JP5607593B2 - Coagulation activity measuring device, measuring chip and measuring method - Google Patents

Description

  The present invention relates to an improvement in a method for measuring a blood coagulation active factor index (eg, blood coagulation time).

  Blood coagulation is one of the important functions in the hemostasis mechanism, and coagulation activity has become an important item in clinical examinations. Blood coagulation is a phenomenon that is balanced by the involvement of various factors, also called cascade reactions. Therefore, blood coagulation factors include various types of proteins and ions, and the factors involved in the endogenous system (VIII, IX, XI, XII) that progress only with the components in plasma and the tissues outside the blood vessels also progress. There are factors (III, VII) involved in the extrinsic system and factors (I, II, V, X) involved in both the intrinsic and extrinsic systems. In the past, blood coagulation was observed by methods such as coagulation time using whole blood (whole blood coagulation time) and blood clot observation, but there are many necessary blood volumes, automation is difficult, and reproducibility is not possible. Due to the problem, an indicator that can screen for an affected blood coagulation factor is now preferred.

  One of the indexes obtained by typical coagulation measurement is prothrombin time (blood clotting time, hereinafter abbreviated as PT). The PT measurement value is affected by factors of VIII, X, V, and II other than Ca ions and tissue thromboplastin. When the PT measurement value is extended, the person who provided the blood to be measured can be hit by a suspicion of a congenital blood coagulation factor deficiency, hepatocyte disorder, vitamin K deficiency, or the like. Another index obtained by a typical coagulometric measurement is activated partial thromboplastin time (hereinafter abbreviated as APTT). Judgment based on APTT measurement values is required after surgery, when heparin is administered to prevent thrombus, which is administered at the time of recovery from congenital blood diseases, infarctions, and the like. APTT is often measured when it is affected by intrinsic factors and has a tendency to bleed. When abnormalities are observed in the APTT measurement value, there is a deficiency of intrinsic factors I, II, V, and X, or a deficiency of factors VIII, IX, XI, and XII that are involved in both intrinsic and extrinsic systems. Conceivable.

  Indices obtained by blood coagulation measurements such as PT and APTT observe the phenomenon that the fibrinogen corresponding to the last part of the cascade reaction is converted into fibrin in terms of time. Basic measurement of PT and APTT is performed using plasma obtained by removing blood cells by centrifugation from blood collected from a subject. An activator is added in advance to plasma to activate coagulation factors contained in the plasma, and then PT or APTT is measured.

  Conventionally, APTT has been measured by a stirring method or a light scattering method after mixing an activator, which is a reagent containing a component that promotes coagulation activation, and a plasma sample in the same manner as PT measurement. It was. The stirring method is a method in which a plasma sample and an activator are introduced together in a test container, stirred with fins, and APTT is measured from temporal changes in stirring resistance and temperature rise. The light scattering method is a method of measuring APTT by mixing a plasma sample and an activator in a test container, irradiating the container with light, and monitoring temporal changes in the amount of scattered light accompanying coagulation. In the methods disclosed in Patent Document 1 and Patent Document 2, in order to obtain a more accurate measurement value, the time change of the measurement value is monitored, and a unique calculation method is used for each measurement device.

Blood coagulation is a phenomenon in which different reaction pathways and many factors are intricately intertwined and a cascade reaction proceeds by a certain trigger. There are various factors for each plasma sample to be measured. For example, when measuring with an absorption spectrum, when the reaction proceeds, multiple peaks are observed, or the peak values become relatively large. When it gets smaller, various changes occur. In such observation, it has been necessary to develop a method for calculating a peak value so that an accurate value can be obtained from many plasma samples.
As described above, in any system, a blood-derived sample is measured in a container, and measurement is not performed in a state where blood flows as in a living body. Further, in a complicated clotting reaction, the reaction proceeds to the level at which the whole plasma sample to be coagulated, that is, the final state level of the clotting reaction, and is observed.

