JP2003075434A - Plasma separation method and apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and an apparatus for efficiently taking out plasma from an extremely small amount of blood that is less than 5 μL. SOLUTION: Blood is developed by a filter 50 comprising filtering films A and B for separating into globule and plasma on the filter 50. Then, the area between a region where the globule is located in the filter 50 and a region where the plasma is located is shut out by an edge-cutting plunger 11, and then only the region where the plasma in the filter 50 is located is pushed out for compression by a plunger 21, thus taking out plasma from the filter 50.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plasma separation method and a plasma separation apparatus for separating serum or plasma from a trace amount of blood.

[0002]

2. Description of the Related Art Analysis for bedside diagnosis (POC (point of care)) in which measurement necessary for medical diagnosis is performed near the patient.
Analysis) and analysis of harmful substances in rivers and wastes at rivers and waste disposal sites, food preparation, harvesting,
Attention is focused on the importance of performing analysis / measurement at or near the site where analysis / measurement is required, such as contamination inspection at each import site (hereinafter collectively referred to as "POC analysis"). Therefore, in recent years, development of a detection method and a device applied to such POC analysis has been emphasized.

In such POC analysis and the like, it is required that analysis can be performed easily, in a short time, and at low cost. Especially, in analysis for medical diagnosis, analysis time is shortened and a sample required for analysis is required. Minimizing the amount is an important issue. For example, in a blood test, if the amount of sample required for analysis is small, the amount of blood collected can be reduced, thus reducing the burden on the patient and expanding the possibility of application to home medical care such as self-analysis by self-collecting blood.

However, when biochemical diagnosis is performed on blood, serum or plasma is used as a sample.
Need to remove blood cells and blood coagulation factors from the blood,
In the case of plasma, it is necessary to remove blood cells from the blood. In the present specification, unless otherwise specified, the term "plasma" also includes serum. Therefore, POC
When measuring blood components at the site where analysis / measurement is required in analysis, etc., an inexpensive, quick and simple blood pretreatment method or a method that can directly measure plasma components with the blood itself is required. Becomes A blood glucose meter has been put into practical use in the latter case, but most of the biochemical diagnostic items still use serum or plasma separated from blood.

As an example of analyzing a blood component by using a small amount of blood, Japanese Patent Publication No. 53-21677 discloses that an analysis mechanism is provided with a filter layer to remove blood cells from the blood.
Further, Japanese Patent No. 2618727 describes a method of quantifying a test component by treating a blood sample of about 5 μL to 30 μL with a blood cell separation layer in an analysis element.

[0006]

However, the above-mentioned conventional example is not related to the treatment of blood less than 5 μL, and is not used for the measurement of a plurality of items from the shape of the analysis mechanism described. That is, in a dry chemistry system in which a blood cell removal layer and a reaction layer are integrated, it is essential to add a mechanism for distributing serum or plasma in performing measurement of a plurality of items, and at present, measurement of a plurality of items is not possible. It is possible. Further, in order to prevent the deviation of the detected value due to the color derived from red blood cells, a light shielding substance is included between the blood cell filtration mechanisms or the blood cell filtration layer is removed at the time of measurement (Japanese Patent Laid-Open No. 61-96466). Issue). However, it has not been possible to separate the serum or plasma from the target micro blood by the above-mentioned operation and to quantify the measurement objects of a plurality of items.

Further, as an example of extracting plasma from blood by performing volumetric filtration, Japanese Patent Laid-Open Nos. 9-196911, 11-194126 and 11-2952 are cited.
Examples thereof include those described in Japanese Patent Laid-Open No. 99 and Japanese Patent Laid-Open No. 2000-180444. Note that volumetric filtration is a function in which a liquid containing particles permeates in the thickness direction of the filtration membrane and sequentially captures smaller particles than larger particles due to the structure inside the membrane. It is to capture and remove particles having various particle sizes contained therein.

However, in the one described in the above publication, since the transfer of blood is performed by the compressive fluid, in order to process a very small amount of blood by reducing the separation mechanism, a delicate pressure difference is applied. There is a drawback that adjustment is necessary and it is expensive to complete the mechanism. In recent years, attention has been focused on the technical field of analyzing an extremely small amount of sample by applying a semiconductor integrated circuit manufacturing technology, and an analysis system called such a micro total analysis system (hereinafter referred to as μTAS) is used for the POC. Attempts have been made to apply it to analysis and the like.

For example, in International Publication WO99-64846 and International Publication WO2001-13127,
Application to medical diagnosis is disclosed. In addition, JP-A-5-
Regarding the drawback of the wet chemistry method described in Japanese Patent No. 80049, that is, the large amount of blood collected and the labor of adjusting and supplying serum or plasma by centrifugation, an extremely small amount (several μL level) of plasma can be obtained by using in combination with μTAS technology. Or it is known that serum is sufficient. In order to utilize this technique, it is necessary to extract the separated trace amount of plasma as a liquid.

