JP2015090293A - Measuring method and measuring apparatus - Google Patents

Abstract

The present invention provides a technique for comprehensively and quantitatively evaluating the quality such as the amount of thrombus and the strength and stability of the thrombus.
A measurement method is a measurement method for measuring blood by flowing blood through a flow channel provided in a microchip and inducing formation of a thrombus in the flow channel, and the blood inflow pressure into the flow channel Is stored in association with the passage of time, an imaging step in which the flow path is imaged with a camera and the obtained image is stored in association with the passage of time, and the amount of thrombus in a predetermined range from the stored image Then, the computer executes a thrombus amount measuring step that stores the time-lapse time in association with the passage of time.
[Selection] Figure 1

Description

  The present invention relates to a measurement method and a measurement apparatus for measuring blood.

  In thrombus formation in arteries (white thrombus) or primary hemostasis, platelet activation and aggregation plays a central role.

  Platelets bind directly or indirectly to collagen present under vascular endothelial cells when the blood vessels are damaged. In environments where blood flow is slow (under low shear stress), GPVI and other collagen receptors are the main direct bonds, and in environments where blood flow is fast (under high shear stress), vWF binds to collagen and binds to vWF. In contrast, platelet GPIbα receptor binds indirectly to collagen. Direct and indirect interaction with collagen activates platelets, and this stimulation releases various platelet-activating substances such as ADP and serotonin from darkly stained granules and α granules.

  These released platelet activating factors activate their own and surrounding platelets. In activated platelets, the fibrinogen receptor GPIIb / IIIa undergoes a structural change to an activated form and becomes a high affinity form for fibrinogen. Activated platelets are successively cross-linked through fibrinogen, which is a dimer, to form platelet aggregates.

  Conventionally, a technique for examining the function of platelets by passing predetermined blood through a capillary having a platelet adhesion surface and observing or measuring the behavior of the blood in the capillary has been proposed (for example, Patent Document 1).

  In addition, monoclonal antibodies against membrane glycoproteins GP IIb / IIIa and GP Ibalpha present on the platelet surface are fluorescently labeled with FITC and the like, and sample blood mixed in whole blood containing red blood cells simulates the damaged blood vessel wall While flowing through a flow chamber 20 using a glass plate 22 solidified by applying a matrix such as collagen or vWF (von Willebrand factor) on the upper surface, using a confocal microscope apparatus, A technique for capturing a moving image has also been proposed (for example, Patent Document 2).

International Publication No. 2010/018833 Pamphlet JP 2004-251630 A

  A conventionally proposed technique is to capture a part of an image of a flow path (capillary, flow chamber) and to support observation by a user. For example, Patent Document 2 describes that three-dimensional imaging is realized by combining a plurality of frames captured by moving the objective lens in the Z direction (optical axis direction). However, quality such as the amount of thrombus and the strength and stability of the thrombus could not be evaluated comprehensively and quantitatively.

  Therefore, an object of the present invention is to provide a technique for comprehensively and quantitatively evaluating the quality such as the amount of thrombus and the strength and stability of the thrombus.

  The measurement method according to the present invention is a measurement method for measuring blood by flowing blood through a flow channel provided in a microchip and inducing formation of a thrombus in the flow channel, and the blood inflow pressure into the flow channel Is stored in association with the passage of time, an imaging step in which the flow path is imaged with a camera and the obtained image is stored in association with the passage of time, and the amount of thrombus in a predetermined range from the stored image Then, the computer executes a thrombus amount measuring step that stores the time-lapse time in association with the passage of time.

  In this way, since the blood inflow pressure and the amount of thrombus in a predetermined range can be stored in association with the passage of time, the user can perform comprehensive and quantitative evaluation based on them. Become. That is, it is possible to provide a technique for comprehensively and quantitatively evaluating the quality such as the amount of thrombus and the strength and stability of the thrombus.

  Furthermore, in the imaging step, the camera is moved in parallel with the flow path, a plurality of images in which at least a part of the imaging target region overlaps are captured, and a step of combining the plurality of images is further performed. Alternatively, the ratio of the amount of thrombus in the flow channel may be calculated based on the synthesized image. In this way, it is possible to expand the observation area of the flow path simulating a blood vessel, and obtain a more reproducible measurement value than when measuring the amount of thrombus based on a local image, and it is accurate. You will be able to perform a simple analysis.

  Also, an apparatus for executing the processing according to the present invention may be provided, or a program for causing a computer to execute the method according to the present invention may be provided.

  The program may be recorded on a computer-readable recording medium. Here, the computer-readable recording medium refers to a recording medium that accumulates information such as data and programs by electrical, magnetic, optical, mechanical, or chemical action and can be read from the computer. . Among such recording media, those removable from the computer include flexible disks, magneto-optical disks, optical disks, flash memories, magnetic tapes, and the like. As recording media fixed to the computer, there are HDD (Hard Disk Drive), SSD (Solid State Drive), ROM (Read Only Memory) and the like.

  According to the present invention, it is possible to provide a technique for comprehensively and quantitatively evaluating the quality such as the amount of thrombus and the strength and stability of the thrombus.