Japanese Patent Publication No. 6-50317 Japanese Patent Publication No. 3-11423

  As described above, the conventional measurement method has a problem in that measurement is not performed in a state where a blood-derived sample flows. Further, since the reaction is advanced to the final state level of the complex coagulation reaction and the blood coagulation parameter (APTT) is obtained, there is a problem that the measurement takes time.

  The present invention has been made in order to solve the above-mentioned problem, and in the measurement for screening an intrinsic clotting abnormality, it is possible to quickly measure an indicator of a clotting active factor while a blood-derived sample is flowing. An object of the present invention is to provide a clotting activity measuring device, a measuring chip and a measuring method.

The coagulation activity measuring device of the present invention includes a refractive index measuring device for measuring a refractive index at the surface of the measuring chip having a channel through which a solution flows, and a buffer solution filled in the channel of the measuring chip. After that, the flow rate of the plasma traveling in the flow path from the change in the refractive index when the plasma sample in which the activator for activating intrinsic coagulation and plasma is mixed is put into the flow path The activated partial thromboplastin time is calculated from the flow rate calculated by the flow rate calculating means based on the known flow rate calculating means for calculating the flow rate and the activated partial thromboplastin time that is an indicator of the coagulation active factor. And a clotting activity factor indicator calculating means.
Moreover, in one structural example of the coagulation activity measuring device of the present invention, the refractive index measuring device is a surface plasmon resonance measuring device.

  Further, the present invention calculates the flow rate of plasma traveling in the flow path based on the refractive index at the surface of the flow path filled with the buffer solution, and calculates the activated partial thromboplastin time which is an index of the clotting activity factor. A measuring chip for an activity measuring device, comprising a gold thin film formed on a substrate surface and a flow path formed on the gold thin film, wherein the gold thin film is a part of which is a living body that inhibits a coagulation reaction. It is covered with a blocking agent for chemical measurement.

  Further, the coagulation activity measurement method of the present invention includes a refractive index measurement step for measuring a refractive index at the surface of the measurement chip having a flow path through which the solution flows, and a buffer solution in the flow path of the measurement chip. After the filling, the plasma that progresses in the flow path from the change in the refractive index when the plasma sample mixed with the activator that activates intrinsic coagulation and the plasma is put into the flow path Based on the known relationship between the flow rate calculation step for calculating the flow rate of the plasma and the activated partial thromboplastin time which is an indicator of the coagulation activation factor, the activated partial thromboplastin time from the flow rate calculated in the flow rate calculation step And a clotting activity factor index calculating step.

  According to the present invention, the activated partial thromboplastin time, which is an indicator of the coagulation active factor, can be obtained while the coagulation reaction is in progress, so that the measurement time can be shortened compared to the conventional case. In the present invention, it is possible to obtain a coagulation ability with a very high flow rate. Further, in the present invention, since an index having a higher resolution than the number of seconds can be provided, it is possible to measure the coagulation activity parameter with higher accuracy.

  In the present invention, the coagulation activity is physically and biochemically controlled by covering a part of the gold thin film formed on the substrate surface of the measurement chip with a biochemical measurement blocking agent that inhibits the coagulation reaction. be able to.

It is sectional drawing explaining the principle of this invention. It is a disassembled perspective view which shows the structure of the measuring chip which concerns on embodiment of this invention. It is a top view of the measurement chip concerning an embodiment of the invention. It is a block diagram which shows the structure of the coagulation activity measuring apparatus which concerns on embodiment of this invention. It is a figure which shows an example of the incident angle-reflectance curve obtained by the measurement of a measurement chip | tip in the coagulation activity measuring apparatus which concerns on embodiment of this invention. It is a block diagram which shows the structure of the data processor of the coagulation activity measuring apparatus which concerns on embodiment of this invention. It is a flowchart explaining the measuring method of APTT which concerns on embodiment of this invention. It is a figure which shows the relationship between the image which the CCD camera image | photographed in embodiment of this invention, and the advancing direction of plasma. It is a figure which shows the example of the light intensity profile in 1 line of the image image | photographed with the CCD camera in embodiment of this invention. It is a figure explaining the calculation method of the flow rate of the plasma in embodiment of this invention. It is a figure which shows one example of the relationship between the flow rate of plasma, and the activity of a plasma sample. It is a figure which shows the example of a refractive index change at the time of supplying normal range plasma to a measurement chip | tip. It is a figure which shows the example of a refractive index change at the time of supplying abnormal region plasma to a measurement chip.

