Thrombelastogram heparin detection reagent and application thereof
By immobilizing heparinase with aminopolysaccharides, the problems of heparinase adsorption on the inner wall of the sample cup and detachment of lyophilized reagents were solved, thus achieving high efficiency and accuracy in thromboelastography detection.
Patent Information
- Application Number
- CN202512049477.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-31
- Publication Date
- 2026-05-19
AI Technical Summary
In existing technologies, heparinase adsorption on the inner wall of the sample cup leads to reduced activity, and lyophilized reagents are prone to detachment during storage and transportation, affecting the repeatability and accuracy of detection.
Aminopolysaccharides are used as base reagents. Heparinase is immobilized by forming hydrogen bonds and molecular entanglement with the inner wall of the sample cup, thus protecting its activity and allowing it to be released rapidly during detection.
This improved the accuracy and repeatability of the test, reduced the adsorption loss of heparinase, and ensured the stability of the test results.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of medical device technology, specifically to a thromboelastography heparin detection reagent and its application. Background Technology
[0002] Thromboelastography (TEG) is a comprehensive method for assessing the dynamic processes of coagulation and fibrinolysis. It provides a complete evaluation of factors such as the formation rate, dissolution state, firmness, stability, elasticity, and coagulation factors, platelet count, and function of fibrin clots. The thromboelastography heparinase cup provides qualitative and quantitative results after heparinase neutralizes heparin in the blood. Compared to traditional methods, it offers advantages such as reflecting the overall coagulation process, high sensitivity for heparin-based drugs, and the ability to detect low molecular weight heparin. It is an important method for evaluating the efficacy of heparin, low molecular weight heparin, and other heparin-based anticoagulants in heparin-treated patients, as well as assessing heparin residues. Clinicians can adjust treatment plans promptly based on the test results, improving treatment success rates.
[0003] When using thromboelastography (TEG) with heparinase cups, the heparinase in the cups degrades heparin, low molecular weight heparin, and other heparin-based anticoagulants in the blood sample, causing blood clotting. By comparing the R2 value of the heparinase cup with the R1 value of a regular cup, the presence of residual heparin in the blood sample can be determined. This method reflects the body's true baseline coagulation function after the direct effects of heparin have been removed, and is used to assess the level of residual heparin in the body and to identify the cause of bleeding.
[0004] In current technology, heparinase is loaded into the sample cup via lyophilization. However, due to process limitations, two problems arise: First, during detection, the inner wall of the sample cup strongly adsorbs heparinase and other proteins. The adsorbed heparinase molecules lose their original free movement and cannot fully participate in the detection reaction, resulting in the actual amount of enzyme that plays a role being far lower than the theoretical value. This interferes with heparinase activity and reduces the repeatability and accuracy of the detection. Second, during storage and transportation, the lyophilized reagent is prone to detaching from the bottom of the cup or even spilling out, reducing the amount of reagent in the sample cup. During detection, insufficient reagent directly leads to incomplete reaction, and the difference in reagent amount causes inconsistent reaction degrees in different detections. Ultimately, this significantly reduces the repeatability of the detection results, and the accuracy deviates from the true value, providing misleading information for clinical diagnosis or experimental analysis. Summary of the Invention Therefore, the technical problem to be solved by the present invention is to overcome the defects in the prior art where the adsorption of the inner wall of the sample cup interferes with the activity of heparinase and the heparinase reagent is easily detached during storage and transportation, resulting in a reduction in quantity, thereby providing a thromboelastography heparin detection reagent and its application.
[0005] Therefore, the present invention provides the following technical solution: In a first aspect, the present invention provides a thromboelastography heparin detection reagent, comprising a base reagent and a heparinase reagent; wherein the base reagent is an aminopolysaccharide substance.
[0006] In one optional embodiment, the aminopolysaccharide is at least one of aminoglucan, hyaluronic acid, chondroitin sulfate of porcine or bovine origin, dermatan sulfate, and keratin sulfate.
[0007] In one optional embodiment, the mass concentration of the aminopolysaccharide is 1 mg / mL to 50 mg / mL.
[0008] In one optional embodiment, the molecular weight of the aminopolysaccharide is 5,000 to 800,000.
[0009] In one optional embodiment, the molecular weight of the aminoglucan is 50,000 to 200,000.
