Platelet evaluation method, platelet measurement method, and platelet evaluation apparatus

Abstract

To provide a novel platelet evaluation index and platelet evaluation method that can directly and easily evaluate the activation ability of platelets in a sample. [Solution] The following steps: A platelet aggregation inhibition step involves adding a platelet aggregation inhibitor to a platelet-containing sample. A platelet activation step is performed by adding a platelet activating substance to the aforementioned sample. A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. An evaluation step in which the platelet activation ability in the sample is evaluated using the number of granular platelets A and / or the number of degranular platelets B as indicators. A platelet evaluation method, including the following.

Description

Technical Field

[0001] The present invention relates to a platelet evaluation method, a platelet measurement method, and a platelet evaluation device. More specifically, it relates to a method for evaluating the activation ability of platelets in a sample, as well as a platelet measurement method and a platelet evaluation device therefor.

Background Art

[0002] Platelets are blood cells that mainly play a role in primary hemostasis by forming a hemostatic plug when the blood vessel wall is damaged. Usually, they are disc-shaped anucleate small cells with a diameter of about 2 to 4 μm. However, there are also large platelets equivalent to red blood cells (diameter about 4 to 8 μm) and giant platelets larger than red blood cells (diameter about 8 μm or more).

[0003] In platelets, in addition to mitochondria, Golgi apparatus, lysosomes, glycogen, and the cytoskeleton, as characteristic components of platelets, there are organelles such as α-granules, dense granules, open canalicular system (OCS), and dense tubular system. The granules present in platelets contain coagulation / fibrinolysis factors and growth factors that contribute to the formation of a hemostatic plug. For example, there are dozens of α-granules per platelet, which contain factors such as β-thromboglobulin, platelet factor 4 (PF4), platelet-derived growth factor (PDGF), as well as fibrinogen, von Willebrand factor (VWF), and coagulation factor V. Although platelets are anucleate as described above, for example, the intracellular granules can be stained with azur dye.

[0004] In the process of hemostatic clotting by platelets, first, VWF binds to collagen beneath the damaged and exposed vascular endothelium, and then the GPIb / V / IX complex, a glycoprotein (GP) on the platelet membrane, binds to this, causing platelets to adhere to the site of injury. Furthermore, the binding of collagen to the collagen receptor GPVI on the platelet membrane further strengthens the adhesion of platelets to the aforementioned site of injury. In addition, when these bindings occur, intracellular signals (inside-out signals) change the structure of the glycoprotein GPIIb / IIIa (integrin αIIb / β3) on the platelet membrane, making it active, and platelets aggregate through the binding of this active GPIIb / IIIa to fibrinogen or VWF. Furthermore, the aforementioned morphological changes and the activation of GPIIb / IIIa lead to further activation of platelets through new intracellular signals (outside-in signals). Degranulation releases platelet-activating substances such as adenosine diphosphate (ADP), serotonin, and thromboxane A2 (TXA2). This positive chain reaction mediated by these platelet-activating substances accelerates and strengthens platelet aggregation, ultimately forming a stable aggregate that acts as a hemostatic plug.

[0005] In addition to being used in transfusions as whole blood or platelets, platelets also contain growth factors useful for tissue regeneration, thus playing a role as a scaffold material for tissue regeneration. For this reason, platelet concentrates (PCs) and platelet-rich plasma (PRPs) are clinically applied in various medical fields, such as orthopedics (arthritis, intractable fractures, bone defect treatment; lumbar interbody fusion), plastic surgery (pressure ulcers, burns, wound treatment; angiogenesis and angioplasty), dentistry and oral surgery (jawbone regeneration, alveolar bone regeneration), cosmetic surgery (hair regeneration, improvement of skin wrinkles and sagging), and obstetrics and gynecology (infertility treatment). However, the quality of these preparations has varied due to individual differences in the origin of platelets and differences in preparation methods among hospitals and institutions, which has affected the therapeutic effect.

[0006] A common method for evaluating platelets in the aforementioned preparations is the platelet aggregation test using a transmittance measurement method, which involves adding platelet-activating substances such as ADP or collagen to PRP and utilizing the increase in PRP transparency as platelet aggregation occurs (for example, Non-Patent Literature 1: GVRBORN, Nature, Vol.194, 1962, pp.927-929). However, this test evaluates aggregates consisting of hundreds to tens of thousands of platelets and does not directly evaluate individual platelets. Furthermore, it has problems such as low quantitative accuracy and inconsistent evaluation criteria, leading to variations in evaluation from hospital to hospital or institution.

[0007] On the other hand, one method for evaluating individual platelets is to observe morphological changes using a scanning electron microscope (SEM) (for example, Non-Patent Literature 2: Katsuyasu Saigo et al., Japanese Journal of Transfusion Medicine, Vol. 46, No. 5, 2000, pp. 480-486). However, SEM observation has problems such as requiring complicated procedures and being costly. Furthermore, it is difficult to observe the internal condition of platelets.

[0008] Furthermore, for example, Patent Document 1 (Japanese Patent Publication No. 2012-8044) describes a method for measuring platelet activation ability, in which activated platelets are labeled with a fluorescently labeled anti-CD61 antibody and a fluorescently labeled anti-CD62P antibody, and CD62P (p-secretin), a platelet activation marker, is measured by flow cytometry. However, methods using flow cytometry accompanied by labeling via antibodies, such as this method, have problems such as requiring a large amount of sample, unstable results due to pre-treatment procedures, the need for large equipment, and high costs.

[0009] Furthermore, there has been no standardization of indicators for evaluating platelets. In fact, the quality evaluation of blood products for transfusion distributed domestically employs a complex evaluation that includes many items in addition to the platelet aggregation test mentioned above, such as platelet count test, mean platelet volume (MPV) test, pH test, lactate dehydrogenase (LDH) leakage test, hypotonic shock recovery (HSR) test, p-selectin test, platelet morphology test, appearance test, and white blood cell count test (for example, Non-Patent Literature 3: "Stability Test Results of Unirradiated Platelet Preparations", [online], Japanese Red Cross Society, [Retrieved June 13, 2024], Internet).<https: / / www.jrc.or.jp / mr / product / list / pdf / result_pc-lr100205.pdf> ). In addition, as another indicator for evaluating platelets, for example, Patent Document 2 (Japanese Patent Publication No. 4-235348) describes blocking the aggregation reaction of platelets by treating PRP with an anti-GPIIb / IIIa antibody, and then separating and evaluating its adhesiveness. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2012-8044 [Patent Document 2] Japanese Patent Application Publication No. 4-235348 [Non-patent literature]

[0011] [Non-Patent Document 1] GVRBORN,Nature,Vol.194,1962,p.927-929 [Non-Patent Document 2] Katsuyasu Saigo et al., Japanese Journal of Transfusion Medicine, Vol. 46, No. 5, 2000, pp. 480-486. [Non-Patent Document 3] "Stability Test Results of Platelet Preparations (Unirradiated)", [online], Japanese Red Cross Society, [Accessed June 13, 2024], Internet<https: / / www.jrc.or.jp / mr / product / list / pdf / result_pc-lr100205.pdf> [Overview of the project] [Problems that the invention aims to solve]

[0012] Although various indicators have been used to evaluate platelets, platelets, as described above, become activated and release granules, which further accelerates their activation and leads to the formation of aggregates. In other words, one of the essential activating abilities of platelets is their ability to release such granules (granule-releasing ability), but no indicator has been disclosed to date that can directly and easily evaluate the level of this granule-releasing ability.

[0013] This invention has been made in view of the problems of the prior art described above, and aims to provide a new platelet evaluation index and platelet evaluation method that can directly and easily evaluate the activation ability of platelets in a sample, as well as a platelet measurement method and platelet evaluation apparatus for these purposes. [Means for solving the problem]

[0014] The inventors have diligently conducted research to achieve the above objective and have focused on four types of platelets: (a) platelets that have not been exposed to platelet activators and contain a sufficient amount of granules, (b) platelets that have not been exposed to platelet activators but contain little or no granules, (A) platelets that do not degranulate even when exposed to platelet activators, and (B) platelets that degranulate when exposed to platelet activators. Of these, the inventors have decided to use the proportion of platelets that have the desired activation ability as platelets, that is, platelets that have the ability to release granules and degranulate when exposed to platelet activators, as a new evaluation index for platelets. As a method for measuring this index, the inventors have found a method in which platelets that have actually undergone degranulation (demurgical platelets) can be distinguished from platelets that have not undergone degranulation (granular platelets) by adding a platelet aggregation inhibitor (e.g., GPIIb / IIIa inhibitor, etc.) to a platelet-containing sample to inhibit only platelet aggregation, and platelets that have undergone degranulation can be induced by adding a platelet activator. Furthermore, by employing a method for directly observing granules (for example, observation by staining or analysis by tomography), we discovered that it is possible to directly distinguish between degranulated platelets and rich granulated platelets, and that this can be easily performed, thus completing the present invention.

[0015] In other words, the present invention relates to a method for evaluating platelets, and more specifically, provides the following: [1] The following steps: A platelet aggregation inhibition step involves adding a platelet aggregation inhibitor to a platelet-containing sample. A platelet activation step is performed by adding a platelet activating substance to the aforementioned sample. A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. An evaluation step in which the platelet activation ability in the sample is evaluated using the number of granular platelets A and / or the number of degranular platelets B as indicators. A platelet evaluation method, including the following. [2] The platelet evaluation method according to [1], further comprising a sample preparation step of preparing a smear sample of the sample after the platelet aggregation inhibition step and the platelet activation step, and wherein the measurement of the number A of the granule-rich platelets and the number B of the degranulated platelets in the measurement step is performed on the smear sample. [3] The platelet evaluation method according to [2], wherein the sample preparation step includes a fixing treatment for fixing the sample. [4] The platelet evaluation method according to [1], further comprising a sample supply step of supplying the sample into a microchannel device, and wherein the measurement of the number A of the granule-rich platelets and the number B of the degranulated platelets in the measurement step is performed on the microchannel device. [5] The platelet evaluation method according to [4], wherein the sample supply step includes a fixing treatment for fixing the sample. [6] The platelet evaluation method according to any one of [1] to [5], further comprising a staining step of staining the granules of platelets after the platelet aggregation inhibition step and the platelet activation step and before the measurement step. [7] The platelet evaluation method according to [6], wherein the staining is staining with an azur dye. [8] The platelet evaluation method according to any one of [1] to [7], wherein the platelet activation step is performed after the platelet aggregation inhibition step, or the platelet aggregation inhibition step and the platelet activation step are performed simultaneously. [9] The method further comprises a control measurement step of performing the measurement step on the sample without performing the platelet aggregation inhibition step and the platelet activation step to measure the number b of degranulated platelets with a low or no granule content and the number a of granule-rich platelets other than the degranulated platelets. In the evaluation step, the activation ability of platelets in the sample is evaluated using the effective platelet rate calculated by the following formula: effective platelet rate = aB / {(a + b)(A + B)} as an index. The platelet evaluation method according to any one of [1] to [8].