It is a schematic diagram which shows an example of a blood measuring device. It is a schematic diagram which shows the 1st board | substrate of the microchip which concerns on a 1st example. It is a schematic diagram which shows the 2nd board | substrate of the microchip which concerns on a 1st example. It is a schematic diagram which shows the microchip which concerns on a 1st example. It is a schematic diagram which shows the 1st board | substrate of the microchip which concerns on a 2nd example. It is a schematic diagram which shows the 2nd board | substrate of the microchip which concerns on a 2nd example. It is a schematic diagram which shows the microchip which concerns on a 2nd example. It is a functional block diagram which shows an example of an information processing part. It is a figure for demonstrating the imaging range on the microchip which concerns on a 1st example. It is a figure for demonstrating the imaging range on the microchip which concerns on a 2nd example. It is an apparatus block diagram which shows an example of a computer. It is a processing flowchart which shows an example of a recording process. It is a processing flowchart which shows an example of an analysis process. It is a figure for demonstrating the synthesis | combination of image data. It is a figure for demonstrating the synthesis | combination of image data. It is a figure for demonstrating the synthesis | combination of image data. It is a figure which shows the other example of the synthesis | combination of image data. It is a figure which shows 1 frame which concerns on the other example of the synthesis | combination of image data. 3 is a graph showing changes in blood pressure according to Example 1. 6 is a diagram illustrating an example of a composite image according to Embodiment 1. FIG. 6 is a graph showing changes in blood pressure according to Example 2. 6 is a diagram illustrating an example of an image of control blood according to Example 2. FIG. It is a figure which shows an example of the image of the aspirin addition blood which concerns on Example 2. FIG. 6 is a diagram illustrating an example of an OS-1 added blood image according to Example 2. FIG. 6 is a diagram illustrating an example of a composite image of control blood according to Example 2. FIG. It is a figure which shows an example of the synthesized image of the aspirin addition blood which concerns on Example 2. FIG. It is a figure which shows an example of the synthesized image of the OS-1 addition blood which concerns on Example 2. FIG.

  Hereinafter, embodiments for carrying out the present invention will be described with reference to the drawings. The configuration of the present embodiment is an exemplification, and the present invention is not limited to the following configuration. In the present embodiment, “blood” includes whole blood and platelet-rich plasma.

<System configuration of blood measuring device>
FIG. 1 is a schematic diagram illustrating an example of a blood measurement device 1 according to the present embodiment. 1 includes a reservoir 101, a pump 102, a pressure sensor 103, a camera 104, a drive device 105, an information processing unit 106, and a discharge pipe 107, and a microchip at a predetermined position. 2 can be placed. The microchip 2 includes a flow path (also referred to as “capillary”) 201 simulating a blood vessel, an inflow port 211, an outflow port 212, and the like.

  The reservoir 101 is a blood storage unit that stores anticoagulated blood. The reservoir 101 is connected to the inflow port 211 of the microchip 2. The pump 102 is connected to the reservoir 101. The pump 102 presses mineral oil into the upper layer of the reservoir 101, and causes blood to flow into the microchip 2 from the lower layer of the reservoir 101.

  The pressure sensor 103 is an electronic pressure sensor using, for example, a stainless steel or silicon diaphragm. The pressure sensor 103 is connected to the path from the reservoir 101 to the pump 102 and can measure the inflow pressure of mineral oil into the reservoir 101. The inflow pressure of mineral oil to the reservoir 101 can be said to be the inflow pressure of blood to the microchip 2. The pressure sensor 103 is connected to the information processing unit 106, and the information processing unit 106 can record the measured inflow pressure in the storage device.

The camera 104 is an imaging device such as a CCD (Charge Coupled Device) camera equipped with a microscope device, and can capture a still image or a moving image. The camera 104 according to the present embodiment is a so-called area sensor camera. In addition, the camera 104 captures a micrograph or a moving image of the channel 201 of the microchip 2. Note that the camera 104 may image the microchip 2 from vertically above, or may image the microchip 2 from vertically below. Moreover, you may make it provide a light source (not shown) in arbitrary positions.

The driving device 105 is an actuator such as a servo motor, and moves the position of the camera 104. The drive device 105 is, for example, parallel to the microchip 2 and substantially along the flow path (ie, substantially along the direction of blood flow) or along a direction substantially perpendicular to the flow path (ie, of the flow path). The camera 104 can be moved (along the width direction) to change the imaging position of the camera 104.

  The information processing unit 106 is a computer including a processor, a storage device, an input / output device, and the like, and is connected to the pressure sensor 103 and the camera 104. Further, the information processing unit 106 records the blood inflow pressure measured by the pressure sensor 103 over time, or synthesizes a plurality of images (also referred to as “frames”) captured while moving the camera 104. An image exceeding the size of the imaging range 104 is generated, or the area of the thrombus included in the captured image is calculated.

  The discharge tube 107 is connected to the outlet 212 of the microchip 2 and discharges blood out of the blood measurement device 1.