[Principle of the Invention]
In the present invention, an activator, which is a reagent containing a component that promotes the activation of blood coagulation, and a plasma sample are mixed in advance, and the plasma sample mixed with the activator is introduced into a microchannel to determine the flow rate. Obtain blood clotting parameters. In order to obtain the flow velocity in the microchannel, the position of the surface of the microchannel is subdivided, and the temporal change in the refractive index at each position is observed.

FIGS. 1A to 1C are cross-sectional views illustrating the principle of the present invention, showing a plasma sample traveling in the flow channel of a measurement chip filled with a buffer solution.
As shown in FIG. 1A, the flow path 1001 formed on the substrate 1000 of the measurement chip is filled with a buffer solution 1002.

  When a plasma sample 1003 is introduced into the flow channel 1001, the plasma sample 1003 receives the activity of the reagent, and proceeds in the flow channel 1001 while causing the coagulation reaction to proceed (FIG. 1B). In the present invention, the clotting activity is evaluated as the flow rate while the fluidity of the plasma sample 1003 is maintained. Since the fluidity is maintained, when the buffer solution 1004 is introduced after a certain time after the plasma sample 1003 is introduced, the phenomenon that the plasma sample 1003 is pushed out by the buffer solution 1004 and washed away can be observed (FIG. 1 (C )).

  In the APTT measurement that can measure the intrinsic coagulation activity, a foreign substance corresponding to the subendothelial tissue is mixed as an activator. It is said that a positively charged protein of factor XII comes into contact with the negatively charged surface of this foreign substance and becomes electrically neutral, which is activated. Furthermore, this activated factor XII (the activated factor is a and XIIa) forms a complex with blood prikallikrein, high molecular weight kininogen, and factor XI, and prikallikrein is active against kallikrein It becomes. Kallikrein further activates factor XII in the presence of macromolecular kininogen.

Factor XII activates factor XI. When Ca ions coexist there, factor IX is activated, and IXa forms a complex with factor VIII and calcium ions on the phospholipid surface, and then factor X To activate factor X. Furthermore, factor VIII acts as a cofactor of factor IX, is activated by thrombin to become VIIIa, and the production of Xa is accelerated.
APTT is activated at 37 ° C by adding tissue factors that activate ellagic acid, lipids, and thrombin as foreign substances to the plasma sample as the first stage, and Ca ions are added at the second stage to coagulate. Measured with acceleration.

In the present invention, the flow rate of the activated plasma sample as it passes through the microchannel is measured. The sample activated in the two stages forms fibrin, which is the final stage of coagulation, while flowing through the flow path. On the other hand, since a reaction called fibrinolysis is also activated and there is a flow, it is difficult to reach a reaction in which polymerized fibrin further polymerizes to form a high molecular weight fibrin. As a result, in a plasma sample having a high APTT activity, viscosity does not increase when passing through the flow path, and polymer particles generated by fibrin formation may pass through the flow path.
As a result, the higher the plasma with higher clotting activity, the greater the speed of traveling through the channel. Conversely, in plasma with low clotting activity, the flow rate is small. The flow rate, which is the progression rate of the plasma sample, is obtained, and an index correlated with the APTT that is currently generally obtained.