[0010] In one optional embodiment, the molecular weight of the hyaluronic acid is 40,000 to 800,000; preferably 200,000 to 400,000.
[0011] In one optional embodiment, the molecular weight of the porcine or bovine chondroitin sulfate is 5,000-50,000.
[0012] In one alternative embodiment, the dermatin sulfate has a molecular weight of 10,000-40,000.
[0013] In one alternative embodiment, the keratin sulfate has a molecular weight of 5,000 to 100,000.
[0014] In one optional embodiment, the heparinase reagent includes heparinase, sugars, proteins, antioxidants, preservatives, and buffer salts.
[0015] In one optional embodiment, the mass concentration of the heparinase is 10 U to 200 U / mL; preferably 10 U / mL.
[0016] In one optional embodiment, the mass concentration of the sugar is 10 mg / mL to 100 mg / mL; preferably 20 mg / mL.
[0017] In one optional embodiment, the mass concentration of the protein is 5 mg / mL to 100 mg / mL; preferably 20 mg / mL.
[0018] In one optional embodiment, the antioxidant has a mass concentration of 0.1 mg / mL to 5 mg / mL; preferably 1 mg / mL.
[0019] In one optional embodiment, the mass concentration of the preservative is 0.01 mg / mL to 1 mg / mL; preferably 0.5 mg / mL.
[0020] In one optional embodiment, the buffer salt has a mass concentration of 10 mM to 100 mM and a pH of 7-8.
[0021] In one optional embodiment, the sugar is at least one selected from sucrose, trehalose, maltose, lactose, and galactose; preferably maltose.
[0022] In one optional embodiment, the protein is at least one of bovine serum albumin, fetal bovine serum, and casein; preferably bovine serum albumin.
[0023] In one optional embodiment, the antioxidant is at least one selected from ascorbic acid, sodium ascorbate, calcium ascorbate, isoascorbic acid, sodium isoascorbate, and calcium isoascorbate; preferably isoascorbic acid.
[0024] In one optional embodiment, the preservative is at least one of sodium azide, sodium benzoate, and Proclin 300; preferably Proclin 300.
[0025] In one optional embodiment, the buffer salt is at least one of phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane buffer.
[0026] Secondly, the present invention provides the application of the above-mentioned thromboelastography heparin detection reagent in the preparation of thromboelastography heparinase cups.
[0027] In one alternative implementation, the application includes the following steps: Step S1: Preparation of substrate reagents; Step S2: Preparation of heparinase reagent; Step S3: Dispense the base reagent and dry; Step S4: Dispense the heparinase reagent and dry it to obtain the final product.
[0028] In one alternative embodiment, step S1 involves dissolving the aminopolysaccharide in water.
[0029] In one optional embodiment, step S2 involves dissolving heparinase, sugars, proteins, and preservatives in a buffer salt solution, and adding an antioxidant before use.
[0030] In one optional embodiment, the volume of the product dispensed in step S3 is 45-55 μL, and the product is dried at room temperature.
[0031] In one optional embodiment, the volume of the product dispensed in step S4 is 25-35 μL, and the drying is performed by vacuum freeze drying.
[0032] The technical solution of this invention has the following advantages: This invention provides a thromboelastography heparin detection reagent, comprising a base reagent and a heparinase reagent; the base reagent is an aminopolysaccharide. The combination of these two reagents in this invention not only effectively immobilizes the heparinase reagent in the sample cup, reducing the adsorption of heparinase on the inner wall of the sample cup and protecting heparinase activity, but also allows for rapid release of the heparinase reagent during detection.
[0033] This invention also provides the application of the aforementioned thromboelastography heparin detection reagent in the preparation of thromboelastography heparinase cups. The aminopolysaccharides in the base reagent, with their abundant hydroxyl and amino groups, form strong hydrogen bonds with polar sites on the inner wall of the sample cup (such as hydroxyl and carboxyl groups on glass or plastic surfaces), allowing them to be uniformly and firmly adsorbed and fixed onto the inner wall of the sample cup. The heparinase reagent is used to protect the activity of heparinase and ensure its rapid release into the sample during detection. When the heparinase reagent is introduced into the system, the aminopolysaccharides play a role again. On one hand, the polar groups in the aminopolysaccharide molecules form new hydrogen bonds with the polar amino acid residues in the heparinase reagent molecules (such as the hydroxyl groups of serine and the amide groups of asparagine), further shortening the distance between them through intermolecular electrostatic attraction. On the other hand, the aminopolysaccharides typically have a long-chain molecular structure, which intertwines with the molecular chains of the heparinase reagent. This molecular entanglement not only increases the contact area between the two but also restricts the free movement of the heparinase reagent through steric hindrance. Combined with hydrogen bonding, this creates a synergistic effect, fixing the heparinase reagent onto the aminopolysaccharide coating. This provides a stable reaction environment for subsequent biochemical reactions, effectively improving detection efficiency and accuracy. Detailed Implementation
[0034] 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.