As a practical method for separating a trace amount of plasma, there is a method of treating with air as a medium using a pressure pump or a vacuum pump with a filtration membrane, or a method of treating with a small centrifuge. . However, since the required blood volume is 0.5 mL to several mL, an inexpensive mechanism that can easily separate plasma from an extremely small amount of blood is desired.

Therefore, it is an object of the present invention to solve the problems of the prior art as described above and to provide a method and apparatus for extracting plasma as a liquid from an extremely small amount of blood of less than 5 μL.

[0012]

In order to solve the above-mentioned problems, the present invention has the following constitution. That is, the plasma separation method of the present invention develops blood with a separation member composed of a filtration membrane, separates blood cells and serum or plasma on the separation member, and the blood cells of the separation member are positioned. After blocking the movement of the blood cells and the movement of the serum or plasma between the region where the blood serum and the plasma are located, the serum or plasma of the separation member is It is characterized in that only the located region is squeezed to take out the serum or the plasma from the separating member.

Further, the plasma separating apparatus of the present invention comprises a separating member composed of a filtration membrane for expanding blood to separate into blood cells and serum or plasma, and a region of the separating member where the blood cells are located. And a region where the serum or plasma is located, an edge cutting means for blocking movement of the blood cells and movement of the serum or plasma between the region, and the serum or plasma of the separation member. And a squeezing unit for squeezing only the region located to extract the serum or plasma from the separating member.

In such a plasma separation method and plasma separation apparatus, blood is filtered by the separation member to separate blood cells from serum or plasma, and only the portion of the separation member that holds serum or plasma is squeezed. Serum or plasma can be efficiently extracted as a liquid (10% or more of serum or plasma contained in blood) from an extremely small amount of blood of less than 5 μl.

Further, if the thickness of the separating member is made to be gradually smaller in the direction of blood development, serum or plasma can be taken out without containing air bubbles. Further, the separation member is composed of two or more layers of filtration membranes, of which the uppermost filtration membrane captures blood cells and the lowermost filtration membrane holds serum or plasma. The ratio of Vu to the volume Vl of the lowermost filtration membrane (Vu / V
When l) is set to 1 to 3, serum or plasma can be taken out without containing air bubbles. The ratio is 1 to
2 is more preferable and 1 to 1.5 is even more preferable.

Therefore, by combining the plasma separation method and the plasma separation apparatus of the present invention with the μTAS technique, it is possible to perform POC analysis and the like on a plurality of items of blood components.

[0017]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of a plasma separation method and a plasma separation apparatus according to the present invention will be described in detail with reference to the drawings. The present embodiment shows an example of the present invention, and the present invention is not limited to this embodiment. The plasma separation device of the present embodiment includes a filter made of a filtration membrane, a filter holder for storing the filter,
It consists of an edging plunger that divides the filter into two regions, and an extrusion plunger that squeezes the filter to remove plasma. The filter corresponds to the separating member which is a constituent of the present invention, the edging plunger corresponds to the edging means which is a constituent of the present invention, and the pushing plunger corresponds to the squeezing means which is a constituent of the present invention.

[Filtration Membrane and Filter] Any membrane can be applied to the filtration membrane as long as it is a membrane capable of volumetric filtration. For example, a microporous membrane having a fibril structure or a sheet formed by processing ultrafine fibers can be used. As the material, polymer fibers such as cellulose, polyethylene, polypropylene, polyester, polyamide and glass fibers are preferable. It is possible to use a commercially available filter membrane generally used for blood cell separation.

The filter may be composed of a single-layer filtration membrane, but it is preferable to be composed by laminating two or more filtration membranes having substantially the same water permeability. By doing so, blood is filtered in the stacking direction and separated into blood cells and plasma, the blood cells are captured by the upper filter membrane, and the plasma reaching the lower filter membrane is developed in the plane direction of the filter membrane. Therefore, it is possible to further reduce the bubbles in the collected plasma.

In the case where the filter is composed of a single-layer filtration membrane, if the shape of the filtration membrane is such that the thickness gradually decreases in the direction of permeation of plasma, the same as above. The effect is obtained. When various kinds of reactions are carried out by moving a very small amount of liquid in the flow channel, the presence of bubbles causes disturbance of liquid movement. Therefore, in order to enable analysis by μTAS, it is required that the plasma used does not contain bubbles.

The amount of blood spotted on the filtration membrane is less than 80% of the volume of the filtration membrane, more preferably less than 70%, even more preferably less than 60%. The blood spotted on the upper surface of the filtration membrane permeates the filtration membrane due to the capillary phenomenon, and the lower surface is filled with the liquid. At this time, blood cells are mainly captured in the upper part of the filtration membrane, and plasma is mainly retained in the lower part. The ratio (Vu / Vl) of the volume Vu of the upper part that captures blood cells to the volume Vl of the lower part that holds plasma is 1
-3 are preferable, 1-2 are more preferable, and 1-1.5 are especially preferable. The reason for this is to eliminate the infiltration of blood cells into the lower part and to allow the plasma to penetrate to the lower surface.
That is, when the ratio exceeds 3, the plasma develops slowly, and when it is less than 1, the rate of blood cells reaching the lower part increases and the recovery rate of plasma decreases.