[Embodiment]
Hereinafter, embodiments of the present invention will be described with reference to the drawings. 2 is an exploded perspective view showing the structure of the measuring chip according to the embodiment of the present invention, FIG. 3 is a plan view of the measuring chip, and FIG. 4 is a block diagram showing the configuration of the coagulation activity measuring apparatus according to the embodiment of the present invention. FIG.
The measuring chip 100 of the present embodiment has a substantially rectangular parallelepiped outer shape and has a laminated structure. That is, a transparent plate 101 of a rectangular plate made of BK7 glass or plastic having a horizontal size of 16 mm × 16 mm and a thickness of 1 mm, and the outer shape in plan view are formed substantially the same as the substrate 101, A double-sided sealing sheet 102 made of, for example, a resin, and an acrylic casing 103 that is formed on the sheet 102 so that the outer shape in plan view is substantially the same as that of the substrate 101.

  On the substrate 101, a gold thin film 104 having a thickness of about 50 nm is formed by sputtering. Further, a blocking agent 105 for measuring biochemistry derived from milk, fish, or chemical synthesis is formed on a part of the surface of the gold thin film 104 to cover the surface of the gold thin film 104. In the measurement chip 100 of the present embodiment, spots of the blocking agent 105 composed of proteins were formed at intervals of 350 μm. The spot of the blocking agent 105 is formed so as to be positioned in an observation region described later when the sheet 102 is laminated on the substrate 101. The spot of the blocking agent 105 functions as a physical obstacle as well as a chemical obstacle in the coagulation reaction. A blocking agent containing a protein is suitable because it easily forms a spot.

After forming a spot of the blocking agent 105 on the surface of the gold thin film 104, the substrate 101 is washed with water, and the sheet 102 is laminated on the gold thin film 104 of the substrate 101. A circular hole 106 is formed at a position of the sheet 102 corresponding to an inlet described later. In addition, the sheet 102 penetrates the sheet 102 in the plate thickness direction (vertical direction in FIG. 2) and communicates with the circular hole 106, and the sheet 102 penetrates the sheet 102 in the plate thickness direction and communicates with the channel 107. A flow path 108 and a suction flow path 109 that penetrates the sheet 102 in the plate thickness direction and communicates with the flow path 108 are formed. An area where the flow path 107 is formed is an observation area.
In the present embodiment, the spot of the blocking agent 105 is formed on the surface of the gold thin film 104. However, in the present invention, a part of the gold thin film 104 blocks the coagulation reaction. As long as it is covered with the agent, it is not always necessary to form spots.

  The flow paths 107 to 109 formed in the sheet 102 in this way are connected from the circular hole 106 to the flow path 107 at the center of the measurement chip, and further connected to the suction flow path 109 at the periphery of the measurement chip via the flow path 108. It has become. The cross-sectional dimensions of the flow paths 107 and 108 are set to dimensions that cause capillary action on the plasma to be measured. In addition, the sheet 102 and the flow paths 107 to 109 are made of a material, a height of the flow path, a width of the flow path, and a flow rate so that the flow paths 107 to 109 remain in the flow paths 107 to 109 after being filled with the buffer solution. The surface condition of the road is preferable.

  An acrylic casing 103 is laminated on such a sheet 102. In the acrylic casing 103, an inlet 110, which is a circular hole for introducing a buffer solution and plasma to be measured, is formed so as to penetrate the acrylic casing 103 in the plate thickness direction. The inlet 110 is formed at a position where the upper end is opened to the outside air and the lower end communicates with the circular hole 106 when the acrylic casing 103 is laminated on the sheet 102.

  Further, the acrylic casing 103 is formed with a large number of through holes 111 so as to penetrate the acrylic casing 103 in the thickness direction. These many through holes 111 are formed at positions where the upper end is opened to the outside air and the lower end communicates with the flow path 108 or the suction flow path 109 when the acrylic casing 103 is laminated on the sheet 102. It is not formed in the portion of the flow path 107 that is the observation region. The diameter of the through-hole 111 is set to a value that causes capillary action on the plasma to be measured, for example, about 100 microns. In this way, by combining the substrate 101, the sheet 102, and the acrylic casing 103, the measuring chip 100 having a capillary flow type channel for introducing plasma by capillary action is completed.

