JP2532707B2 - Blood circuit, blood measuring apparatus and blood measuring method using the same - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a blood circuit, and a blood measuring device and a measuring method using the blood circuit.

(Prior Art) Measuring and evaluating the functions of erythrocytes, leukocytes, and platelets, which are formed components in blood, is extremely important for health care, diagnosis and treatment of diseases. Therefore, conventionally, the ability of blood to pass through a membrane having micropores such as Nuclepore filter or nickel mesh filter has been investigated for the purpose of measuring erythrocyte deformability. In addition, a method of measuring the change in the turbidity of the platelet suspension due to the aggregation has been used to measure the platelet aggregation ability. In addition, the measurement of leukocyte activity corresponds to several aspects of leukocyte activity by the Boyden chamber method, particle phagocytosis test,
Chemiluminescence measurement methods have been used. This leukocyte activity is particularly important in infectious diseases, immunotherapy, immunosuppressive therapy and the like.

(Problems to be solved by the invention) However, all of the above-mentioned measuring methods have problems such as inefficiency, low reproducibility, and low quantifying ability, and are not effective measuring methods suitable for importance. It can't happen. Also,
The conventional method for measuring platelet aggregability requires time and effort for sample preparation,
The sensitivity is not enough. Further, the above-mentioned conventional technique for measuring red blood cell deformability is unreliable because the hole or groove is clogged by the formed material in the blood sample during the measurement.

On the other hand, the present inventors previously developed a new blood filter composed of fine grooves processed on a silicon substrate,
And by developing a red blood cell deformability measuring device using it, the diameter and shape of the pores are non-uniform, the direction of red blood cells when entering the pores is not uniform, and the deformation process cannot be observed.
The problems of conventional erythrocyte deformability measurement methods, such as the meaning of the index is not clear, have been largely solved (Japanese Patent Application No. 63-283687).

Further, in the device using the blood filter invented by the present inventors, since the groove passage velocity of each red blood cell is directly measured and used as an index, the result itself is not affected by clogging. However, the clogging itself cannot be prevented yet. Therefore, the number of times the filter can be used is limited, which is a major obstacle to putting the device into practical use.

Further, conventionally, in order to prevent interference of blood cells of other types, it has been performed to separate and measure only a single type of blood cell fraction from a blood sample, but such a method requires a great deal of labor. Not only that, however, the degeneration of blood cells or the degeneration due to the separation treatment cannot be prevented during that time, which reduces the physiological or diagnostic value of the result.

In addition, it is important to completely separate and measure the passive movement of blood cells due to the difference in hydrostatic pressure and the active movement of blood cells due to stimulation of physiologically active substances, and the effect of mechanical stress on blood cells is important for research and diagnosis. However, there is currently no way to quantitatively study this type of problem. The above-mentioned device invented by the inventors of the present invention enables such a study by providing multiple filters, but it has not been possible to follow up the measurement of the effect of mechanical stress on individual blood cells. Absent.

In addition, until now, there has been no effective means for measuring and studying the flow condition of each blood cell when the flow path constitutes a network.

Therefore, it is an object of the present invention to provide a novel blood circuit that solves the problem of next-to-row, and a blood measuring apparatus and a measuring method using the same.

1) To effectively quantify and measure leukocyte activity.

2) The platelet aggregation ability should be measured more simply and with higher sensitivity than in the prior art.

3) When measuring the erythrocyte deformability, prevent pores or grooves from being clogged by formed components in the blood sample, thereby increasing the reliability of the measurement.

4) To be able to measure erythrocyte deformability, leukocyte activity, and platelet agglutination ability without separating each blood cell fraction from a blood sample.

5) Minimize the interference of blood cells of other species in the above measurement 4.

6) The migration of specific blood cells due to the action of only the physiologically active substance can also be measured.

7) Follow-up measurement of changes in the above-mentioned functional characteristics of each blood cell due to mechanical stress.

8) Measuring the flow distribution of each blood cell in the flow channel network.