Further, the size of the filtration membrane can be changed according to the volume of blood to be treated. For example, when treating 50 μL of blood, a filtration membrane having a volume about 50 times that of treating 1 μL of blood may be used. The planar shape of the filtration membrane is not particularly limited, such as a quadrangle, a triangle, and a circle, as long as the liquid can be pushed out from the portion holding the plasma.

In the case where two or more layers of filter membranes are laminated to form a filter, there is no particular need for adhesion between the filter membranes as long as the space allows the liquid to penetrate. However, the bonding may be performed by a generally known bonding method so as not to disturb the capillary action of the liquid. Further, an anticoagulant may be applied to the filtration membrane for use. Anticoagulants include heparin, citrate, ethylenediaminetetraacetic acid (EDTA)
Etc. The anticoagulant treatment of the filtration membrane can be performed, for example, by immersing the filtration membrane in a solution anticoagulant and drying.

Further, a member that is easily deformed (hereinafter referred to as a peripheral member) so as to facilitate blocking between the region where the blood cells are captured and the region where the plasma is held in the filtration membrane.
Is preferably arranged around the filtration membrane. The type of material of such a member is not particularly limited as long as the liquid does not permeate and is easily deformed. For example, a closed-cell foam is suitable because it has excellent deformability.

As the specific closed-cell foam, a resin foam such as a polyethylene foam, a polypropylene foam or a rubber foam is preferable, and a soft foam is more preferable. Further, a soft foam having a small cell size is preferable because the blocking can be performed with a weak force. The bubble diameter is preferably 300 μm or less, more preferably 200 μm or less, and particularly preferably 100 μm or less.

[About Filter Holder] The filtration membrane and peripheral members are housed in a container (filter holder). The material of this container is not particularly limited, and can be used without any problem as long as it can be processed into a free shape such as glass, metal and organic polymer. Preferably, a polymer material whose thickness and shape can be freely processed by molding or the like, specifically, polystyrene, styrene-
Selected from the group consisting of styrene resins such as acrylonitrile copolymer, methacrylic resins such as polymethylmethacrylate, methylmethacrylate-styrene copolymer, polycarbonate, polysulfone, polyethersulfone, polyetherimide, polyarylate, polymethylpentene. It is recommended to use a polymer

Further, a 1,3-cyclohexadiene polymer is also preferably used. As the 1,3-cyclohexadiene polymer, a homopolymer can be used, but a copolymer can also be used. Examples of this copolymer include 1,3-butadiene, isoprene, and 1,3.
-Conjugated diene-based monomers such as pentadiene and 1,3-hexadiene, vinyl aromatic-based monomers such as styrene, α-methylstyrene, p-methylstyrene, 1,3-dimethylstyrene, vinylnaphthalene and vinylstyrene, methyl methacrylate , Acrylonitrile, methyl vinyl ketone, polar vinyl monomers such as α-methyl cyanoacrylate, ethylene oxide, propylene oxide, cyclic lactones, cyclic lactams, cyclic siloxanes and other cyclic monomers, or ethylene, α-olefin-based monomers It can be united. The copolymerization ratio is 1,3-weight ratio.
Cyclohexadiene monomer / comonomer = 75/25
-100/0 is preferable.

The shape of the container is not particularly limited as long as it has a structure capable of accommodating the above-mentioned filtration membrane and capable of storing separated liquid plasma, and is flat. In this case, considering the volume required for storage, the thickness is preferably 10 mm or less, more preferably 5 mm or less. The container may be separated from the analytical element using μTAS or the like, or may be an integral type that is connected to the analytical element through a continuous flow path.

Further, when the plasma separation device is produced as a single unit, a portion for storing plasma separated from blood (hereinafter, referred to as
It is preferable to cover the plasma reservoir) with a membrane to add a structure that prevents plasma from leaking to the outside. For this film,
For membranes that allow gas to pass through but not liquid, that is, flat plates, sheets, membranes made of hydrophobic organic polymers or inorganic materials,
It is possible to use one having a large number of small holes of about 0.1 μm to 1 mm.

The average diameter of the pores of this porous membrane is 0.0
The pore size is preferably 1 to 5 μm, and the pore size is more preferably 0.05 to 0.5 μm, considering that the smaller the pore size is, the smaller the amount of permeated air is and the availability. This film is preferably adhered to the container by an adhesive method such as double-sided tape except for the area where air escapes. Then, an adhesion method that allows the blood plasma to be easily peeled off is preferable. However, this is not the case when plasma is collected from the plasma storage tank by making a hole in the membrane using a needle or the like.