[10] It further includes a sample preparation step of preparing a smear specimen of a sample for which the platelet aggregation inhibition step and the platelet activation step have not been performed, and the measurement of the number a of the granule-rich platelets and the number b of the degranulated platelets in the control measurement step is performed on the smear specimen. The platelet evaluation method according to [9].

[11] The sample preparation step includes a fixation treatment for fixing the sample. The platelet evaluation method according to

[10] .

[12] It further includes a sample supply step of supplying a sample for which the platelet aggregation inhibition step and the platelet activation step have not been performed into a microchannel device, and the measurement of the number a of the granule-rich platelets and the number b of the degranulated platelets in the control measurement step is performed on the microchannel device. The platelet evaluation method according to [9].

[13] The sample supply step includes a fixation treatment for fixing the sample. The platelet evaluation method according to

[12] .

[14] The degranulated platelets are platelets in which the contained granules are concentrated in the center of the cell or the ratio of the granules occupying the cytoplasm is 10% or less in terms of area in the two-dimensional image. The platelet evaluation method according to any one of [1] to

[13] .

[15] The degranulated platelets are platelets in which the number of contained granules is 5 or less or the ratio of the granules occupying the cytoplasm is 10% or less in terms of area in the two-dimensional image. The platelet evaluation method according to any one of [1] to

[13] .

[16] It is a measurement method for the platelet evaluation method according to any one of [1] to

[15] , The following steps: A platelet aggregation inhibition step of adding a platelet aggregation inhibitor to a sample containing platelets, A platelet activation step of adding a platelet activator to the sample, A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. A platelet counting method, including the following.

[17] Apparatus for platelet evaluation methods described in [2] and [4], and any one of [3], [5] to

[15] which is subordinate to [2] or [4], An imaging means for capturing an image of the smear specimen or an image of the microfluidic device, A detection means for detecting platelets from an image captured by the imaging means, A determination means for determining whether the platelets detected by the detection means are degranulating platelets or rich granulating platelets. A calculation means that measures the number of degranulated platelets B and the number of rich platelets A determined by the discrimination means and calculates the ratio of the number of rich platelets A and / or the ratio of the number of degranulated platelets B. Output means for outputting the value calculated by the calculation means, A platelet evaluation device, including a platelet evaluation device.

[18] The platelet evaluation apparatus according to

[17] , wherein the imaging in the imaging means is imaging by a microscope equipped with a camera.

[19] The platelet evaluation apparatus according to

[17] or

[18] , wherein the detection in the detection means is detection by a machine learning model.

[20] The platelet evaluation apparatus according to any one of

[17] to

[19] , wherein the discrimination in the discrimination means is discrimination by a machine learning model. [twenty one] The platelet evaluation apparatus according to any one of

[17] to

[20] , wherein the detection means and the discrimination means together perform the simultaneous detection of platelets from an image captured by the imaging means and the determination of whether they are degranulated platelets or rich platelets, and the detection and determination in the means are performed by a machine learning model. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide a novel platelet evaluation index and platelet evaluation method that can directly and easily evaluate the activation ability of platelets in a sample, as well as a platelet measurement method and platelet evaluation apparatus for these purposes.

[0017] These new evaluation indices and platelet evaluation methods, as well as the platelet measurement methods used for them, are useful for quality evaluation of blood products such as platelet preparations (PCs) and platelet-rich plasma (PRPs), and for monitoring the efficacy of antiplatelet drugs. Furthermore, since the platelet evaluation method, platelet measurement method, and platelet evaluation device of the present invention can be applied to small amounts of sample, they are also useful as POCT (point of care testing: a real-time testing system performed in a medical setting). In addition, since image-based automatic judgment can be used for the discrimination and measurement of granular platelets and degranular platelets, automation is easily possible. [Brief explanation of the drawing]

[0018] [Figure 1] This is a schematic diagram showing one embodiment (101) of the microfluidic device according to the present invention. [Figure 2] This is a schematic diagram showing one embodiment (102) of the microfluidic device according to the present invention. [Figure 3] This is a conceptual diagram showing one embodiment of the processing sequence of the platelet counting device of the present invention, as described in Embodiment 1. [Figure 4] This is a conceptual diagram showing one embodiment of the processing sequence of the platelet counting device of the present invention, aspect 2. [Figure 5] This is a conceptual diagram showing one embodiment of the processing sequence of the platelet counting device of the present invention, aspect 3. [Figure 6] This is a conceptual diagram showing one embodiment of the processing sequence of the platelet evaluation device of the present invention. [Modes for carrying out the invention]

[0019] The present invention will be described in detail below with reference to its preferred embodiments.

[0020] <Platelet evaluation methods, platelet measurement methods> The present invention involves the following steps: A platelet aggregation inhibition step involves adding a platelet aggregation inhibitor to a platelet-containing sample. A platelet activation step is performed by adding a platelet activating substance to the aforementioned sample. A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. An evaluation step in which the platelet activation ability in the sample is evaluated using the number of granular platelets A and / or the number of degranular platelets B as indicators. The present invention provides a platelet evaluation method, which includes the platelet aggregation inhibition step, the platelet activation step, and the measurement step for the platelet evaluation method. In this specification, the platelet evaluation method and the platelet measurement method of the present invention may be collectively referred to simply as "the method of the present invention."

[0021] (platelet) In the method of the present invention, the "platelets" that are the subject of measurement and evaluation are usually disc-shaped, anucleated small cells with a diameter of approximately 2 to 4 μm, but may also be large platelets (diameter of approximately 4 to 8 μm) that are equivalent to red blood cells, or giant platelets (diameter of approximately 8 μm or more) that are larger than red blood cells.

[0022] Platelets contain three types of granules: lysosomes, α-granules, and dark-stained granules (δ-granules). These granules are released outside the platelet membrane through a process called "degranulation" caused by the action of platelet-activating substances. The granules contained in platelets are sometimes referred to as azur granules, which are stained with azur dye. In this invention, the "granules" that are the target of measurement below specifically refer to these azur granules.

[0023] (sample) The "sample" used in the method of the present invention is not particularly limited as long as it is a sample that may contain platelets as the subject of evaluation. Examples include blood (whole blood, platelets, etc.) from humans and non-human animals, and separations and / or concentrates of such blood (platelet preparations (PC), platelet-rich plasma (PRP), etc.). Examples of non-human animals include mammals such as chimpanzees, monkeys, cattle, pigs, horses, sheep, mice, rats, and rabbits. Among these, the sample used in the present invention is preferably a human-derived sample that is the subject of diagnosis and treatment in the medical field or clinical testing field.

[0024] The aforementioned sample may be frozen or may contain a preservation solution, etc. The preservation solution may be an aqueous solution containing at least one selected from the group consisting of sodium citrate, citric acid, glucose, and sodium phosphate, for example, ACD-A solution or CPD solution is preferred.

[0025] Furthermore, while the method of the present invention allows for the use of whole blood or other samples as is, the sample used in the method of the present invention may be appropriately diluted or suspended with a diluent. Examples of such diluents include the preservation solution, physiological saline, and known buffers (sodium phosphate buffer, Tris buffer, CFB buffer, glycine buffer, Good's buffer (MES buffer, MOPS buffer, PIPES buffer, HEPES buffer, tricine buffer, bicine buffer, etc.)). Moreover, the method of the present invention may also use a sample that has been fractionated by centrifugation or the like after dilution or suspension with the diluent, as long as platelets to be evaluated are present.

[0026] (Platelet aggregation inhibitor) In the present invention, "platelet aggregation inhibitory substance" refers to a substance that can inhibit platelet aggregation and does not inhibit activation by the platelet activating substance, depending on the type of platelet activating substance described below. For example, platelet aggregation can be suppressed by blocking the receptor function of GPIIb / IIIa using the following GPIIb / IIIa inhibitors as platelet aggregation inhibitors, and platelets can be activated with the following platelet activators (preferably ADP and serotonin); platelet aggregation can be suppressed by inhibiting COX (cyclooxygenase), inhibiting the arachidonic acid pathway, and inhibiting the production of TXA2 (thromboxane A2) using COX inhibitors such as aspirin (acetylsalicylic acid) as platelet aggregation inhibitors, and platelets can be activated with the following platelet activators (preferably ADP and serotonin); platelet aggregation can be suppressed by blocking the P2Y12 receptor by ADP using P2Y12 receptor antagonists (clopidogrel, prasagrel, ticlogrel, efavalenz, etc.), and platelets can be activated with the following platelet activators (preferably serotonin).

[0027] Therefore, examples of platelet aggregation inhibitors according to the present invention include GPIIb / IIIa inhibitors, COX inhibitors (such as aspirin (acetylsalicylic acid)), and P2Y12 receptor antagonists (such as clopidogrel, prasagrel, ticlogrel, and efavalenz), and may be one of these or a combination of two or more. Furthermore, the platelet aggregation inhibitor according to the present invention may be in the form of a composition containing this substance along with a buffering agent or the like. Among these, the GPIIb / IIIa inhibitor is preferred as the platelet aggregation inhibitor according to the present invention.

[0028] [GPIIb / IIIa inhibitors] GPIIb / IIIa is a glycoprotein (GP) belonging to the integrin family, also known as integrin αIIb / β3. GPIIb / IIIa is a complex formed by GPIIb (αIIb) and GPIIIa (β3) in a Ca-ion-dependent ratio of 1:1. GPIIb / IIIa is the most abundant receptor on the platelet membrane, with approximately 80,000 molecules per platelet. It acts as a receptor for fibrinogen and von Willebrand factor (VWF), and platelets aggregate through binding to these substances.

[0029] The "GPIIb / IIIa inhibitor" used in the platelet aggregation inhibition step of the method of the present invention refers to an inhibitor that can inhibit the receptor function of GPIIb / IIIa and does not inhibit activation by the platelet activator, depending on the type of platelet activator described below. Examples of such GPIIb / IIIa inhibitors include decalcifying agents and GPIIb / IIIa antagonists.

[0030] Examples of the decalcifying agent include chelating agents, and in the present invention, aminopolycarboxylic acids are preferred as chelating agents, for example, ethylenediaminetetraacetic acid (EDTA) salts (ethylenediaminetetraacetic acid dipotassium (EDTA-2K), ethylenediaminetetraacetic acid disodium (EDTA-2Na), etc.), diaminopropanol tetraacetic acid salt, and ethylenediaminedipropionate salt, and it may be one of these or a combination of two or more of them.

