JPWO2009110089A1 - Body fluid component analyzer - Google Patents

Abstract

Provided is an analytical instrument for analyzing a specific component of a body fluid that can analyze a plurality of items simultaneously with a single instrument using a very small amount of sample, and that the cost required for the analysis is low. When a whole blood sample is applied to the sample supply port 5, only a blood cell component is removed by the etching film 412 and transferred to the measurement chamber 33, and the other is agglomerated in which specific components are contained in the glass fiber filter 411. Aggregates are generated by the reagent, and blood cell components and aggregates are removed by the etching film 412 and transferred to the measurement chamber 32. Therefore, since the plasma containing all the components and the plasma from which a specific component has been removed are transferred to separate measurement chambers, a plurality of items can be analyzed simultaneously.

Description

  The present invention relates to an analytical instrument for measuring biological fluid components, particularly lipoprotein components such as cholesterol (hereinafter referred to as HDL-C) in high-density lipoprotein (hereinafter referred to as HDL).

  HDL-C has an anti-atherogenic effect and is important as a protective factor for coronary artery disease, and low HDL-C blood is counted as one of the main risk factors for coronary artery disease. Therefore, measurement of HDL-C is useful when analysis of risk factors in arteriosclerotic diseases and abnormal lipid metabolism are assumed.

  Conventionally, when measuring cholesterol in a specific lipoprotein such as HDL-C, first, the lipoprotein to be measured is separated by means such as ultracentrifugation or electrophoresis, and then the lipid component is measured. A so-called fractionation method has been adopted. In recent years, a so-called direct method has also been adopted in which HDL-C is selectively measured using a difference in specificity of a surfactant for lipoprotein without requiring fractionation.

  However, the fractionation method requires a large-scale device for performing the analysis, and requires the skill of the operator to obtain a highly accurate analysis result, resulting in a high cost for the analysis. There is a problem of becoming. In the direct method, when measuring a body fluid component without diluting, the inaccuracy of a measured value due to a nonspecific reaction to cholesterol in lipoproteins other than HDL may be a problem.

On the other hand, there is known a technique for analyzing a body fluid component by spotting a small amount of whole blood sample on a test piece and measuring optical characteristics after reacting with a reagent (see Patent Documents 1 and 2). However, the above method is simple, but when it is applied to HDL-C analysis, pretreatment such as fractionation is required before the sample is applied to the test piece, or the sample is applied to the test piece. After that, since it is necessary to perform a multi-step reaction under precise control, there is a problem that the advantage of a simple method cannot be utilized. Furthermore, in order to analyze about several items including HDL-C, there exists a problem that it is necessary to prepare a separate sample and analysis instrument for every item.
JP-A-8-114539 JP 2002-2224090 A

  Therefore, analysis can be performed with simple operations, high-precision analysis results can be obtained quickly, the cost required for analysis is low, and multiple items can be simultaneously measured using a single drop of sample. There has been a need for an analytical instrument for analyzing components of body fluid that can be analyzed.

  The present invention has been made in order to solve the above-mentioned problems. The invention according to claim 1 is directed to a sample supply port through which a sample is supplied, abutting the sample supply port, and a predetermined amount from the sample. A plate-shaped filtration member that removes particles having a diameter or larger, an aggregation reagent that is provided in the filtration member and reacts with a specific component contained in a body fluid to generate an aggregate, and is in contact with the filtration member, so that the filtration A plurality of receiving ports for receiving the sample that has passed through the member; a plurality of measuring chambers for measuring the sample; and extending from each of the measuring chambers and communicating with any one of the plurality of receiving ports A region 1 in which the sample supply port is projected onto the filtration member and a region 2 in which one of the plurality of reception ports is projected onto the filtration member do not overlap, Region 3 between region 1 and region 2 or region 2 The agglutinating reagent is present, and the agglutinating reagent is not present in the region 5 including the region 4 and the region 1 in which another one of the plurality of receiving ports is projected onto the filtration member. Is a body fluid component analyzing instrument characterized by

  According to the first aspect of the present invention, when the sample of whole blood is applied to the sample supply port, the sample is laterally developed in the surface of the filtration member, and a part of the sample is a coagulation reagent provided in the filtration member. The specific components contained in the body fluid come into contact with each other, and the aggregate is separated and filtered by the filtering member and flows into the one receiving port, and then reaches the measurement chamber through the flow path. That is, since the sample introduced into the measurement chamber communicating with the one receiving port is one in which the specific component is removed, the measurement chamber performs measurement on items other than the specific component. Preferred. On the other hand, the sample that reaches the other one of the receiving ports does not come into contact with the agglutinating reagent, so that a specific component is not removed from the sample introduced into the measurement chamber. Therefore, it is possible to introduce a sample from which a specific component has been removed and a sample containing the specific component into separate measurement chambers, and to analyze a body fluid component that enables simultaneous measurement of multiple items. An instrument can be provided.

  The invention according to claim 2 is the body fluid component analyzing instrument according to claim 1, wherein the region 1 and the region 4 overlap.

  According to the invention described in claim 2, the sample that contacts the agglutination reagent among the supplied samples is expanded in the in-plane direction in the filtering member, and then aggregates are generated by contact with the agglutination reagent. Aggregates are separated by the filter member and reach the receiving port, and then transferred to the measurement chamber. On the other hand, the sample that does not come into contact with the agglutinating reagent does not spread laterally and proceeds only by the thickness of the filter member and then reaches the receiving port. Therefore, the sample should be transferred to the measurement chamber with minimal loss of fluid components. Therefore, it is possible to provide a highly efficient and reliable analysis device for a body fluid component.

  The invention described in claim 3 is the body fluid component analyzing instrument according to claim 1 or 2, wherein the flow path has water repellency.

  According to the third aspect of the present invention, since the flow path has water repellency, the sample present in the filtration member does not naturally flow into the flow path, and the time required for completing the agglutination reaction. The sample can be held in the filter member until it has elapsed. Then, after completion of the agglutination reaction, the sample can be transferred to the measurement chamber by performing pressurization or suction operation. That is, the user can control the timing of transferring the sample to the measurement chamber.

