JPH0679026B2 - Cell function measuring device and method - Google Patents

Description

TECHNICAL FIELD The present invention relates to an apparatus for applying shear stress to a cell suspension and continuously measuring a minute response of cells due to shear stress. More specifically, the present invention relates to, for example, a device that applies shear stress to a platelet suspension and continuously measures minute platelet aggregation due to shear stress, platelet shape change, platelet release reaction, and the like. It is widely used for the diagnosis and treatment of congenital diseases such as thromboasthenia and von Willebrand disease which show changes, and acquired diseases such as myocardial infarction, thrombosis and arteriosclerosis. Furthermore, the device according to the present invention is widely applied to the development of a drug for the purpose of antiplatelet action.

[Prior Art] Various devices have been developed for measuring the function of cells using a cell suspension. In particular, Born and O'Brien were the first to use a specific device for measuring platelet function. devised apparatus (Born, GVR: Nature 194, 927 (1962)
And O'Brein, JR: J.Clin.Path. 15 , 446 (1962)) and S
Device devised by alzman (Salzman, EW: J.Lab.C
lin.Med., 62 , 724 (1963)) and is widely used for clinical examination.

The former (hereinafter referred to as the “first conventional example”) is for investigating the aggregation function of platelets, and includes a portion including a glass cylindrical container with a diameter of about 5 mm for measuring the translucency of the platelet suspension, and the translucent light. It consists of a recorder that translates degrees into electrical signals and moves a pen to draw a cohesion curve. In this device, 0.2 to 0.3 ml of platelet suspension separated from blood by centrifugation is put in a glass cylindrical container, and agglutinating substances such as adenosine diphosphate, collagen, epinephrine, ristocetin, and thrombin are added to the container. When a platelet suspension is stirred by a magnetic stirrer placed in a cylindrical container in advance, platelets rapidly form aggregates, and along with this, the translucency of the platelet suspension increases and the change is continuously observed. It can be recorded as a flocculation curve.

The latter (hereinafter referred to as the second conventional example) is for investigating the adhesion function of platelets, and blood is passed through a tube filled with glass beads by utilizing the property that platelets easily adhere to the glass surface. The change in the number of platelets before and after is measured. This device consists of a tube with an inner diameter of 2 mm and a length of about 15 cm filled with glass beads having a diameter of 0.3 to 0.4 mm, and an empty tube used for collecting control blood. One end of these two tubes is connected with a blood collection needle and a three-way stopcock, and the other end of both tubes is connected with a 2.5 ml plastic syringe.
Further, a blood suction device is connected to the two syringes, and blood is collected at a constant speed.

Also, whereas, academic reports platelet suspension by adding 1 dyne / cm ² of about shear stress was continuously record changes in platelet (Klose, HJ, Rieger, H.and Schid-Schonbein, N .: Thr
ombosis Res. 7 , 261 (1975)), which discloses an experimental device. This experimental device (hereinafter referred to as the third
The conventional example) consists of a translucent concave liquid tank and a rotating body with a diameter of 5 cm that has a conical surface that makes an angle of 3 degrees with the bottom surface of the liquid tank, and the gap between the rotating body and the bottom surface of the liquid tank. Two optical fibers for introducing the irradiation light from the flow direction of the platelet suspension introduced in the above and for extracting the transmitted light are respectively provided on the outer side surface of the liquid tank. Further, a halogen lamp stabilizing light source is provided at one end of the light introduction optical fiber, and a photodetector to which a recorder is connected is provided at one end of the light introduction optical fiber.

Furthermore, the improved device of the above-mentioned third conventional example (hereinafter referred to as four conventional examples) is also reported (Rieger, H., Baier, H., Sch.
roder, H., Wurzinger, L., Schid-Schonbein, H.and Blasbe
rg, P.: Thrombosis Res. 17 , 589 (1980)).
The device of the fourth conventional example is a rotary double-cylindrical viscometer comprising an inner cylinder having a light source and an outer cylinder rotatably arranged around the inner cylinder in order to shorten the optical path length in the platelet suspension as much as possible. It has a shape like. Generally, the rotating cylinder double cylinder type viscometer has a drawback that a secondary flow due to Taylor vortex is apt to occur, but the device of the fourth conventional example avoids this drawback by rotating the outer cylinder. Is a feature.

Furthermore, a device similar to the third conventional example, in which a light source and a light receiver are installed in the vertical direction of the flow direction of the platelet suspension to shorten the optical path flow to about 1 mm (hereinafter referred to as the fifth device).
Reported (conventional example) (Frojmovic, MM: Biorheology 1
2, 193 is (1975)).

[Problems to be Solved by the Invention] A fundamental problem of the first conventional example in which an aggregation-inducing substance is added to a platelet suspension and the function of platelets is examined from the obtained aggregation curve is the stimulation of platelets. Is not physiological. For example, in a clinical test using a device according to this, it is general that the concentration of adenosine diphosphate in the platelet suspension is set to 2 μM to 10 μM, but such high concentration of adenosine 2 in blood in vivo is used. It is completely unknown whether phosphoric acid causes platelet aggregation. In addition, ristocetin, which is one of the aggregation-inducing substances, does not exist in the living body. Furthermore, although many researchers have reported that platelets are activated under the influence of the flow state of blood, in this first conventional example, the factor representing the flow state is the rotation of the stirrer. It can only be defined as an ambiguous thing that does not have the generality of numbers.
Furthermore, since the optical path length in the platelet suspension liquid is only about 5 mm, it is not possible to detect a change in transmitted light amount due to minute aggregation of platelets in response to a slight stimulus that occurs in the living body. Since the first conventional example has the above problems, it is widely used for clinical examinations,
It is said that there are many cases where the results measured in this conventional example do not match the clinical symptoms.

In addition, the second conventional example in which the adhesiveness of platelets is measured by utilizing the adsorptivity of the platelets to the glass surface sees the non-physiological phenomenon of adhesion to the glass surface, and thus the first conventional method described above. It has the same problem as the example. Also, the second
In the conventional example, the flow state of blood, which greatly affects the adhesion of platelets, is completely different from that in the living body because it flows through the gap between the glass beads. Therefore, the results obtained in this conventional example often do not reflect the original function of platelets.

