Activated blood coagulation detection reagent as well as preparation method and application thereof
By using a functional system composed of kaolin, dispersant, fixative, binder and humectant, the problems of water insolubility and stability of kaolin are solved, and the stability and repeatability of activated coagulation test reagent are achieved, which is suitable for thromboelastography activated coagulation test.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-04
- Publication Date
- 2026-03-13
AI Technical Summary
Existing activated coagulation test reagents suffer from poor repeatability and accuracy due to the water insolubility and stability issues of kaolin. While ellagic acid substitution offers some improvement, it is costly and prone to decomposition, and the poor rehydration properties of the three-dimensional network structure affect utilization.
The functional system, composed of kaolin, dispersant, fixative, linker and humectant, forms a stable dispersion system through electrostatic repulsion, electrostatic attraction and hydrogen bonding. Combined with buffer solution to maintain a suitable pH value, it ensures the stability and repeatability of coagulation reaction.
This method achieves uniform dispersion and long-term stability of kaolin particles, improves the repeatability and accuracy of coagulation reactions, and meets the stability requirements of clinical testing.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of coagulation testing, specifically to an activated coagulation testing reagent, its preparation method, and its application. Background Technology
[0002] Thromboelastography (TEG) is a key method for assessing overall coagulation function. Its TEG assay reagent typically consists of the endogenous activator kaolin and calcium ions. Kaolin activates the endogenous coagulation pathway to initiate the coagulation process, while calcium ions replenish the calcium ions consumed during sodium citrate anticoagulation. The two work synergistically to monitor the overall coagulation profile.
[0003] However, on the one hand, the water insolubility of kaolin makes it difficult to form a uniform and stable dispersion system in reagents. On the other hand, since the amount of kaolin in the reagent is usually small, the moisture in the reagent is easily evaporated during long-term storage, leading to kaolin precipitation or even drying out. These problems not only significantly reduce the repeatability of activation detection results but may also cause inaccurate results.
[0004] To address the challenges of water insolubility and stability of kaolin, existing technologies propose using ellagic acid as an endogenous activator instead of kaolin. While ellagic acid can partially solve the water solubility problem, it is expensive and photosensitive, leading to decomposition or activity reduction during storage and use, affecting the repeatability and accuracy of test results. Another existing technology improves the suspension of kaolin through a three-dimensional network structure; however, the poor rehydration properties of this structure make it difficult to swell and dissolve upon reconstitution with water, resulting in ineffective release and dispersion of the encapsulated kaolin particles, thus affecting the utilization rate of kaolin and consequently impacting the repeatability and accuracy of test results. Summary of the Invention
[0005] Therefore, the technical problem to be solved by the present invention is to overcome the defects of poor repeatability and accuracy of activated coagulation test reagents using kaolin in the prior art, thereby providing an activated coagulation test reagent, its preparation method and application.
[0006] In a first aspect, the present invention provides an activated coagulation detection reagent, comprising the following components: Kaolin, dispersant, fixative, binder, humectant, and buffer solution; The dispersant is selected from positively charged amino acid polymers; The fixative is selected from acrylic acid copolymers containing nitrogen heterocycles; The linker is selected from at least one of dialdehyde, epoxy-containing compounds, and compounds containing bis(succinimide) ester groups; The moisturizer is selected from polyhydroxy compounds; The activated coagulation test reagent contains 0.1 mg / mL to 0.2 mg / mL of kaolin, 0.1% to 5% of dispersant, 0.1% to 2% of fixative, 0.01% to 1% of binder, 0.1% to 20% of humectant, and the remainder is buffer solution.
[0007] Furthermore, the dispersant is selected from at least one of the following: homopolymer of lysine, homopolymer of arginine, homopolymer of histidine, copolymer of lysine and arginine, copolymer of lysine and histidine, copolymer of arginine and histidine, and copolymer of lysine and arginine and histidine.