  According to a fourth aspect of the present invention, the flow path and the measurement chamber are formed by laminating a water-permeable and air-permeable ventilation plate and a gas-permeable and water-impermeable sealing plate. 3. The body fluid component analyzing instrument according to claim 1 or 2.

  According to the fourth aspect of the present invention, since the flow path has water impermeability, the sample existing in the filter member does not naturally flow into the flow path, and the time required for the completion of the agglutination reaction is reached. The sample can be held in the filter member until it has elapsed. Then, after completion of the agglutination reaction, the sample can be transferred from the separation means to the measurement chamber by performing pressurization or suction operation. Furthermore, since the walls forming the flow channel and the measurement chamber have air permeability, air accumulated in the flow channel is discharged to the outside through this wall, so that an air vent hole is not necessary.

  The invention according to claim 5 is characterized in that the agglutination reagent reacts with a lipoprotein component other than the high-density lipoprotein to produce an aggregate, and a cholesterol quantification reagent is provided in the measurement chamber communicating with the one receiving port. The body fluid component analyzing instrument according to any one of claims 1 to 4, which is provided.

  According to the fifth aspect of the present invention, a part of the supplied sample that comes into contact with the agglutinating reagent is transferred to the measurement chamber after removing lipoprotein components other than HDL, and cholesterol is measured in the measurement chamber. Since the amount is measured, it is possible to analyze only HDL-C. On the other hand, for samples that do not come into contact with the agglutinating reagent, samples that have not been treated at all are transferred to the measurement chamber. Can be analyzed. That is, it is possible to simultaneously analyze HDL-C and other items using a single drop of sample.

  According to a sixth aspect of the present invention, the filtering member is formed by laminating a glass fiber filter paper disposed on the supply port side and an etching film disposed on the receiving port side, and the aggregating reagent is the glass It is contained in the fiber filter paper, The body fluid component analysis instrument according to any one of claims 1 to 5.

  According to the invention described in claim 6, when the whole blood sample is applied to the sample supply port, the blood cell component in the sample is permeated by the glass fiber filter paper when the sample is laterally developed in the in-plane direction of the glass fiber filter paper. Because the sample is inhibited and the sample comes into contact with the agglutination reagent at a high plasma component ratio, the agglutination reaction of a specific component in the sample proceeds rapidly. When the sample passes through the etching film, first, the aggregate having a small particle size is captured, and then a blood cell component having a large particle size is captured. Therefore, smooth filtration with less risk of clogging is realized, and specific components and blood cells are surely removed from the sample, so that highly accurate analysis is possible.

  In the invention according to claim 7, in the glass fiber filter, the content density of the aggregating reagent varies along the film thickness direction, and the surface on the etching film side contains the surface more than the surface on the sample supply port side. 7. The body fluid component analysis instrument according to claim 6, wherein the body fluid component analysis instrument has a distribution shape with a high density.

  According to the seventh aspect of the present invention, when a sample of whole blood is applied to the sample supply port, when the sample proceeds in the film thickness direction of the glass fiber filter paper, the penetration of blood cell components in the sample is inhibited by the glass fiber. In addition, since the sample comes into contact with the agglutination reagent in a state in which the plasma component ratio is high and the agglutination reaction proceeds rapidly, the time required for analysis can be shortened.

  According to an eighth aspect of the present invention, the filtering member includes a glass fiber filter paper disposed on the supply port side, an etching film disposed on the receiving port side, and between the glass fiber filter paper and the etching film. 6. A reagent holding means made of a material having a liquid absorption property is laminated, and the agglutinating reagent is contained in the reagent holding means. This is a device for analyzing body fluid components.

  According to the eighth aspect of the present invention, since the reagent holding means containing the agglutinating reagent can be arranged independently from the glass fiber filter paper, the sample is put into the agglutinating reagent after the plasma component ratio is high. The contact can be ensured.

  The invention according to claim 9 is characterized in that the hole diameter of the etching film in the region 2 is smaller than the hole diameter of the etching film in the region 1. It is an analytical instrument.

  According to the invention described in claim 9, by setting the pore size of the etching film in the region 2 to be smaller than the particle size of the generated aggregate, a specific component is present from a part of the sample that comes into contact with the aggregation reagent. Can be removed reliably. On the other hand, by setting the hole diameter of the etching film in the region 1 to be larger than the particle size of the body fluid component to be measured, it is possible to minimize the lack of the body fluid component in the remaining sample that is not in contact with the agglutinating reagent. That is, highly accurate analysis can be realized.

  According to a tenth aspect of the present invention, the filter member is an asymmetric porous membrane having a pore size distribution in which the pore diameter of one surface is larger than the pore diameter of the other surface, and the asymmetric porous membrane is the one surface. The body fluid component analyzing instrument according to any one of claims 1 to 5, wherein the agglutinating reagent is contained in the asymmetric porous membrane.

  According to the invention described in claim 10, when a sample of whole blood is applied to the sample supply port, blood cell components in the sample are captured by a gap having a relatively large pore size in the asymmetric pore size membrane, and a specific component in the sample is obtained. Is agglomerated by the agglutinating reagent contained in the asymmetric pore diameter membrane, and the aggregate is trapped by the pores of the asymmetric pore diameter membrane having a relatively small pore size, so that smooth filtration with less risk of clogging is realized and specified from the sample. The components and blood cells can be surely removed and used for measurement. That is, highly accurate analysis can be realized.

  In the invention according to claim 11, the asymmetric pore diameter membrane is such that the content density of the agglutinating reagent changes along the film thickness direction, and the surface on the receiving port side contains the surface more than the surface on the sample supply port side. 11. The body fluid component analyzing instrument according to claim 10, wherein the analyzer has a distribution shape with a high density.