On the other hand, a characteristic of the third, fourth and fifth conventional examples is that the flow state can be defined by a general factor called shear stress. For diseases such as myocardial infarction, thrombosis, arteriosclerosis, etc., the reaction of platelets under a flowing state plays an important role. , Has been studied and discussed in many scientific literatures for many years. Nevertheless, the third, fourth and fifth conventional examples having these excellent features have not been applied to clinical examination at all, unlike the first and second conventional examples. This is because the devices according to the third, fourth, and fifth conventional examples have the problems described below, so that the mechanism of platelet aggregation and reaction due to shear stress can be elucidated and correspondence with each disease can be achieved. Because it was not done.

First, the third conventional example is an apparatus for measuring the aggregation of platelets at a low shear stress of about 100 sec ^-1 , and uses a rotating body whose angle between the conical surface and the bottom surface of the liquid tank is 3 degrees. There is.
Therefore, the uniform shear stress that can be applied to a platelet suspension having a viscosity of about 1 cP using this device is limited to about 500 sec ^-1 . The flow state in which a shear stress higher than this value is applied becomes turbulent flow, and the shear stress applied to the platelet suspension becomes non-uniform. Further, in this conventional example, since the platelet suspension is irradiated with halogen lamp light having a wavelength distribution as a light source without using an optical filter or a spectroscope, the transmitted light detected depends on the protein in the blood. It is easily affected by light absorption and light scattering by lipids. Also, in a coarse dispersion system such as a platelet suspension, the relationship between particle size and light scattering changes intricately depending on the wavelength of light. Therefore, even if the platelet suspension is directly irradiated with light having a wavelength distribution, it is difficult to capture the minute aggregation of platelets due to shear stress.

The fourth conventional example is a double cylindrical tube in which the optical path length is shortened to improve the drawbacks of the third conventional example.
However, in this conventional example, since the optical path length is similar to or shorter than that of the first conventional example, coarse aggregation of platelets can be seen, but subtle changes in platelets due to physiologically slight stimulation are measured. You cannot do it.

Furthermore, in the fifth conventional example, since the optical path length is extremely short at 1 mm, it is completely unsuitable for the measurement of minute changes in platelets.

The present invention focuses on the above-mentioned problems of the prior art, adds shear stress to a cell suspension, and continuously measures a minute response of cells due to shear stress, in particular, shear on a platelet suspension. A device for continuously measuring minute platelet reactions due to shear stress by applying stress, which is applied to the diagnosis and treatment of diseases showing changes in platelet function and the development of drugs for the purpose of antiplatelet action. An object of the present invention is to provide a device and a method for measuring cell function using the device.

[Means for Solving the Problems] The above object can be achieved by the present invention described below.

That is, the present invention is (a) a cylindrical sample chamber consisting of a bottom plate and side faces, and the irradiation light introduction path and the transmitted light derivation path are set so that the optical path length in the sample introduced into the sample room is 1 cm or more. A sample chamber having a side surface, (b) A cell function measuring device having a convex conical surface rotator that forms an angle of 2 degrees or less with respect to a bottom plate of the sample chamber, and (c) a transparent side surface A cylindrical liquid tank having (d) a drive device that engages with the rotating body and rotationally drives the rotating body, (e) a control mechanism that controls the driving device, (f) the irradiation light introducing path A light source that engages and provides sufficient light to the cell suspension, (g) a light receiver that engages with the transmitted light lead-out path and detects light that has transmitted through the cell suspension or light generated from the cell suspension,
And (h) A cell function measuring device equipped with a recorder that engages with the light receiver and continuously records a signal obtained by the light receiver.

Furthermore, the present invention provides a method for measuring the function of cells using the above device.

Here, in the device of the present invention, the cell function to be measured is a reaction of cells directly or indirectly induced by shear stress, specifically, cell aggregation, release of a substance,
For example, there is a change in form.

Further, in the device of the present invention, the shear stress generated in the gap between the outer peripheral side surface of the rotating body and the inner side surface of the sample chamber, which applies the shear stress to the cell suspension, is the conical surface of the rotating body and the bottom surface of the sample chamber. It must be set so as not to exceed the value of the shear stress obtained in the gap of the above. However, if the inner diameter of the sample chamber is extremely increased, the amount of the cell suspension as the sample increases, which is not preferable. . Therefore, the inner diameter of the sample chamber is
The gap between the outer surface of the rotating body and the inner surface of the liquid tank is 0.2 mm.
It is preferable to set it to 2 mm. In addition, it is preferable to use a liquid tank having a translucent side surface inside the sample chamber from the viewpoint of easy handling. Further, it is preferable that the liquid tank is composed of a non-translucent bottom plate and a translucent side surface for accurate measurement. When the liquid tank is used, the distance between the outer surface of the rotating body and the inner surface of the liquid tank is preferably 0.2 mm to 2 mm. Furthermore, when a liquid tank is used, the material in contact with the irradiation light introduction path and the transmitted light derivation path on the side surface of the liquid tank has good transparency to the introduced light and the derived light, respectively. It must be retained. For example, when the target light is in the visible region, inexpensive acrylic polymers and plastics such as polystyrene are preferable, and when the target light is in the ultraviolet or infrared region, quartz glass is preferable.

Further, in the device of the present invention, the radius of the rotating body that applies shear stress to the cell suspension is preferably 3 cm or less,
0.7 cm to 2.0 cm is more preferable, and the angle formed by the conical surface of the rotating body and the plane perpendicular to the axis of the rotating shaft of the rotating body is 2
Or less, preferably 0.3 to 1.5 degrees.

Furthermore, in the apparatus of the present invention, it is preferable to use optical fibers for the irradiation light introduction path and the transmitted light extraction path that are provided so as to communicate with the sample chamber. Further, as the material of the optical fiber, inexpensive plastic is preferable when the transmitted light is in the visible region, and quartz glass is preferable when the light is in the ultraviolet or infrared region.

In the device of the present invention, the light source used by engaging with the irradiation light introduction path is selected depending on the cell function to be measured, but a light source such as a halogen lamp or a tungsten lamp to which an interference filter is attached, or , A laser, etc., which have excellent monochromaticity, are preferable,
It is also possible to introduce the light into the optical fiber of the irradiation light introduction path by using an optical fiber with more than one kind of light source.

Furthermore, in the device of the present invention, the optical receiver includes
Photovoltaic detectors such as photodiodes are preferable, but photomultiplier tubes are more preferable when measuring weak light such as fluorescence. When two or more types of light are irradiated, or when two or more types of fluorescence are measured, an optical filter such as an interference filter is used to transmit only the light of the target wavelength and the optical fiber to the receiver. It is preferable to guide each light.