  Next, the microchip 2 will be described with reference to FIGS. 2A to 3C. 2A to 2C are schematic views showing a first example of the microchip 2. FIG. 2A is a plan view showing an example of the first substrate 200 on the surface of which grooves serving as the flow paths 201 of the microchip 2 are formed. The cross-sectional shape of the groove is arbitrary, such as a concave shape, a U shape, and a V shape. Since the platelet aggregate is fragile, the depth of the groove is desirably 10 to 200 μm or less in order to measure the pressure increase. Moreover, it is preferable that the width | variety of a groove | channel is 10 micrometers-3 mm. Further, at least a part of the channel 201 is coated with a blood coagulation factor such as collagen and tissue thromboplastin to form a platelet-adhesive surface 202 (coagulation factor coat portion). Specifically, the coagulation factor is applied near the center of the flow path 201 as shown in FIG. 2A, for example. Note that the platelet easy-adhesion surface may extend over the entire length of the flow path 201.

  FIG. 2B is a plan view showing an example of the second substrate 210 provided with through holes that serve as the inlet 211 and the outlet 212 of the microchip 2. The positions of the through hole serving as the inflow port 211 and the through hole serving as the outflow port 212 are respectively the first end of the flow path 201 on the first substrate 200 when stacked with the first substrate 200. And a position corresponding to the second end of the flow path 201.

  FIG. 2C is a plan view showing an example of the microchip 2 in which the first substrate 200 and the second substrate 210 are stacked. An inlet 211 and an outlet 212 are located at the end of the flow channel 201. The broken line indicates that the channel 201 exists inside the microchip 2. The channel 201 is provided with a platelet easy-adhesion surface 202 coated with a coagulation factor.

  3A to 3C are schematic diagrams showing a second example of the microchip 2. FIG. FIG. 3A is a plan view showing an example of the first substrate 200 on the surface of which grooves serving as the flow paths 201 of the microchip 2 are formed. The cross-sectional shape of the groove is arbitrary, such as a concave shape, a U shape, and a V shape. Since the platelet aggregate is fragile, the depth of the groove is desirably 10 to 200 μm or less in order to measure the pressure increase. Moreover, it is preferable that the width | variety of a groove | channel is 10 micrometers-3 mm.

  In addition, a plurality of portions extending along the direction of blood flow are formed in a part between the first end (inlet side end) and the second end (outlet side end) of the channel 201. The channel dividing wall 203 is provided to form a channel dividing unit 204 that divides the width of the channel into a plurality of channels. That is, the flow path dividing unit 204 is provided with a plurality of flow path dividing walls 203 in a comb shape.

Further, the interval between the flow path dividing walls 203 is desirably 200 μm or less. If the width is 200 μm or less, when platelet aggregates are formed, the internal pressure can be increased without being blown into the bloodstream even under high blood flow and high shear stress. Further, in the flow path dividing unit 204, the flow path 2
The width of 01 is desirably divided into five or more by the flow path dividing wall 203. That is, if the width of the flow path is divided into five or more, the blockage of each divided flow path is averaged, and data with little variation is easily obtained. The microchip 2 according to the second example is assumed to have 25 flow path dividing walls 203.

  The shape of the flow path dividing wall 203 is not particularly limited as long as the width of the flow path 201 can be divided into a plurality of parts. Further, the flow path dividing wall 203 may be subjected to a treatment such that the surface roughness (center line average surface roughness: Ra) is 10 to 200 nm in order to improve the adhesion of the platelet aggregate.

  Further, when blood of a healthy person is flowed through the microchip channel at a constant speed to form platelet aggregates in the channel dividing part, the blood inflow pressure rises to about 80 kPa. Therefore, the pressure resistance of the microchip is preferably 80 kPa or more.

  It should be noted that a plurality of parts at a predetermined interval in the width direction of the flow path are provided upstream of the flow path dividing section 204, that is, a part between the flow path dividing wall 203 and the first end (inlet side end) A columnar body (not shown) may be installed. The plurality of columnar bodies function as impurity inflow prevention portions. The interval between the columnar bodies (the length of the gap) may be any length that allows blood to pass but not allow impurities to pass, but is preferably 10 to 200 μm. The shape of the columnar body is not particularly limited, and examples thereof include a cylinder and a polygonal column.

  FIG. 3B is a plan view showing an example of the second substrate 210 provided with through holes to be the inlet 211 and the outlet 212 of the microchip 2. The positions of the through hole serving as the inflow port 211 and the through hole serving as the outflow port 212 are respectively the first end of the flow path 201 on the first substrate 200 when stacked with the first substrate 200. And a position corresponding to the second end of the flow path 201. In addition, a blood coagulation factor such as collagen is coated on the back surface of the second substrate 210 at a position where it is covered with the flow path dividing wall 203 on the first substrate 200 when the first substrate 200 is laminated. By doing so, the platelet easy-adhesion surface 202 (coagulation factor coat part) is formed. Specifically, as shown in FIG. 3C, the collagen is applied in a wide manner at a position covering the flow path dividing wall 203 for safety. The platelet easy-adhesion surface may extend over the entire length of the flow path.