  As shown in FIG. 4, the coagulation activity measuring device includes a prism 1, a light source 2 such as an LED, a polarizing plate 3, a condenser lens 4, a CCD camera 5, and a data processing device 6. In the present embodiment, a coagulation activity measurement device is realized using a surface plasmon resonance measurement device as a refractive index measurement device, and the configuration shown in FIG. 4 is the same as that of the surface plasmon resonance measurement device.

  Here, the measurement principle of the surface plasmon resonance measurement apparatus will be briefly described. When the light emitted from the monochromatic light source 2 passes through the polarizing plate 3, only p-polarized light passes through it. The p-polarized light is collected by the condenser lens 4 and enters the hemispherical prism 1. The measurement chip 100 is placed on the upper surface of the prism 1 so that the substrate 101 is in contact with the prism 1, and p-polarized light is incident on the measurement chip 100 from the substrate 101 side. In this way, the p-polarized light is incident on the measurement chip 100 via the prism 1, and the intensity change of the reflected light from the measurement chip 100 is detected by the CCD camera 5.

  The light emitted from the light source 2 becomes an evanescent wave at the boundary between the prism 1 and the gold thin film 104 of the measurement chip 100. On the other hand, surface plasmon waves are generated on the surface of the gold thin film 104. When the evanescent wave and the surface plasmon wave have the same incident angle, the evanescent wave is used to excite the surface plasmon wave, and the amount of light measured as reflected light decreases. At this time, when the intensity of the reflected light is measured by the CCD camera 5, as shown in FIG. 5, a decrease in reflectance is observed at an incident angle at which resonance between the evanescence wave and the surface plasmon wave occurs. Since the surface plasmon resonance angle at which the resonance between the evanescence wave and the surface plasmon wave occurs depends on the refractive index of the substance to be measured that is in contact with the gold thin film 104 of the measurement chip 100, the substance to be measured such as an antibody is fixed on the gold thin film 104. Then, the refractive index of the antibody changes due to the binding with the antigen, and the angle at which the valley appears changes slightly. By measuring this change, the substance to be measured can be quantified. The above is the measurement principle of the surface plasmon resonance measurement apparatus.

  FIG. 6 is a block diagram showing a configuration of the data processing device 6 of the coagulation activity measuring device according to the present embodiment. The data processing device 6 includes a control unit 60 that controls the entire coagulation activity measurement device, a storage unit 61 that stores a program of the control unit 60, and a user who uses the coagulation activity measurement device gives instructions to the device. An input unit 62 for displaying information and a display unit 63 for displaying information to the user.

  The control unit 60 processes an image captured by the CCD camera 5 to obtain a light intensity profile, obtains a change in refractive index from the data of the light intensity profile, and determines the flow rate of plasma from the change in refractive index. And a APTT calculation unit 66 (coagulation active factor index calculation means) for calculating APTT (activated partial thromboplastin time) from the plasma flow rate.

Next, a method for measuring APTT using a coagulation activity measuring apparatus will be described in detail. FIG. 7 is a flowchart for explaining an APTT measurement method.
First, the plasma to be measured and the APTT measurement reagent (activator) are each warmed at 37 ° C. The APTT measurement reagent used here is actin FSL manufactured by Siemens, and contains rabbit brain-derived cepharine, soybean-derived lipid, and ellagic acid.

  The measuring chip 100 is placed on the plane of the prism 1 of the coagulation activity measuring apparatus (step S1 in FIG. 7), and 4 μL of the Oren buffer solution is put into the measuring chip 100 from the inlet 110 (step S2). The buffer solution is filled into the flow paths 107 to 109 through the inlet 110 and the circular hole 106. Of the flow paths 107 to 109 formed in the sheet 102, the flow path 107 in the observation region is designed to be the narrowest, so that the buffer solution remains in the flow path 107.