The optical path length is the sample chamber (inside the liquid tank if a liquid tank is used).
Is the shortest distance from the optical inlet to the optical outlet of the cell suspension in, it is necessary to be 1 cm or more, preferably 1 cm
It is 4 cm or less and more preferably 2 cm or more and 3 cm or less. 1
This is because the ratio of transmitted light amount can be largely changed with respect to the change of the number of crystal plates, and the high sensitivity can be obtained by setting it to be cm or more. It is preferable that the positions of the light guide entrance and the light guide exit are set so as to avoid the center and to make the optical path length long.

In the apparatus of the present invention, in order to examine the change in the shape of platelets or the change in calcium concentration in the platelets by fluorescence measurement
It is also preferable to provide a scattered light lead-out path on the side surface of the sample chamber.

In the apparatus of the present invention, it is preferable to adjust the distance between the inner bottom surface of the sample chamber and the conical surface of the rotating body before use.
For example, at least the conical surface tip of the rotating body is made of a magnetic material, and an eddy current sensor is provided on the bottom wall side of the sample chamber,
The distance is detected by the sensor. Further, in order to measure the distance easily, a cylindrical magnetic body typified by stainless steel may be embedded and used at the tip of the conical surface rotator.

When measuring the shape change of platelets, 1000 sec ^-1
It is preferable to carry out shear stress below or to add a platelet aggregation inhibitor such as prostaglandin E ₁ .

Further, in the device of the present invention, the drive device for rotationally driving the rotating body is preferably a DC motor incorporating an encoder. By using the device of the present invention, 20
A uniform shear stress can be applied up to 0 dyne / cm ² .

Furthermore, the control mechanism engaged with this drive device not only rotates the drive device at a constant speed, but also controls the rotation of the drive device in accordance with a program created by the user, and 0 sec ^{-1 in} one measurement. The response of cells to shear stress can be investigated over a wide range of up to 20000 sec ^-1 .

In addition, when measuring platelet function, Indo-1AM (Grynkiewi) is a dye that easily penetrates the cell membrane, accumulates in the cell membrane, and binds to calcium ions in the cytoplasm to generate fluorescence.
cz, G., Poenie, M. and Tsien, RV: J.Biol.Chem., 260 , 34
40 (1986)) and the like, and by irradiating the platelet suspension with the excitation light of the dye in addition to the light for measuring aggregation from the light introduction path,
Simultaneously with the measurement of platelet aggregation due to shear stress, changes in the intracellular cytoplasmic calcium ion concentration, which is important in the regulation of platelet suspension function, can be continuously measured.

Furthermore, by using the device of the present invention, 1000 sec ^-1
Or a low shear stress region of 4000Sec ^-1, with a 8000Sec ^-1 or more high shear stress regions, by measuring the platelet aggregation,
Abnormalities of glycoproteins on the plasma membrane of platelets, which are closely related to platelet function, or abnormalities of plasma proteins bound to the glycoproteins can be easily examined.

That is, the device of the present invention applies shear stress, which is a physiologically slight stimulus, to cells, and has a feature that weak changes in cells to it can be measured with high sensitivity. In the meantime, it became possible to discover the phenomena described below.

Many studies have been conducted on the aggregation of platelets due to shear stress, but there are many unclear points about the mechanism.
Using the apparatus of the present invention, in a range of shear stress of 0 sec ^-1 to 20000Sec ^-1, the result of examining the relationship between shear stress and platelet aggregation, the aggregation of the platelets, on the platelet membrane glycoprotein with these Plasma proteins that bind to glycoproteins are deeply involved,
and shear stress sec ^-1 to 4000Sec ^-1, at the 80 dyne / cm ² or more shear stress, it was found that the mechanism is different. Up to now, 10 or more types of glycoproteins on the cell membrane of platelets (hereinafter abbreviated as GP) have been recognized, and it is said that they are closely related to functions such as adhesion and aggregation of platelets. Especially in these GPs, GPIb has the ability to adhere to platelets and GPIIb and G
It has been reported that the PIIIa complex (hereinafter abbreviated as GPIIb / IIIa) is closely related to the platelet aggregation ability. On the other hand, in plasma proteins, it has been reported that von Willebrand factor is closely related to platelet adhesion and fibrinogen is closely related to platelet aggregation.

First, as a result of diligent examination of glycoproteins on the platelet membrane and platelet aggregation due to shear stress, the following facts were revealed. As shown in Example 2, treatment of platelet suspension with monoclonal antibodies of GPIb, a shear stress of 1,000 sec ^-1 to 4000Sec ^-1, platelet aggregation is promoted, while in 8000Sec ^-1 or more shear stress, platelet Aggregation is completely suppressed, and further, when platelet suspension is treated with GPIIb / IIIa monoclonal antibody, at shear stress of 1000 sec ^{-1 to} 4000 sec ^-1 , platelet aggregation is completely suppressed, but 8000 sec ^-1 or more. The shear stress was found to be incomplete in suppressing platelet aggregation. Furthermore, as shown in Example 2, 1000 sec in Bernard-Soulier syndrome patients with GPIb deficiency.
^At shear stress of ^-1 to 4000 sec ^-1 , platelets normally aggregate, whereas at shear stress of 8000 sec ^-1 or more, platelets do not aggregate at all. On the other hand, in patients with GPIIb / IIIa deficient thromboasthenia, at shear stress of 1000 sec ^{-1 to} 4000 sec ^-1 , platelets do not aggregate at all, but at shear stress of 8000 sec ^-1 or more, platelets slightly aggregate. Also found.