(Means for Solving the Problems) In order to achieve such an object, the present invention replaces the conventional method in which all of the blood sample passes through the fine groove, and replaces the fine groove in a direction substantially orthogonal to a large flow path. By forming a part of the blood sample from a large flow path to a fine groove, it is adapted to the shape of red blood cells, white blood cells or platelets on the substrate by applying semiconductor microfabrication technology. The fine grooves having various shapes and sizes are formed with high precision. The number of blood cells contained in even a part of a blood sample is extremely large, and a sufficient number of blood cells can be measured.

In this way, in order to guide the blood sample from the large flow channel to the fine groove, the inlet side and the outlet side of the groove, that is, the portion which becomes the large flow channel for flowing the blood sample, and the groove parallel to this channel and By a hydrostatic pressure difference or a physiological activity with respect to a part which becomes another flow path communicating with this flow path (a physiologically inert fluid such as physiological saline is usually flown into this another flow path). It suffices to cause a difference in concentration of substances.

Further, in the present invention, it is also shown that the narrow portion is provided in multiple stages in the groove.

(Activity) The activity of leukocytes is the sum of various reactions such as migration, phagocytosis, secretion of physiologically active substances, and the contraction and movement of intracellular contracting proteins are involved in all the reactions. . On the other hand, the ability of leukocytes to actively or passively pass through the groove, including groove occlusion, remarkably changes depending on the contraction and motility state of the contracting protein in the cell. Therefore, the ability of leukocytes to actively or passively cross the groove or groove occlusion is an appropriate indicator of the activity of leukocytes. Similarly, in platelet aggregation, contraction and movement of intracellular contracting proteins are the main reaction, and therefore, the ability of platelets to pass through the groove or groove occlusion by the platelet aggregate is a good index here. For white blood cells and platelets, the amount of change in groove passage ability including groove occlusion after stimulation with a certain amount of physiologically active substance can be used as an index.

In this method, in which a blood sample is caused to flow through a fine groove channel provided in a direction substantially orthogonal to a large flow channel, most of the sample is caused to flow along the large flow channel, and only a small portion of the blood sample is finely divided. It is possible to lead to the groove. Therefore, for example, in the case of a fine groove having an inlet of a combined shape of erythrocytes, a formed component larger than white blood cells or erythrocytes, for example, an aggregate of blood cells cannot enter the groove even if it comes near the inlet, and a blood sample It will be swept away by the main stream of and go away from the ditch entrance. In this way it is prevented that white blood cells or particles larger than red blood cells block the groove. At that time, inflow of platelets smaller than red blood cells cannot be prevented, but platelets do not impede passage of red blood cells. Similarly, in the case of a groove having an inlet shaped to match white blood cells, red blood cells and platelets pass freely, but do not affect the passage of white blood cells. Further, by fluorescently coloring each blood cell or liquid component with a fluorescent substance, it becomes extremely easy to distinguish between different types of blood cells and between blood cells and surrounding liquid. In this way, by devising the method of flowing the blood sample, the shape of the groove inlet, and the measurement method, it is possible to prevent the inflow of blood cells or tangible components with a larger diameter, while at the same time, the ability of the blood cells to be measured to pass through the groove including groove occlusion. Can be selectively measured. In addition, three types of groove circuit networks and measurement methods that are suitable for red blood cells, white blood cells, and platelets are arranged in parallel,
By allowing a blood sample to flow in each of the above methods, it becomes possible to simultaneously and quickly measure red blood cells, white blood cells, and platelets in the blood sample.

On the other hand, the above-described flow of the blood sample makes it possible to measure the migration of specific blood cells due to only the concentration difference of the physiologically active substance. That is, by providing a concentration difference of the physiologically active substance instead of the hydrostatic pressure difference between the groove inlet side and the outlet side,
Only blood cells that can recognize the difference in concentration of the physiologically active substance migrate into the groove. The above object can be achieved by measuring the number and the passage time.

Further, by providing the narrowed portion in multiple stages in the same groove, not only can blood cells passing therethrough be traced, but also changes occurring in the passage process can be traced at the same time.

How to distribute each material component of blood between different groove networks,
The distribution status of each formed component of blood in the same circuit network is a new index that has never existed before.