[0008] In some alternative embodiments, the molecular weight of the dispersant is 2000-50000.
[0009] Furthermore, the fixative is selected from at least one of poly(4-vinylpyridine) copolyacrylic acid and poly(4-vinylpyrazole) copolyacrylic acid.
[0010] In some alternative embodiments, the molecular weight of the fixative is 2000-5000.
[0011] Furthermore, the binder is selected from at least one of glutaraldehyde and adipaldehyde.
[0012] Furthermore, the binder is selected from at least one of ethylene glycol glycidyl ether, glycerol glycidyl ether, bisphenol A diglycidyl ether, tetra-arm polyethylene glycol glycidyl ether, and poly(dimethylsiloxane) diglycidyl ether.
[0013] Furthermore, the linker is bis-PEG5-succinimide ester.
[0014] In some alternative embodiments, the molecular weight of the binder is 100-5000.
[0015] Furthermore, the moisturizer is selected from at least one of glycerin, propylene glycol, sorbitol, and hyaluronic acid.
[0016] Furthermore, the buffer solution is selected from at least one of 4-hydroxyethylpiperazine ethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, 3-(N-morpholino)propanesulfonic acid buffer, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer.
[0017] Furthermore, the pH of the buffer solution is 7.3-7.5.
[0018] Secondly, the present invention also provides a method for preparing the above-mentioned activated coagulation detection reagent, comprising the following steps: The components are mixed to obtain the activated coagulation test reagent.
[0019] Furthermore, the process of mixing the components includes: Kaolin and buffer solution are mixed and sonicated. Then, dispersant, fixative and humectant are added and stirred for the first time. Finally, binder is added and stirred for the second time.
[0020] In some optional embodiments, the components are mixed and then dispensed to obtain an activated coagulation test reagent; the dispensed volume is 15 μL-25 μL.
[0021] In some alternative implementations, the ultrasound duration is 1-3 hours.
[0022] In some alternative embodiments, the ultrasonic power is 200~400W.
[0023] In some alternative embodiments, the temperature of the first stirring is 20°C-30°C, the time is 12-16 hours, and the speed is 800-1000 rpm.
[0024] In some alternative embodiments, the temperature of the second stirring is 20°C-30°C, the time is 2-6 hours, and the speed is 800-1000 rpm.
[0025] Thirdly, the present invention also provides the application of the above-mentioned activated coagulation detection reagent or the activated coagulation detection reagent prepared by the above-mentioned activated coagulation detection reagent preparation method in thromboelastography activated coagulation detection.
[0026] In some alternative implementations, the method of application includes the following steps: The anticoagulated whole blood containing sodium citrate buffer is first mixed with activated coagulation test reagent, and then second mixed with calcium ion solution for detection.
[0027] In some optional embodiments, the concentration of the sodium citrate buffer solution is 10⁵ to 10⁹ mmol / L.
[0028] In some alternative embodiments, the calcium ion solution is selected from at least one of calcium chloride solution, calcium nitrate solution, and calcium gluconate solution.
[0029] In some optional embodiments, the volume ratio of anticoagulated whole blood containing sodium citrate buffer to activated coagulation test reagent is 1:(0.015-0.025).
[0030] In some optional embodiments, the ratio of the total volume of anticoagulated whole blood containing sodium citrate buffer to the volume of activated coagulation test reagent to the volume of calcium ion solution is (320-340):(15-25), and the concentration of calcium chloride solution is 0.1mol / L-0.3mol / L.
[0031] Fourthly, the present invention also provides an activated coagulation test kit, comprising the activated coagulation test reagent described above or an activated coagulation test reagent prepared by the preparation method of the activated coagulation test reagent described above.
[0032] In some alternative embodiments, a calcium chloride solution and / or a sodium citrate solution are also included.
[0033] In some alternative embodiments, the concentration of the calcium chloride solution is 0.1 mol / L to 0.3 mol / L.
