JPH11133027A - Instrument for analyzing component in red blood corpuscle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an instrument for analyzing components in red blood corpuscles which needs no dilution and hemolytic operations before the supply of a sample and is simple in structure. SOLUTION: A sample feed part 1 and an analysis part 5 are connected with each other at a passage 3. A hemolytic means 2 is provided at the sample feed part 1 or between the sample feed part and the analysis part 5. While a sample including red blood corpuscles is moved from the sample feed part 1 to the analysis part 5, the red blood corpuscles are hemolyzed and components in the red blood corpuscles are diluted with other sample components and analyzed at the analysis part 5. The hemolytic means 2 used is a filter. The sample including the red blood corpuscles is forcibly passed through the filter by negative pressure so as to be hemolyzed. An average diameter of holes of the filter is preferably 7 μm or smaller.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an analytical instrument used for analyzing the internal components of red blood cells.

[0002]

2. Description of the Related Art Glycohemoglobin (hemoglobin A1c), in which glucose is bound to hemoglobin by a non-enzymatic reaction, is one of the inner components of red blood cells.
It reflects the average blood glucose level two months ago. Therefore, the concentration of hemoglobin A1c is an important index for diagnosing diabetes,
It is widely used for adult disease screening and treatment guidance.

[0003] In clinical medical examinations, hemoglobin A
The measurement of 1c is performed by a number of methods such as an HPLC method based on ion exchange chromatography and an immunoassay method.

Since hemoglobin A1c is a component in red blood cells, a hemolysis operation is indispensable, and even if a hemolysis operation is performed, the concentration may be too high and accurate measurement may not be possible. Therefore, the sample is hemolyzed and further diluted before the blood is supplied to the analysis tool. Further, a diluent is placed on an analysis tool, and after the blood is supplied, hemolysis is performed simultaneously with dilution with the diluent.

[0005]

It is troublesome to perform a hemolysis operation or a dilution operation before supplying a sample to an analysis tool. Too much burden. In addition, the method of arranging the diluting solution on the analysis tool complicates the structure of the analysis tool,
There is a problem that the manufacturing cost increases.

Accordingly, an object of the present invention is to provide an instrument for analyzing components in red blood cells which does not require a dilution operation and a hemolysis operation before supplying a sample and has a simple structure.

[0007]

To achieve the above object, the present invention provides an instrument for analyzing components in red blood cells, comprising a sample supply section and an analysis section, wherein the two are connected by a flow path. The sample supply unit or a hemolysis means between the sample supply unit and the analysis unit, when moving a sample containing red blood cells from the sample supply unit to the analysis unit, to lyse the red blood cells and other components in the red blood cells Dilute with the sample component and analyze in the analysis unit.

[0008] As described above, the analysis device of the present invention lyses red blood cells therein, and dilutes components in red blood cells with other components of the sample. Therefore, it is not necessary to perform a hemolysis operation and a dilution operation before supplying the sample to the analysis tool, and the structure of the analysis tool is simple.

In the analytical device of the present invention, not all erythrocytes need to be lysed, and some may be lysed.

[0010] In the analytical device of the present invention, it is preferable that the hemolysis means is a filter, and the sample containing red blood cells is hemolyzed by forcibly passing the filter through the filter by a pressure. The average pore size of the filter is preferably 7 μm or less.

[0011] In the analytical device of the present invention, the object to be analyzed is not particularly limited as long as it is a component in erythrocytes. It is preferably at least one selected from the group consisting of phosphorus.

Since the analytical device of the present invention is particularly useful for measuring hemoglobin A1c, the hemoglobin measuring unit and the reagent disposing unit are arranged in this order in the middle of the flow path between the hemolysis means and the analyzing unit. It is preferable that a labeled anti-hemoglobin A1c antibody is disposed in the reagent disposing portion, and an immobilized anti-hemoglobin A1c antibody is disposed in the analyzing portion.

[0013]

Next, an embodiment of the present invention will be described with reference to the drawings.

