JPH08220089A - Hematocrit value measuring method and testing implement - Google Patents

Abstract

PURPOSE: To rapidly measure the hematocrit value by adding water-soluble indicating substance to total blood sample, absorbing the substance added plasma to a dry gel made of an absorber, and detecting the concentration of the substance introduced into the gel. CONSTITUTION: A predetermined quantity of total blood is drip-adhered from a developing layer 1 side. The blood is uniformly spread by the operation of the layer 1. After spreading, the plasma in the blood is dispersed in the water-soluble indicating substance in a colored layer 2, and the substance is dissolved in the plasma. The plasma in which the substance is dissolved in a measuring layer 3 made of a dry gel medium is impregnated into the medium, and the layer 3 is swelled. Since the absorptivity of the layer 3 is decided according to hematocrit, the substance concentration in the layer 3 corresponds to the hematocrit. Light reflected fine particles are mixed to be dispersed in the layer 3, and the substance concentration in the layer 3 is detected by the light reflection from the direction B of a support 4.

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a rapid method for measuring hematocrit level in whole blood and a test device using the method. The hematocrit value in whole blood (hereinafter, simply referred to as hematocrit) indicates the volume percentage of red blood cells in whole blood. The value is used as an index for knowing the degree of anemia, polycythemia, etc., or is used as a method for correcting the measured value of a whole blood test piece that may give an incorrect value under the influence of hematocrit. . As a conventional method for measuring hematocrit, a method of obtaining from the number of red blood cells in a fixed volume and an average red blood cell volume, a method of performing precipitation by centrifuging whole blood, a method of obtaining from an electric resistance value of whole blood, There is a method of obtaining by spreading. Further, as a method for measuring the hematocrit value with a test piece type without using a large-sized device, Japanese Patent Publication No. 63-3260 discloses a method using a gel medium containing a color change indicator. However, the above-mentioned methods (1) to (3) require special large-sized equipment, and require complicated measuring operations, which requires time and labor for the measurement and is complicated. On the other hand, although the method of (1) is simple and convenient so that it can be processed into a test piece, this method and the test piece show that the indicator substance in the wet gel migrates into the whole blood sample. Since it uses the "diffusion phenomenon", it has a drawback that it takes time to produce a result. An object of the present invention is to provide a method for measuring the hematocrit level in whole blood, which method can be applied to a simple test tool and can quickly produce a result, and It is to provide a test tool to which the method is applied. [0006] In view of the above-mentioned prior art methods, as a result of researching a method and a test tool capable of obtaining a quicker result, the above-mentioned problems can be solved as follows. It turned out to be resolved. That is, a method of measuring a hematocrit value in a whole blood sample, wherein a water-soluble indicator substance that is optically detectable and does not denature the whole blood sample is added to the whole blood sample. A substance-added whole blood sample is brought into contact with a fixed amount of the surface of a dry gel medium composed of an absorbent to swell the gel medium by allowing the gel medium to absorb a serum or plasma component containing a water-soluble indicator substance,
Optically detecting the amount of the water-soluble indicator substance that has entered the gel medium together with the serum or plasma component. A test tool for measuring a hematocrit value can be prepared by using the above method. As one embodiment, a test device for measuring a hematocrit value in a whole blood sample,
On a transparent or semi-transparent support, a measurement layer made of a dry gel medium containing a light-reflecting agent and a colored layer containing a water-soluble indicator are provided in this order, whole blood. It is a test tool for measuring the hematocrit value in a sample. If the area of the test device is large, the development of the whole blood sample will be unsuccessful and unevenness is likely to occur. Therefore, a development layer known in the art may be provided on the colored layer. The spreading layer is not necessary when the area of the test device is small. The absorption rate of the gel medium (an index of how much the gel absorbs liquid and swells) changes due to the fact that red blood cells in a whole blood sample inhibit the absorption of plasma or components into the gel. . That is, the less red blood cells, the higher the absorption rate of plasma in whole blood into the gel medium, and the larger the amount of