Thromboelastometry composite activated coagulation test reagent, preparation method thereof and thromboelastometry activated coagulation test kit

Abstract

The present application belongs to the technical field of in vitro diagnostic reagents, and particularly relates to a thromboelastography composite activated coagulation detection reagent, a preparation method thereof and a thromboelastography activated coagulation detection kit. Compared with the prior art, the present application uses two activators, tannic acid and kaolin, is compounded, uses tannic acid to solve the problem of precipitation, and uses kaolin to supplement the traditional endogenous activation effect, has better synergistic effect, uses a biological buffer, a water-soluble polymer, an osmotic buffer and a preservative as a composite stabilizer, further improves the stability of the reagent, and further uses a freeze-drying protective agent to freeze dry to solve the problems of liquid reagent transportation and storage, avoid the problems of easy dryness and evaporation failure of the liquid reagent.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostic reagent technology, and particularly relates to a thromboelastography-activated coagulation detection reagent, its preparation method, and a thromboelastography-activated coagulation detection kit. Background Technology

[0002] Thromboelastography (TEG) is a diagnostic technique that comprehensively assesses coagulation function by simulating the blood clotting process. It can dynamically analyze the entire process from coagulation initiation and clot formation to dissolution, helping doctors determine whether a patient has a coagulation disorder, hypercoagulable state, or hyperfibrinolysis. It is often used to guide individualized medication in surgery, trauma, or anticoagulation therapy.

[0003] Currently, thromboelastography-activated coagulation assays (endogenous activation) often use ellagic acid or kaolin alone, but these methods have the following problems:

[0004] Kaolin is prone to sedimentation: In automated testing, the precipitation of traditional kaolin suspensions leads to uneven concentration, affecting the accuracy of the results;

[0005] Insufficient ellagic acid activity: Ellagic acid alone may not be able to completely replace the activating effect of kaolin, especially at low concentrations, which prolong clotting time;

[0006] Liquid reagents require a cold chain for transportation. Ice packs cannot guarantee that the reagent performance will not be affected. Furthermore, liquid reagents have a short shelf life; they can only be stored for one year at 2-8°C. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a thromboelastography-activated coagulation detection reagent with high stability, its preparation method and thromboelastography-activated coagulation detection kit.

[0008] This invention provides a thromboelastography composite activated coagulation detection reagent, comprising: a composite activator, a suspending agent, a biological buffer, a water-soluble polymer, an osmotic buffer, and a lyophilization protectant;

[0009] The composite activator includes ellagic acid and kaolin.

[0010] The mass ratio of ellagic acid to kaolin is (20~60):1;

[0011] The mass ratio of the composite activator to the suspending agent is (0.01~0.1):(0.5~5).

[0012] The mass ratio of the composite activator to the biological buffer is (0.01~0.1):(0.05~0.2).

[0013] The mass ratio of the composite activator to the water-soluble polymer is (0.01~0.1):(3~8).

[0014] The ratio of the composite activator to the osmotic buffer is (0.01~0.1) g : (0.3~0.8) mmol;

[0015] The mass ratio of the composite activator to the lyophilization protectant is (0.01~0.1):(3.5~37.5).

[0016] Preferably, the freeze-drying protectant comprises a freeze-drying skeleton material, polyethylene glycol, saccharides and bovine serum albumin; the mass ratio of the freeze-drying skeleton material, polyethylene glycol, saccharides and bovine serum albumin is (2~6):(2~8):(2~8):(1~3).

[0017] Preferably, the suspending agent is selected from one or more of dextran, carbomer, and cellulose-based suspending agents;

[0018] The biological buffer is selected from one or more of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate and 3-morpholinopropanesulfonic acid;

[0019] And / or, the water-soluble polymer is selected from polyethylene glycol and / or polyvinylpyrrolidone;

[0020] And / or, the osmotic buffer is selected from one or more of betaine, mannitol, glycine and trehalose;

[0021] And / or, the freeze-dried skeleton material is selected from mannitol and / or sorbitol;

[0022] And / or, the sugar is selected from one or more of trehalose, sucrose and lactose.

[0023] Preferably, it also includes a preservative; the mass ratio of the composite activator to the preservative is (0.01~0.1):(0.01~0.05).

[0024] Preferably, the preservative is selected from one or more of sodium azide, Proclin, potassium sorbate, methylparaben, gentamicin sulfate, and thimerosal.

[0025] Preferably, the mass ratio of the composite activator to the suspending agent is (0.01~0.1):2;

[0026] The mass ratio of the composite activator to the biological buffer is (0.01~0.1):0.1;

[0027] The mass ratio of the composite activator to the water-soluble polymer is (0.01~0.1):5;

[0028] The mass ratio of the composite activator to the osmotic buffer is (0.01~0.1) g: 0.5 mmol;

[0029] The mass ratio of the composite activator to the freeze-drying protectant is (0.01~0.1):7~25.

[0030] Preferably, the mass ratio of ellagic acid to kaolin is 40:1;

[0031] And / or, the mass ratio of the composite activator to the lyophilization protectant is (0.01~0.1):16;

[0032] And / or, the mass of the composite activator is 0.04% to 0.4% of the mass of the thromboelastography composite activated coagulation test reagent.

[0033] The present invention also provides a method for preparing the above-mentioned thromboelastography-based activated coagulation detection reagent, comprising the following steps:

[0034] S1) Ellagic acid, kaolin, suspending agent, biological buffer, water-soluble polymer, penetrating buffer, preservative and solvent are mixed and wet ball-milled to obtain a pre-ground slurry;

[0035] S2) The pre-ground slurry is subjected to high-pressure homogenization to obtain a homogeneous solution;

[0036] S3) The homogenized solution is subjected to ultrasonic treatment to obtain an activated solution;

[0037] S4) The activation solution is mixed with the lyophilization protectant solution and then lyophilized to obtain the thromboelastography composite activated coagulation detection reagent;

[0038] Alternatively, it may include the following steps:

[0039] A1) Ellagic acid, kaolin and a portion of solvent were sequentially subjected to wet ball milling, high-pressure homogenization and ultrasonic treatment to obtain nano-sized slurry;

[0040] A2) Mix the nano-sized slurry, suspending agent, biological buffer, water-soluble polymer, permeation buffer, preservative and the remaining solvent to obtain an activated solution;

[0041] A3) The activation solution and the lyophilization protectant solution are mixed and then freeze-dried to obtain the thromboelastography composite activated coagulation detection reagent.

[0042] Preferably, the diameter of the grinding media in the wet ball milling in steps S1) and A1) is 0.1~0.5 mm; the wet ball milling time is 6~24 h; and the wet ball milling speed is 300~500 rpm.

[0043] In step A1), the mass ratio of kaolin, grinding media and partial solvent is 1:(5~20):(3~8).

[0044] The pressure of high-pressure homogenization in steps S2) and A1) is 1000~1500 bar; the number of high-pressure homogenization cycles is 5~10.

[0045] The frequency of the ultrasonic treatment in steps S3) and A1) is 10~30 kHz; the power of the ultrasonic treatment is 400~600 W; the ultrasonic treatment is intermittent ultrasonic treatment; the single treatment time of the intermittent ultrasonic treatment is 1~10 s, the interval time is 1~10 s, and the total treatment time is 30~60 min.