[0031] Examples of the GPIIb / IIIa antagonist include RGD-containing peptides, anti-GPIIb / IIIa antibodies, and other antagonists (e.g., tirofiban (Merk), epifibatide (Merk), etc.), and may be one of these or a combination of two or more.

[0032] The RGD-containing peptide is a peptide containing a three-residue RGD sequence of arginine-glycine-aspartic acid (RGD), which is known as a ligand for integrin αIIb / β3. The RGD-containing peptide according to the present invention may have a total of 1 to 8 amino acids (preferably 1 to 4) added to the amino and / or carboxyl terminals of the RGD sequence. The added amino acids are preferably at least one selected from the group consisting of serine (S), glycine (G), proline (P), aspartic acid (D), lysine (K), asparagine (N), and threonine (T). Furthermore, the RGD-containing peptide according to the present invention may be linear or cyclic, and may have polymers such as sugars or polyethylene glycol (PEG) added to it. Such RGD peptides can be produced by conventionally known methods or similar methods, and commercially available ones may also be used as appropriate.

[0033] The aforementioned anti-GPIIb / IIIa antibody can be any antibody capable of inhibiting the function of GPIIb / IIIa as a receptor, and can be produced by conventionally known methods or similar methods, for example, by the hybridoma method or the recombinant DNA method. Commercially available antibodies (e.g., Abciximab (Eli Lilly)) may also be used as appropriate. In this invention, "antibody" includes not only complete antibodies but also their functional fragments (Fab, F(ab')2, Fab', variable region fragment (Fv), disulfide bonded Fv, single-stranded Fv (scFv), sc(Fv)2, diabody, and polymers thereof, etc.). Furthermore, "antibody" includes all classes and subclasses of immunoglobulins and may be either a polyclonal antibody or a monoclonal antibody.

[0034] Among these, the GPIIb / IIIa inhibitor used in the platelet aggregation inhibition step of the method of the present invention is preferably at least one selected from the group consisting of the chelating agent and the GPIIb / IIIa antagonist, and more preferably at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA) salt and the RGD-containing peptide.

[0035] (platelet activator) The "platelet activating substance" used in the platelet activation step of the method of the present invention refers to a substance that can induce degranulation in platelets and does not cause platelet aggregation under the above-mentioned inhibition of platelet aggregation (for example, under the inhibition of GPIIb / IIIa). Although platelet degranulation is mainly induced by signals (outside-in signals) generated by structural changes of GPIIb / IIIa, for example, even if this system is inhibited by the GPIIb / IIIa inhibitor, platelets can still induce degranulation through a system mediated by other platelet activating substances.

[0036] Examples of such platelet-activating substances include adenosine diphosphate (ADP), collagen, epinephrine, conbulxin, serotonin, vasopressin, carbazochrome, blood coagulation factors (FVIII, FIX), thrombin, ethanesylate, prostaglandin metabolites (thromboxane A2 (TXA2)), norepinephrine, ristocetin, and analogues thereof that can bind to their respective receptors. These may be used individually or in combination of two or more, and are selected according to the platelet aggregation inhibitor. Furthermore, the platelet-activating substance according to the present invention may be in the form of a composition containing this substance along with a buffering agent or the like.

[0037] Among these, the platelet activating substance used in the platelet activation step of the method of the present invention is preferably at least one selected from the group consisting of adenosine diphosphate (ADP) and serotonin. However, when the platelet aggregation inhibitor is a P2Y12 receptor antagonist, the platelet activating substance is preferably serotonin.

[0038] (Platelet aggregation inhibition process) In the method of the present invention, as a platelet aggregation inhibition step, the platelet aggregation inhibition substance is added to a sample containing (or potentially containing) platelets as an inhibitory treatment, for example, by blocking the receptor function of GPIIb / IIIa with the GPIIb / IIIa inhibitor, thereby inhibiting the aggregation of platelets.

[0039] The amount of platelet aggregation inhibitor added to the sample should be sufficient to suppress platelet aggregation of platelets in the sample, and can be appropriately adjusted by those skilled in the art depending on the type and concentration of the sample and the type of platelet aggregation inhibitor. Therefore, it is not possible to generalize, but to give one example, when adding EDTA salt (preferably EDTA-2Na) to PRP, it is preferable that the amount of EDTA salt be 0.001 to 1 M relative to the total volume of the solution containing PRP and EDTA salt solution, and more preferably 0.005 to 0.5 M. Also, when adding GPIIb / IIIa antagonist (preferably tirofiban) to PRP, it is preferable that the amount of GPIIb / IIIa antagonist (preferably tirofiban) be 0.01 to 100 μM relative to the total volume of the solution containing PRP and GPIIb / IIIa antagonist solution (preferably tirofiban solution), and more preferably 0.1 to 10 μM. Other conditions for the aforementioned inhibitory treatment (time, temperature, pH, etc.) can be appropriately adjusted by those skilled in the art depending on the type and amount of platelet aggregation inhibitor added.

[0040] (Platelet activation process) In the method of the present invention, the platelet activation step involves an activation treatment in which the platelet activating substance is added to a sample containing (or potentially containing) platelets, thereby inducing platelet degranulation. The platelet activation step according to the present invention is preferably performed after the platelet aggregation inhibition step, but the platelet aggregation inhibition step and the platelet activation step may be performed simultaneously. In this case, the platelet aggregation inhibitor and the platelet activating substance may be added to the sample as a composition containing them.

[0041] The amount of platelet activating substance added to the sample should be sufficient to induce degranulation of platelets in the sample, and can be appropriately adjusted by those skilled in the art depending on the type and concentration of the sample and the type of platelet activating substance. Therefore, it is not possible to generalize, but for example, when adding ADP to PRP, it is preferable that the amount of ADP be 0.001 to 0.5 mM relative to the total volume of the solution containing PRP and ADP solution, and more preferably 0.001 to 0.1 mM. Furthermore, depending on the type of sample, if animal blood other than human blood is used, it is preferable to increase the concentration of ADP to about 10 to 100 times that when human blood is used, depending on the animal species. Other conditions of the activation treatment (time, temperature, pH, etc.) can be appropriately adjusted by those skilled in the art depending on the type and amount of platelet activating substance added.

[0042] (Specimen preparation process) In the method of the present invention, it is preferable to include a sample preparation step after the platelet aggregation inhibition step and the platelet activation step, or, if the control measurement step described below is performed, a sample for which the platelet aggregation inhibition step and the platelet activation step have not been performed, in accordance with the measurement method described below, and as necessary, a sample for observing platelets. Such a sample is preferably, for example, a smear.

[0043] The method for preparing the specimen can be carried out as appropriate by conventionally known methods or similar methods. For example, the sample after the platelet aggregation inhibition step and the platelet activation step, or, in the case of the control measurement step described below, the sample before the platelet aggregation inhibition step and the platelet activation step have been performed, can be diluted with the diluent as needed, applied to a transparent observation substrate such as a glass slide, dried, and then preferably fixed by a fixation treatment to fix it on the transparent observation substrate, thereby preparing the smear specimen. The material of the transparent observation substrate is not limited to glass such as soda-lime glass or borosilicate glass, but may also be a resin such as PET.

[0044] The aforementioned fixation treatment is a process that dehydrates the sample and denatures the proteins in the sample, and tends to preserve the morphology of platelets more stably than smear specimens prepared by coating and drying alone. For this reason, it is preferable that the specimen preparation process according to the present invention includes such a fixation treatment. The method of such a fixation treatment is not particularly limited, and conventionally known methods or similar methods can be used as appropriate. For example, after drying, a fixative such as methanol or ethanol can be applied, or the specimen can be immersed in the fixative and dried. Furthermore, if the method of the present invention includes the following staining step, the fixative can be pre-mixed with a staining solution containing a dye for the following staining (hereinafter, sometimes simply referred to as "staining solution"), and the staining and fixation treatment can be carried out simultaneously, which is preferable from the viewpoint of shortening the processing time. The methods of coating, immersion, and each drying are not particularly limited, and conventionally known methods or similar methods can be used as appropriate. The drying may be done at room temperature or by air drying, but to accelerate it, it may be heated to, for example, 20-24°C, or an inert gas such as nitrogen gas may be applied.

[0045] (dying process) In the measurement methods described below, examples of platelet observation methods include observation by three-dimensional optical diffraction tomography (Jinkyoung Chung et al, Scientific Reports volume 11, Article number: 10511 (2021)) and observation by staining.

[0046] In the method of the present invention, among these, observation by staining is preferred, and more specifically, the method of the present invention preferably includes a staining step to stain platelet granules on the sample after the platelet aggregation inhibition step and the platelet activation step, or on the sample before the platelet aggregation inhibition step and the platelet activation step are performed, if the control measurement step described below is performed. The granules to be stained here are azur granules as described above. Therefore, the staining method is not particularly limited as long as it is a method that can stain such azur granules, that is, staining with azur dye, and other dyes may be combined.

[0047] Examples of staining methods using the aforementioned azure dye include Romanowsky stains such as Giemsa staining, Wright staining, Wright-Giemsa staining, May-Grünwald staining, and May-Grünwald-Giemsa staining (May-Giemsa staining). Among these staining methods, from the viewpoint of being able to stain platelet granules particularly clearly, May-Grünwald staining or May-Grünwald-Giemsa staining (May-Giemsa staining) is preferred as the staining method according to the present invention.

[0048] In the method of the present invention, the staining can be carried out as appropriate by conventionally known methods or similar methods, depending on the type of staining. Furthermore, in the method of the present invention, in accordance with the measurement method described below, it is preferable to either prepare a sample in the sample preparation step as necessary, or supply the sample to the microfluidic device described below and then stain it using a method appropriate to the sample or microfluidic device; or to stain in the staining step and then prepare the sample in the sample preparation step, or supply the sample to the microfluidic device described below, and it is more preferable to prepare the sample (preferably a smear) and then stain it using a method appropriate to the sample.

[0049] For example, a method for staining the smear specimens may involve applying each sample onto a glass slide, drying it, preferably performing the fixation treatment, immersing the glass slide in the staining solution, washing it with a washing solution as needed, and then drying it. Alternatively, the fixation treatment and staining may be performed simultaneously by including the fixation solution in the staining solution. Examples of the washing solution include clear solutions such as phosphate buffer and physiological saline.

[0050] (Sample supply process) Furthermore, in the method of the present invention, for example, a sample after the platelet aggregation inhibition step and the platelet activation step have been performed, or, in the case of the control measurement step described below, a sample before the platelet aggregation inhibition step and the platelet activation step have been performed, can be diluted with the diluent as needed and supplied into a microfluidic device, and the inside of the channel can be observed, thereby replacing the measurement on the smear specimen with a measurement on the microfluidic device.