Next, as a result of examining plasma proteins and platelet aggregation due to shear stress, as shown in Example 2, when the platelet suspension was treated with a monoclonal antibody against the GPIIb / IIIa binding site of von Willebrand factor, 1000 sec ^{-1 to} 4000 sec.
The ^-1 shear stress, whereas platelet aggregation is not suppressed at all, in 8000Sec ^-1 or more shear stress, platelet aggregation was found to be completely suppressed. Further, as shown in Example 2, in patients with von Willebrand's disease lacking von Willebrand factor, the shear stress of 1,000 sec ^-1 to 4000Sec ^-1, platelets are aggregated successfully, 8000Sec ^-1
It becomes equal to or larger than the shear stress, and that platelets are not completely aggregated, further, in patients with afibrinogenemia lacking fibrinogen, the shear stress of 1,000 sec ^-1 to 4000Sec ^-1, although aggregation of platelets does not occur at all On the other hand, it was discovered that at shear stress of 8000 sec ^-1 or higher, platelets aggregated without any abnormalities. As described above, as a result of these earnest studies using the device of the present invention, it was clarified that there are two mechanisms for platelet aggregation due to shear stress. Ie 1000
platelet aggregation induced by low shear stress region of the sec ^-1 to 4000Sec ^-1 is due to binding by interaction GPIIb / IIIa which is present in the fibrinogen and platelet membrane on a plasma protein, also, 8000Sec ^- It was discovered that the platelet aggregation induced in the high shear stress region of ¹ or more is due to the binding of the plasma protein von Willebrand factor by GPIb and GPIIb / IIIA. This important finding provides a simple and accurate method for measuring the two major functions of platelets, the aggregation ability and the adhesion ability. In other words, by measuring platelet aggregation induced by shear stress, 1000 sec ^{-1 to} 4000 s
In the low shear stress region of ec ^-1 , the conventionally known platelet aggregation ability can be measured, and in the high shear stress region of 8000 sec ^-1 or more, the so-called platelet adhesion ability can be measured.

Next, a desirable example of the device of the present invention will be described with reference to the drawings. In addition, the device of this example is designed especially for measuring the function of platelets in the platelet suspension.

FIG. 1 is a schematic view of an apparatus for measuring platelet function according to one embodiment of the present invention, and FIG. 2 is a sectional view of a portion of the apparatus shown in FIG. 1 that applies shear stress to a cell suspension. FIG. 3 is a plan view of a concave sample chamber.

Cylindrical liquid tank 1 into which a sample platelet suspension is introduced
As shown in FIG. 2, it is composed of a transparent and colorless side wall 35 of sufficiently polished acrylic resin and a non-translucent bottom plate 36 of acrylic resin having a sufficiently polished surface in contact with the platelet suspension. . Further, a cylindrical recess 38 is formed on the back surface of the bottom plate, and an eddy current displacement sensor 33 is installed.

Inside the liquid tank 1, there is installed a non-translucent, conical surface rotating body 2 of acrylic resin, which is a non-translucent, convex surface facing the bottom plate 36, and whose surface is sufficiently polished. At the tip of the cone, a stainless steel cylinder 31 with a well-polished surface
Is embedded. Further, a shaft hole 34 is provided in the upper portion of the rotating body 2, and the output shaft of the motor 4 with coreless encoder (in FIG. 1) is inserted into the shaft hole 34. Further, the motor 4 is joined to a precise height adjustment stage 9 fixed to the base plate 42 via a motor fixing plate 41, and a coarse movement dial 10 and a fine movement dial attached to the height adjustment stage 9 are attached. By rotating 11 the gap between the rotor 2 and the bottom plate 36 of the liquid tank 1 is adjusted, and the distance of the gap is determined by the eddy current displacement sensor 33 of the rotor 2. The position of the stainless steel cylindrical body 31 embedded in the tip of the cone is sensed, and the sensor
It is displayed on the rotating body height indicator 27 on the base plate 42 connected by 33 and the cord 39.

The bottom surface of the stainless steel cylindrical body 31 is not a conical surface for facilitating detection by the sensor 33, and may be a flat surface partially or entirely. Further, the motor 4 is a motor drive control device 5 connected to the motor 4 by a cord 17.
According to the program created in advance,
Driven.

On the other hand, as shown in FIG. 2, the liquid tank 1 is held in a concave portion of a sample chamber 3 made of stainless steel, and further fixed by a liquid tank fixing pin 30 provided above the concave portion of the sample chamber 3. To be done. In addition, a cylindrical tubular irradiation light introducing path 28 is formed so as to penetrate slightly above the concave portion of the sample chamber 3, and a cylindrical tubular transmission light guide is also formed on an extension line of the light introducing path 28. An exit path 29 is formed so as to penetrate therethrough, and a fan-shaped slit-like scattered light exit path 40 (in FIG. 3) is formed between the light entrance path 28 and the light exit path 29.
Further, in the light introducing path 28 and the light guiding path 29, a single core light guiding optical fiber 14 in which a core to which a plug of an FC connector is connected is formed of quartz, and the same light guiding optical fiber is used.
15 and 15 are respectively inserted, and these two optical fibers are fixed by two optical fiber fixing pedestals 32 and 26 to which an FC type connector adapter (not shown) is attached. Further, also in the slit-like scattered light derivation path 40 described above,
The core 16 to which the plug of the FC connector is connected is inserted with the scattered light derivation fiber 16 made of quartz.
16 is fixed by an optical fiber guide 25 to which an FC connector connector is mounted, and a stand 24 which supports the optical fiber guide 25 and is fixed to a base plate 42.

The back side (in FIG. 1) of the sample chamber 3 is fixed to a pedestal 43, and the pedestal 43 is fixed to the upper disk of the fine movement rotary stage 12 fixed to the base plate 42. The upper disk of the fine movement rotary stage 12 is the fine movement stage.
The fine movement dial 13 attached to the lower disk of 12 rotates to rotate the sample chamber 3, and at the same time, the sample chamber 3 rotates. As a result, the scattered light is guided from an arbitrary angle within the slit width of the scattered light lead-out path 40. The scattered light is led out through the outgoing optical fiber 16.

An optical fiber fixing pedestal 32 having an FC connector adapter for fixing the optical fiber 14 for introducing light is a multi-core having an FC connector plug in which 12 ferrous quartz optical fibers are filled in one ferrule. An optical fiber 44 is connected, and the optical fiber 44 is divided into two multi-core fibers 18 and 19 each having six fibers. The ends of these multi-core optical fibers 18 and 19 are also filled in one ferrule, and are connected to the FC connector adapter of the light source unit 6 by the CF connector plug. In the light source unit 6 (in FIG. 4), the end face output is 1.6 mW when connecting a quartz optical fiber with a wavelength of 632.8 nm and a core diameter of 125 μ and a length of 2 m, which is used for examining the aggregation of platelets.
And a He-Ne gas laser 45, and an excitation light source of a mercury-xenon lamp 46 attached with an ultraviolet light transmission filter 47 with an output of 100 W used for the purpose of measuring the change of calcium ion concentration inside the platelets.

