Composition, device and detection method for measuring number of platelets

Abstract

The invention relates to the technical field of platelet aggregation function detection, in particular to a composition, a device and a detection method for measuring the number of platelets. The composition for measuring the number of platelets provided by the invention comprises the following components: a platelet activator and optical particles, and the optical particles are coated with a bioactive material combined with a platelet surface receptor. The platelet activator and the optical particles coated with the bioactive material combined with the platelet surface receptor are compounded, in the presence of a blood sample, the platelets activated by the platelet activator aggregate with the optical particles, an optical signal is changed, and then the change of the number of the platelets can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of platelet aggregation function detection, and in particular to a composition, device and detection method for determining the number of platelets. Background Art

[0002] Platelet aggregation function is the process of platelets adhering to each other to form platelet plugs, which is often used to evaluate the function of blood coagulation or hemostasis. Under normal circumstances, when blood vessels are damaged, platelets will be activated and quickly adhere to the site of vascular damage. Subsequently, by releasing a series of chemical substances (such as ADP, thromboxane A2, etc.) and expressing adhesion molecules, platelets aggregate to form primary hemostatic plugs to prevent excessive bleeding. This function is essential for maintaining normal blood circulation and preventing post-traumatic blood loss. If the platelet aggregation function is abnormally enhanced, it may lead to an increased risk of thrombosis, which may in turn cause cardiovascular diseases such as myocardial infarction and stroke. On the contrary, if the platelet aggregation function is weakened, it may lead to bleeding tendency.

[0003] Currently, turbidimetry is mostly used to detect platelet aggregation function, but the effect of platelet number on platelet aggregation function is ignored, resulting in inaccurate test results of platelet aggregation function. Summary of the invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the defect in the prior art that the platelet aggregation function test results are inaccurate due to the inability to determine the platelet number, thereby providing a composition, device and detection method for determining the platelet number.

[0005] Another technical problem to be solved by the present invention is to overcome the defect of complicated platelet aggregation function detection steps in the prior art, thereby providing a composition, device and detection method for determining the number of platelets.

[0006] In one aspect, the present invention provides a composition for measuring the number of platelets, comprising the following components: a platelet activator and optical particles, wherein the optical particles are coated with a bioactive material that binds to a platelet surface receptor.

[0007] In order to activate platelets and make the platelet count more accurate, the embodiments of the present invention provide a platelet activator including at least one of an endogenous activator, an exogenous activator or a bioactive substance. Preferably, the platelet activator is at least one of adenosine diphosphate, adenosine diphosphate sodium salt, collagen, arachidonic acid, arachidonic acid sodium salt, ristocetin, epinephrine, and thrombin receptor activating peptide. Preferably, the concentration of the platelet activator in the composition is 2.5 to 4300 μg / mL based on the volume of the container containing the composition.

[0008] In some embodiments, the bioactive material includes at least one of proteins, polypeptides or amino acids and their derivatives that bind to platelet surface receptors. Preferably, the bioactive material is at least one of fibrinogen, fibronectin, vitronectin, collagen, laminin, and von Willebrand factor.

[0009] The size of the optical particles is 1 nm-100 μm, and the optical characteristic peak of the optical particles is greater than or equal to 700 nm. Based on the volume of the container containing the composition, the concentration of the optical particles is 5×10 5 / mL~5×10 10 Pieces / mL.

[0010] In some embodiments, the composition further comprises at least one of a stirrer or an auxiliary material. Preferably, the stirrer comprises at least one of a stainless steel ball, a ferrite, and a neodymium iron boron magnet. Preferably, the auxiliary material comprises at least one of a buffer salt, a protein, a carbohydrate, a surfactant, a high molecular polymer, or a chlorinated metal salt.

[0011] In some embodiments, the buffer salt is at least one of phosphate, hydroxyethylpiperazineethanesulfonic acid buffer salt, 2-(N-morpholine)ethanesulfonic acid buffer salt, and GOOD's buffer salt.

[0012] In some embodiments, the protein in the auxiliary material is at least one of gelatin, fetal bovine serum, bovine serum albumin, and human albumin.

[0013] In some embodiments, the carbohydrate is a sugar, and the sugar includes at least one of sucrose, trehalose, dextran, maltose, lactose, and galactose.

[0014] In some embodiments, the surfactant is a nonionic surfactant, and the nonionic surfactant is at least one of Tween 20, Tween 80, Triton 100, GEROPON T-77 and TETRONIC 1307.

[0015] In some embodiments, the high molecular weight polymer is at least one of polyvinyl pyrrolidone, polyvinyl alcohol, and cellulose.

[0016] In some embodiments, the halogenated metal salt is at least one of sodium chloride, potassium chloride, and lithium chloride.

[0017] On the other hand, the present invention also provides a platelet aggregation function detection device, comprising a detection area, wherein the detection area comprises a detection unit, and the detection unit comprises the above-mentioned composition for determining the number of platelets.

[0018] In some of the embodiments, the detection unit includes at least three detection chambers, and the number of optical particles in each detection chamber is arranged in a gradient.

[0019] In some embodiments, the detection device further includes a sample tube storage area, and a puncture needle is disposed in the sample tube storage area.

[0020] In some embodiments, the detection device further comprises a sample heating zone for heating the blood sample, and the sample heating zone flows the heated blood sample into the detection zone.

[0021] In some of the embodiments, the detection area further includes a control unit, which includes an optical module for providing a background signal for detecting optical signal characteristic parameters of platelet aggregation function.

[0022] Preferably, the control unit comprises at least one blank detection chamber.

[0023] In some of the embodiments, an optical module is provided in the detection chamber of the detection unit and / or the blank detection chamber of the control unit, and the optical module is used to emit light of a specific wavelength to one side of each detection chamber, receive the transmitted light on the opposite side of the detection chamber, and obtain an optical signal.

[0024] In some of the embodiments, each of the detection chambers is further provided with a stirring module, and the stirring module is used to control the stirring rate of the stirrer in each detection chamber.

[0025] In some embodiments, the detection device also includes a heating module, a liquid driving module and a timing module, wherein the heating module is used to heat the sample in the sample heating area and maintain the temperature of the detection area during platelet aggregation function detection, the liquid driving module is used to drive the blood sample in the sample tube storage area to the detection area and discharge the gas in the detection area, and the timing module is used to record the detection time of the platelet aggregation function.

[0026] On the other hand, the present invention also provides a method for detecting platelet aggregation function. The detection method is based on the above-mentioned platelet aggregation function detection device. The blood sample is injected into the detection area, and the platelet count is measured based on the relationship between the number gradient of optical particles in each detection chamber in the detection area and the gradient of the corresponding optical signal characteristic parameters obtained by detection.

[0027] In some of the embodiments, before the blood sample is injected into the detection area, a step of heating the blood sample is included, and the heating temperature is 36-40°C.

[0028] In some embodiments, the characteristic parameter of the optical signal is at least one of the maximum value of the optical signal, the time to reach the maximum optical signal, the area under the optical signal curve, the maximum slope of the curve formed by the optical signal, and the time to reach the maximum slope of the curve formed by the optical signal.

[0029] When there are three detection chambers, when the maximum optical characteristic parameter of the second detection chamber is less than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is low, and the test result is output based on the optical signal of the first detection chamber 1;

[0030] If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber 1, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, the platelet count is considered normal, and the test result is output based on the optical characteristic parameter of the second detection chamber;

[0031] If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is greater than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is too high, and the test result is output based on the optical characteristic parameter of the third detection chamber.

[0032] The technical solution of the present invention has the following advantages:

[0033] 1. The present invention provides a composition for measuring the number of platelets, comprising the following components: a platelet activator and optical particles, wherein the optical particles are coated with a bioactive material that binds to platelet surface receptors. The present invention uses a platelet activator and optical particles coated with a bioactive material that binds to platelet surface receptors. In the presence of a blood sample, platelets activated by the platelet activator aggregate with the optical particles, resulting in changes in the optical signal, thereby obtaining changes in the number of platelets.

[0034] 2. A platelet aggregation function detection method provided by the present invention, wherein a blood sample is injected into a detection area, and an optical characteristic parameter is obtained by detection of a detection unit in the detection area to output the platelet aggregation function, wherein the detection unit contains the above-mentioned composition for determining the number of platelets. The present invention utilizes the aggregation reaction between activated platelets and optical particles coated with receptor-binding bioactive materials, and the change in the number of platelets can be intuitively obtained through optical signals, thereby determining whether the platelet aggregation function needs to be corrected according to the number of platelets to obtain accurate platelet aggregation function.

[0035] 3. The present invention provides a platelet aggregation function detection device, comprising a detection area, wherein the detection area comprises a detection unit, wherein the detection unit contains the above-mentioned composition for determining the number of platelets. The detection device provided by the present invention is a microfluidic card, which is easy and fast to operate and has a short detection time. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 It is a process flow chart of the method for detecting platelet aggregation function of the present invention;

[0038] Figure 2 is a schematic diagram of the structure of a detection device for platelet aggregation function in an embodiment of the present invention;

[0039] Figure 3 Schematic diagram of the structure of a detection device for platelet aggregation function in another embodiment of the present invention

[0040] Reference numerals:

[0041] 1-sample tube storage area; 11-puncture needle;

[0042] 2-testing area; 21-first test room; 22-second test room;

[0043] 23-third detection chamber; 24-blank detection chamber;

[0044] 3-sample heating area; 4-exhaust port; 5-gas outlet. DETAILED DESCRIPTION

[0045] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0046] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0047] See also Figure 1The present invention provides a platelet aggregation function detection method, which transports a blood sample to a detection area for detection, and outputs a platelet aggregation function detection result based on the optical properties obtained by the detection.

[0048] The detection time is 2-3 minutes.

[0049] The present invention also provides a platelet aggregation function detection device, comprising a detection area, wherein the detection area comprises a detection unit and a control unit.

[0050] The detection unit is used to calibrate platelet aggregation function.

[0051] The control unit is used for detecting background signals to avoid detection errors of the detection unit.

[0052] In some embodiments, the detection unit includes at least 3 detection chambers. The more detection chambers are set, the more accurate the platelet count will be, and the more accurate the output platelet aggregation function will be.

[0053] The volume of each detection chamber is 80-120μL.

[0054] Each detection chamber contains a composition for measuring the number of platelets, wherein the composition comprises a platelet activator and optical particles coated with peptides binding to glycoprotein receptors on the surface of platelets.

[0055] Based on the volume of the detection chamber, the concentration of the platelet activator is 2.5-4300 μg / mL, and the concentration of the optical particles is 5×10 5 / mL~5×10 10 Pieces / mL.

[0056] In some of the embodiments, the number of optical particles in each detection chamber is arranged in a gradient.

[0057] In order to obtain optical signals, in some embodiments, an optical module is provided in each detection chamber, and the optical module is used to emit light of a specific wavelength to one side of each detection chamber, receive transmitted light on the opposite side of the detection chamber, and obtain optical signals.

[0058] In some embodiments, the detection unit includes a first detection chamber, a second detection chamber, and a third detection chamber, wherein the number of optical particles in the first detection chamber is 1×10 7 The number of optical particles in the second detection chamber is 2×10 7 The number of optical particles in the third detection chamber is 3×10 7 indivual.

[0059] The present embodiment does not limit the position of each detection chamber, as long as the number of optical particles between the detection chambers is gradient. In some embodiments, the number of optical particles in the first detection chamber is 5×10 7 The number of optical particles in the second detection chamber is 10×10 7 The number of optical particles in the third detection chamber is 15×10 7 indivual.

[0060] In some embodiments, the detection unit includes a first detection chamber, a second detection chamber, a third detection chamber, and a fourth detection chamber, wherein the number of optical particles in the first detection chamber is 1×10 7 The number of optical particles in the second detection chamber is 2×10 7 The number of optical particles in the third detection chamber is 3×10 7 The number of optical particles in the fourth detection chamber is 4×10 7 indivual.

[0061] In some embodiments, the detection unit includes a first detection chamber, a second detection chamber, a third detection chamber, a fourth detection chamber, and a fifth detection chamber, wherein the number of optical particles in the first detection chamber is 5×10 6 The number of optical particles in the second detection chamber is 1×10 7 The number of optical particles in the third detection chamber is 3×10 7 The number of optical particles in the fourth detection chamber is 2×10 7 The number of optical particles in the fifth detection chamber is 4×10 7 indivual.

[0062] In some embodiments, the control unit includes a blank detection chamber, which does not contain any reagent and is used for detecting background signals.

[0063] In some embodiments, the platelet activator can be at least one of adenosine diphosphate, adenosine diphosphate sodium salt, collagen, collagen protein, arachidonic acid, arachidonic acid sodium salt, ristocetin, epinephrine, and thrombin receptor activating peptide;

[0064] The preparation method of the optical particles coated with the binding peptide of the platelet surface glycoprotein receptor can be a physical adsorption or chemical coupling method.

[0065] Specifically, the physical adsorption method is to dilute the optical particles into a 1 mg / mL suspension with 10 mM PBS at pH 7.4, and then dissolve the binding peptide of the platelet surface glycoprotein receptor into a 10 mg / mL solution with 10 mM PBS at pH 7.4. Take 1 volume of the optical particle suspension and the binding peptide solution, vortex mix, and stir overnight at room temperature. Centrifuge and remove the supernatant. Add twice the volume of the supernatant 10 mg / mL bovine serum albumin solution, stir and seal at room temperature for 30 minutes. Centrifuge and remove the supernatant to obtain the optical particles coated with the binding peptide of the platelet surface glycoprotein receptor. Dilute with a suitable buffer to a suitable concentration for detection.

[0066] The chemical coupling method is as follows: dilute the optical particles into a 1 mg / mL suspension with 10 mM pH 4.0 PBS, take 1 volume of 10 mg / mL EDC solution (dissolved in 10 mM pH 4.0 PBS) and 10 mg / mL NHS solution (dissolved in 10 mM pH 4.0 PBS), vortex mix, and activate at room temperature for 30 min. Centrifuge and remove the supernatant. Dissolve the binding peptide of the platelet surface glycoprotein receptor into a 10 mg / mL solution with 10 mM pH 7.4 PBS, redisperse the optical particles with 1 volume of 10 mM pH 7.4 PBS, add 1 volume of the binding peptide solution, vortex mix, and stir at room temperature for 4 h. Centrifuge and remove the supernatant. Add twice the volume of the supernatant 10 mg / mL bovine serum albumin solution, stir and block at room temperature for 30 min. Centrifuge and remove the supernatant to obtain the optical particles coated with the binding peptide of the platelet surface glycoprotein receptor. Dilute to a suitable concentration with a suitable buffer for detection.

[0067] More specifically, in order to make the binding peptide coated with the optical particles match with each detection chamber, in some embodiments, the amount of each reagent in the preparation method of the optical particles coated with the binding peptide of the platelet surface glycoprotein receptor is 100mL / m 3 , where mL / m 3 It is the ratio of the volume of each reagent to the volume of each detection chamber. For example, when the volume of each detection chamber is 100 μL, 1 volume is 0.01 mL.

[0068] The binding peptides of the platelet surface glycoprotein receptors include fibrinogen, fibronectin, vitronectin, collagen, laminin, von Willebrand factor (vWF), or polypeptides based on the amino acid sequences of these proteins for binding to platelet surface receptors and related derivatives.

[0069] The size of the optical particles is 1 nm-100 μm. The optical characteristic peak of the optical particles is above 700 nm, preferably 700-1000 nm.

[0070] The composition further comprises a stirring bar and auxiliary materials.

[0071] The stirring bar can be a stainless steel ball, a ferrite, or a neodymium iron boron magnet. The number of stirring bars in each detection chamber is at least one, preferably one, two, three, four, five or ten.

[0072] The auxiliary material is at least one of buffer salt, protein, carbohydrate, surfactant, high molecular polymer or halogenated metal salt.

[0073] The buffer salt includes at least one of phosphate buffer (PBS), hydroxyethylpiperazineethanesulfonic acid buffer (HEPES), 2-(N-morpholino)ethanesulfonic acid buffer (MES), and GOOD's buffer salt.

[0074] The concentration of buffer salt is 10-500 nM.

[0075] The protein includes at least one of gelatin, fetal bovine serum, bovine serum albumin and human albumin, and the concentration of the protein is 0.1-10wt%.

[0076] The carbohydrates include sugars, and the sugars include at least one of sucrose, trehalose, dextran, maltose, lactose, and galactose. The concentration of the carbohydrates is 1-30 wt%.

[0077] The surfactant includes at least one of Tween 20, Tween 80, Triton 100, GEROPON T-77 (S5) and TETRONIC 1307 (S9), and the concentration of the surfactant is 0.01-1 wt %.

[0078] The organic polymer includes at least one of polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), and cellulose, and the concentration of the organic polymer is 0.1-10 wt %.

[0079] The inorganic substance includes at least one of sodium chloride, potassium chloride and lithium chloride, and the concentration of the inorganic substance is 0.1-2wt%.

[0080] In order to control the stirring rate of the stirrer in each detection chamber, the detection device is further provided with a stirring module. In this embodiment, the stirring module is arranged at the bottom of the detection area.

[0081] The detection device also includes a sample tube compartment, in which a puncture needle is arranged, and the puncture needle is used to puncture the rubber in the tube cap of the sample tube.

[0082] The sample tube storage area is connected to the detection area, and the sample in the sample tube storage area flows into the detection area for detection.

[0083] The detection device also includes a sample heating area, which is connected to the sample tube storage area and the detection area respectively, and is used to heat the blood sample flowing into the sample heating area, and flow the heated blood sample into the detection area to detect the platelet aggregation function.

[0084] In order to heat the blood sample in the sample heating zone while maintaining the temperature of the detection zone during platelet aggregation function detection, the detection device further includes a heating module. The heating module covers the sample heating zone and the detection zone. Further, the heating module provided by the present invention needs to be arranged away from the detection light path.

[0085] The heating temperature of the sample heating zone and the temperature of the blood sample during the detection process are 36-40°C.

[0086] In some embodiments, the detection device further comprises a liquid driving module, wherein the liquid driving module is used to drive the blood sample into each detection chamber and discharge the gas in each detection chamber from the exhaust port.

[0087] In some of the embodiments, the detection device further comprises a timing module for recording the detection time of the platelet aggregation function.

[0088] Working principle:

[0089] The blood sample enters the detection unit and the control unit respectively. Through the optical properties of the optical particles in each detection chamber in the detection unit and the fibrinogen coated on the optical particles, they can be coupled with the platelets activated by the platelet activator in the blood sample, thereby causing the light signal in the detection unit to change. Since the number of optical particles in each detection chamber in the detection unit is set in a gradient, the platelet number is determined based on the relationship between the number gradient of the optical particles in the detection chamber and the gradient of the corresponding light signal. Specifically, based on the relationship between the optical characteristic parameter results of the blood sample in each detection unit and the number of optical particles, the platelet aggregation function result is output, and the control unit is used as the background parameter to correct the result.

[0090] The more detection rooms are set, the more accurate the identification of platelet count will be, and the output platelet aggregation function will be more accurate.

[0091] When the detection unit has three detection chambers, the judgment method for the result output is that when the maximum optical characteristic parameter of the second detection chamber is less than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is low, and the detection result is output based on the optical signal of the first detection chamber;

[0092] If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, the platelet count is considered normal, and the test result is output based on the optical characteristic parameter of the second detection chamber;

[0093] If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is greater than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is too high, and the test result is output based on the optical characteristic parameter of the third detection chamber.

[0094] The optical characteristic parameters include at least one of the maximum slope of the optical signal, the maximum value of the optical signal, the area under the optical curve, the time to reach the maximum slope, and the time to reach the maximum optical signal.

[0095] Example 1

[0096] See also Figure 2 The present invention provides a platelet aggregation function detection device, including a detection area, wherein the detection area includes a detection unit and a control unit, the detection unit is used to correct the platelet aggregation function detection result, and the control unit is used to detect background signals to avoid detection errors of the detection unit.

[0097] The detection unit includes a first detection chamber 21, a second detection chamber 22, and a third detection chamber 23. The control unit is a blank detection chamber 24. The volume of each detection chamber is 100 μL. An optical module is provided in each detection chamber. The optical module is used to emit light of a specific wavelength to one side of each detection chamber, receive the transmitted light on the opposite side of the detection chamber, and obtain an optical signal. The detection chamber of the detection unit includes a composition for determining the number of platelets, and the blank detection chamber does not contain any reagent.

[0098] The first detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffered salt freeze-dried powder, and optical particles coated with fibrinogen. The number of optical particles is 1×10 7 The second detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 2×10 7 The third detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 3×10 7The size is 50nm and the optical characteristic peak is 700nm; no material is added to the blank detection chamber.

[0099] The detection device is further provided with a stirring module, and the stirring module is used to control the stirring rate of the stirring bar in each detection chamber. In this embodiment, the stirring module is arranged at the bottom of the detection area.

[0100] The detection device further comprises a sample tube compartment 1, in which a puncture needle 11 is arranged, and the puncture needle is used to pierce the rubber in the tube cap of the sample tube to take out the blood sample from the sample tube.

[0101] The detection device further comprises a sample heating zone 3, which continues to heat the blood sample flowing into the zone, and the heating temperature is 37°C.

[0102] The sample tube storage area is connected to the detection unit and the control unit, and the sample in the sample tube storage area flows into each detection chamber and a blank detection chamber to detect the platelet aggregation function.

[0103] The detection device also includes a heating module, a liquid driving module and a timing module.

[0104] The heating module is used to heat the sample in the sample heating zone and maintain the temperature of the detection zone during platelet aggregation function detection.

[0105] The liquid driving module introduces the blood sample into each detection chamber.

[0106] Each detection chamber is also provided with an exhaust port, and the gas exhausted from the exhaust port of each detection chamber is collected at the gas outlet 5, and the liquid driving module exhausts the gas at the gas outlet.

[0107] The timing module is used to record the detection time of platelet aggregation function.

[0108] Example 2

[0109] See also Figure 3 The present invention provides a platelet aggregation function detection device, including a detection area, wherein the detection area includes a detection unit and a control unit, the detection unit is used to correct the platelet aggregation function detection result, and the control unit is used to detect background signals to avoid detection errors of the detection unit.

[0110] The detection unit includes a first detection chamber 21, a second detection chamber 22, and a third detection chamber 23, and the control unit includes a blank detection chamber. The volume of each detection chamber is 100 μL. An optical module is arranged in each detection chamber, and the optical module is used to emit light of a specific wavelength to one side of each detection chamber, receive the transmitted light on the opposite side of the detection chamber, and obtain an optical signal. The detection chamber of the detection unit includes a composition for determining the number of platelets, and the blank detection chamber does not contain any reagent.

[0111] The first detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffered salt freeze-dried powder, and optical particles coated with fibrinogen. The number of optical particles is 1×10 7 The second detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 2×10 7 The third detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 3×10 7 The size is 50nm and the optical characteristic peak is 700nm; no material is added to the blank detection chamber.

[0112] Each of the detection chambers is further provided with a stirring module, and the stirring module is used to control the stirring rate of the stirring bar in each detection chamber. In this embodiment, the stirring module is provided at one side of the bottom of the detection area.

[0113] The detection device further comprises a sample tube compartment 1, in which a puncture needle 11 is arranged, and the puncture needle is used to puncture the rubber in the tube cap of the sample tube.

[0114] The sample tube storage area is connected to the detection unit and the control unit, and the sample in the sample tube storage area flows into each detection chamber and a blank detection chamber to detect the platelet aggregation function.

[0115] The detection device is further provided with a stirring module, and the stirring module is used to control the stirring rate of the stirring bar in each detection chamber. In this embodiment, the stirring module is arranged at the bottom of the detection area.

[0116] The detection device also includes a heating module, a liquid driving module and a timing module. The heating module is used to heat the sample in the sample heating area and maintain the temperature of the detection area during the platelet aggregation function detection. The liquid driving module is used to drive the blood sample in the sample tube storage area to the target area. The timing module is used to record the detection time of the platelet aggregation function.

[0117] Example 3

[0118] The present embodiment provides a platelet aggregation function detection method, using the detection device of Example 1, inserting a blood sample into a sample tube compartment, the puncture needle in the sample tube compartment pierces the rubber in the sample tube cap, and the blood sample flows into the sample heating zone, so that the temperature of the blood sample is maintained at 37°C, and the blood sample is divided into four parts, which are respectively injected into the first detection chamber, the second detection chamber, the third detection chamber and the blank detection chamber. After 2 minutes, the maximum optical signals of the first detection chamber, the second detection chamber and the third detection chamber are read, and the optical signal parameters of the blank detection chamber are read at the same time as the background signal.

[0119] After reading the optical signal characteristic parameters of each detection chamber, the correction basis is: when the optical signal characteristic parameter of the second detection chamber is less than 1 / 2 of the optical signal characteristic parameter of the first detection chamber, and the optical signal characteristic parameter of the third detection chamber is less than 2 / 3 of the optical signal characteristic parameter of the second detection chamber, it is considered that the platelet count is low, and the test result is output based on the optical signal of the first detection chamber 1;

[0120] If the optical signal characteristic parameter of the second detection chamber is greater than 1 / 2 of the optical signal characteristic parameter of the first detection chamber 1, and the optical signal characteristic parameter of the third detection chamber is less than 2 / 3 of the optical signal characteristic parameter of the second detection chamber, the platelet count is considered normal, and the test result is output based on the optical signal of the second detection chamber;

[0121] If the optical signal characteristic parameter of the second detection chamber is greater than 1 / 2 of the optical signal characteristic parameter of the first detection chamber, and the optical signal characteristic parameter of the third detection chamber is greater than 2 / 3 of the optical signal characteristic parameter of the second detection chamber, it is considered that the platelet count is high, and the test result is output based on the optical signal of the third detection chamber.

[0122] Among them, the optical signal characteristic parameter in this embodiment is the maximum value of the optical signal.

[0123] Example 4

[0124] This embodiment provides a platelet aggregation function detection method, the device, steps, parameters and result determination method used are the same as those in Example 3, the characteristic parameters of the optical signal of each detection chamber read are the maximum slope of the optical signal and the components of the composition of each detection chamber of the detection unit are different, and the composition of the detection chamber of the detection unit is specifically as follows:

[0125] The first detection chamber contains 2.5 μg / mL collagen, 2 stainless steel beads, 20 nM HEPES buffered salt freeze-dried powder, and optical particles coated with fibronectin. The number of optical particles is 1×10 7The second detection chamber contains 2.5 μM collagen, 2 stainless steel beads, 20 nM HEPES buffer salt freeze-dried powder, and optical particles coated with fibronectin. The number of optical particles is 2×10 7 The third detection chamber contains 2.5 μM collagen, 2 stainless steel beads, 20 nM HEPES buffer salt freeze-dried powder, and optical particles coated with fibronectin. The number of optical particles is 3×10 7 The size of the particles is 1 nm, and the optical characteristic peak is 700 nm; no material is added to the blank detection chamber.

[0126] Example 5

[0127] This embodiment provides a platelet aggregation function detection method, and the device, steps, parameters and result determination method used are the same as those in Example 3, except that the optical signal characteristic parameter read from each detection chamber is the time to reach the maximum optical signal, and the components of the composition of each detection chamber of the detection unit are different. The specific composition is as follows:

[0128] The first detection chamber contains 4300 μg / mL epinephrine, 3 stainless steel beads, 500 nM MES buffer salt freeze-dried powder, and optical particles coated with collagen. The number of optical particles is 2×10 6 The second detection chamber contains 4300 μg / mL epinephrine, 3 stainless steel beads, 500 nM MES buffer salt freeze-dried powder, and optical particles coated with collagen. The number of optical particles is 4×10 6 The third detection chamber contains 4300 μg / mL epinephrine, 3 stainless steel beads, 500 nM MES buffer salt freeze-dried powder, and optical particles coated with collagen. The number of optical particles is 6×10 6 The size of the particles is 100 nm, and the optical characteristic peak is 1000 nm; no material is added to the blank detection chamber.

[0129] Example 6

[0130] This embodiment provides a platelet aggregation function detection method, and the device, steps, parameters and result determination method used are the same as those in Example 3, except that the optical signal characteristic parameter read from each detection chamber is the area under the optical signal curve, and the components of the composition of each detection chamber of the detection unit are different. The composition of each detection chamber is as follows:

[0131] The first detection chamber contains 100 μg / mL thrombin receptor activating peptide, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder, 10 wt% gelatin, 1 wt% Tween 80, 30 wt% glucose, 5 wt% PVP, 2 wt% sodium chloride, and optical particles coated with von Willebrand factor. The number of optical particles is 1×10 5 The second detection chamber contains 100 μg / mL thrombin receptor activating peptide, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder, 10 wt% gelatin, 1 wt% Tween 80, 30 wt% glucose, 5 wt% PVP, 2 wt% sodium chloride, and optical particles coated with von Willebrand factor. The number of optical particles is 4×10 5 The third detection chamber contains 100 μg / mL thrombin receptor activating peptide, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder, 10 wt% gelatin, 1 wt% Tween 80, 30 wt% glucose, 5 wt% PVP, 2 wt% sodium chloride, and optical particles coated with von Willebrand factor. The number of optical particles is 8×10 5 The size is 50nm and the optical characteristic peak is 700nm; no material is added to the blank detection chamber.

[0132] Example 7

[0133] This embodiment provides a platelet aggregation function detection method, and the device, steps, parameters and result determination method used are the same as those of Example 3, except that the optical signal characteristic parameter read from each detection chamber is the maximum slope of the curve formed by the optical signal, and the components of the composition of each detection chamber of the detection unit are different. The composition of each detection chamber of the detection unit is as follows:

[0134] The first detection chamber contains 100 μg / mL ristocetin, 1 ferrite, 100 nM PBS buffer salt freeze-dried powder, 0.1 wt% bovine serum albumin, 0.01 wt% Tween 20, 1 wt% lactose, 10 wt% PVA, 0.1 wt% potassium chloride, and optical particles coated with vitronectin. The number of optical particles is 1×10 7 The second detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 2×10 7The third detection chamber contains 10 μM adenosine diphosphate, 1 stainless steel bead, 100 nM PBS buffer salt freeze-dried powder and optical particles coated with fibrinogen. The number of optical particles is 3×10 7 The size is 50nm and the optical characteristic peak is 700nm; no material is added to the blank detection chamber.

[0135] Example 8

[0136] This embodiment provides a platelet aggregation function detection method, and the device, steps, parameters and result determination method used are the same as those in Example 3, except that the optical signal characteristic parameter read from each detection chamber is the time to reach the maximum slope of the curve formed by the optical signal.

[0137] Experimental Example 1

[0138] A volunteer with low platelet count (platelet count 5×10 10 / L) of whole blood sample from the cubital vein was drawn into 5 tubes, 2mL / tube.

[0139] Among them, 4 tubes were tested for platelet aggregation function by optical transmission turbidimetry (LTA method) using the gold standard method (3 tubes were standard experimental groups and 1 tube was a control group), and 1 tube was tested by the detection method of Example 3 of the present invention as an experimental group.

[0140] Among them, the specific steps of the standard experimental group of the gold standard method are: centrifuge 3 tubes of blood samples at 200g centrifugal force for 10 minutes to obtain platelet-rich plasma (PRP), combine the 3 tubes of PRP, centrifuge PRP at 1000g centrifugal force for 5 minutes, centrifuge platelets to the bottom of the tube, discard 3 / 4 of the supernatant, gently shake and mix the remaining PRP and platelets, so that the platelets are evenly dispersed, and obtain PRP after the platelet number is adjusted. Centrifuge the blood collection tube after taking PRP at 1500g centrifugal force for 15 minutes to obtain platelet-poor plasma (PPP). According to the LTA instrument operation, first detect the transmittance of PPP, then replace the test sample with PRP to continue to monitor the transmittance of the reaction system, add the activator, and continue to monitor the transmittance of the PRP sample for 5 minutes. After adjusting the platelet number, the platelet aggregation function test was performed. The result showed that the platelet aggregation rate was 85%, which was greater than the threshold of 52%, and the conclusion was obtained that the platelet aggregation function of the blood sample was normal.

[0141] The specific steps of the control group of the gold standard method are as follows: centrifuge a tube of blood sample at 200g centrifugal force for 10 minutes to obtain platelet-rich plasma (PRP). Centrifuge for another 15 minutes at 1500g centrifugal force to obtain platelet-poor plasma (PPP). According to the LTA instrument operation, first detect the transmittance of PPP, then replace the test sample with PRP to continue monitoring the transmittance of the reaction system, add an activator, and continue to monitor the transmittance of the PRP sample for 5 minutes. The aggregation rate of the obtained blood sample is 15%, which is less than the threshold value of 52%, and it is concluded that the platelet aggregation function of the blood sample is weak.

[0142] The specific steps of the experimental group are: using 1 tube of blood sample to detect the platelet aggregation function using the detection method of Example 1, wherein the maximum optical signal of the first detection chamber is 340, the maximum optical signal of the second detection chamber is 142, the maximum optical signal of the third detection chamber is 81, and the optical signal of the blank detection chamber is 10. It can be seen that the optical signal of the second detection chamber is less than 1 / 2 of the optical signal of the first detection chamber, and the optical signal of the third detection chamber is less than 2 / 3 of the optical signal of the second detection chamber, and the platelet count is low. Therefore, the maximum optical signal of the first detection chamber is used as the accurate output of the detection result, wherein the detection result in the blank detection chamber is 10, and the difference between the maximum optical signal of the first detection chamber and the background signal is 330. Therefore, the output result is 330, which is greater than the threshold value of 208, and the platelet aggregation function is normal.

[0143] The threshold value of this embodiment is the aggregation rate of the LTA method×400, that is, the generally recognized threshold value of the LTA method is 52%. Therefore, the threshold value of this embodiment is 52%×400=208.

[0144] Experimental Example 2

[0145] A volunteer with low platelet count (platelet count 3.5×10 11 / L) of whole blood sample from the cubital vein was drawn into 4 tubes, 2mL / tube.

[0146] Among them, 3 tubes were tested for platelet aggregation function by optical transmission turbidimetry (LTA method) using the gold standard method (3 tubes were standard experimental groups and 1 tube was a control group), and 1 tube was tested by the detection method of Example 3 of the present invention as an experimental group.

[0147] The specific steps of the standard experimental group of the gold standard method are as follows: take 1 tube of blood sample and centrifuge it at 200g centrifugal force for 10 minutes to obtain platelet-rich plasma (PRP). The remaining samples in this tube and the other tube of samples are centrifuged at 1500g centrifugal force for 15 minutes to obtain platelet-poor plasma (PPP). Take 1 volume of PRP and 1.75 volumes of PPP, mix them and obtain PRP after platelet number adjustment. According to the LTA instrument operation, first detect the transmittance of PPP, then replace the test sample with PRP to continue to monitor the transmittance of the reaction system, add the activator, and continue to monitor the transmittance of the PRP sample for 5 minutes. After adjusting the platelet number, the platelet aggregation function test was performed. The result showed that the platelet aggregation rate was 28%, which was less than the threshold value of 52%, and it was concluded that the platelet aggregation function of the blood sample was weak.

[0148] The specific steps of the control group of the gold standard method are as follows: centrifuge a tube of blood sample at 200g centrifugal force for 10 minutes to obtain platelet-rich plasma (PRP). Continue centrifugation at 1500g centrifugal force for 15 minutes to obtain platelet-poor plasma (PPP). According to the LTA instrument operation, first detect the transmittance of PPP, then replace the test sample with PRP to continue monitoring the transmittance of the reaction system, add the activator, and continue to monitor the transmittance of the PRP sample for 5 minutes. The blood sample aggregation rate was 71%, which was greater than the threshold of 52%, and it was concluded that the platelet aggregation function of the blood sample was normal.

[0149] Tube 1 uses the detection method of Example 2 to detect platelet aggregation function, wherein the maximum slope of the optical signal of the first detection chamber is 2.1, the maximum slope of the optical signal of the second detection chamber is 1.5, the maximum slope of the optical signal of the third detection chamber is 1.2, and the maximum slope of the optical signal of the blank detection chamber is 0.18. It can be seen that the maximum slope of the second detection chamber is greater than 1 / 2 of the maximum slope of the first detection chamber, and the maximum slope of the third detection chamber is greater than 2 / 3 of the maximum slope of the second detection chamber. It is considered that the platelet count is too high. Therefore, the maximum optical signal of the third detection chamber is used as the accurate output test result, and the result is 102, which is less than the threshold value of 208, and the platelet aggregation function is weak.

[0150] The threshold value of this embodiment is the aggregation rate of the LTA method×400, that is, the generally recognized threshold value of the LTA method is 52%. Therefore, the threshold value of this embodiment is 52%×400=208.

[0151] Experimental Example 3

[0152] Using the method of Experimental Example 1, the platelet aggregation function was tested on the blood samples of 10 volunteers, and the following results were obtained, see Table 1.

[0153] Table 1 Platelet aggregation function test results

[0154]

[0155] According to Table 1, from the test results of platelet aggregation function, the platelet aggregation function obtained by the detection method provided by the present invention is different from that of the control group, but there is no difference with the standard experimental group, which proves that the detection method provided by the present invention can accurately obtain the correct parameters of platelet detection function.

[0156] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A composition for measuring the number of platelets, characterized in that: The following components are included: Platelet activators and optical particles, The optical particles are coated with a bioactive material that binds to platelet surface receptors.

2. The composition for measuring platelet count according to claim 1, characterized in that: The composition further comprises at least one of a stirrer or an auxiliary material; and / or, The platelet activator comprises at least one of an endogenous activator, an exogenous activator or a bioactive substance; and / or, The bioactive material comprises at least one of proteins, polypeptides or amino acids and their derivatives that bind to platelet surface receptors; and / or, The size of the optical particles is 1 nm-100 μm, and the optical characteristic peak of the optical particles is greater than or equal to 700 nm.

3. The composition for measuring platelet count according to claim 2, characterized in that: The platelet activator is at least one of adenosine diphosphate, adenosine diphosphate sodium salt, collagen, arachidonic acid, arachidonic acid sodium salt, ristocetin, epinephrine, and thrombin receptor activating peptide; and / or, The stirrer comprises at least one of a stainless steel ball, a ferrite, and a neodymium iron boron magnet; and / or, The auxiliary material includes at least one of buffer salt, protein, carbohydrate, surfactant, high molecular polymer or chloride metal salt; and / or, The bioactive material is at least one of fibrinogen, fibronectin, vitronectin, collagen, laminin, and von Willebrand factor; and / or, based on the volume of the container containing the composition, the concentration of the platelet activator in the composition is 2.5 to 4300 μg / mL; and / or, Based on the volume of the container containing the composition, the concentration of the optical particles is 5×10 5 / mL~5×10 10 Pieces / mL.

4. The composition for measuring platelet count according to claim 3, characterized in that: The buffer salt is at least one of phosphate, hydroxyethylpiperazineethanesulfonic acid buffer salt, 2-(N-morpholine)ethanesulfonic acid buffer salt, and GOOD's buffer salt; and / or, The protein in the auxiliary material is at least one of gelatin, fetal bovine serum, bovine serum albumin, and human albumin; and / or, The carbohydrate is a sugar, and the sugar includes at least one of sucrose, trehalose, dextran, maltose, lactose, and galactose; and / or, the surfactant is a nonionic surfactant, and the nonionic surfactant is at least one of Tween 20, Tween 80, Triton 100, GEROPON T-77 and TETRONIC 1307; and / or, The high molecular polymer is at least one of polyvinyl pyrrolidone, polyvinyl alcohol and cellulose; and / or, The halogenated metal salt is at least one of sodium chloride, potassium chloride and lithium chloride.

5. A platelet aggregation function detection device, characterized in that: comprising a detection zone, the detection zone comprising a detection unit, The detection unit comprises the composition for determining the number of platelets according to any one of claims 1 to 4.

6. The platelet aggregation function detection device according to claim 5, characterized in that: The detection unit comprises at least three detection chambers, and the number of optical particles in each detection chamber of the detection unit is arranged in a gradient; and / or, It also includes a sample tube storage area, in which a puncture needle is arranged; and / or, It also includes a sample heating area for heating the blood sample, and the sample heating area flows the heated blood sample into the detection area; and / or, The detection area also includes a control unit, which is used to provide a background signal for detecting the optical signal characteristic parameters of platelet aggregation function, and the control unit includes at least one blank detection chamber.

7. The platelet aggregation function detection device according to claim 6, characterized in that: Each detection chamber of the detection unit and / or the control unit is provided with an optical module, and the optical module is used to emit light of a specific wavelength to one side of each detection chamber, and receive the transmitted light at the opposite side of the detection chamber to obtain an optical signal; and / or The detection device also includes a heating module, a liquid driving module and a timing module. The heating module is used to heat the sample in the sample heating area and maintain the temperature of the detection area during platelet aggregation function detection. The liquid driving module is used to drive the blood sample in the sample tube storage area to the detection area, and to discharge the gas in the detection area. The timing module is used to record the detection time of platelet aggregation function; and / or, Each of the detection chambers is also provided with a stirring module, and the stirring module is used to control the stirring rate of the stirring bar in each detection chamber.

8. A method for detecting platelet aggregation function using the platelet aggregation function detection device according to any one of claims 5 to 7, characterized in that: A blood sample is injected into the detection zone, and the platelet count is measured based on the relationship between the number gradient of the optical particles in each detection chamber in the detection zone and the gradient of the corresponding optical signal characteristic parameter obtained by detection.

9. The method for detecting platelet aggregation function according to claim 8, characterized in that: The detection unit comprises at least three detection chambers, and the number of optical particles in each detection chamber is arranged in a gradient; And / or, before the blood sample is injected into the detection area, the step of heating the blood sample is included, and the heating temperature is 36-40° C.; The characteristic parameter of the optical signal is at least one of the maximum value of the optical signal, the time to reach the maximum optical signal, the area under the optical signal curve, the maximum slope of the curve formed by the optical signal, and the time to reach the maximum slope of the curve formed by the optical signal.

10. The method for detecting platelet aggregation function according to claim 9, characterized in that: When there are three detection chambers, when the maximum optical characteristic parameter of the second detection chamber is less than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is low, and the test result is output based on the optical signal of the first detection chamber; If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is less than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, the platelet count is considered normal, and the test result is output based on the optical characteristic parameter of the second detection chamber; If the maximum optical characteristic parameter of the second detection chamber is greater than 1 / 2 of the maximum optical characteristic parameter of the first detection chamber, and the maximum optical characteristic parameter of the third detection chamber is greater than 2 / 3 of the maximum optical characteristic parameter of the second detection chamber, it is considered that the platelet count is too high, and the test result is output based on the optical characteristic parameter of the third detection chamber.

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