The blood or its components flowing through the blood circuit are collected at the outlet end and, if necessary, returned to the original state or conveyed to another measuring system.

(Example) Hereinafter, the structure of the present invention will be described in detail based on an example shown in the drawings. FIG. 1 schematically shows the configuration of the blood measuring apparatus of the present invention. This device consists of three types of blood circuits 1, 2 and 3 that allow blood cells to pass, and measuring devices 26, 27 and 28 that measure the size and speed of blood cells that pass through the grooves of each circuit 1, 2 and 3. Devices 39, 40 for signal processing of measured values and displaying their frequency distribution
, A liquid supply path 31 for supplying a blood sample to each of the circuits 1, 2, 3; a device 4 for injecting a blood sample into the liquid supply path 31; and liquid transfer pumps 5, 6
And a pressure measuring device 13,1 for measuring the liquid pressure in the liquid supply passages 31,32.
4,15,16,17,18 and variable resistance device 19,20,21,22,23,24
, 18, the control unit 25 for controlling the liquid delivery pumps 5, 6 and the pressure measuring devices 13, ..., 18 and the flow path resistance varying devices 19, .., 24, and the recovery tank 33 for recovering blood after passing through the blood circuit. , 34,35,36,37,3
8.It is mainly composed of a device 7,8,9,10,11,12 in the middle of each flow path 31 for adding and mixing a fluorescent substance or a physiologically active substance. The measurement and the platelet aggregation measurement can be performed simultaneously. In this example, three types of blood circuits 1, 2 and 3 for measuring erythrocyte deformability, measuring leukocyte activity and measuring platelet aggregation are used separately. The present invention is not particularly limited to this, and in some cases, it is possible to use one substrate on which the above-mentioned three types or other blood circuits for measurement are formed. In this case, the liquid supply channels 31, 32
, And pressure measuring devices 13, ..., 18 and recovery tanks 33 ,.

In the device, the blood sample is supplied by the injection device 4 to the liquid supply path.
It is put in 31 and is sent to each circuit 1, 2 and 3 by riding on the liquid flow from the liquid feed pump 5. The pressure of the liquid in the liquid supply passage 31 is measured by the pressure measuring device 13, 15, 17 immediately before each circuit 1, 2, 3. The blood sample flows through the blood flow paths in the circuits 1, 2, and 3 along the groove entrance surface, and then is collected through the flow path resistance variable devices 19, 21, and 23. On the other hand, the liquid supply passage 3 is provided in the other passage in each circuit 1, 2, 3.
The physiological saline solution is sent through the 2 and the solution sending pump 6, and is made to flow along the groove outlet surface. The pressure of the liquid in the liquid supply passage 32 is also measured by the pressure measuring device 14, 16, 18 near the inlet of each circuit 1, 2, 3. Then, based on the measured values of the pressures at the circuit inlet side and the outlet side, the liquid feed pumps 5, 6 and the flow path resistance variable devices 19, 20, ...
24 is controlled by the controller 25, and a predetermined hydrostatic pressure difference is set between the groove entrance surface and the exit surface. As a result, a part of the blood sample passes through the groove 65 and is used for measurement. If necessary, a fluorescent substance and a physiologically active substance are added to the blood sample before reaching the circuits 1, 2, and 3. Blood cells that pass through the grooves of the circuits 1, 2, and 3 are projected onto the microscope enlarged projection plane of the measuring devices 26, 27, and 28, and their size and groove passage speed are measured. Fluorescence observations are made as needed. Further, the physiologically active substance is added to the physiological saline solution flowing through the groove outlet side as needed so that the diffusion of the physiologically active substance can occur from the groove outlet side to the inlet side. The spiral flow paths 47, 48, 49, 50, 51, 52 downstream of the respective addition devices 7, 8, ..., 12 are for ensuring mixing of the addition substances. The outputs of the pressure measuring devices 13, 14, ..., 18 on the inlet side and the outlet side of the circuits 1, 2, 3 are controlled by the control unit 25.
To the flow path resistance variable device 19,2 based on the measured value.
A control signal is output from the control unit 25 to 0, ..., 24.