  And the control part 60 of the data processor 6 starts flow velocity measurement (step S3). Next, 20 μL each of plasma warmed to 37 ° C. and APTT measurement reagent (activator) are added to the tube and mixed by pipetting, and the mixed plasma sample is warmed at 37 ° C. for a certain period of time. 20 μL of a 0.025 mol / L calcium ion solution is added to the plasma sample and mixed by pipetting, and 4 μL of the plasma sample mixed with the calcium ion solution is charged into the measuring chip 100 from the inlet 110 (step S4). . The plasma sample is introduced into the flow path 107 through the inlet 110 and the circular hole 106.

  After 90 seconds from the introduction of the plasma sample, 4 μL of Oren buffer is further introduced into the measuring chip 100 from the inlet 110 (step S5). The plasma sample proceeds through the flow path 107 so as to be pushed out by the additionally supplied buffer solution.

The control unit 60 ends the measurement when the calculation of the plasma flow velocity V is completed (step S6). Then, the control unit 60 calculates APTT from the flow velocity V (step S7). The control unit 60 stores the calculated APTT in the storage unit 61 and causes the display unit 63 to display the APTT. This completes the measurement.
Each measurement chip 100 is measured only once, and the measurement chip 100 that has finished the measurement is discarded.

Next, a method for calculating the plasma flow velocity V will be described. The CCD camera 5 of the coagulation activity measuring device detects reflected light from the measuring chip 100 and outputs grayscale image data.
The image processing unit 64 of the data processing device 6 takes in the grayscale image data output from the CCD camera 5, processes the grayscale image data, and obtains a light intensity profile for each line of the image.

FIG. 8 is a diagram showing the relationship between the image taken by the CCD camera 5 and the direction of plasma travel, and FIG. 9 is a diagram showing an example of the light intensity profile on the Yn line of the image.
The X direction in FIG. 8 corresponds to the PX direction in FIGS. 2 to 4, and the Y direction in FIG. 8 corresponds to the PY direction in FIGS. 2 to 4. This Y direction corresponds to the direction of plasma progression. In the example of FIG. 8, the image taken by the CCD camera 5 is made up of 480 pixels with coordinates 0 to 479 in the X direction and 480 pixels with coordinates 0 to 479 in the Y direction. Yn in FIG. 8 indicates the Y coordinate of a certain line on the image.

  In the image captured by the CCD camera 5, a bright (high reflectance) region and a dark (low reflectance) region appear in accordance with the reflectance of light at various points on the measurement chip 100. The image processing unit 64 processes the grayscale image data to obtain a light intensity profile as shown in FIG. 9 for each line of the image. As described above, the X direction in FIG. 8 corresponds to the PX direction in FIGS. 2 to 4, and therefore corresponds to the incident angle of light with respect to the gold thin film 104 of the measurement chip 100. Therefore, in FIG. 9, the position where the light intensity is the weakest, that is, the position where the light is absorbed most, corresponds to the surface plasmon resonance angle.

  In the coagulation activity measuring apparatus that observes the refractive index change, data reflecting the refractive index is observed for each line of the CCD camera 5. Therefore, in the flow path 107 at the position of the observation region, when the plasma progresses in the activator, the refractive index changes, and at what timing the refractive index due to the plasma progression for each line of the CCD camera 5 It will be recorded if a change has occurred.

  The flow velocity calculation unit 65 of the data processing device 6 obtains a change in the refractive index caused by the progress of plasma from the time series data of the light intensity profile of the observation region obtained by the image processing unit 64, and calculates the refractive index. Plasma flow velocity V is calculated from the change. Since the surface plasmon resonance angle depends on the refractive index of plasma in contact with the gold thin film 104 of the measuring chip 100, the change in the surface plasmon resonance angle indicates that the refractive index has changed due to the progress of plasma. Therefore, if the time when the surface plasmon resonance angle changes (the time when the refractive index change occurs) is read along the Y direction in FIG. 8 from the time series data of the light intensity profile in the observation region, the time change of the refractive index change. Can be requested. The slope of the change in refractive index with time indicates the plasma flow velocity V.