  According to the eleventh aspect of the invention, when a whole blood sample is applied to the sample supply port, the blood cell component in the sample is first captured by a gap having a relatively large pore diameter in the asymmetric pore diameter membrane. In contact with the agglutinating reagent at a high ratio, the agglutination reaction proceeds rapidly, and the agglomerates are trapped by voids having a relatively small pore size. Therefore, smooth filtration with less risk of clogging is realized, and specific components and blood cells can be reliably removed from the sample and used for measurement. That is, highly accurate analysis can be realized.

  The invention according to claim 12 is characterized in that the agglutination reagent contains metal ions, and a chelating agent is disposed in a part of the measurement chamber or the flow path. It is an analytical instrument for the body fluid component described.

  According to the twelfth aspect of the present invention, since the color reaction is stabilized in the presence of the metal ion by the chelating agent and the turbidity caused by the metal ion can be prevented, a highly accurate analysis can be realized. Can do.

  The invention according to claim 13 comprises a plurality of circular measurement chambers, wherein the circular measurement chambers are arranged on one axis, and the flow path is perpendicular to the circular radial direction and the one axis. The body fluid component analyzing instrument according to claim 1, wherein the analyzer is extended from the circular measuring chamber.

  According to the invention described in claim 13, the optical measuring device can measure the absorbance of a plurality of measurement chambers by scanning on one axis, and the time required for analyzing a plurality of items can be reduced. can do.

  The invention according to claim 14 is provided with a plurality of elliptical or oval measuring chambers, and the elliptical or oval measuring chambers coincide with a short axis of the elliptical or oval as one axis. The body fluid component analysis according to claim 1, wherein the flow path extends from the measurement chamber along a major axis of the ellipse or ellipse. It is an instrument.

  According to the fourteenth aspect of the present invention, the optical measurement device scans on one axis while avoiding a singular region generated in the vicinity of the coupling portion between the flow path and the measurement chamber. Can be measured, the time required for analysis of a plurality of items can be shortened, and highly accurate analysis can be realized.

  According to the present invention, a sample from which a specific component has been removed from a drop of a sample and a sample containing the specific component can be introduced into separate measurement chambers, and a plurality of items can be analyzed simultaneously. It is possible to provide a body fluid component analysis instrument that can be used. That is, a single body fluid sample and a single instrument can be used for highly accurate analysis of a plurality of items, and the cost required for the analysis is accordingly reduced.

It is sectional drawing which shows the analytical instrument which concerns on the 1st Embodiment of this invention. It is a disassembled perspective view which shows the analytical instrument which concerns on the 1st Embodiment of this invention. It is sectional drawing which shows the analytical instrument which concerns on the 2nd Embodiment of this invention. It is a disassembled perspective view which shows the analytical instrument which concerns on the 3rd Embodiment of this invention. It is sectional drawing which shows the cross section AA of the analytical instrument shown in FIG. It is a disassembled perspective view which shows the analytical instrument which concerns on the 3rd Embodiment of this invention. It is a disassembled perspective view which shows the analytical instrument which concerns on the 3rd Embodiment of this invention. In the analytical instrument which concerns on the 4th Embodiment of this invention, it is the front view of the main-body part seen except the 1st plate. In the conventional analytical instrument, it is the front view of the main-body part seen except the 1st plate. It is drawing which shows the correlation of the measuring method using the analytical instrument which concerns on the 1st Embodiment of this invention, and a control method.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Main-body part 11 1st plate 12 2nd plate 13 3rd plate 21 Flow path 31 Measurement chamber 4 Filtration means 41 Filtration member 411 Glass fiber filter paper 412 Etching film 415 Asymmetric pore diameter film 42 Support tool 5 Sample supply port 61 Receptor port 7 Aggregation Reagent retention range 8 Specific area 9 Axis

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

(First embodiment)
1 and 2 show a first embodiment of an analytical instrument according to the present invention. 1 is a cross-sectional view, and FIG. 2 is an exploded perspective view. This embodiment is a mode for measuring HDL-C based on a whole blood sample.

  As shown in FIGS. 1 and 2, the analytical instrument according to the present embodiment includes a main body 1 and a filtering means 4, and the main body 1 includes a first plate 11, a second plate 12, and a third plate 13. Are bonded through an adhesive layer (not shown) such as a double-sided tape. Although the filtering means 4 has the sample supply port 5, since it is used only when supplying the sample into the main body 1, it is configured to be separable from the main body 1 in this embodiment. It may be adhered.

  The first plate 11 is provided with a through hole 211 having a receiving port 61 at a position substantially concentric with the sample supply port 5. The second plate 12 is also provided with a through hole 212 at a position substantially concentric with the sample supply port 5, a through hole 311 is provided at a position separated from the sample supply port 5, and the groove 213 communicates with the two through holes 212 and 311. . The third plate 13 is provided with a color reagent holding unit 312 substantially concentric with the through hole 311 and holds a color reagent (not shown). The color reagent holding unit 312 can be formed by providing a concave portion at the position of the third plate 13 and filling the color reagent therein, but it is formed by attaching the color reagent to the flat portion. Is also possible. The through holes 211 and 212 and the groove 213 form the flow path 21, and the through hole 311 and the color reagent holding unit 312 form the measurement chamber 31.

  In the present embodiment, the filtering means 4 is impregnated into the filtering member 41 in which the glass fiber filter paper 411 and the etching film 412 are laminated, the support 42 having an opening while supporting the filtering member 41, and the glass fiber filter paper 411. It is composed of an agglutination reagent (not shown) that is dried and reacts with a lipoprotein component other than HDL to generate an aggregate. The opening of the support tool 42 forms the sample supply port 5. The filter member 41 is disposed such that the glass fiber filter paper 411 faces the sample supply port 5 side and the etching film 412 faces the main body 1 side.

  The glass fiber filter 411 has a content of 50% or more as a borosilicate glass fiber, and is selected based on the plasma recovery efficiency and the blood retention amount, and can have a thickness of 400 μm to 1500 μm. The thickness can be selected according to the amount of plasma desired. In this embodiment, Advantech GA-200 was used.