The hydrophobic organic polymer preferably has a critical surface tension of about 0.04 N / m or less at 20 ° C.,
As an example, polytetrafluoroethylene (PTF
E), silicone, polyethylene, polypropylene, polystyrene, polyvinyl chloride, polycarbonate, polysulfone, polyether sulfone, polyarylate, polymethylpentene, 1,3-cyclohexadiene-based polymer and the like.

When the filtration membrane is attached to the container, it is preferable to fix the periphery of the filtration membrane in order to stably suppress the displacement of the seal portion and the exudation of blood components due to the external force acting during filtration. For this fixing, it is preferable to use a soft material, and it is advisable to use a soft adhesive agent, a sealing agent, a caulking agent, an adhesive material, or the like. Examples thereof include vinyl acetate-based, acrylic-based, olefin-based thermoplastic adhesives, rubber-based adhesives, hot-melt adhesives, emulsion adhesives, silicone-based sealing agents, acrylic-based adhesives,
Examples include rubber-based and silicone-based adhesives, and double-sided tape with adhesive on both sides.

[Regarding Edge Cutting Plunger and Extrusion Plunger] In order to separate plasma from an extremely small amount of blood, it is necessary to use a filter having a volume equal to or less than the volume of blood used. In order to separate plasma from blood without causing hemolysis, it is effective to perform volume filtration using a filtration membrane or the like. Further, in the case of moving a liquid through a compressible fluid such as gas, there is a limit in terms of fine adjustment of the pressure of the compressible fluid to satisfy the condition for avoiding hemolysis. The reason is that if a filter is made small to handle a very small amount of blood, its pressure loss is also necessarily made small.

The filtration membrane was made small, plasma was separated from an extremely small amount (4 μL) of blood, and plasma recovery was attempted using air pressure. As a result, as shown in Comparative Example 1 to be described later, the blood cells and plasma separated on the filtration membrane were mixed again at both pressurization and depressurization, and it was not possible to collect only plasma. In order to solve this problem, as a method of taking out plasma separated from a trace amount of blood as a liquid without hemolysis, the inventors have made earnest studies and found that a "squeeze" structure is effective. That is, in order to eliminate the influence of separated blood cells, a region on the filtration membrane that captures blood cells (hereinafter referred to as a blood cell region) and a region that retains plasma (hereinafter referred to as a plasma region) are separated. The inventors have invented a method and an apparatus for squeezing out by mechanically separating (hereinafter referred to as edging) and crushing a region holding plasma (hereinafter referred to as extrusion).

Since the blood cell region and the plasma region are cut off,
The separated plasma does not return to the blood cell area. Also, the edge cutting operation brings the filtration membrane into a state of being hermetically sealed in the filter holder. Then, by the pushing operation,
The plasma in the filtration membrane is transferred to the storage tank. The edging plunger that performs the edging as described above can reciprocate in the longitudinal direction, and can press the filtration membrane to divide it into two regions. That is, it is possible to crush the filtration membrane and the surrounding peripheral member located around the filtration membrane to block the blood cell region and the plasma region.

The driving force for reciprocating the edging plunger may be a spring, a motor, air pressure, etc., but any driving force capable of adjusting the pressing force may be used. Further, it is preferable that the width of the tip portion (portion that presses the filtration membrane) of the edge cutting plunger is slightly smaller than the sum of the width of the filtration membrane main body and the width of the peripheral members located around the filtration membrane body. That is, the width is wider than the width of the filtration membrane and can sufficiently suppress the peripheral members surrounding the filtration membrane.

Further, it is preferable that the thickness of the tip portion of the edging plunger is as thin as possible in order to divide the filtration membrane with a small force. Specifically, 50
It is 0 μm or less, more preferably 100 μm or less, and further preferably 50 μm or less. Furthermore, it is desirable that the surface of the tip of the edging plunger that contacts the filtration membrane be as smooth as possible. For example, when the filtration membrane is fixed to the filter holder using a double-sided tape, the smoothness equal to or less than the thickness of the double-sided tape can be preferably used.

Next, the extrusion plunger will be described. When the push-out plunger for pushing out as described above can reciprocate in the longitudinal direction like the edge-cutting plunger, the tip of the push-out plunger (portion that squeezes the filtration membrane) is plasma. It should be sized and shaped so that the entire area can be squeezed.