[0034] In some alternative embodiments, the concentration of the sodium citrate solution is 105~109 mmol / L.
[0035] The technical solution of this invention has the following advantages: This invention provides an activated coagulation test reagent, comprising the following components: kaolin, a dispersant, a fixative, a binder, a humectant, and a buffer solution; the dispersant is selected from positively charged amino acid polymers; the fixative is selected from nitrogen-containing heterocyclic acrylic acid copolymers; the binder is selected from epoxy-containing siloxane compounds; the humectant is selected from polyols; the mass concentration of kaolin in the activated coagulation test reagent is 0.1 mg / mL-0.2 mg / mL, the mass fraction of the dispersant is 0.1%-5%, the mass fraction of the fixative is 0.1%-2%, the mass fraction of the binder is 0.01%-1%, the mass fraction of the humectant is 0.1%-20%, and the balance is a buffer solution. Kaolin, as a natural aluminum silicate mineral, is used to activate the coagulation reaction due to its high specific surface area and abundant negatively charged surface. The core function of the dispersant is to break the aggregation between kaolin particles through electrostatic repulsion, allowing each particle to fully contact the coagulation factors in the blood, ensuring the consistency and repeatability of the coagulation reaction. The fixative forms a stable composite layer with the dispersant through electrostatic attraction, tightly coating the surface of the kaolin particles. This prevents the kaolin particles from re-aggregating during long-term storage or use, ensuring the long-term stability of its coagulation activation properties. The linker enhances the binding force between polymer molecules through complexation, constructing a stable polymer network that ensures the uniform suspension of kaolin particles in the polymer matrix, providing a stable microenvironment for the coagulation reaction. The humectant locks in moisture in the system through hydrogen bonding, preventing evaporation. Simultaneously, the humectant interacts with other components in the system (such as polymers and dispersants) to maintain the colloidal stability of the system, preventing a decrease in kaolin activity due to drying. This ensures that the moisture content of the system remains within a suitable range for the coagulation reaction during storage or use. The activity of coagulation factors is extremely sensitive to pH. The core function of the buffer solution is to maintain a stable pH value, providing a suitable acid-base environment for the coagulation reaction. By stabilizing the pH value within the optimal range for coagulation factor activity, the efficient activation of the coagulation reaction by kaolin is guaranteed. In the technical solution of this invention, kaolin, as the core of coagulation activation, forms a functional system with dispersant, fixative, binder, humectant and buffer, which together ensure the stability, repeatability and efficiency of coagulation reaction.
[0036] This invention also provides a method for preparing an activated coagulation test reagent and its application. The method involves mixing the components to obtain the activated coagulation test reagent. The mixing process includes: mixing kaolin with a buffer solution, sonicating, then adding a dispersant, a fixative, and a humectant, followed by a first stirring; finally, adding a binder and performing a second stirring. The activated coagulation test reagent prepared by this method can be used for thromboelastography-based activated coagulation detection, exhibiting advantages such as good stability and high repeatability. Furthermore, thermal acceleration studies show that the activated coagulation test reagent prepared by this method effectively maintains its dispersibility and detection efficacy, providing a better option for clinical coagulation testing. Detailed Implementation
[0037] 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.
[0038] 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.
[0039] Example 1 This embodiment provides an activated coagulation test reagent, comprising the following components: Kaolin, dispersant, fixative, binder, humectant, and buffer solution; the activated coagulation test reagent contains 0.15 mg / mL kaolin, 0.5% dispersant, 0.4% fixative, 0.5% binder, 5% humectant, and 10 mL buffer solution.
[0040] The dispersant was a homopolymer of lysine (polylysine) with a molecular weight of 10,000; the immobilizer was poly(4-vinylpyrazole) copolyacrylic acid with a molecular weight of 5,000; the linker was diglycidyl ether-terminated poly(dimethylsiloxane) with a molecular weight of 2,000; the humectant was glycerol; and the buffer was 4-hydroxyethylpiperazine ethanesulfonic acid buffer (HEPES buffer) with a pH of 7.4 and a concentration of 50 mmol / L of 4-hydroxyethylpiperazine ethanesulfonic acid. All reagents were purchased from Shanghai Maclean Biochemical Technology Co., Ltd.
[0041] This embodiment also provides a method for preparing an activated coagulation test reagent, including the following steps: Kaolin was mixed with buffer solution and sonicated for 2 hours at 300W. Then, dispersant, fixative, and humectant were added, and the mixture was stirred at 25°C for 14 hours at 9000 rpm. Finally, binder was added, and the mixture was stirred at 25°C for another 4 hours at 9000 rpm. The mixture was then dispensed into 1 mL cryovials at a rate of 20 μL per sample to obtain the activated coagulation test reagent.
[0042] Examples 2-17 The activated coagulation test reagents provided in Examples 2-17 are basically the same as those in Example 1, except that the molecular weight or content of each component is adjusted. The specific formulations of Examples 2-17 are shown in Table 1.
[0043] Table 1. Specific formulations for Examples 2-17
[0044] Examples 18-49 The activated coagulation test reagents provided in Examples 18-49 are basically the same as those in Example 1, except that the molecular weight or content of each component is adjusted. The specific formulations of Examples 18-49 are shown in Table 2. In Examples 18-49, the mass concentration of kaolin is 0.15 mg / mL, the molecular weight of the diglycidyl ether-terminated poly(dimethylsiloxane) is 2000, the mass fraction is 0.4%, and the mass fraction of glycerol is 5%.
[0045] Table 2 Specific formulations of Examples 18-49
[0046] Comparative Example 1 This comparative example provides an activated coagulation test reagent, keeping other conditions unchanged in Example 1, and omitting the addition of dispersant, fixative, binder, and humectant in Example 1.
[0047] Comparative Example 2 This comparative example provides an activated coagulation test reagent, keeping other conditions unchanged in Example 1 and omitting the addition of the binder in Example 1.
[0048] Comparative Example 3 This comparative example provides an activated coagulation test reagent, keeping other conditions unchanged in Example 1 and omitting the addition of the humectant in Example 1.
[0049] Comparative Example 4 This comparative example provides an activated coagulation test reagent, keeping other conditions unchanged in Example 1 and omitting the addition of fixative in Example 1.
[0050] Comparative Example 5 This comparative example provides an activated coagulation test reagent, keeping other conditions unchanged in Example 1 and omitting the addition of the dispersant in Example 1.
[0051] Test Example 1 The activated coagulation test reagents from Example 1 and Comparative Examples 1-5 were tested. The test method was as follows: One healthy volunteer was randomly selected, and one tube of whole blood (10 mL, concentration of sodium citrate buffer 109 mmol / L) was drawn from the antecubital vein using a sodium citrate anticoagulation blood collection tube. 1 mL of whole blood was added to 20 μL of the activated coagulation test reagent from Example 1, and the mixture was inverted and mixed 5 times to obtain a sample. 330 μL of the sample was added to a test cup containing 20 μL of calcium chloride solution (concentration 0.2 mol / L), and automatic detection was performed using a thromboelastography instrument. The detection parameters and normal reference ranges are shown in Table 3. The above test steps were repeated twice, for a total of 3 tests. Then, based on the results of the 3 tests, the repeatability (CV, CV = SD / mean) was calculated, and the CV values of the four detection parameters were averaged to obtain the total CV. The activated coagulation test reagents in Comparative Examples 1-5 were tested using the same method, and the test results of the activated coagulation test reagents in Example 1 and Comparative Examples 1-5 are shown in Table 4.
