Patent Application
20090145840
The present invention relates to a blood cell separation membrane for separating blood into blood cells and serum or plasma and to a blood retention tool used for retaining or testing blood.
In a blood test, a sheet-like blood testing tool (also referred to as a “test piece”) to be used per specimen is used for various purposes. Examples of this testing tool include those that retain blood, from which blood to be tested is extracted; and those that are pre-impregnated with a reagent or the like. In the latter testing tools, the measurement can be carried out by, for example, dropping blood on the testing tool so that the blood reacts with the reagent and measuring the reaction by an optical or electrochemical method.
Such a blood testing tool has been used for various purposes in general clinical tests or the like. In addition, it actually is used in remote clinical testing systems, for example. In such a remote clinical testing system, a patient collects blood by himself at home, and the blood testing tool is impregnated with the blood. This then is dried, and the blood testing tool is mailed to a test institute such as a hospital for testing. The patient who mailed the blood then can be informed of the test result by mail or by visiting the hospital.
When the test item is a component of serum or plasma such as blood glucose or the like, blood needs to be separated into blood cells and serum or plasma in the blood testing tool. Thus, a conventional blood testing tool generally is provided with a blood cell separator such as a glass filter.
According to such a blood testing tool, it is possible to prepare a serum/plasma sample by, for example, separating blood into blood cells and serum/plasma by the blood cell separator and allowing the serum/plasma that has passed through the blood cell separator to develop in a development portion by capillary action. Other than this blood testing tool, a blood testing tool using an asymmetric porous membrane with pores whose sizes vary in the thickness direction has been developed recently (e.g., JP 11 (1999)-505327 A). When blood is supplied to the asymmetric porous membrane from the side having larger pores, the blood penetrates in the thickness direction, during which blood cells are separated from the blood. Thus, plasma/serum comes out of the other side, which then can be collected. The blood testing tool having such an asymmetric porous membrane is advantageous in that the clogging of blood cells can be prevented.
However, in such conventional blood testing tools, the blood cell separation might involve hemolysis of the blood cells, so that the serum or the like might contain components of the blood cells. On this account, adding an additive for preventing hemolysis to blood beforehand has been required (e.g., JP 9 (1997)-196908 A). However, this poses a problem in that, although the hemolysis is prevented, it becomes difficult to measure serum/plasma components accurately because the hematocrit (Ht) of the collected serum/plasma is decreased drastically by the influence of the additive.
With the foregoing in mind, it is an object of the present invention to provide a blood cell separation membrane that can separate blood into blood cells and serum/plasma while preventing hemolysis of the blood cells accompanying the blood cell separation, and also to provide a blood retention tool using such a blood cell separation membrane.
In order to achieve the above object, a blood cell separation membrane (also referred to as a “blood separation membrane”) according to the present invention includes a porous membrane for separating blood into blood cells and serum or plasma. The blood cell separation membrane is characterized in that the porous membrane contains at least one hemolysis inhibitor selected from the group consisting of hydrophobic aminocarboxylic acids, proteins derived from silk, Tris, TES, ε-aminohexanoic add, tranexamic add, and heparin.
As described above, the blood cell separation membrane of the present invention can prevent the hemolysis of the blood cells by containing at least one of the above-described types of hemolysis inhibitors. Thus, a serum or plasma sample that has been separated from the blood by passing through the blood cell separation membrane is free from components of the blood cells. As a result, components of the serum and the like can be measured with excellent accuracy. Moreover, the chances that hemoglobin pigment might be contained in the sample due to the hemolysis also can be eliminated, so that it becomes possible to carry out the measurement with respect to the sample directly by an optical method, visual observation, or the like.
Furthermore, by preparing a serum/plasma sample using the blood cell separation membrane of the present invention, it becomes possible to measure various components with high accuracy. The inventors of the present invention conducted keen studies as to the decrease in hematocrit (Ht) and the deterioration of the measurement accuracy with regard to various test items in the case of using the above-described conventional blood cell separation membranes. As a result, the inventors found that the fact that the conventionally used additive for preventing hemolysis is glycine and it generally is contained at a high concentration of about 100 mg to 200 mg per unit volume (cm3) of a blood cell separation membrane is responsible for the above-described influences on the measurement. The inventors also found that, with regard to a test item such as GGT or the like in particular, the deterioration of the measurement accuracy occurs not only because serum or plasma is diluted with glycine but also because glycine denatures components of serum or plasma. In the case where the above-described hemolysis inhibitor is contained instead of glycine as in the blood cell separation membrane of the present invention, the concentration thereof need not be as high as that of glycine, for example. Based on this finding, the inventors found that the use of the above-described hemolysis inhibitor can reduce the influence on a Ht and allows various test items, including GGT, for example, to be measured with high accuracy. As described above, the blood cell separation membrane of the present invention can separate blood into blood cells and serum/plasma while preventing hemolysis of the blood cells accompanying the blood cell separation and thus can provide a serum sample or a plasma sample causing little influence on various analyses.
Next, a blood retention tool according to the present invention includes: a blood cell separation portion for separating blood cells from blood; and a development portion in which serum or plasma contained in the blood develops. The blood cell separation portion in this blood retention tool is the blood cell separation membrane according to the present invention. Since the blood retention tool of the present invention is provided with a blood cell separation membrane producing the above-described effect, it is particularly useful in remote clinical testing systems as described above, for example. The blood retention tool according to the present invention also can be used as a tool for retaining a blood specimen in which blood cells and serum/plasma are separated for the purpose of, for example, sending the specimen by mail or the like. Alternatively, the blood retention tool itself can be used as a blood testing tool for testing an analyte.
As described above, the blood cell separation membrane according to the present invention includes a porous membrane for separating blood into blood cells and serum or plasma. The blood cell separation membrane is characterized in that the porous membrane contains at least one hemolysis inhibitor selected from the group consisting of hydrophobic aminocarboxylic acids, proteins derived from silk, Tris, TES, ε-aminohexanoic acid, tranexamic acid, and heparin.
As the hydrophobic aminocarboxylic acid, a hydrophobic amino acid can be used, for example. More specifically, alanine, valine, leucine, isoleucine, or the like can be used. The proteins derived from silk (hereinafter also referred to as “silk extracts”) refer to hydrolysates of fibroin.
Among the above-described hemolysis inhibitors, valine, leucine, ε-aminohexanoic acid, tranexamic acid, and silk extracts are preferable. These hemolysis inhibitors can produce an effect of separating blood into serum/plasma and blood cells highly efficiently, in addition to the effect of preventing hemolysis as described above. Although the mechanism is unknown, when the blood cell separation membrane contains at least one of these preferable hemolysis inhibitors, the speed at which blood cells penetrate into the blood cell separation membrane decreases, whereas the speed at which serum/plasma penetrates into the same increases. Furthermore, even in the case where blood cells pass through the blood cell separation membrane, the speed at which the blood cells develop in the development portion decreases, whereas the speed at which serum or plasma develops in the same increases, for example. Accordingly, in the development portion, it is possible to make the distance by which the blood cells develop short and the distance by which the serum or plasma develops long (hereinafter such a distance is referred to simply as a “development distance”). That is, the above-described preferable hemolysis inhibitors can achieve conflicting effects, i.e., suppression of the blood cell development and promotion of the serum/plasma development, thereby increasing the developing amount of the serum or plasma only. Therefore, in the blood cell separation membrane containing the above-described preferable hemolysis inhibitor, blood can be separated into blood cells and serum or plasma more easily. Furthermore, even in the case where blood cells pass through the blood cell separation membrane, the influence of the blood cell development on the collection rate of serum/plasma can be reduced, so that the collection rate of serum/plasma can be improved. Thus, the amount of blood to be supplied to the blood cell separation membrane may be smaller, so that the burden on the patient at the time of collecting blood can be reduced, for example.
Furthermore, among the above-described preferable hemolysis inhibitors, valine, tranexamic acid, and ε-aminohexanoic add are particularly preferable. These hemolysis inhibitors can produce the above-described effect sufficiently even though their content (weight) may be about ⅕ to 1/10 of that of glycine as described above, for example.
The hemolysis inhibitors may be used alone or in combinations of at least two kinds thereof. Examples of the combination of these hemolysis inhibitors include the combination of valine and heparin and the combination of tranexamic acid and heparin.
In the present invention, the content (weight) of the hemolysis inhibitor preferably is in the range from 1 mg to 50 mg, more preferably from 5 mg to 40 mg, and particularly preferably from 10 mg to 30 mg per unit volume (cm3) of the porous membrane, for example. When the content of the hemolysis inhibitor is at least 1 mg per unit volume (cm3) of the porous membrane, it is possible to prevent hemolysis sufficiently. It should be noted here that “the volume of the porous membrane” refers to the volume of the porous membrane including the volume of pores.
Specifically, in the case where the hemolysis inhibitor is valine, the content (weight) thereof preferably is in the range from 5 mg to 50 mg, more preferably from 15 mg to 45 mg, and particularly preferably from 30 mg to 40 mg per unit volume (cm3) of the porous membrane, for example.
In the case where the hemolysis inhibitor is leucine, the content (weight) thereof preferably is in the range from 1 mg to 30 mg, more preferably from 5 mg to 25 mg, and particularly preferably 10 mg to 20 mg per unit volume (cm3) of the porous membrane, for example.
In the case where the hemolysis inhibitor is tranexamic acid, the content (weight) thereof preferably is in the range from 5 mg to 50 mg, more preferably from 15 mg to 45 mg, and particularly preferably from 30 mg to 40 mg per unit volume (cm3) of the porous membrane, for example.
In the case where the hemolysis inhibitor is c-aminohexanoic add, the content (weight) thereof preferably is in the range from 5 mg to 50 mg, more preferably from 10 mg to 45 mg, and particularly preferably from 30 mg to 40 mg per unit volume (cm3) of the porous membrane, for example.
The blood separation membrane of the present invention further may contain additives such as pullulan and BSA, in addition to the hemolysis inhibitor. By using such additives and the hemolysis inhibitor in combination, the effect of preventing hemolysis can be further improved.
Although it is preferable that the hemolysis inhibitor is contained over the entire area of the blood cell separation membrane, the hemolysis inhibitor may be contained in, for example, at least one surface of the blood cell separation membrane, in particular, the surface of the blood cell separation membrane to which blood is supplied.
In general, it is desirable that blood cells cannot pass through the porous membrane easily. For example, it is preferable that the porous membrane has pores through which blood cells cannot pass. In the present invention, the “pores through which blood cells cannot pass” are not limited to the pores with smaller sizes than spherical diameters of blood cells, but may be pores through which blood cells eventually cannot pass regardless of the mechanism of preventing blood cells from passing through the pores. Therefore, the pores through which blood cells cannot pass may include pores larger than the spherical diameters of blood cells. Moreover, according to the blood cell separation membrane of the present invention, for example, even when blood cells have passed through the separation membrane as described above, the hemolysis inhibitor can increase the developing speed of serum or plasma and decrease the developing speed of blood cells. In this case, it is not necessary for the porous membrane to prevent blood cells from passing therethrough completely to retain all the blood cells therein, and some of the blood cells may pass through the porous membrane, for example.
More specifically, it is preferable that the porous membrane has pores with a pore size of 0.1 μm to 20 μm, more preferably 1 μm to 10 μm, and particularly preferably 2 μm to 8 μm.
There is no particular limitation regarding the porous membrane, and various materials that have been used conventionally for blood cell separation can be used as the porous membrane. More specifically, it is possible to use a glass filter or an asymmetric porous membrane with a pore size distribution in which an average pore size varies so as to be reduced continuously or discontinuously in the thickness direction, for example.
As the glass filter, those having a low fiber density are preferable, for example, and commercially available glass filters such as a product named “AP25” manufactured by Millipore Corporation can be used, for example.
In the case where the asymmetric porous membrane is used as the porous membrane, the pore size varies in the thickness direction. Thus, for example, as blood moves in the thickness direction inside the porous membrane, fewer and fewer blood cells can pass through the pores, and the blood cells are retained when they reach the portion with pores through which they cannot pass. Therefore, the clogging hardly occurs and blood cells can be separated“quickly and easily. In the present invention, the average pore size varies so as to be reduced discontinuously” means that the average pore size may vary, for example, so as to be reduced in a stepwise manner.
In the asymmetric porous membrane, it is preferable that the maximum pore size is in the range from 10 μm to 300 μm and the minimum pore size is in the range from 0.1 μm to 30 μm. It is more preferable that the maximum pore size is in the range from 100 μm to 200 μm and the minimum pore size is in the range from 1 μm to 10 μm. It is particularly preferable that the maximum pore size is in the range from 150 μm to 200 μm and the minimum pore size is in the range from 1 μm to 5 μm.
The material of the asymmetric porous membrane is not particularly limited, and may be, for example, a resin such as polyester, polysulfone, polyethersulfone, polycarbonate, cellulose acetate, polyamide, polyimide, polystyrene, or the like. The material is not limited to only one of them, and two or more of them may be used in combination. Among them, polysulfone and polyethersulfone are preferable, and polyethersulfone is particularly preferable, for example.
The asymmetric porous membrane may be formed using the above-described various resins. Alternatively, commercially available asymmetric porous membranes, e.g., a product named “BTS-SP” manufactured by U.S. Filter Corporation and a product named “Primecare S/G” manufactured by Spectral Diagnostics, Inc., may be used as the asymmetric porous membrane.
The size of the blood cell separation membrane can be determined as appropriate according to the amount of blood to be supplied or the like, for example. Specifically, when the blood to be supplied is 120 μl, the size length×width×thickness) of the blood cell separation membrane is, for example, in the range between 4 mm×1.5 mm×50 μm and 50 mm×20 mm×2000 μm inclusive, preferably between 5 mm×3 mm×75 μm and 30 mm×15 mm×1250 μm inclusive, and more preferably between 10 mm×5 mm×90 μm and 20 mm×12 mm×1100 μm inclusive. It should be noted that the “length” refers to the dimension in the longitudinal direction of the blood cell separation membrane, and the “width” refers to the dimension in the width direction of the same. The same applies hereinafter.
When a glass filter is used as the blood cell separation membrane, the thickness thereof is, for example, in the range from 200 μm to 2000 μm, preferably from 500 μm to 1000 μm. Furthermore, when the blood to be supplied is 120 μl, the size (length×width×thickness) of the glass filter is, for example, in the range between 4 mm×1.5 mm×100 μm and 50 mm×20 mm×2000 μm inclusive, preferably between 5 mm×3 mm×200 μm and 30 mm×15 mm×1250 μm inclusive, and more preferably between 10 mm×5 mm×250 μm and 20 mm×12 mm×1100 μm inclusive.
When an asymmetric porous membrane is used as the blood cell separation membrane, the thickness thereof is, for example, in the range from 50 μm to 400 μm, preferably from 100 μm to 350 μm. Furthermore, when the blood to be supplied is 40 μl, the size (length×width×thickness) of the asymmetric porous membrane is, for example, in the range between 4 mm×1.5 mm×50 μm and 50 mm×20 mm×400 μm inclusive, preferably between 5 mm×3 mm×100 μm and 30 mm×15 mm×350 μm inclusive, and more preferably between 10 mm×5 mm×200 μm and 20 mm×12 mm×300 μm inclusive.
The method for producing the blood cell separation membrane of the present invention is not particularly limited. For example, the blood cell separation membrane can be produced by immersing the porous membrane in a dispersion or a solution of the hemolysis inhibitor and then drying the porous membrane, or by dropping the dispersion or the like on the porous membrane, allowing the dispersion to penetrate into the porous membrane, and then drying the porous membrane.
The concentration of the hemolysis inhibitor in the dispersion or the solution is, for example, in the range from 0.1 wt % to 5 wt %, preferably from 0.5 wt % to 4 wt %, and more preferably from 2 wt % to 3 wt %.
The porous membrane may be treated so as to be provided with hydrophilicity by being immersed in a treatment solution of, for example, a hydrophilic polymer such as hydroxypropylcellulose (HPC), polyvinyl alcohol (PVA), or carboxymethylcellulose (CMC) before the hemolysis inhibitor is added thereto, because this allows whole blood to penetrate into the porous membrane rapidly. The concentration of the hydrophilic polymer in the treatment solution is, for example, in the range from 0.1 wt % to 50 wt %, and the treatment time is, for example, in the range from 0.1 to 24 hours. As a solvent of the treatment solution, for example, water, various organic solvents, or the like can be used. Examples of the organic solvents include alcohols such as ethanol.
Next, as described above, the blood retention tool according to the present invention includes: a blood cell separation portion for separating blood cells from blood; and a development portion in which serum or plasma contained in the blood develops. The blood retention tool is characterized in that the blood cell separation portion is the blood cell separation membrane according to the present invention.
Embodiments of the blood retention tool according to the present invention include: Embodiment A-1 directed to a laminate-type blood retention tool in which a blood cell separation portion is laminated on a development portion; Embodiments A-2 and A-3 directed to a single-layer blood retention tool in which an asymmetric porous membrane includes a development portion and a blood cell separation portion; and an embodiment directed to a single-layer blood retention tool in which a blood separation membrane includes a blood cell separation portion and a development portion. In the following, Embodiments A-1, A-2, and A-3 will be described specifically.
The present embodiment is directed to a blood retention tool configured so that: a blood cell separation portion is a blood cell separation membrane according to the present invention; a development portion is composed of a porous membrane; and the blood cell separation membrane is laminated on the development portion (the development porous membrane). In this embodiment, a surface of the blood cell separation membrane serves as a blood supply portion.
As the development porous membrane composing the development portion 11, a filter paper, a cellulose acetate membrane, a porous membrane, or a glass fiber membrane can be used, for example. Examples of the material of the porous membrane include resins such as polyester, polysulfone, polyethersulfone, polycarbonate, cellulose acetate, polyamide, polyimide, and polystyrene. Among them, polyethersulfone and polycarbonate are preferable. Also, an asymmetric porous membrane as described above can be use as the development porous membrane. In the case where the asymmetric porous membrane is used as the development portion, even when blood cells pass through the blood cell separation portion, blood cell separation can be carried out further in the development portion by the pore structure of the asymmetric porous membrane. Note here that the above-described porous membranes may be used alone or in combinations of at least two kinds thereof. Among them, a filter paper, a cellulose acetate membrane, a nitrocellulose membrane, a polysulfone porous membrane, a polyester porous membrane, and a polycarbonate porous membrane are preferable, and a filter paper, a polysulfone porous membrane, and a polyester porous membrane are particularly preferable, for example. Moreover, as in the case of the above-described blood cell separation membrane, the development porous membrane also may be treated so as to be provided with hydrophilicty, because this further promotes the development of serum or plasma.
The average pore size of pores of the development porous membrane is not particularly limited as long as serum or plasma is allowed to develop by capillary action, for example. However, it is preferable that the average pore size is in the range from 0.1 μm to 300 μm, more preferably from 1 μm to 100 μm, and particularly preferably from 2 μm to 50 μm. The size of this development porous membrane can be determined as appropriate according to the amount of blood to be supplied or the like, for example. When the blood to be supplied is 40 μl, the size (length×width×thickness) of this development porous membrane is, for example, in the range between 4 mm×1.5 mm×50 μm and 50 mm×20 mm×400 μm inclusive, preferably between 5 mm×3 mm×100 μm and 30 mm×15 mm×350 μm inclusive, and more preferably between 10 mm×5 mm×200 μm and 20 mm×12 mm×300 μm inclusive.
Note here that the hemolysis inhibitor may be contained not only in the blood cell separation membrane but also in the development porous membrane.
The combination of the blood cell separation membrane and the development porous membrane is not particularly limited, and preferable examples thereof include the combination of a glass filter as the blood cell separation membrane and an asymmetric porous membrane as the development porous membrane and the combination of an asymmetric porous membrane as the blood cell separation membrane and a nitrocellulose membrane as the development porous membrane.
In the blood retention tool according to the present embodiment can be produced by laminating the blood cell separation membrane on the development porous membrane. The lamination may be carried out, for example, by merely placing the blood cell separation membrane on the development porous membrane or by positioning the blood cell separation membrane with respect to the development porous membrane and then bonding or attaching by pressure the end portions of these membranes.
Preferably, the development porous membrane is supported by a supporter. This allows a blood testing tool with a sufficient strength to be a obtained regardless of the strength of the development porous membrane and also enables easy handling. As the material for forming the supporter, for example, plastic such as polystyrene, polyethylene terephthalate (PET), polyvinyl chloride, an acrylic resin, acrylonitrile-butadiene-styrene copolymer (ABS), or the like can be used. The material is not limited to only one of them, and two or more of them may be used in combination. When the blood retention tool is to be subjected to the measurement directly by the optical method or the like as will be described later, it is preferable that the supporter is optically transparent. In this case, the supporter may be made of polystyrene, PET, an acrylic resin, or the like, for example.
Next, an example of preparing a serum sample or a plasma sample by adding blood to the blood retention tool 10 will be described with reference to
First, blood is dropped on the blood supply portion 13 on the surface of the blood cell separation portion 12. The blood moves in the thickness direction inside the blood cell separation portion 12, during which the blood is separated into blood cells and serum/plasma. The serum/plasma thus separated reaches the development portion 11 and then develops in a direction parallel to the surface (the direction indicated by the arrow A in
When collecting the serum/plasma sample that has developed, the blood retention tool 10 may be dried by air drying, natural drying, or the like. Thereafter, the development portion 11 may be removed from the blood retention tool 10, and then only the portion in which the serum or the plasma has developed may be cut out from the development portion 11.
A cut piece obtained by cutting or the like is put, for example, in a test tube and an extractant is added thereto, which then is left, thus extracting and collecting serum or plasma. The extractant is not particularly limited as long as it can extract serum or plasma and does not affect the detection of an analyte in the serum or plasma. As the extractant, for example, a buffer solution, a physiological salt solution, purified water, a protein solution, or the like or a mixture thereof may be used. Examples of the buffer solution include various buffer solutions containing phosphoric acid, citric add, hydrochloric acid, acetic acid, or the like, and the pH of the buffer solution is, for example, in the range from 6 to 8. The amount of the extractant to be added is not particularly limited, and can be determined as appropriate according to the size of the cut piece or the like. The amount is, for example, 1 to 1000 times the volume of the cut piece. Furthermore, the time for an extracting process is not particularly limited and is, for example, in the range from 1 to 300 minutes.
Note here that after the serum or the plasma has developed, the serum or the plasma can be collected by cutting out the development portion and then subjecting the cut piece to centrifugation directly.
Using the solution thus collected, an analyte in the serum or plasma can be measured.
Alternatively, it is also possible to analyze the serum or plasma retained in the development portion 11 in this blood retention tool 10 without collecting it using the extractant. In this case, an analytical reagent may be provided in the development portion 11 beforehand to form a reagent portion. Examples of the method of providing the reagent in the development portion 11 include a printing method, an impregnation method, and a spraying method.
The analytical reagent is not particularly limited, and can be determined as appropriate according to the kind of an analyte. Examples of the components of the reagent include various enzymes, buffer materials such as phosphate and carbonate, and color developing agents. More specifically, when the analyte is glucose, the reagent may contain glucoldnase, glucose-6-phosphate dehydrogenase, β-NADP, ATP, a buffer solution, and the like, for example.
Furthermore, when a plurality of reagents are provided in the development portion 11 so as to be parallel to the direction in which the serum or the plasma moves, multiple items can be analyzed in a single blood retention tool. In this case, in order to prevent reagents for the multiple items from being mixed, it is preferable to provide boundary layers between the respective reagent portions by, for example, impregnation of a hydrophobic resin solution.
In the blood retention tool 10 configured as above, when blood is a supplied to the blood retention tool 10 to cause serum or plasma to develop as described above, various analytes react with detection reagents, respectively, in the development portion 11. The analysis can be conducted easily by detecting, for example, color developed through these reactions according to an electrochemical method, an optical method including visual observation), or the like.
The present embodiment is directed to a blood retention tool configured so that: a blood cell separation portion is an asymmetric porous membrane; the asymmetric porous membrane further includes a development portion; and the blood cell separation portion and the development portion are arranged along the surface direction of the asymmetric porous membrane so that the blood cell separation portion is on a side from which blood is supplied and the development portion is on a downstream side in the developing direction of the blood supplied. That is, a single asymmetric porous membrane includes the blood cell separation portion and the development portion arranged along the surface direction, so that blood cell separation and development of serum or plasma can be performed within the single asymmetric porous membrane.
In the asymmetric porous membrane, the maximum pore size is, for example, in the range from 10 μm to 300 μm, preferably from 50 μm to 150 μm, and the minimum pore size is, for example, in the range from 0.1 μm to 30 μm, preferably from 1 μm to 10 μm.
The size of the blood retention tool 20 is not particularly limited as in the above, and can be determined as appropriate according to the amount of a blood specimen to be supplied or the like. When the whole blood to be supplied is about 100 μl, the size length×width×thickness) of the blood retention tool 20 as a whole is, for example, in the range between 4 mm×1.5 mm×50 μm and 60 mm×20 mm×400 μm inclusive, preferably between 5 mm×3 mm×100 μm and 30 mm×15 mm×350 μm inclusive, and more preferably between 10 mm×5 mm×200 μm and 20 mm×12 mm×300 μm inclusive. It should be noted that the “length” refers to the dimension in the longitudinal direction of the blood retention tool, and the “width” refers to the dimension in the width direction of the same.
In the blood retention tool 20 configured as above, the blood cell separation portion 22 and the development portion 21 need not be separated explicitly with a boundary line, for example. In the blood retention tool 20, when blood is dropped on the blood supply portion 23, the blood moves in the thickness direction, during which the blood also develops in the surface direction (the direction indicated by the arrow A in
As described above, this blood retention tool 20 is composed of an asymmetric porous membrane having a single layer structure. Thus, for example, a solution containing the hemolysis inhibitor may be contained only in a portion serving as the blood cell separation portion 22. Alternatively, the entire asymmetric porous membrane may be immersed in the solution so that the entire area of the porous membrane is impregnated with the solution.
Next, an example of preparing a serum sample or a plasma sample by adding blood to the blood retention tool 20 will be described with reference to
First, blood is dropped on the blood supply portion 23. The blood moves in the thickness direction inside the blood cell separation portion 22, during which blood cells are separated from the blood. Serum or plasma moves in the surface direction (the direction indicated by the arrow A in
In the blood retention tool composed of an asymmetric porous membrane including both the blood cell separation portion and the development portion as described in Embodiment A-2, it is preferable that a blood cell blocking portion further is provided, for example, at the boundary between the blood cell separation portion and the development portion in order to improve the efficiency of blood cell separation further. More specifically, a blood cell blocking portion including only pores through which blood cells cannot pass may be formed between the blood cell separation portion and the development portion so as to extend in the width direction of the asymmetric porous membrane. In the blood retention tool provided with the blood cell blocking portion, with respect to the direction in which blood develops when it is supplied thereto, a portion downstream from the blood cell blocking portion serves as a development portion, and the blood cell blocking portion and a portion upstream from the blood cell blocking portion serve as a blood cell separation portion. The blood retention tool configured as above is advantageous in that, for example, even when blood cells contained in the blood move not only in the thickness direction but also in the surface direction, it is possible to prevent the blood cells from entering the development portion reliably by the blood cell blocking portion so that only serum or plasma develops in the development portion. Preferably, a groove is formed so as to extend in the width direction of the asymmetric porous membrane, and a portion between the bottom of the groove and a part of the asymmetric porous membrane surface corresponding to the bottom serves as the blood cell blocking portion. Unless otherwise stated, conditions (the size, the material, etc.) of the blood retention tool according to the present embodiment are the same as those of the blood retention tool according to Embodiment A-2.
In the asymmetric porous membrane, the size of the pores in the blood cell blocking portion 32 is, for example, in the range from 0.1 μm to 50 μm, preferably from 1 μm to 30 μm.
The size of the blood retention tool 30 is not particularly limited as in the above, and can be determined as appropriate according to the amount of a blood specimen to be supplied or the like. When the whole blood to be supplied is about 100 μl, the size (length×width×thickness) of the blood cell blocking portion 32 is, for example, in the range between 0.1 mm×1.5 mm×10 μm and 10 mm×20 mm×400 μm inclusive, preferably between 0.2 mm×3 mm×15 μm and 8 mm×15 mm×350 μm inclusive, and more preferably between 0.3 mm×5 mm×20 μm and 6 mm×12 mm×300 μm inclusive.
The groove can be formed, for example, by compression of a part of the surface of the asymmetric porous membrane. For instance, the groove can be formed by compression through rolling of a disk-shaped roller or by compression using a cutting tool with a dull edge to a degree causing no cut. When the groove is formed by compression, the blood cell blocking portion may include large pores of the asymmetric porous membrane. However, since the pores are deformed or crushed after compression, the blood cells are thus prevented from passing through the blood cell blocking portion.
Alternatively the groove may be formed, for example, by cutting off a part of the porous membrane using a cutting tool such as a cutter. In this case, the size of the groove is the same as in the case described above.
Next, as specific examples of the blood retention tool according to the present invention, those that can be used as a blood testing tool will be described by way of embodiments with reference to
In the blood retention tool 40 configured as above, when blood is dropped on the blood supply portion 43, the blood moves in the direction indicated by the arrow A inside the blood separation membrane 46, during which the blood is separated into blood cells and serum, for example. Since the blood separation membrane 46 contains a hemolysis inhibitor as described above, it is possible to separate the blood into blood cells and serum while preventing the hemolysis of the blood cells. The serum separated moves inside the blood separation membrane 46 to develop in the reagent portion 45, where an analyte contained in the serum reacts with the analytical reagent. The analysis can be conducted by detecting this reaction according to an electrochemical method or an optical method including visual observation), for example.
As shown in
In the blood retention tool 50 configured as above, serum separated in the blood separation membrane 56 develops in the reagent layer 55 laminated on the blood separation membrane 56, so that an analyte contained in the serum reacts with the analytical reagent contained in the reagent layer 55, for example.
In the blood retention tool 60 configured as above, for example, serum separated in the blood separation membrane 66 first develops in the first reagent portion 651, where an analyte contained in the serum reacts with the first analytical reagent. A reaction solution obtained through this reaction further develops in the second reagent portion 652 to react with the second analytical reagent. Then, a reaction solution obtained through this reaction finally develops in the third reagent portion 653 to react with the third analytical reagent. This reaction can be detected in the manner described above.
The blood retention tool 60 with such a configuration is suitable in the case where an analyte is to be measured utilizing a multistep reaction, and the first reagent portion 651, the second reagent portion 652, and the third reagent portion 653 may be provided with appropriate reagents according to the reaction sequence in the multistep reaction.
In the blood retention tool 70 configured as above, for example, serum separated in the blood separation membrane 76 develops in the first reagent layer 751, the second reagent layer 752, and the third reagent layer 753 laminated on the blood separation membrane 76. Analytes contained in the serum respectively react with the analytical reagents contained in the respective reagent layers (751,752, and 753). Thus, multiple test items can be analyzed in a single blood retention tool by, for example, providing the reagent layers with reagents corresponding to the respective items.
As shown in
Note here that, as shown in the sectional view of
As shown in
In the following, an example of a method of analyzing a component of serum through an antigen-antibody reaction using the blood retention tool 100 will be described. In the first reagent portion 151, a labeled first antibody against an analyte (an antigen) and a labeled third antibody against a second antibody that will be described later are provided. In the second reagent portion 152, an unlabeled second antibody against the antigen is immobilized. As the labels of the labeled first antibody and the labeled third antibody, colored latex particles can be used.
First, blood is dropped on the blood supply portion 103. The blood passes through the blood separation membrane 102, during which serum is separated from the blood. The serum thus separated moves in the direction indicated by the arrow A inside the development layer 101. The serum first develops in the first reagent portion 151, where the antigen contained in the serum forms a complex with the labeled first antibody through an antigen-antibody reaction. The serum, which contains the complex, the labeled first antibody not bound to the antigen, and the labeled third antibody, further develops in the second reagent portion 152. In the second reagent portion 152, the complex and the labeled third antibody are bound to the unlabeled second antibody immobilized in the second reagent portion 152 through an antigen-antibody reaction and thus are captured in the second reagent portion 152. Then, the serum, which contains the labeled first antibody not bound to the antigen and also the complex and the labeled third antibody that are not captured in the first detecting portion (the second reagent portion) 152, further develops in the second detecting portion 153.
After that, the measurement can be carried out in the following manner. First, in the first detecting portion (the second reagent portion) 152, the captured complex of the antigen and the labeled first antibody is detected. In the first detecting portion 152, the captured labeled third antibody further is detected, and in the second detecting portion 153, the labeled third antibody not captured in the second reagent portion 152 is detected Note here that the above-described detections can be carried out by measuring an absorbance at a wavelength specific to each of the labels (the colored latex particles) of the labeled first antibody and the labeled third antibody. Based on the result of the detections of the labeled third antibody, the capturing efficiency of the second reagent portion 152 is determined. Then, using the thus-determined capturing efficiency and the result of detection showing the apparent amount of the complex, the actual amount of the complex (i.e., the amount of the antigen) is calculated. By measuring the labeled third antibody as well, the correction using the capturing efficiency becomes possible. Thus, according to the present embodiment, the accuracy of analyzing an analyte (an antigen) can be improved.
A blood retention tool according to Embodiment A-1 as shown in
(Development Porous Membrane)
A commercially available asymmetric porous membrane (product name “Primecare S/G”: Spectral Diagnostics, Inc.) made of polyethersulfone was washed under the following conditions and used as a development porous membrane. This porous membrane had a thickness of 290 μm, a length of 20 mm, and a width of 6 mm. In this porous membrane, the maximum pore size was 200 μm and the minimum pore size was 2 μm. The washing of the asymmetric porous membrane was carried out in the following manner. First, the asymmetric porous membrane was immersed in purified water and left for 10 minutes, after which the purified water was replaced with fresh purified water. This operation was carried out 5 times in total. Thereafter, the asymmetric porous membrane was dried by air drying for half a day, and then further dried in a desiccator for half a day. The asymmetric porous membrane then was used as a development porous membrane.
(Blood Separation Membrane)
Hemolysis inhibitors were dissolved in solvents (distilled water) beforehand to prepare hemolysis inhibitor solutions shown in Table 1 below. Then, 30 μl of each of the hemolysis inhibitor solutions with various concentrations shown in Table 1 was dropped on a commercially available glass filter (product name “AP25”: Millipore Corporation) having a thickness of 1200 μm, a length of 12 mm, and a width of 6 mm, which then was dried. The dried glass filter was used as a blood separation membrane. Note here that the silk extract shown below was a product named “Silk Extract” manufactured by FUKUI KINU SHOJI CO, LTD.
(Method for Producing Blood Retention Tool)
A PET film having a length of 50 mm, a width of 6 mm, and a thickness of 180 μm was provided as a supporter. The development porous membrane was bonded to this PET film with an adhesive. Furthermore, the glass filter was disposed on one end of the development porous membrane as shown in
On the other hand, a blood retention tool according to Comparative Example 1 was produced in the same manner as in Example 1, except that a 5% glycine solution was contained in the glass filter.
(Hemolysis of Blood Cells)
On the glass filter surface of each of the thus-obtained blood retention tools, 100 μl of whole blood (hematocrit: 38%) was dropped to cause the separation of blood cells and plasma and the development of the plasma. Then, after a lapse of 2 minutes, the presence or absence of hemolysis of the blood cells was determined. Note here that the plasma was allowed to develop until a tip of the development portion (the right end portion of the development portion 11 in
As shown in Table 1, hemolysis occurred in the blood retention tool according to Comparative Example 1. In contrast, in the blood retention tools according to Example 1 (1-1 to 1-8), the hemolysis was prevented from occurring, while the concentrations of the hemolysis inhibitor solutions contained in the respective blood retention tools were the same as or lower than that in the blood retention tool according to Comparative Example 1.
Blood retention tools were produced in the same manner as in Example 1, except that various hemolysis inhibitors shown in Table 2 below respectively were contained as hemolysis inhibitors. Using these blood retention tools, the development of blood cells was determined.
(Measurement of Development Distance of Blood Cells)
As the development distance of blood cells, the distance from the end of the development porous membrane on the blood dropping side (the left end portion of the development portion 11 in
As shown in Table 1 and Table 2, hemolysis occurred in the blood retention tool containing the 5% glycine (Comparative Example 2). In contrast, in the blood retention tools according to Example 2 (2-1 to 2-6), the hemolysis was prevented from occurring and besides, the development distances of the blood cells were smaller than that in the blood retention tool according to Comparative Example 2, while the amounts of the hemolysis inhibitors contained in the respective blood retention tools were the same as or smaller than that in the blood retention tool according to Comparative Example 2. These results demonstrate that, according to the blood retention tools of the present example containing the above hemolysis inhibitors, blood cells and plasma can be separated sufficiently due to the suppression of the development of the blood cells and the promotion of the development of the plasma. This increases the developing amount of the plasma, thereby improving the collection rate of the plasma.
Blood retention tools (Example 3-1 and 3-2) were produced in the same manner as in Example 1, except that the following development porous membranes (a filter paper and a cellulose acetate membrane) and blood cell separation porous membranes (glass filters) were used and valine was used as the hemolysis inhibitors. Furthermore, blood retention tools according to Comparative Example 3 (Comparative Examples 3-1 and 3-2) were produced in the same manner as in Example 3-1 or 3-2, except that no valine was contained in the blood cell separation porous membranes. Using these blood retention tools, blood cell separation was carried out and the presence or absence of hemolysis and the development of blood cells were determined in the same manner as in Examples 1 and 2. The results are shown in Table 3 below.