The tip of the push-out plunger is smoothly inclined in the direction of blood development, and the length of the push-out plunger is gradually shortened in the direction of blood development. It is preferable. When the filtration membrane is squeezed with an extrusion plunger having such a shape, the inclined surface of the tip portion squeezes the filtration membrane sequentially from the region close to the edge cutting plunger to the region distant, so that plasma can be efficiently collected. You can

When the push-out plunger can reciprocate in the expanding direction in addition to the reciprocating motion in the longitudinal direction, the push-out plunger is moved in the expanding direction while being pressed against the filtration membrane. Can be moved. Therefore, it is possible to sequentially squeeze from the region close to the edging plunger to the region far from the rim cutting plunger, and the plasma can be efficiently collected. In this case, it is preferable to make the tip of the extrusion plunger a curved convex surface or attach a roller to the tip.

The pressing pressure of the push-out plunger must be such that the plasma does not flow into the blood cell region side beyond the portion blocked by the edge-cutting plunger when the filtration membrane is pressed. Examples of the mechanism for adjusting the pressing pressure include those using an air pressure pump and a screw-in type positioning mechanism. As a squeezing means, instead of using an extruding plunger, a bag whose shape can be freely changed is attached to the edging plunger, and after the edging operation, a fluid is injected into the bag to expand and press the filtration membrane. Then, the plasma may be squeezed out.

In the present embodiment, the tip end portions of both plungers are made of aluminum alloy. However, other materials may be used as long as they have a strength to withstand the pressing pressure of each plunger. For example, as a metal material,
Iron, soft iron, aluminum, etc. are listed. Polymer materials include polycarbonate, acrylic resin, polyethylene,
Examples include polystyrene. (Examples) The present invention will be described more specifically with reference to Examples.

[Regarding Edge Cutting Device and Compressing Device] The configurations of an edge cutting device 10 that cuts between the blood cell region and the plasma region of the filtration membrane and a pressing device 20 that presses the plasma region to extract plasma are shown in FIGS. A description will be given with reference to the front view of each device shown in FIG. Edge trimming device 1 shown in FIG.
Reference numeral 0 is composed of a movable portion 12 to which an edge cutting plunger 11 is attached via a spring 14 and a main body portion 13 fixed to a surface plate or the like (not shown). And the movable part 1
2 can reciprocate up and down with respect to the main body 13. When the movable part 12 is lowered and the edging plunger 11 comes into contact with the filtration membrane, the rim cutting plunger 11 is pressed against the filtration membrane by the spring 14 so that the blood cell region and the plasma region of the filtration membrane are diced. It has become.

Further, in the squeezing device 20 shown in FIG. 2, a movable portion 22 to which a pushing plunger 21 is attached via a spring 24 and a main body portion 2 fixed to a surface plate (not shown) or the like.
3 and 3. The movable portion 22 is capable of reciprocating vertically with respect to the main body portion 23. When the movable portion 22 is lowered and the push-out plunger 21 comes into contact with the filtration membrane, the push-out plunger 2 is pushed by the spring 24.
1 is pressed by the filtration membrane, the plasma region is squeezed, and plasma is taken out.

The edge cutting device 10 and the pressing device 20
Are fixed to a surface plate (not shown) in an arrangement as shown in the plan view of FIG. In addition, the tip end shape of both plungers was wedge-shaped in the case of the edge-cutting plunger 10 as shown in the partially enlarged side view of FIG. 1B, and was rectangular parallelepiped in the case of the extrusion plunger 20.

Further, the size of the plunger was changed according to the size of the filtration membrane. For example, when 50 μL of blood is used, the edge cutting plunger 10 has a width of 10 mm,
The thickness is 100 μm and the extrusion plunger 20 has a width of 1
The thickness was 0 mm and the thickness was 5 mm. When 4 to 5 μL of blood is used, the edge cutting plunger 10 has a width of 5 mm,
The thickness is 100 μm and the extrusion plunger 20 has a width of 5
mm and a thickness of 4 mm. Further, when 1 μL of blood is used, the edge cutting plunger 10 has a width of 5 mm and a thickness of 100 μm, and the extrusion plunger 20 has a width of 5 m.
m and thickness 1 mm.

[About Filter Holder] FIG. 4A shows a plan view of the filter holder 40.
A sectional view is shown in FIG. A 2 mm-thick polymethylmethacrylate (PMMA) flat plate 41 is cut out by cutting, and a rectangular through hole 42 (width 12 m) for accommodating a filtration membrane is cut out.
m, length 20 mm). Then, a groove (depth 100 μm, which communicates with the through hole 42, is formed on one plate surface of the flat plate 41.
(Width 100 μm, length 50 mm) is provided, and the plasma outlet groove 43
And Furthermore, a diameter of 1.5 m at the end of the plasma outlet groove 43
A circular through hole of m was provided to form a plasma storage tank 44.

A 0.3 mm-thick PMMA flat plate 45 and a double-sided tape 46 (double-sided adhesive tape No. 5302A manufactured by Nitto Denki Kogyo Co., Ltd.) are used on the surface of the flat plate 41 having the plasma outlet groove 43. And pasted together to produce a filter holder 40 (thickness: 2.5 mm). [Filtration Membrane and Filter] Filtration Membrane A (What
glass filter membrane GF / D manufactured by man, thickness 1.5 mm)
And a filtration membrane B (a glass filtration membrane F48 manufactured by Whatman)
7-09, thickness 0.35 mm) was laminated to form a filter 50 (blood cell separation element). The contact portion between the two filtration membranes A and B is not adhered.