[0052] Table 3. Detection parameters and normal reference ranges
[0053] In Table 3, R refers to the time required from the start of the test to fibrin formation (coagulation initiation), K refers to the time from the end of the R value to the clot strength reaching an amplitude of 20 mm (reflecting the coagulation formation rate), Angle refers to the angle between the tangent of the rising segment of the coagulation curve and the horizontal line, and MA refers to the maximum strength of the clot (reflecting the stability of the blood clot).
[0054] Table 4. Determination results of activated coagulation assay reagent in Example 1 and Comparative Examples 1-5.
[0055] As can be seen from Table 4, compared with the comparative example, the total CV of the four detection parameters indicates that the detection repeatability of the activated coagulation test reagent in Example 1 is better.
[0056] Test Example 2 The activated coagulation test reagents from Examples 2-17 and Comparative Example 1 were tested. The test method is as follows: the total repeatability (total CV) was calculated using the same method as in Test Example 1, and the test results of the activated coagulation test reagents from Examples 2-17 and Comparative Example 1 were obtained, as shown in Table 5.
[0057] Table 5. Determination results of activated coagulation test reagent in Examples 2-17 and Comparative Example 1
[0058] The activated coagulation test reagents in Examples 18-49 and Comparative Example 1 were applied for testing. The test method is as follows: the total repeatability (total CV) was calculated using the same method as in Test Example 1, and the test results of the activated coagulation test reagents in Examples 18-49 and Comparative Example 1 were obtained, as shown in Table 6.
[0059] Table 6. Determination results of activated coagulation assay reagent in Examples 18-49 and Comparative Example 1
[0060] As can be seen from Tables 5 and 6, compared with Comparative Example 1, Examples 2-49 all showed good total CV results. Therefore, within the range of component types, molecular weights and contents defined by this invention, the repeatability of detection can be improved.
[0061] Test Example 3 It is generally believed that the rate of a chemical reaction doubles for every 10°C increase in temperature; therefore, 3 months at 37°C is equivalent to approximately 22 months at 8°C. The activated coagulation test reagents prepared in Example 1 and Comparative Example 1 were placed in a 37°C oven and tested on day 0, day 14, month 1, month 2, and month 3. One healthy volunteer was randomly selected for each test, and the same method as in Example 1 was used. Each group was tested 5 times. The test results are recorded in Table 7.
[0062] Table 7 Test Results
[0063] As can be seen from Table 7, the total CV value detected in Example 1 performed well throughout the entire testing period, meeting the stability requirements of this type of product stored at 2~8℃ for 18 months on the market.
[0064] Test Example 4 The activated coagulation test reagent prepared in Example 1 and the commercially available activated coagulation test reagent (TEG brand activated coagulation test kit (coagulation method)) were placed in a 50°C oven and tested on day 0 and month 4, respectively. One healthy volunteer was randomly selected for each test, and the same method as in Example 1 was used for testing. Each group was tested 10 times. The test results are recorded in Table 8.
[0065] Table 8 Test Results
[0066] As can be seen from Table 8, the test results of Example 1 are basically consistent with those of commercially available products, with a deviation of no more than ±10%, demonstrating good stability and accuracy.
[0067] 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. An activated coagulation test reagent, characterized in that, Includes the following components: Kaolin, dispersant, fixative, binder, humectant, and buffer solution; The dispersant is selected from positively charged amino acid polymers; The fixative is selected from acrylic acid copolymers containing nitrogen heterocycles; The linker is selected from at least one of dialdehyde, epoxy-containing compounds, and compounds containing bis(succinimide) ester groups; The moisturizer is selected from polyhydroxy compounds; The activated coagulation test reagent contains 0.1 mg / mL to 0.2 mg / mL of kaolin, 0.1% to 5% of dispersant, 0.1% to 2% of fixative, 0.01% to 1% of binder, 0.1% to 20% of humectant, and the remainder is buffer solution.