  FIG. 3C shows an example of the microchip 2 in which the first substrate 200 and the second substrate 210 are laminated so that the groove of the first substrate 200 and the platelet easy-adhesion surface 202 of the second substrate 210 are inside. FIG. A broken line indicates that the channel 201 exists inside the microchip 2.

  The material of the microchip 2 is preferably metal, glass, plastic, silicone or the like. A transparent material is preferable from the viewpoint of use for blood observation (particularly image analysis). Further, from the viewpoint of forming a circuit, plastic is preferable, and transparent plastic is particularly preferable. When the material is made of silicone such as PDMS (polydimethylsiloxane), it can be laminated by pressing the first substrate and the second substrate without using an adhesive or the like because of excellent adhesion between the substrates. . However, when a high pressure is applied to the inside of the microchip 2, it is preferable to use an adhesive. Also, poly 2-methoxyethyl acrylate (PMEA) can easily and effectively suppress blood coagulation at unintended sites. In addition, although the groove | channel and hole provided in the board | substrate of a microchip can also be formed with a cutter or a laser beam, when the material of the microchip 2 is a plastic, it can also be formed by injection molding. If formed by injection molding, it is preferable in that a microchip having a certain quality can be efficiently produced.

  According to the blood measuring device 1, quantitative blood measurement can be performed by measuring the inflow pressure by the pressure sensor 103 connected to the pump 102. Blood measurement can also be performed by observing with the camera 104 platelet activation (platelet adhesion / aggregation, etc.) in the channel 201 of the microchip 2 and the resulting blockage of the channel 201. Furthermore, blood measurement can also be performed by measuring the passage time or passage amount of the blood flow channel 201.

The combined use of visual evaluation by photographing platelet activation in the channel 201 and quantitative examination of platelet activation due to pressure increase is important in comprehensively determining the blood state. For example, in cases where platelets have already been activated in vivo and platelets are consumed and markedly decreased in pathologies such as DIC, the pressure rise and occlusion time are delayed. Even in such a case, by imaging the channel 201 with the camera 104, it is possible to confirm the increase in adhesion and aggregation of platelets to the platelet easy-adhesion surface 202 immediately after the start of the examination, and the state of the patient's platelets. Can be comprehensively determined.

  Specifically, according to the blood measurement device 1 according to the present embodiment, it is possible to record and output a change with time of the inflow pressure and a change with time of the area of the thrombus. Here, it can be said that the area of the thrombus represents the amount of thrombus. The inflow pressure represents not only the amount of thrombus but also the quality of thrombus such as strength and stability. By comprehensively judging these changes, for example, (1) drugs and diseases that reduce thrombus stability, (2) drugs and diseases that prevent thromboembolism, and (3) thrombus formation itself are suppressed. It will be possible to evaluate drugs and diseases in detail. Moreover, the blood measuring apparatus 1 according to the present embodiment generates a composite image (so-called panoramic image) in which a wide range is captured by combining a plurality of images captured while moving the camera in a planar manner. In this way, a reproducible measurement value can be obtained rather than measuring the amount of thrombus based on a local image, and an accurate analysis can be performed.

  FIG. 4 is a functional block diagram illustrating an example of the information processing unit 106. 4 is a so-called computer, and includes an inflow pressure acquisition unit 1061, an information storage unit 1062, an image acquisition unit 1063, an image synthesis unit 1064, an area calculation unit 1065, and an information output unit 1066. Have.

  The inflow pressure acquisition unit 1061 acquires the pressure (inflow pressure) measured by the pressure sensor 103 and stores it in the information storage unit 1062. At this time, the pressure value and the elapsed time are stored in association with each other. Here, the elapsed time may be information indicating the measured date and time, or may be an elapsed time from a predetermined reference time such as a processing start time.

  The image acquisition unit 1063 acquires image data captured by the camera 104 and stores it in the information storage unit 1062. Image data is also stored in association with the passage of time. Here, the camera 104 captures a still image or a moving image at a predetermined cycle. In the present embodiment, each of a plurality of frames included in a moving image is handled as an image (still image). Further, the camera 104 generates a plurality of image data while the driving device 105 moves the position of the camera 104. For example, in the case of the microchip 2 according to the first example, it is assumed that a region 1041 indicated by a thick line in FIG. 5A is an imaging range that can be accommodated in one image data. At this time, image data of the hatched area is continuously (periodically) imaged while the camera 104 is moved along the flow path 201. In the case of the microchip 2 according to the second example, it is assumed that a region 1041 indicated by a thick line in FIG. 5B is an imaging range that can be accommodated in one image data. At this time, image data of the hatched area is continuously (periodically) imaged while moving the camera 104 along the width direction of the flow path 201. In addition, a plurality of image data covering the hatched area is imaged at a period of, for example, 1 to 3 minutes with reference to the processing start time. In this way, it is possible to analyze the change over time of the formed thrombus based on the continuously captured images.