The sizes of the filtration membranes A and B were changed depending on the volume of blood to be treated. When the blood volume is 1 μL, the filtration membrane A has a width of 1 mm and a length of 0.7 mm, and the filtration membrane B has a width of 1 mm.
The length was 2 mm. When the blood volume is 4 μL, the filtration membrane A has a width of 1 mm and a length of 2.8 mm, and the filtration membrane B has a width of 1 m.
m and the length was 8 mm. Further, when the blood volume was 5 μL, the filtration membrane A had a width of 1 mm and a length of 3.5 mm, and the filtration membrane B had a width of 1 mm and a length of 10 mm. In addition, blood volume is 50
In the case of μL, the filtration membrane A had a width of 5 mm and a length of 7 mm, and the filtration membrane B had a width of 5 mm and a length of 20 mm.

Further, in order to confirm the effect of plasma permeating the entire filter 50, a filter having a thickness gradually changing in the direction of blood permeation was prepared. That is, the filtration membrane A for the blood volume of 50 μL was shaved with a razor or the like so that the thickness was gradually reduced by about 150 μm every 2 mm in the length direction (8 stages).

[Regarding Filter Unit] The structure of the filter unit including the filter 50 and the filter holder 40 will be described with reference to the sectional view of FIG. 5A and the plan view of FIG. Further, the plan view of FIG. 5C showing only the filter 50 of the filter unit will also be described with reference to the same. The following dimensions are for a blood volume of 50 μL.

The filtration membrane B is housed in the through hole 42 for the filtration membrane of the filter holder 40 so as to be in contact with the end portion of the plasma extraction groove 43, and is arranged symmetrically with respect to the extension line of the plasma extraction groove 43. did. Filtration membrane B and filter holder 4
For adhesion to the bottom of 0, double-sided tape 46 already on the bottom
Was used. Next, the filtration membrane A was housed in the through hole 42 and placed on the end portion of the filtration membrane B far from the plasma outlet groove 43.
Then, avoiding the filtration membrane A, the double-sided tape 51 is placed on the filtration membrane B.
Glued. Further, a polyethylene independent foam having a width of 12 mm, a length of 20 mm and a thickness of 2 mm (Sanpelka L-4000 manufactured by Sanwa Kako Co., Ltd.) was processed into a substantially U shape as shown in FIG. The lower surface was shaved so that the filtration membrane A and the filtration membrane B could fit into the peripheral member 52, and the peripheral member 52 was housed in the through hole 42 so that the recess 52a of the peripheral member 52 surrounded the filtration membrane A. Then, the lower surface of the peripheral member 52 was adhered to the upper surface of the filtration membrane B with the double-sided tape 51.

In order to bring the outer peripheral surface of the peripheral member 52 into close contact with the inner surface of the through-hole 42, a bond silicon coke acetic acid-free type (manufactured by Konishi Co., Ltd.) is provided between the peripheral member 52 and the filter holder 40 with a thickness of about 1 mm. Intervened. As a result, the peripheral member 52 and the through-hole 4 are inserted from the end of the plasma outlet groove 43.
It is possible to prevent the liquid from leaking through between the inner surface of 2 and the inner surface of 2.

Further, a 0.5 mm-thick silicone rubber sheet 53 (manufactured by Tigers Polymer Co., Ltd.) having a hole 53a for dropping blood having a diameter of about 1.5 mm is provided at the position of the hole 53a for dropping blood. It was attached with a double-sided tape to cover the through hole 42. At this time, a hole 5 for dropping blood
A silicone rubber sheet 53 was arranged so that 3a was located above the filtration membrane A. Finally, the opening of the plasma storage tank 44 that opens on the upper surface of the filter holder 40 has a diameter of 0.1 mm.
PTFE film 54 (ADVANTEC) having pores of μm
The filter unit was completed.

[Method of Preparing Blood with Different Hct] Blood was collected from a normal person to prepare heparinized blood. Specifically, Novo Heparin Injection 1000 (manufactured by Aventis Pharma KK) was added to fresh blood at a concentration of 10 unit / mL for preparation. Plasma containing less platelets was separated from the obtained blood by centrifugation according to a standard method. After that, the blood coming to the sedimentation fraction is counted by the secondary (S) mode of the hemocytometer MAXMA / L-Retic (Coulter),
The blood having a desired Hct value (about 20 to 70%) was prepared by diluting the blood plasma with a small amount of platelets separated previously.