plasma in the gel medium. Conversely, the more red blood cells there are, the lower the absorption rate of plasma in whole blood into the gel medium and the smaller the amount of plasma in the gel medium. Therefore, by measuring the amount of plasma that has entered the gel medium, the degree of inhibition of red blood cells can be known, and as a result, the hematocrit value in the whole blood sample can be measured. Since the original gel medium before the measurement is in a dry state, the plasma component is absorbed into the dry gel medium at a very high speed, and a quick measurement can be realized. The gel medium used in both the method and the test device of the present invention is a commercially available absorbent. This absorbent is a swelling absorbent material having a property that the swelling absorption rate changes with hematocrit, and polyethylene oxide is particularly preferable. The spreading layer of the test device of the present invention is preferably one that can spread the spotted sample uniformly and allow all the components (in this case, whole blood) in the sample to permeate. For example, a water-insoluble fibrous or non-fibrous porous layer whose surface and inside are hydrophilic is used. Regarding the shape and distribution density of the pores, it is suitable that the whole blood component easily penetrates and spreads, and the pore diameter of 3 to 20 μm is suitable. The colored layer of the test device of the present invention contains a water-soluble indicator substance to be added to a whole blood sample, which is optically detectable and does not denature the whole blood sample.
For example, it may be a colored molecule such as food blue dye 1. The method of incorporating the dye into the coloring layer may be a method usually used in this industry, such as a method of impregnating a porous matrix such as a filter paper or a method of layering a material kneaded with a water-soluble binder. . Examples of water-soluble binders include synthetic polymers such as polyvinyl alcohol, polyacrylic acid and polyacrylamide, and natural polymers such as cellulose derivatives and metrolose. These binders are
A single substance may be used, or two or more types may be mixed. The wet thickness of the reagent layer is preferably 1 μm to 1.5 mm, especially 5 to 9 mm.
00 μm is suitable. As for the support of the test device, any support may be used as long as it is transparent or translucent, light-transmitting, inert to reagents, and strong enough to hold a reagent layer or the like. It may be made of various materials. For example, glass, polyester (polyethylene terephthalate, etc.), cellulose ester (cellulose acetate propionate, etc.), synthetic resin film of polymethylmethacrylate, etc. are used. The layers can be laminated by any method that allows transverse movement of water between layers, such as a method in which a water-soluble polymer containing no reagents is adhered as glue or pressure is applied for pressure bonding. The method used by those skilled in the art may be used. Specific examples of the present invention will be described below with reference to the accompanying drawings. The basic structure of the hematocrit measurement test tool of the present invention is as shown in the sectional view of FIG. In this test device, a measurement layer 3 affected by the absorption and swelling rate due to hematocrit is provided on a light-transmissive support 4, and a colored layer 2 is provided thereon.
And the development layer 1 is laminated thereon. In FIG. 1, when measuring the hematocrit value of a whole blood sample, first, a fixed amount of whole blood is spotted from the direction A of the test device, that is, from the spreading layer side. Whole blood is 3-10
Any amount between 0 μl may be used, but 15 μl or more is a suitable amount as a sufficient amount for measurement. The spotted whole blood is spread uniformly by the action of the spreading layer 1 immediately after the spotting.
After spreading, the plasma in whole blood disperses the water-soluble indicator substance in the colored layer 2, and the water-soluble indicator substance dissolves in the plasma. Red blood cells, which are particles, cannot enter the measurement layer 3 made of the dry gel medium, but plasma in which the indicator substance is dissolved permeates into the dry gel medium, and the measurement layer 3
Swells. At this time, the degree of permeation is affected by the red blood cells, so the gel medium swells while being affected by the hematocrit value. Light-reflecting fine particles are mixed and dispersed in the measurement layer 3, and the concentration of the indicator substance in the measurement layer 3 can be detected from the direction B of the support 4 by light reflection. Since the absorption rate of the measurement layer 3 is determined by hematocrit, the concentration of the indicator substance in the measurement layer 3 corresponds to hematocrit. When there are few red blood cells, the gel swells with a large amount of plasma components and is colored dark, and when there are many red blood cells, the gel swells with a small amount of plasma components and is colored lightly. Since the light-reflecting substance is mixed in the gel medium, photometry can be easily performed. EXAMPLES Examples will be shown below. [Preparation of test device for hematocrit measurement] (Measurement layer) -Aquacoke (nonionic polymer absorbent, manufactured by Sumitomo Seika Chemicals, Ltd.) 10% (weight ratio) -Triton X-100 (surfactant, pure Wako) Yaku Co., Ltd.