  It is preferable that the aggregating reagent is concentrated on the surface close to the main body 1 in the glass fiber filter paper 411. This is because the movement of the blood cell component is inhibited by the glass fiber, and the agglutination reaction of the sample can be caused in a state where the plasma ratio is high, and the agglutination reaction proceeds rapidly. After impregnating the glass fiber filter paper 411 with the agglutinating reagent and drying it so as to apply air to the surface close to the main body 1, the solution in which the agglutinating reagent is dissolved moves toward the surface where the moisture evaporates, The glass fiber filter paper 411 in which the agglutinating reagent is concentrated on the surface side close to the main body 1 can be prepared. In the present embodiment, after impregnating with the aggregating reagent, 40 ° C. hot air was applied to the surface of the glass fiber filter 411 close to the main body 1 and dried for 30 minutes.

  Note that, instead of impregnating the glass fiber filter paper 411 with the aggregating reagent, a liquid-absorbing material such as a filter paper impregnated with the aggregating reagent is disposed at an arbitrary position between the etching film 412 and the sample supply port 5. It can also be arranged. By doing so, it becomes easy to control the position of the layer containing the agglutinating reagent. Furthermore, in this case, it is preferable that a liquid-absorbing material such as a filter paper impregnated with the aggregating reagent is interposed between the glass fiber filter paper 411 and the etching film 412. This is because the movement of blood cell components is inhibited by the glass fiber, and the agglutination reaction of the sample can be caused in a state where the plasma ratio is high, and the agglutination reaction proceeds rapidly.

  In the present embodiment, sodium phosphotungstate and magnesium chloride are used as an agglutinating reagent that reacts with a lipoprotein component other than HDL to generate an aggregate. The contents were set to sodium phosphotungstate: 2% w / w and magnesium chloride: 2% w / w when dissolved with respect to the blood volume that the glass fiber filter paper 411 can hold. When the magnesium chloride concentration is fixed at 1% w / w and sodium phosphotungstate is varied in the range of 0% to 4%, if it contains 2% w / w or more, the measured value of HDL-C On the other hand, when the sodium phosphotungstate concentration is fixed at 2% w / w and the magnesium chloride is varied in the range of 0% to 3%, if it contains 1.5% w / w or more, HDL Since it was confirmed that the measured value of -C was stabilized, the above-mentioned formulation was used.

  The etching film 412 is a porous track etching film made of polycarbonate or polyester, and a film thickness of 5 to 20 μm can be used, but the pore diameter is 0.4 based on the efficiency of capturing blood cells and aggregates. The thing of -1.0 micrometer is suitable.

  Next, the main body 1 will be described. A plastic material such as polyethylene terephthalate (PET) or AS resin is suitable for the first plate 11 and the third plate 13 because they are easy to process. However, the present invention is not limited to this as long as a flow path in which the sample does not leak can be formed.

  The second plate 12 is preferably made of a water-impermeable and air-permeable porous material such as polytetrafluoroethylene (PTFE). This is because the sample can be transferred to the measurement chamber 31 without providing an air vent hole or the like. However, it is also possible to use the same plastic material as that of the first plate 11 and the third plate 13 for the second plate 12 if a means for venting air such as an air vent hole is provided. At this time, the second plate 12 and the third plate 13 are formed as an integral plate, a groove is provided as the flow channel 21 in the integral plate, and a recess is provided as the measurement chamber 31, and the first plate 11 and the integral plate are provided. It is also possible to have a two-layer structure composed of Further, if the plate material in which the second plate 12 and the third plate 13 are integrated is a porous material that is impervious to water and air permeable, no air vents are required, and body fluid can be obtained with a small number of parts. It becomes possible to constitute a component analysis instrument.

  On the third plate 13, a color reagent (not shown) is applied and dried in a color reagent holding unit 312 and held.

  Next, the analysis procedure using the instrument according to the present embodiment and the function of the instrument will be described. The analysis procedure is the same in all embodiments described later.

  The filtering means 4 is placed on the main body 1 and the position is determined and set so that the filtrate flows into the receiving port 61. Thereafter, a sample of whole blood is dropped into the sample supply port 5.

  The sample dropped into the sample supply port 5 penetrates into the glass fiber filter paper 411. Here, the glass fiber filter 411 is positively charged because it is close to the plastic etching film 412 and the first plate 11, and the sample traveling through the glass fiber filter 411 is a positively charged glass fiber. Since the filter paper 411 and the negatively charged blood cells are electrostatically coupled to each other, the movement of the blood cells is delayed and reaches a site where the agglutinating reagent is impregnated with a high plasma component ratio. Thereafter, lipoprotein components other than HDL present in plasma react with the agglutinating reagent to produce aggregates. Since the reaction with the condensing reagent occurs at a high plasma component ratio, the agglutination reaction proceeds rapidly.

  In addition, in order for the reaction of the lipoprotein component other than HDL and the agglutinating reagent to proceed, and to produce an agglomerate having a size that can be separated and filtered, the sample stays in the glass fiber filter paper 411 for a certain period of time. There is a need to. Here, the second plate 12 is made of a porous material that is impermeable and breathable, that is, the flow path 21 has water repellency, so that no capillary action occurs. Therefore, the sample does not naturally flow into the channel 21 unless the pressure operation or the suction operation is performed after the sample is dropped. This allows the sample to remain in the glass fiber filter 411 for a time sufficient to produce agglomerates that can be separated and filtered. Even when each plate is not a water-impermeable and air-permeable porous material, it is possible to prevent the sample from naturally flowing into the flow channel 21 if the water repellent treatment is separately applied to the flow channel 21.

  After a sufficient time for the agglutination reaction to be completed, the sample retained in the glass fiber filter paper 411 proceeds toward the flow path 21 through the receiving port 61 by performing a pressurizing operation or a suction operation. . At that time, an aggregate having a small particle size is first separated and filtered by the etching film 412, and then a blood cell component having a large particle size is separated and filtered to realize smooth filtration. As a filtrate, a plasma component containing only HDL flows into the flow path 21. Here, the method of the pressurizing operation and the suction operation is arbitrary, but when a porous material is used for the second plate 12, for example, the pressurizing operation is provided with an elastic plug covering the sample supply port 5. The suction operation is realized by connecting a pump to a part of the second plate 12 and performing suction.