  The image synthesis unit 1064 synthesizes a plurality of images with an image in which the position of the flow channel 201 of the microchip 2 that is a subject (also referred to as “object to be imaged”) overlaps at least partially, and the information storage unit 1062 Remember me. Note that the method for synthesizing the image is not particularly limited, but it is desirable to perform the pixel normalization cross correlation method. It is also possible to shoot and synthesize still images while moving the camera so that the images overlap each other by a predetermined ratio (for example, about one third or more, preferably about three quarters). In addition, when combining frames of a moving image, the moving distance for each frame can be estimated in advance based on the moving speed of the camera and the number of frames taken per unit time (frame rate). Therefore, it is also possible to extract the frames so that the images to be combined overlap each other by a predetermined ratio and apply the pixel normalized cross correlation method. The composite image is also stored in association with the passage of time. Since the composite image includes a plurality of images, for example, the date / time or elapsed time of the image captured earliest is stored as the elapsed time. The image composition unit 1064 generates a composite image representing the hatched area in FIGS. 5A and 5B.

  The area calculation unit 1065 identifies a thrombus from the synthesized image, calculates the area of the region where the thrombus is imaged, and stores it in the information storage unit 1062. The area is stored in association with the composite image or the passage of time. Here, a red blood region and a white thrombus (white thrombus) region are imaged in the flow path 201 of the microchip 2 that is the object to be imaged. For example, the area calculation unit 1065 may extract a portion where the luminance of the image is discontinuous as an edge based on a predetermined threshold (binarization level), and specify the region where the thrombus is imaged. In the microchip 2 according to the present embodiment, the end of the flow path is imaged black. Since a light and dark gap occurs in the shadow (black) portion formed at the end of the flow path, it is effective for identifying the boundary between the flow path and the white thrombus in the image analysis. The absolute value of the thrombus area may be calculated, or the ratio of the thrombus area in the entire flow path may be calculated. The area of the thrombus is calculated based on the number of pixels included in the image, for example. In this way, the user can not only observe the image but also perform quantitative measurement.

  The information output unit 1066 reads out information from the information storage unit 1062 and outputs the information. The output information is information indicating a change in pressure value and information indicating a change in thrombus area. Further, a composite image of the flow path may be displayed. The user can evaluate a disease and a medicinal effect based on the output information.

  The blood measurement device 1 shown in FIG. 4 is realized by, for example, a computer executing a predetermined program.

<Device configuration>
FIG. 6 is an apparatus configuration diagram illustrating an example of a computer. The information processing unit 106 is a computer as shown in FIG. The computer is a CPU (Central Processing Unit) 1
001, main storage device 1002, external storage device 1003, communication IF (Interface) 1004
, Input / output IF (Interface) 1005, drive device 1006, and communication bus 1007. The CPU 1001 performs processing according to the present embodiment by executing a program (also referred to as “software” or “application”). The main storage device 1002 caches programs and data read by the CPU 1001 and develops a work area of the CPU. Specifically, the main storage device is a RAM (Random Access Memory), a ROM (Read Only Memory), or the like. The external storage device 1003 stores programs executed by the CPU 1001, information used in the present embodiment, and the like. Specifically, the external storage device 1003 is an HDD (Hard-disk Drive), an SSD (Solid State Drive), a flash memory, or the like. The main storage device 1002 and the external storage device 1003 work as the information storage unit 1062 of the information processing unit 106. The communication IF 1004 transmits / receives data to / from other computers. The communication IF 1004 is specifically a wired or wireless network card or the like. The input / output IF 1005 is connected to the input / output device and accepts input from the user or outputs information to the user. Specifically, the input / output device is a keyboard, a mouse, a display, various sensors or a touch panel, a pressure sensor 103, a camera 104, and the like. The drive device 1006 reads data recorded on a storage medium such as a magnetic disk, a magneto-optical disk, and an optical disk, and writes data to the storage medium. The above components are connected by a communication bus 1007. A plurality of these components may be provided, or some of the components (for example, the communication IF 1004 and the drive device 1006) may not be provided. The input / output device may be configured integrally with the computer. Then, the program executed in this embodiment is provided via a portable storage medium readable by the drive device 1006, a portable external storage device 1003 such as a flash memory, a communication IF 1004, and the like. It may be. Then, the CPU 1001 causes the computer as described above to work as the information processing unit 106 by executing the program.

<Recording process>
FIG. 7 is a process flow illustrating an example of a recording process performed by the information processing unit 106. The inflow pressure acquisition unit 1061 of the information processing unit 106 continuously receives data indicating the pressure value from the pressure sensor 103 and stores the data in the information storage unit 1062 (FIG. 7: S1). Further, the image acquisition unit 1063 of the information processing unit 106 acquires image data from the camera 104 at a predetermined cycle and stores it in the information storage unit 1062 (S2). Imaging of image data is started, for example, every 1 to 3 minutes. For example, in the case of the microchip 2 according to the first example illustrated in FIG. 2, images are continuously captured while the camera 104 is translated along the flow path 201. In the case of the microchip 2 according to the second example illustrated in FIG. 3, images are continuously captured while the camera 104 is translated along a direction perpendicular to the flow channel 201 (width direction of the flow channel 201). Although S1 and S2 have been described in order for convenience, they may be executed in parallel (that is, in multitasking). Thereafter, the information processing unit 106 determines whether to end the process (S3). For example, when the end of processing is instructed by the user via the input / output IF, it is determined that the processing is to be ended when a predetermined time has elapsed from the start of processing, when there is no blood remaining in the reservoir 101, or the like. The If it is determined that the process is to be terminated (S3: YES), the recording process is terminated. On the other hand, when it is determined not to end the process (S3: NO), the process proceeds to S1 and the process is repeated.