[0051] Furthermore, when using the microfluidic device, the platelet activation step and / or the staining step, and more preferably the platelet aggregation inhibition step, can also be performed on the microfluidic device as needed. Additionally, when using the microfluidic device, the control measurement step and the staining step for that step can be performed in parallel. This makes it possible to implement the method of the present invention more simply, quickly, and efficiently.

[0052] Such a microfluidic device eliminates the need for complex operations and allows for efficient measurement even with small amounts of sample. Therefore, it is preferable that the method of the present invention includes a sample supply step of supplying the sample (a sample after the platelet aggregation inhibition step and the platelet activation step have been performed, or a sample before the platelet aggregation inhibition step and the platelet activation step have been performed) to such a microfluidic device.

[0053] The microfluidic device according to the present invention comprises a microfluidic channel and a series of channels including a sample supply port and an outlet port, and may also include channels other than the microfluidic channel, such as tanks or openings, as long as the sample can communicate within the device by capillary action of the microfluidic channel or external pressure. The microfluidic device also comprises a measurement section which is the position for measuring platelets in the measurement step or control measurement step described below, and the measurement section may be a part of the microfluidic channel that sequentially measures one or more platelets passing through it, a plate-shaped tank or the like that measures the platelets pooled therein together in the same image, or it may have an opening that allows it to come into contact with the outside air.

[0054] As for the aforementioned microfluidic channels, since each blood cell in the blood is approximately 2 to 22 μm in diameter, and platelets are approximately 2 to 4 μm in diameter, it is preferable that the width and / or depth of the microfluidic channels be 1 mm or less (preferably 50 μm to 1 mm in width and 10 μm to 1 mm in depth) so that the sample does not stagnate or aggregate in the channel, forms a stable flow, and mixes uniformly with the reagents described below as needed. The material of the microfluidic device may be glass, PDMS (dimethylpolysiloxane), resin, LTCC (Low Temperature Co-fired Ceramics), etc., but is not limited thereto. Furthermore, some or all of the device may be treated with hydrophobic or hydrophilic processing, or coated with ion charging. This makes it possible to control the movement speed of the sample, for example, or to fix the sample to the measurement unit.

[0055] The method for supplying each of the aforementioned samples into the microfluidic device is not particularly limited. For example, the sample can be diluted with the diluent as needed to make an aqueous solution, injected or dropped into the supply port using a variable volume pipette, and external pressure can be applied as needed to cause the sample to flow into the flow channel (supply) by capillary action or external pressure.

[0056] Furthermore, when the platelet activation step and / or the staining step, and more preferably the platelet aggregation inhibition step, are performed on the microfluidic device, the microfluidic device may further include, for example, a containment section (e.g., a tank) filled with reagents to be added to the sample, such as the platelet aggregation inhibitor, the platelet activating substance, and the staining solution, as well as an inert gas, as needed, such as a fixative and washing solution, or a supply port for supplying each of the reagents and the inert gas; a mixing section (e.g., a circular mixing tank, a zigzag structure, or a channel with an adjusted inflow angle); and a sealing plug or vent for controlling each of the mixing steps and the movement within the channel.

[0057] Figure 1 shows a schematic diagram of one embodiment (101) of a microfluidic device in which each of these parts is connected by microfluidic channels. In the sample supply process using the microfluidic device (101) shown in Figure 1, for example, a sample is injected or dropped into the supply port (1) using a variable volume pipette, and external pressure is applied as needed to cause the sample to flow into the channel by capillary action or external pressure. Of these, the sample that flows into the lower channel in Figure 1 comes into contact with the platelet aggregation inhibitor supplied from the platelet aggregation inhibitor containment section or supply port (16), and is mixed in the platelet aggregation inhibitor mixing section (17, 18). Next, it comes into contact with the platelet activator supplied from the platelet activator containment section or supply port (19), and is mixed in the platelet activator mixing section (20). Next, it comes into contact with the staining solution supplied from the staining solution containment section or supply port (14), is mixed in the staining solution mixing section (15), reaches the measurement section (13), and is discharged from the outlet (12) together with each reagent. Platelets in the sample that reach the measurement unit (13) can be measured in the following measurement step as a sample after the platelet aggregation inhibition step, platelet activation step, and staining step have been performed. Of the sample that flows into the supply port (1), the sample that flows into the upper flow path in Figure 1 comes into contact with the staining solution supplied from the staining solution containment unit or supply port (24), is mixed in the staining solution mixing unit (25), reaches the measurement unit (23), and is discharged from the outlet (22) together with each reagent. Platelets in the sample that reach the measurement unit (23) can be measured in the following control measurement step as a control sample after the staining step has been performed but the platelet aggregation inhibition step and platelet activation step have not been performed.

[0058] In the microfluidic device, it is desirable to use a staining solution with a lower concentration than usual in order to clearly distinguish the stained platelets from the background solution (including the staining solution) during observation (preferably by microscopic observation) in the measurement section (13, 23) after the staining process. Furthermore, if observation becomes difficult due to the movement of platelets in the background solution, it is possible to easily observe the platelets and the granules within them by, for example, increasing the viscosity of the solution to suppress the movement of platelets. For this viscosity improvement, it is preferable to use a polymer that is colorless, transparent, and does not affect optical observation, and it is more preferable to add methylcellulose or polyvinyl alcohol to the solution.

[0059] However, the microfluidic device according to the present invention is not limited to this, and may also have configurations that do not include some of the containment sections (14, 16, 19, 24 in Figure 1) filled with reagents to be added to the sample, or the mixing sections (15, 17, 18, 20, 25) (for example, a configuration that does not include 16 to 18 in Figure 1, or a configuration that does not include 16 to 20 in Figure 1 (in these cases, the sample after the platelet activation step or platelet aggregation inhibition step is supplied to the lower channel in Figure 1), or a configuration that does not include 14, 15, 24, 25 in Figure 1 (in this case, the sample supply port (1) also serves as the staining solution supply port (14, 24), and the sample and staining solution are supplied with a time difference), or a configuration that further includes supply ports for the fixative and washing solutions).

[0060] Furthermore, when using the microfluidic device, the fixation process for fixing the sample to the measurement unit can also be performed on the microfluidic device. From the viewpoint of further improving the accuracy in the measurement process described below, it is preferable that the sample supply process according to the present invention includes such a fixation process. The fixation process is the same as the fixation process described in the sample preparation process, and can be performed, for example, by drying the sample that has reached the measurement unit, and then bringing the fixative solution to the measurement unit and drying it. Alternatively, when the staining process is performed on the microfluidic device, the fixative solution can be included in the staining solution beforehand, and the staining and fixation processes can be performed simultaneously.

[0061] As a microfluidic device capable of performing the fixation process, for example, the measuring section may have an opening that allows it to come into contact with the outside air in order to dry the fixative or the staining solution containing the fixative by air drying. It may also further have a gas supply port for introducing an inert gas such as nitrogen gas, or the sample supply port may also serve as the gas supply port. Furthermore, as a microfluidic device, it is preferable that the measuring section is pre-coated with a positively charged substance such as polylysine or polyethyleneimine. Such a coating makes it possible to further improve the adhesion of platelets to the measuring section and to further suppress the outflow of platelets from the measuring section.

[0062] Figure 2 shows a schematic diagram of one embodiment (102) of a microfluidic device capable of performing such a fixation process. In the microfluidic device (102) shown in Figure 2, the sample supply port (1) also serves as the supply port (14, 24) for the fixative and staining solution, or the staining solution containing the fixative solution. The fixative and staining solutions are supplied from the supply port (1) after the sample reaches the measurement section (13', 23') and is dried. In addition, in the microfluidic device (102), the bottom surface of the measurement section (13', 23') is coated, and the top surface is open and in contact with the outside air (shaded area).

[0063] The sample supply process, which includes a fixation treatment using the microfluidic device (102) shown in Figure 2, involves, for example, injecting or dropping the sample into the supply port (1) using a variable volume pipette, and applying external pressure as needed, thereby causing the sample to flow into the flow channel by capillary action or external pressure. Of these, the sample that flows into the lower flow channel in Figure 2 comes into contact with the platelet aggregation inhibitor supplied from the platelet aggregation inhibitor containment section or supply port (16), is mixed in the platelet aggregation inhibitor mixing section (17, 18), then comes into contact with the platelet activator supplied from the platelet activator containment section or supply port (19), is mixed in the platelet activator mixing section (20), and reaches the measurement section (13'). In the microfluidic device (102), the measurement section (13') is structured to be in contact with the outside air, thereby promoting the drying (air drying) of the sample and shortening the time required for fixation. Furthermore, in order to accelerate drying and further shorten the drying time, the measurement unit (13') may be heated to, for example, 20-24°C using a heater or the like, or the supply port (1) may also serve as a gas supply port (26), from which an inert gas such as nitrogen gas may be introduced to further accelerate the drying. After the sample in the measurement unit (13') has dried, the fixative solution is then introduced from the supply port (1, which also serves as the fixative solution supply port (14)) and brought to the measurement unit (13'). After drying and fixing it in the same manner as above, the staining solution is introduced from the supply port (1, which also serves as the staining solution supply port (14)) and brought to the measurement unit (13') for staining. Here, fixation and staining may be performed simultaneously using a staining solution that contains the fixative solution. Furthermore, as needed, washing solution is introduced from the supply port (1) and brought to the measurement unit (13'), where excess staining solution is discharged from the outlet (12) for washing. This process fixes the sample in the measurement unit (13') after the platelet aggregation inhibition, platelet activation, and staining processes, allowing it to be measured using the measurement process described below. Of the sample that flows into the supply port (1), the sample that flows into the upper channel shown in Figure 2 reaches the measurement unit (23') directly.The measurement unit (23') also has a structure that is exposed to the outside air, similar to the measurement unit (13'), so that these processes are carried out in parallel with the drying, fixation, staining, and washing described above. A control sample that has not undergone the platelet aggregation inhibition process and the platelet activation process, but has undergone the staining process, is fixed to the measurement unit (23') and can be measured in the control measurement process described below.

[0064] (Measurement process) In the method of the present invention, after the platelet aggregation inhibition step and the platelet activation step, more preferably after the specimen preparation step or the sample supply step and / or the staining step, a measurement step is performed to measure the number B of degranulated platelets and the number A of granular platelets other than the degranulated platelets. For example, with staining with azur dye, the granules contained in platelets (azur granules) are stained purple to purplish-red, the rest of the cytoplasm is stained gray to light blue, and destroyed platelets are not identified, so the number of platelets present, as well as the number, location, and / or content of granules within the platelets present can be measured.