[0046] The freeze-drying process includes a pre-freezing stage, a sublimation stage, and a drying stage. The temperature of the pre-freezing stage is -50°C, and the pre-freezing time is 2-6 hours. The sublimation stage is a gradient sublimation, with a gradient of 5°C to 10°C. The holding time for each gradient in the gradient sublimation is 1-3 hours. The vacuum degree of the sublimation drying is 120-180 bar. The temperature of the drying stage is 15°C to 25°C, and the drying time is 1-3 hours. The vacuum degree of the drying stage is 30-80 bar.

[0047] The present invention also provides a thromboelastography-activated coagulation detection kit, comprising the above-mentioned thromboelastography-activated coagulation detection reagent.

[0048] Compared with existing technologies, this invention uses a combination of ellagic acid and kaolin as activators. Ellagic acid solves the precipitation problem, while kaolin supplements the traditional endogenous activation effect, resulting in a better synergistic effect. At the same time, a suspending agent is used to better suspend the activator in the solution, improving the stability of the reagent. In addition, a biological buffer, a water-soluble polymer, and an osmotic buffer are used as composite stabilizers to further improve the stability of the reagent. Furthermore, freeze-drying with a freeze-drying protectant solves the problems of transporting and storing liquid reagents, and also avoids the problems of liquid reagents being easy to dry out and evaporate. Attached Figure Description

[0049] Figure 1 This is a correlation analysis graph showing the results of R-value testing of blood samples using deep-well plate-packaged reagents and test cup-packaged reagents in Embodiment 2 of the present invention, and the results of R-value testing using blood transfusion reagents.

[0050] Figure 2 This is a correlation analysis graph showing the results of Angle value testing of blood samples using deep-well plate-packaged reagents and test cup-packaged reagents in Embodiment 2 of the present invention, and the results of Angle value testing using blood transfusion reagents.

[0051] Figure 3 This is a correlation analysis graph showing the results of MA value testing of blood samples using deep-well plate-packaged reagents and test cup-packaged reagents in Embodiment 2 of the present invention, and the results of MA value testing using blood transfusion reagents. Detailed Implementation

[0052] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0053] This invention provides a thromboelastography-based composite activated coagulation assay reagent, comprising: a composite activator, a suspending agent, a biological buffer, a water-soluble polymer, an osmotic buffer, and a lyophilization protectant; the composite activator comprises ellagic acid and kaolin; the mass ratio of ellagic acid to kaolin is (20~60):1; the mass ratio of the composite activator to the suspending agent is (0.01~0.1):(0.5~5); the mass ratio of the composite activator to the biological buffer is (0.01~0.1):(0.05~0.2); the mass ratio of the composite activator to the water-soluble polymer is (0.01~0.1):(3~8); the mass ratio of the composite activator to the osmotic buffer is (0.01~0.1) g:(0.3~0.8) mmol; and the mass ratio of the composite activator to the lyophilization protectant is (0.01~0.1):(3.5~37.5).

[0054] In one specific embodiment of the present invention, the mass ratio of ellagic acid to kaolin is (20~60):1; optionally, the mass ratio of ellagic acid to kaolin is 20:1, 30:1, 40:1, 50:1, 60:1 or any two of the above values.

[0055] In one specific embodiment of the present invention, the mass ratio of ellagic acid to kaolin is 40:1.

[0056] This invention does not impose any special restrictions on the sources of ellagic acid and kaolin. In one specific embodiment of this invention, the ellagic acid is preferably sourced from Sinopharm Group Pharmaceutical Co., Ltd., and the kaolin is sourced from Sigma.

[0057] In one specific embodiment of the present invention, the suspending agent is preferably one or more of dextran, carbomer, and cellulose suspending agents; the cellulose suspending agent is preferably sodium carboxymethyl cellulose and / or methyl cellulose.

[0058] In a specific embodiment of the present invention, the mass ratio of the composite activator to the suspending agent is preferably (0.01~0.1):(1~4), more preferably (0.01~0.1):(1~3), even more preferably (0.01~0.1):(1.5~2.5), even more preferably (0.01~0.1):2, even more preferably (0.02~0.08):2, and even more preferably (0.04~0.06):2.

[0059] In one specific embodiment of the present invention, the biological buffer is preferably one or more of tris(hydroxymethyl)aminomethane (Tris), 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), PBS, and 3-morpholinopropanesulfonic acid (MPOS), more preferably tris(hydroxymethyl)aminomethane. The biological buffer helps maintain the pH stability of the system.

[0060] In a specific embodiment of the present invention, the mass ratio of the composite activator to the biological buffer is preferably (0.01~0.1):(0.08~0.15), more preferably (0.01~0.1):(0.08~0.12), even more preferably (0.01~0.1):0.1, even more preferably (0.02~0.08):0.1, even more preferably (0.04~0.06):0.1, and most preferably 0.05:0.1.

[0061] In a specific embodiment of the present invention, the water-soluble polymer is preferably polyethylene glycol and / or polyvinylpyrrolidone, more preferably polyethylene glycol; the molecular weight of the polyethylene glycol is preferably 4000-8000; optionally, the molecular weight of the polyethylene glycol is 4000, 5000, 6000, 7000, 8000 or any two of the above values. The water-soluble polymer can act as a steric stabilizer in the activated solution, reducing protein aggregation or precipitation by increasing solution viscosity and intermolecular repulsion.

[0062] In a specific embodiment of the present invention, the mass ratio of the composite activator to the water-soluble polymer is preferably (0.01~0.1):(3~7), more preferably (0.01~0.1):(4~6), even more preferably (0.01~0.1):(4.5~5.5), even more preferably (0.01~0.1):5, even more preferably (0.02~0.08):5, even more preferably (0.04~0.06):5, and most preferably 0.05:5.

[0063] In one specific embodiment of the present invention, the osmotic buffer is preferably one or more of betaine, mannitol, glycine, and trehalose, more preferably betaine. The osmotic buffer maintains an isotonic environment, preventing cell or protein inactivation due to changes in osmotic pressure.

[0064] In a specific embodiment of the present invention, the mass ratio of the composite activator to the osmotic buffer is preferably (0.01~0.1) g:(0.3~0.7) mmol, more preferably (0.01~0.1) g:(0.4~0.6) mmol, even more preferably (0.01~0.1) g:(0.45~0.55) mmol, even more preferably (0.01~0.1) g:0.5 mmol, even more preferably (0.02~0.08) g:0.5 mmol, even more preferably (0.04~0.06) g:0.5 mmol, and most preferably 0.05 g:0.5 mmol.

[0065] In a specific embodiment of the present invention, the mass ratio of the composite activator to the freeze-drying protectant is preferably (0.01~0.1):(4~35), more preferably (0.01~0.1):(5~32), even more preferably (0.01~0.1):(5~30), even more preferably (0.01~0.1):(7~25), even more preferably (0.01~0.1):(10~20), even more preferably (0.01~0.1):16, even more preferably (0.02~0.08):16, even more preferably (0.04~0.06):16, and most preferably 0.05:16.