(Embodiment 1) FIG. 1 shows an example of the analysis tool of the present invention. FIG. 1A is a plan view of the analysis tool, and FIG. 1B is a cross-sectional view in the II direction of FIG. 1A.

[0015] As shown in the figure, the analytical device is in the shape of a rectangular plate, and has one end (the right end in the figure)
A sample supply port 1 is formed, and an analysis section 5 is formed substantially at the center, and these are connected to each other by a flow path 3. The filter 2 is disposed below the sample supply port 1 so as to block the flow path 3. A flow path extends from the analysis section 5 to the other end (the left end in the figure) of the analysis instrument, and a surplus sample reservoir 6 is formed in the middle of the flow path. It communicates with 7. A reagent film 4 is disposed in the analysis section 5. Here, examples of the reagent film include a film in which a detection reagent is impregnated in a carrier such as a membrane filter, and a film in which the detection reagent is directly fixed to the analysis unit 5 with a binder. When measuring with reflected light as with a densitometer, the upper part of the analysis unit 5 is usually transparent so that light is transmitted. When measuring with transmitted light, the upper part of the analysis unit 5 is used. The lower part is also transparent.

The filter 2 is not particularly limited as long as it can lyse red blood cells when passing them. As described above, a membrane filter, a glass filter, a sintered body or the like having an average pore diameter of 7 μm or less can be used.
The preferred range of the average pore size is 0.1 to 5 μm. The material of the membrane filter is not particularly limited, and examples thereof include polycarbonate, polysulfone, nylon, and nitrocellulose. The thickness of the membrane filter is usually 10 to 400 μm,
Preferably it is in the range of 20 to 300 μm. The size of the membrane filter is appropriately determined depending on the size of the flow path and the sample supply port. The membrane filter is preferably subjected to a hydrophilic treatment. The hydrophilization treatment can be performed by, for example, a method in which the filter is immersed in a hydrophilic polymer solution or a surfactant solution and then dried.

The type of the reagent film can be appropriately selected according to the object to be analyzed. For example, when glucose is to be analyzed, the reagent film is composed of glucose oxidase (GOD), peroxidase (POD),
Aminoantipyrine and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TO
The four reagents (OS) are impregnated in a carrier such as a membrane filter, or the four reagents are directly fixed to the analysis unit 5 with a binder such as polyvinyl alcohol (PVA). This produces a dye that absorbs at a light wavelength of 555 nm. When hemoglobin is analyzed, hemoglobin has an optical wavelength of 5
Since it exhibits characteristic absorption at 40 nm and at a light wavelength of 577 nm, it is not necessary to arrange a reagent film if this is used (oxyhemoglobin method), but measurement is performed by arranging a reagent containing potassium ferricyanide, potassium cyanide and potassium bicarbonate. (Cyanmethemoglobin method).

This analytical tool can be manufactured by laminating synthetic resin films of a predetermined shape. Examples of the type of the synthetic resin include an acrylonitrile-butadiene-styrene copolymer (ABS resin), polystyrene, polyethylene terephthalate (PET), and an acrylic resin. Lamination of the film may be performed by using an adhesive or laminating by heating and pressing.

In this analytical device, the filter 2 is disposed in the flow path immediately below the sample supply port.
The present invention is not limited to this. For example, FIG.
As shown in (2), the sample supply port 1 may be covered with a filter 2. As shown in FIG. 2B, two filters 2a and 2b may be stacked to cover the sample supply port 1. In this case, since the outer filter 2a functions as a pre-filter, the outer filter 2a
The average pore diameter of a is preferably larger than the average pore diameter of the inner filter 2b.

The size of the analytical device is not particularly limited, and is usually 15 to 100 mm in overall length and 5 in overall width.
６０60 mm, overall thickness 1-5 mm. Also, the size of each part is not particularly limited, and usually, the diameter of the sample supply port is 1 to 10 mm, the width of the channel is 1 to 10 mm,
The flow path depth is 0.03 to 2 mm, and the diameter of the analysis section is 2 to 10
mm, the diameter of the excess specimen reservoir is 3 to 30 mm, and the diameter of the air outlet is 0.1 to 2 mm.