[0065] The measurement method is not particularly limited, but since platelets are plate-shaped, such measurements can be performed in a two-dimensional image, and it is preferable to perform them on the platelet smear. Furthermore, from the viewpoint of efficiency and speed, it is also preferable to perform the measurements on the microfluidic device (for example, the measurement unit). As for the measurement method on the platelet smear or on the microfluidic device, it is simpler and more preferable to perform the measurements within the field of view of a microscope or within a microscope image.

[0066] In the method of the present invention, platelets with a low or no granule content are defined as "degranulated platelets," and a group of observed platelets (platelet groups) are divided into these degranulated platelets and the remaining "granular platelets." Therefore, granular platelets according to the present invention refer to platelets with a higher granule content than the degranulated platelets. The number of platelets in the platelet group (the number of platelets constituting the population) when dividing the platelets into degranulated platelets and granular platelets is preferably 100 or more, and more preferably 100 to 1000.

[0067] The criteria for distinguishing between degranulated platelets and rich granulated platelets can be appropriately set depending on the type of sample and the purpose of evaluating platelets. For example, degranulated platelets are defined as platelets in which the granules they contain are concentrated in the center of the cell (centralization phenomenon is observed), or in which the proportion of granules in the cytoplasm is 50% or less (more preferably 20% or less, even more preferably 10% or less) in the area of ​​the two-dimensional image, while rich granulated platelets are defined as all others, i.e., in which granules occupy the majority of the cytoplasm, and the proportion of granules in the cytoplasm is greater than 10% (for example, 11% or more, 21% or more, more preferably 51% or more). In particular, it is preferable to define degranulating platelets as platelets in which central concentration is observed or in which the proportion of granules in the cytoplasm is 10% or less in area in a two-dimensional image, and rich granulating platelets as all other platelets, that is, platelets in which central concentration is not observed and in which the proportion of granules in the cytoplasm is greater than 10% (e.g., 11% or more) in area in a two-dimensional image. Here, it can be said that the granules contained in a platelet are concentrated in the center of the cell (central concentration is observed) if, for example, the granules contained in the platelet are distributed within a range with a diameter of less than half the diameter of the platelet, inside the cell margin, and the granules fuse together and are observed as clumps.

[0068] As another preferred criterion for distinguishing (degranulating) platelets from degranulous platelets, for example, it is preferable to define degranulous platelets as platelets with 5 or fewer granules, or platelets in which granules occupy 50% or less of the area in the two-dimensional image (more preferably 20% or less, even more preferably 10% or less), and rich granular platelets as the others, i.e., platelets with 6 or more granules, and in which the proportion of granules occupying the cytoplasm exceeds 10% of the area in the two-dimensional image (for example, 11% or more, 21% or more, more preferably 51% or more). In particular, it is even more preferable to define degranulous platelets as platelets with 5 or fewer granules, or platelets in which granules occupy 10% or less of the area in the two-dimensional image, and rich granular platelets as the others, i.e., platelets with 6 or more granules, and in which the proportion of granules occupying the cytoplasm exceeds 10% of the area in the two-dimensional image (for example, 11% or more).

[0069] The distinction between degranulated platelets and rich granular platelets may be performed, for example, by the naked eye under a microscope based on the above criteria, or it can be automated by employing the detection algorithm or discrimination algorithm described later.

[0070] (Evaluation process) In the platelet evaluation method of the present invention, after the measurement step, the activation ability of platelets in the sample is evaluated as an evaluation step, using the number of granular platelets A and / or the number of degranular platelets B as indicators.

[0071] The number of degranulated platelets B measured after the platelet aggregation inhibition step and the platelet activation step includes the number of platelets that degranulated upon exposure to the platelet activating substance, i.e., platelets with the desired granule-releasing ability. Furthermore, the number of granular platelets A measured after the platelet aggregation inhibition step and the platelet activation step indicates the number of platelets that did not degranulate even upon exposure to the platelet activating substance. Therefore, the number of granular platelets A and / or the number of degranulated platelets B can be used as indicators of granule-releasing ability, representing the activation ability of platelets in the sample.

[0072] When using the number of granular platelets A and / or the number of degranulated platelets B as indicators, for example, the percentage of platelets with granule-releasing ability can be expressed using the following formulas: B / (A+B) and B / A (the percentage of B), or conversely, the percentage of platelets without granule-releasing ability can be expressed using the following formulas: A / (A+B) and A / B (the percentage of A), but these are not the only indicators that can be used.

[0073] Furthermore, the number of degranulated platelets B mentioned above may also include platelets with a low or no granule content even without exposure to the platelet activating substance, that is, platelets that had a low granule count or were in a degranulated state even before exposure to the platelet activating substance. Therefore, the method of the present invention further includes a step of similarly measuring the number of degranulated platelets and the number of granular platelets separately for samples (control samples) in which at least the platelet activation step, preferably the platelet aggregation inhibition step and the platelet activation step have not been performed, and it is preferable to correct the proportion of A or B mentioned above in the evaluation step using these as controls. More specifically, a preferred embodiment of the method of the present invention further includes a control measurement step in which the sample is not subjected to the platelet aggregation inhibition step and the platelet activation step, but the measurement step, more preferably the sample preparation step or the sample supply step, and / or the staining step and the measurement step are performed, and the number of degranulated platelets b and the number of granular platelets a other than the degranulated platelets are measured.

[0074] If the control measurement step is further included, then in the evaluation step, for example, the following formula: Effective platelet percentage = aB / {(a+b)(A+B)} The effective platelet rate calculated by this method represents the percentage of platelets that have the ability to release granules (granule-releasing ability) by platelet-activating substances, and this value can be used as a specific indicator, but is not limited to this.

[0075] The evaluation method based on the above indicators may be a relative evaluation between samples or with a standard sample. For example, the obtained indicators can be compared with a predetermined cutoff value, and samples with values ​​higher than the cutoff value can be evaluated as having high or low platelet activation ability (granule release ability). The cutoff value is not particularly limited and can be set as appropriate depending on the type of sample and the purpose of evaluating platelets.

[0076] <Device for platelet measurement> The platelet measurement method of the present invention can also be automated. Therefore, the present invention provides the following means as an apparatus for the platelet evaluation method or platelet measurement method of the present invention: A platelet aggregation inhibitory means for adding a platelet aggregation inhibitory substance to a platelet-containing sample. A platelet activation means for adding a platelet activating substance to the aforementioned sample, A measuring means for measuring the number of degranulated platelets B, which have a low or no granule content, and the number of granulated platelets A, which are other than the degranulated platelets, after the platelet aggregation inhibitory means and the platelet activation means. The present invention also provides a platelet measuring device equipped with the above. The platelet measuring device of the present invention may further include a specimen preparation means for preparing a specimen and a staining means for staining platelet granules after the platelet aggregation inhibitory means and the platelet activation means, and before the measurement means, and may further include an evaluation means for evaluating the platelet activation ability in the sample using the number of granular platelets A and / or the number of degranulated platelets B as indicators, and a control measurement means for measuring the number of degranulated platelets b and the number of granular platelets a.

[0077] The platelet aggregation inhibitory means and the platelet activation means may be combined into a single means, in which case the platelet measuring device of the present invention may further include a mixing means for pre-mixing the platelet aggregation inhibitory substance and the platelet activation substance. Examples of the platelet aggregation inhibitory means, the platelet activation means, and the mixing means include, for example, supply means for a sample, platelet aggregation inhibitory substance, platelet activation substance, and diluent as needed (sensor, valve, pump, tank, nozzle, bottle, cartridge, filter, actuator, etc.), as well as mixing means (shaker, rotating device, suction / discharge stirring device, etc.) and temperature control means (heater, sensor, etc.).

[0078] If the platelet measurement device of the present invention further comprises the staining means, the staining means may include, for example, supply and discharge means (sensors, valves, pumps, tanks, nozzles, bottles, cartridges, filters, actuators, etc.) for a staining solution or washing solution containing a staining dye, and diluent if necessary, as well as mixing means (shakers, rotating devices, etc.) and drying means (heaters, fans, etc.). Furthermore, the sample that has passed through the platelet aggregation inhibitory means and the platelet activation means may be subjected to the staining means after preparing the above-mentioned specimen outside the device if necessary, but it is preferable for the platelet measurement device of the present invention to further comprise the specimen preparation means, and in this case, it is even more preferable to further comprise fixing means for fixing the sample. Examples of the specimen preparation means include, for example, in the case of a smear specimen, means for guiding the sample (flow channel tube, pump, nozzle, etc.) that has passed through the platelet aggregation inhibitory means and the platelet activation means, and drying means (heater, fan, etc.). Examples of the fixation means include means for supplying fixative solution (sensor, valve, pump, tank, nozzle, bottle, cartridge, filter, actuator, etc.), heating means (heater, etc.), and inert gas supply means (sensor, valve, pump, tank, nozzle, filter, actuator, etc.). In conventional methods of preparing smear specimens from samples by hand, the consistency and reproducibility of the smear specimen depend on the skill of the specimen preparer. In recent years, automation technology has advanced, and for example, a smear specimen preparation device (for example, the automated blood smear analyzer DI-60 (manufactured by Simex Corporation)) has been developed, and such a device may be used as the specimen preparation means.

[0079] In one embodiment of a platelet measurement device using such a smear specimen, for example, the sample is injected into the device, to which a platelet aggregation inhibitor is added by the platelet aggregation inhibitor means, and a platelet activating substance is added by the platelet activation means. Next, a smear specimen is prepared by the specimen preparation means, and a staining solution is added by the staining means. Then, an image of the smear specimen is captured by the imaging means (e.g., a microscope), and the number of each platelet A and B is measured by the measurement means, and the platelet activation ability is evaluated based on these (Embodiment 1). Furthermore, without adding the platelet aggregation inhibitor and platelet activating substance, the number of each platelet a and b may be measured by the control measurement means, and the platelet activation ability may also be evaluated based on these. Here, regarding the manner of preparing the smear specimen and adding the staining solution, it is preferable that the sample is dried by adding an inert gas or the like, then fixed by adding a fixative by the fixation means, and then staining solution is added by the staining means, and preferably a washing solution is further added (Embodiment 2). Figure 3 shows a conceptual diagram illustrating the processing sequence of Embodiment 1 of the platelet measurement device using the aforementioned smear specimen, and Figure 4 shows a conceptual diagram illustrating the processing sequence of Embodiment 2. However, the platelet measurement device according to the present invention is not limited to these. For example, as described above, the addition of the platelet aggregation inhibitor and the platelet activator may be simultaneous, the order of adding the staining solution and preparing the smear specimen may be reversed, and for example, the evaluation of platelet activation ability may not be performed in the device.