  After reaching the measurement chamber 31, the sample reacts with the color reagent held in the color reagent holding unit 312. Here, the first plate 11 and the third plate 13 are configured to be transparent at the position of the measurement chamber 31. Therefore, after the time necessary for completing the color reaction has elapsed, the measurement chamber is detected by detecting the incident light irradiated from the external light source toward the measurement chamber 31 and the transmitted light transmitted through the measurement chamber 31. The absorbance at 31 can be measured, and specific components in the sample can be quantified based on the obtained absorbance. Here, in the above-described embodiment, since the absorbance is measured by measuring the transmitted light, both the first plate 11 and the third plate 13 are configured to be transparent at the position of the measurement chamber 31. However, when the absorbance is measured using reflected light, only one of the plates may be configured to be transparent.

  In the case where the glass fiber filter paper 411 is impregnated with the agglutinating reagent in advance to cause the reaction, the concentration of the agglutinating reagent with respect to the sample varies depending on the amount of the dropped sample, the sample penetration, the reagent dissolution, and the like. The aggregating reagent contains Mg2 +, which is a divalent metal ion. However, if impregnated with a sufficient amount of the aggregating reagent for the reaction, in some cases, it becomes a considerably excessive state, in which case it is not used for the agglutination reaction. Since the surplus divalent metal ions flow into the measurement chamber 31 as they are, they may be contained in large amounts during the color reaction. When the color reaction proceeds while containing a high concentration of divalent metal ions in this way, there is a risk that a phenomenon that adversely affects the reaction system, such as insolubilization or turbidity of cholesterol dehydrogenase, may occur.

  Here, when a chelating agent such as ethylenediaminetetraacetic acid is added to the color reagent, it forms a complex with the metal ion, so the adverse effect of the divalent metal ion on the color reaction is removed, and the color reaction is stabilized. High-precision measurement is possible.

  It should be noted that instead of measurement with transmitted light, measurement with reflected light can be used, and instead of optical measurement, electrodes can be provided in the measurement chamber for electrical measurement.

  Here, in order to confirm the effect of the analytical instrument according to the present invention, samples were analyzed using the analytical method using the analytical instrument according to the present embodiment and the control method, and the results were compared. The control method was a direct measurement method (N-assay L HDL Nittobo B-type / Hitachi-7020), and the analysis target was HDL-C in blood. In order to obtain a correlation coefficient, HDL-C values were measured for 30 samples by the method according to the present invention and the control method, respectively.

  The color reagent used in the analytical instrument according to the present invention is prepared as follows. In addition, the following density | concentration is a density | concentration at the time of reaction.

Preparation of color reagent
Cholesterol esterase (CE) 200 U / mL
Cholesterol dehydrogenase (CHDH) 150 U / mL
Diaphorase (DI) 200 U / mL
Nicotinamide adenine dinucleotide (NAD) 20 mM
Water-soluble tetrazolium salt (WST-4) 60 mM
Surfactant (Triton (registered trademark) X-100) 1.0% w / v
Good buffer (TAPS) pH 8.5 200 mM
Ethylenediaminetetraacetic acid trisodium 50 mM

  After dropping the sample on the analytical instrument according to the present embodiment and waiting for 1 minute, a pressurizing operation was performed from the sample supply port 5, and the sample was transferred to the measurement chamber 31. Then, after 5 minutes from the time when the sample was transferred to the measurement chamber 31, the absorbance at a wavelength of 635 nm was measured.

  Table 1 and FIG. 10 show the comparison results between the measurement values obtained by the analysis method using the analysis instrument according to the present embodiment and the measurement values obtained by the control method.

  From this result, it was shown that the analysis method using such an instrument has a very good correlation with the control method.

(Second Embodiment)
FIG. 3 shows a cross-sectional view of a second embodiment of the analytical instrument according to the present invention. As in the first embodiment, this embodiment is also a mode for measuring HDL-C from a whole blood sample.

  The configuration other than the filtering means 4 is the same as that of the first embodiment. Therefore, the filtering means 4 will be mainly described below.

  Here, the filtering means 4 includes a filtration member 41 made of an asymmetric pore diameter membrane 415, a support 42 having an opening while supporting the filtration member 41, an asymmetric pore diameter membrane 415 impregnated and dried, and lipoprotein components other than HDL. It comprises an agglutinating reagent (not shown) that reacts to produce an aggregate. The opening of the support tool 42 forms the sample supply port 5.

  The asymmetric pore diameter membrane 415 is made of polysulfone or polyether sulfone and has a structure in which the pore diameter changes in the film thickness direction. The asymmetric pore diameter membrane 415 is selected based on the trapping efficiency of blood cells and aggregates. It is preferable that the pore diameter is 30 μm, the small pore diameter side is 0.5 to 1.0 μm, and the film thickness is 150 μm to 400 μm. Moreover, it arrange | positions so that the surface by the side of a small hole may face the main-body part 1 side.

  The specification of the agglutinating reagent impregnated in the asymmetric pore diameter membrane 415 is the same as that in the first embodiment. The agglutination reagent is contained in the asymmetric pore diameter membrane 415, but the presence of the agglomeration reagent in the vicinity of the surface on the small pore diameter side of the asymmetric pore diameter membrane 415 can lead the sample to an agglutination reaction in a state where the plasma ratio is high. It is preferable. When the asymmetric pore diameter membrane 415 is impregnated with the agglutinating reagent and then dried so as to apply air to the surface on the small pore diameter side, the solution in which the agglutinating reagent is dissolved moves toward the surface where the moisture evaporates. As a result, an asymmetric pore diameter film 415 can be created in such a manner that the concentration is present on the small pore diameter side.