<Analysis processing>
FIG. 8 is a processing flow illustrating an example of analysis processing performed by the information processing unit 106. First, the image synthesis unit 1064 of the information processing unit 106 synthesizes an image and stores the synthesized image in the information storage unit 1062 (FIG. 8: S11). The image synthesizing unit 1064 synthesizes a plurality of images with an image in which the position of the flow channel 201 that is the imaging target overlaps at least partially, and stores the synthesized image in the information storage unit 1062. The composite image is stored in association with information indicating the passage of time. The composite image according to this step is generated for each group of images periodically captured. Therefore, the increase / decrease in the amount of thrombus can be seen from the change with time of the thrombus included in the plurality of composite images.

  For image synthesis, for example, images captured so that some areas overlap as shown in FIG. 9A and FIG. 9B are overlapped with pixel matching cross-correlated parts as shown in FIG. 9C by pixel normalization cross-correlation. This is done by connecting. FIG. 9A to FIG. 9C are examples of images captured along the blood flow direction of the flow channel 201 for the microchip 2 according to the first example. When shooting moving images, the moving distance for each frame is estimated from the moving speed and moving distance of the camera and the number of frames of the moving image, and the frames are extracted so that the frames overlap each other by a predetermined ratio. It is possible to create a connected image from a moving image. By repeating such connection, a composite image can be obtained in which a wide range of the channel can be observed.

  In the case of the microchip 2 according to the second example, a plurality of image data captured along the width direction of the flow path 201 are combined. FIG. 10 shows an example of a composite image in the case of the microchip 2 according to the second example. In the example of FIG. 10, a part of the flow path 201 is imaged so that one end (the end in the blood flow direction) of the flow path dividing wall 203 provided in the flow path 201 is substantially at the center. Similar to the case of the microchip 2 according to the first example, the image data can be synthesized based on the shape of the boundary between the blood portion and the thrombus portion. FIG. 11 shows one frame of a moving image actually taken. In this way, local thrombus formation can be observed in detail in the moving image. In addition, the degree of overall thrombus formation can be confirmed from the synthesized image as shown in FIG. Detailed observation of thrombus formation is possible by local observation and overall observation.

  Next, the area calculation unit 1065 identifies a thrombus from the synthesized image, calculates the area of the region where the thrombus is imaged, and stores it in the information storage unit 1062 (S12). The area is stored in association with the composite image or the passage of time. Here, a red blood region and a white thrombus (white thrombus) region are imaged in the flow path 201 of the microchip 2 that is the object to be imaged. For example, the area calculation unit 1065 may extract a portion where the luminance of the image is discontinuous as an edge and specify the region where the thrombus is imaged. The absolute value of the thrombus area may be calculated, or the ratio of the thrombus area in the entire flow path may be calculated. The ratio of the thrombus region is calculated as, for example, the ratio of the number of white pixels to the total number of pixels in the channel. In addition, since the edge part of a flow path is imaged as a black area | region with a CCD camera, an area is calculated for the inside of the said black area | region.

  Then, the information output unit 1066 reads and outputs information from the information storage unit 1062 (S13). The output information is information indicating a change in pressure value and information indicating a change in thrombus area. Further, a composite image of the flow path may be displayed. The user can evaluate a disease and a medicinal effect based on the output information. For example, the pressure value is output by drawing a graph with the pressure value on the vertical axis and the elapsed time on the horizontal axis. Moreover, you may superimpose and display the line graph showing the change of the area of a thrombus on the same graph. Further, by pointing the position on the graph with a cursor or the like, a composite image of the flow path at the elapsed time corresponding to the position may be displayed.

  According to such an analysis process, it is possible to support evaluation of a disease and a drug effect by a user. In particular, in this embodiment, since the amount of thrombus is measured using a composite image, a reproducible measurement value can be obtained and accurate analysis can be performed compared to the case of measuring the amount of thrombus based on a local image. Will be able to. Next, the evaluation of diseases and drug efficacy will be described.

<Evaluation of disease and efficacy>
Blood containing corn trypsin inhibitor (CTI), an inhibitor of blood coagulation factor 12, is flowed through a capillary coated with collagen and tissue factor to form a mixed white thrombus composed of fibrin and platelets. When abciximab that suppresses the GpIIb / IIIa receptor is added to blood, thrombus formation itself is suppressed, so that the start of pressure increase is delayed in a concentration-dependent manner, and the pressure lower area is also suppressed accordingly. Since thrombus formation itself is suppressed, the thrombus area is also significantly suppressed.

