JP2002350428A - Method and apparatus for separating plasma or serum from whole blood - Google Patents

Abstract

(57) [Problem] To provide a method, apparatus and test carrier for separating plasma or serum from whole blood, in which erythrocytes can be rapidly separated, have high separation ability, and have little hemolysis. A method for separating plasma or serum from whole blood by contacting whole blood with a fiber-containing layer and retaining red blood cells by the fiber-containing layer, wherein the fiber-containing layer is a layer containing glass fibers And using a glass fiber coated with butanol-containing propanol and / or acrylamide as the glass fiber, thereby suppressing the hemolysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and an apparatus for separating plasma or serum from whole blood by a fiber-containing layer, and to a test carrier containing the apparatus for separating plasma or serum.

[0002]

2. Related Art In clinical chemistry tests or clinical immunity tests, many rapid tests for qualitative and quantitative diagnosis have developed in the field of hospital-related tests using blood as a sample.

[0003] Although some of these clinical tests can be tested on whole blood, it is necessary to utilize serum or plasma separated from a blood sample to obtain accurate test results. Otherwise, impurities in the red blood cells or blood sample will interfere with the measurement methods reflected light, transmitted light and luminescence, affecting the measurement.

Conventionally, centrifugation has been used to separate plasma or serum from whole blood. However, this method is time-consuming, and it is difficult to collect serum or plasma after separation of a small amount of whole blood sample,
And the need for ancillary equipment that is not generally available outside the examination room.

[0005] For these reasons, point-of-care tests, such as those performed in bedside and emergency tests, that are performed outside the laboratory by specialists other than laboratory technicians, Centrifugation is completely unsuitable.

Many techniques have been devised and proposed to avoid these problems. In these techniques, a number of materials were generally used to form the filter. As these filter materials, membrane materials having an appropriate pore size such as paper, woven fabric, glass and synthetic fiber have been conventionally known.

For example, US Pat. S. P. 4,81
No. 6,224 discloses a method of laminating glass fiber filter paper of a specific size and a porous membrane. Also,
U.S. Pat. S. P. No. 4,753,776 relates to an apparatus for separating plasma from red blood cells by bringing whole blood into contact with a filter made of glass fiber capable of carrying an erythrocyte agglutinating agent, and the thickness of the glass fiber filter. Teaches a method of defining a diameter, during which whole blood is passed through and capillary-separated plasma is passed to a capillary flow device. However, in both methods, the practical separated yield of plasma is usually 25% or less, and easily clogged.

Next, US Pat. S. P. 5,665,
238, a high-yield separation is achieved by separating serum in a layer containing 1 to 40% of the polysaccharide mannitol and 0.1 to 15% of albumin in a fiber filter made of glass or the like having a pore size of 3 μ or less. The disadvantages of this method are that there is no consideration for hemolysis and that the separation time is long.

Another method is disclosed in US Pat. S. P.
3,146,163 and DE-A-
1498577 describes a method of applying whole blood to a hemagglutinin-coated material, such as phytohemagglutinin, for the separation of plasma, with plastic and cardboard as possible carrier materials. Such fibrous substances are mentioned.

Japanese Patent Application Publication No. 61-118661 discloses a method for separating whole blood, which comprises applying whole blood to a lectin-impregnated matrix and isolating plasma or serum. Use absorbent materials having a relative resistance to fluid flow of up to 40%. In addition, mention may be made of fibrous structures or matrices having a fibrous structure and resistance to the smallest possible fluid flow, with preferred materials being exemplified by cotton and viscose fibers and cellulosic materials. However, a clear disadvantage of this method is that the separated plasma or serum must be washed from the matrix with a diluent.

Japanese Patent Application Publication No. 64-21362 discloses another apparatus for separating plasma or serum from whole blood. The device includes an absorbent matrix that has been treated with a reagent that aggregates blood cells, and thrombin or lectin is mentioned as a reagent that aggregates blood cells. Hydrophobic powders, sponge and clay materials, fibers and polymers are recommended as absorbent matrices, fiber-containing papers are also recognized as polymers, and filter papers are preferred matrices.

Japanese Patent Application Publication No. Sho 61-207966
Teaches a reagent for separating plasma or serum from whole blood using an absorbent matrix impregnated with lectin as a separation layer.