  Next, the operation of the analytical instrument according to the present embodiment will be described.

  The whole blood sample dripped into the sample supply port 5 penetrates into the asymmetric pore diameter membrane 415, but the blood cell component contained in the whole blood sample is captured by a gap having a relatively large pore diameter in the asymmetric pore diameter membrane 415, thereby Blood cell components are removed from the sample. The plasma of the sample moves to the vicinity of the surface on the small pore diameter side. Here, the agglutinating reagent is concentrated in the vicinity of the surface on the small pore diameter side, and reacts with lipoprotein components other than HDL present in plasma to produce aggregates. This agglomerate is trapped by a void having a relatively small pore diameter in the asymmetric pore diameter membrane 415. That is, smooth filtration with less risk of clogging is realized, and the filtrate flowing into the receiving port 61 becomes plasma containing only HDL.

  In order for the reaction between the lipoprotein component other than HDL and the agglutinating reagent to proceed to produce an aggregate that is large enough to be separated and filtered, plasma stays in the asymmetric pore membrane 415 for a certain period of time. There is a need to. Here, as in the first embodiment, the second plate 12 is made of a porous material that is impermeable and air permeable, that is, the channel 21 has water repellency, so that no capillary phenomenon occurs. Therefore, plasma does not naturally flow into the flow path 21 unless a pressure operation or a suction operation is performed after dropping the sample. This allows plasma to remain in the asymmetric pore membrane 415 for a time sufficient to produce aggregates that can be separated and filtered. Even when each plate is not a water-impermeable and air-permeable porous material, it is possible to prevent the sample from naturally flowing into the flow channel 21 if the water repellent treatment is separately applied to the flow channel 21.

  After a sufficient time for the agglutination reaction to be completed, by performing a pressurizing operation or a suction operation, plasma containing only HDL flows into the flow path 21 through the receiving port 61 and is then transferred to the measurement chamber 31. For measurement.

(Third embodiment)
4 to 7 show an exploded perspective view and a cross-sectional view of a third embodiment of the analytical instrument according to the present invention. This embodiment is a preferred embodiment in the case where a plurality of different items are analyzed simultaneously from a single drop of sample, and one of the analysis items is HDL-C.

  As shown in FIGS. 4 and 5, the analytical instrument according to the present embodiment includes a main body 1 and a filtering means 4, and the main body 1 includes a first plate 11, a second plate 12, and a third plate 13. Are bonded through an adhesive layer (not shown) such as a double-sided tape. Although the filtering means 4 has the sample supply port 5, since it is used only when supplying the sample into the main body 1, it is configured to be separable from the main body 1 in this embodiment. It may be adhered.

  The material and specifications of each component in the present embodiment are the same as those in the first embodiment. Therefore, only the structurally different points and the effects resulting therefrom will be described below.

  The filtration means 4 includes a plate-like filtration member 41, a support 42 having the sample supply port 5 while supporting the filtration member 41, and held by the filtration member 41, and reacts with lipoprotein components other than HDL to aggregate. And an agglutinating reagent (not shown). In addition, the support member 42 is provided with a filtration member accommodating portion 421 on the opposite side to the sample supply port 5, and the filtration member 41 is fitted in the filtration member accommodation portion 421.

  The first plate 11 is provided with a through-hole 221 having a receiving port 62 and a through-hole 231 having a receiving port 63, and the second plate 12 has through-holes 222, 232, 321, 331 and these communicating with each other. Grooves 223 and 233 are provided. The third plate 13 is provided with color reagent holding portions 322 and 332. Here, the sample supply port 5 and the receiving port 63 are substantially concentric, and the receiving port 62 and the receiving port 63 are separated from each other. That is, the region where the sample supply port 5 is projected onto the filtration member 41 and the region where the reception port 63 is projected onto the filtration member 41 are substantially the same, while the region where the sample supply port 5 is projected onto the filtration member 41 and the reception port 62. Does not overlap with the region projected onto the filter member 41. The second plate 12 is provided with a through hole 232 at a position substantially concentric with the through hole 231, a through hole 331 is provided at a position separated from the through hole 232, and the groove 233 communicates the two through holes 232 and 331. is doing. Further, a through hole 222 is provided at a position substantially concentric with the through hole 221, a through hole 321 is provided at a position separated from the through hole 222, and the groove 223 communicates the two through holes 222 and 321. The third plate 13 is provided with color reagent holding portions 322 and 332 substantially concentric with the through holes 321 and 331, and holds a color reagent (not shown). The color reagent holding portions 322 and 332 can be formed by providing a concave portion at the position of the third plate 13 and filling the color reagent therein, but the color reagent is attached to the flat portion. It is also possible to do. The through holes 221 and 222 and the groove 223 constitute the flow path 22, and the through holes 231 and 232 and the groove 233 constitute the flow path 23. Further, the through hole 321 and the color reagent holding unit 322 constitute the measurement chamber 32, and the through hole 331 and the color reagent holding unit 332 constitute the measurement chamber 33.

  Here, the filter member 41 can also be configured by laminating a glass fiber filter paper 411 and an etching film 412 as in the first embodiment. As in the second embodiment, the filter member 41 is an asymmetric pore diameter film. It is also possible to configure. However, the size of the filtering member 41 occupies a range that covers both the two receiving ports 62 and 63 of the first plate 1. Hereinafter, the case where the filter member 41 is configured by laminating the glass fiber filter paper 411 and the etching film 412 will be described. However, the same effect can be obtained even when the filter member 41 is configured by an asymmetric pore diameter film in the second embodiment. It is clear from the explanation.

  The agglutination reagent that reacts with a lipoprotein component other than HDL to produce an agglomerate is impregnated only around the area where the receiving port 62 is projected onto the glass filter paper 411, while the area where the receiving port 63 is projected onto the glass filter paper 411 Is not impregnated. The hatched portion shown in FIG. 4 is the aggregation reagent holding range 7.

  Next, the operation of the analytical instrument according to the present embodiment will be described with reference to FIGS.