The pump connected to the analysis tool is not particularly limited, and for example, a micro syringe, a syringe, a syringe pump, an aspirator, a tube pump, a roller pump, an air pump and the like can be used.

Next, an example of use of this analysis tool will be described. First, a pump is connected to the air outlet 7 via a hose or the like. Then, a sample (such as whole blood) is supplied to the sample supply port 1.
Drops, the sample stops on the filter 2. In this state, when the pump is started to generate a suction pressure, the sample passes through the filter 2 and is forcibly sucked into the channel 3. During this passage, the red blood cells are lysed, the components in the red blood cells are eluted, and are appropriately diluted with other analyte components (such as plasma). Then, the sample is introduced into the analyzer 5 by forced aspiration. In this introduction, the surplus sample is further discharged to the surplus sample accumulating section 6 through the flow path. And the analysis unit 5
In the above, the reagent in the reagent film 4 reacts with the components in the red blood cells, and the reagent film 4 is colored. As described above, when hemoglobin is measured by the oxyhemoglobin method, the reagent film 4 is not required. Then, the analysis tool having the color of the reagent film 4 is attached to an optical measuring instrument such as a densitometer, and the coloration is measured to measure the concentration of the components in the red blood cells.

(Embodiment 2) FIG. 3 shows an example of an analysis tool provided with a suction pressure generating chamber. FIG. 2A is a plan view of the analysis tool, and FIG. 2B is a cross-sectional view taken along the line II-II of the plan view.

In this analytical tool, a suction pressure generating chamber 8 is formed on an air outlet 7, and a liquid-blocking gas-permeable membrane 9 is disposed in a flow path below the air outlet 7. Is the configuration,
The material, size, and the like are the same as those of the analysis tool of the first embodiment.

The pressure generating chamber 8 is made of an elastic material. After the pressure generating chamber 8 is pressurized and compressed, the pressure is released to restore the pressure generating chamber 8 to its original shape, thereby generating a pressure. The size of the suction pressure generating chamber 8 is determined as appropriate depending on the size of the entire analytical tool, and the like.
The maximum diameter is 3 to 15 mm and the height is 0.5 to 3 mm.
In addition, as the elastic material, for example, the constituent resin of the above-described analysis tool can be mentioned.

The liquid blocking gas permeable membrane 9 is for preventing the sample from flowing into the suction pressure generating chamber 8.
For example, a hydrophobic porous membrane is used. Examples of the hydrophobic porous membrane include a polyethylene porous membrane, a polypropylene porous membrane, and a polytetrafluoroethylene porous membrane. Examples of the hydrophobic porous membrane suitable for the present invention include Celgard (trade name, manufactured by Hoechst Celanese) and Hypore (trade name, manufactured by Asahi Kasei Corporation). The average pore size of the hydrophobic porous membrane is usually 0.1 to 1 μm.
m, preferably 0.3 to 0.7 μm. Further, the thickness of the hydrophobic porous membrane is usually 10 to 100 μm. Such a hydrophobic porous membrane can be produced by preparing a film of the hydrophobic resin and stretching the film uniaxially or biaxially.

An example of the use of this analysis tool is as follows. That is, first, the suction pressure generating chamber 8 is pressurized and compressed with a finger or the like. In this state, when a sample (such as whole blood) is dropped into the sample supply port 1, the sample stops on the filter 2. In this state, when the pressure is released to generate a suction pressure, the sample is forcibly aspirated into the channel 3 through the filter 2. The subsequent operation is the same as in the first embodiment.

(Embodiment 3) FIG. 4 shows that hemoglobin A1
The example of the analysis tool which makes c an analysis object is shown. Figure (A)
FIG. 3 is a plan view of the analysis tool, and FIG. 3B is a cross-sectional view of the analysis tool taken along the line III-III in the plan view.