On the other hand, one end of an optical fiber 20 having plugs of an FC type connector at both ends is connected to an optical fiber fixing pedestal 26 having an FC type connector adapter for fixing the optical fiber 15 for optical derivation. The plug on the other end of the
Connected to the FC connector adapter. Further, the scattered-fiber derivation optical fiber 16 fixed by the optical fiber guide 25 and the stand 24 is attached to the optical fiber guide 25.
FC-type connector adapter for one ferrule
A multi-core optical fiber 23 having an FC connector filled with twelve quartz optical fibers at its tip is connected. The optical fiber 23
Is divided into two multi-core optical fibers 21 and 22, 6 each,
The tip of each multi-core optical fiber is also filled in one ferrule, and the FC connector plug is used to
Each is connected to the FC connector adapter.

In the light receiver 7 (in FIG. 5), one photodiode 48, two photomultiplier tubes 49a and 49b, these photodetectors and an electronic circuit 50 for data analysis are incorporated. There is. An interference filter 51 with a center wavelength of 633 nm is attached to the FC connector adapter to which the optical fiber 20 is connected.
Further, the photodiode is connected. Further, the FC connectors to which the optical fibers 21 and 22 are connected are respectively connected to a photomultiplier tube, and on the front surface of the photocathode of one of the photomultiplier tubes, an interference filter 52a having a center wavelength of 410 nm, and the other The photocathode of the photomultiplier tube has a central wavelength of 480 nm.
Interference filters 52b are attached respectively.
Also, a recorder 8 having two pens is connected to the light receiver 7, and changes in the turbidity of the platelet suspension due to platelet aggregation and changes in the calcium ion concentration inside the platelets are recorded in the recorder 8. It is recorded continuously.

In this example, the liquid tank 1 in which the eddy current sensor 33 is incorporated
Therefore, it is possible to use the sample chamber 3 which has the liquid tank insertion portion 37 at the center of the sample chamber 3 but does not have the liquid tank insertion portion 37.

Next, quantification of platelet aggregation due to shear stress will be described. In order to quantitatively evaluate the platelet aggregation state, it is necessary to generalize the obtained aggregation curve and devise some index. The first widely used in clinical practice now
In the conventional platelet aggregation measuring apparatus, the platelet aggregation rate is defined as follows.

Aggregation rate (%) = I _L −I _PRP / (I _PPP −I _PRP ) × 100 (1) where Il is the amount of transmitted light during aggregation, I _PPP is the amount of transmitted light of PPP, I
_PRP represents the amount of transmitted light of PRP before aggregation. This indicator is
It greatly changes depending on the optical path length in the cell, the turbidity of plasma, and the number of platelets before aggregation. Further, it is not suitable for generalization because it is not proportional to the rate of change of the number of non-aggregated platelets. In the device of the present invention, which has a feature that can sometimes detect platelet aggregation with high sensitivity,
These problems become apparent because the difference in the amount of transmitted light between PPP and PRP becomes extremely large. As a result of diligent examination, in this device, the relationship between the number of platelets and the amount of transmitted light complies with Lambert Beer's law, and further, the change in the amount of transmitted light due to aggregation is caused mainly by the change in the number of non-aggregated platelets I found a thing. Then, we devised an index defined by the following formula.

Using this index, it is possible to express the aggregation rate of platelets as the rate of change of the number of non-aggregated platelets, and moreover, regardless of the optical path length in the cell, the turbidity of plasma, the number of platelets before aggregation, Aggregation can be expressed quantitatively. Further, in the present invention, a convex conical surface rotating body that makes an angle of 2 degrees or less is used with respect to the bottom plate of the sample chamber, but if it exceeds 2 degrees, the flow state of the sample cannot maintain a laminar flow. This is because it becomes impossible to apply uniform shear stress to the resultant sample.

[Operation] In the apparatus of the present invention configured as described above, by a suitable rotary drive device and a control mechanism engaged with the drive device,
A rotating body having a conical surface is rotated in a cylindrical liquid tank, and shear stress is applied to the cell suspension introduced into the gap between the rotating body and the bottom surface of the liquid tank while maintaining a laminar flow state. During this time, the cell suspension is irradiated with appropriate light from the light introduction path communicating with the sample chamber, and the minute response of the cells to shear stress such as release reaction is transferred from the light exit path communicating with the sample chamber to the light receiver. , Is guided as desired light through an optical filter and continuously recorded as a change in the intensity of this light.

[Example] Example 1 In the above-mentioned apparatus, using the following outline dimensions of the main part,
The measurement was performed.

The radius of the rotating body 2 is 1.5 cm, and the radius of the stainless steel cylinder is
2.5 mm, the angle formed by the conical surface of the rotating body 2 and the plane perpendicular to the axis of the rotating shaft of the rotating body 2 is 1.0 degree.
The inner diameter of the liquid tank 1 is 3.1 cm, the outer diameter of the liquid tank 1 is 3.4 cm, and the depth of the recess for introducing the platelet suspension in the liquid tank 1 is 1.3 cm. The thickness of the bottom plate 36 of the liquid tank 1 is 2 mm, and the thickness of the bottom plate 36 where the eddy current displacement sensor 33 is inserted is 0.5 mm.
The inner diameter of the concave liquid tank holding portion of the sample chamber 3 is 3.41 cm, and the outer diameter of the sample chamber is 5.5 cm. In the sample chamber 3, the positions of the light introducing path 28 and the light introducing path 29 are the center line (in FIG. 3) of the circle of the concave liquid tank holding part and the center of each of the light introducing path 28 and the light guiding path 29. The connecting lines are set to be parallel and the distance between these two straight lines is set to 0.8 cm. The width of the slit of the scattered light lead-out path 40 is determined by the angle formed by the line connecting both ends of the lead-out path 40 and the center of the concave liquid tank holding portion of the sample chamber 3.
It is set to 120 degrees. Optical fiber 14, 1
5, 16 and 20 core diameter is 1mm, clad diameter is 1.035m
m. The optical fibers 18, 19, 21, 22, 23 and 44 have a core diameter of 0.200 mm and a cladding diameter of 0.230 mm. The height of the height adjustment stage 9 is 15.5 cm, and the diameter of the disc of the fine movement rotation stage is 9 cm. The vertical length of the base plate 42 is 20 cm, and the horizontal length thereof is 25 cm.

Next, the operation of the above-described apparatus will be described while explaining the process of actually measuring the platelet function in the platelet suspension.