On the other hand, when blood to which t-PA as a thrombolytic agent is added is used, a thrombus is formed and a pressure increase starts, but thereafter no continuous pressure increase occurs. Therefore, the pressure increase start does not change greatly, and the lower area of the pressure decreases. However, the reduction in thrombus area is limited.
When binding to VWF-GPIbalpha is inhibited, the thrombus formation is particularly inhibited under high shear stress, and the final blockage of the capillary is specifically inhibited. For example, congenital bleeding disorders such as von Willebrand disease and Bernard syndrome fall under this case.
In this case, since the pressure rise start and the pressure lower area are both extended, a suppression pattern of pressure data similar to abciximab is measured.

However, abciximab greatly suppresses the thrombus area, whereas the decrease in the thrombus area when VWF-GPIbalpha is inhibited is mild. In this way, it is possible to identify and evaluate diseases and drug effects that are difficult to distinguish by analyzing only the pressure or thrombus area, using the blood measuring apparatus 1 that combines the analysis of pressure and thrombus area.

In addition, when a drug that reduces the strength and stability of the thrombus, such as aspirin that has an aggregation-inhibiting action or t-PA that has a thrombus-dissolving action, the maximum pressure is reduced, but the change in the thrombus area is small. . In the case of a disease in which the final occlusion due to thrombus formation is strongly suppressed, such as VWD (von Willebrand disease), the start of pressure increase is significantly delayed, but the change in the thrombus area is small. . In addition, in the case of a drug or disease in which thrombus formation itself is significantly inhibited, such as a GPIIb / IIIa inhibitor or platelet asthenia (GP-2 deficiency), both the onset of pressure increase and the thrombus area are remarkably suppressed.

  There are individual differences in drug efficacy. For example, when chronological changes in inflow pressure (flow channel pressure) were observed for multiple healthy individuals, multiple aspirin patients, and multiple aspirin and clopidogrel combination patients, aspirin-resistant patients and antiplatelets In patients with drug resistance, the inflow pressure hardly changes (does not decrease).

  The blood measuring apparatus 1 can store and output the inflow pressure and the thrombus area in association with the passage of time. That is, it is possible to support detailed evaluation of a disease or a drug effect by a user. It should be noted that a predetermined threshold value is set in advance for parameters such as a pressure rise start time, maximum pressure, and thrombus area, and the blood measuring device is used to determine whether there is a suspected disease or drug effect based on the measured value and the threshold value. One information processing unit 106 may determine and output the information.

[Example 1]
A capillary having a width of 300 μm, a depth of 80 μm and a length of 1.5 cm was coated with type I collagen (Nitta Gelatin Co., Ltd.) and tissue thromboplastin (Sysmex Corporation). Then, blood with CTI (50 μg / ml) added to the capillary was perfused for 30 minutes at a flow rate of 4 μl / min, and pressure occlusion due to thrombus was continuously measured.

  FIG. 12 is a graph showing a pressure pattern when a blood sample is perfused. (1) indicated by a solid line indicates a pressure change in the case of control blood to which no drug is added. (2) indicated by a broken line indicates a change in pressure when a blood sample to which abciximab (6 μg / ml) was added was perfused. (3) shown with a dashed-dotted line has shown the pressure change when the blood sample which added t-PA (6000 IU / ml) was perfused. (4) indicated by a two-dot chain line indicates a change in pressure when a blood sample to which a GPIb inhibitor: OS-1 (1600 nM) was added was perfused.

  When the pressure waveform is analyzed in comparison with the control blood (1), it can be seen that in (2) and (4), the pressure rise is completely suppressed and a very similar pressure pattern is taken. On the other hand, in (3), it can be seen that the start of the pressure increase does not change greatly, and thereafter the pressure increase rate is suppressed by thrombolysis.

In addition, in order to calculate the thrombus area, the camera was moved along the capillary at a speed of 0.2 mm / second, and a moving image of the coated portion of collagen and tissue thromboplastin was taken. The frame rate of the moving image was 29.2 frames / second. The shooting period was every 3 minutes. Then, a still image was extracted for each frame from the captured moving image, and the still image was reconstructed as a synthesized image by normalized correlation in pixel units. FIG. 13 is an example of a composite image 6 minutes after the start of measurement. From such a composite image, the edge of the white thrombus portion was extracted at a binarization level set as a threshold for the portion inside the capillary, excluding the region outside the capillary. Thereafter, the ratio of white thrombus in the entire capillary was calculated and used as the thrombus area. Table 1 shows the ratio of the thrombus area 6 minutes after the start of measurement for the same blood samples (1) to (4) as in FIG.

As can be seen from Table 1, in (2), the thrombus area is remarkably suppressed, but in (3) and (4), the decrease in thrombus area is limited. On the other hand, as can be seen from FIG. 12, (2) and (4) cannot be distinguished by the pressure increase. Moreover, as shown in Table 1, it is difficult to distinguish (3) and (4) in the thrombus area. However, according to the blood measuring apparatus 1, it is possible to quantitatively and comprehensively evaluate by combining the pressure measurement by the thrombus and the thrombus area calculation, and it becomes possible to analyze the characteristics of the thrombus in detail. That is, in this system, the user can distinguish the blood samples (1) to (4) by using the thrombus strength and the thrombus amount.