In this analytical device, a hemoglobin measuring section 10 and a reagent arranging section 11 are formed in this order in the flow path 3 between the sample supply port 1 and the analyzing section 5. Other than this,
The configuration, material, size, and the like are the same as those of the analysis tool of the first embodiment.

The hemoglobin measuring section is formed to measure the concentration of hemoglobin A1c with respect to hemoglobin, and irradiates light to measure the absorbance at 540 nm or 577 nm.

The reagent arranging section 11 is one in which a reagent is merely arranged in the middle of the flow path 3. Here, a labeled anti-hemoglobin A1c antibody is usually placed. In the analysis section 5, an immobilized anti-hemoglobin A1c antibody is provided. The above two antibodies can be prepared by a conventional method, and can be immobilized on an analysis part by a conventional method. The label is not particularly limited as long as it can be measured by an optical measurement method or the like. Hereinafter, usable antibodies, labeling methods, and immobilized antibodies will be described.

(Antibody used) Immobilized anti-hemoglobin A1c
Examples of the antibody include hemoglobin A manufactured by DAKO.
1c monoclonal antibodies can be used. As the labeled anti-hemoglobin A1c antibody, an anti-hemoglobin A1c polyclonal antibody manufactured by Iwki can be used.

(Labeling Method) In the preparation of a labeled anti-hemoglobin A1c antibody, latex, colloidal gold, or the like can be used as a label. As the latex, for example, a carboxyl group-bonded latex (particle size: 200 nm) manufactured by Bang can be used, which can be bound to an antibody by a carbodiimide bond. As the gold colloid, for example, gold colloid (particle size: 30 nm) manufactured by Biocell can be used, and this can be bound to the antibody by physical adsorption.

(Immobilized Antibody) The immobilized antibody can be prepared, for example, as follows. That is, first, the membrane (made of nitrocellulose, nylon, etc.) is
After applying the antibody solution at a rate of about 1 μg / cm ² using a dispensing applicator manufactured by DOT, for example, at about 40 ° C. for about 20
Allow to dry for minutes. About 5% by weight of this membrane
It is immersed in bovine serum albumin (BSA) solution or Block Ace (trade name, manufactured by Snow Brand Milk Products Co., Ltd.) for about one hour to perform blocking, and dried at about 40 ° C. for about 20 minutes using a trans oven or the like. Then, this membrane is arranged in the analysis unit 5. In order to increase the application amount of the antibody, a complex of dextran and the antibody may be applied.

An example of the use of this analysis tool is as follows. That is, first, a pump is connected to the air outlet 7 via a hose or the like. When a sample (such as whole blood) is dropped into the sample supply port 1, the sample stops on the filter 2. In this state, when the pump is started to generate a suction pressure, the sample passes through the filter 2 and is forcibly sucked into the channel 3. During this passage, the erythrocytes are lysed to elute the components in the erythrocytes (hemoglobin and hemoglobin A1c, etc.) and are appropriately diluted by other analyte components (plasma, etc.). Then, the sample is passed through the reagent placement unit 11 by forced aspiration. In this passage, hemoglobin A1c
Is bound by a labeled anti-hemoglobin A1c antibody. Then, when the sample is further introduced into the analysis unit 5 by the pressure reduction,
The hemoglobin A1c to which the labeled anti-hemoglobin A1c antibody has bound also binds to the immobilized anti-hemoglobin A1c antibody and is fixed to the analysis unit 5. The surplus sample is further discharged to the surplus sample reservoir 6 through the flow path. Next, the pump is stopped, and the analysis tool is mounted on an optical measuring device such as a densitometer. Then, the hemoglobin measuring unit 10 and the analyzing unit 5 are irradiated with light to measure the absorbance of hemoglobin and the absorbance of the label, respectively. Then, based on the total hemoglobin, hemoglobin A1c
Calculate the concentration.

[0036]

Next, an embodiment will be described.

