 5 to 50 μm has a ratio (L / D) of a blood flow path length (L) to a blood flow path diameter (D) of 0.15 to 6. A method for separating plasma or serum, wherein a filter for separating plasma or serum loaded in a container having an inlet and an outlet is used, and the average linear processing speed of blood is 1 to 300 cm / min. 11. The pressure difference between the filter inlet and outlet is 0.0
11. The pressure is 1 to 5 kg / cm ^2.
The method for separating plasma or serum according to the above.