From the elbow vein of a healthy adult, use a siliconized 20-gauge needle, and use 3.8% sodium citrate solution / whole blood = 1
Collect in a plastic container at a ratio of / 9. This blood
Platelet-rich plasma (below
After obtaining PRP), the remaining red blood cell suspension is centrifuged at 1500 G for 10 minutes to produce platelet poor plasma (hereinafter abbreviated as PPP). Create a PRP with the numbers adjusted appropriately. P that adjusted this platelet count
Take 0.3 ml of RP, put it near the center of the bottom plate 36 of the liquid tank 1, and then move the coarse adjustment dial 10 and the fine adjustment dial of the height adjustment stage 9.
By rotating the rotor 11, the rotor 2 is lowered onto the bottom plate 36, and the eddy current displacement sensor 33 that senses the distance between the center of the bottom plate 36 and the stainless steel cylinder 31 embedded in the conical tip of the rotor. The value output to the rotating body height indicator 27
Set to 40 μm. Then, with shear stress in the range of ⁰ sec ^{-1 to} 20000 sec ^-1 , the transmitted light having a wavelength of 633 nm that is added to the PRP for 5 minutes and is incident from the light introducing optical fiber 14 is guided from the light guiding optical fiber 15. Is detected by the light receiver 7,
When platelets aggregate, the amount of transmitted light increases, and the state of the change is drawn on the recorder 8. In Figure 6, the healthy adult five shear stress 1800sec ^-1, 3600sec ^-1, 7200sec ^-1 and 108
The respective platelet aggregation curves at 00 sec ^-1 are shown. In general the figure, platelet aggregation, than shear stress of 3600 sec ^-1 and 7200sec ^-1, more of a shear stress of 1800 sec ^-1 and 10800Sec ^-1 It can be seen that a marked.

Example 2 Using the same apparatus as in Example 1, an ordinary platelet suspension (sample 1), a platelet suspension obtained by treating a glycoprotein GPIb, which is closely related to platelet adhesion on the platelet cell membrane, with a monoclonal antibody (sample 2), the complex of glycoprotein GPIIb / IIIa (hereinafter GPIIb / IIIa), which is closely related to the platelet aggregation ability on the platelet cell membrane.
IIIa) platelet suspension treated with monoclonal antibody (Sample 3), platelet suspension treated with monoclonal antibody to GPIIb / IIIa binding site of von Willebrand factor involved in platelet adhesion (Sample 4), GPIb Suspension collected from a patient with Bernard-Soulier syndrome with deficiency of glucose (Sample 5), platelet suspension collected from a patient with thromboasthenia with GPIIb / IIIa deficiency (Sample 6), von lacking von Willebrand factor For each of the platelet suspension collected from a patient with Wilbrand's disease (Sample 7) and the platelet suspension collected from a patient with fibrinogen-deficient afibrinogenemia (Sample 8),
Platelet aggregation at shear stress of 18 dyne / cm ² and 108 dyne / cm ² was measured in the same manner as in Example 1.

As shown in FIG. 8, as compared with FIG. 7 (Sample 1), Sample 2 (platelet suspension in which GPIb was treated with a monoclonal antibody) promoted platelet aggregation at a low shear stress of 1800 sec ⁻¹ , while At high shear stress of 10800 sec ^-1 , platelet aggregation is completely suppressed. As also shown in Figure 9 for a sample 3 (platelet suspension to the GPIIb / IIIa was treated with monoclonal antibody), in the low shear stress of 1800 sec ^-1 platelet aggregation is completely suppressed, the high shear stress 10800Sec ^-1 , Slight platelet aggregation occurs. Sample 4 As shown in FIG. 10 in (platelet suspension was treated with monoclonal antibody to the binding site of the GPIIb / IIIa of von Willebrand Factor), at the low shear stress of 1800 sec ^-1, platelet aggregation is not inhibited, 10800Sec ^{- At} high shear stress of ¹ , platelet aggregation is almost completely suppressed. Also, as shown in FIG.
In (GPIb platelet suspension were collected from a patient of Bernard Sue Rie syndrome deficient), the low shear stress of 1800 sec ^-1, whereas platelets aggregate normally, in a high shear stress 10800Sec ^-1, platelets Does not aggregate at all. Sample 6 (GP
In a platelet suspension collected from a patient with thromboasthenia deficient in IIb / IIIa), platelets do not aggregate at a low shear stress of 1800 sec ^-1 , as shown in FIG.
At high shear stress of ⁰ sec ^-1 , platelets are slightly aggregated.
Furthermore, in sample 7 (platelet suspension collected from a patient with von Willebrand disease lacking von Willebrand factor), platelets normally aggregate at a low shear stress of 1800 sec ^-1 , as shown in FIG. ^At high shear stress of ^-1 , platelets do not aggregate at all. Finally sample 8
In (platelet suspension collected from a fibrinogen-deficient afibrinogen-deficient patient), as shown in FIG. 14, at a low shear stress of 1800 sec ^-1 , no platelet aggregation occurs, whereas at 10800 sec. ^At high shear stress of ^-1 , platelets aggregate without abnormalities.

As can be seen from the above results, by measuring platelet aggregation in the low shear stress region and high shear stress region, abnormalities of GPIb, GPIIb / IIIa, fibrinogen, and von Willebrand factor, which are closely related to the function of platelets, were detected. It can be seen that is easy to find.

Example 3 In the apparatus of Example 1, by connecting a measurement system composed of a photon counter, a GP-IB interface and a microcomputer to the two photomultiplier tubes, the calcium ion concentration in platelets was changed by shear stress. Was measured.

First, a calcium ion buffer solution is prepared as follows. 60 mM ethylene glycol bis (β-aminoethyl ether) -N, N, N ', N'-tetraacetic acid (hereinafter EGT
A) and 12 mM of 3- (N-morpholino) propanesulfonic acid (hereinafter abbreviated as MOPS) to a solution of 120 mM
Potassium chloride (hereinafter abbreviated as KCl), 12 mM MOPS and 1
Add to a solution consisting of ₂ mM calcium chloride (hereinafter abbreviated as CaCl ₂ ) and confirm that 100 mM KCl, 10
mM MOPS and 10 mM EGTA calcium salt (hereinafter K ₂ CaEGTA
Abbreviated) to prepare a solution of pH 7.20. Also, 120m
A solution consisting of M KCl and 12 mM MOPS, 60 mM EGTA and 12 mM
1/5 volume of MOPS solution, 100mM KCl, 10m
A solution of pH 7.20 consisting of M MOPS and 10 mM EGTA potassium salt (hereinafter abbreviated as K ₂ H ₂ EGTA) is prepared. Using these two adjusted solutions, 0nM, 37.3nM, 100nM, 226n
Make a buffer solution of calcium ions at concentrations of M, 604 nM, 1 mM.