[0035] 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.
[0036] All reagents used were purchased from Beijing Innocare Technology Co., Ltd.
[0037] The ordinary cup reagents used in the following examples and comparative examples were all prepared using the following methods: ① Reagent 1: Dissolve 12 mg of kaolin in 40 mL of physiological saline, stir at room temperature for 24 h, and the final concentration of kaolin is 0.3 mg / mL. The aliquot volume is 10 μL / tube. ② Reagent 2: Dissolve 221.96 mg of calcium chloride in 10 mL of physiological saline, stir at room temperature for 24 h, and the final concentration of calcium chloride is 0.2 M. The aliquot volume is 1 mL / tube.
[0038] Example 1 This embodiment provides a thromboelastography heparin detection reagent and its application in the preparation of thromboelastography heparinase cups.
[0039] The heparinase cup of this invention comprises: ③ Base reagent: Dissolve 50 mg of hyaluronic acid (molecular weight 100,000) in 10 mL of purified water, stirring at room temperature for 24 h to achieve a final hyaluronic acid concentration of 5 mg / mL; ④ Heparinase reagent: Dissolve heparinase (final concentration 10 U / mL), maltose (final concentration 20 mg / mL), bovine serum albumin (final concentration 20 mg / mL), and Proclin 300 (final concentration 0.5 mg / mL) in 10 mL of 50 mM pH 7.4 phosphate buffer, stirring at room temperature for 24 h to dissolve. Before use, add isoascorbic acid (final concentration 1 mg / mL) and stir at room temperature for 5 min to dissolve. The sample cup for heparin detection reagent is treated with a nitrogen-based plasma cleaner for 1 min, then left to stand for 30 min. Add reagent ③ at a dispensing volume of 50 μL / cup and air dry at room temperature for 24 h. Then, add reagent ④ at a dispensing volume of 30 μL / cup, place the test cup in a vacuum freeze dryer, freeze at -50℃ for 30 min, then turn on the vacuum and vacuum dry for 24 h. Remove the sample cup, add the cup lid, and seal it with plastic to obtain the heparinase cup of this invention.
[0040] The control reagents were commercially available TEG heparinase cups (control 1, heparinase content of 50 U / mL) and the liquid reagent of the heparin detection reagent of the present invention (control 2, non-lyophilized state).
[0041] Both the thromboelastography instrument and the testing cup are products of Beijing Ruizhen Biotechnology Co., Ltd.
[0042] Ten volunteers using heparin were selected, and whole blood was drawn from the antecubital vein. One tube of anticoagulated whole blood was drawn from each volunteer, and sodium citrate was used for anticoagulation (10 mL / tube). Heparinase cups prepared according to this invention and controls were simultaneously used for comparative testing. During testing, three sets of ordinary reagent test cups and three sets of heparinase reagent test cups were loaded onto a thromboelastography instrument. 20 μL of ② was added to each of the six cups. 1 mL of sodium citrate-anticoagulated whole blood was taken from each of the three ① aliquot tubes, mixed by inversion, and then 330 μL of sample was sequentially transferred from each ① aliquot tube to one set of ordinary reagent test cups and one set of heparinase reagent test cups, and monitoring was started sequentially for 30 minutes. 30 μL of ④ was added to the heparinase reagent test cup of control 2. The R value was recorded after monitoring.
[0043] The test results are shown in Table 1 below. As can be seen from the table, the deviations of the present invention and control 2 from control 1 are all within ±6.5%, indicating that the present invention can achieve the same detection effect as commercially available products with a lower heparinase dosage. Moreover, lyophilization did not have a significant impact on the detection results of the heparinase detection reagent of the present invention. Therefore, it can be seen that the heparinase activity of the present invention is effectively maintained after lyophilization.