[0080] Furthermore, the platelet measurement apparatus of the present invention may be a microfluidic device in whole or in part, and in this case, it may further include a flow channel supply means (sensor, valve, pump, tank, nozzle, bottle, cartridge, filter, actuator, etc.) for supplying the sample to the microfluidic device. The microfluidic device is as described above. The microfluidic device may include the platelet activation means and / or the staining means, more preferably the platelet aggregation inhibition means, and may further include the fixation means for fixing the sample. This makes it possible to carry out a sample supply process including a platelet aggregation inhibition step, a platelet activation step, a staining step, and a fixation process as described above. The microfluidic device in this case is also as described above.

[0081] In one embodiment of a platelet measurement device using such a microfluidic device, for example, by supplying the sample to the microfluidic device of the device, the sample moves within the channels of the device, a platelet aggregation inhibitor, a platelet activator, and a staining solution are added, and an image of the measurement section is captured by the following imaging means (e.g., a microscope), and the platelet activation ability is evaluated by the evaluation means based on this image. Furthermore, the number of each platelet a and b may be measured for a sample in which the platelet aggregation inhibitor and platelet activator are not added, and only the staining solution is added, and the platelet activation ability may also be evaluated based on this. A conceptual diagram showing the processing sequence (embodiment 3) of the platelet measurement device using the microfluidic device is shown in Figure 5. However, the platelet measurement device according to the present invention is not limited thereto, and for example, the addition of the staining solution may include drying the sample, adding a fixative, staining solution, and washing solution, as in embodiment 2 (Figure 4) of the platelet measurement device using a smear specimen described above, and can be appropriately changed according to the configuration of the microfluidic device as described above, and for example, the evaluation of the platelet activation ability may not be performed in the device.

[0082] The aforementioned measurement means and the control measurement means, when provided with the aforementioned measurement means, include, for example, an imaging means (microscope, camera, sensor, image recording device, etc.) for imaging the smear specimen or the microfluidic device, and an image analysis means (program, CPU, transmitting / receiving device, recording device, input / output device, etc.) for detecting platelets from the image obtained based on predetermined criteria, distinguishing between degranulated platelets and rich platelets, and measuring their numbers. The criteria for distinguishing between degranulated platelets and rich platelets are as described above and can be programmed in advance.

[0083] If the platelet measuring device of the present invention further includes the evaluation means, such evaluation means may include, for example, a calculation means for calculating an index percentage (percentage of A, percentage of B, effective platelet rate, etc.) from the number of granular platelets A (and optionally a) and degranulated platelets B (and optionally b) obtained by the measurement means, and an output means (calculation program, CPU, transmitting / receiving device, recording device, output device, etc.) for outputting the same. Furthermore, the device may further include a determination means (calculation program, CPU, transmitting / receiving device, recording device, input / output device, etc.) for determining whether the index is high or low by comparing it with a predetermined cutoff value.

[0084] The configuration of the platelet measuring device of the present invention is not limited to the above, and may be divided and combined into one or more of the above means as appropriate, and may further include control means (CPU, etc.) for controlling each of the above means, a storage device, a network connection port, a motherboard, input ports such as USB (keyboard, mouse input, etc.), a power supply, etc.

[0085] <Platelet evaluation device> Furthermore, the present invention provides an apparatus for the platelet evaluation method of the present invention described above, An imaging means for capturing an image of the smear specimen or an image of the microfluidic device, A detection means for detecting platelets from an image captured by the imaging means, A determination means for determining whether the platelets detected by the detection means are degranulating platelets or rich granulating platelets. A calculation means that measures the number of degranulated platelets B and the number of rich platelets A determined by the discrimination means and calculates the ratio of the number of rich platelets A and / or the ratio of the number of degranulated platelets B. Output means for outputting the value calculated by the calculation means, The present invention also provides an apparatus (hereinafter referred to as a "platelet evaluation apparatus") equipped with the above. The platelet evaluation apparatus of the present invention is one embodiment of an apparatus comprising at least an imaging means and an image analysis means among the measurement means described in the platelet measurement apparatus of the present invention above, and at least a calculation means and an output means among the evaluation means, and can also be used as an apparatus constituting the platelet measurement apparatus of the present invention. The smear specimen and microfluidic device to be used in the platelet evaluation apparatus of the present invention are as described above, and may be prepared or supplied in the platelet measurement apparatus of the present invention above, or may be prepared or supplied separately outside of the apparatus.

[0086] The platelet evaluation device of the present invention preferably further includes means for performing the control measurement step. That is, the platelet evaluation device of the present invention also includes the imaging means, the detection means, and the discrimination means for a smear specimen prepared from a specimen in which the platelet aggregation inhibition step and the platelet activation step were not performed, or for a microfluidic device to which the same specimen was supplied, and the calculation means is a means for further measuring the number b of degranulated platelets and the number a of rich granulated platelets determined by the discrimination means, preferably a means for calculating the effective platelet rate calculated by the following formula: effective platelet rate = aB / {(a+b)(A+B)}, and it is more preferable that the value output by the output means is the effective platelet rate. In this case, the measurement of the number of degranulated platelets B and the number of rich granulated platelets A in the measurement unit on the smear of the sample or on the microfluidic device after the platelet aggregation inhibition step and the platelet activation step, and the measurement of the number of degranulated platelets b and the number of rich granulated platelets a in the measurement unit on the smear of the sample or on the microfluidic device before the platelet aggregation inhibition step and the platelet activation step have been performed, may be performed simultaneously and in parallel, or one may be performed first and the other may be performed separately.

[0087] More specifically, the imaging means may include, for example, a microscope equipped with a camera. The microscope may be, for example, a phase-contrast microscope or a differential interference microscope when the smear specimen or the sample on the measurement unit of the microfluidic device to be imaged is unstained, and a bright-field microscope when the imaged specimen is stained. The camera may be any known imaging device that can be incorporated into the optical system of the microscope, such as a CCD camera, a CMOS camera, or a hyperspectral camera. Furthermore, the imaging means may also include, for example, imaging moving means for moving the smear specimen, the microfluidic device, the microscope, and the camera vertically (Z-axis) and / or horizontally (XY-axis). Such imaging moving means may include a revolving nosepiece, a motor, and automatic control devices (sensors, etc.) thereof. Furthermore, the microscope may also be equipped with, as needed, illumination for the microscope (LED, halogen lamp, etc.); insertion and ejection devices for the smear specimen (e.g., glass slide) or microfluidic device; output means (display, output port, etc.) and storage means (semiconductor memory, magnetic memory, optical memory, etc.) for the captured image; and an automatic oil injection device for the oil immersion objective lens.

[0088] The detection means is preferably real-time object detection. Examples of real-time object detection algorithms include sliding window detection algorithms; region proposal detection algorithms such as R-CNN (Region CNN) and Cascade R-CNN; and end-to-end detection algorithms such as Faster R-CNN, YOLO (You Only Look Once), SSD (Single Shot Multibox Detector), RetinaNet, EfficientDet, CenterNet, DETR (DEtection Transformers), M2Det, and Swin Transformer. Among these, detection algorithms using machine learning models (more preferably supervised machine learning models) are preferred, for example, Faster R-CNN, YOLO, and Swin Transformer are preferred, but the system is not limited to these. By employing a machine learning model, for example, by performing machine learning (preferably deep learning) on ​​images containing red blood cells other than platelets and images of platelets, platelets can be identified and detected from images captured by the imaging means.

[0089] Specific examples of such detection means include, for instance, a processor (CPU, GPU, etc.) and main memory (semiconductor memory, magnetic memory, etc.) equipped with the detection algorithm, and may further include a network connection port, means for outputting detection results (display, output port, etc.) and means for storing them (semiconductor memory, magnetic memory, optical memory, etc.).

[0090] The discrimination means is preferably discrimination by real-time object discrimination. The algorithm for real-time object discrimination is, for example, not limited to, discrimination algorithms using machine learning models such as YOLO, Faster R-CNN, ResNet, VGG, DenseNet, DETR (DEtection Transformers), and Swin Transformer (preferably supervised machine learning models). By employing the machine learning model, for example, by machine learning images of degranulated platelets and images of rich platelets, it is possible to distinguish between degranulated platelets and rich platelets from the images captured by the imaging means. The preferred criteria for distinguishing between degranulated platelets and rich platelets are basically as described above, but in this case, the machine learning may preferably be deep learning, and discrimination may be performed based on the criteria explored by it.

[0091] Specific examples of such discrimination means include, for instance, a processor (CPU, GPU, etc.) and main memory (semiconductor memory, magnetic memory, etc.) equipped with the discrimination algorithm, and may further include a network connection port, means for outputting detection results (display, output port, etc.) and means for storing them (semiconductor memory, magnetic memory, optical memory, etc.).

[0092] Furthermore, the detection means and discrimination means may also be, for example, employ algorithms such as YOLO, Faster R-CNN, DETR (DEtection Transformers), and Swin Transformer, so that they combine the functions of each other to perform detection and discrimination at once, resulting in a "means for simultaneously detecting platelets from images captured by the imaging means and determining whether they are degranulated platelets or rich granular platelets." In this case, for example, by using machine learning on images containing red blood cells other than platelets, images of degranulated platelets, and images of rich granular platelets, the imaging means can distinguish and detect degranulated platelets and rich granular platelets from the images captured by the imaging means. In this case as well, the preferred criteria for distinguishing between degranulated platelets and rich granular platelets are basically as described above, but the machine learning may preferably be deep learning, and the discrimination may be performed based on the criteria explored by it.

[0093] The calculation means is not particularly limited as long as it can count the number of degranulated platelets B (and b if necessary) and the number of rich platelets A (and a if necessary) determined by the discrimination means and calculate an index ratio (the ratio of A, the ratio of B, the effective platelet rate, preferably the effective platelet rate).

[0094] The detection means, the discrimination means, and the calculation means are preferably executed on a PC equipped with a GPU, and more preferably on a small single-board computer (such as Jetson (NVIDIA), Raspberry Pi (Raspberry Pi Ltd), Arduino, etc.). Such a PC may further include a storage device (such as semiconductor memory or magnetic memory), a network connection port, input / output means (such as a mouse, keyboard, display, input / output ports, or printer), a power supply, etc.

[0095] In one embodiment of such a platelet evaluation apparatus, for example, a smear (e.g., a glass slide) or a microfluidic device after the sample supply step is inserted into the apparatus, its position is adjusted as needed by the imaging moving means, and an image is captured by the imaging means. From the captured image, the detection means and determination means (or means that combine both) detect and determine degranulated platelets and rich granulated platelets, respectively, and the measurement means measures the number A and B (and numbers a and b as needed) of each platelet and records them as needed. From the obtained numbers, the calculation means calculates a desired ratio, the result is output by the output means (e.g., screen display), and recorded as needed, and the inserted smear or microfluidic device is ejected from the apparatus. A conceptual diagram showing the processing sequence of such a platelet evaluation apparatus is shown in Figure 6, but the platelet measurement apparatus according to the present invention is not limited thereto.