An analytical tool as shown in FIG. 5 was prepared.
FIG. 2A is a plan view of the analysis tool, and FIG.
It is IV-IV direction sectional drawing in the said top view. This analytical tool is obtained by laminating a transparent PET plate (length 30 mm, width 15 mm, thickness 0.18 mm) 14 a and a white PET plate (size is the same as above) 14 b via a spacer (double-sided tape) 13. is there. Reference numeral 5 denotes an analysis unit, which irradiates light (arrow) to this part as shown in the figure. Also, a little end from the center of the transparent PET plate 14a (right side in the figure)
The sample supply port 1 is formed by piercing a portion biased toward the sample. The transparent PET 14a including the sample supply port 1
Half of the surface (right half in the figure) is covered with the porous film 2. Further, from the side opposite to the sample supply port 1 side, the flow path 3
Is connected to the micro syringe 12. The size of this analysis tool is 30 mm in length and 15 mm in width. Channel 3
Has a width of 4 mm and a length of 17 mm, and has a depth of 120 μm and 240 μm by the spacer 13.
m. The size of the porous membrane 2 is 15 mm both vertically and horizontally, and the thickness is as shown in the table below. The diameter of the sample supply port 1 is 3 mm.

(Examples 1 to 9) In this analytical device,
Using the porous membranes shown in Tables 1 and 2 below, hemolysis of red blood cells and dilution of components in red blood cells were examined. That is, first, whole blood is dropped on a portion corresponding to the sample supply port 1 on the porous membrane 2, and aspirated into the channel 3 by the micro syringe 12.
It was moved to the analysis unit 5. The state of hemolysis and dilution of red blood cells at the time of this suction was examined by visual observation. The results are also shown in Tables 1 and 2 below. In the column of results in the table below, if dilution or hemolysis was successfully performed,
で indicates.

(Table 1) Porous membrane type Porous membrane structure Channel depth (μm) Result (Example 1) BTS55 (polysulfone) single layer 240 Dilution: ○ Hemolysis made by Memtech: ○ Pore diameter 0.2 μm (Implementation Example 2) BTS5 (polysulfone) monolayer 240 dilution: o hemolysis made by Memtech Co., Ltd .: pore size 1.2 μm (Example 3) Diotaine A (nylon) monolayer 240 dilution: o hemolysis made by foil Corporation: o pore size 1.25 μm (Example 4) High flow membrane (nitrocellulose) monolayer 240 dilution: ○ Hemolysis manufactured by Millipore Corporation: ○ Pore diameter 3 μm Use after hydrophilization treatment (* 1) (Example 5) Lower layer: BTS55 2 layers 240 dilution: ○ Upper layer: Cytosec II (* 2) Hemolysis: ○ 0.6 mm thickness manufactured by Kellman Science * 1: 1% by weight surfactant (Tween 20, manufactured by Nacalai Tesque) After immersion in the solution, it was dried. * 2: Composite of glass fiber and cellulose

(Table 2) Porous membrane type Porous membrane structure Flow channel depth (μm) Result (Example 6) Lower layer: BTS55 2 layers 240 Dilution: ○ Upper layer: Cytoseff (* 1) Hemolysis: ○ Thickness 0 0.3 mm (Example 7) Lower layer: Biotainein A 2 layer 240 Dilution: ○ Pore diameter 1.2 μm Hemolysis: ○ Upper layer: Cytosafe (* 1) 0.3 mm in thickness (Example 8) Lower layer: Biotainein A 2 layer 240 Dilution: ○ Pore size 1.2 μm Hemolysis: ○ Upper layer: Biotamine A Pore size 5.0 μm (Example 9) BTS55 monolayer 120 Dilution: ○ Hemolysis: ○ * 1: Composite of glass fiber and cellulose

From these results, it can be seen that by forcing whole blood through the porous membrane by forced aspiration, hemolysis of erythrocytes and dilution of components in erythrocytes were completed.

Next, in the fifth and ninth embodiments, the reflection absorbance was measured using a flying spot scanner CS-9.
000 (manufactured by Shimadzu Corporation). The results are shown in the graph of FIG. 6 for Example 5 and in the graph of FIG. 7 for Example 9. Also, the reflection absorbance was measured under the same conditions except that the porous film was not used, and this was used as a control. In FIGS. 5 and 6, the curve A represents the measurement result of the control, and the curve B represents the measurement result of the example.