  The whole blood sample dripped into the sample supply port 5 penetrates into the glass fiber filter paper 411. Here, the glass fiber filter 411 is positively charged because it is close to the plastic etching film 412 and the first plate 1.

  The sample spreads in the plane in the glass filter paper 411, and a part of the sample reaches the agglutinating reagent holding range 7 located at the upper part of the receiving port 62. At this time, since the positively charged glass filter paper 411 and the negatively charged blood cells are electrostatically coupled, the movement of the blood cells is delayed, the sample comes into contact with the agglutination reagent in a state where the plasma ratio is high, and the agglutination reaction proceeds rapidly. . Here, as in the first embodiment, since the channel 22 has water repellency, capillary action does not occur, and the sample does not naturally flow into the channel 22. Therefore, after taking a sufficient time to complete the agglutination reaction, the sample retained in the glass fiber filter paper 411 passes through the receiving port 62 and proceeds through the flow path 22 by performing a pressurizing operation or a suction operation. . At that time, blood cell components and aggregates are separated and filtered by the etching film 412, and a plasma component containing only HDL flows into the flow path 22, reaches the measurement chamber 32, and is used for analysis related to HDL.

  On the other hand, since a part of the sample that has reached the upper part of the receiving port 63 does not react with the agglutinating reagent, only the blood cell component is separated and filtered by the etching film 412 by performing a pressure operation or a suction operation. After the plasma components including all lipoprotein components flow into the flow path 23, they reach the measurement chamber 33 and are used for measurement. That is, here, it is used for analysis of items other than HDL-C.

  In order for the sample flowing into the receiving port 62 to contain no lipoprotein components other than HDL, there is an aggregating reagent in the region where the receiving port 62 is projected onto the glass filter paper 411, or the receiving port 62 is projected onto the glass filter paper 411. The agglutination reagent only needs to be present in a region between the formed region and the region where the sample supply port 5 is projected onto the glass filter paper 411. On the other hand, in order for the sample flowing into the receiving port 63 to contain all the lipoprotein components, a region including the region where the sample supply port 5 is projected onto the glass filter paper 411 and the region where the receiving port 63 is projected onto the glass filter paper 411 are included. In addition, there should be no agglutinating reagent. By comprising in this way, one drop sample can be divided into the sample which does not contain lipoprotein components other than HDL, and the sample which contains all the lipoprotein components, and can each be transferred to a separate measurement chamber.

  In FIG. 6, the flow path 23 communicating with the receiving port 63 branches into three, and these communicate with the three measurement chambers 33. Since the sample flowing in from the receiving port 63 has not undergone the agglutination reaction, it contains all the lipoprotein components. Therefore, in the measurement chamber 33, it is possible to simultaneously measure a plurality of items other than HDL-C, such as glucose and neutral fat. On the other hand, since the measurement chamber 32 communicates with the receiving port 62 and the sample flowing into the receiving port 62 does not contain lipoprotein components other than HDL, it can be suitably used for the measurement of HDL-C.

  Further, the filter member 41 in FIG. 6 has two types of etching films 413 and 414. As described above, since the whole blood sample that has reached the upper part of the receiving port 63 does not react with the agglutinating reagent, only the blood cell component is separated and filtered by the etching film 414 after passing through the pressurizing operation and the suction operation. A plasma component containing a lipoprotein component flows into the receiving port 63. On the other hand, the whole blood sample that has spread horizontally on the glass filter paper 41 and reached the upper portion of the receiving port 62 comes into contact with the agglutinating reagent in a high plasma ratio, and the agglutination reaction proceeds rapidly. After that, the blood cell component and the aggregate are separated and filtered by the etching film 413, and the plasma component from which the lipoprotein component other than HDL is removed flows into the receiving port 61.

  That is, the etching film 413 needs to filter out not only blood cell components but also aggregates, and the pore size must be smaller than the aggregates to be generated in order to capture the aggregates. On the other hand, the etching film 414 is required to pass all body fluid components to be analyzed other than blood cell components. However, when the same pore diameter as the etching film 413 is applied, even the body fluid component to be analyzed is separated and filtered. There are (for example, neutral fat). Therefore, it is preferable to apply different hole diameters to the etching films 413 and 414 so that the hole diameter of the etching film 414 is larger than the hole diameter of the etching film 413. This is because the sample flowing into the receiving port 63 can be transferred to the measurement chamber 33 without capturing any body fluid component to be analyzed. Note that the hole diameter of the etching film should be appropriately selected according to the size of the aggregate to be generated. However, if an etching film having a hole diameter of 0.4 μm is selected as the etching film 413, the aggregate can be captured and etched. If a membrane having a pore diameter of 0.8 μm is selected for the membrane 414, it is possible to pass neutral fat without trapping, which is preferable.

  As described above, in this embodiment, a plurality of items including HDL-C can be simultaneously analyzed from a single sample. Although FIGS. 4 to 6 show the analytical instruments in which the measurement chambers are arranged in a row, it is preferable that the measurement chambers 32 and 33 are arranged in a row when measuring a plurality of items simultaneously. When the sample is transferred to the measurement chambers 32 and 33 and the absorbance is measured, the optical periodic measurement device such as the irradiation light generation device and the optical sensor scans on one axis to measure the absorbance in a plurality of measurement chambers. This is because the time required for analyzing a plurality of items can be shortened. On the other hand, as shown in FIG. 7, an analytical instrument having a single filtering means 4 in the center and a plurality of flow paths 24 and measurement chambers 34 communicating with the receiving port 64 so as to surround it is also created. This is preferable in that it can analyze a large number of items in a small space.

(Fourth embodiment)
FIG. 8 shows a front view of the main body 1 viewed from the fourth embodiment of the analytical instrument according to the present invention except for the first plate. This embodiment is an embodiment suitable for simultaneously performing analysis on a plurality of different items from a single drop of sample. Since any of the first to third embodiments described above can be applied to the configuration other than the measurement chamber and the channel, the configuration and operation of the measurement chamber and the channel will be mainly described below. To do.