[0044] Table 1 Detection results of Example 1
[0045] Examples 2-3 The differences between Examples 2 and 3 and Example 1 are shown in Table 2 below: Table 2. Differences between Examples 2 and 3 and Example 1
[0046] Examples 4-11 The differences between Examples 4-11 and Example 1 are shown in Table 3 below: Table 3. Differences between Examples 4-11 and Example 1
[0047] The control reagent used was Control 1 from Example 1, and the detection was performed according to the detection method in Example 1. The detection results are shown in Tables 4-7 below: Table 4. Detection results of Examples 2-4
[0048] Table 5. Detection results of Examples 5-7
[0049] Table 6. Detection results of Examples 8-10
[0050] Table 7 Detection results of Example 11
[0051] Comparative Example 1 In this comparative example, all other conditions remained unchanged in Example 1, and the addition of the substrate reagent in Example 1 was omitted. Tests were performed according to the method in Example 1, and the results are shown in the table below.
[0052] The test results are shown in Table 8 below. It can be seen from the table that Comparative Example 1 has the adverse effect of impaired activity of heparinase due to the lack of base reagent. Therefore, the deviation of Comparative Example 1 from Example 1 is mostly greater than ±5%, indicating that the base reagent of the present invention has a good sealing effect.
[0053] Table 8 Detection results of Comparative Example 1
[0054] Experimental Example 1 The fixation effect of the detection reagent of the present invention was compared by simulating an accidental drop during transportation. The heparinase cup of the present invention in Examples 1-11 and the commercially available TEG heparinase cup in Control 1 of Example 1 were dropped from a height of 1.5m in a free fall, and the drop was repeated 50 times.
[0055] For each reagent test, five volunteers using heparin were selected. Whole blood was drawn from the antecubital vein, and two tubes of anticoagulated whole blood were drawn from each volunteer, with sodium citrate anticoagulation (10 mL / tube). Repeatability tests (each sample was repeated 5 times) were performed simultaneously using the reagents prepared in Examples 1-11 of this invention and Control 1. Simultaneously, comparative tests were performed using reagents that had not been dropped (row B in the table below). For specific testing methods, refer to Example 1.
[0056] The test results are shown in Table 9 below. As can be seen from the table, the CV values of the ordinary cup test reagent without drop treatment are all within 5%. Compared with the heparinase cup of control 1, the heparinase cup prepared by this invention has smaller mean CV values of R1, R2, and (R1-R2) after simulated drop treatment. This result indicates that the heparinase cup test reagent obtained by this invention has a good fixation effect within the test cup.
[0057] Table 9 Detection results of Experiment Example 1
[0058] Experiment Example 2: Simulating an accidental drop during transportation to investigate the fixation effect of the substrate reagent on the heparinase reagent. The heparinase cups from Examples 1-11 and Comparative Example 1 were used for testing. They were dropped from a height of 1.5m in a free-fall manner, and the drop was repeated 50 times.
[0059] For each reagent test, five volunteers using heparin were selected. Whole blood was drawn from the antecubital vein, with two tubes of anticoagulated whole blood drawn from each volunteer, using sodium citrate anticoagulation (10 mL / tube). Repeatability tests (5 times per sample) were performed simultaneously using the heparin test reagents from Examples 1-11 and Comparative Example 1. Comparative tests were also performed using reagents that had not been dropped (row B in the table below). Specific testing methods are detailed in Example 1.
[0060] The test results are shown in Table 10 below. It can be seen from the table that: due to the lack of fixation effect of the substrate treatment reagent, the repeatability of R2 and (R1-R2) of Comparative Example 1 is worse than that of all embodiments of the present invention, indicating that the substrate reagent of the present invention has a good fixation effect.
[0061] Table 10 Detection results of Experiment Example 2
[0062] Experiment Example 3: High Temperature Accelerated Experiment The heparinase cups of the present invention (Examples 1-11) and the commercially available TEG heparinase cup (Control 1) were placed in a 37°C oven and tested on day 0, day 14, month 1, month 2, and month 3. High temperature was used to simulate the effect of long-term storage at room temperature in a shorter time. It is generally believed that the chemical reaction rate doubles for every 10°C increase in temperature; therefore, 3 months at 37°C is equivalent to approximately 22 months at room temperature (8°C). These results meet the stability requirement of commercially available products of this type for 18 months of storage at 2-8°C.