0.1% (weight ratio) ・ Titanium dioxide fine particles (light reflecting agent, manufactured by Wako Pure Chemical Industries, Ltd.)
10% (weight ratio) The above substances were mixed in methanol, coated on a transparent polyethylene terephthalate film with a wet thickness of 50 μm and dried to form a measurement layer on the support. (Colored layer) -Edible blue dye No. 1 (manufactured by Tokyo Chemical Industry Co., Ltd.) 30 mg-Metrose (# 4000, manufactured by Shin-Etsu Chemical Co., Ltd.) 2.5
% (Weight ratio) -Triton X-100 (surfactant, Wako Pure Chemical Industries, Ltd.)
0.1% (weight ratio) was mixed and coated on a polyethylene terephthalate film different from the measurement layer with a wet thickness of 100 μm and dried to obtain a colored layer. The fibrous material which became the spreading layer and was wet with water was brought into contact with the colored layer on the film to transfer the colored layer to the material. Peel off the film and stack the colored layer and the development layer.
The above-mentioned measurement layer was pressure-bonded to the applied film. After pressure bonding, the completed film was cut into 7 × 7 mm to obtain a test tool. [Preparation of sample] Whole blood containing the anticoagulant heparin was centrifuged to separate it into red blood cells and plasma components, which were mixed at various ratios to obtain five types of samples with different hematocrit values shown in Table 1. It was The assay of these mixed samples was performed by dispensing the mixed sample into a capillary tube, centrifuging, and then examining the hematocrit value. [Preparation of calibration curve] Samples with different hematocrit values 10μ
1 was spotted on the test tool from the direction A in FIG. After spotting
Using a colorimeter (Σ90, manufactured by Nippon Denshoku Industries Co., Ltd.), the reflectance at a wavelength of 640 nm was measured from the direction B in FIG. The reflectance was converted into a K / S value. The measurement time was 180 seconds at 10-second intervals after spotting. The results are shown in Table 1, and the obtained correlation diagram (calibration curve) is shown in Graph 1. [Calibration of Calibration Curve] 10 μl of fresh human blood containing heparin as an anticoagulant was spotted on the above test device. The K / S value was calculated 60 seconds later and found to be 0.0734. From the calibration curve of Graph 1, the hematocrit value was 40.8%. When the hematocrit value of the fresh human blood from the same origin was determined by centrifugation, it was 41%. From this, it can be seen that a good calibration curve was obtained from the method according to the present invention and the test device based thereon. [Table 1]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic sectional view of a hematocrit value measuring test tool according to the present invention. 1: Development layer 2: Colored layer 3: Measurement layer 4: Light transmissive support A: Whole blood sample spotting direction B: Reflectance measurement direction Graph 1 shows the relationship between hematocrit value and reflectance according to the method of the present invention. It is a correlation diagram. Vertical axis: K / S value Horizontal axis: Hematocrit value

Claims (1)

Claim: What is claimed is: 1. A method for measuring a hematocrit value in a whole blood sample, wherein a water-soluble indicator that is optically detectable and does not denature the whole blood sample is used as the whole blood sample. And a certain amount of this water-soluble indicator-added whole blood sample is brought into contact with the surface of a dry gel medium composed of an absorbent so that the serum or plasma component containing the water-soluble indicator is absorbed in the gel medium. A method for measuring a hematocrit value in a sample of whole blood, which comprises swelling the gel medium with the method described above, and optically detecting the amount of the water-soluble indicator substance that has entered the gel medium together with the serum or plasma component. 3. The measuring method according to claim 1, wherein the polymer absorbent is polyethylene oxide. 3. A test device for measuring a hematocrit value in a whole blood sample, comprising a measurement layer made of a dry gel medium made of an absorbent having a light reflecting agent mixed on a light transmitting support. A test device for measuring a hematocrit value in a whole blood sample, characterized in that a colored layer containing a water-soluble indicator substance is provided in this order. 4. The test device according to claim 3, wherein the polymer absorbent is polyethylene oxide.