[0066] In a specific embodiment of the present invention, the freeze-drying protectant comprises a freeze-drying skeleton material, polyethylene glycol, saccharides, and bovine serum albumin; the freeze-drying protectant comprises a freeze-drying skeleton material, polyethylene glycol, saccharides, and bovine serum albumin; the preferred mass ratio of the freeze-drying skeleton material, polyethylene glycol, saccharides, and bovine serum albumin is (2~6):(2~8):(2~8):(1~3), more preferably (3~5):(3~8):(3~8):(1~3), even more preferably (3~5):(4~6):(4~6):(1~3), even more preferably (3.5~4.5):(4.5~5.5):(4.5~5.5):(1.5~2.5), and most preferably 4:5:5:2.

[0067] The thromboelastography-based activated coagulation assay reagent provided by this invention is a lyophilized agent. During the pre-freezing stage, the lyophilized framework material, polyethylene glycol, and sugars in the cryoprotectant work synergistically to reduce ice crystal damage and maintain protein structure. During the drying stage, the sugars in the cryoprotectant and the lyophilized framework material work synergistically to form a stable glassy state and prevent dehydration and deformation. During long-term storage, the sugars in the cryoprotectant and BSA work synergistically to inhibit protein aggregation and surface adsorption.

[0068] In one specific embodiment of the present invention, the freeze-drying framework material is preferably mannitol and / or sorbitol. The freeze-drying framework material can form a porous, stable solid scaffold during the freeze-drying process, facilitating reconstitution after freeze-drying. It can also improve the mechanical strength of the freeze-dried cake, preventing collapse or structural damage during the drying stage, and provides low-temperature protection, partially reducing physical damage to proteins caused by ice crystals.

[0069] In a specific embodiment of this invention, the polyethylene glycol (PEG) can be any PEG well-known to those skilled in the art, without any particular limitation. The molecular weight of the PEG is preferably 6000-8000. PEG within this molecular weight range better meets the reagent requirements in terms of solubility, viscosity, and safety, jointly ensuring the stability of the reagent and the accuracy of the detection results. In this invention, the PEG includes, but is not limited to, PEG 6000 and / or PEG 8000. Adding PEG to the freeze-drying protectant can reduce the aggregation or denaturation of protein molecules through surface coating, stabilizing the protein conformation. Simultaneously, it can inhibit ice crystal growth, reducing mechanical damage to proteins during freezing (synergistically with freeze-drying framework materials, especially mannitol), and can also improve resolubility, increasing the dissolution rate of the freeze-dried product.

[0070] In one specific embodiment of the present invention, the sugar is preferably one or more of trehalose, sucrose, and lactose. Adding sugar to the freeze-drying protectant has the following effects: water molecule replacement: replacing water molecules on the protein surface during dehydration, maintaining its native conformation ("water replacement hypothesis"); vitrification protection: synergistically forming an amorphous glassy state with mannitol, reducing phase separation and protein denaturation; antioxidant: partially protecting sensitive molecules from oxidative damage.

[0071] Adding bovine serum albumin (BSA) to cryoprotectants has the following effects: surface protection: adsorbs onto protein surfaces, reducing adsorption loss at gas-liquid interfaces or container surfaces; competitive inhibition of aggregation: protects target proteins (especially low-concentration proteins) by occupying potential aggregation sites; filler: increases the quality of freeze-dried solids and improves the appearance of freeze-dried cakes.

[0072] In one specific embodiment of the present invention, the thromboelastography-based activated coagulation assay reagent further includes a preservative; the preservative is preferably one or more of sodium azide, Proclin, potassium sorbate, methylparaben, gentamicin sulfate, and thimerosal, more preferably sodium azide. Preservatives can prevent sample contamination by inhibiting the growth of bacteria and fungi (commonly used for long-term storage of antibodies and protein solutions).

[0073] In a specific embodiment of the present invention, the mass ratio of the composite activator to the preservative is preferably (0.01~0.1):(0.01~0.04), more preferably (0.01~0.1):(0.02~0.03), even more preferably (0.01~0.1):0.025, even more preferably (0.02~0.08):0.025, even more preferably (0.04~0.06):0.025, and most preferably 0.05:0.025.

[0074] In one specific embodiment of the present invention, the mass of the composite activator is preferably 0.04% to 0.4% of the mass of the thromboelastography composite activated coagulation test reagent; optionally, the mass of the composite activator is preferably 0.04%, 0.06%, 0.08%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4% of the mass of the thromboelastography composite activated coagulation test reagent, or a range between any two of the above values.

[0075] In one specific embodiment of the present invention, the pH value of the thromboelastography composite activated coagulation detection reagent is preferably 7.4 to 7.6.

[0076] In one specific embodiment of the present invention, the thromboelastography-based activated coagulation detection reagent is a lyophilized powder.

[0077] This invention employs a combination of ellagic acid and kaolin as activators. Ellagic acid solves the precipitation problem, while kaolin supplements the traditional endogenous activation effect, resulting in a better synergistic effect. At the same time, a suspending agent is used to better suspend the activator in the solution, improving the stability of the reagent. In addition, a composite stabilizer consisting of a biological buffer, a water-soluble polymer, an osmotic buffer, and a preservative is used to further improve the stability of the reagent. Furthermore, freeze-drying with a lyophilization protectant solves the problems of transporting and storing liquid reagents, and also avoids the problems of liquid reagents being prone to drying out and evaporation.

[0078] The present invention also provides a method for preparing the above-mentioned thromboelastography composite activated coagulation detection reagent, comprising the following steps: S1) mixing ellagic acid, kaolin, suspending agent, biological buffer, water-soluble polymer, osmotic buffer, preservative and solvent and wet ball milling to obtain a pre-ground slurry; S2) homogenizing the pre-ground slurry under high pressure to obtain a homogenized solution; S3) ultrasonically treating the homogenized solution to obtain an activated solution; S4) mixing the activated solution with a lyophilization protectant solution and freeze-drying to obtain the thromboelastography composite activated coagulation detection reagent.

[0079] In this invention, there are no special restrictions on the source of any raw materials; they can be commercially available. The ellagic acid, kaolin, suspending agent, biological buffer, water-soluble polymer, osmotic buffer, preservative, solvent, and lyophilization protectant in the solution are all as described above and will not be repeated here.

[0080] Ellagic acid, kaolin, suspending agent, biological buffer, water-soluble polymer, permeation buffer, preservative, and solvent are mixed and wet-milled to obtain a pre-ground slurry. The solvent can be any solvent well-known to those skilled in the art and is not particularly limited, but water is preferred, and purified water is more preferred. The grinding media used in the wet ball milling is any grinding media well-known to those skilled in the art and is not particularly limited, including but not limited to zirconia balls. The diameter of the grinding media is preferably 0.1~0.5 mm; optionally, the diameter of the grinding media is preferably 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or any two of the above values. The mass ratio of the total mass of the composite activator (ellagic acid and kaolin) to the mass of the grinding media is preferably 1:(5~20), more preferably 1:(5~15), even more preferably 1:(8~12), and most preferably 1:10. The rotation speed of the wet ball milling is preferably 300~500 rpm; optionally, the rotation speed of the wet ball milling is 300 rpm, 400 rpm, or 500 rpm. The time for wet ball milling is preferably 6 to 24 hours; optionally, the time for wet ball milling is 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or any two of the above values.