[0096] The above-mentioned device is a platelet evaluation device used for the platelet evaluation method of the present invention described above, but it is not limited to being used for other purposes. For example, the device described as a platelet evaluation device can also be used as a device for measuring the number of granular platelets and degranular platelets in a smear prepared from a sample (preferably peripheral blood) in which the platelet aggregation inhibition step and the platelet activation step were not performed, or in a microfluidic device to which the sample was supplied, and calculating and outputting their ratios. This makes it possible, for example, to easily and quickly obtain the ratio of granular platelets and / or degranular platelets in a smear prepared from a patient's peripheral blood or in a microfluidic device and use it as an indicator for platelet evaluation.

[0097] <Kit> The present invention also provides a platelet evaluation method, a platelet measurement method, a platelet measurement device, or a kit for use with the platelet evaluation device described above. The kit of the present invention preferably contains the platelet aggregation inhibitor and the platelet activating substance, and more preferably further contains the staining dye or the staining solution. The platelet aggregation inhibitor and the platelet activating substance may be independent of each other, or they may be a composition containing both, and each may be a composition containing other components or solvents as needed. In the kit of the present invention, the platelet aggregation inhibitor, the platelet activating substance, and their compositions, as well as the dye or staining solution, may each be a solid (powder, etc.) or a concentrate so that they can be diluted and used independently at the time of use.

[0098] Furthermore, the kit may further include at least one selected from the group consisting of: a sample preservation solution; a sample diluent; diluents for the platelet aggregation inhibitor, the platelet activator, the composition, and the dye; the fixative; the washing solution; a transparent substrate for observation (slide glass, plate, etc.) for preparing the smear specimen; the microfluidic device; and instructions for use of the kit. [Examples]

[0099] The present invention will be described more specifically below based on examples, but the present invention is not limited to the following examples.

[0100] <Test Example 1> (Preparation of rat blood samples) Blood was collected from rats in a syringe pre-filled with 2 mL of ACD-A solution (aqueous solution of 2.20 w / v% sodium citrate hydrate, 0.80 w / v% citric acid hydrate, and 2.20 w / v% glucose) to a total blood volume of 10 mL. Blood collected on the day of collection (within 4 hours of collection) was designated as the "day-of-collection blood." The day-of-collection blood was centrifuged at 200 G for 10 minutes to separate it into a lower layer containing blood cells and an upper layer containing platelets. The upper layer was collected from near the interface between the lower and upper layers (buffy coat) to prepare platelet-rich plasma (PRP) from the day-of-collection blood. Furthermore, the remaining upper layer was centrifuged at 1,000 G for 5 minutes, and the resulting upper layer was collected to prepare platelet-poor plasma (PPP) from the day-of-collection blood.

[0101] Furthermore, blood was collected from another rat using a syringe filled with 2 mL of the ACD-A solution, in the same manner as above, to obtain a total blood volume of 10 mL. This blood was then stored at room temperature for 48 hours to obtain standing blood. From this standing blood, platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared, respectively, in the same manner as with the blood collected on the same day.

[0102] (Example 1) (1) Blood PRP on the day 5 μL of platelet-rich plasma (PRP) prepared as described above was spread onto a glass slide and air-dried to prepare a control smear (control specimen).

[0103] Furthermore, the same volume of 100 mM EDTA-2Na solution was added to the PRP of the same day's blood prepared as described above, and the mixture was shaken at 37°C for 15 minutes to inhibit GPIIb / IIIa. After the inhibition treatment, the ACD-A solution was added to the treatment solution and stirred, and the supernatant was removed by centrifugation to wash the platelets. The platelet-poor plasma (same-day blood PPP) prepared as described above was added to the same volume as the same-day blood PRP used in this preparation, and the mixture was resuspended to obtain GPIIb / IIIa-inhibited PRP. Next, 10 μL of 550 μM ADP solution was added to 100 μL of GPIIb / IIIa-inhibited PRP and the mixture was shaken at 37°C for 5 minutes to obtain platelet-activated PRP. Next, 5 μL of platelet-activated PRP was spread onto a glass slide and air-dried to prepare a smear specimen (post-reaction specimen) for measurement.

[0104] The prepared control specimens and post-reaction specimens were first immersed in a staining bottle containing May-Geemsa solution (Muto Chemical Co., Ltd.) for 4 minutes and allowed to stand. Then, they were immersed in a staining bottle containing May-Geemsa solution diluted with an equal volume of 1 / 150 M phosphate buffer (pH 6.4) for 3 minutes and allowed to stand. The May-Geemsa solution contains approximately 99% methanol as a fixative (the same applies below). Next, after washing with water, the specimens were immersed in a staining bottle containing diluted Giemsa solution (Muto Chemical Co., Ltd.) for 20 minutes and allowed to stand. After washing with water, they were immediately air-dried to obtain May-Geemsa stained specimens. The preparation of blood PRP and staining were performed on the same day.

[0105] Each stained specimen was observed using a light microscope, and of the 500 or more platelets on each specimen, platelets with low or no granule content (degranulated platelets) and other platelets (granulated platelets) were classified according to the following criteria: Degranulated platelets: Platelets in which a phenomenon of centralization of stained granules is observed, or the number of stained granules is 5 or less, or the proportion of stained granules in the cytoplasm is 10% or less in area on the microscopic field of view. Granular platelets: Platelets in which the centralization phenomenon of stained granules is not observed, the number of stained granules is 6 or more, and the proportion of stained granules in the cytoplasm exceeds 10% in terms of the area of ​​the microscopic field of view. The plates were classified, and the number of each type was measured. The results are shown in Table 1 below.

[0106] [Table 1]

[0107] Let a be the number of granular platelets measured in the control sample, A be the number of granular platelets measured in the post-reaction sample, b be the number of degranulated platelets measured in the control sample, and B be the number of degranulated platelets measured in the post-reaction sample. Then, the following formula applies: Control granule content = a / (a+b) Degranulation rate after reaction = B / (A+B) Effective platelet percentage = (Control rich granule percentage) × (Post-reaction degranulation percentage) = aB / {(a+b)(A+B)} The effective platelet rate was calculated using the following method. As a result, in the same-day blood PRP, the control rich granule rate (469 / (469+53)) was 0.898 (89.8%), and the post-reaction degranulation rate (458 / (101+458)) was 0.819 (81.9%). Furthermore, the effective platelet rate (469×458 / {(469+53)(101+458)}), which indicates the percentage of platelets capable of releasing granules in response to platelet activators, was calculated to be 0.736 (73.6%).

[0108] (2) Neglected blood PRP 5 μL of platelet-rich plasma (PRP) prepared from blood left to stand as described above was spread onto a glass slide and air-dried to prepare a control smear (control specimen).

[0109] Furthermore, the same volume of 100 mM EDTA-2Na solution was added to the PRP prepared above, and the mixture was shaken at 37°C for 15 minutes to inhibit GPIIb / IIIa. After the inhibition treatment, the ACD-A solution was added to the treated solution and stirred, and the supernatant was removed by centrifugation to wash the platelets. The platelet-poor plasma (PPP) prepared above from the blood was added so that the total volume was the same as the PRP used in this preparation, and the mixture was resuspended to obtain GPIIb / IIIa-inhibited PRP. Next, 10 μL of 550 μM ADP solution was added to 100 μL of GPIIb / IIIa-inhibited PRP, and the mixture was shaken at 37°C for 5 minutes to obtain platelet-activated PRP. Next, 5 μL of platelet-activated PRP was spread onto a glass slide and air-dried to prepare a smear specimen (post-reaction specimen) for measurement.

[0110] Except for using the prepared control and post-reaction specimens, May-Giemsa staining and measurement of each platelet count were performed in the same manner as described in (1) of the blood PRP on the day of the reaction. The results are shown in Table 2 below.

[0111] [Table 2]

[0112] In the same manner as described in (1) above for PRP of blood collected on the day of treatment, the control rich granule rate, the degranulation rate after reaction, and the effective platelet rate were calculated. As a result, for PRP of blood that had been left standing, the control rich granule rate was 0.460 (46.0%), the degranulation rate after reaction was 0.671 (67.1%), and the effective platelet rate was calculated to be 0.309 (30.9%). As described above, it is known that the performance of platelets (aggregation ability, etc.) decreases in platelets in blood that has been left standing. Similarly, in the comparison of effective platelet rates obtained by the method of the present invention, it was confirmed that the effective platelet rate in blood that had been left standing decreased (decreased by more than 40%) compared to blood collected on the day of treatment. Therefore, the effectiveness of adopting the percentage of platelets that have the ability to release granules by platelet activating substances (granule release ability) (e.g., effective platelet rate) as an index for evaluating platelets has been demonstrated. According to the method of the present invention, this can be measured easily by direct observation of cells.

[0113] <Test Example 2> (Preparation of human blood samples) Blood was collected from healthy volunteers in a syringe pre-filled with 2 mL of ACD-A solution (aqueous solution of 2.20 w / v% sodium citrate hydrate, 0.80 w / v% citric acid hydrate, and 2.20 w / v% glucose) to obtain a total blood volume of 10 mL. Blood collected on the day of collection (within 4 hours of collection) was designated as the "daily blood." The "daily blood" was centrifuged at 200G for 10 minutes to separate it into a lower layer containing blood cells and an upper layer containing platelets. The upper layer was collected from near the interface between the lower and upper layers (buffy coat) to prepare platelet-rich plasma (PRP) from the "daily blood."

[0114] Furthermore, blood was collected from another healthy volunteer using a syringe filled with 2 mL of the ACD-A solution, in the same manner as above, to obtain a total blood volume of 10 mL. This blood was then stored at room temperature for 48 hours to obtain standing blood. Platelet-rich plasma (PRP) was prepared from this standing blood in the same manner as the blood collected on the same day.

[0115] (Example 2) (1) Blood PRP on the day 5 μL of platelet-rich plasma (PRP) prepared as described above was spread onto a glass slide and air-dried to prepare a control smear (control specimen).

[0116] Furthermore, 240 μL of the PRP of the blood sample prepared above was transferred to a separate container, 5 μL of 100 μg / mL tirofiban solution was added, and the mixture was shaken at 37°C for 10 minutes to inhibit GPIIb / IIIa, thereby obtaining GPIIb / IIIa-inhibited PRP. Next, 5 μL of 500 μM ADP solution was added to the GPIIb / IIIa-inhibited PRP and the mixture was shaken at 37°C for 5 minutes to obtain platelet-activated PRP. Then, 5 μL of platelet-activated PRP was spread onto a glass slide and air-dried to prepare a smear (post-reaction specimen) for measurement.

[0117] The prepared control and post-reaction specimens were first immersed in a staining bottle containing May-Geemsa solution (Muto Chemical Co., Ltd.) for 4 minutes and allowed to stand. Then, they were immersed in a staining bottle containing May-Geemsa solution diluted with an equal volume of 1 / 150 M phosphate buffer (pH 6.4) for 3 minutes and allowed to stand. Next, after washing with water, they were immersed in a staining bottle containing diluted Giemsa solution (Muto Chemical Co., Ltd.) for 20 minutes and allowed to stand. After washing with water, they were immediately air-dried to obtain May-Geemsa stained specimens. The preparation of blood PRP and staining were performed on the same day.

[0118] Each stained specimen was observed using a light microscope, and of the 200 or more platelets on each specimen, platelets with low or no granule content (degranulated platelets) and other platelets (granulated platelets) were classified according to the following criteria: Degranulated platelets: Platelets in which a phenomenon of centralization of stained granules is observed, or the number of stained granules is 5 or less, or the proportion of stained granules in the cytoplasm is 10% or less in area on the microscopic field of view. Granular platelets: Platelets in which the centralization phenomenon of stained granules is not observed, the number of stained granules is 6 or more, and the proportion of stained granules in the cytoplasm exceeds 10% in terms of the area of ​​the microscopic field of view. The plates were classified, and the number of each type was measured. The results are shown in Table 3 below.

[0119] [Table 3]

[0120] Let a be the number of granular platelets measured in the control sample, A be the number of granular platelets measured in the post-reaction sample, b be the number of degranulated platelets measured in the control sample, and B be the number of degranulated platelets measured in the post-reaction sample. Then, the following formula applies: Control granule content = a / (a+b) Degranulation rate after reaction = B / (A+B) Effective platelet percentage = (Control rich granule percentage) × (Post-reaction degranulation percentage) = aB / {(a+b)(A+B)} The effective platelet rate was calculated using the following method. As a result, in the same-day blood PRP, the control rich granule rate (187 / (187+14)) was 0.930 (93.0%), and the post-reaction degranulation rate (223 / (34+223)) was 0.868 (86.8%). Furthermore, the effective platelet rate (187×223 / {(187+14)(34+223)}), which indicates the percentage of platelets capable of releasing granules in response to platelet activators, was calculated to be 0.807 (80.7%).

[0121] (2) Neglected blood PRP 5 μL of platelet-rich plasma (PRP) prepared from blood left to stand as described above was spread onto a glass slide and air-dried to prepare a control smear (control specimen).

[0122] Furthermore, 240 μL of the blood PRP prepared above was transferred to a separate container, 5 μL of 100 μg / mL tirofiban solution was added, and the mixture was shaken at 37°C for 10 minutes to inhibit GPIIb / IIIa, thereby obtaining GPIIb / IIIa-inhibited PRP. Next, 5 μL of 500 μM ADP solution was added to the GPIIb / IIIa-inhibited PRP and the mixture was shaken at 37°C for 5 minutes to obtain platelet-activated PRP. Then, 5 μL of platelet-activated PRP was spread onto a glass slide and air-dried to prepare a smear (post-reaction specimen) for measurement.

[0123] Except for using the prepared control and post-reaction specimens, May-Giemsa staining and measurement of each platelet count were performed in the same manner as described in (1) of the blood PRP on the day of the reaction. The results are shown in Table 4 below.

[0124] [Table 4]

[0125] In the same manner as described in (1) above for PRP of blood collected on the day of administration, the control rich granule rate, the degranulation rate after the reaction, and the effective platelet rate were calculated. As a result, for PRP of blood that had been left standing, the control rich granule rate was 0.735 (73.5%), the degranulation rate after the reaction was 0.541 (54.1%), and the effective platelet rate was calculated to be 0.398 (39.8%). Thus, in human blood samples, similar to the rat blood samples in Test Example 1, it was confirmed that the effective platelet rate decreased (decreased by more than 40%) in blood that had been left standing compared to blood collected on the day of administration. Therefore, in human blood samples as well, the effectiveness of adopting the percentage of platelets that have the ability to release granules by platelet activators (granule release ability) (e.g., effective platelet rate) as an indicator for evaluating platelets was demonstrated. [Industrial applicability]

[0126] As described above, the present invention makes it possible to provide a novel platelet evaluation index and platelet evaluation method that can directly and easily evaluate the activation ability of platelets in a sample, as well as a platelet measurement method and platelet evaluation apparatus for these purposes.

[0127] These new evaluation indicators, the platelet evaluation method of the present invention, and the platelet measurement method for them are useful for quality evaluation of blood products such as platelet preparations (PCs) and platelet-rich plasma (PRPs), and for monitoring the drug effects of antiplatelet drugs. Furthermore, since the platelet evaluation method, platelet measurement method, and platelet evaluation device of the present invention can be applied to small amounts of sample, they are also useful as POCT (point of care testing: a real-time testing system performed in a medical setting). In addition, since image-based automatic judgment can be used for the discrimination and measurement of granular platelets and degranular platelets, automation is easily possible. [Explanation of symbols]

[0128] 101, 102…Microfluidic device; 1…Supply port; 12…Outlet (Sample outlet after platelet aggregation inhibition process and / or platelet activation process); 22…Outlet (Control sample outlet); 13, 13'…Measurement unit (Sample measurement unit after platelet aggregation inhibition process and / or platelet activation process); 23, 23'…Measurement unit (Control sample measurement unit); 14, 24…Staining solution storage unit or supply port; 15, 25…Staining solution mixing unit; 16…Platelet aggregation inhibitor storage unit or supply port; 17, 18…Platelet aggregation inhibitor mixing unit; 19…Platelet activation unit storage unit or supply port; 20…Platelet activation unit mixing unit; 26…Gas supply port

Claims

1. The following steps: A platelet aggregation inhibition step involves adding a platelet aggregation inhibitor to a platelet-containing sample. A platelet activation step is performed by adding a platelet activating substance to the aforementioned sample. A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. An evaluation step in which the platelet activation ability in the sample is evaluated using the number of granular platelets A and / or the number of degranular platelets B as indicators. A platelet evaluation method, including the following.

2. The platelet evaluation method according to claim 1, further comprising a sample preparation step of preparing a smear of the sample after the platelet aggregation inhibition step and the platelet activation step, wherein the measurement of the number of granular platelets A and the number of degranular platelets B in the measurement step is performed on the smear.

3. The platelet evaluation method according to claim 2, wherein the specimen preparation step includes a fixation process for fixing the sample.

4. The platelet evaluation method according to claim 1, further comprising a sample supply step of supplying the sample into a microfluidic device, wherein the measurement of the number of rich granular platelets A and the number of degranular platelets B in the measurement step is performed on the microfluidic device.

5. The platelet evaluation method according to claim 4, wherein the sample supply step includes a fixation process for fixing the sample.

6. The platelet evaluation method according to claim 1, further comprising a staining step of staining platelet granules after the platelet aggregation inhibition step and the platelet activation step, and before the measurement step.

7. The platelet evaluation method according to claim 6, wherein the staining is staining with azure dye.

8. The platelet evaluation method according to claim 1, wherein the platelet activation step is performed after the platelet aggregation inhibition step, or the platelet aggregation inhibition step and the platelet activation step are performed simultaneously.

9. The aforementioned sample is further subjected to a control measurement step, in which the measurement step is performed without performing the platelet aggregation inhibition step and the platelet activation step, and the number of degranulated platelets with low or no granule content (b) and the number of granulated platelets other than the degranulated platelets (a) are measured. In the evaluation step described above, the platelet activation ability in the sample is evaluated using the effective platelet rate calculated by the following formula: effective platelet rate = aB / {(a+b)(A+B)} as an indicator. The platelet evaluation method according to claim 1.

10. The platelet evaluation method according to claim 9, further comprising a sample preparation step of preparing a smear of a sample in which the platelet aggregation inhibition step and the platelet activation step have not been performed, wherein the measurement of the number of granular platelets a and the number of degranular platelets b in the control measurement step is performed on the smear.

11. The platelet evaluation method according to claim 10, wherein the specimen preparation step includes a fixation process for fixing the sample.

12. The platelet evaluation method according to claim 9, further comprising a sample supply step of supplying a sample in which the platelet aggregation inhibition step and the platelet activation step have not been performed into a microfluidic device, wherein the measurement of the number of rich granular platelets a and the number of degranular platelets b in the control measurement step is performed on the microfluidic device.

13. The platelet evaluation method according to claim 12, wherein the sample supply step includes a fixation process for fixing the sample.

14. The platelet evaluation method according to claim 1, wherein the degranulated platelets are platelets in which the granules they contain are concentrated in the center of the cell, or in which the proportion of granules in the cytoplasm is 10% or less in terms of area in a two-dimensional image.

15. The platelet evaluation method according to claim 1, wherein the degranulated platelets contain five or fewer granules, or the proportion of granules in the cytoplasm is 10% or less in area in a two-dimensional image.

16. A measurement method for a platelet evaluation method according to any one of claims 1 to 15, The following steps: A platelet aggregation inhibition step involves adding a platelet aggregation inhibitor to a platelet-containing sample. A platelet activation step is performed by adding a platelet activating substance to the aforementioned sample. A measurement step is performed after the platelet aggregation inhibition step and the platelet activation step, to measure the number B of degranulated platelets with a low or no granule content and the number A of granulated platelets other than the degranulated platelets. A platelet counting method, including the following.

17. Apparatus for platelet evaluation method according to claim 2 or 4, An imaging means for capturing an image of the smear specimen or an image of the microfluidic device, A detection means for detecting platelets from an image captured by the imaging means, A determination means for determining whether the platelets detected by the detection means are degranulating platelets or rich granulating platelets. A calculation means that measures the number of degranulated platelets B and the number of rich platelets A determined by the discrimination means and calculates the ratio of the number of rich platelets A and / or the ratio of the number of degranulated platelets B. Output means for outputting the value calculated by the calculation means, A platelet evaluation device equipped with the following features.

18. The platelet evaluation apparatus according to claim 17, wherein the imaging in the imaging means is imaging by a microscope equipped with a camera.

19. The platelet evaluation apparatus according to claim 17, wherein the detection in the detection means is detection by a machine learning model.

20. The platelet evaluation apparatus according to claim 17, wherein the discrimination in the discrimination means is discrimination by a machine learning model.

21. The platelet evaluation apparatus according to claim 17, wherein the detection means and the discrimination means together are means for simultaneously detecting platelets from an image captured by the imaging means and determining whether they are degranulated platelets or rich platelets, and the detection and discrimination in the means are detection and discrimination by a machine learning model.

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