2. The activated coagulation test reagent according to claim 1, characterized in that, The dispersant is selected from at least one of the following: homopolymer of lysine, homopolymer of arginine, homopolymer of histidine, copolymer of lysine and arginine, copolymer of lysine and histidine, copolymer of arginine and histidine, and copolymer of lysine and arginine and histidine. Optionally, the molecular weight of the dispersant is 2000-50000.
3. The activated coagulation test reagent according to claim 1, characterized in that, The fixative is selected from at least one of poly(4-vinylpyridine) copolyacrylic acid and poly(4-vinylpyrazole) copolyacrylic acid; Optionally, the molecular weight of the fixative is 2000-5000.
4. The activated coagulation test reagent according to claim 1, characterized in that, The binder is selected from at least one of glutaraldehyde and adipaldehyde; and / or, The binder is selected from at least one of ethylene glycol glycidyl ether, glycerol glycidyl ether, bisphenol A diglycidyl ether, tetra-arm polyethylene glycol glycidyl ether, and poly(dimethylsiloxane) diglycidyl ether; and / or, The binder is bis-PEG5-succinimide ester; Optionally, the molecular weight of the binder is 100-5000.
5. The activated coagulation test reagent according to claim 1, characterized in that, The moisturizer is selected from at least one of glycerin, propylene glycol, sorbitol, and hyaluronic acid.
6. The activated coagulation test reagent according to claim 1, characterized in that, The buffer solution is selected from at least one of 4-hydroxyethylpiperazine ethanesulfonic acid buffer, tris(hydroxymethyl)aminomethane hydrochloride buffer, 3-(N-morpholino)propanesulfonic acid buffer, and N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid buffer. And / or, the pH of the buffer solution is 7.3-7.
5.
7. The method for preparing the activated coagulation detection reagent according to any one of claims 1-6, characterized in that, Includes the following steps: The components are mixed to obtain the activated coagulation test reagent.
8. The method for preparing the activated coagulation detection reagent according to claim 7, characterized in that, The process of mixing the components includes: Kaolin was mixed with buffer solution and sonicated. Then, dispersant, fixative and humectant were added and stirred for the first time. Finally, binder was added and stirred for the second time. Optionally, the ultrasound duration is 1-3 hours; Optionally, the ultrasonic power is 200~400W; Optionally, the temperature of the first stirring is 20℃-30℃, the time is 12-16h, and the speed is 800~1000rpm; Optionally, the temperature of the second stirring is 20℃-30℃, the time is 2-6h, and the speed is 800~1000rpm.
9. The application of the activated coagulation detection reagent according to any one of claims 1-6 or the activated coagulation detection reagent prepared by the preparation method according to claim 7 or 8 in thromboelastography activated coagulation detection, characterized in that, The method of application includes the following steps: The anticoagulated whole blood containing sodium citrate buffer is first mixed with the activated coagulation test reagent, and then second mixed with calcium ion solution for detection. Optionally, the concentration of the sodium citrate buffer solution is 10⁵~10⁹ mmol / L; Optionally, the calcium ion solution is selected from at least one of calcium chloride solution, calcium nitrate solution, and calcium gluconate solution; Optionally, the volume ratio of anticoagulated whole blood containing sodium citrate buffer to activated coagulation test reagent is 1:(0.015-0.025). Optionally, the ratio of the total volume of anticoagulated whole blood containing sodium citrate buffer to the volume of activated coagulation test reagent to calcium ion solution is (320-340):(15-25), and the concentration of calcium chloride solution is 0.1mol / L-0.3mol / L.
10. An activated coagulation test kit, characterized in that, The activated coagulation test reagent includes the activated coagulation test reagent according to any one of claims 1-6 or the activated coagulation test reagent prepared by the preparation method according to claim 7 or 8; Optionally, it may also include calcium chloride solution and / or sodium citrate solution; Optionally, the concentration of the calcium chloride solution is 0.1 mol / L to 0.3 mol / L; Optionally, the concentration of the sodium citrate solution is 105~109 mmol / L.