[0081] The pre-ground slurry is subjected to high-pressure homogenization to obtain a homogenized solution. The pressure of the high-pressure homogenization is preferably 1000-1500 bar; optionally, the pressure is 1000 bar, 1100 bar, 1200 bar, 1300 bar, 1400 bar, 1500 bar, or any two of the above values. The number of high-pressure homogenization cycles is preferably 5-10; optionally, the number of cycles is 5, 6, 7, 8, 9, or 10. High-pressure homogenization can further reduce the particle size.

[0082] The homogenized solution is subjected to ultrasonic treatment to obtain an activated solution; the frequency of the ultrasonic treatment is preferably 10~30kHz; optionally, the frequency of the ultrasonic treatment is 10 kHz, 20 kHz, 30 kHz or any two of the above values; the power of the ultrasonic treatment is preferably 400~600 W; optionally, the power of the ultrasonic treatment is 400 W, 450 W, 500 W, 550 W, 600 W or any two of the above values; the ultrasonic treatment is preferably intermittent to avoid overheating; the single treatment time of the intermittent ultrasonic treatment is preferably 1~10 s; optionally, the single treatment time is 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s or any two of the above values; the interval time between the single treatments of the intermittent ultrasonic treatment is preferably 1~10 s; optionally, the interval time is 1 s, 2 s, 3 s, 4 s, 5 s, 6 s, 7 s, 8 s, 9 s, 10 s or any two of the above values; The total processing time of the ultrasonic treatment is preferably 30 to 60 minutes; alternatively, the total processing time of the ultrasonic treatment is 30 minutes, 40 minutes, 50 minutes, 60 minutes, or any two of the above values.

[0083] The mass concentration of the composite activator in the activation solution is preferably 0.01% to 0.1%; optionally, the mass concentration of the composite activator in the activation solution is 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or any two of the above values.

[0084] In one specific embodiment of the present invention, the particle size of the activation solution is less than or equal to 100 nm and the PDI (polydispersity index) is less than 0.2.

[0085] The activation solution is mixed with the lyophilization protectant solution, and then freeze-dried to obtain a thromboelastography composite activated coagulation detection reagent. The lyophilization protectant solution comprises a lyophilized matrix material, polyethylene glycol, saccharides, bovine serum albumin, and water. The mass of the lyophilized matrix material is 2% to 6% of the mass of the lyophilization protectant solution. Optionally, the mass of the lyophilized matrix material is 2%, 3%, 4%, 5%, 6%, or any two of the above values. The mass of the polyethylene glycol is 2% to 8% of the mass of the lyophilization protectant solution. Optionally, the mass of the polyethylene glycol is 2% to 8% of the mass of the lyophilization protectant solution. The mass of the sugar is 2%, 3%, 4%, 5%, 6%, 7%, 8%, or any two of the above values; the mass of the sugar is 2% to 8% of the mass of the lyophilization protectant solution; optionally, the mass of the sugar is 2%, 3%, 4%, 5%, 6%, 7%, 8%, or any two of the above values; the mass of the bovine serum albumin is 1% to 3% of the mass of the lyophilization protectant solution; optionally, the mass of the bovine serum albumin is 1%, 1.5%, 2%, 2.5%, 3%, or any two of the above values.

[0086] In one specific embodiment of the present invention, the mass ratio of the activating solution to the lyophilization protectant solution is 1:(0.5~1.5); optionally, the mass ratio of the activating solution to the lyophilization protectant solution is 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5 or any two of the above ratios.

[0087] The freeze-drying process includes a pre-freezing stage, a sublimation stage, and a drying stage. The temperature of the pre-freezing stage is -50°C. The pre-freezing time is preferably 2-6 hours. Optionally, the pre-freezing time is 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, or any two of the above values. The sublimation stage is a gradient sublimation. The gradient of the gradient sublimation is 5°C to 10°C. The holding time of each gradient in the gradient sublimation is independently 1-3 hours. Optionally, the holding time of each gradient in the gradient sublimation is independently 1 hour, 2 hours, 3 hours, or any two of the above values. The vacuum degree of the sublimation drying is preferably 120-180 bar. Optionally, the vacuum degree of the sublimation drying is 120 bar, 140 bar, 150 bar, 160 bar, or 180 bar. The temperature of the drying stage is preferably 15°C to 25°C; optionally, the temperature of the drying stage is 15°C, 20°C, 25°C, or any two of the above values; the drying time is preferably 1 to 3 hours; optionally, the drying time is 1 hour, 2 hours, 3 hours, or any two of the above values; the vacuum level of the drying stage is preferably 30 to 80 bar; optionally, the vacuum level of the drying stage is 30 bar, 40 bar, 50 bar, 60 bar, 70 bar, 80 bar, or any two of the above values.

[0088] In a specific embodiment of the present invention, the temperature of the pre-freezing stage is -50°C; the pre-freezing stage is preferably 4 h; the sublimation stage is gradient sublimation; the gradient of the gradient sublimation is 5°C to 10°C; the holding time of each gradient in the gradient sublimation is independently 2 h; the vacuum degree of the sublimation drying is preferably 150 bar; the temperature of the drying stage is preferably 20°C; the drying stage is preferably 2 h; and the vacuum degree of the drying stage is preferably 50 bar.

[0089] This invention also provides another method for preparing a thromboelastography-based composite activated coagulation test reagent, comprising the following steps: A1) Ellagic acid, kaolin, and a portion of the solvent are sequentially subjected to wet ball milling, high-pressure homogenization, and ultrasonic treatment to obtain a nano-sized slurry; A2) The nano-sized slurry, suspending agent, biological buffer, water-soluble polymer, permeation buffer, preservative, and the remaining solvent are mixed to obtain an activation solution; A3) The activation solution is mixed with a lyophilization protectant solution and then freeze-dried to obtain the thromboelastography-based composite activated coagulation test reagent.

[0090] Ellagic acid, kaolin, and a portion of solvent are sequentially subjected to wet ball milling, high-pressure homogenization, and ultrasonic treatment to obtain a nano-sized slurry. The grinding media used in the wet ball milling can be any grinding media known to those skilled in the art, without special limitations, including but not limited to zirconia balls. The diameter of the grinding media is preferably 0.1~0.5 mm; optionally, the diameter is preferably 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or any two of the above values. The mass ratio of kaolin, grinding media, and a portion of solvent is preferably 1:(5~20):(3~8), more preferably 1:(5~15):(4~6), even more preferably 1:(8~12):(4~6), and most preferably 1:10:5. The rotation speed of the wet ball milling is preferably 300~500 rpm; optionally, the rotation speed is 300 rpm, 400 rpm, 500 rpm, or any two of the above values. The wet ball milling time is preferably 6~24 minutes. h; Optionally, the wet ball milling time is 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h or any two of the above values.