As the control unit 25, it is preferable to adopt a generally known computer.

2 (a) and 2 (b) respectively show an example of the configuration of the blood circuit of the present invention. It is composed at least of a first substrate 60 having dents or grooves forming a flow path or a groove on the surface thereof, and a second substrate 61 having a flat surface bonded to the surface of the first substrate 60. The embodiment shown in FIG. 2 (a) is
The first substrate 60 is provided with two vertically elongated depressions 62, 63 which are parallel to each other, and each depression 6 is formed in a wall portion 64 which partitions the depressions 62, 63.
A groove 65 is provided in a direction substantially orthogonal to the flow path formed by 2, 63. The recesses 62, 63 are provided with an inlet 66 and an outlet 67 at both ends thereof, and a fluid is introduced from the inlet 66 to form the recesses 62.
Alternatively, it is provided so that 63 is discharged from the outflow port 67. The inflow port 66 and the outflow port 67 penetrate the first substrate 60 in the thickness direction, and the liquid supply path 31 of the blood measuring apparatus of FIG.
Or connected to 32. For example, as shown in FIG. 2 (d), a base plate 68 is joined or pressure-bonded under the first substrate 60, and drawing channels 69, 70 for connecting the liquid supply path 31 or 32 to the base plate 68. Is formed.

In addition, the embodiment shown in FIG.
One recess 62 for flowing a liquid containing a blood sample and two recesses 63 for flowing a fluid not containing a blood sample are provided in parallel with each other. A wall 64 that divides each of the depressions 62, 63 from each other.
As shown in the enlarged view of FIG. 2C, a large number of fine grooves 65 are provided in the recesses 62, 63 in a direction substantially orthogonal to the flow paths. In FIGS. 2A and 2B, reference numeral 50 indicates the flow of the blood sample in these blood circuits, and reference numeral 51.
Indicates the flow of physiological saline.

The first substrate 60 having such a shape is not particularly limited, but a substrate composed of a silicon single crystal that is easy to perform fine processing and is relatively inert to blood is preferably used. On the silicon single crystal plate, the above-mentioned depressions 62, 63, the groove 65, etc. are formed by etching, photolithography or the like used in semiconductor manufacturing.

On the first substrate 60, a second contact surface is a second surface.
The substrate 61 is bonded or pressure-bonded, and a flow path is formed in the space formed by the depressions 62 and 63 and the groove 65 at the bonding or pressure-bonding portion between the first substrate 60 and the second substrate 61. still,
It is desirable that this second substrate 61 be transparent so that optical observation of blood passing through the flow path can be easily performed.
For example, Pyrex glass or the like is used.

FIGS. 3 (a) to 3 (d) show an example of a method of measuring the size and passage speed of blood cells passing through the groove 65 of each blood circuit 1, 2, 3 on the microscope projection plane. This measurement method uses a photo sensor, and the wall part 64 on the microscope projection plane is used.
The change in the amount of light detected by photosensors 71, 72 such as a photocell or a photomultiplier having an opening of the photocathode that is attached or patterned at the portions corresponding to the inlet side and the outlet side of the groove 65 is used. It measures the size and passage speed of blood cells 73. In the embodiment of FIG. 3 (a), a fluorescent substance that is not incorporated into blood cells 73 is added to cause the liquid component 74 to emit light, and the blood cells 73 are observed as dark areas. Changes in the outputs of the photosensors 71 and 72 due to the passage of blood cells at that time are shown in FIG. In the embodiment shown in FIG. 3 (c), a fluorescent substance taken into the blood cells 73 is added to cause the blood cells 73 themselves to emit light, and the blood cells are observed as bright areas. Changes in the outputs of the photosensors 71 and 72 accompanying the passage of blood cells at that time are shown in FIG. 3 (d).
In either case, the height V of the peak of the current flowing through the photosensors 71 and 72 indicates the size of the blood cell, and the interval T between the peaks indicates the passage time. Distance between photo sensors 71 and 72 (distance)
Is constant, the blood cell passage speed can be obtained from the time T from peak to peak. Further, the blood cells 73 are deformed at the entrance of the groove 65. Because of that time, the peak width on the groove entrance side 75 becomes wider than that on the exit side 76. Therefore, the peak width difference DD ′ represents the time required for deformation. In this way, it becomes possible to separately obtain the deformation time of the blood cells 73 from the passage time.