Japanese Patent Application Publication No. 2-140147 discloses immobilizing a polycation on an absorbent solid material so as to form a cationic surface to which erythrocytes bind upon contact with whole blood. Preferred carrier materials include paper.

Japanese Patent Application Publication No. 60-36961 discloses the separation of plasma from whole blood using an absorbent porous material impregnated with a substance having several polar groups. For example, paper, pads and fibers are generally mentioned.

European Patent Application Publication EP-A-030580
No. 3 discloses an apparatus for separating blood cells from a erythrocyte-containing body fluid consisting of a fiber-containing filtration layer containing a hemagglutinating antibody and, if desired, a lectin, or a glass fiber filtration layer and a lectin.

Among the known whole blood separation methods described above, a method in which whole blood is brought into contact with a fiber-containing layer having an erythrocyte agglutinating action and red blood cells are retained by the fiber-containing layer can be said to be practically effective. However, a problem with using this type of fiber-containing layer is that hemolysis occurs when erythrocytes are brought into contact with the fiber-containing layer.

On the other hand, US Pat. S. P. 5,262,
No. 067 is a good method which can coat a glass fiber layer containing an erythrocyte agglutinating substance with polyvinyl alcohol or polyvinyl alcohol / polyvinyl acetate to suppress hemolysis to a plasma component separated from whole blood to such an extent that practical use is not affected. However, there is a problem that the separation time is required.

Hemolysis releases hemoglobin from red blood cells, thus decolorizing plasma or serum. Such bleaching can significantly interfere with the colorimetric test, so that the smaller the amount of red blood cell hemolysis caused by the glass, the less the inhibitor will have an effect on the measurement.

[0019]

SUMMARY OF THE INVENTION The present inventors have found the possibility of separating undiluted blood cell components, especially red blood cells, from plasma or serum, and to ensure that no lysis occurs during this separation. It has been surprisingly found that the separation speed at the time of the separation is high and that the separation can be performed in a point-of-care test in a clinical test. Strikingly, by using a fiber-containing layer comprising glass fibers coated with butoxypropanol or butoxymethylacrylamide as a plasma or serum separation layer, undiluted whole blood cells from plasma or serum can be substantially free of hemolysis. The inventors have found that they are suitable for separating components very well, and arrived at the present invention.

The present invention has been made in view of the above-mentioned problems, and provides a method, an apparatus and a test for separating plasma or serum from whole blood which has a high separation ability, a high separation ability, and a low hemolysis in addition to the rapid separation of red blood cells. It is intended to provide a carrier.

[0021]

According to the present invention, there is provided a method for separating plasma or serum from whole blood according to the present invention, wherein whole blood is brought into contact with a fiber-containing layer and red blood cells are retained by the fiber-containing layer. By separating plasma or serum from whole blood, wherein the fiber-containing layer is constituted by a layer containing glass fiber, and the glass fiber is coated with propanol and / or acrylamide having a butoxy group. Is used to suppress hemolysis.

The apparatus for separating plasma or serum from whole blood of the present invention is an apparatus for separating plasma or serum from whole blood by contacting the whole blood with a fiber-containing layer and retaining red blood cells by the fiber-containing layer. The fiber-containing layer is a layer containing glass fibers coated with butanol-containing propanol and / or acrylamide, thereby suppressing hemolysis.

The test carrier of the present invention comprises a plasma or serum separation layer for separating plasma or serum from a blood sample component, and a test zone for testing the separated plasma or serum. A test carrier for performing the measurement of, wherein the plasma or serum separation layer is configured to contain the above-described plasma or serum separation device, thereby suppressing hemolysis.

The glass fiber used in the present invention is preferably borosilicate glass having an average pore size of 3 to 6 μm.
As a commercially available glass fiber membrane that meets this specification,
GF / D type (Whatman) is suitable.
However, when this glass fiber is used alone, hemolysis occurs in the process of separating whole blood, and the separation time varies depending on the lot.