[0091] The conditions for high-pressure homogenization and ultrasonic treatment are the same as those described above, and will not be repeated here.

[0092] The nano-sized slurry is mixed with a stabilizer solution to obtain an activated solution. In a specific embodiment of the present invention, the particle size of the activated solution is less than or equal to 100 nm and the PDI (polydispersity index) is less than 0.2.

[0093] The conditions for mixing the activation solution and the freeze-drying protectant solution and the freeze-drying process are the same as those described above, and will not be repeated here.

[0094] The present invention also provides the application of the above-mentioned thromboelastography-activated coagulation detection reagent in the preparation of a thromboelastography-activated coagulation detection kit.

[0095] The present invention also provides a thromboelastography-activated coagulation detection kit, comprising the above-mentioned thromboelastography-activated coagulation detection reagent.

[0096] In a specific embodiment of the present invention, the thromboelastography-activated coagulation test kit further includes a deep-well plate or a test cup; the thromboelastography-activated coagulation test reagent is disposed in the deep-well plate or test cup and sealed with an aluminum-plastic film; the deep-well plate is preferably a deep-well plate; the deep-well plate may be a 24-well plate, a 48-well plate or a 96-well plate.

[0097] In one specific embodiment of the present invention, the thromboelastography activation coagulation detection kit further includes a calcium chloride solution; the concentration of the calcium chloride solution is 0.2 mol / L.

[0098] This invention places the thromboelastography-activated coagulation assay reagent in 24, 48, and 96-well plates, resulting in lower costs and greater suitability for industrial production. The aluminum-plastic film sealing ensures closed-loop testing, minimizing human error and making it suitable for hospitals of varying levels and sample sizes. Placing the reagent in a test cup also makes it suitable for hospitals with smaller sample volumes, thus overcoming sample size limitations. The thromboelastography-activated coagulation assay kit provided by this invention is compatible with the PCD 8000 fully automated thromboelastography system independently developed by Shandong Xinhua Medical Instrument Co., Ltd., enabling fully automated testing and simplifying the operation process.

[0099] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a thromboelastography-activated coagulation detection reagent and its preparation method, and a thromboelastography-activated coagulation detection kit provided by the present invention.

[0100] All reagents used in the following examples are commercially available.

[0101] (a) Component screening

[0102] 1.1 Raw material screening

[0103] The screening of activators is shown in Table 1 below, which examines the effectiveness of different activators and their ratios.

[0104] Table 1. Types and sources of activators

[0105]

[0106] The main components of the stabilizer were screened as shown in Table 2 below. The effects of different stabilizers on the product results were tested. The composition in this table refers to the concentration in the solution after being prepared with the activator.

[0107] Table 2 Composition of stabilizers

[0108]

[0109] The screening of lyophilization protectant solutions is shown in Table 3 below. The solvent is purified water. The effects of different lyophilization protectants on product results were tested.

[0110] Table 3 Composition of the lyophilization protectant solution

[0111]

[0112] 1.2 Screening Results

[0113] Kaolin and ellagic acid from different manufacturers were prepared into solutions with purified water and tested using TEG from Blood Technologies, Inc. (hereinafter referred to as Blood Technologies). The results were compared with the Blood Technologies Activated Coagulation Test Kit (Coagulation Method) (Control Reagent) using the same blood sample to screen the feasibility of using them as the main raw materials. The results are shown in Table 4.

[0114] Table 4 Screening Results

[0115]

[0116] The experimental results showed that the relative deviation between the R value of 0.05% Sinopharm kaolin and the blood test results reached 21%; the relative deviation between the R value of 0.005% Sigma ellagic acid and the blood test results reached 12.9%; while the deviations of R, K, Angle, MA, and CI of 0.05% Sigma kaolin and 0.005% Sinopharm ellagic acid from the reagent test results were all below ±10%, indicating good accuracy. Therefore, Sigma kaolin and Sinopharm ellagic acid were selected as the main raw materials.

[0117] Sigma kaolin and Sinopharm ellagic acid were mixed in purified water at different ratios. The total mass concentration of kaolin and ellagic acid in the mixed solution was 0.05%. A blood sample was tested at the same time, and the results were compared with those of blood testing reagent. The test results are shown in Table 5.

[0118] Table 5. Test results of different mixing ratios

[0119]

[0120] The results showed that when different mixing ratios of the composite activating reagent were prepared for testing, the total mass concentration of kaolin and ellagic acid in the mixed solution was 0.05%, and the mixing ratio of Sigma kaolin and Sinopharm ellagic acid of 1:40 was closest to the test results of the blood technology reagent.

[0121] Sigma kaolin and Sinopharm ellagic acid were mixed at a ratio of 1:40 and formulated into solutions A, B, and C with different stabilizers. The total mass concentration of the activator in solutions A, B, and C was 0.05%. The stabilizer composition in solution A was stabilizer 1 in Table 2, i.e., PEG content of 0.5%, sodium azide content of 0.025%, and sodium chloride content of 0.9%. The stabilizer composition in solution B was stabilizer 2 in Table 2, i.e., Tris content of 0.1%, PEG content of 5%, sodium azide content of 0.025%, and pH value of 7.5. The stabilizer composition in solution C was stabilizer 3 in Table 2, i.e., Tris content of 0.1%, PEG content of 5%, betaine content of 5 mM, sodium azide content of 0.025%, and pH value of 7.5.

[0122] Solutions A, B, and C were subjected to Level II quality control tests (Shandong Xinhua Medical Instrument Co., Ltd., Lu Medical Device Registration Certificate No. 20212400223) for repeatability and real-time stability (placement temperature 2℃~8℃) to screen their feasibility as stabilizers. The test results are shown in Table 6.

[0123] Table 6. Repeatability and Real-Time Stability Test Results

[0124]

[0125]

[0126] The test results above show that when the stabilizer composition in the solution is stabilizer 3, the repeatability is still within 10% after 3 months. However, when the stabilizer composition in the solution is stabilizer 1 and stabilizer 2, the repeatability gradually decreases over time. Therefore, the stabilizer 3 formulation is selected.

[0127] The final selection was a 1:40 mixture of Sigma kaolin and Sinopharm ellagic acid, with stabilizer 3 as the stabilizer component in the solution.

[0128] (II) Process Screening

[0129] Screening of nano-process parameters

[0130] (1) Wet ball milling: Sigma kaolin and Sinopharm ellagic acid were mixed in a ratio of 1:40 and wet ball milled. The total mass ratio of kaolin and ellagic acid, and the mass ratio of zirconia balls to water were 1:10:5. Suspension agent dextran and stabilizer were added. After preparation, the total mass concentration of kaolin and ellagic acid in the solution was 0.05%. The composition and content of stabilizer are shown in stabilizer 3 in Table 2. The mass concentration of dextran in the solution was 0.2%. The experiment was designed as shown in Table 7. The particle size and PDI were tested using a nanoparticle size and zeta potential analyzer (DLS). The quality control material Level 2 was tested using thromboelastography. The results are shown in Table 7.