  When the sample flows into the measurement chambers 32 and 33 provided with the color reagent, the flow velocity is rapidly reduced at the joint between the flow paths 22 and 23 and the measurement chambers 32 and 33, so that the entire measurement chamber is colored. However, the unique region 8 having a different coloration degree from other regions is likely to be generated in the vicinity of the connection point between the measurement chamber and the flow path. When measuring the absorbance, if the specific region 8 is irradiated with light, the correct absorbance may not be measured.

  Also, as shown in FIG. 9, if the angle at which the sample flows into each measurement chamber is different, the position of the singular region 8 is different for each measurement chamber. Therefore, even if one axis 9 is scanned, one measurement is performed. In the chamber, the absorbance can be measured by removing the specific region 8, but in another chamber, there is a possibility that the absorbance in the specific region 8 is measured. Therefore, if the flow path extends from the circular measurement chamber in a circular radial direction and perpendicular to the axis 9, the singular regions in all the measurement chambers are located in the same manner, and the optical measurement device is Even when scanning on one axis, the absorbance can be measured by removing the singular point in all measurement chambers.

  Further, as shown in FIG. 8, by making the shape of the measurement chambers 32 and 33 oval, an area other than the singular area 8 is enlarged without increasing the width of the analytical instrument, that is, without increasing the length of the scanning path. Therefore, the degree of freedom of the position of the axis scanned by the optical measuring device is increased, and the influence of the error in the position of the axis 9 is reduced. That is, stable measurement accuracy can be realized. In this case, the measurement chambers 32 and 33 are arranged in a line with the ellipse minor axis aligned with the axis 9, and the flow paths 22 and 23 are arranged to extend from the measurement chamber along the major axis of the ellipse. If this is done, it is preferable in that a large area other than the specific area 8, that is, an area where correct absorbance can be measured can be secured. Although an elliptical measurement chamber is shown in FIG. 7, the same effect can be obtained with an elliptical shape. The shape and size of the measurement chamber can be appropriately determined according to the optical measurement device.

Claims (14)

A sample supply port through which a sample is supplied;
A plate-like filtration member that contacts the sample supply port and removes particles having a predetermined diameter or more from the sample;
An agglutinating reagent that is provided in the filtration member and reacts with a specific component contained in a body fluid to generate an aggregate;
A plurality of receiving ports that contact the filter member and receive the sample that has passed through the filter member;
A plurality of measurement chambers in which measurement of the sample is performed;
Extending from each of the measurement chambers, and composed of a flow path communicating with any one of the plurality of receiving ports,
The region 1 where the sample supply port is projected onto the filtering member and the region 2 where one of the plurality of receiving ports is projected onto the filtering member do not overlap,
In the region 3 or the region 2 between the region 1 and the region 2, the aggregation reagent is present,
Analysis of a body fluid component characterized in that the agglutinating reagent does not exist in a region 5 including the region 4 and the region 1 in which another one of the plurality of receiving ports is projected onto the filtration member. Instruments. 2. The body fluid component analysis instrument according to claim 1, wherein the region 1 and the region 4 overlap each other. The body fluid component analysis instrument according to claim 1 or 2, wherein the flow path has water repellency. The flow path and the measurement chamber are impermeable and breathable ventilation plates;
3. The body fluid component analysis instrument according to claim 1, wherein the body fluid component analysis instrument is formed by laminating an impermeable and impermeable sealing plate. The agglutination reagent reacts with a lipoprotein component other than high-density lipoprotein to produce an aggregate,
The body fluid component analysis instrument according to any one of claims 1 to 4, wherein a cholesterol quantification reagent is provided in the measurement chamber communicating with the one receiving port. The filter member is a glass fiber filter disposed on the supply port side,
An etching film disposed on the receiving port side is laminated,
The body fluid component analysis instrument according to any one of claims 1 to 5, wherein the agglutinating reagent is contained in the glass fiber filter paper. The glass fiber filter has a distribution shape in which the content density of the aggregating reagent varies along the film thickness direction, and the surface on the etching film side has a distribution shape with the content density higher than the surface on the sample supply port side. 7. The body fluid component analysis instrument according to claim 6. The filter member is a glass fiber filter disposed on the supply port side,
An etching film disposed on the receiving port side;
Intervened between the glass fiber filter paper and the etching film, a reagent holding means made of a material having a liquid absorption property is laminated,
The body fluid component analysis instrument according to any one of claims 1 to 5, wherein the agglutinating reagent is contained in the reagent holding means. 9. The body fluid component analysis instrument according to claim 6, wherein the hole diameter of the etching film in the region 2 is smaller than the hole diameter of the etching film in the region 1. The filtration member is an asymmetric porous membrane having a pore size distribution in which the pore size of one surface is larger than the pore size of the other surface,
The asymmetric porous membrane is disposed with the one surface facing the sample supply port side,
The body fluid component analysis instrument according to any one of claims 1 to 5, wherein the agglutinating reagent is contained in the asymmetric porous membrane. The asymmetric pore diameter membrane has a distribution shape in which the content density of the agglutinating reagent changes along the film thickness direction, and the content density is higher on the surface on the receiving port side than on the surface on the sample supply port side. 11. The body fluid component analyzing instrument according to claim 10, The agglutination reagent comprises a metal ion;
The analytical device for body fluid components according to any one of claims 1 to 11, wherein a chelating agent is disposed in a part of the measurement chamber or the flow path. A plurality of circular measuring chambers,
The circular measuring chamber is arranged on one axis;
The body fluid component analysis instrument according to any one of claims 1 to 12, wherein the flow path extends from the circular measurement chamber in the circular radial direction and perpendicular to the one axis. A plurality of the elliptical or oval measuring chambers are provided,
The elliptical or elliptical measuring chambers are arranged in a row with the elliptical or elliptical minor axis aligned with one axis,
The body fluid component analysis instrument according to any one of claims 1 to 12, wherein the flow path extends from the measurement chamber along the major axis of the ellipse or ellipse.

Images