As shown in the graphs of FIG. 6 and FIG. 7, in Examples 5 and 9 in which whole blood was passed through the porous membrane by forced aspiration, 540 nm and 577 nm were used.
A clear absorption peak of hemoglobin was confirmed. On the other hand, in the control, the hemoglobin concentration was too high, and the absorption peak of hemoglobin could not be confirmed.

[0044]

As described above, the analysis device of the present invention comprises a sample supply unit and an analysis unit, and the two components are connected by a flow path. By having hemolysis means between this and the analysis unit,
When a sample containing red blood cells is moved from the sample supply unit to the analysis unit, the red blood cells are lysed, the components in the red blood cells are diluted with other sample components, and analyzed by the analysis unit. For this reason, if the analysis tool of the present invention is used, there is no need to perform a dilution operation and a hemolysis operation before supplying a sample, and it is possible to quickly and easily analyze components in red blood cells such as hemoglobin A1c.
Since a filter or the like can be used as the hemolysis means, the analysis tool of the present invention can have a simple structure and can be manufactured at low cost. That is, since the use of the analysis tool of the present invention makes it possible to process a large number of samples at low cost without imposing a burden on a measurer, the analysis tool of the present invention, for example, processes a large number of samples. It is useful in the medical field where there is a need.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of an embodiment of an analysis tool according to the present invention, wherein A is a plan view and B is a cross-sectional view.

FIG. 2 is a cross-sectional view showing an arrangement of a porous membrane in a sample supply port of an analytical device of the present invention, wherein A shows an example using one porous membrane, and B shows two porous membranes. Are shown below.

FIG. 3 is a view showing a configuration of another embodiment of the analysis tool of the present invention, wherein A is a plan view and B is a cross-sectional view.

FIG. 4 is a diagram showing a configuration of still another embodiment of the analysis tool of the present invention, wherein A is a plan view and B is a cross-sectional view.

FIG. 5 is a view showing a configuration of still another embodiment of the analysis tool of the present invention, wherein A is a plan view and B is a cross-sectional view.

FIG. 6 is a graph showing the results of measuring the absorbance of hemoglobin in an example of the present invention.

FIG. 7 is a graph showing the results of measuring the absorbance of hemoglobin in an example of the present invention.

[Explanation of symbols]

 Reference Signs List 1 sample supply port 2 filter 3 flow path 4 reagent film 5 analysis section 6 excess sample storage section 7 air outlet

Claims (5)

[Claims] An instrument for analyzing components in red blood cells, comprising a sample supply unit and an analysis unit, both of which are connected by a flow path, wherein a hemolysis means is provided between the sample supply unit or this and the analysis unit. A red blood cell component analysis tool for lysing red blood cells, diluting red blood cell components with another sample component, and analyzing the red blood cell components with another sample component when the sample containing red blood cells is moved from the sample supply unit to the analysis unit. 2. The analysis tool according to claim 1, wherein the hemolyzing means is a filter, and the sample containing red blood cells is hemolyzed by forcibly passing the filter through the filter by a pressure. 3. The analytical device according to claim 2, wherein the filter has an average pore size of 7 μm or less. 4. The analysis target is at least one selected from the group consisting of hemoglobin, iron, glucose, hemoglobin A1c, urea nitrogen, uric acid, creatinine, creatine, neutral fat, magnesium and phosphorus. 4. The analytical tool according to any one of items 3 to 5. 5. A hemoglobin measuring section and a reagent arranging section are installed in this order in the middle of a flow path between the hemolytic means and the analyzing section, and the reagent arranging section has a labeled anti-hemoglobin A1.
c antibody is placed, and an immobilized anti-hemoglobin A1c antibody is placed in the analysis section, and the analysis target is hemoglobin A1.
The analysis tool according to any one of claims 1 to 3, which is c.