Butoxypropanol, which is a propanol and / or acrylamide having a butoxy group as a functional group, made of this glass fiber, for example, 1-butoxy-
By using 2-propanol and / or butoxymethyl acrylamide, for example, one coated with N- (butoxymethyl) acrylamide, hemolysis in the separation process can be suppressed to a practically acceptable amount. At the same time, separation of plasma or serum from blood cells starts and there is no delay time for whole blood to develop in the glass fiber, and rapid separation can be performed.

The lower limit of the amount of propanol and / or acrylamide having butoxy groups to glass fiber is 0.05% by weight, preferably 0.1% by weight, more preferably 0.2% by weight, and the upper limit is 0.2% by weight. Value is 3.0% by weight, preferably 2.0% by weight, and preferably 1.5% by weight.
% By weight.

When an acrylamide-based or propanol-based substance is used as the coating, hemolysis of dimethylacrylamide, N-hydroxyacrylamide, N-N'-methylene-bis (acrylamide), N-isopropylacrylamide and propanol is not effective. There was a delay in the development time, and there was no significant difference from other separation membrane techniques, indicating that acrylamide and / or propanol with a butoxy group was effective.

In the present invention, the glass fiber filter paper is preferably impregnated with a solution of the coating substance. For example, the impregnating solution is applied to the glass fiber filter paper or is immersed in the impregnation liquid.

The amount of the impregnating solution is preferably in the range of 0.3% by weight to 2.0% by weight. If the amount by weight is out of this range, the capillary force between the glass fibers is affected, and the elution of hemolysis occurs. , Causing a delay in the development of whole blood.

In the present invention, N- (butoxymethyl) acrylamide or 1-butoxy-2-glass is used to separate plasma or serum from whole blood in such a manner as to cover the entire surface of the glass. Coat with a propanol layer.

As the method, glass fiber filter paper is N-
It is immersed in (butoxymethyl) acrylamide or 1-butoxy-2-propanol impregnating solution, taken out, and dried at room temperature to 90 ° C. Drying time is 50 ° C
There is no practical problem in the range of 25 to 80 minutes in the range of -90 ° C.

As the fiber-containing layer of the present invention, glass fibers may be used as described above. For example, glass fibers containing known erythrocyte agglutinating substances such as lectins and antibodies may be used.

[0033]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One embodiment of the present invention will be described below with reference to the accompanying drawings, but the illustrated examples are illustrative only and various modifications may be made without departing from the technical idea of the present invention. Needless to say, this is possible.

FIG. 1 is a cross-sectional view showing one embodiment of the test carrier of the present invention, in which a strip used for immunochromatography is used as a test carrier, and the plasma or serum separating device of the present invention is used for its sample pad. Is shown.

In FIG. 1, reference numeral 10 denotes a test carrier, which has a long support plate 12 made of plastic or the like.
The plasma or serum separation layer 1 is provided on the upper surface of one end of the support plate 12.
4 are provided. As the plasma or serum separation layer 14, a fiber-containing layer having the above-mentioned red blood cell agglutinating action and containing glass fibers coated with butoxypropanol and / or butoxymethylacrylamide is used.

Reference numeral 16 denotes a conjugate pad made of glass fiber or the like. The distal end of the plasma or serum separation layer 14 is adjacent to the proximal end of the conjugate pad 16 provided on the support plate 12. It is provided so as to cover. The conjugate pad 16 contains an antibody or antigen labeled with latex, colloidal gold, or the like.

Reference numeral 18 denotes a developing layer serving as a test area, which is formed of a nitrocellulose film or the like. The spreading layer 18 is provided at the center of the upper surface of the support plate 12, and is constituted by a solid-phase band 20, for example, a capture zone 20a and a control zone 20b. This solid-phase band 20
Contains antibodies or antigens. Reference numeral 22 denotes a water-absorbing layer made of glass fiber or the like provided on the upper surface of the other end of the support plate 12 and moves unreacted substances that have not reacted in the solid-phase band 20 to the outside of the solid-phase band 20 by a water-absorbing action. Work.

The operation of the test carrier 10 will be described below. First, when a blood sample component and a reaction solution are dropped on the plasma or serum separation layer 14, red blood cells in the blood sample component are retained by the glass fibers in the plasma or serum separation layer 14, and plasma or serum is separated. .

The separated plasma or serum component and the reaction solution cause an antigen-antibody reaction with the labeled antibody or antigen such as latex or colloidal gold on the conjugate pad 16, and the generated reactant and unreacted material are traversed. Move to the direction, that is, the development layer 18.

The reactant reacts with the antibody or antigen in the solid-phase band 20 in the solid-phase band 20 of the developing layer 18 to form an antigen-antibody reaction to form a measurement complex colored by the labeling substance. Analysis of blood sample components by visually checking the density of the dye based on the amount of the labeling substance in the measurement complex in the solid phase band 20, or expressing the color density of the solid phase band 20 by reflected light by the intensity of the dye absorption light An object can be measured.

Alternatively, the latex itself is also colored with a substance having fluorescence or luminescence, and the amount of the label in the solid phase band is determined from the amount of fluorescence or luminescence, and expressed as a qualitative or quantitative value.

The unreacted substances that did not react in the solid phase band 20 of the developing layer 18 are removed by the water absorbing layer 22.
Out of the solid-phase band 20 of FIG.

FIG. 2 is a cross-sectional view showing another embodiment of the test carrier of the present invention. The plasma or serum is separated from whole blood using capillary force, and then the plasma or serum is taken into a capillary and the capillary is separated. 455 nm with a fine capillary
An example of a test carrier when bilirubin having an absorption wavelength is directly colorimetrically measured from plasma or serum is shown.

In the figure, reference numeral 30 denotes a test carrier, which is a long capillary 3 made of glass or transparent plastic.
Two. At one end of the capillary 32, an attachment portion 34 is formed by removing a part of the capillary 32.

The mounting portion 34 has a plasma or serum separating layer 36
Is provided. The plasma or serum separation layer 36 is formed of the same fiber-containing layer as the plasma or serum separation layer 14 in FIG. The portion of the capillary 32 other than the portion where the plasma or serum separation layer 36 is provided functions as a test area 38 as shown in FIG.

With the above configuration, first, when a blood sample component is dropped on the plasma or serum separation layer 36, red blood cells in the blood sample component are retained by the glass fibers in the plasma or serum separation layer 36, and the plasma or serum is separated. Is done.

The separated plasma or serum is introduced into the capillary 32, ie, the test area 38 by capillary force, irradiated with light from the top or bottom of the capillary 32, detected on the bottom or top, and the bilirubin concentration is determined by the absorbance at 455 nm. Measure.

[0048]

EXAMPLES The present invention will be described in more detail with reference to the following examples. However, it is needless to say that these examples are illustrative and should not be construed as limiting.

(Production Example 1) A 0.5% aqueous solution of N- (butoxymethyl) acrylamide (manufactured by Wako Pure Chemical Industries, Ltd.)
After dipping glass fiber filter paper GF / D (Whatman, thickness 675 μm, pore size average 5.7 μm) cut into a size of 4 mm in width and 15 mm in length, dried at 70 ° C. for 30 minutes, plasma or A serum separation pad was manufactured.

(Production Example 2) A 0.5% aqueous solution of 1-butoxy-2-propanol (manufactured by Wako Pure Chemical Industries, Ltd.) was 4 m wide.
m, glass fiber filter paper GF cut to a size of 15 mm in length
/ D (Whatman, thickness 675 μm, pore size average 5.7 μm), and then dipped at 70 ° C.
After drying for minutes, a plasma or serum separation pad was produced.

(Examples 1 and 2) Separation characteristics of plasma or serum: Plasma or serum separation pad made of glass fiber of the present invention produced in Production Examples 1 and 2 (Example 1: 0.5% N- (butoxymethyl) The plasma or serum separation ability of acrylamide-containing glass fiber (Example 2: glass fiber containing 0.5% 1-butoxy-2-propanol) and the amount of hemoglobin in the separated plasma or serum were measured.

Method for measuring plasma or serum separation ability: width 4 mm
× 80 μL of blood having a hematocrit of 56% was dropped on the end of the above-mentioned plasma or serum separation pad having a length of 15 mm with a pipette, and the area ratio of the blood cell portion to be separated on the plasma or serum separation pad and the plasma or serum portion was measured.

Measurement of Separation Time: Blood sample components were dropped on a plasma or serum separation pad, and the time required for blood cells to move through the pad and stop was visually measured.