An example of a groove of the blood circuit is shown in FIGS.
The following describes how these properties are separated and measured. Red blood cells, white blood cells, and platelets in the blood sample uniformly reach the entrance surface of each groove, but those that enter the groove and those that do not enter are divided at the groove entrance. Figure 4 (a)
An example of the groove 65 for measuring red blood cell deformability is shown in FIG. This groove 65
Is 4 μm deep and 10 μm wide before and after the narrow V-shaped groove 77A.
An inlet side groove 75A and an outlet side groove 76A, which are rectangular grooves of m, are provided. Therefore, the diameter is 8 μm and the thickness is 2 μm.
The disc-shaped red blood cells 73A are put in the inlet side groove 75A, but the spherical white blood cells 73B having a diameter of 6 to 10 μm cannot enter the groove 75A. Therefore, the white blood cells 73B will not flow away from the groove 75A and be blocked by the main flow of the blood sample orthogonal to the groove. The red blood cells 73A oriented by entering the rectangular inlet side groove 75A are then deformed and pass through the V-shaped groove 77A.
The passage speed may be considered to be proportional to the deformability of red blood cells 73A, and the former is an appropriate index for the latter. The size of the red blood cells can be accurately obtained from the projected image of the disk surface of the oriented red blood cells passing through the rectangular inlet side groove 75A and outlet side groove 76A. From these measurements obtained for individual red blood cells 73A, by taking the size and passage velocity of the red blood cells 73A on the X and Y axes, respectively, and by taking the frequency for them on the Z axis,
The functional characteristics of red blood cells and their distribution are displayed three-dimensionally. FIG. 7 is a three-dimensional display example of such a histogram of blood cell size and groove passage time. By making the size and velocity histograms of blood cells three-dimensional in this way, it is possible to make a more sophisticated determination as compared with the case where only the abnormality of the conventional form is detected two-dimensionally.
Under normal illumination and microscopy, red blood cells 73A and surrounding liquid 74
If the distinction between and is not clear, fluorescence observation is performed. At this time, labeling red blood cells 73A with a fluorescent substance is time-consuming and adversely affects red blood cell deformability. Therefore, as shown in FIG. 3 (a), the liquid component 74 is fluorescently colored,
The method of observing red blood cells 73A as a shadow or a dark area is used.

FIG. 4 (b) shows an example of a groove for measuring leukocyte activity. In this leukocyte activity measuring groove, a V-shaped groove 77B and trapezoidal inlet-side grooves 75B and outlet-side grooves before and after the V-shaped groove 77B are provided.
The dimension of 76B is large, for example, the depth of the groove is 10 μm. The white blood cells 73B enter the inlet side groove 75B, and then are deformed to V
Pass through shaped groove 77B. The method of measuring and displaying the size and passage speed of the white blood cells 73B is the same as that of the red blood cells 73A. Red blood cell 73A
Can freely pass through these grooves 75B, 77B, 76B, but does not impede the passage of white blood cells. The white blood cells 73B and the red blood cells 73A can be easily discriminated from each other, but for the purpose of further clarifying the distinction, observation by a fluorescence method is performed. White blood cells 73B are easily fluorescent-stained with dyes such as acridine orange and acridine red, and are observed as bright areas as shown in FIG. 3 (c). When leukocytes 73B are stimulated by physiologically active substances to start active cell motility, the groove passage speed is significantly reduced. This is because the external force and the intracellular force come to compete with each other. FIGS. 5 (a) and 5 (b) schematically show changes in the groove passage ability of the white blood cells 73B. The activity of white blood cells is quantified by this groove-passing ability and its change. If a hydrostatic pressure difference is not provided between the inlet side and the outlet side of the groove 65, and instead a concentration difference of the physiologically active substance is provided, the white blood cells 73B start migration and the groove 65B
Will actively pass through. The number of white blood cells passing through the groove 65 and the speed of passage under these conditions are also indicators of the white blood cell activity.