[Example 2]
In Example 2, 25 channels each having a width of 40 μm were arranged in a comb shape at equal intervals as a channel dividing portion in a capillary having a width of 2 mm and a depth of 40 μm. In addition, collagen was coated on the channel division part. Then, blood to which 50 μg / ml hirudin was added was perfused at a flow rate of 18 ul / min. FIG. 14 is a graph showing a pressure pattern when a blood sample is perfused. The blood sample (1) shown by the solid line in FIG. 14 shows the pressure change of the control blood without addition. The blood sample (2) indicated by a broken line is aspirin (200 ug / ml) added blood. A blood sample (3) indicated by a one-dot chain line is a pressure waveform of blood added with OS-1 (100 nM). As shown in FIG. 14, in the case of aspirin (2) and OS-1 (3), an increase in pressure was suppressed as compared with the case of control blood (1).

  In addition, every 30 seconds, a moving image was taken for the comb portion while moving the camera at a speed of 0.2 mm / second in a direction perpendicular to the blood flow of the capillary. The frame rate of the moving image was 29.2 frames / second. In addition, a still image was extracted for each frame from the moving image, and a composite image was reconstructed by normalization correlation in units of pixels.

  FIG. 15 is an image of control blood (1) taken 3 minutes after the start of measurement. FIG. 16 is an image of aspirin-added blood (2) 3 minutes after the start of measurement. FIG. 17 is an image of OS-1 added blood (3) 3 minutes after the start of measurement. 18 to 20 are composite images of blood samples (1) to (3), respectively.

When the image is analyzed, it can be seen that in the control blood (1) and the aspirin-added blood (2), platelet thrombus is formed in the entire capillary and no clear difference is observed. Moreover, it is difficult to understand the details of the state of the thrombus from the composite image of the OS-1 added blood (3). Regarding the OS-1 added blood (3), by analyzing the moving image before synthesis, it is understood that thrombus is formed but the final capillary blockage is suppressed.

  As described above, in this system, the user can grasp the characteristics of thrombus formation by using the enlarged image or moving image of the local thrombus and the composite image of the entire capillary. In combination with the pressure waveform, it was possible to identify the detailed mechanism of action of the antiplatelet drug and the characteristics of the disease.

<Others>
In addition, platelets can be fluorescently labeled with quinacrine or the like, and in that case, the luminance per unit area due to fluorescence development can be measured by image analysis to quantify the measurement results and obtain them as data. is there.

  The microchip 2 may be placed on a heater (not shown). If the microchip 2 is heated to about 37 ° C. with a heater, the measurement process can be performed under conditions close to the living body.

  In the above embodiment, the image data is synthesized based on the shape of the boundary between the blood portion and the thrombus portion. However, the synthesis of the image data is not limited to such a method. For example, when thrombus does not occur so much, the characteristic change that is the basis of synthesis may not be clearly displayed. Based on the speed at which the camera 104 is moved by the driving device 105 and the period at which the image data is captured, the corresponding positions between the image data may be specified, and the image data may be synthesized.

DESCRIPTION OF SYMBOLS 1 Blood measuring apparatus 101 Reservoir 102 Pump 103 Pressure sensor 104 Camera 105 Drive apparatus 106 Information processing part 107 Discharge pipe 2 Microchip 200 1st board | substrate 201 Flow path 202 Platelet easy-adhesion surface 203 Flow path dividing wall 204 Flow path dividing part 210 Second substrate 211 Inlet 212 Outlet

Claims (3)

A method of measuring blood by flowing blood through a channel provided in a microchip and inducing formation of a thrombus in the channel,
Storing the inflow pressure of the blood into the flow path in a storage device in association with the passage of time;
An imaging step of capturing the flow path with a camera and storing the obtained image in the storage device in association with the passage of time;
Calculating the amount of thrombus in a predetermined range from the stored image, and storing the thrombus amount in association with the passage of time;
The measurement method that the computer executes. In the imaging step, the camera is moved substantially parallel or substantially perpendicular to the flow path to capture a plurality of images in which at least a part of the imaging target region overlaps,
Further performing the step of combining the plurality of images;
The measurement method according to claim 1, wherein, in the thrombus amount measurement step, a thrombus amount in the predetermined range is calculated based on the synthesized image. A measurement device that causes blood to flow through a channel provided in a microchip and induces thrombus formation in the channel, and measures the blood,
A pressure acquisition unit that stores the inflow pressure of the blood into the flow path in a storage device in association with the passage of time;
An image acquisition unit that images the flow path with a camera and stores the obtained image in the storage device in association with the time lapse;
A thrombus amount measuring unit for calculating the amount of thrombus in a predetermined range from the stored image and storing it in the storage device in association with the passage of time;
A measuring apparatus comprising: