A composition, device and method for determining the number of platelets

Abstract

This invention relates to the field of platelet aggregation function detection technology, specifically to a composition, apparatus, and detection method for determining platelet count. The composition provided by this invention for determining platelet count includes 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. This invention employs a combination of a platelet activator and optical particles coated with 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, causing a change in the light signal, thereby enabling the determination of changes in platelet count.

Description

Technical Field

[0001] This invention relates to the field of platelet aggregation function detection technology, and specifically to a composition, apparatus and detection method for determining platelet count. Background Technology

[0002] Platelet aggregation is the process by which platelets adhere to each other to form platelet emboli, often used to evaluate blood clotting or hemostasis. Normally, when a blood vessel is damaged, platelets are activated and rapidly adhere to the site of injury. Subsequently, through the release of a series of chemicals (such as ADP and thromboxane A2) and the expression of adhesion molecules, platelets aggregate to form primary hemostatic emboli, preventing excessive bleeding. This function is crucial for maintaining normal blood circulation and preventing post-traumatic blood loss. Abnormally enhanced platelet aggregation may increase the risk of thrombosis, potentially leading to cardiovascular diseases such as myocardial infarction and stroke. Conversely, weakened platelet aggregation may lead to a bleeding tendency.

[0003] Currently, most methods use turbidimetry to detect platelet aggregation function, but this ignores the influence of platelet count on platelet aggregation function, resulting in inaccurate test results. 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 count, thereby providing a composition, device and detection method for determining the platelet count.

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

[0006] On one hand, the present invention provides a composition for determining platelet count, 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.

[0007] To activate platelets and improve platelet count accuracy, embodiments of the present invention provide a platelet activator comprising at least one of endogenous activators, exogenous activators, or bioactive substances. Preferably, the platelet activator is at least one of adenosine diphosphate (ATP), sodium adenosine diphosphate, collagen, arachidonic acid, sodium arachidonic acid, ristoctylmycin, adrenaline, and thrombin receptor activating peptide. Preferably, the concentration of the platelet activator in the composition is 2.5–4300 μg / mL, based on the volume of the container holding the composition.

[0008] In some embodiments, the bioactive material comprises at least one of a protein, polypeptide, or amino acid and its derivatives that bind to platelet surface receptors. Preferably, the bioactive material is at least one of fibrinogen, fibronectin, fibronectin, collagen, laminin, and von Willebrand factor.

[0009] The optical particles have a size of 1 nm to 100 μm, and their optical characteristic peaks are greater than or equal to 700 nm. The concentration of the optical particles, based on the volume of the container holding the composition, is 5 × 10⁻⁶. 5 Cells / mL ~ 5×10 10 per mL.

[0010] In some embodiments, the composition further includes at least one of a stir bar or an adjuvant. Preferably, the stir bar includes at least one of stainless steel balls, ferrite, and neodymium iron boron magnets. Preferably, the adjuvant includes at least one of a buffer salt, protein, carbohydrate, surfactant, polymer, or metal chloride salt.

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

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

[0013] In some embodiments, the carbohydrate is a sugar, including 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 polymer is at least one of polyvinylpyrrolidone, polyvinyl alcohol, and cellulose.

[0016] In some embodiments, the halide 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 detection device for platelet aggregation function, including a detection area, the detection area including a detection unit, the detection unit including the above-mentioned composition for determining platelet count.

[0018] In some embodiments, the detection unit includes at least three detection chambers, each containing a gradient number of optical particles.

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

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

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

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

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

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

[0025] In some embodiments, the detection device further 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 zone and maintain the temperature of the detection zone during platelet aggregation function detection. The liquid driving module is used to drive the blood sample in the sample tube area to the detection zone and to expel the gas in the detection zone. The timing module is used to record the detection time of platelet aggregation function.

[0026] On the other hand, the present invention also provides a method for detecting platelet aggregation function. This method is based on the above-mentioned platelet aggregation function detection device, in which a blood sample is injected into the detection area, and the platelet count is determined according to 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 embodiments, before the blood sample is injected into the detection area, a step of heating the blood sample is included, with the heating temperature being 36-40°C.

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

[0029] When there are three detection chambers, if 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, then the platelet count is considered to be low, and the detection 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, then the platelet count is considered normal, and the detection 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, then the platelet count is considered to be too high, and the detection result is output based on the optical characteristic parameter of the third detection chamber.

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

[0033] 1. This invention provides a composition for determining platelet count, 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. This invention uses a combination of 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, causing a change in the light signal, thereby enabling the determination of changes in platelet count.

[0034] 2. This invention provides a method for detecting platelet aggregation function. A blood sample is injected into a detection area, and the platelet aggregation function is output by detecting optical characteristic parameters within the detection unit. The detection unit contains the aforementioned composition for determining platelet count. This invention utilizes the aggregation reaction between activated platelets and optical particles coated with receptor-binding bioactive materials. Changes in platelet count can be directly observed through optical signals, thereby determining whether platelet aggregation function needs correction based on platelet count to obtain accurate platelet aggregation function results.

[0035] 3. This invention provides a device for detecting platelet aggregation function, comprising a detection area, wherein the detection area includes a detection unit, and the detection unit contains the aforementioned composition for determining platelet count. The detection device provided by this invention is a microfluidic card, which is simple and fast to operate, and has a short detection time. Attached Figure Description

[0036] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 This is a process flow diagram of the platelet aggregation function detection method of the present invention;

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

[0039] Figure 3 This is a schematic diagram of the platelet aggregation function detection device in another embodiment of the present invention.

[0040] Figure label:

[0041] 1-Sample tube compartment area; 11-Puncture needle;

[0042] 2-Testing area; 21-First testing room; 22-Second testing room;

[0043] 23 - Third testing room; 24 - Blank testing room;

[0044] 3-Sample heating zone; 4-Exhaust port; 5-Air outlet. Detailed Implementation

[0045] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0046] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0047] See Figure 1This invention provides a method for detecting platelet aggregation function, which involves transporting a blood sample to a detection area for testing and outputting the platelet aggregation function detection result based on the optical characteristics obtained from the detection.

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

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

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

[0051] The control unit is used for background signal detection to avoid detection errors in the detection unit.

[0052] In some embodiments, the detection unit includes at least three detection chambers. The more detection chambers, the more accurate the identification of platelet counts and the more accurate the output platelet aggregation function.

[0053] Each testing chamber has a volume of 80-120 μL.

[0054] Each testing chamber contains a composition for determining platelet count, the composition comprising a platelet activator and optical particles of a binding peptide coated with a platelet surface glycoprotein receptor.

[0055] Based on the volume of the testing chamber, the concentration of platelet activator is 2.5-4300 μg / mL, and the concentration of the optical particles is 5 × 10⁻⁶. 5 Cells / mL ~ 5×10 10 per mL.

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

[0057] In order to acquire optical signals, in some embodiments, each detection chamber is provided with an optical module, which is used to emit light of a specific wavelength to one side of each detection chamber and receive the transmitted light on the opposite side of the detection chamber, and acquire 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 1×10⁻⁶. 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] This embodiment does not limit the location of each detection chamber, as long as the number of optical particles in each detection chamber is gradient. In some embodiments, the number of optical particles in the first detection chamber is 5 × 10⁻⁶. 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 1×10⁻⁶. 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 × 10⁻⁶. 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. 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 × 10⁻⁶. 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 that contains no reagents for the detection of background signals.

[0063] In some embodiments, the platelet activator may be at least one of adenosine diphosphate, sodium adenosine diphosphate, collagen, collagen protein, arachidonic acid, sodium arachidonic acid, ristoctidine, adrenaline, and thrombin receptor activating peptide.

[0064] The optical particles coated with platelet surface glycoprotein receptor binding peptides can be prepared by physical adsorption or chemical coupling.

[0065] Specifically, the physical adsorption method involves diluting the optical particles to a 1 mg / mL suspension using 10 mM pH 7.4 PBS, and then dissolving the platelet surface glycoprotein receptor-binding peptide in 10 mM pH 7.4 PBS to a 10 mg / mL solution. One volume of the optical particle suspension and the peptide-binding solution is vortexed and stirred overnight at room temperature. The supernatant is removed by centrifugation. Twice the volume of the supernatant is added to a 10 mg / mL bovine serum albumin solution, and the mixture is stirred and blocked at room temperature for 30 min. The supernatant is removed by centrifugation, yielding the optical particles coated with the platelet surface glycoprotein receptor-binding peptide. These are then diluted to an appropriate concentration with a suitable buffer for detection.

[0066] The chemical coupling method is as follows: The optical particles were diluted to a suspension of 1 mg / mL with 10 mM pH 4.0 PBS. One 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) were mixed by vortexing and activated at room temperature for 30 min. The supernatant was removed by centrifugation. The platelet surface glycoprotein receptor binding peptide was then dissolved to a solution of 10 mg / mL with 10 mM pH 7.4 PBS. The optical particles were redispersed with one volume of 10 mM pH 7.4 PBS, and one volume of the binding peptide solution was added. The mixture was vortexed and stirred at room temperature for 4 h. The supernatant was removed by centrifugation. Twice the volume of the supernatant in 10 mg / mL bovine serum albumin solution was added, and the mixture was stirred and blocked at room temperature for 30 min. The supernatant was removed by centrifugation, yielding the optical particles coated with the platelet surface glycoprotein receptor binding peptide. The particles were diluted to an appropriate concentration with a suitable buffer for detection.

[0067] More specifically, to ensure that the binding peptides coated with optical particles are compatible with each detection chamber, in some embodiments, the amount of each reagent used in the preparation method of optical particles coated with platelet surface glycoprotein receptor binding peptides is 100 mL / m 3 , where mL / m 3 This 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 platelet surface glycoprotein receptor binding peptides include fibrinogen, fibronectin, fibronectin, collagen, laminin, von Willebrand factor (vWF), or polypeptides and related derivatives based on the amino acid sequences of these proteins that bind to platelet surface receptors.

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

[0070] The composition also includes a stir bar and excipients.

[0071] The stir bar can be a stainless steel ball, ferrite, or neodymium iron boron magnet. Each testing chamber has at least one stir bar, preferably one, two, three, four, five, or ten.

[0072] The excipients are at least one of buffer salts, proteins, carbohydrates, surfactants, polymers, or metal halide salts.

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

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

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

[0076] Carbohydrates include sugars, including at least one of sucrose, trehalose, dextran, maltose, lactose, and galactose, and the concentration of 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 polyvinylpyrrolidone (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 0.1-2 wt%.

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

[0081] The detection device also includes a sample tube compartment, in which a puncture needle is provided for puncturing the rubber in the sample tube cap.

[0082] The sample collection area is connected to the detection area, and the sample from the sample collection area flows into the detection area for detection.

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

[0084] To heat the blood sample in the sample heating zone while maintaining the temperature of the detection zone during platelet aggregation function testing, the detection device further includes a heating module. The heating module covers both the sample heating zone and the detection zone; furthermore, the heating module provided by this invention needs to be positioned away from the detection optical path.

[0085] The heating temperature of the sample heating zone and the temperature of the blood sample during the testing process are 36-40℃.

[0086] In some embodiments, the detection device further includes a liquid-driven module. The liquid-driven module is used to drive the blood sample into each detection chamber and to expel gas from each detection chamber through an exhaust port.

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

[0088] Working principle:

[0089] Blood samples are fed into the detection unit and control unit respectively. The optical properties of the optical particles in each detection chamber of the detection unit and the fibrinogen coated on the optical particles can couple with the platelets in the blood sample activated by the platelet activator, thereby causing a change in the light signal in the detection unit. Since the number of optical particles in each detection chamber of the detection unit is set in a gradient, the platelet count is determined according to the relationship between the gradient of the number of optical particles in the detection chamber and the gradient of the corresponding light signal. Specifically, based on the relationship between the optical property parameters of the blood sample in each detection unit and the number of optical particles, the platelet aggregation function result is output, and the result is corrected by using the control unit as background parameters.

[0090] The more testing chambers there are, the more accurate the identification of platelet counts will be, and the more accurate the output of platelet aggregation function will be.

[0091] When the detection unit has 3 detection chambers, the method for judging the output result is as follows: if 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, then the platelet count is considered to be 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, then the platelet count is considered normal, and the detection 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, then the platelet count is considered to be too high, and the detection 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 following: 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 Figure 2 The present invention provides a detection device for platelet aggregation function, including a detection area, the detection area including a detection unit and a control unit, the detection unit being used to correct the platelet aggregation function detection results, and the control unit being 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. Each detection chamber has a volume of 100 μL. Each detection chamber is equipped with an optical module. The optical module is used to emit light of a specific wavelength to one side of each detection chamber and receive the transmitted light on the opposite side of the detection chamber to obtain an optical signal. The detection chamber of the detection unit contains a composition for measuring platelet count, while the blank detection chamber does not contain any reagents.

[0098] The first detection chamber contains 10 μM adenosine diphosphate, one stainless steel bead, 100 nM PBS buffered lyophilized powder, and optical particles coated with fibrinogen, with a particle count of 1 × 10⁻⁶. 7 The first detection chamber contains one 50nm particle with an optical characteristic peak at 700nm; the second detection chamber contains 10μM adenosine diphosphate, one stainless steel bead, 100nM PBS buffered lyophilized powder, and 2×10 optical particles coated with fibrinogen. 7 Each particle has a size of 50 nm and an optical characteristic peak at 700 nm. The third detection chamber contains 10 μM adenosine diphosphate, one stainless steel bead, 100 nM PBS buffered lyophilized powder, and optical particles coated with fibrinogen, with a particle count of 3 × 10⁻⁶. 7Each sample has a size of 50 nm and an optical characteristic peak of 700 nm; no material is added to the blank detection chamber.

[0099] The detection device is also equipped with a stirring module, which is used to control the stirring rate of the stir bar in each detection chamber. In this embodiment, the stirring module is located at the bottom of the detection area.

[0100] The detection device also includes a sample tube compartment 1, in which a puncture needle 11 is provided. The puncture needle is used to puncture the rubber in the sample tube cap to remove the blood sample from the sample tube.

[0101] The detection device also includes a sample heating zone 3, which continues to heat the blood sample flowing into the zone at a temperature of 37°C.

[0102] The sample collection area is connected to the detection unit and the control unit. The samples from the sample collection area flow into each detection chamber and the blank detection chamber for platelet aggregation function detection.

[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 area and maintain the temperature of the detection area during platelet aggregation function detection.

[0105] The liquid-driven module introduces blood samples into each testing chamber.

[0106] Each testing chamber is also equipped with an exhaust port. The gas discharged from the exhaust ports of each testing chamber is collected at the exhaust port 5, and the liquid drive module discharges the gas at the exhaust port.

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

[0108] Example 2

[0109] See Figure 3 The present invention provides a detection device for platelet aggregation function, including a detection area, the detection area including a detection unit and a control unit, the detection unit being used to correct the platelet aggregation function detection results, and the control unit being 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. The control unit includes a blank detection chamber. Each detection chamber has a volume of 100 μL. Each detection chamber is equipped with an optical module. The optical module is used to emit light of a specific wavelength to one side of each detection chamber and receive the transmitted light on the opposite side of the detection chamber to acquire an optical signal. The detection chamber of the detection unit contains a composition for measuring platelet count, while the blank detection chamber does not contain any reagents.

[0111] The first detection chamber contains 10 μM adenosine diphosphate, one stainless steel bead, 100 nM PBS buffered lyophilized powder, and optical particles coated with fibrinogen, with a particle count of 1 × 10⁻⁶. 7 The first detection chamber contains one 50nm particle with an optical characteristic peak at 700nm; the second detection chamber contains 10μM adenosine diphosphate, one stainless steel bead, 100nM PBS buffered lyophilized powder, and 2×10 optical particles coated with fibrinogen. 7 Each particle has a size of 50 nm and an optical characteristic peak at 700 nm. The third detection chamber contains 10 μM adenosine diphosphate, one stainless steel bead, 100 nM PBS buffered lyophilized powder, and optical particles coated with fibrinogen, with a particle count of 3 × 10⁻⁶. 7 Each sample has a size of 50 nm and an optical characteristic peak of 700 nm; no material is added to the blank detection chamber.

[0112] Each of the aforementioned detection chambers is also equipped with a stirring module, which is used to control the stirring rate of the stir bar in each detection chamber. In this embodiment, the stirring module is located on one side of the bottom of the detection area.

[0113] The detection device also includes a sample tube compartment 1, in which a puncture needle 11 is provided, which is used to puncture the rubber in the sample tube cap.

[0114] The sample collection area is connected to the detection unit and the control unit. The samples from the sample collection area flow into each detection chamber and the blank detection chamber for platelet aggregation function detection.

[0115] The detection device is also equipped with a stirring module, which is used to control the stirring rate of the stir bar in each detection chamber. In this embodiment, the stirring module is located 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 platelet aggregation function detection. The liquid driving module is used to drive the blood sample in the sample tube area to the target area. The timing module is used to record the detection time of platelet aggregation function.

[0117] Example 3

[0118] This embodiment provides a method for detecting platelet aggregation function. Using the detection device of Embodiment 1, a blood sample is inserted into the sample tube chamber. The puncture needle in the sample tube chamber punctures the rubber in the sample tube cap, allowing the blood sample to flow into the sample heating zone, maintaining the temperature of the blood sample at 37°C. The blood sample is divided into four portions and injected into the first detection chamber, the second detection chamber, the third detection chamber, and the blank detection chamber, respectively. After 2 minutes, the maximum optical signal of the first detection chamber, the second detection chamber, and the third detection chamber is read, and the optical signal parameters of the blank detection chamber are read as the background signal.

[0119] After reading the optical signal characteristic parameters of each detection chamber, the correction basis is as follows: if 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, then the platelet count is considered to be low, and the detection 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, then the platelet count is considered normal, and the detection 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, then the platelet count is considered to be too high, and the detection result is output based on the optical signal of the third detection chamber.

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

[0123] Example 4

[0124] This embodiment provides a method for detecting platelet aggregation function. The apparatus, steps, parameters, and result determination methods used are the same as in Embodiment 3. The optical signal characteristic parameters read from each detection chamber are the maximum slope of the optical signal and the different compositions of the components in each detection chamber of the detection unit. The specific compositions of the detection chambers of the detection unit are as follows.

[0125] The first detection chamber contains 2.5 μg / mL collagen, two stainless steel beads, 20 nM HEPES buffered lyophilized powder, and optical particles coated with fibronectin, with a particle count of 1 × 10⁻⁶. 7The first detection chamber contains 1 nm in size and 700 nm in optical characteristic peak; the second detection chamber contains 2.5 μM collagen, 2 stainless steel beads, 20 nm HEPES buffered salt lyophilized powder, and optical particles coated with fibronectin, with a particle count of 2 × 10⁻⁶. 7 Each particle has a size of 1 nm and an optical characteristic peak at 700 nm. The third detection chamber contains 2.5 μM collagen, 2 stainless steel beads, 20 nm HEPES buffered salt lyophilized powder, and optical particles coated with fibronectin, with a particle count of 3 × 10⁻⁶. 7 Each sample has a size of 1 nm and an optical characteristic peak of 700 nm; no material is added to the blank detection chamber.

[0126] Example 5

[0127] This embodiment provides a method for detecting platelet aggregation function. The apparatus, steps, parameters, and result determination methods are the same as in Embodiment 3. The difference lies in that the optical signal characteristic parameters read from each detection chamber are the time to reach the maximum optical signal, and the composition of the composition of each detection chamber in the detection unit is different. The specific composition is as follows.

[0128] The first detection chamber contains 4300 μg / mL adrenaline, 3 stainless steel beads, 500 nM MES buffered lyophilized powder, and collagen-coated optical particles, with a particle count of 2 × 10⁻⁶. 6 The first detection chamber contains 100 nm in size and 1000 nm in optical characteristic peak; the second detection chamber contains 4300 μg / mL adrenaline, 3 stainless steel beads, 500 nM MES buffered salt lyophilized powder, and collagen-coated optical particles, with a particle count of 4 × 10⁻⁶. 6 Each particle has a size of 100 nm and an optical characteristic peak at 1000 nm. The third detection chamber contains 4300 μg / mL adrenaline, 3 stainless steel beads, 500 nM MES buffered lyophilized powder, and collagen-coated optical particles, with a particle count of 6 × 10⁻⁶. 6 Each sample has a size of 100 nm and an optical characteristic peak of 1000 nm; no material is added to the blank detection chamber.

[0129] Example 6

[0130] This embodiment provides a method for detecting platelet aggregation function. The apparatus, steps, parameters, and result determination methods are the same as in Embodiment 3. The difference lies in that the optical signal characteristic parameters read from each detection chamber are the areas under the optical signal curves, and the composition of the composition of each detection chamber in the detection unit is different. The specific composition of each detection chamber is as follows.

[0131] The first testing chamber contains 100 μg / mL thrombin receptor activating peptide, one stainless steel bead, 100 nM PBS-buffered lyophilized 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, with a particle count of 1 × 10⁻⁶. 5 The first detection chamber contains 100 μg / mL thrombin receptor activating peptide, 1 stainless steel bead, 100 nM PBS buffered salt lyophilized 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, with a particle count of 4 × 10⁻⁶. 5 Each particle, 50 nm in size, has an optical characteristic peak at 700 nm. The third detection chamber contains 100 μg / mL thrombin receptor activating peptide, one stainless steel bead, 100 nM PBS buffered lyophilized 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, with a particle count of 8 × 10⁻⁶. 5 Each sample has a size of 50 nm and an optical characteristic peak of 700 nm; no material is added to the blank detection chamber.

[0132] Example 7

[0133] This embodiment provides a method for detecting platelet aggregation function. The apparatus, steps, parameters, and result determination methods are the same as in Embodiment 3. The difference lies in that the optical signal characteristic parameters read from each detection chamber are the maximum slope of the curve formed by the optical signal, and the composition of the composition of each detection chamber in the detection unit is different. The specific composition of each detection chamber in the detection unit is as follows.

[0134] The first detection chamber contains 100 μg / mL ristocetine, one ferrite, 100 nM PBS-buffered lyophilized 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 hylocinin, with a particle count of 1 × 10⁻⁶. 7 The first detection chamber contains one 50nm particle with an optical characteristic peak at 700nm; the second detection chamber contains 10μM adenosine diphosphate, one stainless steel bead, 100nM PBS buffered lyophilized powder, and 2×10 optical particles coated with fibrinogen. 7Each particle has a size of 50 nm and an optical characteristic peak at 700 nm. The third detection chamber contains 10 μM adenosine diphosphate, one stainless steel bead, 100 nM PBS buffered lyophilized powder, and optical particles coated with fibrinogen, with a particle count of 3 × 10⁻⁶. 7 Each sample has a size of 50 nm and an optical characteristic peak of 700 nm; no material is added to the blank detection chamber.

[0135] Example 8

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

[0137] Experimental Example 1

[0138] One volunteer with a low platelet count (platelet count 5 × 10⁻⁶) was selected. 10 Five tubes of whole blood samples were drawn from the elbow vein (number of samples per L), with 2 mL per tube.

[0139] Four tubes were used to detect platelet aggregation function using the gold standard method, optical transilluminance turbidimetry (LTA method) (three tubes were the standard experimental group and one tube was the control group), and one tube was used to detect platelet aggregation function using the detection method of Example 3 of this invention.

[0140] The standard experimental group of the gold standard method involved centrifuging three tubes of blood samples at 200g for 10 minutes to obtain platelet-rich plasma (PRP). After combining the three PRP tubes, the PRP was centrifuged again at 1000g for 5 minutes to collect platelets. Three-quarters of the supernatant was discarded, and the remaining PRP and platelets were gently shaken to ensure even platelet dispersion, resulting in platelet-adjusted PRP. The blood collection tube containing PRP was then centrifuged at 1500g for 15 minutes to obtain platelet-poor plasma (PPP). Following the LTA method instrument operation, the transmittance of PPP was first measured. Then, the test sample was replaced with PRP, and the transmittance of the reaction system was monitored again. An activator was added, and the transmittance of the PRP sample was monitored for 5 minutes. After adjusting the platelet count, platelet aggregation function was tested. The results showed a platelet aggregation rate of 85%, exceeding the threshold of 52%, indicating normal platelet aggregation function in the blood sample.

[0141] The specific steps for the control group of the gold standard method were as follows: One tube of blood sample was centrifuged at 200g for 10 minutes to obtain platelet-rich plasma (PRP). Then, it was centrifuged again at 1500g for 15 minutes to obtain platelet-poor plasma (PPP). Following the LTA method instrument operation, the transmittance of PPP was first measured. Then, the test sample was replaced with PRP, and the transmittance of the reaction system was monitored again. An activator was added, and the transmittance of the PRP sample was monitored for 5 minutes. The resulting blood sample aggregation rate was 15%, less than the threshold of 52%, indicating weak platelet aggregation function in the blood sample.

[0142] The specific steps of the experimental group were as follows: Platelet aggregation function was tested using the detection method described in Example 1 on one tube of blood sample. The maximum optical signal values ​​were 340 in the first detection chamber, 142 in the second detection chamber, 81 in the third detection chamber, and 10 in the blank detection chamber. It was found that the optical signal in the second detection chamber was less than half that of the first detection chamber, and the optical signal in the third detection chamber was less than two-thirds that of the second detection chamber, indicating a low platelet count. Therefore, the maximum optical signal in the first detection chamber was used as the accurate output result. The detection result in the blank detection chamber was 10. The difference between the maximum optical signal in the first detection chamber and the background signal was 330. Therefore, the output result was 330, which is greater than the threshold of 208, indicating normal platelet aggregation function.

[0143] In this embodiment, the threshold is the aggregation rate of the LTA method × 400. That is, the generally accepted threshold of the LTA method is 52%. Therefore, the threshold of this embodiment is 52% × 400 = 208.

[0144] Experiment Example 2

[0145] One volunteer with a low platelet count (platelet count 3.5 × 10⁻⁶) was selected. 11 Four tubes of whole blood samples were drawn from the elbow vein (number of samples per L), with 2 mL per tube.

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

[0147] The standard experimental procedure for the gold standard method is as follows: One tube of blood sample is centrifuged at 200g for 10 minutes to obtain platelet-rich plasma (PRP). The remaining sample in this tube and another sample are centrifuged at 1500g for 15 minutes to obtain platelet-poor plasma (PPP). One volume of PRP and 1.75 volumes of PPP are mixed to obtain platelet-adjusted PRP. Following the LTA method instrument operation, the transmittance of PPP is first measured, then the test sample is replaced with PRP, and the transmittance of the reaction system is monitored again. An activator is added, and the transmittance of the PRP sample is monitored for 5 minutes. After adjusting the platelet count, platelet aggregation function is tested. The results show a platelet aggregation rate of 28%, which is less than the threshold of 52%, indicating weak platelet aggregation function in the blood sample.

[0148] The specific steps for the control group of the gold standard method were as follows: One tube of blood sample was centrifuged at 200g for 10 minutes to obtain platelet-rich plasma (PRP). Then, it was centrifuged again at 1500g for 15 minutes to obtain platelet-poor plasma (PPP). Following the LTA method instrument operation, the transmittance of PPP was first measured. Then, the test sample was replaced with PRP, and the transmittance of the reaction system was monitored again. An activator was added, and the transmittance of the PRP sample was monitored for 5 minutes. The blood sample aggregation rate was found to be 71%, which is greater than the threshold of 52%, indicating that the platelet aggregation function of the blood sample is normal.

[0149] Platelet aggregation function was tested using the detection method described in Example 2. The maximum slope of the optical signal in the first detection chamber was 2.1, the maximum slope of the optical signal in the second detection chamber was 1.5, the maximum slope of the optical signal in the third detection chamber was 1.2, and the maximum slope of the optical signal in the blank detection chamber was 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. Therefore, the platelet count is considered to be high. Thus, the maximum optical signal of the third detection chamber is used as the accurate output detection result, which is 102, less than the threshold of 208, indicating that the platelet aggregation function is weak.

[0150] In this embodiment, the threshold is the aggregation rate of the LTA method × 400. That is, the generally accepted threshold of the LTA method is 52%. Therefore, the threshold of this embodiment is 52% × 400 = 208.

[0151] Experimental Example 3

[0152] Using the method described in Experiment 1, platelet aggregation function was tested on blood samples from 10 volunteers, and the results are shown in Table 1.

[0153] Table 1. Results of platelet aggregation function test

[0154]

[0155] As shown in Table 1, the platelet aggregation function obtained by the detection method provided by this invention differs from that of the control group, but not from that of the standard experimental group. This proves that the detection method provided by this invention can accurately obtain the correct parameters of platelet aggregation function.