FIG. 4 (c) shows an example of a groove for measuring platelet aggregation ability. In this groove, an inlet-side groove 75C, a V-shaped groove 77C, and an outlet-side groove 76C, which are sized to match the platelet diameter of about 3 μm, are provided. Red blood cells 73A and white blood cells 73B cannot enter the inlet side groove 75C, and only platelets 73C pass through [Fig. 5 (c)]. When platelets 73C are aggregated by the physiologically active substance, it becomes difficult to pass through the groove as shown in FIG. 5 (d). The size of the platelet aggregate and the groove passage speed are good indicators of the platelet aggregation ability.

FIG. 6 shows a structure in which V-shaped grooves 77D and 77E are provided in multiple stages in the same groove. For each blood cell passing through the groove 65, the passing speed of the V-shaped grooves 77D and 75E at each stage is measured. The intermediate groove 78D and the outlet side groove 7D are obtained by taking as a reference the passing speed of the groove 77D of the first stage, which is obtained by the photosensors (not shown) arranged in the inlet side groove 75D and the intermediate groove 78D.
The change in the passing speed of each step can be obtained from the relationship with the passing speed of the V-shaped groove 77E of the next step obtained by the 6D photo sensor (not shown). This change reflects the effect of previous groove passage or deformation on the functional properties of blood cells.

In addition, in each of the above-mentioned examples, only one specific example is provided for each of the shapes, depths, and sizes of the grooves for red blood cells, white blood cells, and platelets, but the shape of these grooves is the shape of each blood cell. Various modifications are possible depending on the purpose of measurement. In addition, it is possible to form a plurality of types of blood circuits on separate substrates or in a single substrate.

(Effects of the invention) Since the present invention is configured as described above, (1) the size distribution of red blood cells and the frequency distribution of deformability, the number distribution of white blood cells can be rapidly increased without separating each blood cell fraction from a blood sample. The size and activity or the frequency distribution of the degree of response to stimuli, the size of platelets and the agglutination ability or the frequency distribution of agglutinates can be measured. (2) In addition, the conventional blood image shows each blood cell in blood. In contrast to the morphological blood image based on the measured value of the number and the distribution of its size, the device of the present invention provides the function of each blood cell, that is, the functional image of blood, and can be used for various diseases. The morphological picture of the blood changes only after the symptoms have progressed considerably, whereas the functional changes of the blood are likely to appear early. In addition, functional changes in blood are expected to strongly reflect differences in pathological conditions.

Therefore, the device of the present invention contributes to early diagnosis and precise diagnosis of various diseases.

[Brief description of drawings]

FIG. 1 is a diagram showing the configuration of the apparatus of the present invention. FIGS. 2 (a) and 2 (b) are views showing an example of the configuration of the first substrate constituting the blood circuit of the present invention, and FIG. 2 (c) is an A in FIGS. 2 (a) and 2 (b). FIG. 2 (d) is an enlarged view of a portion, and is a vertical sectional view showing an embodiment assembled as a blood circuit. FIGS. 3 (a) to 3 (d) are diagrams showing a method for obtaining the size and passage time of blood cells passing through the groove, and FIG. 3 (a) is a diagram showing blood cells passing through the groove in the blood circuit as the surrounding liquid. Fig. 3 (b) shows the change in the output of the photosensor at that time, and Fig. 3 (c) shows the blood cells passing through the groove in the blood circuit. FIG. 3 (d) is a diagram showing the change in the output of the photosensor at that time, which is observed by fluorescent emission. FIG. 4 (a) is a diagram showing an example of the shape of the red blood cell deformability measuring groove, FIG. 4 (b) is a diagram showing an example of the shape of the white blood cell activity measuring groove, and FIG. 4 (c) is a platelet. It is a figure which shows the groove | channel for aggregating ability measurement. 5 (a) and 5 (b) are diagrams schematically showing the relationship between leukocyte activity and groove passage ability, and FIGS. 5 (c) and 5 (d) are schematic diagrams showing the relationship between platelet aggregation and groove passage ability. FIG. FIG. 6 is a view showing a groove in which a plurality of narrow portions are provided in the same groove. FIG. 7 is a diagram showing a display example of a histogram of the size of each blood cell and groove passage time. 1 ... Blood circuit for measuring red blood cell deformability, 2 ... Blood circuit for measuring leukocyte activity, 3 ... Blood circuit for measuring platelet aggregation ability, 60 ... First substrate, 61 ... Second substrate, 62 , 63 …… Dimple, 64 …… Wall, 65 …… Groove, 75A, 75B, 75C, 75D …… Inlet side groove, 76A, 76B, 76C, 76D …… Outlet side groove, 77A, 77B, 77C, 77D, 77E …… V-shaped groove, 78E …… intermediate groove, 73 …… blood cells, 73A …… red blood cells, 73B …… white blood cells, 73C …… platelets, 74 …… liquid components.