[Confirmation of blood cell arrival tip position in blood with different Hct] For the purpose of estimating the pressing position of the edging plunger 11, the tip position of blood cells in various Hct blood was confirmed. 50 μL of already prepared blood with Hct of 20-70%,
Each volume of 5 μL, 4 μL, and 1 μL was prepared, and spotted on the filter unit (the upper surface of the filtration membrane A) for each volume described above to separate blood into blood cells and plasma. The results are shown in Table 1. The separation position in Table 1 is the ratio of the distance that the plasma separated from blood spreads on the filtration membrane B to the length of the filtration membrane B. In addition, as a result of 10 times of control experiments using 50 μL of Hct 45% blood, the experimental error was 10 in terms of CV value.
%Met.

[0057]

[Table 1]

From the results shown in Table 1, when 5 μL or less of blood is used, the pressing position of the trimming plunger 11 is about 45% of the length of the filtration membrane B from the plasma outlet groove 43, and the blood is to be obtained. It was found that it can be changed depending on the amount of plasma. When using a blood volume of more than 5 μL,
The holding position of the edge-cutting plunger 11 is the plasma outlet groove 4
It was found from 3 that if the length was within approximately 35% of the length of the filtration membrane B, it could be changed according to the amount of plasma to be obtained.

[Confirmation of Blood Development Time in Blood with Different Hct] In the above confirmation test of the blood cell arrival tip position, after spotting the blood on the filtration membrane A, the end portion of the filtration membrane B (plasma outlet groove 4
The time for plasma to penetrate up to the portion (contact with 3) was measured.
The results are shown in Table 1. From this result, it was found that when 5 μL or less of blood was used, the development of blood was completed within 15 seconds and the extrusion of plasma could be started. Also, 5 μL
It was found that when the blood having a volume of more than 100 μm was used, the development of blood was completed within 100 seconds and the extrusion of plasma could be started.

[Confirmation of Plasma Recovery Amount in Blood with Different Hct] From the filter unit holding the separated plasma used in the above-mentioned confirmation test of the blood cell arrival tip position, plasma was extracted using the squeezing device 20. I took it out. Then, the amount and recovery rate of the obtained plasma were confirmed. The results are shown in Table 2. The blood volume was 4 μL and 1 μL only.

[0061]

[Table 2]

From the results, it was found that 10% or more of the plasma amount calculated by the Hct value can be recovered. [Confirmation of Bubbles in Recovered Plasma] The inside of the plasma outlet groove 43 during the plasma recovery operation was observed from below using a digital microscope manufactured by KEYENCE CORPORATION. As a result, FIG.
As shown in (a) of the above, plasma containing no bubbles could be obtained. Note that this image shows the case where the blood volume is 50 μL. Further, although the image shows a part of the groove, no bubbles were present over the entire area of the groove. Furthermore, similar results were obtained when the blood volume was other.

[Test with Filter Membrane whose Thickness is Gradually Decreased] Blood is separated in the same manner as described above except that the above-mentioned filter membrane with the thickness gradually reduced is used as the filter 50. went. In this test, 50 μL of blood with Hct of 40% was dropped on the upper surface of the filtration membrane,
Separated while observing from above. When the plasma reaches the end surface of the filtration membrane (the portion in contact with the plasma outlet groove 43),
The infiltration state of plasma on the upper and lower surfaces of the filtration membrane was observed.

As a result, as shown in FIG. 7 (a), the filter membrane having a gradually decreasing thickness has discoloration due to the infiltration of plasma on the lower surface of the filter membrane. It was found that plasma was infiltrated throughout. (Comparative Example 1) After separating blood in the same manner as above using the same filter unit as in the example, the separated plasma was collected from the filter by pressurizing and depressurizing with air. The blood volume was 4 μL and the filtration membrane used was 4 μL. The pressurization was performed manually by dropping blood and then sticking a 1 mL syringe (made by Terumo Corp.) with a double-sided tape on the blood dropping hole. The depressurization was performed manually by attaching a 1 mL syringe to the plasma storage tank with double-sided tape.

As a result, blood cells were mixed in the plasma in the plasma outlet groove. This is because the blood cells separated on the filtration membrane have permeated into the plasma region by the pressurizing and depressurizing operations. (Comparative Example 2) Separation of blood and collection of plasma were performed in the same manner as in Example except that the filter was composed of only the filtration membrane A. However, the size of the filtration membrane A was adjusted to the size of the filtration membrane B in the example. And the blood volume is 50μ
It was set to L.

First, after completion of the separation, the infiltration state of plasma in the filtration membrane was confirmed. As a result, as shown in FIG. 7B, a white portion derived from the color of the filtration membrane was observed in the lower portion of the lower surface of the filtration membrane, and it was confirmed that plasma did not penetrate into the portion. Was done. Next, plasma was collected from the filtration membrane using a pressing device. As a result, bubbles having a diameter of about 20 μm (see FIG. 6 (b)) and bubbles having a diameter of about 200 μm (see FIG. 6 (c)) are introduced into the plasma discharge groove into which the plasma has been sent.
There were many.