[0156] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for detecting platelet aggregation function using a platelet aggregation function detection device for purposes other than disease diagnosis and / or disease treatment, characterized in that, The platelet aggregation function detection device includes a detection area, which includes a detection unit. The detection unit includes a composition for determining platelet count, and the composition includes the following components: Platelet activators and optical particles, The optical particles are coated with bioactive materials that bind to platelet surface receptors. The concentration of the optical particles is 5 × 10⁻⁶ based on the volume of the container holding the composition. 5 Cells / mL ~5×10 10 cells / mL; The detection unit includes three detection chambers, and the number of optical particles in each detection chamber of the detection unit is set in a gradient. The detection method includes: A blood sample is injected into the detection area, and the platelet count is determined based on the relationship between the number gradient of optical particles in each detection chamber of the detection area and the gradient of the corresponding optical signal characteristic parameters obtained from the detection. If 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, then the platelet count is considered low, and the detection 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, then the platelet count is considered normal, and the detection 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, then the platelet count is considered to be too high, and the detection result is output based on the optical characteristic parameter of the third detection chamber. The optical signal characteristic parameter is the maximum value of the optical signal.

2. The method for detecting platelet aggregation function according to claim 1, characterized in that, Before the blood sample is injected into the testing area, a step of heating the blood sample is also included, with the heating temperature being 36-40℃.

3. The method for detecting platelet aggregation function according to claim 1, characterized in that, The composition further includes at least one of a stir bar or an excipient; and / or, The platelet activator includes at least one of endogenous activators, exogenous activators, or bioactive substances; and / or, The bioactive material includes at least one of a protein, polypeptide, or amino acid and its derivatives that bind to platelet surface receptors; and / or, The optical particles have a size of 1nm-100μm and an optical characteristic peak greater than or equal to 700nm.

4. The method for detecting platelet aggregation function according to claim 3, characterized in that, The platelet activator is at least one of the following: adenosine diphosphate, sodium adenosine diphosphate, collagen, arachidonic acid, sodium arachidonicate, ristoctylmycin, adrenaline, thrombin receptor activating peptide, fibrinogen, fibronectin, hydrin, collagen, laminin, and von Willebrand factor; and / or, The stir bar comprises at least one of stainless steel balls, ferrite, and neodymium iron boron magnets; and / or, The excipients include at least one of buffer salts, proteins, carbohydrates, surfactants, polymers, or metal chloride salts; and / or, The bioactive material is at least one of fibrinogen, fibronectin, fibronectin, collagen, laminin, and von Willebrand factor; and / or, The concentration of platelet activator in the composition is 2.5 to 4300 μg / mL, based on the volume of the container holding the composition.

5. The method for detecting platelet aggregation function according to claim 4, characterized in that, The buffer salt is at least one selected from phosphate, hydroxyethylpiperazine ethanesulfonic acid buffer salt, and 2-(N-morpholino)ethanesulfonic acid buffer salt; and / or, The protein in the excipients is at least one of gelatin, fetal bovine serum, bovine serum albumin, and human albumin; and / or, The carbohydrates are sugars, including 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 selected from Tween 20, Tween 80, Triton 100, Geropon T-77, and TETRONIC 1307; and / or, The polymer is at least one of polyvinylpyrrolidone, polyvinyl alcohol, and cellulose; and / or, The halide metal salt is at least one of sodium chloride, potassium chloride, and lithium chloride.

6. The method for detecting platelet aggregation function according to claim 1, characterized in that, The detection device further includes a sample compartment area, wherein a puncture needle is disposed within the sample compartment area; and / or, The detection device further includes a sample heating zone for heating the blood sample, wherein the heated blood sample flows into the detection zone; and / or, The detection area also includes a control unit, which is used to provide a background signal for the optical signal characteristic parameters of platelet aggregation function detection. The control unit includes at least one blank detection chamber.

7. The method for detecting platelet aggregation function according to claim 6, characterized in that, Each detection chamber of the detection unit and / or the control unit is equipped with an optical module, which is used to emit light of a specific wavelength into one side of each detection chamber and receive the transmitted light on the opposite side of the detection chamber to acquire 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 testing. The liquid-driven module is used to drive the blood sample from the sample tube compartment to the detection area and to expel the gas from the detection area. The timing module is used to record the detection time of platelet aggregation function; and / or, Each of the aforementioned detection chambers is also equipped with a stirring module, which is used to control the stirring rate of the stir bar in each detection chamber.

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