[0063] For each test, one healthy volunteer was selected to have whole blood drawn from their antecubital vein. One tube (10 mL) was drawn using a sodium citrate anticoagulant blood collection tube. The testing method is the same as in Example 1.
[0064] The test results are shown in Table 11 below. As can be seen from the table, the heparinase cup of the present invention has good thermal stability.
[0065] Table 11 Detection results of Experiment Example 3
[0066] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A thromboelastography heparin detection reagent, characterized in that, It includes a base reagent and a heparinase reagent; the base reagent is an aminopolysaccharide.
2. The thromboelastography heparin detection reagent according to claim 1, characterized in that, The aminopolysaccharide is at least one of aminoglucan, hyaluronic acid, chondroitin sulfate of porcine or bovine origin, dermatin sulfate, and keratin sulfate.
3. The thromboelastography heparin detection reagent according to claim 2, characterized in that, The mass concentration of the aminopolysaccharide is 1 mg / mL to 50 mg / mL; And / or, the molecular weight of the aminopolysaccharide is 5,000 to 800,000.
4. The thromboelastography heparin detection reagent according to claim 2, characterized in that, The molecular weight of the aminoglucan is 50,000 to 200,000; And / or, the molecular weight of the hyaluronic acid is 40,000 to 800,000; preferably 200,000 to 400,000; And / or, the molecular weight of the porcine or bovine chondroitin sulfate is 5,000 to 50,000; And / or, the molecular weight of the dermatin sulfate is 10,000 - 40,000; And / or, the molecular weight of the keratin sulfate is 5,000 - 100,000.
5. The thromboelastography heparin detection reagent according to claim 1, characterized in that, The heparinase reagent includes heparinase, sugars, proteins, antioxidants, preservatives, and buffer salts.
6. The thromboelastography heparin detection reagent according to claim 5, characterized in that, The mass concentration of the heparinase is 10 U to 200 U / mL; preferably 10 U / mL. And / or, the mass concentration of the sugar is 10 mg / mL to 100 mg / mL; preferably 20 mg / mL; And / or, the mass concentration of the protein is 5 mg / mL to 100 mg / mL; preferably 20 mg / mL; And / or, the mass concentration of the antioxidant is 0.1 mg / mL to 5 mg / mL; preferably 1 mg / mL; And / or, the mass concentration of the preservative is 0.01 mg / mL to 1 mg / mL; preferably 0.5 mg / mL; And / or, the mass concentration of the buffer salt is 10mM to 100mM, and the pH is 7-8.
7. The thromboelastography heparin detection reagent according to claim 5 or 6, characterized in that, The sugar is at least one selected from sucrose, trehalose, maltose, lactose, and galactose; preferably maltose. And / or, the protein is at least one of bovine serum albumin, fetal bovine serum, and casein; preferably bovine serum albumin; And / or, the antioxidant is at least one of ascorbic acid, sodium ascorbate, calcium ascorbate, isoascorbic acid, sodium isoascorbate, and calcium isoascorbate; preferably isoascorbic acid; And / or, at least one of the preservatives sodium azide, sodium benzoate, and Proclin 300; preferably Proclin 300; And / or, the buffer salt is at least one of phosphate buffer, 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and tris(hydroxymethyl)aminomethane buffer.
8. The use of the thromboelastography heparin detection reagent according to any one of claims 1 to 7 in the preparation of thromboelastography heparinase cups.
9. The application according to claim 8, characterized in that, Includes the following steps: Step S1: Preparation of substrate reagents; Step S2: Preparation of heparinase reagent; Step S3: Dispense the base reagent and dry; Step S4: Dispense the heparinase reagent and dry it to obtain the final product.
10. The application according to claim 9, characterized in that, Step S1 involves dissolving the aminopolysaccharide substance in water; And / or, step S2 involves dissolving heparinase, sugars, proteins, and preservatives in a buffer salt solution, and adding an antioxidant before use; And / or, the volume of the dispensing in step S3 is 45-55 μL, and the drying is carried out at room temperature; And / or, the volume of the dispensing in step S4 is 25-35 μL, and the drying is performed by vacuum freeze drying.