Next, to the buffer solution having these calcium ion concentrations, an aqueous solution of the dye Indo-1, which emits specific fluorescence by binding with calcium ions, was added to the buffer solution at 6 /
A sample is prepared by adding 1000 volumes. Take 0.3 ml of each of these samples, and in the same manner as in Example 1, liquid tank 1
And the rotating body 2 is lowered. Subsequently, the rotating body is driven at an appropriate number of rotations, and by the above-mentioned measurement system,
Two-wavelength fluorescence of Indo-1 generated by the ultraviolet light incident from the optical fiber 14 for introducing light, that is, fluorescence of 410 nm wavelength from Indo-1 combined with calcium ions, and Indo-1.
-1 Measure the fluorescence of a single substance at a wavelength of 480 nm. Regarding the relationship between calcium ion concentration and these fluorescence intensities,
The following formula has been reported in Grynkiewicz et al.

Ca ²⁺ = Kd (▲ S ⁴⁸⁰ _indo-1 ▼ / ▲ S ⁴⁸⁰ _indo-1Ca ▼
(R−Rmin) / (Rmax−R) where Ca ²⁺ is the calcium ion concentration, Kd is the calcium ion dissociation constant of Indo-1 bound with calcium ions, and ▲ S ⁴⁸⁰ _indo-1 ▼ is the calcium ion concentration. Fluorescence intensity at a wavelength of 480 nm when set to 0, ▲ S ⁴⁸⁰ _indo-1Ca ▼ when calcium ions are bound to all the calcium ions in Indo-1 (calcium ion concentration 1 m
(Corresponding to the case of using a buffer solution of M), the fluorescence intensity at a wavelength of 480 nm, Rmin is a wavelength of 41 when the calcium ion concentration is 0.
Fluorescence intensity ratio of 0 nm and 480 nm, Rmax is a fluorescence intensity ratio of wavelength 410 nm and 480 nm when calcium ion is in a large excess, and R is 410 nm and 480 when fluorescence of each calcium ion buffer solution is measured.
It is a fluorescence intensity ratio of nm. In this relational expression, the fluorescence ratio R of each sample
By substituting for Kd, a calibration curve is created.

Subsequently, a sample of platelet suspension is prepared as follows. First, in the same manner as in Example 1, PRP is prepared by collecting from healthy adults. Acid citrate glucose solution (Acid Citrate Dextrose) was added to this PRP so that the final concentration was 20%, and centrifugation was performed at 800 G for 7 minutes to precipitate platelets. After removing this supernatant, HEPES-Tyrode buffer (4 mM N-2-hydroxyethylpiperazine-
A solution consisting of N'-ethylsulfate, 137 mM sodium chloride, 2.7 mM potassium chloride, 3.3 mM sodium phosphate, 1 mM magnesium chloride, 5.6 mM glucose, 0.35 mg / ml bovine serum albumin). , Platelet count is 10 × 10
Adjust so that it becomes ⁴ cells / μl. Further, Indo-1A obtained by converting the carboxyl group of Indo-1 into acetoxymethyl ester to impart cell membrane permeability to this platelet suspension.
M 1 mM dimethylsulfoxide solution was added and Indo-1A
Adjust so that the final concentration of M is 5 μM, and leave at 37 ° C. for 30 minutes. In addition, acid citrate glucose solution was added to this platelet suspension to a final concentration of 20%, and the solution was added again.
Centrifuge at 800 G for 7 minutes to pellet the platelets. Then, after removing the supernatant, the platelets are suspended in a HEPES-Tyrode buffer solution and adjusted so that the platelet count is 10 × 10 ⁴ cells / μl, which is used as a sample for measuring calcium concentration in platelets. Fibrinogen and von Willebrand factor were added to the final concentration of 20μ
It is added so as to be g / ml, 0.3 ml as a sample is put in the liquid tank 1, and the fluorescence intensity is measured by the same procedure as in the case of preparing the calibration curve described above. Figure 15 shows shear stress 1800 sec ^-1 and 1
In the case of 0800sec ^-1 , the fluorescence intensity ratio R of wavelength 410nm and 480nm
The changes were compared. As can be seen from the figure, low shear stress does not change the calcium ion concentration in platelets, whereas high shear stress increases the calcium ion concentration.

Example 4 In the apparatus of Example 1, by newly connecting a measuring system composed of an optical power meter, a GP-IB interface and a microcomputer to a photodiode for measuring a change in the amount of transmitted light, a newly devised platelet aggregation was obtained. We examined the effectiveness of the indicators.

First, a vinyl chloride tube having an outer diameter of 1.35 mm was inserted into the carotid artery of a rabbit weighing about 3 kg, and blood was collected in a plastic container at a ratio of 3.8% sodium citrate aqueous solution / whole blood = 1/9. Then, in the same manner as in Example 1, PPP and P
Platelet count 42 by making RP and diluting PRP with PPP
Platelet suspensions of × 10 ⁴ cells / μl, 21 × 10 ⁴ cells / μl, and 14 × 10 ⁴ cells / μl were designated as Sample 1, Sample 2, and Sample 3, respectively. First, shear stress of 300 sec ^-1 was applied to these samples for 15 seconds so as not to cause platelet aggregation, and
After measuring the amount of transmitted light of PRP, the shear stress was increased to 1800 sec ⁻¹ for 15 seconds to obtain platelet aggregation data. further
The amount of transmitted light of the PPP is measured, and the aggregation curve obtained by the conventional equation (1) is shown by the aggregation rate (Fig. 16) and the aggregation curve newly obtained by the equation (2) is shown by the aggregation rate (Fig. 17). ) Was drawn using a microcomputer, and the maximum aggregation rate was calculated for each. After the measurement, each platelet was immediately fixed with formalin, the number of non-aggregated platelets was measured by an automatic platelet counter, and the change rate of the number of non-aggregated platelets was calculated from the value with the number of platelets before aggregation. As a result, sample A is 65%,
Sample B was 76% and sample C was 80%, which were in very good agreement with the agglomeration rate at the measurement end point of each sample obtained from FIG.
On the other hand, as shown in FIG. 16, when expressed by the conventional aggregation rate, the aggregation rate at the measurement end point obtained from the aggregation curve is
Well below that value obtained from an automatic platelet counter. Further, in terms of the conventional aggregation rate, the illusion that the aggregation of platelets becomes more active is given to the measurer as the number of platelets before aggregation decreases.