  A method for calculating the flow velocity V will be described with reference to FIG. FIG. 10 is a diagram for visually explaining a method of calculating the flow velocity V, and is a diagram illustrating an example of the relationship between the time when the refractive index change occurs and the pixel position in the Y direction. FIG. 10 shows data at the X coordinate (for example, X = 230) corresponding to the flow path 107 in the observation region. P in FIG. 10 indicates the pixel position in the Y direction where the surface plasmon resonance angle has changed, that is, the pixel position where the refractive index has changed. The gradient y / t (pixel / sec) of the time change of the refractive index change indicates the plasma flow velocity V. One pixel in the Y direction in the image captured by the CCD camera 5 corresponds to, for example, 10 μm when converted to an actual distance on the flow path 107. Therefore, the blood flow velocity V (μm / sec) of plasma can be obtained by slope (pixel / sec) × actual distance (μm / pixel), that is, y / t × 10 (μm / sec). In this way, the flow rate calculation unit 65 of the data processing device 6 can calculate the plasma flow rate V.

  Needless to say, since the plasma flow velocity V changes with time, the slope of the change in refractive index with time also changes with time. Here, it is only necessary to calculate the plasma flow velocity V, that is, the initial velocity, obtained from the refractive index change at a plurality of points after the time when the refractive index change first occurs as the plasma progresses.

Next, the APTT calculation method in step S7 will be described. The APTT calculation unit 66 of the control unit 60 refers to the storage unit 61 in which a known relationship between the plasma flow velocity V and APTT is registered in advance, and calculates the APTT from the flow velocity V based on this relationship.
First, how to obtain the relationship between the plasma flow velocity V and APTT will be described. In order to obtain the relationship between the plasma flow velocity V and APTT, as described in step S2 of FIG. 7, a measurement chip in which a buffer is filled in the flow path is used, and the coagulation activity value and APTT are known in this measurement chip. The plasma flow rate V may be calculated using the coagulation activity measurement apparatus of the present embodiment after introducing plasma. As the activity of the control plasma used at this time, the average value of the factor activity described in the data attached to the reagent is used, and the control plasma is diluted with oren buffer to adjust the activity value. did. As the control plasma, two types of plasma, normal range N plasma and abnormal range P plasma, were used. By such measurement, the relationship between the plasma flow velocity V and the plasma activity value as shown in FIG. 11 can be obtained. As a result of the flow rate measurement, it was found that the higher the plasma activity, the faster the flow rate. The relationship between the flow rate and the activity value has good linearity as shown in FIG.

Since the APTT of the control plasma is a known value that can be obtained from the attached data, the following equation for obtaining the APTT from the flow velocity V can be obtained based on the measurement result shown in FIG.
APTT [sec] = − 0.0413 × V [μm / sec] +107.04
... (1)

The expression (1) obtained in advance is registered in the storage unit 61 of the data processing device 6. The APTT calculation unit 66 calculates APTT from the plasma flow velocity V using Equation (1).
In order to adjust the activity of the control plasma, it was diluted with an orene buffer, but in order to evaluate whether this dilution rate affects the flow rate, the flow rate was measured in a plasma sample to which no active agent was added. As a result, in the plasma sample to which no active agent was added, the flow rate was faster as the dilution rate was higher (that is, the activity in the plasma sample was lower). Therefore, it can be said that the plasma has been subjected to the activity of the APTT measurement reagent, and thus has a property that the flow rate is high in the plasma sample having high activity. In addition, the activity and flow of the plasma sample with the APTT measurement reagent were observed under a microscope. When a plasma sample is put into the microchannel, it is observed that a suspended substance having a size of about several tens of μm flows. When activated by adding an APTT measurement reagent to the plasma sample, There was no stagnation of the flow.

  On the other hand, after the plasma sample was added, observation was also performed by adding a buffer solution. FIG. 12 is a diagram showing an example of a change in refractive index when normal range plasma having an activity of about 97% is input to the measurement chip, and FIG. 13 is a diagram showing refraction when an abnormal range plasma having an activity of about 16% is input to the measurement chip. It is a figure which shows the example of a rate change. (A), (B), and (C) in FIGS. 12 and 13 indicate the states of FIGS. 1 (A), 1 (B), and 1 (C), respectively. As shown in FIG. 12 and FIG. 13, the refractive index measured by the refractive index measuring device is such that the measurement chip 100 is loaded with only the Oren buffer (A) and the plasma sample is loaded (B ) And the state (C) in which the aurene buffer solution is added after the coagulation measurement.