It is considered that when the plasma was extruded by the extruding plunger, the plasma was extruded while entraining the air remaining inside the filtration membrane, so that bubbles of various diameters were present in the plasma. .

[0068]

As described above, according to the present invention, 5 μL
Plasma can be efficiently extracted as a liquid from an extremely small amount of blood less than. Further, the plasma can be taken out so as not to contain air bubbles.

[Brief description of drawings]

FIG. 1 is a front view showing the configuration of an edge cutting device that constitutes the plasma separation device of the present invention.

FIG. 2 is a front view showing the configuration of a squeezing device that constitutes the plasma separation device of the present invention.

FIG. 3 is a plan view illustrating the arrangement of a rim cutting device and a squeezing device that form the plasma separation device of the present invention.

FIG. 4 is an explanatory diagram showing a configuration of a filter holder that constitutes the plasma separation device of the present invention.

FIG. 5 is an explanatory diagram showing the configuration of a filter unit that constitutes the plasma separation device of the present invention.

FIG. 6 is a view showing bubbles in separated plasma.

FIG. 7 is a diagram showing a state of plasma membrane infiltration of a filtration membrane.

[Explanation of symbols]

10 Edge cutting device 11 Edge Plunger 20 Squeezing device 21 Extrusion plunger 50 filters A, B filtration membrane

   ─────────────────────────────────────────────────── ─── Continued front page    F-term (reference) 2G045 BA08 BB04 CA25 GA01                 2G052 AA30 AD06 AD26 CA05 EA03                       EA13 EA17 JA07

Claims (12)

[Claims] 1. A method for separating serum or plasma from a trace amount of blood of less than 5 μl without centrifugation, which is capable of recovering 10% or more of serum or plasma contained in the blood. Method. 2. A region in which blood is spread by a separation member composed of a filtration membrane and separated into blood cells and serum or plasma on the separation member, and a region of the separation member where the blood cells are located,
After blocking the movement of the blood cells and the movement of the serum or plasma between the area where the serum or plasma is located, only the area where the serum or plasma is located of the separation member A method for separating plasma, comprising squeezing the blood to extract the serum or the plasma from the separation member. 3. The blood plasma separation method according to claim 2, wherein the thickness of the separation member is gradually reduced in the blood development direction. 4. The separation member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane holds the serum or the plasma. The ratio (Vu / Vl) between the volume Vu of the uppermost filtration membrane and the volume Vl of the lowermost filtration membrane is 1 to 3; The plasma separation method described. 5. The separation member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane retains the serum or the plasma. The ratio (Vu / Vl) of the volume Vu of the filtration membrane of the uppermost layer and the volume Vl of the filtration membrane of the lowermost layer is 1 to 2. The plasma separation method described. 6. The separation member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane holds the serum or the plasma. The ratio (Vu / Vl) of the volume Vu of the filtration membrane of the uppermost layer and the volume Vl of the filtration membrane of the lowermost layer is 1 to 1.5. 3. The plasma separation method according to item 3. 7. A device for separating serum or plasma from a trace amount of blood of less than 5 μl without centrifugation, which is capable of recovering 10% or more of serum or plasma contained in the blood. apparatus. 8. A separation member composed of a filtration membrane, which expands blood to separate blood cells and serum or plasma, a region of the separation member in which the blood cells are located, and the serum or plasma. A region where the blood cells are moved between the region where it is located and an edging means that blocks the movement of the serum or the plasma, and only the region where the serum or the plasma is located of the separation member is squeezed. A plasma separating apparatus comprising: a squeezing unit that extracts the serum or the plasma from the separating member. 9. The plasma separation apparatus according to claim 8, wherein the thickness of the separation member is gradually reduced in the blood development direction. 10. The separation member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane retains the serum or the plasma. And the ratio (Vu / Vl) between the volume Vu of the uppermost filtration membrane and the volume Vl of the lowermost filtration membrane.
Is 1 to 3, and Claim 8 or Claim 9 characterized by the above-mentioned.
The plasma separation apparatus according to. 11. The separation member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane retains the serum or the plasma. And the ratio (Vu / Vl) between the volume Vu of the uppermost filtration membrane and the volume Vl of the lowermost filtration membrane.
Is 1 or 2, and Claim 8 or Claim 9 characterized by the above-mentioned.
The plasma separation apparatus according to. 12. The separating member is composed of two or more layers of the filtration membrane, of which the uppermost filtration membrane captures the blood cells and the lowermost filtration membrane retains the serum or the plasma. And the ratio (Vu / Vl) between the volume Vu of the uppermost filtration membrane and the volume Vl of the lowermost filtration membrane.
Is 1 to 1.5, The plasma separation apparatus according to claim 8 or 9, wherein.