[0131] Table 7 Wet ball milling process parameters and test results

[0132]

[0133]

[0134] The experimental results showed that when zirconia balls (0.1~0.5 mm in diameter) were added to the solution and the mixture was ball-milled for 6~24 hours at a speed of 300~500 rpm, the relative deviations of R, Angle, MA and blood transfusion reagent were within ±10%.

[0135] (2) High-pressure homogenization: The pre-ground slurry (i.e., the slurry after wet ball milling in the above wet ball milling) was circulated through a high-pressure homogenizer, and then the suspending agent dextran and stabilizer were added. The composition and content of the stabilizer in the solution after preparation were stabilizer 3 in Table 2. The total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran in the solution was 0.2%. The particle size was further reduced by high-pressure homogenization. The specific experimental design and the test results of quality control 2 are shown in Table 8.

[0136] Table 8. High-pressure homogenization process parameters and test results

[0137]

[0138]

[0139] The results showed that at pressures of 500 bar and 2000 bar, and at 2 and 15 cycles, the relative deviations from the blood test results of the control reagent exceeded ±10% to varying degrees. When the pre-ground slurry was circulated through a high-pressure homogenizer (1000~1500 bar) 5~10 times, the relative deviations of R, Angle, and MA from the blood test reagent were within ±10%.

[0140] (3) Ultrasonic-assisted dispersion: The slurry homogenized under high pressure was ultrasonically treated, and then the suspending agent dextran and stabilizer were added. The composition and content of the stabilizer in the solution after preparation were stabilizer 3 in Table 2. The total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran in the solution was 0.2%. The ultrasonic treatment was performed by intermittent ultrasonic treatment with a probe (20kHz, 500W) (on / off cycle 5 s / 5 s) to avoid overheating. The specific time experimental design and blood sample test results are shown in Table 9.

[0141] Table 9. Ultrasonic-assisted dispersion process parameters and test results

[0142]

[0143] The test results show that when using probe ultrasound (20 kHz, 500 W) for intermittent treatment (on / off cycle 5 s / 5 s) for 15 minutes and 75 minutes, the relative deviations of R value and Angle from the control reagent exceeded ±10%. When the time was 30~60 minutes, all parameters were within the required range of relative deviation. Therefore, the specified time for using probe ultrasound (20 kHz, 500 W) for intermittent treatment (on / off cycle 5 s / 5 s) is 30~60 minutes.

[0144] (III) Screening of freeze-drying protectants and freeze-drying processes

[0145] Analysis of the effect of lyophilization protectant on results

[0146] Sigma kaolin and Sinopharm ellagic acid were mixed in a 1:40 ratio with an appropriate amount of water and subjected to a nano-process (zirconia balls with a diameter of 0.3 mm, the total mass of kaolin and ellagic acid, and the mass ratio of zirconia balls to water were 1:10:5, ball milling for 6 hours at 400 rpm, high-pressure homogenization at 1000 bar for 5 cycles, and intermittent ultrasonic treatment (5 s / 5 s on / off cycle) for 30 minutes using a probe (20 kHz, 500 W)). After adding suspending agent dextran and stabilizer, the total mass concentration of kaolin and ellagic acid in the solution was 0.05%. The composition and content of the stabilizer are shown in Stabilizer 3 in Table 2. The mass concentration of dextran in the solution was 0.2%. The solution was then mixed with freeze-drying protectant solutions A, B, and C in a 1:1 ratio and injected into 24, 48, and 96 deep-well plates for freeze-drying, 80 µL per well; and directly freeze-dried into test cups, 28 µL per test cup. µL was used to test its effect on the accuracy of blood sample testing after product freeze-drying.

[0147] After mixing, freeze-drying was carried out according to the same freeze-drying procedure: pre-freezing temperature -50℃ for 4 h, after pre-freezing, sublimation was carried out in a gradient of 5℃~10℃, with the vacuum degree controlled at 150 bar, and the temperature was maintained at -40℃, -30℃, -20℃, -10℃, 0℃ and 10℃ for 2 h, and 20℃ for 2 h, and the vacuum degree was reduced to 50 bar.

[0148] Deep well plate packaging: After reconstitution with 1 mL of blood sample, no undissolved precipitate was observed, and the appearance after reconstitution met the requirements. 20 µL of CaCl2 was added to an empty test cup, and 340 µL of the reconstituted blood sample solution was added to the test cup for testing. The results are shown in Table 10.

[0149] Test cup packaging: Add 20 µL of CaCl2 to the test cup, and add 340 µL of blood sample to the test cup for testing.

[0150] Table 10. Effect of different lyophilization protectants on test results

[0151]

[0152]

[0153] The results of the on-machine test show that, after lyophilization using the three lyophilization protectant solutions in Table 10, the relative deviations of the repeatability of lyophilization protectant solution C from the control reagent are all within 10% in terms of R, Angle, and MA. This formula meets the product design requirements.

[0154] Based on the above screening results, a Sigma kaolin and Sinopharm ellagic acid were selected in a mixing ratio of 1:40. The stabilizer composition and content in the solution were stabilizer 3 as shown in Table 2. The total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran in the solution was 0.2%. The solution was not freeze-dried and was designated as reagent A.

[0155] The solution was prepared by mixing Sigma kaolin and Sinopharm ellagic acid in a 1:40 ratio. The stabilizer composition and content were as shown in Table 2 (Stabilizer 3). The total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran was 0.2%. The solution was then mixed with lyophilization protectant solution C in a 1:1 ratio and lyophilized according to the following procedure: pre-freezing at -50℃ for 4 hours; after pre-freezing, sublimation was carried out by increasing the temperature in increments of 5-10℃, maintaining a vacuum of 150 bar; holding at -40℃, -30℃, -20℃, -10℃, 0℃, and 10℃ for 2 hours each, and at 20℃ for 2 hours, before reducing the vacuum to 50 bar. This reagent is reagent B.

[0156] Sigma kaolin and Sinopharm ellagic acid were mixed in a 1:40 ratio with an appropriate amount of water and subjected to nano-processing (zirconia balls with a diameter of 0.3 mm, total mass of kaolin and ellagic acid, mass ratio of zirconia balls to water of 1:10:5, ball milling for 6 hours at 400 rpm, high-pressure homogenizer at 1000 bar for 5 cycles, and intermittent ultrasonic treatment (20 kHz, 500 W) with a probe (5 s / 5 s on / off cycle) for 30 minutes). Then, suspending agent dextran and stabilizer were added to prepare the solution. The stabilizer composition and content in the prepared solution were as shown in Table 2 (Stabilizer 3), the total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran in the solution was 0.2%. The solution was then mixed with freeze-drying protectant solution C in a 1:1 ratio and freeze-dryed according to the following procedure: pre-freezing temperature -50℃ for 4 hours, followed by sublimation at a temperature gradient of 5℃~10℃, with the vacuum degree controlled at 150℃. The temperature was maintained at -40°C, -30°C, -20°C, -10°C, 0°C, and 10°C for 2 hours each, and at 20°C for 2 hours, with the vacuum level reduced to 50 bar. This reagent is reagent C.

[0157] The real-time stability of the three reagents A, B, and C at 25℃ for 0, 1, and 3 months was tested. Aging tests were also conducted at 50℃ for 5, 10, 14, 19, and 21 weeks (where 50℃ for 5, 10, 14, 19, and 21 weeks represents 6, 12, 18, 24, and 27 months at 25℃, respectively). The repeatability of these tests was compared with that of the control reagent, and the results are shown in Table 11.

[0158] Table 11 Stability Test Results

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] The stability test data above show that, since reagent A is a liquid phase (and has not undergone nano-processing), its R value CV exceeds 10 after 1 month at 25°C; reagent B has not undergone nano-processing, but it is lyophilized, and its R value CV exceeds 10 after 12 months; reagent C (which has undergone nano-processing and is lyophilized) still has good stability after 27 months, comparable to that of blood transfusion reagents.

[0165] Therefore, the final selection was to use a 1:40 mixture of Sigma kaolin and Sinopharm ellagic acid, with an appropriate amount of water, for nano-processing (zirconia balls diameter 0.3, total mass of kaolin and ellagic acid, mass ratio of zirconia balls to water 1:10:5, ball milling for 6 hours at 400 rpm, high-pressure homogenizer at 1000 bar for 5 cycles, and intermittent ultrasonic treatment (20 kHz, 500W) for 30 minutes with a 5 s / 5 s on / off cycle). Then, a suspending agent and stabilizer were added and the solution was prepared. The stabilizer composition and content in the prepared solution were as shown in Table 2 (Stabilizer 3), the total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran in the solution was 0.2%. The solution was then mixed with freeze-drying protectant solution C at a 1:1 ratio and freeze-dryed according to the freeze-drying procedure: pre-freezing temperature -50℃, 4... After pre-freezing, the temperature is increased and sublimated in a gradient of 5℃~10℃, with the vacuum degree controlled at 150 bar. The temperature is maintained at -40℃, -30℃, -20℃, -10℃, 0℃ and 10℃ for 2 hours, and at 20℃ for 2 hours. The vacuum degree is then reduced to 50 bar.

[0166] Example 1

[0167] Sigma kaolin and Sinopharm ellagic acid were mixed in a mass ratio of 1:40 and an appropriate amount of water was added. The mixture was then subjected to a nano-process (zirconia balls with a diameter of 0.3 mm; the total mass of kaolin and ellagic acid, and the mass ratio of zirconia balls to water were 1:10:5; ball milling for 6 hours at 400 rpm; high-pressure homogenization at 1000 bar for 5 cycles; and intermittent ultrasonic treatment using a probe (20 kHz, 500W) with an on / off cycle of 5 seconds). (s) 30 minutes), then add the suspending agent dextran and stabilizer and prepare the solution. The stabilizer composition and content in the prepared solution are stabilizer 3 in Table 2. The total mass concentration of kaolin and ellagic acid in the solution is 0.05%, and the mass concentration of dextran in the solution is 0.2%. Then mix the solution with the lyophilization protectant solution C at a ratio of 1:1, and dispense 80µL / well into a 96-well deep plate and 28µL / cup into test cups, respectively. Perform lyophilization according to the following procedure: pre-freeze at -50℃ for 4 hours. After pre-freezing, sublimate by increasing the temperature in a gradient of 5℃~10℃, with the vacuum degree controlled at 150 bar. Hold at -40℃, -30℃, -20℃, -10℃, 0℃ and 10℃ for 2 hours, and hold at 20℃ for 2 hours, and then reduce the vacuum degree to 50 bar. After lyophilization, test the reagent performance.

[0168] Deep well plate packaging: Add 1 mL of water to reconstitute the quality control 2 solution, add 1 mL of the reconstituted quality control 2 solution to the deep well plate, add 20 µL of CaCl2 to an empty test cup, take 340 µL of the solution in the deep well plate and add it to the test cup for testing. The results are shown in Table 12.

[0169] Test cup packaging: Add 1 mL of water to reconstitute the quality control solution 2, add 20 µL of CaCl2 to the test cup, take 340 µL of the quality control solution and add it to the test cup for direct testing. The results are shown in Table 12.

[0170] Table 12 Test results of quality control products with different packaging forms

[0171]

[0172] The test results show that the test results of the two packaging forms of the reagent are similar to those of the blood test reagent, indicating that both methods of lyophilizing the reagent into a deep well plate, reconstitute it and then test it, and lyophilizing the reagent into a test cup and testing it directly are feasible.

[0173] Example 2

[0174] Sigma kaolin and Sinopharm ellagic acid were mixed at a mass ratio of 1:40 and an appropriate amount of water was added. The mixture underwent nano-processing (zirconia balls with a diameter of 0.3 mm; the total mass of kaolin and ellagic acid, and the mass ratio of zirconia balls to water were 1:10:5; ball milling for 6 hours at 400 rpm; high-pressure homogenizer at 1000 bar for 5 cycles; and intermittent ultrasonic treatment (20 kHz, 500W, on / off cycle 5 s / 5 s) for 30 minutes). Then, dextran, a suspending agent, and a stabilizer were added to prepare the solution. The stabilizer composition and content in the prepared solution were as shown in Table 2 (Stabilizer 3). The total mass concentration of kaolin and ellagic acid in the solution was 0.05%, and the mass concentration of dextran was 0.2%. The solution was then mixed with lyophilization protectant solution C at a 1:1 ratio and dispensed into 96-well and 28-well plates at 80 µL / well, respectively. Dispense µL / cup into test cups and perform lyophilization according to the above procedure: pre-freeze at -50℃ for 4 hours; after pre-freezing, sublimate by increasing the temperature in increments of 5℃ to 10℃, maintaining a vacuum of 150 bar; hold at -40℃, -30℃, -20℃, -10℃, 0℃, and 10℃ for 2 hours each, and at 20℃ for 2 hours, then reduce the vacuum to 50 bar. After lyophilization, test the reagent properties.

[0175] Deep well plate packaging: Add 1 ml of blood sample to the deep well plate, add 20 µL of CaCl2 to an empty test cup, and add 340 µL of blood sample from the deep well plate to the test cup for testing.

[0176] Test cup packaging: Add 20 µL of CaCl2 to the test cup, then add 340 µL of blood sample to the test cup for direct testing.

[0177] Following the above experimental method and using the blood transfusion reagent, 50 samples were tested to analyze the correlation between R, Angle, and MA. The experimental results are shown in Table 13.

[0178] Table 13 Blood sample test results for different packaging formats

[0179]

[0180]

[0181] Using the blood sample R-value test results from the hematology reagent as the x-axis and the blood sample R-value test results from the deep-well plate packaged reagent and the test cup packaged reagent in Example 2 as the y-axis, a scatter plot was drawn, and correlation analysis was performed. Figure 1 As shown. By Figure 1 It can be seen that, based on the testing of 50 clinical samples, the correlation R with hematology was... 2 The values ​​are 0.9948 and 0.9989, respectively. 2All values ​​were greater than 0.98, indicating that the reagents prepared in this application were in good consistency with blood samples and fully met the requirements of clinical trials.