From the above results, it can be seen that the aggregation rate calculated by the equation (2) is very effective as an index that accurately and quantitatively indicates the aggregation state of platelets.

[Effect of the Invention] The device of the present invention applies a wide range of shear stress to a cell suspension.
It can be applied uniformly in a laminar flow state, and the minute response of cells accompanying it can be measured with about 1000 times the sensitivity of the conventional one. Further, by using the device of the present invention, it is possible to easily investigate the abnormality of specific glycoproteins and plasma proteins on the platelet membrane, which are closely related to platelet function. Further, the use of the device of the present invention is extremely useful in the diagnosis and treatment of diseases showing changes in platelet function, and in the development of drugs for the purpose of antiplatelet action.

[Brief description of drawings]

1 is a schematic view of an apparatus for measuring platelet function according to one embodiment of the present invention, FIG. 2 is a sectional view of a portion of the apparatus shown in FIG. 1 that applies shear stress to a cell suspension, and FIG. The figure is a plan view of a concave sample chamber. FIG. 4 is a plan view of the inside of the light source section, and FIG. 5 is a plan view of the inside of the light receiver. FIG. 6 is a platelet aggregation curve obtained in Example 1 using the apparatus according to the present invention shown in FIG. 7 to 14 are platelet aggregation curves obtained by Example 2 using the apparatus according to the present invention shown in FIG. 1, and FIG. 7 shows a normal platelet suspension,
Figure 8 shows platelet suspension treated with GPIb monoclonal antibody, Figure 9 shows platelet suspension treated with GPIIb / IIIa monoclonal antibody, and Figure 10 shows von Willebrand factor GP.
Platelet suspension treated with a monoclonal antibody against the binding site of IIb / IIIa, Fig. 11 is a platelet suspension collected from a patient with Bernard-Soulier syndrome, Fig. 12 is a platelet suspension collected from a patient with thromboasthenia, Figure 13: Platelet suspension from a patient with von Willebrand disease, 14th
The figure shows measurements of platelet suspensions collected from patients with afibrinogenemia. FIG. 15 shows the results of measuring changes in calcium ion concentration in platelets obtained in Example 3 using the apparatus according to the present invention shown in FIG. FIGS. 16 and 17 are platelet aggregation curves obtained by Example 4 using the apparatus according to the present invention shown in FIG. 1, and FIG. 16 is an aggregation curve represented by a conventional aggregation rate. , And FIG. 17 is the aggregation curve expressed by the newly devised aggregation rate. 1: Liquid tank 2: Rotating body 3: Sample chamber 4: Motor with coreless encoder 5: Motor drive control device 6: Light source unit 7: Light receiver 8: Recorder 9: Height adjustment stage 10: Coarse movement dial 11: Fine adjustment dial 12: Fine adjustment rotary stage 13: Fine adjustment dial 14: Optical fiber for introducing light 15: Optical fiber for extracting light 16: Optical fiber for extracting scattered light 17: Motor code 18: Multicore optical fiber 19: Multicore optical fiber 20: Single-core optical fiber 21: Multi-core optical fiber 22: Multi-core optical fiber 23: Multi-core optical fiber 24: Stand 25: Optical fiber guide 26: Optical fiber fixed base 27: Rotating body height indicator 28: Light introduction path 29: Light guide path 30: Liquid tank fixing pin 31: Stainless steel cylindrical body 32: Optical fiber fixed pedestal 33: Eddy current displacement sensor 34: Shaft hole 35: Liquid tank side wall 36: Liquid tank bottom plate 37: Liquid tank insertion part 38: Sensor insertion part 39: Sensor code 40: Scattered light guide path 41: Motor fixing plate 42: Base plate 43: Pedestal 44: Multi-core optical fiber IVA 45: He-Ne gas laser 46: Mercury-xenon lamp 47: Ultraviolet light transmission filter 48: Photodiode 49a: Photomultiplier tube 49b: Photomultiplier tube 50: Electronic circuit 51: Interference filter 52a: Interference filter 52b: Interference filter

Claims (7)

[Claims] (A) A cylindrical sample chamber composed of a bottom plate and side faces, and an optical path length in the sample introduced into the sample chamber is 1c.
Cell function measurement with a sample chamber having irradiation light introduction path and transmitted light extraction path on the side so that the distance becomes m or more, and (b) a convex conical surface rotator that makes an angle of 2 degrees or less with respect to the bottom plate of the sample chamber. apparatus. 2. A cylindrical sample chamber consisting of a bottom plate and side faces, and the optical path length in the sample introduced into the sample chamber is 1c.
A sample chamber having an irradiation light introduction path and a transmitted light extraction path on its side surface so that the distance is at least m, (b) a convex conical surface rotating body that forms an angle of 2 degrees or less with respect to the bottom plate of the sample chamber, and A cell function measuring device having a cylindrical liquid tank having an optical side surface. 3. The cell function measuring device according to claim 1 or 2, further comprising: (d) a drive device that engages with the rotating body and rotationally drives the rotating body; and (e) control for controlling the driving device. Mechanism, (f) a light source that engages with the irradiation light introduction path and provides sufficient light to the cell suspension, and (g) receives light that engages with the transmitted light derivation path and detects light transmitted through the cell suspension. And (h) a cell function measuring device, comprising: (h) a recorder that engages with the light receiver and continuously records a signal obtained by the light receiver. 4. The cell function measuring device according to claim 1, 2 or 3, wherein a scattered light lead-out path is provided on a side surface of the sample chamber. 5. The platelet aggregation ability and adhesion ability are measured by using the cell function measuring apparatus according to claim 1, 2, 3 or 4 to measure the aggregation induced by applying shear stress to the platelets. How to measure. 6. Using the cell function measuring apparatus according to claim 1, 2, 3 or 4, wherein, in the shear stress of 1,000 sec ^-1 to 4000Sec ^-1, by measuring the aggregation of platelets, measured platelet aggregation how to. 7. Using cell function measuring apparatus according to claim 1, 2, 3 or 4, wherein the low shear stress region of of 1,000 sec ^-1 to 4000sec ^-1, 8000sec ^-1 or more in the high shear region, platelets A method for measuring platelet aggregation ability and adhesion ability by measuring the aggregation of the.