Claims (12)

(57) [Claims] 1. A plurality of dents each having an inflow port at one end and an outflow port at the other end are arranged in parallel, and the inflow port and the outflow port are connected to a wall portion that defines the cavities. In a direction substantially orthogonal to a straight line, a first substrate having minute grooves communicating with each other and a second substrate having a flat surface bonded or pressure-bonded to the surface of the first substrate are formed. A blood circuit having a space formed by the recess and the groove as a flow path in a joint portion or a crimping portion of the first substrate and the second substrate. 2. A passage for each blood cell in a channel formed by this groove by adjusting any or all of the width, depth or shape of the groove to the size and shape of any of red blood cells, white blood cells or platelets. 2. The blood circuit according to claim 1, wherein blood cells that have different resistances or that can pass through a flow path formed by the groove are limited. 3. The blood circuit according to claim 1, wherein a plurality of kinds of grooves are arranged among three kinds of grooves adapted to red blood cells, white blood cells and platelets. 4. The blood circuit according to claim 1, wherein the groove has a plurality of narrow portions provided in multiple stages. 5. The blood circuit according to claim 1, wherein the second substrate is transparent. 6. The blood circuit according to claim 1, wherein the first substrate is made of silicon single crystal. 7. A blood sample injection device is connected to the inlet of one depression of the blood circuit according to claim 1, and physiological saline is injected into the inlet of the depression arranged in parallel with this depression. A blood measuring apparatus, wherein the apparatus is connected, and a pressure generating source having a control device is further provided in the vicinity of the inflow port and / or the outflow port of each depression. 8. An optical system for irradiating light to a flow path portion formed by a groove communicating between dents arranged in parallel, and light reflected from the flow path portion or light emitted from the flow path portion. The blood measuring apparatus according to claim 7, further comprising a measuring system for measuring the variable of. 9. The blood circuit or the blood measuring apparatus according to claim 1, wherein a hydrostatic pressure difference is provided between the parallel depressions of the blood circuit to connect the parallel depressions. The flow of blood is caused in the flow channel formed by, and the increase or decrease in the number of each formed component of blood in each depression thereafter or the clogging condition of the groove flow passage due to each formed component of blood is measured, and A blood measuring apparatus, characterized in that the flow characteristic or activity of each formed element of blood is obtained. 10. The blood circuit or the blood measuring apparatus according to claim 1, wherein a difference in concentration of the physiologically active substance is provided between the parallel depressions of the blood circuit so that the gap between the parallel depressions is increased. The movement of white blood cells is caused through the flow path formed by the groove connecting the two, and the increase or decrease in the number of each leukocyte fraction in each depression after that or the occlusion state of the groove flow path due to the white blood cell is measured. A blood measuring device characterized by determining the migration ability and the adhesion ability of a fraction. 11. The blood circuit or blood measuring device according to claim 1, wherein
11. A blood measurement method, which comprises performing the blood measurement according to 10 on a blood sample after being exposed to a physiologically active substance. 12. The blood measurement according to claim 9 or 10,
A method for measuring blood, characterized in that either a blood cell or a liquid component is fluorescently colored with a fluorescent substance.