[0182] Using the blood sample Angle value test results from the hematology reagent as the x-axis and the blood sample Angle value test results from the deep-well plate packaged reagent and the test cup packaged reagent in Example 2 as the y-axis, a scatter plot was drawn, and correlation analysis was performed. Figure 2 As shown. By Figure 2 It can be seen that the correlation R between 50 clinical samples and hematological techniques was [missing information]. 2 The values ​​are 0.9969 and 0.9963 respectively. 2 All values ​​were greater than 0.98, indicating that the reagents prepared in this application were in good consistency with blood samples and fully met the requirements of clinical trials.

[0183] Using the MA value test results of blood samples obtained with hematology reagents as the x-axis and the MA value test results of blood samples obtained with deep-well plate-packaged reagents and test cup-packaged reagents in Example 2 as the y-axis, a scatter plot was drawn, and correlation analysis was performed. Figure 3 As shown. Fifty clinical samples were tested, and the correlation R with hematology was determined. 2 The values ​​are 0.9939 and 0.9936 respectively. 2 All values ​​were greater than 0.98, indicating that the reagents prepared in this application were in good consistency with blood samples and fully met the requirements of clinical trials.

[0184] In summary, this invention combines ellagic acid and kaolin as a composite activator, optimizing the ratio to achieve a better synergistic effect. Ellagic acid solves the precipitation problem, while kaolin supplements the traditional endogenous activation effect. A composite stabilizer (such as Tris 0.1% + PEG 5% + betaine 5 mM + sodium azide 0.025%) is used to replace the traditional buffer solution, adjusting the pH to 7.5±0.1. The lyophilization protectant solution (mannitol 4% + PEG 5% + trehalose 5% + BSA 2%) is also optimized. By combining with calcium chloride to form a lyophilized powder, this invention addresses the industry pain points of transporting and storing liquid reagents (no cold chain required, shelf life extended to 24 months at room temperature (25°C)). It also avoids the problem of liquid reagents easily drying out: small volumes (10~40 μL) of liquid activators are prone to evaporation and inactivation during long-term storage. It can be placed in 24, 48, or 96-well deep-well plates (low cost, more suitable for industrial production, and different packaging options available depending on sample volume, meeting the needs of hospitals of different levels), and sealed with aluminum-plastic film, ensuring closed-loop detection and avoiding human error. After lyophilization in test cups, direct testing is possible, making it suitable for hospitals with small sample volumes; thus, this invention is not limited by sample size. It is compatible with the PCD 8000 thromboelastography system independently developed by Shandong Xinhua Medical Instrument Co., Ltd., simplifying the operation process for chemical detection.

[0185] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A thrombelastography composite activated coagulation test reagent, characterized by, The complex activator, suspending agent, biological buffer, water-soluble polymer, penetration buffer and freeze-drying protective agent are composed of; The complex activator is composed of ellagic acid and kaolin; The mass ratio of the ellagic acid and kaolin is 40:1; The mass ratio of the complex activator and suspending agent is (0.04-0.06):2; The mass ratio of the complex activator and biological buffer is 0.05:0.1; The mass ratio of the complex activator and water-soluble polymer is 0.05:5; The mass ratio of the complex activator and penetration buffer is 0.05 g:0.5 mmol; The mass ratio of the complex activator and freeze-drying protective agent is 0.05:16; The freeze-drying protective agent is composed of freeze-drying skeleton material, polyethylene glycol, sugar substance and bovine serum albumin; the mass ratio of the freeze-drying skeleton material, polyethylene glycol, sugar substance and bovine serum albumin is 4:5:5:2; The suspending agent is selected from one or more of dextran, carbopol and cellulose suspending agent; The biological buffer is selected from one or more of tris(hydroxymethyl)aminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate and 3-morpholinopropanesulfonic acid; The water-soluble polymer is selected from polyethylene glycol and / or polyvinylpyrrolidone; The penetration buffer is selected from one or more of betaine, mannitol, glycine and trehalose; The freeze-drying skeleton material is selected from mannitol and / or sorbitol; The sugar substance is selected from one or more of trehalose, sucrose and lactose; The thrombelastogram complex activated coagulation detection reagent is a freeze-dried powder; The preparation method of the thrombelastogram complex activated coagulation detection reagent comprises the following steps: S1) mixing ellagic acid, kaolin, suspending agent, biological buffer, water-soluble polymer, penetration buffer, preservative and solvent for wet ball milling to obtain a pre-milling slurry; S2) high-pressure homogenization of the pre-milling slurry to obtain a homogenized solution; S3) ultrasonic treatment of the homogenized solution to obtain an activated solution; S4) mixing the activated solution and freeze-drying protective agent solution, and freeze-drying to obtain a thrombelastogram complex activated coagulation detection reagent; Alternatively, comprising the following steps: A1) sequentially wet ball milling, high-pressure homogenization and ultrasonic treatment of ellagic acid, kaolin and part of the solvent to obtain a nano-sized slurry; A2) mixing the nano-sized slurry, suspending agent, biological buffer, water-soluble polymer, penetration buffer, preservative and the remaining solvent to obtain an activated solution; A3) mixing the activated solution and freeze-drying protective agent solution, and freeze-drying to obtain a thrombelastogram complex activated coagulation detection reagent; The pressure of high-pressure homogenization in steps S2) and A1) is 1000-1500 bar; the number of high-pressure homogenization is 5-10 times; The frequency of ultrasonic treatment in steps S3) and A1) is 10-30 kHz; the power of ultrasonic treatment is 400-600 W; the ultrasonic treatment is intermittent ultrasonic treatment; the single treatment time of the intermittent ultrasonic treatment is 1-10 s, the interval time is 1-10 s, and the total treatment time is 30-60 min; The freeze-drying comprises a pre-freezing stage, a sublimation stage and a drying stage; the temperature of the pre-freezing stage is -50℃; the time of the pre-freezing stage is 2-6 h; the sublimation stage is gradient sublimation; the gradient of the gradient sublimation is 5-10℃; the holding time of each gradient in the gradient sublimation is independently 1-3 h; the vacuum degree of the sublimation drying is 120-180 bar; the temperature of the drying stage is 15-25℃; the time of the drying stage is 1-3 h; and the vacuum degree of the drying stage is 30-80 bar.

2. The thromboelastography composite activated coagulation assay test reagents of claim 1, wherein, The preservative is selected from one or more of sodium azide, Proclin, potassium sorbate, methyl paraben, gentamicin sulfate and thiomersal.

3. The thromboelastography composite activated coagulation assay test reagents of claim 2, wherein, The preservative is selected from one or more of sodium azide, Proclin, potassium sorbate, methyl paraben, gentamicin sulfate and thiomersal.

4. The thromboelastography composite activated coagulation assay test reagents of claim 1, wherein, The diameter of the grinding medium in the wet ball milling in step S1) and step A1) is 0.1-0.5 mm; the time of the wet ball milling is 6-24 h; and the rotating speed of the wet ball milling is 300-500 rpm. The mass ratio of kaolin, grinding medium and part of solvent in step A1) is 1:(5-20):(3-8).

5. A thromboelastography activated coagulation test kit, characterized by, The thrombelastography composite activated coagulation detection reagent comprises the thrombelastography composite activated coagulation detection reagent according to any one of claims 1-4.

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