(Filter Paper)
(Cellulose Acetate Membrane)
(Blood Cell Separation Porous Membrane)
A glass filter (product name “AP25”: Millipore Corporation) having a thickness of 1200 μm was used.
A shown in Table 3, hemolysis occurred in the blood retention tools of Comparative Examples 3-1 and 3-2 in which no valine was contained in the glass filters. In contrast, hemolysis did not occur in the blood retention tools of both Examples 3-1 and 3-2. Moreover, the development distance of the blood cells in the blood retention tool of Example 3-1 was shorter than that in the blood retention tool of Comparative Example 3-1, and the development distance of the blood cells in the blood retention tool of Example 3-2 was shorter than that in the blood retention tool of Comparative Example 3-2. These results demonstrate that, according to the blood retention tools of Example 3, the development of blood cells can be suppressed, so that only the developing amount of plasma can be increased and thus the collection rate of plasma can be improved, as in the case of the blood retention tools of Example 2. It should be noted that the reason why the development distance of the blood cells in the blood retention tool of Examples 3-1 was different from that in the blood retention tool of Example 3-2 is that the types of the development porous membranes used therein were different.
Blood retention tools were produced in the same manner as in Example 1, except that valine solutions with various concentrations (5%, 2%, and 1%) and a 2% leucine solution respectively were used as the hemolysis inhibitors. With regard to each hemolysis inhibitor, ten blood retention tools were produced Using these blood retention tools, plasma was collected and components contained in the collected plasma were measured (Example 4).
Furthermore, blood retention tools according to Comparative Example 4 were produced in the same manner as in Example 1, except that glycine solutions with predetermined concentrations (20%, 5%, and 0%) were contained in the glass filters, respectively. With regard to each glycine solution, ten blood retention tools were produced.
A whole blood specimen was dropped on each of the blood retention tools to cause the separation of blood cells and the development of plasma. After the plasma had developed, a portion of the development porous membrane where only the plasma had developed was cut out to obtain a cup piece of 10 mm×6 mm. With regard to each hemolysis inhibitor, ten cut pieces were collected, and they were centrifuged (15,000 g, 10 min) directly to collect the plasma. Using the thus-collected plasma as a sample, the amounts of respective components were measured. Furthermore, as a control test, the amounts of the respective components were measured using the plasma obtained by centrifuging the above whole blood specimen (3000 g, 10 min) as a sample.
(Measurement of Amounts of Components)
Amounts of the respective components were measured with an autoanalyzer (product name Chtachi 7070′: Hitachi, Ltd.), using the following commercially available kits according to their application methods. In the respective measurements, purified water was used as a blank.
1. High-Density Lipoprotein Cholesterol BL-C)
2. Amylase (AMY)
3. Albumin (ALB)
4. Glutamic-Oxaloacetic Transaminase (GOT)
5. Gamma Glutamyl aanspeptidase (GGT)
6. Glutamic-Pyruvic aansaminase (GPT)
7. Triglyceride (TG)
8. Lactate Dehydrogenase (LDH)
9. Total Protein (TP)
10. Ibtal Bilirubin (T-Bil)
11. Creatinine (CRE)
12. Total Cholesterol (TC)
13. Alkaline Phosphatase (ALP)
14. Urea Nitrogen (BUN)
15. Uric Add (UA)
16. Fructosamine MA)
17. Glucose (Glu)
18. Creatinine Phosphokinase (CPK)
Table 4 below shows the results of the measurements, in which the measured values obtained in Example 4 and Comparative Example 4 are indicated as relative values (percentages on average) with respect to the measured values obtained in the control test as 100%.
As shown in Table 4, according to the blood retention tools of Comparative Example 4 containing glycine, variations were observed in the measurement accuracy of the respective components. In particular, with regard to GGT, the relative value with respect to the control test was only about 50%. In contrast, according to the blood retention tools containing valine or leucine, excellent measurement results were obtained with regard to the respective components including GGT. Moreover, hemolysis was prevented in all the blood retention tools of Example 4, whereas the occurrence of hemolysis was observed in the blood retention tools of Comparative Example 4 containing the 5% glycine solution. Furthermore, the blood retention tools of Comparative Example 4 containing the 20% glycine solution exhibited small relative values with regard to all the components. The reason for this is considered to be that blood cells shrank due to the high concentration of glycine, so that the volume of the plasma components increased accordingly, thereby diluting the plasma components. These results demonstrate that the blood retention tools according to the present invention can prevent hemolysis and also can prepare a sample causing little influence on the measurement of the respective components.
As described above, according to the blood cell separation membrane of the present invention, it is possible to separate blood into blood cells and serum/plasma while preventing the occurrence of hemolysis of the blood cells, so that a serum or plasma sample can be prepared efficiently. Moreover, a sample prepared by the blood cell separation membrane of the present invention has little influence on measurement systems of various components. Thus, highly accurate measurement becomes possible by using such a sample. Therefore, the blood cell separation membrane and the blood retention tool of the present invention are useful in fields such as clinical medicine as described above, for example.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2003-063847 | Mar 2003 | JP | national |
| 2003-090472 | Mar 2003 | JP | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10548434 | Sep 2005 | US |
| Child | 12370080 | US |