Measurement of hemoglobin amount: width 4 mm × length 1
Hematocrit 56% on 5mm plasma or serum separation pad
Is dropped by a pipette, and a plasma or serum is collected by applying a capillary to a plasma or serum portion to be separated on a plasma or serum separation pad. Using a BH meter (manufactured by Sanko Junyaku) capable of measuring the amount of hemoglobin in the collected plasma or serum using a capillary, the absorbance is measured at a wavelength of 575 nm,
The hemoglobin concentration was calculated from the absorbance using hemoglobin control (Hemocon-N) (15.0 g / dL) (aswell) as a reference. The results are shown in Table 1.

(Comparative Examples 1 to 5) Glass fiber filter paper GF / D
(Whatman) (Comparative Example 1) Glass fiber containing 1% mannitol and 0.1% albumin (Comparative Example 2), glass fiber containing 2% polyvinyl alcohol (Comparative Example 3), glass fiber containing polybrene (Comparative Example 4) )as well as,
A plasma- or serum-separation pad made of glass fiber was produced in the same manner as in Example 1 using 2% polyvinyl alcohol and polybrene-containing glass fiber (Comparative Example 5), and plasma or serum separation characteristics, plasma or serum separation ability, and hemoglobin were obtained in the same procedure. The amount was measured, and the results are shown in Table 1 together with Examples 1 and 2.

[0056]

[Table 1]

As is clear from the results in Table 1, the plasma or serum separation pads of the present invention (Examples 1 and 2) are different from the conventional plasma or serum separation pads (Comparative Examples 1 to 5).
It has been found that it has characteristics that it has the ability to separate erythrocytes and has little hemolysis, and that the separation time of plasma or serum is quick.

In Examples 1 and 2 described above, 0.5% N
-(Butoxymethyl) acrylamide and 0.5% 1-
An example in which butoxy-2-propanol was independently contained in the glass fiber was shown, but it was confirmed that the same results as in Examples 1 and 2 were obtained when both were mixed at the same ratio.

[0059]

As described above, according to the present invention, when plasma or serum is separated from whole blood, erythrocytes can be rapidly separated, the separation ability is high, and the hemolysis is small. Achieved.

[Brief description of the drawings]

FIG. 1 is a design diagram of a test carrier containing a plasma or serum separation layer of the present invention.

FIG. 2 is a design diagram of a test carrier containing a plasma or serum separation layer of the present invention.

[Explanation of symbols]

10, 30: test carrier, 12: support plate, 14, 36:
Plasma or serum separation layer, 16: conjugate pad, 1
8: development layer (test area), 20: solid phase band, 20a: capture zone, 20b: control zone, 2
2: water absorption layer, 32: capillary, 34: mounting part, 3
8: Test area.

Claims (5)

[Claims] 1. A method for separating plasma or serum from whole blood by contacting whole blood with a fiber-containing layer and retaining red blood cells by the fiber-containing layer, wherein the fiber-containing layer contains glass fibers. A method for separating plasma or serum from whole blood, characterized in that hemolysis is suppressed by using a glass fiber formed of a layer and coated with butanol-containing propanol and / or acrylamide as the glass fiber. 2. The method according to claim 2, wherein the propanol and / or acrylamide having the butoxy group is butoxypropanol and / or acrylamide.
Or the butoxymethyl acrylamide. 3. An instrument for separating plasma or serum from whole blood by contacting whole blood with a fiber-containing layer and retaining red blood cells by the fiber-containing layer, wherein the fiber-containing layer has a butoxy group. An apparatus for separating plasma or serum from whole blood, wherein hemolysis is suppressed by forming a layer containing glass fibers coated with propanol and / or acrylamide. 4. The method of claim 1, wherein the propanol and / or acrylamide having a butoxy group is a butoxypropanol and / or acrylamide.
4. The device according to claim 3, wherein the device is butoxymethylacrylamide. 5. A blood plasma or serum separation layer for separating plasma or serum from a blood sample component, and a test area for testing the separated plasma or serum for measuring an analyte of the blood sample component. A test carrier characterized in that the plasma or serum separation layer contains the apparatus for separating plasma or serum according to claim 3 or 4, thereby suppressing hemolysis. .