  When a buffer solution is introduced after the plasma sample is introduced, adsorbate that cannot be washed away with the buffer solution remains on the channel surface, regardless of whether the activity is as high as 97% or as low as 16%. Therefore, there is a possibility that fibrin or the like generated by the activity of the APTT reagent is deposited on the substrate surface of the flow path.

  The data processing device 6 according to the present embodiment can be realized by a computer having a CPU, a storage device, and an external interface, and a program for controlling these hardware resources. In this computer, a program for realizing the coagulation activity measurement method of the present invention is provided in a state of being recorded on a recording medium such as a flexible disk, a CD-ROM, a DVD-ROM, or a memory card. The CPU writes the program read from the recording medium into the storage device, and executes the processing described in this embodiment according to the program.

  The present invention can be applied to a coagulation activity measuring apparatus that measures an indicator of a blood coagulation activity factor.

  DESCRIPTION OF SYMBOLS 1 ... Prism, 2 ... Light source, 3 ... Polarizing plate, 4 ... Condensing lens, 5 ... CCD camera, 6 ... Data processing device, 60 ... Control part, 61 ... Memory | storage part, 62 ... Input part, 63 ... Display part, 64 ... Image processing unit, 65 ... Flow velocity calculation unit, 66 ... APTT calculation unit, 100 ... Measuring chip, 101 ... Substrate, 102 ... Sheet, 103 ... Acrylic housing, 104 ... Gold thin film, 105 ... Blocking agent, 106 ... Circular hole , 107, 108... Channel, 109... Suction channel, 110.

Claims (5)

A refractive index measuring device for measuring a refractive index at the surface of the flow channel of the measurement chip including the flow channel through which the solution flows;
After the buffer of the measurement chip is filled with a buffer solution, the refractive index when the plasma sample in which the activator that activates intrinsic coagulation and plasma are mixed is put into the channel. A flow rate calculating means for calculating a flow rate of plasma traveling in the flow path from the change;
A clotting activity factor index calculating means for calculating an activated partial thromboplastin time from a flow rate calculated by the flow rate calculating means based on a known relationship between the plasma flow rate and an activated partial thromboplastin time which is an index of a clotting activity factor; A coagulation activity measuring device comprising: In the coagulation activity measuring device according to claim 1,
The refractive index measuring device is a surface plasmon resonance measuring device. Measurement for a coagulation activity measurement device that calculates the flow velocity of plasma traveling in the flow path based on the refractive index at the surface of the flow path filled with buffer solution and calculates the activated partial thromboplastin time, which is an indicator of the coagulation activity factor A chip,
A gold thin film formed on the substrate surface;
A flow path formed on the gold thin film,
A measuring chip, wherein the gold thin film is partially covered with a biochemical measurement blocking agent that inhibits a coagulation reaction. A refractive index measurement step for measuring a refractive index at the surface of the flow channel of the measurement chip including the flow channel through which the solution flows;
After the buffer of the measurement chip is filled with a buffer solution, the refractive index when the plasma sample in which the activator that activates intrinsic coagulation and plasma are mixed is put into the channel. A flow rate calculating step for calculating a flow rate of plasma traveling in the flow path from the change;
A clotting activity factor index calculating step for calculating an activated partial thromboplastin time from the flow rate calculated in the flow rate calculating step based on a known relationship between the plasma flow rate and an activated partial thromboplastin time that is an index of a clotting activity factor; A coagulation activity measurement method comprising: In the coagulation activity measuring method according to claim 4,
In the refractive index measuring step, the refractive index is measured by a surface plasmon resonance measuring device, and the coagulation activity measuring method is characterized.